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Bioengineering (Basel, Switzerland) Jun 2024Biophysical factors play a fundamental role in human embryonic development. Traditional in vitro models of organogenesis focused on the biochemical environment and did... (Review)
Review
Biophysical factors play a fundamental role in human embryonic development. Traditional in vitro models of organogenesis focused on the biochemical environment and did not consider the effects of mechanical forces on developing tissue. While most human tissue has a Young's modulus in the low kilopascal range, the standard cell culture substrate, plasma-treated polystyrene, has a Young's modulus of 3 gigapascals, making it 10,000-100,000 times stiffer than native tissues. Modern in vitro approaches attempt to recapitulate the biophysical niche of native organs and have yielded more clinically relevant models of human tissues. Since Clevers' conception of intestinal organoids in 2009, the field has expanded rapidly, generating stem-cell derived structures, which are transcriptionally similar to fetal tissues, for nearly every organ system in the human body. For this reason, we conjecture that organoids will make their first clinical impact in fetal regenerative medicine as the structures generated ex vivo will better match native fetal tissues. Moreover, autologously sourced transplanted tissues would be able to grow with the developing embryo in a dynamic, fetal environment. As organoid technologies evolve, the resultant tissues will approach the structure and function of adult human organs and may help bridge the gap between preclinical drug candidates and clinically approved therapeutics. In this review, we discuss roles of tissue stiffness, viscoelasticity, and shear forces in organ formation and disease development, suggesting that these physical parameters should be further integrated into organoid models to improve their physiological relevance and therapeutic applicability. It also points to the mechanotransductive Hippo-YAP/TAZ signaling pathway as a key player in the interplay between extracellular matrix stiffness, cellular mechanics, and biochemical pathways. We conclude by highlighting how frontiers in physics can be applied to biology, for example, how quantum entanglement may be applied to better predict spontaneous DNA mutations. In the future, contemporary physical theories may be leveraged to better understand seemingly stochastic events during organogenesis.
PubMed: 38927855
DOI: 10.3390/bioengineering11060619 -
Genes Jun 2024Chickpea () is a major food legume providing high quality nutrition, especially in developing regions. Chickpea wilt ( f. sp. ) causes significant annual losses....
Chickpea () is a major food legume providing high quality nutrition, especially in developing regions. Chickpea wilt ( f. sp. ) causes significant annual losses. Integrated disease management of Fusarium wilt is supported by resistant varieties. Relatively few resistance genes are known so there is value in exploring genetic resources in chickpea wild relatives. This study investigates the inheritance of Fusarium wilt resistance (race 2) in recombinant inbred lines (RILs) from a cross between a cultivated susceptible chickpea variety (Gokce) and a wild resistant line (Kayat-077). RILs, parents, resistant and susceptible tester lines were twice grown in the greenhouse with inoculation and disease symptoms scored. DNA was extracted from dried leaves and individuals were single nucleotide polymorphism (SNP) genotyped. SNPs were placed on the reference chickpea genome and quantitative trait locus (QTL) mapping was performed. Significant QTL regions were examined using PulseDB to identify candidate genes. The results showed the segregation of Fusarium wilt resistance conforming to a single gene inheritance. One significant QTL was found at the start of chromosome 8, containing 138 genes, three of which were disease-resistance candidates for chickpea breeding.
Topics: Cicer; Fusarium; Disease Resistance; Quantitative Trait Loci; Plant Diseases; Polymorphism, Single Nucleotide; Chromosome Mapping; Plant Breeding
PubMed: 38927754
DOI: 10.3390/genes15060819 -
Genes Jun 2024Although guidelines exist for identifying mixtures, these measures often occur at the end-point of analysis and are protracted. To facilitate early mixture detection, we...
Although guidelines exist for identifying mixtures, these measures often occur at the end-point of analysis and are protracted. To facilitate early mixture detection, we integrated a high-resolution melt (HRM) mixture screening assay into the qPCR step of the forensic workflow, producing the integrated Quantifiler Trio-HRM assay. The assay, when coupled with a prediction tool, allowed for 75.0% accurate identification of the contributor status of a sample (single source vs. mixture). To elucidate the limitations of the developed qPCR-HRM assay, developmental validation studies were conducted assessing the reproducibility and samples with varying DNA ratios, contributors, and quality. From this work, it was determined that the integrated Quantifiler Trio-HRM assay is capable of accurately identifying mixtures with up to five contributors and mixtures at ratios up to 1:100. Further, the optimal performance concentration range was found to be between 0.025 and 0.5 ng/µL. With these results, evidentiary-like DNA samples were then analyzed, resulting in 100.0% of the mixture samples being accurately identified; furthermore, every time a sample was predicted as a single source, it was true, giving confidence to any single-source calls. Overall, the integrated Quantifiler Trio-HRM assay has exhibited an enhanced ability to discern mixture samples from single-source samples at the qPCR stage under commonly observed conditions regardless of the contributor's sex.
Topics: Humans; Forensic Genetics; Real-Time Polymerase Chain Reaction; DNA; DNA Fingerprinting; Reproducibility of Results; Microsatellite Repeats
PubMed: 38927704
DOI: 10.3390/genes15060768 -
Genes Jun 2024Human endogenous retroviruses (HERVs) are DNA transposable elements that have integrated into the human genome via an ancestral germline infection. The potential... (Review)
Review
Human endogenous retroviruses (HERVs) are DNA transposable elements that have integrated into the human genome via an ancestral germline infection. The potential importance of HERVs is underscored by the fact that they comprise approximately 8% of the human genome. HERVs have been implicated in the pathogenesis of neurodegenerative diseases, a group of CNS diseases characterized by a progressive loss of structure and function of neurons, resulting in cell death and multiple physiological dysfunctions. Much evidence indicates that HERVs are initiators or drivers of neurodegenerative processes in multiple sclerosis and amyotrophic lateral sclerosis, and clinical trials have been designed to target HERVs. In recent years, the role of HERVs has been explored in other major neurodegenerative diseases, including frontotemporal dementia, Alzheimer's disease and Parkinson's disease, with some interesting discoveries. This review summarizes and evaluates the past and current research on HERVs in neurodegenerative diseases. It discusses the potential role of HERVs in disease manifestation and neurodegeneration. It critically reviews antiretroviral strategies used in the therapeutic intervention of neurodegenerative diseases.
Topics: Humans; Endogenous Retroviruses; Neurodegenerative Diseases; Amyotrophic Lateral Sclerosis; Animals
PubMed: 38927681
DOI: 10.3390/genes15060745 -
Genes May 2024The integration of target capture systems with next-generation sequencing has emerged as an efficient tool for exploring specific genetic regions with a high resolution...
The integration of target capture systems with next-generation sequencing has emerged as an efficient tool for exploring specific genetic regions with a high resolution and facilitating the rapid discovery of novel alleles. Despite these advancements, the application of targeted sequencing methodologies, such as the myBaits technology, in polyploid oat species remains relatively unexplored. In this study, we utilized the myBaits target capture method offered by Daicel Arbor Biosciences to detect variants and assess their reliability for variant detection in oat genomics and breeding. Ten oat genotypes were carefully chosen for targeted sequencing, focusing on specific regions on chromosome 2A to detect variants. The selected region harbors 98 genes. Precisely designed baits targeting the genes within these regions were employed for the target capture sequencing. We employed various mappers and variant callers to identify variants. After the identification of variants, we focused on the variants identified via all variants callers to assess the applicability of the myBaits sequencing methodology in oat breeding. In our efforts to validate the identified variants, we focused on two SNPs, one deletion and one insertion identified via all variant callers in the genotypes KF-318 and NOS 819111-70 but absent in the remaining eight genotypes. The Sanger sequencing of targeted SNPs failed to reproduce target capture data obtained through the myBaits technology. Similarly, the validation of deletion and insertion variants via high-resolution melting (HRM) curve analysis also failed to reproduce target capture data, again suggesting limitations in the reliability of the myBaits target capture sequencing using short-read sequencing for variant detection in the oat genome. This study shed light on the importance of exercising caution when employing the myBaits target capture strategy for variant detection in oats. This study provides valuable insights for breeders seeking to advance oat breeding efforts and marker development using myBaits target capture sequencing, emphasizing the significance of methodological sequencing considerations in oat genomics research.
Topics: Avena; High-Throughput Nucleotide Sequencing; Plant Breeding; Polymorphism, Single Nucleotide; Genome, Plant; Genomics; Genotype; Sequence Analysis, DNA
PubMed: 38927635
DOI: 10.3390/genes15060700 -
Genes May 2024Several years of research into the small circular DNA molecules called SPHINX and BMMF (SPHINX/BMMF) have provided information on several areas of research, medicine,... (Review)
Review
Several years of research into the small circular DNA molecules called SPHINX and BMMF (SPHINX/BMMF) have provided information on several areas of research, medicine, microbiology and nutritional science. But there are still open questions that have not yet been addressed. Due to the unclear classification, evolution and sources of SPHINX/BMMF, a risk assessment is currently not possible. However, risk assessment is necessary as SPHINX/BMMF are suspected to be involved in the development of cancer and neurodegenerative diseases. In order to obtain an overview of the current state of research and to identify research gaps, a review of all the publications on this topic to date was carried out. The focus was primarily on the SPHINX/BMMF group 1 and 2 members, which is the topic of most of the research. It was discovered that the SPHINX/BMMF molecules could be integral components of mammalian cells, and are also inherited. However, their involvement in neurodegenerative and carcinogenic diseases is still unclear. Furthermore, they are probably ubiquitous in food and they resemble bacterial plasmids in parts of their DNA and protein (Rep) sequence. In addition, a connection with bacterial viruses is also suspected. Ultimately, it is still unclear whether SPHINX/BMMF have an infectious capacity and what their host or target is.
Topics: Humans; Animals; DNA, Circular; Neurodegenerative Diseases; Neoplasms
PubMed: 38927614
DOI: 10.3390/genes15060678 -
Biology Jun 2024The proliferation of large green macroalgae in marine environments has led to the occurrence of green tides, particularly in the South Yellow Sea region of China, where... (Review)
Review
The proliferation of large green macroalgae in marine environments has led to the occurrence of green tides, particularly in the South Yellow Sea region of China, where has been identified as the primary species responsible for the world's largest green tide events. Allelopathy among plants is a critical factor influencing the dynamics of green tides. This review synthesizes previous research on allelopathic interactions within green tides, categorizing four extensively studied allelochemicals: fatty acids, aldehydes, phenols, and terpenes. The mechanisms by which these compounds regulate the physiological processes of green tide algae are examined in depth. Additionally, recent advancements in the rapid detection of allelochemicals are summarized, and their potential applications in monitoring green tide events are discussed. The integration of advanced monitoring technologies, such as satellite observation and environmental DNA (eDNA) analysis, with allelopathic substance detection is also explored. This combined approach addresses gaps in understanding the dynamic processes of green tide formation and provides a more comprehensive insight into the mechanisms driving these phenomena. The findings and new perspectives presented in this review aim to offer valuable insights and inspiration for researchers and policymakers.
PubMed: 38927336
DOI: 10.3390/biology13060456 -
Biomolecules Jun 2024Lung cancer is the leading cause of cancer deaths globally, necessitating effective early detection methods. Traditional diagnostics like low-dose computed tomography...
Lung cancer is the leading cause of cancer deaths globally, necessitating effective early detection methods. Traditional diagnostics like low-dose computed tomography (LDCT) often yield high false positive rates. gene methylation has emerged as a promising biomarker. This study aimed to develop and validate a novel semi-nested real-time PCR assay enhancing sensitivity and specificity for detecting methylation using extendable blocking probes (ExBPs). The assay integrates a semi-nested PCR approach with ExBPs, enhancing the detection of low-abundance methylated DNA amidst unmethylated sequences. It was tested on spiked samples with varied methylation levels and on clinical samples from lung cancer patients and individuals with benign lung conditions. The assay detected methylated DNA down to 0.01%. Clinical evaluations confirmed its ability to effectively differentiate between lung cancer patients and those with benign conditions, demonstrating enhanced sensitivity and specificity. The use of ExBPs minimized non-target sequence amplification, crucial for reducing false positives. The novel semi-nested real-time PCR assay offers a cost-effective, highly sensitive, and specific method for detecting methylation, enhancing early lung cancer detection and monitoring, particularly valuable in resource-limited settings.
Topics: Humans; Lung Neoplasms; DNA Methylation; Homeodomain Proteins; Real-Time Polymerase Chain Reaction; Biomarkers, Tumor; Sensitivity and Specificity
PubMed: 38927132
DOI: 10.3390/biom14060729 -
BMC Biotechnology Jun 2024Mammalian display is an appealing technology for therapeutic antibody development. Despite the advantages of mammalian display, such as full-length IgG display with...
BACKGROUND
Mammalian display is an appealing technology for therapeutic antibody development. Despite the advantages of mammalian display, such as full-length IgG display with mammalian glycosylation and its inherent ability to select antibodies with good biophysical properties, the restricted library size and large culture volumes remain challenges. Bxb1 serine integrase is commonly used for the stable genomic integration of antibody genes into mammalian cells, but presently lacks the efficiency required for the display of large mammalian display libraries. To increase the Bxb1 integrase-mediated stable integration efficiency, our study investigates factors that potentially affect the nuclear localization of Bxb1 integrase.
METHODS
In an attempt to enhance Bxb1 serine integrase-mediated integration efficiency, we fused various nuclear localization signals (NLS) to the N- and C-termini of the integrase. Concurrently, we co-expressed multiple proteins associated with nuclear transport to assess their impact on the stable integration efficiency of green fluorescent protein (GFP)-encoding DNA and an antibody display cassette into the genome of Chinese hamster ovary (CHO) cells containing a landing pad for Bxb1 integrase-mediated integration.
RESULTS
The nucleoplasmin NLS from Xenopus laevis, when fused to the C-terminus of Bxb1 integrase, demonstrated the highest enhancement in stable integration efficiency among the tested NLS fusions, exhibiting over a 6-fold improvement compared to Bxb1 integrase lacking an NLS fusion. Subsequent additions of extra NLS fusions to the Bxb1 integrase revealed an additional 131% enhancement in stable integration efficiency with the inclusion of two copies of C-terminal nucleoplasmin NLS fusions. Further improvement was achieved by co-expressing the Ran GTPase-activating protein (RanGAP). Finally, to validate the applicability of these findings to more complex proteins, the DNA encoding the membrane-bound clinical antibody abrilumab was stably integrated into the genome of CHO cells using Bxb1 integrase with two copies of C-terminal nucleoplasmin NLS fusions and co-expression of RanGAP. This approach demonstrated over 14-fold increase in integration efficiency compared to Bxb1 integrase lacking an NLS fusion.
CONCLUSIONS
This study demonstrates that optimizing the NLS sequence fusion for Bxb1 integrase significantly enhances the stable genomic integration efficiency. These findings provide a practical approach for constructing larger libraries in mammalian cells through the stable integration of genes into a genomic landing pad.
Topics: Animals; CHO Cells; Integrases; Cricetulus; Nuclear Localization Signals; Cell Nucleus; Serine; Green Fluorescent Proteins; Cricetinae; Xenopus laevis
PubMed: 38926833
DOI: 10.1186/s12896-024-00871-4 -
Scientific Reports Jun 2024Excess amounts of histones in the cell induce mitotic chromosome loss and genomic instability, and are therefore detrimental to cell survival. In yeast, excess histones...
Excess amounts of histones in the cell induce mitotic chromosome loss and genomic instability, and are therefore detrimental to cell survival. In yeast, excess histones are degraded by the proteasome mediated via the DNA damage response factor Rad53. Histone expression, therefore, is tightly regulated at the protein level. Our understanding of the transcriptional regulation of histone genes is far from complete. In this study, we found that calcineurin inhibitor treatment increased histone protein levels, and that the transcription factor NFATc1 (nuclear factor of activated T cells 1) repressed histone transcription and acts downstream of the calcineurin. We further revealed that NFATc1 binds to the promoter regions of many histone genes and that histone transcription is downregulated in a manner dependent on intracellular calcium levels. Indeed, overexpression of histone H3 markedly inhibited cell proliferation. Taken together, these findings suggest that NFATc1 prevents the detrimental effects of histone H3 accumulation by inhibiting expression of histone at the transcriptional level.
Topics: NFATC Transcription Factors; Histones; Calcineurin; Humans; Cell Proliferation; Gene Expression Regulation; Promoter Regions, Genetic; Signal Transduction; Transcription, Genetic; Calcium
PubMed: 38926604
DOI: 10.1038/s41598-024-65769-9