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Ras-related Rap1A GTPase is implicated in pancreas β-cell insulin secretion and is stimulated by the cAMP sensor Epac2, a guanine exchange factor and activator of Rap1 GTPase. In this study, we examined the differential proteomic profiles of pancreata from C57BL/6 Rap1A-deficient (Null) and control wild-type (WT) mice with nanoLC-ESI-MS/MS to assess targets of Rap1A potentially involved in insulin regulation. We identified 77 overlapping identifier proteins in both groups, with 8 distinct identifier proteins in Null versus 56 distinct identifier proteins in WT mice pancreata. Functional enrichment analysis showed four of the eight Null unique proteins, ERO1-like protein β (Ero1lβ), triosephosphate isomerase (TP1), 14-3-3 protein γ, and kallikrein-1, were exclusively involved in insulin biogenesis, with roles in insulin metabolism. Specifically, the mRNA expression of Ero1lβ and TP1 was significantly (p < 0.05) increased in Null versus WT pancreata. Rap1A deficiency significantly affected glucose tolerance during the first 15–30 min of glucose challenge but showed no impact on insulin sensitivity. Ex vivo glucose-stimulated insulin secretion (GSIS) studies on isolated Null islets showed significantly impaired GSIS. Furthermore, in GSIS-impaired islets, the cAMP-Epac2-Rap1A pathway was significantly compromised compared to the WT. Altogether, these studies underscore an essential role of Rap1A GTPase in pancreas physiological function. [ABSTRACT FROM AUTHOR]

In vitro and ex-vivo target identification strategies often fail to predict in vivo efficacy, particularly for glioblastoma (GBM), a highly heterogenous tumor rich in resistant cancer stem cells (GSCs). An in vivo screening tool can improve prediction of therapeutic efficacy by considering the complex tumor microenvironment and the dynamic plasticity of GSCs driving therapy resistance and recurrence. This study proposes lipid nanoparticles (LNPs) as an efficient in vivo CRISPR-Cas9 gene editing tool for target validation in mesenchymal GSCs. LNPs co-delivering mRNA (mCas9) and single-guide RNA (sgRNA) were successfully formulated and optimized facilitating both in vitro and in vivo gene editing. In vitro, LNPs achieved up to 67 % reduction in green fluorescent protein (GFP) expression, used as a model target, outperforming a commercial transfection reagent. Intratumoral administration of LNPs in GSCs resulted in ∼80 % GFP gene knock-out and a 2-fold reduction in GFP signal by day 14. This study showcases the applicability of CRISPR-Cas9 LNPs as a potential in vivo screening tool in GSCs, currently lacking effective treatment. By replacing GFP with a pool of potential targets, the proposed platform presents an exciting prospect for therapeutic target validation in orthotopic GSCs, bridging the gap between preclinical and clinical research. [Display omitted] • LNPs an efficient in vitro transfection tool of CRISPR-Cas9 RNAs in GSCs, an alterantive to commercial transfecion reagents. • The CRISPR-Cas9 LNP platform enables efficient in vivo gene editing of a difficult to treat model of mesenchymal GSCs. • Proposing CRISPR-Cas9 LNPs as an in vivo screening tool for therapeutic target identification in GSCs. [ABSTRACT FROM AUTHOR]

Clubroot disease is a severe threat to Brassica crops globally, particularly in western Canada. Genetic resistance, achieved through pyramiding clubroot resistance (CR) genes with different modes of action, is the most important strategy for managing the disease. However, studies on the CR gene functions are quite limited. In this study, we have conducted investigations into the temporal, structural, and interacting features of a newly cloned CR gene, Rcr1, using CRISPR/Cas9 technology. For temporal functionality, we developed a novel CRISPR/Cas9-based binary vector, pHHIGR-Hsp18.2, to deliver Rcr1 into a susceptible canola line (DH12075) and observed that early expression of Rcr1 is critical for conferring resistance. For structural functionality, several independent mutations in specific domains of Rcr1 resulted in loss-of-function, highlighting their importance for CR phenotype. In the study of the interacting features of Rcr1, a cysteine protease gene and its homologous allele in canola were successfully disrupted via CRISPR/Cas9 as an interacting component with Rcr1 protein, resulting in the conversion from clubroot resistant to susceptible in plants carrying intact Rcr1. These results indicated an indispensable role of these two cysteine proteases in Rcr1-mediated resistance response. This study, the first of its kind, provides valuable insights into the functionality of Rcr1. Further, the new vector pHHIGR-Hsp18.2 demonstrated an inducible feature on the removal of add-on traits, which should be useful for functional genomics and other similar research in brassica crops.

Introduction: Mycobacterium tuberculosis (Mtb), the main cause of tuberculosis (TB), has brought a great burden to the world's public health. With the widespread use of Mtb drug-resistant strains, the pressure on anti-TB treatment is increasing. Anti-TB drugs with novel structures and targets are urgently needed. Previous studies have revealed a series of CYPs with important roles in the survival and metabolism of Mtb. However, there is little research on the structure and function of CYP138. Methods: In our study, to discover the function and targetability of CYP138, a cyp138-knockout strain was built, and the function of CYP138 was speculated by the comparison between cyp138-knockout and wild-type strains through growth curves, growth status under different carbon sources, infection curves, SEM, MIC tests, quantitative proteomics, and lipidomics. Results and discussion: The knockout of cyp138 was proven to affect the Mtb's macrophage infection, antibiotics susceptibility, and the levels of fatty acid metabolism, membrane-related proteins, and lipids such as triacylglycerol. We proposed that CYP138 plays an important role in the synthesis and decomposition of lipids related to the cell membrane structure as a new potential anti-tuberculosis drug target. [ABSTRACT FROM AUTHOR]

Background and aim: CRISPR-Cas9, enable precise DNA manipulation via RNA-guided breaks. KHDC3L and PEG3 genes are vital; CRISPR-Cas9 studies their roles in reproduction, development, and gene regulation. Focusing on KHDC3L's impact on PEG3 promoter in HCT116 cells, insights into gene functions and disease mechanisms emerge, informing potential therapies. Method: This study aims to design sgRNAs for the KHDC3L gene using CRISPR tools involved ranking, off-target evaluation, and cloning. HCT116 cells were cultured, synchronized, and transfected with sgRNAs using lipofectamine. Successful transfections were confirmed by fluorescence microscopy. Clonal expansion followed, with DNA extracted and genotyped using PCR and Sanger sequencing. Bisulfite conversion analyzed DNA methylation, employing restriction enzymes for CpG site analysis. Statistical significance (p≤0.05) was assessed using SPSS software. Results: The neighboring regions exhibited significant genomic changes. The designed sgRNAs were cloned into the PX458 plasmid, directing Cas9 to create double-strand breaks (DSBs) in KHDC3L exon 3. Transfected cells showed around 65% efficiency. Gap-PCR confirmed knock-out in 3 out of 17 clones. COBRA analysis revealed allele-specific CpG island methylation in PEG3, indicating the impact of KHDC3L knock-out on PEG3 promoter methylation and expression. Conclusion: The study demonstrates increased PEG3 promoter methylation upon KHDC3L deletion, indicating its role in modulation. Knockout correlates with reduced cell proliferation and colony formation, suggesting KHDC3L's role in promoting cell growth. The gene's relevance in PEG3 regulation and potential therapeutic implications are underscored, though further mechanistic insights are warranted.

CRISPR-Cas9, enable precise DNA manipulation via RNA-guided breaks. KHDC3L and PEG3 genes are vital; CRISPR-Cas9 studies their roles in reproduction, development, and gene regulation. Focusing on KHDC3L's impact on PEG3 promoter in HCT116 cells, insights into gene functions and disease mechanisms emerge, informing potential therapies. This study aims to design sgRNAs for the KHDC3L gene using CRISPR tools involved ranking, off-target evaluation, and cloning. HCT116 cells were cultured, synchronized, and transfected with sgRNAs using lipofectamine. Successful transfections were confirmed by fluorescence microscopy. Clonal expansion followed, with DNA extracted and genotyped using PCR and Sanger sequencing. Bisulfite conversion analyzed DNA methylation, employing restriction enzymes for CpG site analysis. Statistical significance (p≤0.05) was assessed using SPSS software. The neighboring regions exhibited significant genomic changes. The designed sgRNAs were cloned into the PX458 plasmid, directing Cas9 to create double-strand breaks (DSBs) in KHDC3L exon 3. Transfected cells showed around 65% efficiency. Gap-PCR confirmed knock-out in 3 out of 17 clones. COBRA analysis revealed allele-specific CpG island methylation in PEG3, indicating the impact of KHDC3L knock-out on PEG3 promoter methylation and expression. The study demonstrates increased PEG3 promoter methylation upon KHDC3L deletion, indicating its role in modulation. Knockout correlates with reduced cell proliferation and colony formation, suggesting KHDC3L's role in promoting cell growth. The gene's relevance in PEG3 regulation and potential therapeutic implications are underscored, though further mechanistic insights are warranted. [ABSTRACT FROM AUTHOR]

Ras-related Rap1A GTPase is implicated in pancreas β-cell insulin secretion and is stimulated by the cAMP sensor Epac2, a guanine exchange factor and activator of Rap1 GTPase. In this study, we examined the differential proteomic profiles of pancreata from C57BL/6 Rap1A-deficient (Null) and control wild-type (WT) mice with nanoLC-ESI-MS/MS to assess targets of Rap1A potentially involved in insulin regulation. We identified 77 overlapping identifier proteins in both groups, with 8 distinct identifier proteins in Null versus 56 distinct identifier proteins in WT mice pancreata. Functional enrichment analysis showed four of the eight Null unique proteins, ERO1-like protein β (Ero1lβ), triosephosphate isomerase (TP1), 14-3-3 protein γ, and kallikrein-1, were exclusively involved in insulin biogenesis, with roles in insulin metabolism. Specifically, the mRNA expression of Ero1lβ and TP1 was significantly (p < 0.05) increased in Null versus WT pancreata. Rap1A deficiency significantly affected glucose tolerance during the first 15–30 min of glucose challenge but showed no impact on insulin sensitivity. Ex vivo glucose-stimulated insulin secretion (GSIS) studies on isolated Null islets showed significantly impaired GSIS. Furthermore, in GSIS-impaired islets, the cAMP-Epac2-Rap1A pathway was significantly compromised compared to the WT. Altogether, these studies underscore an essential role of Rap1A GTPase in pancreas physiological function.

The 3,4‐dihydroxyphenylalanine (DOPA) melanin is one of the important virulence factors for Cryptococcus neoformans, which may trigger immune responses in the host. While the production of DOPA melanin is catalyzed by laccase that is predominantly encoded by LAC1 gene. Therefore, regulating the genetic expression of C. neoformans is conducive to exploring the impact of interested molecules on the host. In this work, we established two systems that were constructed quickly and easily for the knock‐down/knock‐out of LAC1 gene: RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats CRISPR‐Cas9. The RNAi system was constructed by pSilencer 4.1‐CMV neo plasmid and short hairpin RNA to achieve effective transcriptional suppression. The CRISPR‐Cas9 system was used the PNK003 vectors to obtain a stable albino mutant strain. The results of phenotype, quantitative real‐time polymerase chain reaction, transmission electron microscope, and spectrophotometry were used to assess the ability of melanin production. As a result, the RNAi system displayed attenuation of transcriptional suppression when the transformants continuously passed on new plates. However, the transcriptional suppression of long loop in short hairpin RNA was more powerful and lasted longer. An albino strain produced by CRISPR‐Cas9 was completely unable to synthesize melanin. In conclusion, strains with different capacities of melanin production were obtained by RNAi and CRISPR‐Cas9 systems, which might be useful for exploring the linear relation between melanin and immunoreaction of the host. In addition, the two systems in this article might be convenient to quickly screen the possible trait‐regulating genes of other serotypes of C. neoformans. [ABSTRACT FROM AUTHOR]

In this study, the function of a non-ribosomal peptide synthetase-like (NRPSlike) encoding gene AOL_s00188g306 (g306) was investigated to reveal the association between NRPS and nematocidal activity in the nematode-trapping fungus Arthrobotrys oligospora. Sequence analysis indicated that the encoded product of g306 is an adenylation domain of non-ribosomal peptide synthetases and extended short-chain dehydrogenase/reductase domain-containing proteins, and displays a wide substrate spectrum. The Δg306 mutants were more sensitive to chemical stressors than the wild type. Disruption of g306 impeded the nematocidal efficiency of A. oligospora. Metabolomics analysis showed that secondary metabolite biosynthesis and lipid metabolism were altered in the mutants. The phenotypic changes in the mutants can be attributed to the downregulation of various metabolites, including fatty acyls, prenol lipids, steroidsand steroid derivative, and amino acid derivatives, identified in the present study. This study investigated the association between the non-ribosomal polypeptideencoding gene g306 and nematicidal activity in A. oligospora, providing a reference for resolving the predation mechanism of nematode-trapping fungus. [ABSTRACT FROM AUTHOR]

Abstract Background We previously identified Leucine-rich repeats and immunoglobulin-like domains 1 (Lrig1) as a marker of long-term neurogenic stem cells in the lateral wall of the adult mouse brain. The morphology of the stem cells thus identified differed from the canonical B1 type stem cells, raising a question about their cellular origin. Thus, we investigated the development of these stem cells in the postnatal and juvenile brain. Furthermore, because Lrig1 is a known regulator of quiescence, we also investigated the effect(s) of its deletion on the cellular proliferation in the lateral wall. Methods To observe the development of the Lrig1-lineage stem cells, genetic inducible fate mapping studies in combination with thymidine analog administration were conducted using a previously published Lrig1 T2A-iCreERT2 mouse line. To identify the long-term consequence(s) of Lrig1 germline deletion, old Lrig1 knock-out mice were generated using two different Lrig1 null alleles in the C57BL/6J background. The lateral walls from these mice were analyzed using an optimized whole mount immunofluorescence protocol and confocal microscopy. Results We observed the Lrig1-lineage labeled cells with morphologies consistent with neurogenic stem cell identity in postnatal, juvenile, and adult mouse brains. Interestingly, when induced at postnatal or juvenile ages, morphologically distinct cells were revealed, including cells with the canonical B1 type stem cell morphology. Almost all of the presumptive stem cells labeled were non-proliferative at these ages. In the old Lrig1 germline knock-out mice, increased proliferation was observed compared to wildtype littermates without concomitant increase in apoptosis. Conclusions Once set aside during embryogenesis, the Lrig1-lineage stem cells remain largely quiescent during postnatal and juvenile development until activation in adult age. The absence of premature proliferative exhaustion in the Lrig1 knock-out stem cell niche during aging is likely due to a complex cascade of effects on the adult stem cell pool. Thus, we suggest that the adult stem cell pool size may be genetically constrained via Lrig1.

Abstract Background Saccharomyces cerevisiae generally consumes glucose to produce ethanol accompanied by the main by-products of glycerol, acetic acid, and lactic acid. The minimization of the formation of by-products in S. cerevisiae was an effective way to improve the economic viability of the bioethanol industry. In this study, S. cerevisiae GPD2, FPS1, ADH2, and DLD3 genes were knocked out by the Clustered Regularly Interspaced Short Palindromic Repeats Cas9 (CRISPR-Cas9) approach. The mechanism of gene deletion affecting ethanol metabolism was further elucidated based on metabolic flux and transcriptomics approaches. Results The engineered S. cerevisiae with gene deletion of GPD2, FPS1, ADH2, and DLD3 was constructed by the CRISPR-Cas9 approach. The ethanol content of engineered S. cerevisiae GPD2 Delta FPS1 Delta ADH2 Delta DLD3 Delta increased by 18.58% with the decrease of glycerol, acetic acid, and lactic acid contents by 22.32, 8.87, and 16.82%, respectively. The metabolic flux analysis indicated that the carbon flux rethanol in engineered strain increased from 60.969 to 63.379. The sequencing-based RNA-Seq transcriptomics represented 472 differential expression genes (DEGs) were identified in engineered S. cerevisiae, in which 195 and 277 genes were significantly up-regulated and down-regulated, respectively. The enriched pathways of up-regulated genes were mainly involved in the energy metabolism of carbohydrates, while the down-regulated genes were mainly enriched in acid metabolic pathways. Conclusions The yield of ethanol in engineered S. cerevisiae increased with the decrease of the by-products including glycerol, acetic acid, and lactic acid. The deletion of genes GPD2, FPS1, ADH2, and DLD3 resulted in the redirection of carbon flux.

Background: We previously identified Leucine-rich repeats and immunoglobulin-like domains 1 (Lrig1) as a marker of long-term neurogenic stem cells in the lateral wall of the adult mouse brain. The morphology of the stem cells thus identified differed from the canonical B1 type stem cells, raising a question about their cellular origin. Thus, we investigated the development of these stem cells in the postnatal and juvenile brain. Furthermore, because Lrig1 is a known regulator of quiescence, we also investigated the effect(s) of its deletion on the cellular proliferation in the lateral wall. Methods: To observe the development of the Lrig1-lineage stem cells, genetic inducible fate mapping studies in combination with thymidine analog administration were conducted using a previously published Lrig1T2A-iCreERT2 mouse line. To identify the long-term consequence(s) of Lrig1 germline deletion, old Lrig1 knock-out mice were generated using two different Lrig1 null alleles in the C57BL/6J background. The lateral walls from these mice were analyzed using an optimized whole mount immunofluorescence protocol and confocal microscopy. Results: We observed the Lrig1-lineage labeled cells with morphologies consistent with neurogenic stem cell identity in postnatal, juvenile, and adult mouse brains. Interestingly, when induced at postnatal or juvenile ages, morphologically distinct cells were revealed, including cells with the canonical B1 type stem cell morphology. Almost all of the presumptive stem cells labeled were non-proliferative at these ages. In the old Lrig1 germline knock-out mice, increased proliferation was observed compared to wildtype littermates without concomitant increase in apoptosis. Conclusions: Once set aside during embryogenesis, the Lrig1-lineage stem cells remain largely quiescent during postnatal and juvenile development until activation in adult age. The absence of premature proliferative exhaustion in the Lrig1 knock-out stem cell niche during aging is likely due to a complex cascade of effects on the adult stem cell pool. Thus, we suggest that the adult stem cell pool size may be genetically constrained via Lrig1. [ABSTRACT FROM AUTHOR]

Peropsin is a non-visual opsin in both vertebrate and invertebrate species. In mammals, peropsin is present in the apical microvilli of retinal pigment epithelial (RPE) cells. These structures interdigitate with the outer segments of rod and cone photoreceptor cells. RPE cells play critical roles in the maintenance of photoreceptors, including the recycling of visual chromophore for the opsin visual pigments. Here, we sought to identify the function of peropsin in the mouse eye. To this end, we generated mice with a null mutation in the peropsin gene (Rrh). These mice exhibited normal retinal histology, normal morphology of outer segments and RPE cells, and no evidence of photoreceptor degeneration. Biochemically, Rrh-/- mice had ∼2-fold higher vitamin A (all-trans-retinol (all-trans-ROL)) in the neural retina following a photobleach and 5-fold lower retinyl esters in the RPE. This phenotype was similar to those reported in mice that lack interphotoreceptor retinoid-binding protein (IRBP) or cellular retinol-binding protein, suggesting that peropsin plays a role in the movement of all-trans-ROL from photoreceptors to the RPE. We compared the phenotypes in mice lacking both peropsin and IRBP with those of mice lacking peropsin or IRBP alone and found that the retinoid phenotype was similarly severe in each of these knock-out mice. We conclude that peropsin controls all-trans-ROL movement from the retina to the RPE or may regulate all-trans-ROL storage within the RPE. We propose that peropsin affects light-dependent regulation of all-trans-ROL uptake from photoreceptors into RPE cells through an as yet undefined mechanism.

Background: Saccharomyces cerevisiae generally consumes glucose to produce ethanol accompanied by the main by-products of glycerol, acetic acid, and lactic acid. The minimization of the formation of by-products in S. cerevisiae was an effective way to improve the economic viability of the bioethanol industry. In this study, S. cerevisiae GPD2, FPS1, ADH2, and DLD3 genes were knocked out by the Clustered Regularly Interspaced Short Palindromic Repeats Cas9 (CRISPR-Cas9) approach. The mechanism of gene deletion affecting ethanol metabolism was further elucidated based on metabolic flux and transcriptomics approaches. Results: The engineered S. cerevisiae with gene deletion of GPD2, FPS1, ADH2, and DLD3 was constructed by the CRISPR-Cas9 approach. The ethanol content of engineered S. cerevisiae GPD2 Delta FPS1 Delta ADH2 Delta DLD3 Delta increased by 18.58% with the decrease of glycerol, acetic acid, and lactic acid contents by 22.32, 8.87, and 16.82%, respectively. The metabolic flux analysis indicated that the carbon flux rethanol in engineered strain increased from 60.969 to 63.379. The sequencing-based RNA-Seq transcriptomics represented 472 differential expression genes (DEGs) were identified in engineered S. cerevisiae, in which 195 and 277 genes were significantly up-regulated and down-regulated, respectively. The enriched pathways of up-regulated genes were mainly involved in the energy metabolism of carbohydrates, while the down-regulated genes were mainly enriched in acid metabolic pathways. Conclusions: The yield of ethanol in engineered S. cerevisiae increased with the decrease of the by-products including glycerol, acetic acid, and lactic acid. The deletion of genes GPD2, FPS1, ADH2, and DLD3 resulted in the redirection of carbon flux. [ABSTRACT FROM AUTHOR]

Copyright of Mycosystema is the property of Mycosystema Editorial Board and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

[Objective] The regulation of GATA transcription factor Ac-nsdD in Aspergillus cristatus of Fu brick tea was further researched.[Method] The The characteristic of Ac-NsdD protein sequence of A. cristatus was analyzed by genome sequencing and annotation and phylogenetic construction. The Ac-nsdD deletion mutant was constructed by homologous recombination via Agrobacterium tumefaciens-mediated transformation, and the phenotype of the A. cristatus mutant was analyzed. [Result] Based on the genome sequencing and annotation, A. cristatus Ac-nsdD encoded a putative transcription factor with a conserved DNA-binding domain, ZnF GATA. Phylogenetic analysis showed that A. cristatus Ac-nsdD gene was closely related to other homologs from filamentous fungal species. The Ac-nsdD gene deletion mutant strain during in vitro cultivation in M3 Y medium displayed the number of cleistothecium decreased significantly when compared with their wild type.Interestingly, during in vitro cultivation in M3 Y medium containing 5% NaCl, the mutant strain showed the amount of cleistothecium was decreased, the amount of conidiation was significantly increased, growth rate slower, pigment formation in the middle of the medium was significantly increased. Furthermore, during in vitro cultivation in M3 Y medium containing 17% NaCl, the Ac-nsdD gene mutant showed the amount of cleistothecium was reduced to nearly zero, the amount of conidiation was decreased, growth rate slower. [Conclusion]. The Ac-nsdD gene in a salt-free environment might have a positive effect on sexual development in A. cristatus. In response to low salt-induced osmotic stress, Ac-nsdD gene might positively regulate sexual reproduction, negatively regulate asexual development, maintain a developmental balance between asexual and sexual sporulation, be involved in pigment biosynthesis. The Ac-nsdD gene might negatively regulate sexual development, asexual development and vegetative growth in response to high salt-induced osmotic stress. This study provides laboratory generated data support for further study of the signaling pathways and downstream-regulated genes associated with Ac-nsdD gene, and for common and unique signal transduction pathways in sexual and asexual development in filamentous fungi. [ABSTRACT FROM AUTHOR]

Abstract This study aimed to investigate the effects of cofD gene knock-out on the synthesis of coenzyme F420 and production of methane in Methanobrevibacter ruminantium (M. ruminantium). The experiment successfully constructed a cofD gene knock-out M. ruminantium via homologous recombination technology. The results showed that the logarithmic phase of mutant M. ruminantium (12 h) was lower than the wild-type (24 h). The maximum biomass and specific growth rate of mutant M. ruminantium were significantly lower (P

Among the four different types of thyroid cancer, treatment of medullary thyroid carcinoma poses a major challenge because of its propensity of early metastasis. To further investigate the molecular mechanisms of medullary thyroid carcinoma and discover candidates for targeted therapies, we developed a new mouse model of medullary thyroid carcinoma based on our CGRPCreER mouse line. This system enables gene manipulation in parafollicular C cells in the thyroid, the purported cells of origin of medullary thyroid carcinoma. Selective inactivation of tumor suppressors, such as p53, Rb, and Pten, in mature parafollicular C cells via an inducible Cre recombinase from CGRPCreER led to development of murine medullary thyroid carcinoma. Loss of Pten accelerated p53/Rb-induced medullary thyroid carcinoma, indicating interactions between pathways controlled by tumor suppressors. Moreover, labeling differentiated parafollicular C cells by CGRPCreER allows us to follow their fate during malignant transformation to medullary thyroid tumor. Our findings support a model in which mutational events in differentiated parafollicular C cells result in medullary thyroid carcinoma. Through expression analysis including RNA-Seq, we uncovered major signaling pathways and networks that are perturbed following the removal of tumor suppressors. Taken together, these studies not only increase our molecular understanding of medullary thyroid carcinoma but also offer new candidates for designing targeted therapies or other treatment modalities.

The CRISPRi system using dCas9 Sth1 from Streptococcus thermophilus developed for Mycobacterium tuberculosis and M. smegmatis was modified to allow gene knock-out in M. abscessus. Efficacy of the knock-out system was evaluated by applying deletions and insertions to the mps1 gene. A comparative genomic analysis of mutants and wild type validated the target specificity. • Streptococcus thermophilus CRISPR/Cas (Cas9 Sth1) used for gene knock-out in Mycobacterium abscessus. • A large variety of deletion size obtained with CRISPR/Cas9 Sth1 in M. abscessus. • High gene target specificity provided by CRISPR/ Cas9 Sth1 in M. abscessus. [ABSTRACT FROM AUTHOR]

Yeast studies usually focus on exploring diversity in terms of a specific trait (such as growth rate, antibiotic resistance, or fertility) among extensive strains. Microfluidic chips improve these biological studies in a manner of high throughput and high efficiency. For a population study of yeast, it is of great significance to set a proper initial cell density for every strain under specific circumstances. Herein, we introduced a novel design of chip, which enables users to load cells in a gradient order (six alternatives) of initial cell density within one channel. We discussed several guidelines to choose the appropriate chamber to ensure successful data recording. With this chip, we successfully studied the growth rate of yeast strains under a mating response, which is crucial for yeasts to control growth behaviors for prosperous mating. We investigated the growth rate of eight different yeast strains under three different mating pheromone levels (0.3 μM, 1 μM, and 10 μM). Strains with, even, a six-fold in growth rate can be recorded, with the available data produced simultaneously. This work has provided an efficient and time-saving microfluidic platform, which enables loading cells in a pattern of multi-cell densities for a yeast population experiment, especially for a high-throughput study. Besides, a quantitatively analyzed growth rate of different yeast strains shall reveal inspiring perspectives for studies concerning yeast population behavior with a stimulated mating pheromone. [ABSTRACT FROM AUTHOR]

[Display omitted] • CRTS (4-ketolase/3-hydroxylase) was in dicyclic pathway of astaxanthin biosynthesis. • CRTYB (lycopene beta-cyclase) played a role in dicyclic and monocyclic pathway. • CRTS and CRTYB were possible rate-limiting enzymes of carotenoids biosynthesis. Phaffia rhodozyma is a potential industrial source of natural astaxanthin. Dicyclic and monocyclic pathways of carotenoid synthesis are important nodes in Phaffia rhodozyma , but the relationship between the two pathways and astaxanthin production remains unclear. Eight genes related to dicyclic and monocyclic pathways in three different strains of P. rhodozyma were analysed, and the relationship between expression and astaxanthin biosynthesis was explored. Among these genes, CRTS genes (R = 0.69, P < 0.05) and CRTYB (R = 0.75, P < 0.05) showed the closest correlations with carotenoid and astaxanthin biosynthesis, respectively. To further study these relationships in detail, the CRTYB and CRTS genes were knocked out by homologous recombination. After CRTYB knockout, astaxanthin was decreased to an undetectable level. This result suggested that CRTYB plays a role in dicyclic and monocyclic pathways. Meanwhile, the CRTS gene was in the dicyclic pathway of astaxanthin biosynthesis, and its knockout promoted the monocyclic pathway and resulted in a 25 % increase in astaxanthin production at 120 h. The possible rate-limiting enzymes were the enzymes encoding CRTS and CRTYB by regression analysis. Knockout of the CRTS and CRTYB genes was helpful in understanding the synthetic pathway of astaxanthin and significant to the industrial application of astaxanthin production. [ABSTRACT FROM AUTHOR]

Gene Doctoring is a genetic modification technique for E. coli and related bacteria, in which the Red-recombinase from bacteriophage λ mediates chromosomal integration of a fragment of DNA by homologous recombination (known as recombineering). In contrast to the traditional recombineering method, the integrated fragment for Gene Doctoring is supplied on a donor plasmid rather than as a linear DNA. This protects the DNA from degradation, facilitates transformation, and ensures multiple copies are present per cell, increasing the efficiency and making the technique particularly suitable for strains that are difficult to modify. Production of the donor plasmid has, until recently, relied on traditional cloning techniques that are inflexible, tedious, and inefficient. This protocol describes a procedure for Gene Doctoring combined with Golden Gate assembly of a donor plasmid, using a custom-designed plasmid backbone, for rapid and simple production of complex, multi-part assemblies. Insertion of a gene for superfolder green fluorescent protein, with selection by tetracycline resistance, into E. coli strain MG1655 is used as an example but in principle the method can be tailored for virtually any modification in a wide range of bacteria., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective To investigate the function of general chaperones in the secretion of two-partner-secretion (TPS) protein across the inner membrane to the periplasm of Gram-negative bacteria. Methods The general chaperones were knocked-out and the strains were used to prepare spheroplast to see the effects on the secretion of FhaB*, which is a substrate of the representative model TPS substrates FhaB/FhaC system. Results Single knock-out of chaperones Skp,DegP,PpiD,YfgM and double knock-out of PpiD/YfgM has no or little effect on the secretion of FhaB* into the periplasm, while single knock-out of SurA or double knock-out of Skp/DegP severely affect the secretion of FhaB*. Conclusions There may be two molecular chaperone pathways mediated by SurA or by both SKP and DegP in Gram-negative bacteria, which can mediate the substrate protein of the two-partner-secretion system secretes into the peripheral space.

Abstract Background Trichophyton rubrum is an obligate human parasitic fungus and responsible for approximately 80–90% of dermatomycosis in human. Molecular genetic manipulations of this pathogen are challenging and available tools and protocols are only rudimentary. We adapt molecular genetics methods of well established fungal model organism, to knock out genes in T. rubrum. For the adaptation, crucial modifications are necessary. With the implementation of in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, it is possible to adapt molecular genetic methods, to knock out genes in T. rubrum. Results The gene knock-out method is based on integration of a selection marker into the target site, to interrupt the gene translation. The target gene gets preassigned by the homologous sequence of the in vitro synthesized Cas9-sgRNA ribonucleoprotein complex. To develop the method, we first isolated and characterized a T. rubrum strain with a high amount of microconidia. Next, we developed a transformation protocol, whereby the Cas9-sgRNA ribonucleoprotein gets delivered into the fungal protoplast by the PEG method. We knocked out the URA3 gene and resulted, as predicted, uracil auxotrophic strains. These strains can be used for specific gene knock-outs by reintegrating the URA3 fragment and selection on uracil lacking cultivation media. Exemplary, we knocked out the TRP3 gene and got the predicted phenotype, tryptophan auxotrophic strains. The mutation had been verified by sequencing. Conclusions We developed a method, based on in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, for target specific gene knock-outs in T. rubrum. We knocked out the Ura3 gene and resulted uracil auxotrophic strains. These strains were used for target specific gene knock-outs by reintegrating the Ura3 fragment into the target gene site to interrupt the gene transcription. The developed method allows to adapt sophisticate gene manipulation methods of model fungal species to non-model species.

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Retinal diseases are the primary reasons for severe visual defects and irreversible blindness. Retinal diseases are also inherited and acquired. Both of them are caused by mutations in genes or disruptions in specific gene expression, which can be treated by gene-editing therapy. Clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) system is a frontier of gene-editing tools with great potential for therapeutic applications in the ophthalmology field to modify abnormal genes and treat the genome or epigenome-related retinal diseases. The CRISPR system is able to edit and trim the gene include deletion, insertion, inhibition, activation, replacing, remodeling, epigenetic alteration, and modify the gene expression. CRISPR-based genome editing techniques have indicated the enormous potential to treat retinal diseases that previous treatment was not available for them. Also, recent CRISPR genome surgery experiments have shown the improvement of patient's vision who suffered from severe visual loss. In this article, we review the applications of the CRISPR-Cas9 system in human or animal models for treating retinal diseases such as retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), age-related macular degeneration (AMD), proliferative diabetic retinopathy (PDR), and proliferative vitreoretinopathy (PVR), then we survey limitations of CRISPR system for clinical therapy. [ABSTRACT FROM AUTHOR]

Copyright of Experimental Technology & Management is the property of Experimental Technology & Management Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

This study aimed to investigate the effects of cofD gene knock-out on the synthesis of coenzyme F420 and production of methane in Methanobrevibacter ruminantium (M. ruminantium). The experiment successfully constructed a cofD gene knock-out M. ruminantium via homologous recombination technology. The results showed that the logarithmic phase of mutant M. ruminantium (12 h) was lower than the wild-type (24 h). The maximum biomass and specific growth rate of mutant M. ruminantium were significantly lower (P < 0.05) than those of wild-type, and the maximum biomass of mutant M. ruminantium was approximately half of the wild-type; meanwhile, the proliferation was reduced. The synthesis amount of coenzyme F420 of M. ruminantium was significantly decreased (P < 0.05) after the cofD gene knock-out. Moreover, the maximum amount of H2 consumed and CH4 produced by mutant were 14 and 2% of wild-type M. ruminantium respectively. In conclusion, cofD gene knock-out induced the decreased growth rate and reproductive ability of M. ruminantium. Subsequently, the synthesis of coenzyme F420 was decreased. Ultimately, the production capacity of CH4 in M. ruminantium was reduced. Our research provides evidence that cofD gene plays an indispensable role in the regulation of coenzyme F420 synthesis and CH4 production in M. ruminantium. [ABSTRACT FROM AUTHOR]

Yeast studies usually focus on exploring diversity in terms of a specific trait (such as growth rate, antibiotic resistance, or fertility) among extensive strains. Microfluidic chips improve these biological studies in a manner of high throughput and high efficiency. For a population study of yeast, it is of great significance to set a proper initial cell density for every strain under specific circumstances. Herein, we introduced a novel design of chip, which enables users to load cells in a gradient order (six alternatives) of initial cell density within one channel. We discussed several guidelines to choose the appropriate chamber to ensure successful data recording. With this chip, we successfully studied the growth rate of yeast strains under a mating response, which is crucial for yeasts to control growth behaviors for prosperous mating. We investigated the growth rate of eight different yeast strains under three different mating pheromone levels (0.3 μM, 1 μM, and 10 μM). Strains with, even, a six-fold in growth rate can be recorded, with the available data produced simultaneously. This work has provided an efficient and time-saving microfluidic platform, which enables loading cells in a pattern of multi-cell densities for a yeast population experiment, especially for a high-throughput study. Besides, a quantitatively analyzed growth rate of different yeast strains shall reveal inspiring perspectives for studies concerning yeast population behavior with a stimulated mating pheromone.

Despite its convenience and precision, CRISPR-based gene editing approaches still suffer from off-target effects and low efficiencies, which are partially rooted in Cas9, the nuclease component of the CRISPR/Cas9 system. In this study, we showed how mouse genome editing efficiency can be improved by constitutive and inheritable expression of Cas9 nuclease. For this goal, a transgenic mouse line expressing the Cas9 protein (Cas9-mouse) was generated. For in vitro assessment of gene editing efficiency, the Cas9-mice were crossed with the EGFP-mice to obtain mouse embryonic fibroblasts (MEF) expressing both EGFP and Cas9 (MEFCas9-EGFP). Transfection of these cells with in vitro transcribed (IVT) EGFP sgRNA or phU6-EGFPsgRNA plasmid led to robust decrease of Mean Fluorescent Intensity (MFI) to 8500 ± 1025 a.u. and 13,200 ± 1006 a.u. respectively. However, in the control group, in which the MEFEGFP cells were transfected with a pX330-EGFPsgRNA plasmid, the measured MFI was 16,800 ± 2254 a.u. For in vivo assessment, the Cas9-zygotes at two pronuclei stage (2PN) were microinjected with a phU6-HhexsgRNA vector and the gene mutation efficiency was compared with the wild-type (WT) zygotes microinjected with a pX330-HhexsgRNA plasmid. The analysis of born mice showed that while the injection of Cas9-zygotes resulted in 43.75% Hhex gene mutated mice, it was just 15.79% for the WT zygotes. In conclusion, the inheritable and constitutive expression of Cas9 in mice provides an efficient platform for gene editing, which can facilitate the production of genetically-modified cells and animals. [ABSTRACT FROM AUTHOR]

Introduction: Mycobacterium tuberculosis ( Mtb ), the main cause of tuberculosis (TB), has brought a great burden to the world's public health. With the widespread use of Mtb drug-resistant strains, the pressure on anti-TB treatment is increasing. Anti-TB drugs with novel structures and targets are urgently needed. Previous studies have revealed a series of CYPs with important roles in the survival and metabolism of Mtb . However, there is little research on the structure and function of CYP138., Methods: In our study, to discover the function and targetability of CYP138, a cyp138 -knockout strain was built, and the function of CYP138 was speculated by the comparison between cyp138 -knockout and wild-type strains through growth curves, growth status under different carbon sources, infection curves, SEM, MIC tests, quantitative proteomics, and lipidomics., Results and Discussion: The knockout of cyp138 was proven to affect the Mtb 's macrophage infection, antibiotics susceptibility, and the levels of fatty acid metabolism, membrane-related proteins, and lipids such as triacylglycerol. We proposed that CYP138 plays an important role in the synthesis and decomposition of lipids related to the cell membrane structure as a new potential anti-tuberculosis drug target., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Lu, Chen, Shao, Sun, Li, Lu, You and Yang.)

Tuberculosis (TB) is one of deadly transmissible disease that causes death worldwide; however, only 10% of people infected with Mycobacteriumtuberculosis develop disease, indicating that host genetic factors may play key role in determining susceptibility to TB disease. In this way, the analysis of gene expression profiling of TB infected individuals can give us a snapshot of actively expressed genes and transcripts under various conditions. In the present study, we have analyzed microarray data set and compared the gene expression profiles of patients with different datasets of healthy control, latent infection, and active TB. We observed the transition of genes from normal condition to different stages of the TB and identified and annotated those genes/pathways/processes that have important roles in TB disease during its cyclic interventions in the human body. We identified 488 genes that were differentially expressed at various stages of TB and allocated to pathways and gene set enrichment analysis. These pathways as well as GSEA’s importance were evaluated according to the number of DEGs presents in both. In addition, we studied the gene regulatory networks that may help to further understand the molecular mechanism of immune response against the TB infection and provide us a new angle for future biomarker and therapeutic targets. In this study, we identified 26 leading hubs which are deeply rooted from top to bottom in the gene regulatory network and work as the backbone of the network. These leading hubs contains 31 key regulator genes, of which 14 genes were up-regulated and 17 genes were down-regulated. The proposed approach is based on gene-expression profiling, and network analysis approaches predict some unknown TB-associated genes, which can be considered (or can be tested) as reliable candidates for further (in vivo/in vitro) studies.

AXL, a member of the TAM family, is a promising therapeutic target due to its elevated expression in advanced hepatocellular carcinoma (HCC), particularly in association with acquired drug resistance. Previously, RNA interference was used to study its role in cancer, and several phenotypic changes, including attenuated cell proliferation and decreased migration and invasion, have been reported. The mechanism of action of AXL in HCC is elusive. We first studied the AXL expression in HCC cell lines by real-time PCR and western blot and showed its stringent association with a mesenchymal phenotype. We then explored the role of AXL in mesenchymal SNU475 cells by CRISPR-Cas9 mediated gene knock-out. AXL-depleted HCC cells displayed drastic phenotypic changes, including increased DNA damage response, prolongation of doubling time, G2 arrest, and polyploidization in vitro and loss of tumorigenicity in vivo. Pharmacological inhibition of AXL by R428 recapitulated G2 arrest and polyploidy phenotype. These observations strongly suggest that acute loss of AXL in some mesenchymal HCC cells is lethal and points out that its inhibition may represent a druggable vulnerability in AXL-high HCC patients.

Copyright of Firat Universitesi Sağlik Bilimleri Tip Dergisi is the property of Firat Universitesiu, Saglik Bilimleri Enstitusu and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Mycosystema is the property of Mycosystema Editorial Board and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Background: Trichophyton rubrum is an obligate human parasitic fungus and responsible for approximately 80–90% of dermatomycosis in human. Molecular genetic manipulations of this pathogen are challenging and available tools and protocols are only rudimentary. We adapt molecular genetics methods of well established fungal model organism, to knock out genes in T. rubrum. For the adaptation, crucial modifications are necessary. With the implementation of in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, it is possible to adapt molecular genetic methods, to knock out genes in T. rubrum. Results: The gene knock-out method is based on integration of a selection marker into the target site, to interrupt the gene translation. The target gene gets preassigned by the homologous sequence of the in vitro synthesized Cas9-sgRNA ribonucleoprotein complex. To develop the method, we first isolated and characterized a T. rubrum strain with a high amount of microconidia. Next, we developed a transformation protocol, whereby the Cas9-sgRNA ribonucleoprotein gets delivered into the fungal protoplast by the PEG method. We knocked out the URA3 gene and resulted, as predicted, uracil auxotrophic strains. These strains can be used for specific gene knock-outs by reintegrating the URA3 fragment and selection on uracil lacking cultivation media. Exemplary, we knocked out the TRP3 gene and got the predicted phenotype, tryptophan auxotrophic strains. The mutation had been verified by sequencing. Conclusions: We developed a method, based on in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, for target specific gene knock-outs in T. rubrum. We knocked out the Ura3 gene and resulted uracil auxotrophic strains. These strains were used for target specific gene knock-outs by reintegrating the Ura3 fragment into the target gene site to interrupt the gene transcription. The developed method allows to adapt sophisticate gene manipulation methods of model fungal species to non-model species. [ABSTRACT FROM AUTHOR]

Copyright of Mycosystema is the property of Mycosystema Editorial Board and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Temporally-regulated maternal RNA translation is essential for embryonic development, with defective degradation resulting in stalled 2-cell embryos. We show that DDX1, a DEAD box protein implicated in RNA transport, may be a key regulator of maternal RNA utilization. DDX1 protein localizes exclusively to cytoplasmic granules in both oocytes and early stage mouse embryos, with DDX1 requiring RNA for retention at these sites. Homozygous knockout of Ddx1 causes stalling of mouse embryos at the 2–4 cell stages. These results suggest a maternal RNA-dependent role for DDX1 in the progression of embryos past the 2–4 cell stage. The change in appearance of DDX1-containing granules in developing embryos further supports a role in temporally-regulated degradation of RNAs. We carried out RNA-immunoprecipitations (RNA-IPs) to identify mRNAs bound to DDX1 in 2-cell embryos, focusing on 16 maternal genes previously shown to be essential for embryonic development past the 1- to 2-cell stages. Five of these RNAs were preferentially bound by DDX1: Ago2 , Zar1 , Tle6, Floped and Tif1α. We propose that DDX1 controls access to subsets of key maternal RNAs required for early embryonic development. • DDX1 localizes to cytoplasmic granules in early stage embryos. • DDX1 knockout embryos stall at the 2- to 4-cell stages. • DDX1 co-localizes with stress granule markers TIA-1 and TIAR. • DDX1 localization to cytoplasmic granules is RNA dependent. • DDX1 binds to maternal RNAs that are essential for early embryonic development. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is one of deadly transmissible disease that causes death worldwide; however, only 10% of people infected with Mycobacterium tuberculosis develop disease, indicating that host genetic factors may play key role in determining susceptibility to TB disease. In this way, the analysis of gene expression profiling of TB infected individuals can give us a snapshot of actively expressed genes and transcripts under various conditions. In the present study, we have analyzed microarray data set and compared the gene expression profiles of patients with different datasets of healthy control, latent infection, and active TB. We observed the transition of genes from normal condition to different stages of the TB and identified and annotated those genes/pathways/processes that have important roles in TB disease during its cyclic interventions in the human body. We identified 488 genes that were differentially expressed at various stages of TB and allocated to pathways and gene set enrichment analysis. These pathways as well as GSEA's importance were evaluated according to the number of DEGs presents in both. In addition, we studied the gene regulatory networks that may help to further understand the molecular mechanism of immune response against the TB infection and provide us a new angle for future biomarker and therapeutic targets. In this study, we identified 26 leading hubs which are deeply rooted from top to bottom in the gene regulatory network and work as the backbone of the network. These leading hubs contains 31 key regulator genes, of which 14 genes were up-regulated and 17 genes were down-regulated. The proposed approach is based on gene-expression profiling, and network analysis approaches predict some unknown TB-associated genes, which can be considered (or can be tested) as reliable candidates for further (in vivo / in vitro) studies. [ABSTRACT FROM AUTHOR]

Clustered regularly interspaced short palindromic repeats associated with Cas9 (CRISPR/Cas9) system is a powerful and efficient tool for molecular modification in the genome using programable RNAs. It holds a wide application in plant and animal biotechnology for enhancing multiple traits of interest. Here, we attempted to gather and review the recent progress and provide an overview of the application benefits of this technology in aquatic animal genomes. In this respect, several studies were performed on different species (marine and freshwater) investigating the growth bioprocess trait, wherein, the majority of them focused on somatic axis suppressors such as mstn (myostatin) knock-outs and their effects on the differentiation and proliferation of somatic cells. Regarding the reproduction feature, the retrieved studies provide insights into germ cells, sex reversal, and fertility mechanisms with more focus on zebrafish and tilapia compared to other species. In turn, a generation of de novo strains more resistant to viral infections has been successfully attended to proving the practical ability of this technology in enhancing the immunity of certain species such as grass carp. Furthermore, multiple experiments were performed to explore the pigmentation particularity and understand the melanin biosynthesis pathway via tyr (tyrosinase) gene knock-out in different freshwater species. To investigate the metabolism processes, researchers attempted to grasp the molecular mechanisms underlying polyunsaturated fatty acids biosynthesis and lipid metabolism in Atlantic salmon and channel catfish. In another aspect, earlier studies were undertaken in cyprinids to produce intramuscular bone-free populations as an important trait of interest. Finally, we examined the possible challenges and perspectives for the future use of this technology in the research field. Overall, the application of CRISPR/Cas9 system is an effective approach to manipulating target genes and enhancing economic traits in different aquatic animal species, thereby strengthening genetic breeding programs. • Inducing mutagenesis to enhance different biological traits via CRISPR/Cas9. • Microinjection is the main used technique for CRISPR/Cas9 delivery. • Challenges and perspectives for the future use of this editing tool in the research field. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (Mtb) protein tyrosine phosphatase (PtpA) has so far been known to control intracellular survival of mycobacteria; whereas the ATP synthase which is essential for mycobacterial growth has recently been contemplated in developing a breakthrough anti-TB drug, diarylquinoline. Since both of these enzymes have been established as validated drug targets; we report a robust and functional relationship between these two enzymes through a series of experiments using Mtb H37Ra. In the present study we report that the mycobacterial ATP synthase alpha subunit is regulated by PtpA. We generated gene knock-out for the enzyme PtpA and subjected to determine the mycobacterial replication and the proteome profile of wild type, mutant (Δ ptpA) and complemented (Δ ptpA : ptpA) strains of Mtb H37Ra. A substantial amount of decrease in the protein level of ATP synthase alpha subunit (AtpA) in case of mutant H37Ra was observed, while the levels of the enzyme were either increased or remained unchanged, in wild type and in the complemented strains. • Tyrosine Phosphatase A (PtpA) increases intracellular survival of Mtb H37Ra. • ATP synthase is the essential enzyme which is regulated by PtpA. • Levels of alpha subunit of ATP synthase is notably reduced in ptpA KO H37Ra. • First report showing that PtpA interacts with alpha subunit of ATP synthase in Mtb H37Ra. [ABSTRACT FROM AUTHOR]

Rats make an excellent model system for studying xenotransplantation since, like mice pluripotent stem cell lines, such as embryonic stem cells and induced pluripotent stem cells as well as gene knock-outs are also available for rats, besides rats have larger organs. The emergence of new genome-editing tools combined with stem cell technology, has revolutionized biomedical research including the field of regenerative medicine. The aim of this manuscript is to provide an overview of the recent progresses in stem cell-derived organ regeneration involving "gene knock-out" and "blastocyst complementation" in the rat model system. Knocking-out Pdx1, Foxn1, and Sall1 genes have successfully generated rat models lacking the pancreas, thymus, and kidney, respectively. When allogeneic (rat) or xenogeneic (mouse) pluripotent stem cells were microinjected into blastocyst-stage rat embryos that had been designed to carry a suitable organogenetic niche, devoid of specific organs, the complemented blastocysts were able to develop to full-term chimeric rat offspring containing stem cell-derived functional organs in their respective niches. Thus, organs with a tridimensional structure can be generated with pluripotent stem cells in vivo, accelerating regenerative medical research, which is crucial for organ-based transplantation therapies. However, to address ethical concerns, public consent after informed discussions is essential before production of human organs within domestic animals. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9 system has been developed as a highly efficient genome editing technology to specifically induce mutations in a few aquaculture species. In this study, we described induction of targeted gene (namely tyrosinase, tyr) mutations in large-scale loach Paramisgurnus dabryanus, an important aquaculture fish species and a potential model organism for studies of intestinal air-breathing function, using the CRISPR/Cas9 system. Tyr gene in large-scale loach was firstly cloned and then its expressions were investigated. Two guide RNAs (gRNAs) were designed and separately transformed with Cas9 in the loach. 89.4% and 96.1% of injected loach juveniles respectively displayed a graded loss of pigmentation for the two gRNAs, in other words, for target 1 and target 2. We classified the injected loach juveniles into five groups according to their skin color phenotypes, including four albino groups and one wild-type-like group. And one of them was clear albino group, which was of high ornamental and commercial value. More than 50 clones for each albino transformant with a visible phenotype in each target were randomly selected and sequenced. Results obtained here showed that along with the increase of pigmentation, wild-type alleles appeared in the injected loach juveniles more often and insertion/deletion alleles less frequently. This study demonstrated that CRISPR/Cas9 system could be practically performed to modify large-scale loach tyr to produce an albino mutant of high ornamental and commercial value, and for the first time showed successful use of the CRISPR/Cas9 system for genome editing in a Cobitidae species. [ABSTRACT FROM AUTHOR]

Abstract Type IV secretion system (T4SS) is a specialized nanomachine that is utilized for the pathogenicity of gram-negative bacteria. However, the role of T4SS genes in virulence of rice bacterial brown stripe pathogen Acidovorax oryzae (Ao) strain RS-2 is not clear, which contains T4SS gene cluster based on genome-wide analysis. Here we compared the virulence-related phenotypes between the wild-type strain RS-2 and nine T4SS mutants, which were constructed in this study. Results indicated that mutation of pilT, pilM, pilQ, or pilZ3 genes not only significantly reduced bacterial virulence, but also caused a reduction of 20.4–62.0% in biofilm formation and 37.7–47.7% reduction in motility, but had no effect on exopolysaccharide (EPS) production or extracellular enzymatic activities when compared to the wild type. The four T4SS genes had a differential effect on bacterial growth after 24 h post-incubation. The complemented strains of the four T4SS mutants restored similar virulence symptom as the wild type. In addition, no change was observed in bacterial virulence by mutation of the other five T4SS genes. Totally, these results demonstrated that T4SS played vital roles in bacterial virulence, motility and biofilm formation in plant pathogen Ao strain RS-2. [ABSTRACT FROM AUTHOR]

We have constructed two plasmids that can be used for cloning as templates for PCR-based gene disruption, mutagenesis and the construction of DNA chromosome translocation cassettes. To our knowledge, these plasmids are the first vectors that confer resistance to ampicillin, kanamycin and hygromycin B in bacteria, and to geneticin (G418) and hygromycin B in Saccharomyces cerevisiae simultaneously. The option of simultaneously using up to three resistance markers provides a highly stringent control of recombinant selection and the almost complete elimination of background resistance, while unique restriction sites allow easy cloning of chosen genetic material. Moreover, we successfully used these new vectors as PCR templates for the induction of chromosome translocation in budding yeast by the bridge-induced translocation system. Cells in which translocation was induced carried chromosomal rearrangements as expected and exhibited resistance to both, G418 and hygromycin B. These features make our constructs very handy tools for many molecular biology applications.

The properties of most systems composed of many interacting elements are neither determined by the topology of the interaction network alone, nor by the dynamical laws in isolation. Rather, they are the outcome of the interplay between topology and dynamics. In this paper, we consider four different types of systems with critical dynamic regime and with increasingly complex dynamical organization (loosely defined as the emergent property of the interactions between topology and dynamics) and analyze them from a structural and dynamic point of view. A first noteworthy result, previously hypothesized but never quantified so far, is that the topology per se induces a notable increase in dynamic organization. A second observation is that evolution does not change dramatically the size distribution of the present dynamic groups, so it seems that it keeps track of the already present organization induced by the topology. Finally, and similarly to what happens in other applications of evolutionary algorithms, the types of dynamic changes strongly depend upon the used fitness function.

The Type VI secretion system (T6SS) is a class of macromolecular machine that is required for the virulence of gram-negative bacteria. However, it is still not clear what the role of T6SS in the virulence of rice bacterial brown stripe pathogen Acidovorax avenae subsp. avenae (Aaa) is. The aim of the current study was to investigate the contribution of T6SS in Aaa strain RS2 virulence using insertional deletion mutation and complementation approaches. This strain produced weak virulence but contains a complete T6SS gene cluster based on a genome-wide analysis. Here we compared the virulence-related phenotypes between the wild-type (RS-2) and 25 T6SS mutants, which were constructed using homologous recombination methods. The mutation of 15 T6SS genes significantly reduced bacterial virulence and the secretion of Hcp protein. Additionally, the complemented 7 mutations ΔpppA, ΔclpB, Δhcp, ΔdotU, ΔicmF, ΔimpJ, and ΔimpM caused similar virulence characteristics as RS-2. Moreover, the mutant ΔpppA, ΔclpB, ΔicmF, ΔimpJ and ΔimpM genes caused by a 38.3~56.4% reduction in biofilm formation while the mutants ΔpppA, ΔclpB, ΔicmF and Δhcp resulted in a 37.5~44.6% reduction in motility. All together, these results demonstrate that T6SS play vital roles in the virulence of strain RS-2, which may be partially attributed to the reductions in Hcp secretion, biofilm formation and motility. However, differences in virulence between strain RS-1 and RS-2 suggest that other factors may also be involved in the virulence of Aaa. [ABSTRACT FROM AUTHOR]

Random boolean networks are a model of genetic regulatory networks that has proven able to describe experimental data in biology. Random boolean networks not only reproduce important phenomena in cell dynamics, but they are also extremely interesting from a theoretical viewpoint, since it is possible to tune their asymptotic behaviour from order to disorder. The usual approach characterizes network families as a whole, either by means of static or dynamic measures. We show here that a more detailed study, based on the properties of system's attractors, can provide information that makes it possible to predict with higher precision important properties, such as system's response to gene knock-out. A new set of principled measures is introduced, that explains some puzzling behaviours of these networks. These results are not limited to random Boolean network models, but they are general and hold for any discrete model exhibiting similar dynamical characteristics. [ABSTRACT FROM AUTHOR]