Mutant Genes Research Articles

The vacuolar phosphatases PAP26 and HIIA2.1 hydrolyze 5'-, 3'-, and 2'-nucleotides derived from RNA degradation.

The vacuole is an important site for RNA degradation. Autophagy delivers RNA to the vacuole, where the vacuolar T2 RNase Ribonuclease 2 (RNS2) plays a major role in RNA catabolism. The presumed products of RNS2 activity are 3'-nucleoside monophosphates (3'-NMPs). Vacuolar phosphatases that carry out 3'-NMP hydrolysis are required to metabolize 3'-NMPs, but the specific players remain unknown. Using a mutant of RNS2 and mutants of the Autophagy-Related Genes 5 and 9 (atg5 and atg9), we confirmed that 3'-NMPs are products of vacuolar RNS2-mediated RNA degradation in Arabidopsis (Arabidopsis thaliana). Moreover, we identified Purple Acid Phosphatase 26 (PAP26) and Haloacid Dehalogenase (HAD) IIA2.1 (HIIA2.1) as vacuolar 3'-NMP phosphatases. Based on phylogenetic analysis, we propose systematic nomenclature for HADIIA enzymes. PAP26 and HIIA2.1 differ in their NMP specificity and activity in vitro. However, the hiia2.1 pap26 double mutant, but generally not the respective single mutants, accumulates 3'-NMPs in addition to 5'-NMPs and, surprisingly, also 2'-NMPs. These findings suggest that PAP26 and HIIA2.1 have overlapping NMP substrate spectra in vivo. Excess 3'- and 2'-NMPs accumulate in plants exposed to a prolonged night, presumably because carbon limitation enhances autophagy-mediated vacuolar RNA degradation. We conclude that vacuolar RNA catabolism releases 3'-NMPs as well as 2'-NMPs through RNS2 and other RNases that also generate 5'-NMPs. PAP26 and HIIA2.1 are required to dephosphorylate these NMPs so that they can enter general nucleotide metabolism outside the vacuole.

The effect of 4-phenylbutyrate and sodium 4-phenylbutyrate on genetic mutation diseases: A meta-analysis.

To determine the efficacy of 4-phenylbutyrate (4-PB) or sodium 4-phenylbutyrate (SPB) in treating diseases caused by genetic mutations. We searched PubMed, Web of Science, Cochrane Library, and EMBASE for studies of patients with genetic mutations treated with 4-PB or SPB. All data were tested using RStudio software. 4-PB or SPB corrected the "functional" production of mutant genes (0.88 [95% confidence interval {CI}: 0.73-1.00], P = .45, I2 = 0%), restored mRNA transcription of mutant genes (0.50 [95% CI: 0.18-0.82], P = .13, I² = 47%), and improved symptoms (0.89 [95% CI: 0.78-1.00], P = .99, I² = 0%) and biochemical laboratory values (1.00 [95% CI: 0.89-1.00], P = .11, I² = 33%) in patients with inherited genetic diseases. 4-PB or SPB can be used to treat genetic diseases. However, they must be validated in high-quality randomized controlled trials before clinical use.

WD40 proteins PaTTG1 interact with both bHLH and MYB to regulate trichome formation and anthocyanin biosynthesis in Platanus acerifolia.

Trichome development and anthocyanin accumulation are regulated by a complex regulatory network, the MBW complexes consist of MYB, bHLH, and WD40 transcription factors. In this study, two sequences, named PaTTG1.1, and PaTTG1.2, were cloned and functionally characterized from Platanus acerifolia. Quantitative real-time PCR results showed that PaTTG1 genes were expressed in the trichomes and red leaves. Overexpression of PaTTG1.1 and PaTTG1.2 genes in Arabidopsis ttg1 mutants restored the phenotypes of ttg1 mutants that were glabrous and lacked purple anthocyanins in hypocotyls and seeds. In transgenic plants, the expression levels of the trichome regulation-related genes AtCPC, AtTRY, AtETC1, AtMYB23, and AtGL2, as well as early and late biosynthetic genes related to anthocyanin biosynthesis, were significantly upregulated. The results of the yeast two-hybrid showed that PaTTG1.1 and PaTTG1.2 proteins could physically interact with both bHLH and R2R3-MYB transcription factors from Arabidopsis and P. acerifolia. Taken together, the results presented in this study suggest that the two PaTTG1 genes share similar functions in the regulation of trichomes and anthocyanins in different species. However, there may be some differences in their regulatory mechanisms.

Mechanism of female CHH caused by compound heterozygous mutations in the LHB gene.

Luteinizing hormone (LH) plays a crucial role in the postnatal development and maturation of gonads. Inactivating mutations of the luteinizing hormone beta subunit (LHB)gene are extremely rare and can result in congenital hypogonadotropic hypogonadism (CHH). We conducted DNA sequencing on an 18-year-old female patient with undetectable LH and clinical symptoms of CHH. Pulsatile GnRH was administered to promote puberty development. In vitro construction of mutant genes, confocal microscopy, and protein functional assays were used to investigate the effects of genetic variants on hormone function and secretion. Experiments were conducted in HEK293T cells to examine the colocalization and dimerization of LH subunits, as well as to measure intracellular and extracellular LH concentrations. Compound heterozygous mutations of c.252C>G (p.F84L) and c.364G>A (p.G122S) were found in the patient's genome. Pulsatile GnRH therapy was effective in promoting puberty development and ovulation. The LH alpha subunit was found to co-localize with both mutant beta subunits after immunofluorescence staining, and immunoprecipitation detected the dimerization of the LH alpha subunit with both mutant beta subunits. Higher intracellular LH concentrations and lower extracellular LH concentrations compared to the wild type indicate secretion dysfunction for LH. Compound heterozygous mutations of c.252C>G (p.F84L) and c.364G>A (p.G122S) in the LHB gene may lead to CHH for female patient. These mutations do not impair the expression and dimerization of the alpha and beta subunits, but they do prevent the secretion of LH. The study expands our understanding of the clinical manifestation of LHB gene mutations in females and provides a treatment for these patients.

Comparative genomics analysis of the reason for 12C6+ heavy-ion irradiation in improving Fe3O4 nanoparticle yield of Acidithiobacillus ferrooxidans.

The Fe3O4 nanoparticle synthesized by Acidithiobacillus ferrooxidans have a broad practical value, while the low yield limits their commercial application. Herein, we employed a 12C6+ heavy-ion beam to induce mutagenesis of A. ferrooxidans BYM and successfully screened a mutant BYMT-200 with a 1.36 mg/L Fe3O4 nanoparticle yield, which could stably inherit over many generations based on assessing cell magnetism and Fe3O4 nanoparticle synthesis. Comparative genome analysis detected 14 mutation sites, causing six synonymous mutations, one missense mutation, and one nonsense mutation. We further annotated the genes involved in the mutation, such as hcp, hsdM, yghU, K7B00_11365, and K7B00_11355, which are responsible for the substantial changes in the Fe3O4 nanoparticle yield of A. ferrooxidans. Additionally, we performed a pan-genome analysis to understand how these genes regulate Fe3O4 nanoparticle synthesis. The core genome of 2376 orthologous clusters was identified and visualized by progressive Mauve alignment and OrthoVenn. A total of 109 regulatory genes related to iron metabolism were identified, mainly involved in electron transport, iron acquisition, iron storage, and oxidative stress. The mutant genes are closely related to iron-sulfur clusters and oxidative stress. Accordingly, we proposed a hypothetical mechanism for increasing Fe3O4 nanoparticle production in A. ferrooxidans BYMT-200 to withstand high oxidative stress caused by heavy ion radiation. Our study offers significant theoretical guidance for further acquiring the high-yield Fe3O4 nanoparticle-producing bacteria and studying the mechanism of its synthesis.

Mechanisms of Staphylococcus aureus Antibiotics Resistance Revealed by Adaptive Laboratory Evolution.

Infection caused by drug-resistant Staphylococcus aureus is a serious public health and veterinary concern. Lack of a comprehensive understanding of the mechanisms underlying the emergence of drug-resistant strains, it makes S. aureus one of the most intractable pathogenic bacteria. To identify mutations that confer resistance to anti-S. aureus drugs, we established a laboratory-based adaptive evolution system and performed 10 rounds of evolution experiments against 15 clinically used antibiotics. We discovered a panel of known and novel resistance-associated sites after performing whole-genome sequencing. Furthermore, we found that the resistance evolved at distinct rates. For example, streptomycin, rifampicin, fusidic acid and novobiocin all developed significant resistance quickly in the second round of evolution. Intriguingly, the cross-resistance experiment reveals that nearly all drug-resistant strains have varying degrees of increased sensitivity to fusidic acid, pointing to a novel approach to battle AMR. In addition, the in silico docking analysis shows that the evolved mutants affect the interaction of rifampcin-rpoB, as well as the novobiocin-gyrB. Moreover, for the genes we got in the laboratory evolution, mutant genes of clinical isolates of human had significant differences from the environmental isolates and animal isolates. We believe that the strategy and data set in this research will be helpful for battling AMR issue of S. aureus, and adaptable to other pathogenic microbes.

A Review of KRAS Inhibitors and Potential Improvement in The Future

Kirsten rat sarcoma viral (KRAS) oncogene is one of the most common mutant genes in tumors, especially in non-small cell lung cancer, colorectal cancer, and pancreatic cancer. In recent years, great success was achieved in targeting KRAS for the treatment of tumors. So far, two drugs, sotorasib and adagrasib, have gotten through the accelerated approval by the FDA. Even so, current treatments are still far from satisfaction due to the inevitable acquired drug resistance and the diversity of mutant isoforms. Various approaches are being investigated to address these issues, including the combination of multiple medications and the development of drugs with more targets. Overall, targeting KRAS is a promising and effective strategy for the treatment of cancer. For this reason, the article aims at introducing several representative KRAS-targeting drugs and exhibiting the latest research progress. Discussions about the acquired resistance and the potential solutions are also included, hoping to provide inspiration for the development and improvement of new drugs.

Effects of Several Bile Acids on the Production of Virulence Factors by Pseudomonas aeruginosa

The presence of bile acids in the cystic fibrosis patient’s lungs contributes to an increase in the inflammatory response, in the dominance of pathogens, as well as in the decline in lung function, increasing morbidity. The aim of this study is to determine the effects of exposure of Pseudomonas aeruginosa to primary and secondary bile acids on the production of several virulence factors which are involved in its pathogenic power. The presence of bile acids in the bacterial culture medium had no effect on growth up to a concentration of 1 mM. However, a slight decrease in the adhesion index as well as a reduction in the virulence of the bacteria on the HT29 cell line could be observed. In this model, exposure of P. aeruginosa to bile acids showed a significant decrease in the production of LasB and AprA proteases due to the reduction in the expression of their genes. A decrease in pyocyanin production was also observed in relation to the effects of bile acids on the quorum sensing regulators. In order to have an effect on gene expression, it is necessary for bile acids to enter the bacteria. P. aeruginosa harbors two potential homologs of the eukaryotic genes encoding the bile acid transporters NTCP1 and NTCP2 that are expressed in hepatocytes and enterocytes, respectively. By carrying out a comparative BLAST-P between the amino acid sequences of the PAO1 proteins and those of NTCP1 and NTCP2, we identified the products of the PA1650 and PA3264 genes as the unique homologs of the two eukaryotic genes. Exposure of the mutant in the PA1650 gene to chenodeoxycholic acid (CDCA) and lithocholic acid (LCA) showed a less significant effect on pyocyanin production than with the isogenic PAO1 strain. Also, no effect of CDCA on the PA3264 gene mutant was observed. This result indicated that CDCA should enter the bacteria by the transporter produced by this gene. The entry of LCA into bacteria seemed more complex and rather responded to a multifactorial system involving the product of the PA1650 gene but also the products of other genes encoding potential transporters.

Fine-Mapping of Tomato male sterile-24 Locus and Marker Development for ms-24 and Its Alleles, ms-10, ms-35, and ms-36

Male sterility is an essential trait for minimizing costs and ensuring seed purity in the production of tomato hybrid seeds. However, its application in commercial breeding faces challenges such as instability, low stigma exsertion rates, and the lack of molecular markers to facilitate the efficient backcrossing of male sterile loci into elite tomato lines. This study characterized the male sterile-24 (ms-24) mutant, which demonstrated exserted stigmas and complete male sterility. Our histological examination revealed that ms-24 anthers displayed dysfunctional development of the tetrads and tapetum. The ms-24 locus was finely mapped to a 149 kb interval containing 22 putative genes. Among these, Solyc02g079810, also known as MS-10, encodes a basic helix–loop–helix (bHLH) transcription factor essential for tapetum and pollen development. A sequencing analysis revealed an approximately 4.9 kb retrotransposon insertion in the first exon of the MS-10 gene in ms-24. Allelism tests confirmed that ms-24 was allelic to ms-10, which is also allelic to ms-35 and ms-36. The same retrotransposon insertion was also identified in the ms-10 mutant, and a similar insertion of retrotransposon was detected in the second exon of the MS-10 gene in the ms-36 mutant. Based on these retrotransposon sequences, codominant insertion–deletion (InDel) markers, MS-24I and MS-35I, were developed for the precise identification of the ms-24, ms-10, ms-35, and ms-36 alleles. These findings establish a foundational basis for subsequent investigations into the molecular mechanisms underlying male sterility and enhance the selection process of male sterile lines in tomato hybrid breeding programs.

Towards the Albino Mutant Gene in Malus × Domestica Borkh.

Albino mutation is among the most common phenomena that often causes a water imbalance and disturbs physiological functions in higher species of trees. Albinism frequently occurs in hybridized apples, but almost all seedlings die shortly after germination. In this study, a spontaneous albino mutant on Fuji apple trees was obtained. After bud grafting, new albino shoots with greenish-white leaves grew, although they were slender, small, and died easily. Resequencing analysis indicated that a total of 49.37 Gbp clean data of the albino mutant samples was obtained; its Q30 reached 91.43%, the average rate mapped was 93.69%, and genome coverage was 96.47% (at least one base cover). Comparisons of the sequences for the albino mutants revealed 4,817,412 single-nucleotide polymorphisms (SNPs), 721,688 insertion/deletion markers (InDels), and 43,072 structural variations (SVs). The genes with non-synonymous SNPs, InDels, and SVs in CDS were compared with KEGG, GO, COG, NR, and SwissProt databases, and a total of 5700 variant genes were identified. A total of 1377 mutant genes had the GO annotation information. Among these, 1520 mutant genes had the pathway annotation and took part in 123 pathways. A total of 1935 variant genes were functionally classified into 25 COG categories. Further research on these variants could help understand the molecular regulatory mechanism of the apple albino mutant. Similarly, variations in the homologous MdAPG1 (Albino or pale-green mutant 1) gene, which was located on Chromosome 11 and belonged to the S-adenosyl-L-methionine-dependent methyltransferases superfamily, may have led to the generation of this apple albino mutant.

Toward a Quadruplet Codon Mitochondrial Genetic Code.

Nature has evolved to exclusively use a genetic code consisting of triplet nucleotide codons. The translation system, however, is known to be compatible with 4-nucleotide frameshift or quadruplet codons. In this study, we begin to explore the possibility of a genome made up entirely of quadruplet codons using the minimal mitochondrial genome of Saccharomyces cerevisiae as a model system. We demonstrate that mitochondrial tryptophanyl- and tyrosyl-tRNAs with modified anticodons effectively suppress mutant cox3 genes containing a TAG stop or TAGA quadruplet codon, leading to the production of full-length COX3 and a respiratory-competent phenotype. This work provides a method for introducing heterologous tRNAs into the yeast mitochondria for genetic engineering applications and serves as a starting point for the development of a quadruplet codon genetic code.

Identification of Pappa and Sall3 as Gli3 direct target genes acting downstream of cilia signaling in corticogenesis

Abstract The cerebral cortex is critical for advanced cognitive functions and relies on a vast network of neurons to carry out its highly intricate neural tasks. Generating cortical neurons in accurate numbers hinges on cell signaling orchestrated by primary cilia to coordinate the proliferation and differentiation of cortical stem cells. While recent research has shed light on multiple ciliary roles in corticogenesis, specific mechanisms downstream of cilia signaling remain largely unexplored. We previously showed that an excess of early-born cortical neurons in mice mutant for the ciliary gene Inpp5e was rescued by re-introducing Gli3 repressor. By comparing expression profiles between Inpp5e and Gli3 mutants, we here identified novel Gli3 target genes. This approach highlighted the transcription factor gene Sall3 and Pappalysin1 (Pappa), a metalloproteinase involved in IGF signaling, as upregulated genes in both mutants. Further examination revealed that Gli3 directly binds to Sall3 and Pappa enhancers and suppresses their activity in the dorsal telencephalon. Collectively, our analyses provide important mechanistic insights into how primary cilia govern the behavior of neural stem cells, ultimately ensuring the production of adequate numbers of neurons during corticogenesis.

Investigation of choline-binding protein of CbpD in the pathogenesis of Streptococcus suis type 2.

Streptococcus suis serotype 2 (S. suis type 2, SS2) is one of the zoonotic pathogens known to induce meningitis, septicemia, and arthritis in both pigs and humans, resulting in public health concerns. CbpD, also termed CrfP, is one of the choline-binding proteins (CBPs) that was found as a murein hydrolase in SS2 and plays crucial roles in natural genetic transformation under the control of ComRS-ComX regulatory system by a previous study. Nonetheless, the possible functions of CbpD in virulence and pathogenesis in SS2 remain unclear. In this study, a cbpD gene mutant (ΔcbpD) with its complemental strain (cΔcbpD) was constructed and further used to examine the pathogenic roles of CbpD in SS2 infection. The results showed that the CbpD deficiency leads to increased bacterial chain elongation and aggregation with little impact on the growth capability of SS2. The ΔcbpD strain represented more vulnerable to a thermo, acid, or oxidative stress. Elevated adhesion to human epithelial HEp-2 cells, decreased invasion into bEND3.0 cells, and more easily phagocytosed by murine RAW264.7 macrophages of ΔcbpD were found. The virulence of cbpD mutant was attenuated in a mouse infection model. Enhanced susceptibility within mice blood and impaired ability to colonize organs with alleviated histopathological lesions were also demonstrated as compared with wild-type SS2. It is noteworthy that the discrepant expression of multiple virulence-associated factors including serine/threonine phosphorylase Stp, anti-phagocytosis factor of transglutaminase TGase and adhesin of chaperon DnaJ, were examined resulting from the deletion of cbpD. Overall, these findings provided evidence that the CbpD factor contributes to SS2 infection and is involved in bacterial adhesion, invasion, and anti-phagocytosis processes by modulating crucial virulence-associated factors expression.

Biomarker analyses from the phase 3 randomized CLEAR trial: Lenvatinib plus pembrolizumab versus sunitinib in advanced renal cell carcinoma.

In CLEAR, lenvatinib + pembrolizumab (L+P) significantly improved efficacy versus sunitinib in first-line treatment of patients with advanced renal cell carcinoma (RCC). We report results from CLEAR biomarker analyses. PD-L1 immunohistochemistry (IHC) and next-generation sequencing assays (whole exome sequencing/RNA-sequencing) were performed on archival tumor specimens. For IHC-derived/RNA-sequencing analyses, a continuous analysis was performed adjusting by Karnofsky performance status (KPS) score for: PD-L1 CPS versus best overall response (BOR)/PFS; and each gene-signature score (T-cell inflamed gene-expression profile [TcellinfGEP]/non-TcellinfGEP signatures including proliferation and angiogenesis) versus BOR/PFS. Association between mutation status of RCC driver genes and PFS were analyzed for genes for which ≥20 patients per arm had oncogenic alterations. Association of molecular subtypes with outcome was evaluated with baseline KPS adjustments. The set of biomarkers evaluated and statistical significance criteria for PD-L1 CPS, gene signature scores, and molecular subtypes were pre-specified. Within-arm analyses using continuous values showed no association between PD-L1 levels and BOR/PFS for either treatment. PFS hazard ratios between arms were similar regardless of mutant or wildtype subgroups of RCC driver genes (VHL, PBRM1, SETD2, BAP1, KDM5C). No associations between PFS and gene-signature scores were observed for L+P. With sunitinib, high proliferation and MYC signature scores showed shorter PFS; high angiogenesis and microvessel density signature scores showed longer PFS. Six new molecular subtypes were defined. Tumors of patients with favorable/intermediate risk were enriched in angiogenesis and angiogenesis/stromal clusters; those with poor risk were enriched in proliferative and unclassified (low-TcellinfGEP/low-angiogenesis/low-proliferation) clusters. No association between molecular subtypes and PFS for L+P/sunitinib was observed (after adjustment for KPS and gene signatures that were individually associated with PFS). Improvements in ORR and PFS for L+P versus sunitinib in aRCC were observed consistently across a range of biomarker subgroups defined using RCC driver mutations, PD-L1, gene expression signatures, and molecular subtypes.

Rapid and high-throughput identification of thermal-responsive genes based on cDNA-library in Upland Cotton

Severe high temperature (HT) climate significantly impacts cotton quality and yield. Consequently, it is essential to mine thermal-responsive genes and explore the underlying mechanisms of HT response in cotton. In this study, we employed a high-throughput cDNA-library method in conjunction with the ALRS system to screen thermotolerant genes in Upland cotton. As a result, a total of 16,120, 13,216 and 172 effective survival genes were filtered after HT stress exposure (42 °C, 220 rpm) for 48 h, 60 h and 72 h, respectively. Functional annotation and enrichment analysis revealed that 170 common genes were involved in regulatory processes associated with HT stress, and the relevant transcriptome data indicated that the majority of these genes responded to temperature fluctuations. Twenty-one genes were randomly selected for verification, and it was found that these genes could enhance yeast resistance to HT stress. Additionally, we selected mutants of homologous Arabidopsis genes for four candidate genes to validate plant thermotolerance during flowering; the thermotolerances of SALK_201915 and SALK_120540.1 were significantly worse. The results demonstrate that numerous candidate genes identified from the cDNA-library contribute to the highly complex molecular network that governs the response and resistance to HT stress in Upland cotton. The high-throughput heat-screening method utilized in this study was optimized for mining thermotolerant genes including improvement in yeast library construction, screening system, gradient reverse pressure, and sequencing library construction. We hope that this new method can be applied in future studies on stress in cotton and other species.

Mutation-Specific CRISPR Targeting with SaCas9 and AsCas12a Restores Therapeutic Sensitivity in Treatment-Resistant Melanoma.

Background: Melanoma remains one of the most challenging cancers to treat effectively with drug resistant remaining a constant concern, primarily with activating BRAF mutations. Mutations in the BRAF gene appear in approximately 50% of patients, 90% of which are V600E. Two frontline BRAF inhibitors (BRAFi), vemurafenib and dabrafenib, are frequently used to treat unresectable or metastatic BRAF V600E melanoma. Initial response rates are high, but soon thereafter, 70-80% of patients develop resistance to treatment within a year. A major mechanism of resistance is the generation of a secondary Q61K mutation in the NRAS gene. Methods: We have developed an approach in which a CRISPR-Cas complex can be designed to distinguish between mutant genes enabling resistance to standard care in tumor cells and normal genomes of healthy cells. For the first time, we demonstrated the utility of two CRISPR-directed mutation-specific editing approaches to restore BRAFi sensitivity in BRAFV600E/NRASQ61K resistant A375 cells. Results: We utilize an AsCas12a protospacer adjacent motif site created by the NRAS Q61K mutation and the Q61K mutation in the critical seed region of an SaCas9 sgRNA for Q61K-selective targeting. We show here that both approaches allow for effective NRAS targeting of only mutated-Q61K and after CRISPR-directed Q61K-targeting, previously resistant A375 cells are re-sensitized to BRAFi treatment. Conclusion: Our data support the feasibility of the development of CRISPR-Cas therapeutic approaches to the treatment of melanoma. Successful therapeutic CRISPR-directed gene editing would enable both specific and efficient editing of a mutation-specific targeting approach eliminate concern for on- and off-target damage to the genomes of healthy cells.

Clinical and genetic characteristics of patients with Alagille syndrome in China: identification of six novel JAG1 and NOTCH2 mutations.

Alagille syndrome (ALGS) is a rare disease. The variable clinical manifestations make the diagnosis of ALGS difficult. This study aimed to provide a basis for the early diagnosis of ALGS patients whose clinical identification is difficult and to enrich the spectrum of genetic variants implicated in Chinese children with ALGS. From August 2016 to August 2022, 14 children with ALGS were enrolled in this retrospective study. Clinical and related data were obtained from medical records. Among the 14 patients, 11 were males and 3 were females. The age of first manifestation of liver disease mean (Q1, Q3) was 0.4 (0.1, 37.0) months, and the age of diagnosis mean (Q1, Q3) was 5.6 (2.4, 48.5) months. Cholestasis was seen in 14 patients, cardiac defects in eight, characteristic facial features in 11, skeletal abnormalities in six, and renal abnormality in one. Among eight patients who underwent ophthalmological examination, posterior embryotoxon was seen in two. We identified 12 different JAG1 gene mutations and two different NOTCH2 gene variations. Among the mutations detected, six were novel, including c.2849_2850del (p.S950*), c.35_45delGCCCCCTAAGC (p.R12Pfs*57), c.1860delC (p.F621Sfs*122), and c.1293_1294insTAGTAGACA (p.A432*) in JAG1, and c.6040_6041 del (p.L2014Vfs*10) and c.1915+1G>T (splicing) in NOTCH2. The follow-up time mean (Q1, Q3) was 48.5 (11.5, 69.0) months; four patients had delayed growth, eight had pruritus, two had xanthomas, seven had elevated bilirubin, and 13 had elevated transaminase. All patients were stable after medical treatment. ALGS presents a variety of clinical manifestations. Some patients may be misdiagnosed with biliary atresia due to bile duct proliferation in liver biopsies along with biochemical abnormalities. Genetic testing is helpful for early diagnosis. JAG1 and NOTCH2 gene mutant spectra are abundant and there are many novel mutations in Chinese children with ALGS.

Identifying Genes Associated With Proliferation, Immunity and Thrombosis in Paroxysmal Nocturnal Haemoglobinuria.

PIGA mutation cannot fully explain the proliferative advantage of abnormal clones and thrombosis tendency in paroxysmal nocturnal haemoglobinuria (PNH), and additional genes may play a role, justifying further investigation. CD59+ and CD59- peripheral blood mononuclear cells from six PNH patients were sorted and subjected to whole-exon sequencing (WES) and whole-transcriptome sequencing respectively. Six age- and sex-matched healthy volunteers were enrolled as controls. Genes related to proliferation, immunity and thrombosis were selected by gene ontology (GO) analysis. The selected gene mutant alleles were then identified in the WES results for 40 patients and verified by the Sanger method in another 40 PNH patients. CD59+ and CD59- peripheral blood mononuclear cells from seven patients were sorted, and the RNA and protein expression levels of target genes were assessed via quantitative real-time PCR (RT-qPCR), flow cytometry and western blotting. The final selected genes were then related to clinical features to analyse. T-cell activation-related genes were upregulated, whereas platelet degranulation, coagulation, haemostasis, leukocyte proliferation and platelet activation-related genes were downregulated in both CD59+ and CD59- cells. The mRNA or protein expression levels of SELP, FLT1, NRP1 and vWF were either different from those in healthy controls or different between CD59+ and CD59- cells. Moreover, platelet aggregation was greater in patients with mutations in these genes than in patients without such mutations. Except for PIGA, other genes may be involved in the proliferation and coagulopathy that occur in PNH patients.

High-Efficiency Genome Editing in Naturally Isolated Aeromonas hydrophila and Edwardsiella Piscicida Using the CRISPR-Cas9 System.

Aeromonas hydrophila and Edwardsiella piscicida are significant bacterial pathogens in aquaculture, causing severe diseases and tremendous economic losses worldwide. Additionally, both of them can act as opportunistic pathogens in humans, leading to severe infections. Efficient genome editing tools for these pathogens are essential for understanding their pathogenic mechanisms and physiological behaviors, enabling the development of targeted strategies to control and mitigate their effects. In this study, we adapted the CRISPR-Cas9 system for high-efficiency, marker-less genome editing in multiple naturally isolated strains of these two aquaculture pathogens. We developed a streamlined procedure that successfully generated deletion mutants of the aerA gene (encoding for aerolysin, a pore-forming toxin that plays a critical role in the pathogenicity) and the gfp insertion mutants in three naturally isolated A. hydrophila strains. Additionally, we deleted five putative hemolysin-encoding genes in both A. hydrophila ML10-51K and its ∆aerA derivative. The same system was also applied to the naturally isolated E. piscicida S11-285 strain, successfully deleting the ssaV gene (a component of the Type III Secretion System-a critical virulence mechanism in many pathogenic bacteria). The methodologies developed herein could be broadly applied to other pathogenic strains from natural environments, providing valuable tools for studying bacterial pathogenesis and aiding in the development of effective control strategies.

Multi-omics uncovers the potential functions of transcription factor Dp-1 in human digestive cancers

Previous studies have reported the critical effects of TFDP1 on the occurrence and progression of multiple cancers. Nevertheless, these studies were confined to a single cancer type rather than human digestive cancers. Therefore, using various integrated databases available, we conducted a multi-omics analysis of the potential roles of TFDP1 in digestive cancers. Firstly, high TFDP1 mRNA expression levels were observed in all seven digestive cancers, and were closely related to survival differences, pathological features, as well as immune and molecular subtypes. Epigenetic alterations of TFDP1 were evident in the four digestive cancers, and TFDP1 promoter methylation was negatively correlated with TFDP1 mRNA expression. Subsequently, using MuTarget, we identified several mutant genes accountable for TFDP1 overexpression. In addition, function enrichment analysis unveiled that TFDP1 could influence the cell cycle process and the pathways of the cell cycle and FoxO signaling. Intriguingly, our single-cell analysis demonstrated high expression of TFDP1 in blood and immune cells. We also found the statistically significant correlation of TFDP1 expression with six infiltration cell types, four immunosuppressive cell types, and various drugs. Finally, the TIDE results revealed that TFDP1 had a higher predictive ability of TFDP1 than four classical biomarkers and TFDP1 expression was associated with various types of cohorts. Taking the CRISPR screen cohorts, we observed the powerful effect of TFDP1-knockout on MC38 colon cancer cells. These findings could help elucidate the potential functions of TFDP1 and facilitate the development of TFDP1-related diagnostic and therapeutic strategies for different digestive cancers.