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PSMD1 and PSMD2 regulate HepG2 cell proliferation and apoptosis via modulating cellular lipid droplet metabolism
Obesity and nonalcoholic steatohepatitis (NASH) are well-known risk factors of hepatocellular carcinoma (HCC). The lipid-rich environment enhances the proliferation and metastasis abilities of tumor cells. Pre...
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The effect of BACE1-AS on β-amyloid generation by regulating BACE1 mRNA expression
The BACE1 antisense transcript (BACE1-AS) is a conserved long noncoding RNA (lncRNA). The level of BACE1-AS is significantly increased and the level of the BACE1 mRNA is slightly increased in subjects with AD....
Overlapping transcriptional expression response of wheat zinc-induced facilitator-like transporters emphasize important role during Fe and Zn stress
Hexaploid wheat is an important cereal crop that has been targeted to enhance grain micronutrient content including zinc (Zn) and iron (Fe). In this direction, modulating the expression of plant transporters i...
MiR-32-5p influences high glucose-induced cardiac fibroblast proliferation and phenotypic alteration by inhibiting DUSP1
The current study aimed to investigate the effects of miR-32-5p on cardiac fibroblasts (CFs) that were induced with high levels of glucose; we also aimed to identify the potential mechanisms involved in the re...
Correction to: A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone
The original article [1] contains three erroneous mentions of usage of a restriction enzyme— Bst Z17I—in the Methods section as displayed in the following sentences.
The original article was published in BMC Molecular Biology 2018 19 :3
Comparison of miRNA - 101a - 3p and miRNA - 144a - 3p regulation with the key genes of alpaca melanocyte pigmentation
Many miRNA functions have been revealed to date. Single miRNAs can participate in life processes by regulating more than one target gene, and more than one miRNA can also simultaneously act on one target mRNA....
Correction to: MicroRNA-325-3p protects the heart after myocardial infarction by inhibiting RIPK3 and programmed necrosis in mice
The original article [1] contains an error whereby Fig. 7 displays incorrect results; the correct version of Fig. 7 can be viewed ahead in this Correction article and should be considered in place of the origi...
The original article was published in BMC Molecular Biology 2019 20 :17
MicroRNA-325-3p protects the heart after myocardial infarction by inhibiting RIPK3 and programmed necrosis in mice
Receptor-interacting serine-threonine kinase 3 (RIPK3)-mediated necroptosis has been implicated in the progression of myocardial infarction (MI), but the underlying mechanisms, particularly whether microRNAs (...
The Correction to this article has been published in BMC Molecular Biology 2019 20 :18
Giant group I intron in a mitochondrial genome is removed by RNA back-splicing
The mitochondrial genomes of mushroom corals (Corallimorpharia) are remarkable for harboring two complex group I introns; ND5-717 and COI-884. How these autocatalytic RNA elements interfere with mitochondrial ...
Exploration of carbohydrate binding behavior and anti-proliferative activities of Arisaema tortuosum lectin
Lectins have come a long way from being identified as proteins that agglutinate cells to promising therapeutic agents in modern medicine. Through their specific binding property, they have proven to be anti-ca...
Characterization of cadmium-responsive MicroRNAs and their target genes in maize ( Zea mays ) roots
Current research has shown that microRNAs (miRNAs) play vital roles in plant response to stress caused by heavy metals such as aluminum, arsenic, cadmium (Cd), and mercury. Cd has become one of the most hazard...
Identification and validation of reference genes for real-time quantitative RT-PCR analysis in jute
With the availability of genome sequences, gene expression analysis of jute has drawn considerable attention for understanding the regulatory mechanisms of fiber development and improving fiber quality. Gene e...
Small nucleolar RNA Sf-15 regulates proliferation and apoptosis of Spodoptera frugiperda Sf9 cells
Small nucleolar RNAs (snoRNAs) function in guiding 2′- O -methylation and pseudouridylation of ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). In recent years, more and more snoRNAs have been found to play ...
Key genes differential expressions and pathway involved in salt and water-deprivation stresses for renal cortex in camel
Camels possess the characteristics of salt- and drought-resistances, due to the long-time adaption to the living environment in desert. The camel resistance research on transcriptome is rare and deficient, esp...
Development of a novel selection/counter-selection system for chromosomal gene integrations and deletions in lactic acid bacteria
The underlying mechanisms by which probiotic lactic acid bacteria (LAB) enhance the health of the consumer have not been fully elucidated. Verification of probiotic modes of action can be achieved by using sin...
Selection of reference genes for the quantitative real-time PCR normalization of gene expression in Isatis indigotica fortune
Isatis indigotica , a traditional Chinese medicine, produces a variety of active ingredients. However, little is known about the key genes and corresponding expression profiling involved in the biosynthesis pathwa...
MEF2A alters the proliferation, inflammation-related gene expression profiles and its silencing induces cellular senescence in human coronary endothelial cells
Myocyte enhancer factor 2A (MEF2A) plays an important role in cell proliferation, differentiation and survival. Functional deletion or mutation in MEF2A predisposes individuals to cardiovascular disease mainly...
Transcriptomic responses to grazing reveal the metabolic pathway leading to the biosynthesis of domoic acid and highlight different defense strategies in diatoms
A major cause of phytoplankton mortality is predation by zooplankton. Strategies to avoid grazers have probably played a major role in the evolution of phytoplankton and impacted bloom dynamics and trophic ene...
RNA sequencing, selection of reference genes and demonstration of feeding RNAi in Thrips tabaci (Lind.) (Thysanoptera: Thripidae)
Thrips tabaci is a severe pest of onion and cotton. Due to lack of information on its genome or transcriptome, not much is known about this insect at the molecular level. To initiate molecular studies in this ins...
A fragment activity assay reveals the key residues of TBC1D15 GTPase-activating protein (GAP) in Chiloscyllium plagiosum
GTPase-activating proteins (GAPs) with a TBC (Tre-2/Bub2/Cdc16) domain architecture serve as negative regulators of Rab GTPases. The related crystal structure has been studied and reported by other members of ...
HexA is required for growth, aflatoxin biosynthesis and virulence in Aspergillus flavus
Woronin bodies are fungal-specific organelles whose formation is derived from peroxisomes. The former are believed to be involved in the regulation of mycotoxins biosynthesis, but not in their damage repair fu...
Genome-wide identification of brain miRNAs in response to high-intensity intermittent swimming training in Rattus norvegicus by deep sequencing
Physical exercise can improve brain function by altering brain gene expression. The expression mechanisms underlying the brain’s response to exercise still remain unknown. miRNAs as vital regulators of gene ex...
Graphene oxide down-regulates genes of the oxidative phosphorylation complexes in a glioblastoma
Recently different forms of nanographene were proposed as the material with high anticancer potential. However, the mechanism of the suppressive activity of the graphene on cancer development remains unclear. ...
MiRNAs differentially expressed in skeletal muscle of animals with divergent estimated breeding values for beef tenderness
MicroRNAs (miRNAs) are small noncoding RNAs of approximately 22 nucleotides, highly conserved among species, which modulate gene expression by cleaving messenger RNA target or inhibiting translation. MiRNAs ar...
The Dictyostelium discoideum homologue of Twinkle, Twm1, is a mitochondrial DNA helicase, an active primase and promotes mitochondrial DNA replication
DNA replication requires contributions from various proteins, such as DNA helicases; in mitochondria Twinkle is important for maintaining and replicating mitochondrial DNA. Twinkle helicases are predicted to a...
Matrix association region/scaffold attachment region (MAR/SAR) sequence: its vital role in mediating chromosome breakages in nasopharyngeal epithelial cells via oxidative stress-induced apoptosis
Oxidative stress is known to be involved in most of the aetiological factors of nasopharyngeal carcinoma (NPC). Cells that are under oxidative stress may undergo apoptosis. We have previously demonstrated that...
Molecular analysis of NPAS3 functional domains and variants
NPAS3 encodes a transcription factor which has been associated with multiple human psychiatric and neurodevelopmental disorders. In mice, deletion of Npas3 was found to cause alterations in neurodevelopment, as w...
Integration of transcriptome and proteome profiles in glioblastoma: looking for the missing link
Glioblastoma (GB) is the most common and aggressive tumor of the brain. Genotype-based approaches and independent analyses of the transcriptome or the proteome have led to progress in understanding the underly...
Analyses of changes in myocardial long non-coding RNA and mRNA profiles after severe hemorrhagic shock and resuscitation via RNA sequencing in a rat model
Ischemia–reperfusion injury has been proven to induce organ dysfunction and death, although the mechanism is not fully understood. Long non-coding RNAs (lncRNAs) have drawn wide attention with their important ...
Coincidence cloning recovery of Brucella melitensis RNA from goat tissues: advancing the in vivo analysis of pathogen gene expression in brucellosis
Brucella melitensis bacteria cause persistent, intracellular infections in small ruminants as well as in humans, leading to significant morbidity and economic loss worldwide. The majority of experiments on the tr...
Positive cofactor 4 (PC4) contributes to the regulation of replication-dependent canonical histone gene expression
Core canonical histones are required in the S phase of the cell cycle to pack newly synthetized DNA, therefore the expression of their genes is highly activated during DNA replication. In mammalian cells, this...
Evaluation of suitable reference genes for qRT-PCR normalization in strawberry ( Fragaria × ananassa ) under different experimental conditions
Strawberry has received much attention due to its nutritional value, unique flavor, and attractive appearance. The availability of the whole genome sequence and multiple transcriptome databases allows the grea...
Laser capture microdissection for transcriptomic profiles in human skin biopsies
The acquisition of reliable tissue-specific RNA sequencing data from human skin biopsy represents a major advance in research. However, the complexity of the process of isolation of specific layers from fresh-...
Targeting miR-9 in gastric cancer cells using locked nucleic acid oligonucleotides
Gastric cancer is the third leading cause of cancer-related mortality worldwide. Recently, it has been demonstrated that gastric cancer cells display a specific miRNA expression profile, with increasing eviden...
Quantitative profiling of BATF family proteins/JUNB/IRF hetero-trimers using Spec-seq
BATF family transcription factors (BATF, BATF2 and BATF3) form hetero-trimers with JUNB and either IRF4 or IRF8 to regulate cell fate in T cells and dendritic cells in vivo. While each combination of the heter...
pH-mediated upregulation of AQP1 gene expression through the Spi-B transcription factor
Bicarbonate-based peritoneal dialysis (PD) fluids enhance the migratory capacity and damage-repair ability of human peritoneal mesothelial cells by upregulating AQP1. However, little is known about the underly...
A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone
Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided adaptive immune systems are found in prokaryotes to defend cells from foreign DNA. CRISPR Cas9 systems have been modified and employ...
The Correction to this article has been published in BMC Molecular Biology 2019 20 :20
Recommendations for mRNA analysis of micro-dissected glomerular tufts from paraffin-embedded human kidney biopsy samples
Glomeruli are excellent pre-determined natural structures for laser micro-dissection. Compartment-specific glomerular gene expression analysis of formalin-fixed paraffin-embedded renal biopsies could improve r...
Nutrient depletion and TOR inhibition induce 18S and 25S ribosomal RNAs resistant to a 5′-phosphate-dependent exonuclease in Candida albicans and other yeasts
Messenger RNA (mRNA) represents a small percentage of RNAs in a cell, with ribosomal RNA (rRNA) making up the bulk of it. To isolate mRNA from eukaryotes, typically poly-A selection is carried out. Recently, a...
An optimized rapid bisulfite conversion method with high recovery of cell-free DNA
Methylation analysis of cell-free DNA is a encouraging tool for tumor diagnosis, monitoring and prognosis. Sensitivity of methylation analysis is a very important matter due to the tiny amounts of cell-free DN...
Sumoylation in p27kip1 via RanBP2 promotes cancer cell growth in cholangiocarcinoma cell line QBC939
Cholangiocarcinoma is one of the deadly disease with poor 5-year survival and poor response to conventional therapies. Previously, we found that p27kip1 nuclear-cytoplasmic translocation confers proliferation ...
An optimised protocol for isolation of RNA from small sections of laser-capture microdissected FFPE tissue amenable for next-generation sequencing
Formalin-fixed paraffin embedded (FFPE) tissue constitutes a vast treasury of samples for biomedical research. Thus far however, extraction of RNA from FFPE tissue has proved challenging due to chemical RNA–pr...
Physical shearing imparts biological activity to DNA and ability to transmit itself horizontally across species and kingdom boundaries
We have recently reported that cell-free DNA (cfDNA) fragments derived from dying cells that circulate in blood are biologically active molecules and can readily enter into healthy cells to activate DNA damage...
Interaction between NFATc2 and the transcription factor Sp1 in pancreatic carcinoma cells PaTu 8988t
Nuclear factors of activated T-cells (NFATs) have been mainly characterized in the context of immune response regulation because, as transcription factors, they have the ability to induce gene transcription. N...
Splicing arrays reveal novel RBM10 targets, including SMN2 pre-mRNA
RBM10 is an RNA binding protein involved in message stabilization and alternative splicing regulation. The objective of the research described herein was to identify novel targets of RBM10-regulated splicing. ...
Growth arrest specific gene 2 in tilapia ( Oreochromis niloticus ): molecular characterization and functional analysis under low-temperature stress
Growth arrest specific 2 ( gas2 ) gene is a component of the microfilament system that plays a major role in the cell cycle, regulation of microfilaments, and cell morphology during apoptotic processes. However, li...
Identification of G-quadruplex structures that possess transcriptional regulating functions in the Dele and Cdc6 CpG islands
G-quadruplex is a DNA secondary structure that has been shown to play an important role in biological systems. In a previous study, we identified 1998 G-quadruplex-forming sequences using a mouse CpG islands D...
Mitochondrial RNA processing in absence of tRNA punctuations in octocorals
Mitogenome diversity is staggering among early branching animals with respect to size, gene density, content and order, and number of tRNA genes, especially in cnidarians. This last point is of special interes...
Microarray expression profiling in the denervated hippocampus identifies long noncoding RNAs functionally involved in neurogenesis
The denervated hippocampus provides a proper microenvironment for the survival and neuronal differentiation of neural progenitors. While thousands of lncRNAs were identified, only a few lncRNAs that regulate n...
Early growth response protein 1 regulates promoter activity of α -plasma membrane calcium ATPase 2, a major calcium pump in the brain and auditory system
Along with sodium/calcium (Ca 2+ ) exchangers, plasma membrane Ca 2+ ATPases (ATP2Bs) are main regulators of intracellular Ca 2+ levels. There are four ATP2B paralogs encoded by four different genes. Atp2b2 encodes t...
BMC Molecular Biology
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Circulating microRNAs as potential biomarkers of physical activity in geriatric patients with HCV
Circulating microRNAs have been implicated in a diverse array of biological and pathological phenomena. Their potential utility as noninvasive biomarkers for screening and diagnosing various diseases has been ...
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Correction: The primary cilium dampens proliferative signaling and represses a G2/M transcriptional network in quiescent myoblasts
The original article was published in BMC Molecular and Cell Biology 2020 21 :25
Upregulated dual oxidase 1-induced oxidative stress and caspase-1-dependent pyroptosis reflect the etiologies of heart failure
Oxidative stress is implicated in the pathogenesis of heart failure. Dual oxidase 1 (DUOX1) might be important in heart failure development through its mediating role in oxidative stress. This study was design...
Comparing chemical transfection, electroporation, and lentiviral vector transduction to achieve optimal transfection conditions in the Vero cell line
Transfection is an important analytical method for studying gene expression in the cellular environment. There are some barriers to efficient DNA transfection in host cells, including circumventing the plasma ...
High-fat diet enhances cell proliferation and compromises intestinal permeability in a translational canine intestinal organoid model
Emerging evidence underscores the responsiveness of the mammalian intestine to dietary cues, notably through the involvement of LGR5 + intestinal stem cells in orchestrating responses to diet-driven signals. H...
mTOR signaling pathway regulation HIF-1 α effects on LPS induced intestinal mucosal epithelial model damage
Sepsis-induced small-intestinal injury is associated with increased morbidity and mortality. Our previous study and other papers have shown that HIF-1α has a protective effect on intestinal mucosal injury in s...
Long non-coding RNA SOX2OT in tamoxifen-resistant breast cancer
Hormone receptor (HR)-positive breast cancer can become aggressive after developing hormone-treatment resistance. This study elucidated the role of long non-coding RNA (lncRNA) SOX2OT in tamoxifen-resistant (T...
Mice lacking DIO3 exhibit sex-specific alterations in circadian patterns of corticosterone and gene expression in metabolic tissues
Disruption of circadian rhythms is associated with neurological, endocrine and metabolic pathologies. We have recently shown that mice lacking functional type 3 deiodinase (DIO3), the enzyme that clears thyroi...
Optimization of seeding density of OP9 cells to improve hematopoietic differentiation efficiency
OP9 mouse stromal cell line has been widely used to induce differentiation of human embryonic stem cells (hESCs) into hematopoietic stem/progenitor cells (HSPCs). However, the whole co-culture procedure usuall...
Development of an in vitro human alveolar epithelial air-liquid interface model using a small molecule inhibitor cocktail
The alveolar epithelium is exposed to numerous stimuli, such as chemicals, viruses, and bacteria that cause a variety of pulmonary diseases through inhalation. Alveolar epithelial cells (AECs) cultured in vitr...
Mechanical stretch leads to increased caveolin-1 content and mineralization potential in extracellular vesicles from vascular smooth muscle cells
Hypertension-induced mechanical stress on vascular smooth muscle cells (VSMCs) is a known risk factor for vascular remodeling, including vascular calcification. Caveolin-1 (Cav-1), an integral structural compo...
Melatonin reduces lung injury in type 1 diabetic mice by the modulation of autophagy
In recent years, the role of autophagy has been highlighted in the pathogenesis of diabetes and inflammatory lung diseases. In this study, using a diabetic model of mice, we investigated the expression of auto...
TonEBP/NFAT5 expression is associated with cisplatin resistance and migration in macrophage-induced A549 cells
Macrophages promote angiogenesis, metastasis, and drug resistance in several cancers. Similarly, TonEBP/NFAT5 induces metastasis in renal carcinoma and colon cancer cells. However, the role of this transcripti...
Optimizing combination therapy in prostate cancer: mechanistic insights into the synergistic effects of Paclitaxel and Sulforaphane-induced apoptosis
Combination therapies in cancer treatment have demonstrated synergistic or additive outcomes while also reducing the development of drug resistance compared to monotherapy. This study explores the potential of...
CTC together with Shh and Nrf2 are prospective diagnostic markers for HNSCC
The lack of appropriate prognostic biomarkers remains a significant obstacle in the early detection of Head and Neck Squamous Cell Carcinoma (HNSCC), a cancer type with a high mortality rate. Despite considera...
Prioritization of Trypanosoma brucei editosome protein interactions interfaces at residue resolution through proteome-scale network analysis
Trypanosoma brucei is the causative agent for trypanosomiasis in humans and livestock, which presents a growing challenge due to drug resistance. While identifying novel drug targets is vital, the process is dela...
Sumoylation of SAP130 regulates its interaction with FAF1 as well as its protein stability and transcriptional repressor function
Fas-associated factor 1 (FAF1) is a multidomain protein that interacts with diverse partners to affect numerous cellular processes. Previously, we discovered two Small Ubiquitin-like Modifier (SUMO)-interactin...
Loss of Dec1 inhibits alcohol-induced hepatic lipid accumulation and circadian rhythm disorder
Chronic alcohol exposure increases liver damage such as lipid accumulation and hepatitis, resulting in hepatic cirrhosis. Chronic alcohol intake is known to disturb circadian rhythms in humans and animals. DEC...
Association between plasma L-carnitine levels and mitochondrial DNA copy number
Mitochondria are key cytoplasmic organelles in eukaryotic cells that generate adenosine triphosphate (ATP) through the electron transport chain and oxidative phosphorylation. Mitochondrial DNA (mtDNA) copy num...
Effect of Emi1 gene silencing on the proliferation and invasion of human breast cancer cells
Breast cancer is the most common malignant tumour in women. The early silk-splitting inhibitor protein 1 Emi1 is responsible for mediating ubiquitin protein degradation. The present study investigated the effe...
TNFα induces Caspase-3 activity in hematopoietic progenitor cells CD34+, CD33+, and CD41 + of myelodysplastic syndromes
Cytopenia is the primary feature of Myelodysplastic Syndrome, even in the presence of hypercellular bone marrow. TNFα is recognized as both a proinflammatory, and proapoptotic cytokine with a well established ...
From network analysis to experimental validation: identification of regulators of non-muscle myosin II contractility using the folded-gastrulation signaling pathway
The morphogenetic process of apical constriction, which relies on non-muscle myosin II (NMII) generated constriction of apical domains of epithelial cells, is key to the development of complex cellular pattern...
Simple, low-cost, and well-performing method, the outgrowth technique, for the isolation of cells from nasal polyps
Epithelial cells are an important part of the pathomechanism in chronic rhinosinusitis with nasal polyps. It is therefore essential to establish a robust method for the isolation and culture of epithelial cell...
Comprehensive brain tissue metabolomics and biological network technology to decipher the mechanism of hydrogen-rich water on Radiation-induced cognitive impairment in rats
Hydrogen-rich water (HRW) has been shown to prevent cognitive impairment caused by ionizing radiation. This study aimed to investigate the pharmacological effects and mechanisms of HRW on ionizing radiation by...
Mineral elements and adiposity-related consequences in adolescents with intellectual disabilities
Patients with intellectual disabilities are shown to have a limited capacity for cooperation, communication,and other biological consequences, which significantly require a specialized interest in healthcare p...
Glycyrrhizin inhibits LPS-induced inflammatory responses in goat ruminal epithelial cells in vitro
Inflammation plays a crucial role in the progression of Subacute Ruminal Acidosis (SARA). The experiment was designed to investigate anti-inflammatory effects of glycyrrhizin on goats ruminal epithelial cells ...
D-galactose-induced mitochondrial oxidative damage and apoptosis in the cochlear stria vascularis of mice
Age-related hearing loss, known as presbycusis, is the result of auditory system degeneration. Numerous studies have suggested that reactive oxygen species (ROS) and mitochondrial oxidative damage play importa...
Keratin 19 binds and regulates cytoplasmic HNRNPK mRNA targets in triple-negative breast cancer
Heterogeneous nuclear ribonucleoprotein K (HNRNPK) regulates pre-mRNA processing and long non-coding RNA localization in the nucleus. It was previously shown that shuttling of HNRNPK to the cytoplasm promotes ...
A computational peptide model induces cancer cells’ apoptosis by docking Kringle 5 to GRP78
Cells can die through a process called apoptosis in both pathological and healthy conditions. Cancer development and progression may result from abnormal apoptosis. The 78-kDa glucose-regulated protein (GRP78)...
BMP9 maintains the phenotype of HTR-8/Svneo trophoblast cells by activating the SDF1/CXCR4 pathway
Bone morphogenetic protein 9 (BMP9) has been shown to regulate processes such as angiogenesis, endothelial dysfunction, and tumorigenesis. However, the role of BMP9 in preeclampsia (PE) is unclear. The purpose...
Emodin and aloe-emodin, two potential molecules in regulating cell migration of skin cells through the MAP kinase pathway and affecting Caenorhabditis elegans thermotolerance
Emodin and aloe-emodin are two anthraquinones having positive effects in wound healing. However, their mechanism of action of wound healing is not fully understood. The MAP kinase family, which plays an active...
Knockdown of ELF4 aggravates renal injury in ischemia/reperfusion mice through promotion of pyroptosis, inflammation, oxidative stress, and endoplasmic reticulum stress
Renal ischemia/reperfusion (I/R) injury is a major cause of acute kidney injury (AKI). Dysfunction of E74-like ETS transcription factor 4 (ELF4) leads to inflammation. This research intended to look into the f...
Janus Kinase 3 phosphorylation and the JAK/STAT pathway are positively modulated by follicle-stimulating hormone (FSH) in bovine granulosa cells
Janus kinase 3 (JAK3) is a member of the JAK family of tyrosine kinase proteins involved in cytokine receptor-mediated intracellular signal transduction through the JAK/STAT signaling pathway. JAK3 was previou...
Genetic and protein interaction studies between the ciliary dyslexia candidate genes DYX1C1 and DCDC2
DYX1C1 (DNAAF4) and DCDC2 are two of the most replicated dyslexia candidate genes in genetic studies. They both have demonstrated roles in neuronal migration, in cilia growth and function and they both are cytosk...
SUMOylation of PDGF receptor α affects signaling via PLCγ and STAT3, and cell proliferation
The platelet-derived growth factor (PDGF) family of ligands exerts their cellular effects by binding to α- and β-tyrosine kinase receptors (PDGFRα and PDGFRβ, respectively). SUMOylation is an important posttra...
Myogenic differentiation of human myoblasts and Mesenchymal stromal cells under GDF11 on Poly-ɛ-caprolactone-collagen I-Polyethylene-nanofibers
For the purpose of skeletal muscle engineering, primary myoblasts (Mb) and adipogenic mesenchymal stem cells (ADSC) can be co-cultured and myogenically differentiated. Electrospun composite nanofiber scaffolds...
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Computational analysis of missense variant CYP4F2*3 (V433M) in association with human CYP4F2 dysfunction: a functional and structural impact
Cytochrome P450 4F2 (CYP4F2) enzyme is a member of the CYP4 family responsible for the metabolism of fatty acids, therapeutic drugs, and signaling molecules such as arachidonic acid, tocopherols, and vitamin K...
Using RNA-seq to identify suitable housekeeping genes for hypoxia studies in human adipose-derived stem cells
Hypoxic culture conditions have been used to study the impact of oxygen deprivation has on gene expression in a number of disease models. However, hypoxia response elements present in the promoter regions of s...
SCAT8/miR-125b-5p axis triggers malignant progression of nasopharyngeal carcinoma through SCARB1
Nasopharyngeal carcinoma is a tumor with high malignancy and poor prognosis, which severely affects the health of the patients. LncRNAs and microRNAs are crucial for the occurrence and development of nasophary...
ARNTL2 upregulation of ACOT7 promotes NSCLC cell proliferation through inhibition of apoptosis and ferroptosis
Recent studies have reported that the circadian transcription factor aryl hydrocarbon receptor nuclear translocator like 2 (ARNTL2) promotes the metastatic progression of lung adenocarcinoma. However, the mole...
Evolutionary relevance of single nucleotide variants within the forebrain exclusive human accelerated enhancer regions
Human accelerated regions (HARs) are short conserved genomic sequences that have acquired significantly more nucleotide substitutions than expected in the human lineage after divergence from chimpanzees. The f...
The DNA demethylation-regulated SFRP2 dictates the progression of endometriosis via activation of the Wnt/β-catenin signaling pathway
Endometriosis cause decreases in life quality and pelvic pain in reproductive-age women. Methylation abnormalities played a functional role in the progression of endometriosis, this study aimed to explore the ...
Pre-treatment with IL-6 potentiates β-cell death induced by pro-inflammatory cytokines
Type I Diabetes mellitus (T1D) is characterized by a specific destruction of β-cells by the immune system. During this process pro-inflammatory cytokines are released in the pancreatic islets and contribute for β...
Role of the human solute carrier family 14 member 1 gene in hypoxia-induced renal cell carcinoma occurrence and its enlightenment to cancer nursing
Hypoxia is considered a critical contributor to renal cell carcinoma progression, including invasion and metastasis. However, the potential mechanisms by which it promotes invasion and metastasis have not yet ...
Cyclic tensile force modifies calvarial osteoblast function via the interplay between ERK1/2 and STAT3
Mechanical therapies, such as distraction osteogenesis, are widely used in dental clinics. During this process, the mechanisms by which tensile force triggers bone formation remain of interest. Herein, we inve...
Urine-derived mesenchymal stem cells-derived exosomes enhances survival and proliferation of aging retinal ganglion cells
This study was designed to investigate to test the effect of exosomes from urine-derived mesenchymal stem cells (USCs) on the survival and viability of aging retinal ganglion cells (RGCs), and explored the pre...
RPL11 promotes non-small cell lung cancer cell proliferation by regulating endoplasmic reticulum stress and cell autophagy
Abnormal biogenesis and ribosome free function of ribosomal proteins (RPs) is important for tumorgenesis and development. Ribosomal protein L11 (RPL11) is a component of ribosomal 60 S large subunit with diffe...
Sperm capacitation and transcripts levels are altered by in vitro THC exposure
Delta-9-tetrahydrocannabinol (THC) is the primary phytocannabinoid responsible for the psychoactive properties of cannabis and is known to interact with the endocannabinoid system, which is functionally presen...
The dual role of Nrf2 in melanoma: a systematic review
Melanoma is the most lethal type of skin cancer that originates from the malignant transformation of melanocytes. Although novel treatments have improved patient survival in melanoma, the overall prognosis rem...
Hyperoxia exposure upregulates Dvl-1 and activates Wnt/β-catenin signaling pathway in newborn rat lung
Bronchopulmonary dysplasia is a serious and lifelong pulmonary disease in premature neonates that influences around one-quarter of premature newborns. The wingless-related integration site /β-catenin signaling...
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BMC Molecular and Cell Biology
ISSN: 2661-8850
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- Published: 02 March 2023
Methods in molecular biology and genetics: looking to the future
- Diego A. Forero 1 &
- Vaibhav Chand 2
BMC Research Notes volume 16 , Article number: 26 ( 2023 ) Cite this article
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In recent decades, advances in methods in molecular biology and genetics have revolutionized multiple areas of the life and health sciences. However, there remains a global need for the development of more refined and effective methods across these fields of research. In this current Collection, we aim to showcase articles presenting novel molecular biology and genetics techniques developed by scientists from around the world.
A brief overview of the development of methods of molecular biology and genetics
Since ancient times, humankind has recognized the influence of heredity, based on familial resemblance, selective breeding of livestock, and climate-adapted crops. Prior to Gregor Johann Mendel’s work in the nineteenth century, there was no clear scientific theory to explain heredity. Mendel’s work remained essentially theoretical until the discovery of DNA and confirmation of its role as the principal agent of heredity in organisms in the twentieth century [ 1 ]. In addition, the resolution of the DNA structure paved the way for the invention of the Polymerase Chain Reaction (PCR) (by Kary Mullis), nucleotide synthesis [ 2 ] and the Sanger sequencing method [ 3 ] which revolutionized the field of genetics and led to the development of several sub-disciplines, including cytogenetics, biotechnology, bioprocess technology, and molecular biology. Automation of Sanger sequencing led to the Human Genome Project in 1990 [ 1 ], soon followed by sequencing the complete genomes of numerous other species of flora and fauna [ 4 ].
In recent decades, advances in methods in molecular biology and genetics have revolutionized multiple areas of life and health sciences [ 2 ]. As a major example from health sciences, PCR-based methods have advanced our understanding of the aetiology of a myriad of acute and chronic diseases, in addition to allowing the diagnosis of multiple disorders [ 1 , 5 ]. As a recent global application of molecular methods, the PCR-based approaches have led to the processing of hundreds of millions of samples for the analysis of the SARS-CoV-2 virus [ 6 ]. In addition, molecular methods have been key for the creation of multiple companies, products and jobs [ 7 ].
The development of sequencing technologies and their iterative improvements have been instrumental in advancing the understanding of DNA and RNA, their identification, association with various proteins, their covalent modifications, the function of the genes they carry, and the function of the non-coding portion of DNA and RNA in normal and diseased cells, in pathogenic bacteria and viruses, and in plants [ 8 , 9 ]. By producing RNA-based vaccines, we were able to combat the recent SARS-CoV2 pandemic. This was made possible by sequencing and in vitro nucleotide synthesis technologies [ 10 ].
Gene editing technologies, such as restriction endonuclease digestion, transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR-Cas) system, are an additional development in the field of molecular biology that has aided in the understanding of DNA and genes. There is optimism about the use of CRISPR-Cas9 technology in the treatment of a wide variety of diseases, such as cancer, blood-related diseases, hereditary blindness, cystic fibrosis, viral diseases, muscular dystrophy, and Huntington´s disease, due to its precision and its constant improvement, in comparison with other gene-editing technologies [ 15 ].
Need for novel methods in molecular biology and genetics
There is a global need for the development of novel methods for molecular biology and genetics. Particularly, in the area of human health, there is a need for further approaches that facilitate point-of-care molecular analysis (particularly miniaturized and portable platforms), for infectious and non-transmissible diseases [ 11 ], the development of more efficient methods for DNA sequencing [ 3 ], which facilitate cost-effective genome-wide analysis of patients, among others.
In addition, three key factors would also help push this field forward: additional research comparing the performance of different methods for molecular biology [ 12 ], the broader use of reporting standards (such as the Minimum Information for Publication of Quantitative Real-Time PCR Experiments -MIQE-, which describes details of experimental conditions) [ 13 ], and the increased participation of scientists from the Global South.
Although older techniques, such as x-ray crystallography, gene cloning, PCR, and sequencing, have been instrumental in the study of various aspects of genetics, these techniques have several limitations that result in gaps, missing links, and incomplete understanding of the genome. Advances in these techniques are needed to fill in these missing pieces of the puzzle to better comprehend genetics and accelerate the discovery of the causes of various genetically linkeddiseases. From a technological standpoint, the accuracy of sequencing and coverage across the genome remain major issues, especially for GC-rich regions and long homopolymer stretches of DNA. Furthermore, the short read lengths generated by the majority of current platforms severely restrict our ability to accurately characterize large repeat regions, numerous indels, and structural variation, rendering large portions of the genome opaque or inaccurate. Fragmentation of the genome for sequencing continues to be a major source of disruption in the continuity of the correct genomic sequence [ 14 , 15 ].
Recent advances in CRISPR technology provide hope for the medical treatment of cancer and other fatal diseases. Despite significant advances in this field, a number of technical obstacles remain, including off-target activity, insufficient indel or low homology-directed repair (HDR) efficiency, in vivo delivery of the Cas system components, and immune responses. This requires a substantial amount of technological advancement or the creation of new, superior methods to combat severe diseases with minimal side effects [ 14 , 16 ].
Additional considerations
As high-throughput, automated methods commonly produce very large amounts of data, deeper interaction between wet-lab and dry-lab researchers is required, to facilitate the design of efficient assays [ 17 ] and allow effective analysis and interpretation of results. Interdisciplinary collaborations, between biologists, engineers and professionals in the health sciences, might lead to newer and better methods of addressing current and future needs.
Further collaborations between scientists from academia and industry (in addition to researchers from government agencies) [ 18 ] would help to facilitate the development of novel methods, and aid in promoting their implementation around the world. For many countries, the main barrier to the broad use of molecular methods is the high cost of equipment and reagents [ 19 ]. Strategies aimed at lowering costs would be helpful for multiple institutions around the globe. In terms of intellectual property, fair licensing to institutions in the Global South as well as the implementation of Open Innovation and Open Science policies would be appropriate [ 20 ].
Overview of the current collection
In this current Collection, we are calling for articles showcasing novel methods from molecular biology and genetics, written by scientists from around the world. It is our goal to compile a set of articles that will help to address the challenges faced by the fields of molecular biology and genetics and broaden our understanding of genetic disorders and potential treatment strategies. We invite researchers working on such methods to consider submitting to our collection.
Data availability
Not applicable.
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DAF has been previously supported by research grants from Minciencias and Areandina. VC has been previously supported by research grants from NIH and VA.
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DAF is a medical doctor, Ph.D. in Biomedical Sciences and Professor and Research Leader at the School of Health and Sport Sciences, Fundación Universitaria del Área Andina (Bogotá, Colombia). He has worked with multiple methods of molecular biology and genetics and is an author of more than 100 articles in international journals, has been peer reviewer for more than 115 international scientific journals, in addition to being part of editorial boards of several international journals. VC is a Research Assistant Professor in the Department of Biochemistry and Molecular Genetics at the University of Illinois at Chicago. His expertise in Biochemistry, Molecular Biology, Genetics, Oncology, and Cancer Biology is extensive. He is an invited reviewer for more than fourteen international peer review journals and is the author of fourteen articles with high impact.
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Forero, D.A., Chand, V. Methods in molecular biology and genetics: looking to the future. BMC Res Notes 16 , 26 (2023). https://doi.org/10.1186/s13104-023-06298-y
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- aDepartment of Medicine, University of Western Australia, Queen Elizabeth II Medical Centre, Nedlands, Western Australia 6009, bLung Biology Centre, UC San Francisco, San Francisco, CA 94143-0854, USA, cDepartment of Molecular Pharmacology, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA, dDepartment of Internal Medicine, East Carolina University, Greenville, NC 27858-4354, USA, ePulmonary, Allergy, and Critical Care Division, 856 BRB II/III, Philadelphia, PA 19104, USA, fDivision of Allergy and Pulmonary Medicine, Department of Pediatrics, Washington University School of Medicine, St Louis, MO 63110, USA, gPulmonary & Critical Care Medicine Division, Medical University of South Carolina, Charleston, SC 29425, USA
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There have been a number of major advances in molecular biology in the past few years and the aim of this review is to describe some of these advances, focusing on their benefits and limitations when applied to investigating pulmonary disorders. It is written with the practising pulmonary researcher in mind, not as an introduction for the uninitiated. Useful web addresses and a list of references are included to enable interested readers to examine each technique in detail.
New technologies either improve existing techniques or develop new approaches to old questions in order to generate information more quickly, easily, accurately or in a more easily repeatable fashion then existing methods. 1 Some of the most powerful new technologies include polymerase chain reaction (PCR) advances, “difference analysis” (that is, the discovery of different gene expression patterns between different cells), transgenic/gene knockout technology, and gene delivery to tissues/gene therapy.
Advances in PCR technology
Since its introduction in the 1980s PCR has become a standard tool in biomedical research. The equipment (a thermal cycler) and reagents (thermostable polymerases, oligonucleotides, etc) required for PCR are widely available and relatively inexpensive. One advantage of PCR is its extreme sensitivity which makes possible the detection and analysis of low abundance DNAs. This is especially helpful when limited amounts of starting material are available, or when few copies of the target sequence are present. Applications of PCR include the cloning of known and novel genomic DNA and cDNA sequences, DNA sequencing, construction of mutant or chimeric DNAs, and quantification of mRNA and DNA. PCR is also used in certain methods of difference analysis
PCR BASED CLONING AND SEQUENCING METHODS
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Mechanism of action of dihydroquercetin in the prevention and therapy of experimental liver injury.
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1. Introduction
2. mechanism of action of dhq in protection against liver injury, 2.1. ameliorative effects on drug-induced liver injury, 2.1.1. ameliorative effects of acetaminophen (apap)-induced liver injury, 2.1.2. ameliorative effects against carbon tetrachloride (ccl 4 )-induced liver injury, 2.1.3. ameliorative effects against other drug-induced liver injury, 2.2. ameliorative effects on alcoholic liver injury, 2.3. ameliorative effects on fatty liver injury, 2.4. ameliorative effects against autoimmune hepatitis.
Incidence/100,000 | Rrevalence/100,000 | Ref. | Year | Cases |
---|---|---|---|---|
0.8 | —— | [ ] | 1997 | 496 |
3.0 | —— | [ ] | 2007 | 200 |
0.85 | 10.7 | [ ] | 2008 | 473 |
1.68 | 23.9 | [ ] | 2014 | 1721 |
1.1 | 18.3 | [ ] | 2014 | 1313 |
2.0 | 24.5 | [ ] | 2010 | 138 |
0.67 | 11.0 | [ ] | 2013 | 100 |
1.37 | 11.61 | [ ] | 2004 | 13 |
—— | 42.9 | [ ] | 2002 | 77 |
3. Improvement of DHQ Bioavailability
Click here to enlarge figure
4. Prospects and Outlook
Author contributions, institutional review board statement, informed consent statement, data availability statement, acknowledgments, conflicts of interest.
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Wei, H.; Zhao, T.; Liu, X.; Ding, Q.; Yang, J.; Bi, X.; Cheng, Z.; Ding, C.; Liu, W. Mechanism of Action of Dihydroquercetin in the Prevention and Therapy of Experimental Liver Injury. Molecules 2024 , 29 , 3537. https://doi.org/10.3390/molecules29153537
Wei H, Zhao T, Liu X, Ding Q, Yang J, Bi X, Cheng Z, Ding C, Liu W. Mechanism of Action of Dihydroquercetin in the Prevention and Therapy of Experimental Liver Injury. Molecules . 2024; 29(15):3537. https://doi.org/10.3390/molecules29153537
Wei, Hewei, Ting Zhao, Xinglong Liu, Qiteng Ding, Junran Yang, Xiaoyu Bi, Zhiqiang Cheng, Chuanbo Ding, and Wencong Liu. 2024. "Mechanism of Action of Dihydroquercetin in the Prevention and Therapy of Experimental Liver Injury" Molecules 29, no. 15: 3537. https://doi.org/10.3390/molecules29153537
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Integrating authentic research into the classroom
A recent manuscript, published in the journal Biochemistry and Molecular Biology Education, describes how BMB undergraduates engage with authentic research as part of their major requirements. Course-Based Undergraduate Research Experiences (CUREs) allow students to conduct real-world research in the classroom, such as combating the highly destructive plant pathogen Fusarium oxysporum .
A recent manuscript, published in the journal Biochemistry and Molecular Biology Education , describes how undergraduates in the Biochemistry and Molecular Biology (BMB) department engage with authentic research as part of their major requirements.
Course-Based Undergraduate Research Experiences (CUREs) provide students the opportunity to gain research experience by participating in research projects conducted in the classroom. This model offers students more agency over their coursework, helping them feel more personally invested in their scientific studies while increasing their sense of belonging in STEM.
The BMB department has embedded the CURE model into all of their laboratory courses, including one centered around the research topic of the senior author Professor Li-Jun Ma . The Ma lab focuses on a cross-kingdom fungal pathogen Fusarium oxysporum that infects both plants and humans. Long since regarded as a highly destructive plant pathogen, F. oxysporum was recently recognized by the World Health Organization as a high-priority threat to human health.
Students in this course investigate the functions of secreted proteins using the molecular cloning technique, bringing global food security and human health issues to the classroom. The CURE model empowers students to know that their work is making a difference, while also providing researchers with the unique opportunity to gather large amounts of data to advance their ongoing lab research.
The manuscript describes long-standing collaborations among undergraduates, graduate students, Professor Li-Jun Ma, and Senior Lecturer Amy Springer . Reflecting on the challenges of transforming a research project into a CURE, the manuscript offers some possible solutions. “It is widely accepted that Course-based Undergraduate Research Experiences add tremendous value to BMB education,” says Ma. “This manuscript describes a tested working protocol for faculty members who are interested in implementing this type of curriculum.”
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Recent Advances in Cellular and Molecular Bioengineering for Building and Translation of Biological Systems
1 Department of Bioengineering, Northeastern University, 805 Columbus Ave, ISEC 224, Boston, MA 02115 USA
Adam W. Feinberg
2 Departments of Biomedical Engineering & Materials Science & Engineering, Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, PA 15213 USA
3 Departments of Biomedical Engineering & Biological Sciences, Rensselaer Polytechnic Institute, Biotech 2147, 110 8th Street, Troy, NY 12180 USA
In January of 2020, the Biomedical Engineering Society (BMES)- Cellular and Molecular Bioengineering (CMBE) conference was held in Puerto Rico and themed “Vision 2020: Emerging Technologies to Elucidate the Rule of Life.” The annual BME-CMBE conference gathered worldwide leaders and discussed successes and challenges in engineering biological systems and their translation. The goal of this report is to present the research frontiers in this field and provide perspectives on successful engineering and translation towards the clinic. We hope that this report serves as a constructive guide in shaping the future of research and translation of engineered biological systems.
Introduction and Background
In the field of cellular and molecular bioengineering (CMBE), engineering biological systems is one of the fastest-growing areas, especially with recent research breakthroughs simultaneously in multiple fields, including stem cell research, tissue engineering, gene editing, synthetic biology, omics, and biomanufacturing. The expanding toolbox of cutting-edge techniques enables transformative discoveries by the adoption of engineered biological systems for the modeling of a range of processes from development to diseases. However, there remain significant challenges: (i) how to integrate new technologies and novel biological findings to better mimic developmental and pathological processes, (ii) how to translate these engineered systems for applications in drug discovery and clinical practice, and (iii) how to foster new collaborations between scientists, engineers, clinicians, and industry.
To address these challenges, we co-chaired the annual BMES-CMBE conference in Puerto Rico on January 2–6, 2020, themed “ Vision 2020: Emerging technologies to elucidate the rule of life ” (see Appendix for the conference program). As such, we have recruited established and emerging research leaders who have performed innovative research, integrated engineering, and biology to solve complex problems and built a strong connection with the industry to translate research for diagnostics and therapies. The program focused on how novel and advanced techniques and model systems are applied to research in various physiological systems and diseases. To highlight both emerging research and emerging scientists, we honored a talented group of young principal investigators (as “Rising Stars”), postdoctoral fellows, and graduate students for their exciting research. In addition, we reached out to faculty and students at the University of Puerto Rico to encourage participation in the conference and incorporated two training modules on 3D bioprinting and molecular imaging, as well as extending opportunities to present their research and to connect with scientists from all over the world. At the conference, engineers, biologists, and clinicians shared research across disciplines towards the common goal of improving human health. Furthermore, the conference hosted a panel session dedicated to discussing strategies and best practices for translating novel platform technologies to industry and to the clinic in order to improve human health.
From these interactions, we increasingly believe that traditional approaches to answering outstanding biological questions will benefit from an integration of biology and engineering to model physiological and pathological processes. This includes utilizing genetic editing, single-cell analysis, multicellular emerging properties, novel biomaterial, advanced bioreactors, and organ/tissue-on-a-chip, to accurately and precisely drive assembly, formation, and maturation of biological systems. In addition, researchers can be inspired by and build from lessons learned from across diverse fields. Indeed, general principles and engineering approaches may be shared across a variety of biological systems and require venues for communication between researchers from these different fields in order to integrate and advance the field of engineered biological systems.
To highlight the observations and findings from the conference, as co-chairs, here we summarize the collective thoughts and opinions of the participants, including those experts who participated in the panel discussions at the conference (Drs. Nancy Allbritton, David Mooney, Doris Taylor, Valerie Weaver, and Kun Zhang). The goals of this paper are to highlight a selection of emerging research areas that define general principles and shift paradigms in the engineering of biological systems and to provide perspectives on translation. We hope that this paper will play a constructive role in shaping future research in this field.
Emerging Areas in Engineering Biological Systems
Engineered biological systems have become a hot topic in molecular and cellular bioengineering and have great potential to generate significant impacts in drug discovery and health care. First, engineered biological systems are often cost-effective and allow researchers to more quickly evaluate their hypotheses as compared to time-consuming and labor-intensive animal studies and/or carefully designed clinical trials and studies. Second, when properly implemented, engineered biological systems can allow for the screening of drugs and genetic and environmental factors that cause disease. Third, engineered biological systems can be used for mechanistic studies without systematic effects often associated with an in vivo system. Lastly, engineered biological systems may be a critical tool for research studies and scientific discoveries in certain fields, such as human embryonic development, due to ethical concerns.
Over the past few years, there has been significant progress in several frontiers in the engineering of biological systems resulting from advances in techniques and knowledge in several key fields. These new concepts/approaches are likely to continue to develop rapidly and further inspire new ideas on engineering a better biological system. Here we will review some of the important emerging research areas (Table 1 ). It is worthwhile to point out that there are several other fields of research that may be critical for the engineering of biological systems, including real-time measurement or monitoring of outcomes of the biological systems. We exclude them here as these techniques do not necessarily involve bioengineering at the cell and molecular levels, but they are certainly of great importance for developing efficient systems.
Technological approaches for engineering biological systems at various levels
Level | Scientific discipline | Engineering techniques and examples |
---|---|---|
Subcellular | Genetics, epigenetics | Gene editing and synthetic biology to alter cell gene expression and responses to stimuli |
Cellular | Cell biology, cell heterogeneity | Single-cell profiling and -omics approaches for characterizing cellular properties |
Multicellular | Cell-cell and cell-substrate interactions | Emerging and collective cell behavior through cell-cell interactions; Engineering of organoids and embryo-like structures through cell self-assembly |
Tissue/organ | Tissue repair and regeneration | Tissue engineering, organ-on-a-chip, 3D printing, biomanufacturing |
Whole body | Immunology | Immunoengineering using cellular engineering, biomaterial, and nanomedicine approaches |
Synthetic Biology and Gene Editing Approaches
Recent developments in synthetic biology and gene editing technologies have created new possibilities to control cell behavior through the engineering of genetic networks that respond to environmental stimuli. Synthetic biology involves the design and assembly of genetic circuits to create new biological functions. By understanding the native genetic circuits that are usually optimized via evolution and reconstructing them from modular design, bioengineers can use synthetic biology to build systems with novel activity. 4 In the early stage of synthetic biology, simple gene circuits could be built to be precisely executed to recapitulate patterns of certain natural biological systems in bacteria, such as oscillating gene expression networks, multistate toggle switches, logic computation, and intercellular signaling networks. 24 , 31 , 38 Following these early successes, significant progress has been made, such as new modular genetic parts with standardized design and connectivity principles to streamline the construction of novel circuits with greater complexity. 47 , 73 However, most of the early success of synthetic biology is performed in prokaryotic cells, while it remains difficult to directly transfer the methods into eukaryotic cells due to the complexity of the genetic networks. With the discovery of more precise genome engineering tools, such as CRISPR/Cas9, researchers have begun to interrogate and control gene function and network dynamics in eukaryotic cells. 75 CRISPR/Cas9 allows precise targeting of specific genome loci. When genetically engineered to pair with functional domains, this precise control has enabled the construction of multilayered gene circuits with higher-order functions in mammalian cells. 55 , 66
Applying synthetic biology and gene editing in mammalian cells, researchers can now control the stem cell differentiation process and even reprogram somatic cells toward pluripotency or other cell types. Recent research has utilized tools and principles from synthetic biology to recreate natural developmental processes or engineer cell fate in precise ways. Previously, it was shown that ectopic overexpression of transcription factors could reprogram one cell type into another. 107 Transcription factors are considered the master regulators of cell-type specification and can be used to program cell fate decisions. 23 However, this method of overexpressing transcription factors for reprogramming relies on the ectopic expression of randomly inserted genes that can limit the efficiency and kinetics of generating the desired cell type. Consequently, several groups have utilized tools and approaches from synthetic biology to enhance native transcription factors and improve reprogramming efficiency and fidelity. Similarly, direct conversion of somatic cell types, such as the conversion of fibroblasts to skeletal myocytes by MyoD, 43 can also be achieved with synthetic biology combined with CRISPR/Cas9. In a recent study, multiplexed activation of three endogenous pro-neural genes using CRISPR/Cas9-based activators was sufficient to convert mouse fibroblasts to induced neuronal cells. 6 In this case, targeting the endogenous loci more rapidly remodeled the epigenome and induced transcriptional activation of the target loci than the ectopic overexpression of genes. In another study, a synthetic lineage control circuit in human cells was used to coordinate the kinetics of activation and repression of lineage-specific transcription factors by use of looped circuitry. The circuit directed the differentiation of iPSC-derived pancreatic progenitor cells into glucose-sensitive insulin-secreting β -like cells. 89 Similar circuits based on the same downstream signaling cascade were built to sense and respond to environmental pH level, 3 blood dopamine level, 83 and response to injury 44 for the treatment of diabetes, hypertension, or injury, respectively. Lastly, several synthetic circuits have been described to monitor and maintain aspects of metabolic homeostasis in vivo. 46 , 120 , 121
Synthetic biology has also been used to control cellular signaling, gene expression, and phenotype at high spatial and temporal resolution in response to chemical, mechanical, and optical inputs. Natural biological processes often rely on the coordinated action of multiple different inputs to execute complex cellular behaviors. To this end, several groups have successfully built synthetic tools to link precisely controlled extracellular stimuli to intracellular signaling networks to regulate gene expression patterns and cell phenotypes. One example is the engineering of cells to respond to the mechanical environment. It is known that cells use mechanically-sensitive receptors to survey their extracellular microenvironment and generate a corresponding response. Thus, synthetic control of mechanical signaling could provide a means of programming cell behaviors. To this end, a mechanically controlled signal transducer in mammalian cells was developed. 94 Similarly, external inputs to cells have been controlled via light and ultrasonic pulses using synthetic biology approaches. 26 , 56 Genetically encoded actuators that respond to mechanical, chemical, magnetic, or optical inputs provide a diverse set of synthetic tools for control of cellular signaling and gene expression at unprecedented spatial and temporal resolution.
Beyond synthetic circuits in single cells, synthetic biology approaches could enable more elegant designs of engineered tissue constructs by programming logic circuits that assess cell fate and local environmental conditions and compute desired functional outputs or generate measurable signals. 35 , 62 The development of multicellular structures and tissues depends on cell-cell and cell-environment interactions and signaling. Thus, using synthetic biology to engineer the cell-cell interaction will allow controlling the multicellular structure and morphogenesis. For example, a synthetic Notch receptor has been engineered that is capable of mediating contact-dependent cellular signaling. 62 Toda et al. 109 used an engineered cell-communication system adapted from nature called synNotch 62 to mimic the native self-assembly during cell-cell interaction. The authors engineered the cells so that the synNotch sensors regulated the expression of cadherin proteins, which mediate cell-cell adhesion, and so are essential for holding cells together and creating tissue boundaries during development. It was found that cell populations that have different patterns or levels of cadherins can sort themselves into separate groups after being mixed together and can self-assemble into a range of structures in vitro . Other types of synthetic-biology shape control can also be programmed. For instance, cells have been generated that can be artificially polarized such that asymmetric cell-cell contacts can be made. 45 , 58 These patterns—such as stripes, spirals, or the spots on a giraffe—arise during development as a result of biological signaling programs. In the future, these toolkits could be expanded to generate short- and long-distance cell-cell communication alongside a synthetic system that controls all of the shape-changing operations involved in making biological structures. This could eventually give engineers total control when designing shapes that have some of the properties of living multicellular organisms.
Omics, Single-Cell Profiling, and Big Data Approaches
The transcriptional, proteomic, and metabolic profiling of cells and tissues provides important information on the underlying molecular states of biological processes. Previously, the development of high throughput sequencing technology allowed the simultaneous acquisition of a large amount of molecular information. However, these efforts have relied on the bulk profiling of whole tissues, which reflect the averaged expression across a population of cells rather than individual cells. The specific 3D organization of different cell types, each with its unique molecular and cellular phenotypes, has a profound impact on normal function, natural aging, tissue remodeling, and disease progression. Recently, rapid advances in transformative technologies of single-cell profiling 68 , 92 , 106 , 108 and multiplexed spatial analysis of tissues have allowed us to interrogate this complexity at unprecedented scale and single-cell resolution.
These advances have motivated the extraordinary effort to build a high-resolution atlas and 3D maps of entire human tissues and organs. In particular, the NIH Human Biomolecular Atlas Program (HuBMAP) Consortium ( https://hubmapconsortium.org/ ) was established to coordinate these efforts. To achieve spatially resolved, single-cell maps, researchers will use a complementary two-step approach. First, omic assays will be used to generate global genome sequence and gene expression profiles of dissociated single cells or nuclei in a massively parallel manner. The molecular state of each cell will be revealed by single-cell transcriptomic 9 and chromatin accessibility 8 , 14 assays. The transcription factor binding regions from the open chromatin data combined with the gene expression data will be used to construct a computational program to model the regulation of gene expression across the distinct cell types. 15 Second, spatial information will be acquired for various biomolecules such as RNA, 95 protein, 32 metabolites, and lipids in tissue sections, using imaging methodologies such as fluorescent microscopy (confocal, multiphoton, light-sheet), sequential fluorescence in situ hybridization (seqFISH), 25 , 59 imaging mass spectrometry (spatial proteomics), 104 , 110 and imaging mass cytometry (IMC). 11 , 12 , 76 , 91 The extensive single-cell and nucleus profiles obtained will inform in situ modalities, which will provide spatial information for up to hundreds of molecular targets of interest.
These data will allow the computational registration of cell-specific epigenomic or transcriptomic profiles to cells on a histological slide to reveal various microenvironmental states. 70 , 110 The powerful combination of single-cell profiling and multiplexed in situ imaging will provide a pipeline for constructing multi-omics spatial maps for the various human organs and their cellular interactions at a molecular level. To fully comprehend the human body atlas, the HuBMAP Consortium works actively with other ongoing initiatives, including the Human Cell Atlas, 78 , 84 Human Protein Atlas, 40 LifeTime ( https://lifetime-fetflagship.eu/ ), and related NIH-funded consortia that are mapping specific organs including the brain, 21 lungs ( https://www.lungmap.net/ ), kidney ( https://kpmp.org/about-kpmp/ ), and genitourinary ( https://www.gudmap.org/ ) regions). In parallel, NCI is sponsoring the Human Tumor Atlas Network (HTAN) ( https://humantumoratlas.org ) as part of the cancer moon shot project ( https://www.cancer.gov/research/key-initiatives/moonshot-cancer-initiative/funding/upcoming/hta-foa-video ).
During the course of these activities, innovative technologies will be developed to address the limitations of existing state-of-the-art techniques. For example, transformative technologies such as signal amplification in order to analyze molecules at low abundance by exchange reaction (SABER-Signal Amplification By Exchange Reaction), 51 , 88 seqFISH, 25 , 96 , 125 and lumiphore probes. 13 These technologies will be refined to improve multiplexing, sensitivity, and throughput for imaging RNA and proteins across multiple tissues. Furthermore, new mass spectrometry imaging techniques will enable the quantitative mapping of hundreds of lipids, metabolites, and proteins from the same tissue section with high spatial resolution and sensitivity. 122 , 124 To analyze a large amount of data that will be generated from these programs, new computational tools and machine learning algorithms will be developed for data integration across modalities.
In the end, a comprehensive, accessible 3D molecular and cellular atlas of the human body, in health and under various disease conditions, will be made available to the public. Moreover, these programs will produce an unprecedented volume and diversity of datasets for comprehensive data capture, management, mining, modeling, visual exploration, and communication. These data will be highly useful for the generation of new biomedical hypotheses, tissue engineering, the development of robust simulations of spatiotemporal interactions, machine learning of tissue features, and educational purposes. Ultimately, these data will catalyze novel views on the organization of tissues, regarding not only which types of cells are neighboring one another but also the gene and protein expression patterns that define these cells, their phenotypes, and functional interactions.
Emerging and Collective Behavior of Cells
During morphogenetic processes in embryonic development and tissue regeneration, the behavior of cells depends on not only the extracellular matrix (ECM) environment but also the interactions with neighboring cells. 29 The collective behavior and functions of a group of cells is, therefore, not simply a sum of individual cells. Instead, cell clusters may have spatial patterns of cell functions, depending on the locations of individual cells. The cells are known to coordinate and exhibit a swarming behavior in motion. Indeed, the emerging behavior of cells has been noted long ago and believed to underly the importance of the form (or shape) of biological tissues in their functions through the regulation of local cellular proliferation and differentiation. With microfabrication techniques, spatial differences in cell functions were observed and correlated to the distribution of mechanical forces as a result of cell-cell interactions. 63 , 85 The YAP/TAZ signaling may mediate the process. 1 These studies underscore the importance of the integration of biology and mechanics in understanding the self-assembly of biological tissues. A better understanding of the mechanobiology of the relationship between form and function may be critical in designing multicellular tissues or organoids.
Collective cell migration is a very active research field. Using technologies such as particle image velocimetry, traction force microscopy, and monolayer stress microscopy, detailed migration velocity profiles and cellular stresses inside the epithelial layer have been obtained. 53 The cells can undergo laminar motion but also rotate and swirl. Cell proliferation (i.e., the addition of a cell) can lead to a bipolar flow field and propagate to a large field that is far away from the division site. Cell extrusion (i.e., removal of a cell) from the epithelial layer is typically associated with a coordinated movement of cells towards the extrusion site. The movement of the cells will be suppressed when the cell density increases. The change from the fluid-like behavior to the solid-like behavior is called the jamming transition, and the reversal is called the unjamming transition. The transitions are important for development and disease. 69 , 86 Biomechanical modeling has contributed to the understanding of the biophysical mechanisms. A cell vertex-based model predicts that the phase transitions can occur even when cell density remains constant. 5 A single parameter, the target shape index, or the preferred perimeter-to-area ratio, mediates the jamming and unjamming transition. The result suggests a strong cell-cell adhesion may promote a fluid behavior while a strong cortical tension enhances a solid behavior. A further study suggests that for an anisotropic tissue that is often found in development, a cell alignment index is also required to determine the status of the tissue. 114 With a self-propelled Voronoi model that takes into account cell-substrate interactions, the phase transitions can be further regulated by the self-propulsion speed that measures cell motility. 119
Cancer cell collective migration is another interesting topic and has received much attention. For instance, cancer cells can form tubular structures in the tumor without endothelial cells, called vascular mimicry (VM). Multiple factors can affect VM, including hypoxia, matrix type, and the presence of the other cells (such as macrophages and fibroblasts). Recently, with engineered hydrogels, collagen matrices with small pores and short fibers were shown to change the transcription profile and motility of cancer cells and enhance the formation of a multicellular network. 112 Further studies suggest that ECM microstructure regulates cell adhesion possibly through mediating matrix degradability, which is necessary for the cells to attach 3D collagen fibrils. 111 In addition, the regulation of Snail 1 and Notch signaling was found, indicating the emergence of collective cell behaviors. The fundamental understanding of the collective behavior of the cells (i.e., VM and alike) may lead to the rational design of biological systems that will fully utilize the power within the cells and ultimately benefit the biological modeling of development and disease.
Engineering Human Embryo-Like Structures
The engineering of human embryo-like structures represents a great opportunity in the CMBE field that allows studies that are otherwise impossible due to ethical concerns. 30 , 79 , 117 In the United States, approximately 4% of infants suffer from congenital abnormalities, and these birth defects account for 20% of infant deaths annually. Regulatory guidance often requires the use of animal models for teratogen testing, but they are often costly and labor-intensive. In addition, due to well-known human-animal differences, the findings from animals may not be representative of human development outcomes. Therefore, a humanized in vitro model that can recapitulate the developmental process is in high demand.
Earlier efforts, such as control of the clonal size of human pluripotent stem cells (hPSCs), have some success in guiding stem cells into specific lineages. It’s not until recently that developmental structures can be recapitulated with in vitro platforms. One of the first early efforts used mouse embryonic stem cells to demonstrate the principles. 37 When mixed with extraembryonic trophoblast stem cells (TSCs) in a 3D Matrigel scaffold, artificial embryos were created with the formation of the pro-amniotic cavity and characteristic embryo architecture, patterning of the embryonic compartment, and specification of primordial-germ-like cells. Further studies show that without biochemical cues from maternal sources, the hPSCs alone, without the use of TSCs, were able to self-organize into human amnion-like tissue inside the biomaterial. 98 Later, with a microfluidic device, modeling human epiblast and amnion development with hPSCs was successfully demonstrated with high controllability and scalability. 123
Human gastrulation has also been modeled with engineering techniques. These earlier studies using micropatterning techniques reveal that upon the BMP4 treatment, circular micropatterns of hPSCs have distinct three regions that represent the three germ layers found in early embryonic development. 115 With similar microscale patterns, WNT and ACTIVIN stimulation induced an organizer with the expression of transcription factor Goosecoid. These cells, when transplanted into chick embryos, induced and contributed to the formation of a secondary axis. Subsequently, using 3D biomaterials, the hPSCs treated with BMP4 form a luminal structure, which polarizes into regions expressing ectoderm and mesoderm markers, mimicking the anterior-posterior symmetry breaking found in vivo . 100 The full potential of these engineered embryonic-like structures is still actively explored, with great promise for impacting science and society.
Biomanufacturing: 3D Bioprinting and the Translation to Large-Scale Manufacturing
Biomanufacturing is emerging as a major research area within CMBE, as novel results and new technologies look to be translated to industry and the clinic. While the moonshot vision for the field has been the 3D bioprinting of organs for transplant, that reality is still at least a decade away and likely longer given the challenges of bringing tissue-engineered medical products to the market. Instead, 3D bioprinting has emerged as a powerful tool for building more complex in vitro systems that can mimic in vivo conditions and enabled hypothesis testing with the ability to systematically vary physical, mechanical, chemical, and biological properties of the microenvironment. Examples include organ-on-chip, small tissue disease models, and scaffolds for tissue regeneration that leverage the unique capabilities to combine cells, biological materials, and synthetic materials based on computer-aided design models. 61
In terms of in vitro model systems, 3D bioprinting has seen rapid growth because of the ability to fabricate complex 3D microfluidics and engineered tissues using a wide range of cells and biomaterials. 27 For example, digital light processing (DLP) bioprinters have been developed that can use UV and visible wavelengths to photocrosslink synthetic and natural hydrogels with high resolution of ~ 5 μ m. 64 Using well-established hydrogels such as polyethylene glycol diacrylate (PEGDA) and gelatin methacrylate (GelMA), these DLP bioprinters have been used to build complex 3D fluidic networks to replicate structures such as vascular networks and alveoli. 34 Multiphoton microscopes have also been modified into light-based 3D bioprinters to even achieve a higher resolution of < 1 μ m to create true capillary-scale vascular networks. 77 Cells can be integrated into the material being printed, and/or be seeded after scaffold fabrication to create cellularized tissues. Extrusion 3D bioprinting has also seen widespread adoption because it has the flexibility to use many different kinds of biomaterials from thermoplastics, to hydrogels, to microparticle slurries, to photocrosslinkable polymers. Additionally, because the biomaterials and cell-laden bioinks are extruded out of syringes, it is straightforward to use multiple materials and cell types in the same print to increase complexity of the engineered tissues. One of the more recent advances has been extrusion inside of a yield-stress support bath using Freeform Reversible Embedding of Suspended Hydrogels (FRESH) 3D bioprinting inside a gelatin microparticle bath and Sacrificial Writing into Functional Tissue (SWIFT) inside a cell-spheroid support bath. 54 , 99 , 101 This has enabled relatively advanced tissues to be engineered, such as beating ventricle-like heart chambers and large vascularized cardiac tissues differentiated from human pluripotent stem cells. 52 These are just a few examples, and many researchers are working on innovative new biomaterials, printing methods, and engineered tissue constructs.
The growth of 3D bioprinting and the translation towards the clinic and large-scale biomanufacturing is being catalyzed by a combination of industrial, federal, and academic efforts. The US federal government has funded several center-level efforts through the NSF and NIH, primarily focused on academic research into 3D bioprinted tissues and organs. To bring in broader industry participation, the DOD funded the Advanced Regenerative Manufacturing Institute (ARMI) as a public-private partnership, part of the National Network for Manufacturing Innovation (NNMI) initiative. Rather than applied research, ARMI is focused on the technology, manufacturing processes, regulatory framework and work force training required to make tissue engineered medical products (TEMPs) a commercial reality. Direct investment by established companies in the 3D printing industry has also grown with 3D Systems investing in Lung Biotechnology and Desktop Metal acquiring a bioprinting franchise through its acquisition of EnvisionTEC. Many startups have also emerged based on university spinouts such as Aspect Biosystems, Volumetrix, and FluidForm, as well as 3D bioprinter companies including CELLINK, which is the first bioprinting-focused company to exceed a $1B valuation. These investments in research and development, manufacturing, and clinical translation are strong indicators that 3D bioprinted tissues will soon be commercialized for a range of biopharma and medical applications.
Immunoengineering
The field of immune engineering, termed immunoengineering, is progressing at a fast pace, owing to rapid advances in several fields, including immunology, nanomedicine, biomaterial, and tissue engineering. The native immune system is the defense system of our body against pathogens, and it also regulates tissue repair and regeneration. Immunoengineering uses engineering principles and techniques to establish models for the immune system and develop therapeutic solutions for a variety of diseases, such as infection, cancer, diabetes, and inflammatory diseases. 33 , 116
To fight against infectious diseases, scientists have immunoengineered new types of vaccines and constructed them with lipid nanoparticles carrying genetic material (such as mRNA and DNA). The recent development of mRNA vaccines for SARS-CoV-2 is based on intracellular delivery of mRNA encoding a harmless version of the spike proteins present on the surface of the virus. Such vaccines evoke and train the immune system against the virus. 41 , 72 Cancer vaccines are another exciting area of immunoengineering. 81 , 116 This type of therapeutics intends to train the immune system to activate and exert a systemic cellular immune response against cancer. Cellular engineering for adoptive T cell immunotherapy is a hot field and shows great promise in the clinic. 42 , 82 In particular, chimeric antigen receptor T cells can be created through gene editing approaches for treating blood cancers.
Inflammation involves the natural process of wound healing as well as the host tissue responses to implanted tissues and biomaterials. Therefore, modulating inflammatory responses in tissue repair and regeneration is of great interest. One area of research focuses on understanding the role of immune responses on the outcomes of regenerative medicine therapies. Macrophages play a central role in wound healing and host tissue responses. The effects of those cells and their different phenotypes have been evaluated on cells of interest, such as human bone marrow-derived mesenchymal stem cells and hPSC-derived cardiomyocytes. For example, cardiomyocytes co-cultured with macrophages activated with lipopolysaccharide (LPS) and interferon-gamma (IFNγ) or those with interleukin 4 and interleukin 13 had decreased expression of cardiac-related genes. 118 In particular, the expression of BMP2, BMP4, and GATA 4 was affected by the exposure to macrophages and inflammatory signals (i.e., LPS and IFNγ). This study highlights the potential impacts of modeling inflammatory responses on the efficacy of tissue-engineered products or regenerative strategies.
Careful choice of biomaterial properties can harness the power of innate immunity. 102 The responses of macrophages depend on scaffold material and structure (e.g., fiber size and scaffold porosity). 87 The increase of pore size seems to associate with an elevated expression of M2 phenotype or anti-inflammatory responses of these macrophages. 105 A soft substrate results in lower amounts of inflammatory cytokines, while a stiff cell-adhesive surface increases the foreign body response. 7 Modification of the surface topography has been shown to modulate the function of macrophages as well. The elongated cellular shape on the nano- to micro-scale patterns skews macrophages towards an M2 phenotype. 10 , 60 , 113 Therefore, engineering biomaterial properties such as fiber size, pore size, stiffness, and surface topography can serve as a simple means to modulate the immune response. Detailed mechanisms and design principles are, however, unclear and worthy of further investigation.
The biomaterial has also been used for the delivery of immunomodulatory molecules and cells. 18 As biomolecule carriers, biomaterial scaffolds can be designed to physically entrap biomolecules, to change their diffusion properties, and to alter the scaffold degradation profile to determine when the biomolecules are released. The cargo release dynamics can be tailored by using different chemical reactions that affect the cargo-carrier affinity. The release can be designed to be triggered by internal or external factors, such as pH, temperature, and magnetic fields. Biomaterials-assisted cell delivery can provide the cells with an artificial microenvironment to support the survival and function of immune cells. The delivered cells are encapsulated locally, but the secreted therapeutic factors can diffuse out and reach the rest of the body. For example, islet encapsulation in Type 1 Diabetes can allow the insulin to diffuse out the biomaterial system while protecting the cells from the attack of the host immune systems. 17 , 19 , 103
Efforts have also been focused on engineering lymphoid cells and organs, including bone marrow, thymus tissue, and lymph nodes. 49 For instance, recreating the bone marrow niche allows for the maintenance and expansion of the CD34+ cell population. 28 , 65 Recapitulating the interaction of stromal cells (genetically engineered to express DLL1 for Notch activation) and human hematopoietic stem cells (HSCs) enables the long-term maintenance of lymphoid progenitors and improves the efficiency of differentiation and positive selection of human T cells. 93 Activated B cells can be produced from engineered immune organoids mimicking the germinal center. 2 , 67 , 74 , 80 The development of these in vitro systems provides an opportunity for investigating the physiology and pathology of immune systems and for designing and developing novel immunotherapies.
Perspectives on Building Biological Systems
How to build biological systems and how to translate the engineered systems have been two central topics towards developing platforms and strategies for improving human health (Table 2 ). For the former, it often requires deep knowledge in human physiology and disease pathology. The engineering of a system has to capture the main characteristics of human organ or tissue physiology and closely mimic diseased processes at the molecular, cellular, and tissue levels. Due to the variety and complexities of biological systems, there are many different strategies that have been utilized by researchers. Below are a few major considerations in building a sound biological system, highlighted during the panel discussion.
Table 2
Challenges and suggestions for building and translating biological systems
Challenges | Suggestions |
---|---|
How to build a biological system relevant to physiology and diseases? | ∙ Out-of-box thinking for maximal impact ∙ Exploit what is known from basic research ∙ Use a template from nature or an existing model ∙ Keep it simple if possible ∙ Verify it earlier with animal models |
How to accelerate the translation of engineering biological systems? | ∙ Familiar with governmental policies ∙ Collaborate with clinicians ∙ Develop engineering solutions that are marketable. ∙ Manage time and efforts with different paths towards translation |
There is no doubt that the understanding of biological and engineering principles may be critical in designing systems that recapitulate the essence of the physiology of biological tissues/organs and the pathology of associated diseases. This highlights the importance of basic research in engineering biological systems. The knowledge may include the genetic mutations relevant for specific diseases, epigenetic processes that regulate gene expression, biomechanical mechanisms that may involve cytoskeleton and morphological processes, and the crosstalk between various signals and different cell phenotypes. Successful engineering can be achieved when the essential aspects of human physiology and diseases are incorporated in the engineered systems through the appropriate inclusion of factors such as cells, biomaterial, biomechanical constraints/signals, and genetics. In the absence of any critical element, the engineered systems are deemed to fail from the beginning.
Interpretation of the results from an engineered biological system has to be careful. When we utilize the strengths of in vitro biological systems, such as the increased throughput that can accelerate research discovery, please note that the in vitro systems may miss certain characteristics in vivo and/or bring artificial features that do not exist in vivo . Therefore, when designing in vitro systems, the minimal essential requirements for in vitro systems need to be identified so that they can be useful for studying physiology and pathology. When implementing a specific design, be aware of possible drawbacks that may complicate experimental results and data interpretation. Often verification of an in vitro system is accomplished by comparing to in vivo situations (e.g., with an animal model) to examine its physiological and pathological relevance. This verification process is usually performed prior to a large-scale study or screen using the in vitro system. Importantly, any substantial findings from in vitro systems have to be fully verified by animal models and subsequent clinical trials.
Out-of-the-box thinking or being innovative is necessary to make impactful or transformative contributions to the field. The progress of a field becomes stagnant when researchers only focus on trying different parameters under the same framework of thinking. A new idea, concept, or design may bring drastic changes that may be desired. These new thoughts and techniques can be paradigm-shifting and disruptive to the current research practice. When they are widely adopted, they become new standards. In this regard, the engineering of biological systems is, in fact, to bring such a change to existing practice in the field. Therefore, the incorporation of new thinking and new techniques may be the key to generate significant impacts.
The engineered systems don’t have to be overly complicated. If the essential part of biology can be recapitulated, the engineered biosystem can be simple yet effective compared to native biosystems. There are some examples of well-established assays. A transwell assay can establish epithelial monolayers with well-connected junctions as seen in vivo , and a scratch assay simulates cell polarization and migration occurring in the wound-healing process. Recently, hPSC- based organoids, including the human embryo-like structures discussed above, utilize the self-assembly capabilities of differentiating stem cells and demonstrate great similarities in gene expression and tissue structures as native tissues/organs. 48 These organoids provide tremendous opportunities for us to optimize approaches for stem cell differentiation, to understand organ development, and to explore the genetic and environmental factors in defects and diseases.
Mimicking biology or physiology doesn’t need to start from scratch. Using a template from nature is sometimes helpful. One example is the success of using decellularized material or other biomaterial derived from human or animal tissues. These materials preserve the components and structure of native tissues that promote proper cell-ECM interactions and normal cellular and tissue functions. Another example is using animal models (or their modifications) and their cells that may be sufficient to model human physiology or to create biomedical products that will treat human diseases.
Finally, ethical concerns on engineered biological systems shall be properly addressed before any in vitro experiments and translational studies can be conducted. In particular, when we deal with the culture of human pluripotent stem cells, 50 , 57 genetic editing, 16 , 36 synthetic biology approaches, 20 , 90 and development of embryo-like structures, 97 potential ethical and societal impacts have to be carefully evaluated by professional committees. For instance, while the development of embryo-like structures can reveal valuable insights into human embryonic development and birth defects, researchers shall know the existing guideline that may not allow for the culture of human embryo beyond 14 days 97 and ongoing debates whether such a rule shall apply to engineered embryo-like structures. 39 , 71
Perspectives on Translating Engineered Biological Systems
For clinical translation, one has to understand government policies regarding possible products. For tissue/cell engineered products that will be used in human bodies, the Regenerative medicine advanced therapy (RMAT) designation by the FDA (Food and Drug Administration) may be highly relevant. 22 RMAT designation was created under the 21st Century Cures Act at the end of 2016 and provided ways for patients who have a serious or life-threatening disease or condition to receive innovative treatments. RMATs have a wide scope and may include human cell and tissue products, cell therapies, tissue-engineered products, and any combination products. Only around 37% of all applications (55 out of 149) for the RMAT designation have been approved as of October 2020. This highlights the importance for the researchers who work on the bench side to have those policies in mind so that the impact of their research can be maximized and that the products can reach their intended patients in a timely fashion.
During the process of clinical translation, communication between experts in different fields is the key. It may be ideal for engineering-trained researchers to work with clinicians at the very beginning of the projects. The importance perceived by engineers may not be what is really needed in clinical settings. By talking with clinicians, the engineers are ensured to work on real problems and gain insights into pathological conditions. In addition, the collaboration helps engineers create treatment solutions eventually translatable into clinical practice, therefore making impacts on the health of patients. Early collaboration between engineers and clinicians on a project driven by clinical needs may be the best path towards more fruitful outcomes of research translation.
It is important to note that developing medical products or any commercial products may be very different from doing lab research for scientific discoveries. For a commercial product that will be viable in the market, we have to consider the cost of the product and the possible ways to manufacture it in a large quantity. For instance, a human heart has 2-3 billion cells. To obtain cells in this order of magnitude may require extensive cell culture and the use of growth factors to a level that one has to question financial feasibility. Techniques or ideas overcoming these challenges can be valuable. The marketability as an important criterion of the feasibility of an idea shall be in the mind of engineers on day 1.
For academic PIs, there are several considerations of choosing paths toward successful translation. For technologies developed in a bioengineering lab that may have many biomedical applications, it may be wise to choose the direct path that takes the least effort and time so that the technology can generate immediate impact. Also, depending on interests and availability, there are different levels of involvement: licensing, conducting academic clinical trials, and building your own company. Licensing requires much less time involvement but gives you less control of the outcomes of invented technology. Starting your own company gives you great control, but it may take a lot of time and energy, and most startups fail.
The field of engineering biological systems has grown drastically in the past few years, thanks to the rapid progress in several related fields, including gene editing, omics, biomanufacturing, and tissue and cell engineering (including stem cell biology, organoid engineering, and immune engineering). Engineers, biologists, and clinicians are joining forces to tackle the most challenging questions on building such biomimetic systems for drug discovery and clinical translation at several frontiers. The success of the efforts will rely on not only the close integration of biological insights and cutting-edge techniques but also the familiarization of governmental policies and the effective communication between scientists with different expertise as well as among all players in research and development. In the decades to come, we hope to see the tremendous scientific and social impact of this field of research.
Acknowledgements
We would like to thank all the attendees of the 2021 BMES-CMBE conference. In particular, we appreciate the panelists for their expertise and insightful discussions (in alphabetical order): Drs. Nancy Allbritton, David Mooney, Doris Taylor, Valerie Weaver, and Kun Zhang. We would also like to acknowledge funding support from NIH R13 EB029293 (Wan) and NSF CMMI 1933397 (Dai).
Conflict of interest
Guohao Dai, Adam W. Feinberg, and Leo Q. Wan declare that they have no conflicts of interest.
Ethical Standards
No human studies or animal studies were carried out by the authors for this article.
Appendix: Sessions and speakers at the 2020 BMES-CMBE conference
Bmes-cellular and molecular bioengineering (cmbe) conference, “ vision 2020: emerging technologies to elucidate the rule of life ”.
Thursday January 2nd, 2020
Friday January 3rd, 2020
Saturday January 4th, 2020
Sunday January 5th, 2020
Monday January 6th, 2020
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The successful candidate will work in the group of Prof. Dr. Kirsten Jung in the Department of Microbiology at the Faculty of Biology, LMU Munich. This faculty is located in the Biocenter of the High-Tech Campus Martinsried, together with other Life Sciences of LMU, the Max-Planck-Society and the Helmholtz-Center Munich. Core facilities for Genomics, Bioanalytics, Electron Microscopy and Light Microscopy, Proteomics and Metabolomics are available within the Faculty of Biology. We are looking for you: Postdoctoral Research Position in Molecular Microbiology (m/f/x) in Planegg/Martinsried
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The power of scientific collaborations and the future of structural biology
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