Gene Deletion Research Articles

Development of an Efficient CRISPR/Cas9 System in Fusarium verticillioides and Its Application in Reducing Mycotoxin Contamination.

Fusarium verticillioides (F. verticillioides) is a globally recognized and highly impactful fungal pathogen of maize, causing yield losses and producing harmful mycotoxins that pose a threat to human and animal health. However, the genetic tools available for studying this crucial fungus are currently limited in comparison to other important fungal pathogens. To address this, an efficient CRISPR/Cas9 genome editing system based on an autonomously replicating plasmid with an AMA1 sequence was established in this study. First, gene disruption of pyrG and pyrE via nonhomologous end-joining (NHEJ) pathway was successfully achieved, with efficiency ranging from 66 to 100%. Second, precise gene deletions were achieved with remarkable efficiency using a dual sgRNA expression strategy. Third, the developed genome editing system can be applied to generate designer chromosomes in F. verticillioides, as evidenced by the deletion of a crucial 38 kb fragment required for fumonisin biosynthesis. Fourth, the pyrG recycling system has been established and successfully applied in F. verticillioides. Lastly, the developed ΔFUM1 and ΔFUM mutants can serve as biocontrol agents to reduce the fumonisin B1 (FB1) contamination produced by the toxigenic strain. Taken together, these significant advancements in genetic manipulation and biocontrol strategies provide valuable tools for studying and mitigating the impact of F. verticillioides on maize crops.

Genetic or pharmacological GHSR blockade has sexually dimorphic effects in rodents on a high-fat diet

The stomach-derived hormone ghrelin regulates essential physiological functions. The ghrelin receptor (GHSR) has ligand-independent actions; therefore, GHSR gene deletion may be a reasonable approach to investigate the role of this system in feeding behaviors and diet-induced obesity (DIO). Here, we investigate the effects of a long-term (12-month) high-fat (HFD) versus regular diet on obesity-related measures in global GHSR-KO and wild-type (WT) Wistar male and female rats. Our main findings are that the GHSR gene deletion protects against DIO and decreases food intake during HFD in male but not in female rats. GHSR gene deletion increases thermogenesis and brain glucose uptake in male rats and modifies the effects of HFD on brain glucose metabolism in a sex-specific manner, as assessed with small animal positron emission tomography. We use RNA-sequencing to show that GHSR-KO rats have upregulated expression of genes responsible for fat oxidation in brown adipose tissue. Central administration of a novel GHSR inverse agonist, PF-5190457, attenuates ghrelin-induced food intake, but only in male, not in female mice. HFD-induced binge-like eating is reduced by inverse agonism in both sexes. Our results support GHSR as a promising target for new pharmacotherapies for obesity.

Contribution of clinically relevant copy number variations in long QT syndrome: a large cohort study

Abstract Background Copy number variations (CNVs) have increasingly been recognized in the cardiovascular setting. Their role in hypertrophic cardiomyopathy or sudden cardiac death has been described, but their involvement in Long QT syndrome (LQTS) has been scarcely studied. Most of these descriptions are confined to case reports or small series. Purpose The aim of this study is to investigate the proportion of clinically relevant CNVs among a large cohort of index cases referred for genetic testing due to LQTS. Methods Retrospective study in which the presence of likely pathogenic (LP) or pathogenic (P) CNVs was evaluated in a cohort of index cases referred for genetic study with LQTS phenotype. Index cases were genotyped with customized libraries through next generation sequencing (NGS). CNVs analysis was conducted by a proprietary method by means of comparative analysis of normalized read depth data. Clinically relevant (LP or P) CNVs were then confirmed by other techniques. Results 1183 index cases were referred for genetic testing to our laboratory with long QT syndrome phenotype. Some of these index cases were referred for suspicion of Jervell-Lange-Nielsen syndrome (JNLS) or for LQTS associated with congenital deafness (n=10) or Anderson-Tawil syndrome (n=1). Around 27% (n=322) of the index cases had a positive result. CNVs analysis was run on 1071 (91%) of the index cases. Most of the CNVs were considered negative (n=994, 93%), 59 could not be analysed (6%) and in 11 patients (1%), a LP/P CNV was identified. All CNVs but three (72.72%), were confirmed by orthogonal molecular techniques (Sanger sequencing or multiplex ligation-dependent probe amplification (MLPA)). In all, but two, the genetic study was considered positive due to the CNV findings. One of these patients carried a pathogenic lost-of-function (LOF) CNV in the LDLR gene (incidental finding) but the report was negative because he was referred for suspicion of JLNS. In another case a CNV in the DSP gene was identified in mosaicism (30-40%). However, in this case, the positive report was attributed to the presence of an additional pathogenic variant in CACNA1C. Regarding the other index cases with LP/P CNVs: 4 carried a deletion in the KCNH2 gene, 3 carried a deletion in KCNQ1 gene (one of them in homozygosis and was studied for suspicion of JLNS) and 1 carried a duplication in the KCQN1 (LOF variant). In addition, one patient carried a deletion of exon 3 in the RYR2 gene. Even if the patient was referred for suspicion of LQTS, the report was considered positive. These clinically relevant CNVs represented around 3% of the positive genetic results. Conclusions In our large cohort of patients referred for genetic testing for LQTS, around 1% of clinically relevant CNVs were identified. As such large genomic rearrangements underlie a non-neglectable portion of cases, we consider that their analysis should be performed as part of the routine genetic testing.Deletion exons 6-14 in KCNH2 gene (CNV)MLPA with deletion exons 6-14 in KCNH2

Effects of efflux pumps on antifungal activity of chitosan against Candida albicans

ABSTRACT Background Antifungal resistance is a major problem, commonly caused by drug-efflux pump overexpression. To evaluate if chitosan could be effective in drug-resistant Candida infections, we investigated the effects of efflux pumps on antifungal activity of chitosan. Materials and Methods The minimal fungicidal concentration (MFC) of oligomer (7–9 kD) and polymer (900–1,000 kD) chitosan against Saccharomyces cerevisiae and Candida albicans were evaluated by broth and agar dilution methods. The MFCs of S. cerevisiae with single deletion of efflux pump genes, with deletion of seven efflux pumps (AD∆), and AD∆ overexpressing C. albicans efflux pump genes (CDR1, CDR2 and MDR1) were determined. C. albicans with homozygous deletions of CDR1 and of CDR2 were generated using CRISPR-Cas9 system and tested for chitosan susceptibility. Results While deleting any individual efflux pump genes had no effect on chitosan susceptibility, simultaneous deletion of multiple pumps (in AD∆) increased sensitivity to both types of chitosan. Interestingly, the overexpression of CDR1, CDR2 or MDR1 in AD∆ barely affected its sensitivity. Moreover, C. albicans with homozygous deletions of CDR1 and/or CDR2 showed similar sensitivity to wildtype. Conclusion Thus, C. albicans susceptibility to chitosan was not affected by drug-efflux pumps. Chitosan may be a promising antifungal agent against pump-overexpressing azole-resistant C. albicans.

Spinal cord neurone loss and foot placement changes in a rat knock-in model of amyotrophic lateral sclerosis type 8

Abstract Amyotrophic Lateral Sclerosis is an age-dependent cell type-selective degenerative disease. Genetic studies indicate that Amyotrophic Lateral Sclerosis is part of a spectrum of disorders ranging from spinal muscular atrophy to frontotemporal dementia that share common pathological mechanisms. Amyotrophic Lateral Sclerosis type 8 is a familial disease caused by mis-sense mutations in VAPB. VAPB is localised to the cytoplasmic surface of the endoplasmic reticulum where it serves as a docking point for cytoplasmic proteins and mediates inter-organelle interactions with the endoplasmic reticulum membrane. A gene knock-in model of Amyotrophic Lateral Sclerosis type 8 based on the VapBP56S mutation and VapB gene deletion have been generated in the rat. These animals display a range of age-dependent phenotypes distinct from those previously reported in mouse models of Amyotrophic Lateral Sclerosis type 8. A loss of motor neurones in VapBP56S/+ and VapBP56S/P56Sanimals is indicated by a reduction in the number of large choline acetyl transferase-staining cells in the spinal cord. VapB-/-animals exhibit a relative increase in cytoplasmic TDP-43 levels compared to the nucleus, but no large protein aggregates. Concomitant with these spinal cord pathologies VapBP56S/+, VapBP56S/P56S and VapB-/-animals exhibit age-dependent changes in paw placement and exerted pressures when traversing a CatWalk apparatus, consistent with a somatosensory dysfunction. Extra motor dysfunction is reported in half the cases of motor neurone disease, and this is the first indication of an associated sensory dysfunction in a rodent model of Amyotrophic Lateral Sclerosis. Different rodent models may offer complementary experimental platforms with which to understand the human disease.

Immune Deficiency/Dysregulation Associated Lymphoid Proliferations, EBV+ In Persons Living With HIV.

The 5th edition of the World Health Organization Classification of Hematolymphoid Tumors recently defined immune deficiency/dysregulation (IDD)-associated-lymphoid-proliferations in HIV settings, where information is scarce, often gone under or misdiagnosed. To describe the clinical picture, histopathology, and outcomes of IDD-associated-lymphoidproliferations Epstein-Barr virus+ (EBV) in people living with HIV without organ transplantation, antiretroviral therapy (ART) treated. HIV+ patients diagnosed with IDD-associated-lymphoid-proliferations seen at an academic medical center in Mexico from 2016 to 2019 were included. Immunohistochemical studies, in situ hybridization, and polymerase chain reaction analysis for EBV and LMP1 gene deletions were performed and correlated with clinical data. We included 27 patients, all men who have sex with men, median age 36 years (interquartile range [IQR] 22-54). The median baseline CD4+ T cells were 113/mL (IQR 89-243), the CD4+/CD8+ ratio was 0.15 (IQR: 0.09-0.22), and the HIV viral load was 184,280 copies/mL (IQR: 76,000-515,707). Twenty patients (74.07%) had IDD-associated-lymphoid-proliferations hyperplasia plasma cell type EBV+, 3 (11.1%) had hyperplasia mononucleosis-like type (IM-type), 1 patient (3.70%) had florid follicular hyperplasia, 3 (11.1%) IDD-associated-lymphoid-proliferations polymorphic type, and there were 22 cases (81.4%) of synchronic Kaposi Sarcoma. Two patients were diagnosed with Hodgkin lymphoma following a second positron emission tomography-computed tomography scan-guided biopsy. The median follow-up was 228 weeks (IQR 50-269); 6 patients died (22.2%) of causes unrelated to IDD-associated-lymphoid-proliferations related. IDD-associated-lymphoid-proliferations EBV+ occured in severely immunosuppressed HIV+ patients, a high percentage of whom had concomitant Kaposi sarcoma. The prognosis was good in patients treated only with ART.

#3020 A novel copy number analysis identifies human patients with NPHP1 whole gene deletions in previously genetically unsolved cases

Abstract Background and Aims Genome sequencing is rapidly becoming a routine approach to investigating inherited human diseases in clinical practice. Nevertheless, data generated from genome sequencing requires extensive bioinformatic analysis and filtering of variants to determine pathogenicity and remains challenging. Despite well-established workflows and software in place to process raw data, the analysis of genome sequencing data is still complex. Copy Number Variations (CNVs) are increasingly recognised to be pathogenic, especially where this leads to a whole gene being deleted. Software tools to decipher CNVs across genome sequencing datasets are being utilised but are not completely accurate. Method Using a bespoke CNV analysis of Genomics England 100,000 Genomes Project (100 kGP) WGS data from 11754 parent-child trios, we searched for homozygous deletions overlapping with annotated genes, across the 22 autosomes. Results Homozygous ∼150 kb whole gene deletion of NPHP1-MALL on chromosome 1, which aligned with a known region containing segmental duplications (SD), was the most frequent observation among all autosomal recessive (AR) OMIM-morbid genes. Clinical phenotypes were consistent with this genotype including nephronophthisis and retinal dystrophy. We extended our analysis to look for NPHP1 whole gene deletions in 18877 additional singletons within the 100 kGP. This reverse genetics approach identified additional patients, up to this point genetically unsolved, with whole gene deletions and matching phenotypes. The frequency of heterozygous NPHP1 whole gene deletion was assessed in a control cohort of 15440 cancer patients within 100 kGP and was found to be 0.35%. Conclusion In conclusion, NPHP1 was the most frequently observed AR OMIM-morbid gene knockout in our analysis. Homozygous whole gene deletions in NPHP1 may be easily missed using standard WGS pipelines which don't assess fully for CNVs and a targeted read-depth approach to identify CNVs in parent-patient trios and singletons is valuable in identifying these deletions.

#1049 Genetic characteristics of autosomal dominant polycystic kidney disease in a Greek population

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD) is the predominant monogenic renal genetic disorder, and the fourth leading cause of end stage kidney disease. Confirmatory genetic testing defines the disease, provides prognostic information and may support therapeutic management. This study aims to investigate the genetic characteristics of a substantial cohort of ADPKD patients from a single Greek center. Method Ninety-three patients (46 females, 47 males with a mean age of 34 ± 15 years) were enrolled from 59 distinct families attending the outpatient ADPKD clinic at the Nephrology Department of the General Hospital of Athens “G. Gennimatas.” Genetic analysis included targeted next-generation sequencing (tNGS) of over 600 clinically relevant genes, Sanger sequencing, and Multiplex ligation-dependent probe amplification (MLPA). Whole exome and whole genome sequencing (WES and WGS) studies were conducted in 3 patients with negative results. Magnetic Resonance Imaging (MRI) was performed in 74 out of 93 patients, revealing more than 10 renal cysts in all but one patient. Children with a positive family history (FH) displayed at least one cyst in echography, while two without FH had multiple renal cysts. Results Genetic diagnosis was established in 88 of 93 patients (94.5%). Seventy patients (75%), demonstrated single nucleotide variants (SNVs) and large deletions (4 patients) in the PKD1 gene, whereas SNVs and large deletions (5 patients) in the PKD2 gene were identified in 15 (16%) patients. Additionally, mutations in genes other than PKD1 and PKD2 were identified in 3 patients (3.5%). Two cases, involving a father and son, revealed the presence of a SEC61B variant; the father exhibited more than 10 renal cysts and liver cysts in the MRI, while the son displayed only liver cysts. Additionally, a third patient, a woman with nephrolithiasis and over 10 renal cysts in the MRI, exhibited a SLC3A1 variant. Notably, 5 patients (5.5%) with ADPKD phenotype had negative genetic analyses, despite the application of both WES and WGS in three of these patients. Truncating mutations in PKD1 were observed in 43 of 70 cases, (61%), while the rest (39%) had non-truncating mutations. Detected variants were classified as Pathogenic (45.5%), Likely Pathogenic (36%), and Variants of Uncertain Significance (VUS-13.5%, 12 cases), according to the guidelines of the American College of Medical Genetics and Genomics. Reclassification to Likely Pathogenic could be proposed for 6 out of 12 cases with VUS variants for which family segregation was noted. Finally, 52% and 82% of the detected variants have not been previously reported in ClinVar and gnomAD databases, respectively. Conclusion In this ethnically homogenous population, truncating mutations in the PKD1 gene were the predominant genetic cause of ADPKD, followed by PKD1 non-truncating mutations, PKD2 gene variants and other rare or undefined genetic etiologies.

#187 Vasorin (VASN) commands podocyte quiescence and glomerular homeostasis. A novel FSGS mechanism?

Abstract Background and Aims Podocyte dysfunction and loss of quiescence contribute to focal segmental glomerulosclerosis (FSGS) and crescentic glomerulonephritis (CGN). Here, we characterized the role of Vasorin (VASN) in podocytes. Method We combined laser capture microdissection and deep RNA sequencing of human glomeruli, RNAscope-based in situ hybridization, immunostaining and mouse models with Vasn-driven fluorescent reporter, targeted Vasn gene deletion in podocytes and cultures of human podocytes. Results VASN mRNA is strongly expressed in healthy human and mouse glomeruli with a podocyte-specific pattern. Both global and podocyte-specific, constitutive or inducible gene deletion of Vasn in mice induced a nephrotic syndrome and kidney failure after day 14, with podocyte dedifferentiation and loss and severe FSGS lesions, indicating a fundamental and non-developmental role of VASN in podocyte homeostasis. Furthermore, we measured markedly decreased VASN expression in podocytes of patients with FSGS and ANCA-associated CGN but not with Minimal Change Disease (MCD) and Membranous Nephropathy (MN). We thus wondered whether VASN abundance could be a disease modifier for glomerulopathies. Mice with podocyte-specific partial heterozygous deletion of Vasn showed no evident baseline renal abnormalities. Still, they showed significantly more podocyte loss, albuminuria and kidney failure than wild-type mice when challenged by type 1 diabetes with glomerular hyperfiltration or CGN experimental models. Human podocytes transduced with lentiviral VASN-targeting shRNA (shVASN) displayed increased focal adhesion dynamics, resistance to oscillating mechanical stretches, enhanced migratory capacity and epithelial-mesenchymal transition (EMT) markers. Moreover, shVASN podocytes had a higher proliferative capacity, increased cell-cycle reentry and a relative S-phase slow-down. Likewise, transcriptomics analysis found the up-regulation of genes involved in cell metabolism, adhesion and EMT and the down-regulation of genes involved in the S to G2/M phase transition. In vivo, we confirmed that VASN deficiency increased podocyte proliferation in situ and urinary shedding. Conclusion These findings highlight the novel role of VASN as an essential master protein that maintains podocyte quiescence and homeostasis during health and kidney diseases while offering a potential new therapeutic target. VASN control mechanisms driving podocyte cell-cycle reentry and detachment and FSGS. Precise mechanisms of VASN functions in podocytes need further insights into VASN molecular partners.

#2245 ECYSCO, a European cystinosis cohort

Abstract Background and Aims Cystinosis is a rare multisystem lysosomal storage disease due to variants in the CTNS gene, coding for the carrier protein cystinosin, a lysosome membrane transporter, causing cystine accumulation. Specific treatment by cysteamine decreases renal and extrarenal complications frequency in cystinosis patients but data on long-term manifestations of the disease are lacking. The aim of the ECYSCO cohort is to describe the natural history of the disease and the effect of treatments. Methods We set up a European, multi-centre, longitudinal, non-interventional cohort, ECYSCO, that uses observational study methods to collect uniform data. 243 patients with a confirmed diagnosis of cystinosis and followed in 25 French and 5 European centers (Belgium, Italy, Spain and Germany) were included. Data are collected on the secure RaDiCo platform, via an e-CRF (REDCap). Results Data from 177 patients (50.8% male) were analyzed. Median age at diagnosis was 1.3 years [IQ 0.9; 1.8], with earlier diagnosis since the 1980s, but no further improvement in the 2000s. Genetic analysis was available for 158 patients: 46 (31.9%) presented with homozygous 57kb deletion in the CTNS gene, 57 (39.6%) with heterozygous 57kb deletion associated with another variant and 46 (26.4%) with other variants. The type of variant had no impact on the age at diagnosis. Median age at cysteamine start was 1.6 years (IQ 1.0-3.0). An improvement on age at treatment start was observed after the 1990s. All but 7 patients were treated with cysteamine. Sixty-seven patients received immediate release formulation (Cystagon®) and 103 received extended release formulation (Procysbi®). Median white blood cell cystine level was 1.2 nmol ½ cystine/mg protein (IQ 0.6; 2.2). The median duration of treatment was 21.5 years [IQ 11.7; 31.1]. 165 (93,2%) patients also received cysteamine ocular gel, Cystadrops®. Median age at inclusion was 18.4 years (IQ 10.4; 30.6). At that time, 104 patients (57.8%) had reached kidney failure (KF). There was no impact of genotype on age at KF. Median age at KF was 12.9 years [IQ 9.9; 18.0]. A 5-year gain in renal survival was observed after the 1990s. 102 patients (56.7%) received a kidney transplant. Among these transplanted patients: 76 (74.5%) received 1 transplant, 23 (22.5%) received 2 consecutive transplants, and 3 (2.9%) received 3. Median eGFR in the remaining patients was 58.9 ml/min [IQ 40.4; 82.2]. Extrarenal manifestations included hypothyroidism in 29 (16.4%) patients, diabetes mellitus in 15 (8.4%), skeletal manifestations in 89 (53.6%), myopathy in 28 (17.9%), and neurological disorders in 21 (13.5%). Skeletal manifestations involved patients before dialysis/transplantation in 46% of cases. The main complications in these patients were genu valgum (62.9%), genu varum (11.1%), kyphosis (21.2%), fractures (20.4%), and 12.6% required surgery. Patients with myopathy were dialysed or transplanted in 58.8% of cases. Conclusion Cystinosis is a good example of a pediatric disease with multiorgan involvement extending into adult care. More than half of patients are adults and have reached kidney failure even if age at renal replacement therapy start has increased. The high frequency of extra-renal manifestations demonstrates the importance of a multidisciplinary follow up of these patients.

Analysis of the nischarin expression across human tumor types reveals its context-dependent role and a potential as a target for drug repurposing in oncology.

Nischarin was reported to be a tumor suppressor that plays a critical role in breast cancer initiation and progression, and a positive prognostic marker in breast, ovarian and lung cancers. Our group has found that nischarin had positive prognostic value in female melanoma patients, but negative in males. This opened up a question whether nischarin has tumor type-specific and sex-dependent roles in cancer progression. In this study, we systematically examined in the public databases the prognostic value of nischarin in solid tumors, regulation of its expression and associated signaling pathways. We also tested the effects of a nischarin agonist rilmenidine on cancer cell viability in vitro. Nischarin expression was decreased in tumors compared to the respective healthy tissues, most commonly due to the deletions of the nischarin gene and promoter methylation. Unlike in healthy tissues where it was located in the cytoplasm and at the membrane, in tumor tissues nischarin could also be observed in the nuclei, implying that nuclear translocation may also account for its cancer-specific role. Surprisingly, in several cancer types high nischarin expression was a negative prognostic marker. Gene set enrichment analysis showed that in tumors in which high nischarin expression was a negative prognostic marker, signaling pathways that regulate stemness were enriched. In concordance with the findings that nischarin expression was negatively associated with pathways that control cancer growth and progression, nischarin agonist rilmenidine decreased the viability of cancer cells in vitro. Taken together, our study lays a ground for functional studies of nischarin in a context-dependent manner and, given that nischarin has several clinically approved agonists, provides rationale for their repurposing, at least in tumors in which nischarin is predicted to be a positive prognostic marker.

#1453 EZH2 is an epigenetic regulator of renal gluconeogenesis in chronic kidney disease through downregulation of PCK1

Abstract Background and Aims Impaired renal gluconeogenesis has been recently identified as a hallmark of chronic kidney diseases (CKD), and is tightly correlated with the progression of renal tubulointerstitial fibrosis. The methyltransferase enhancer of zeste homolog 2 (EZH2) is an epigenetic regulator playing an important role in renal tubulointerstitial fibrosis. Whether renal gluconeogenesis can be epigenetically regulated is currently unknown. In the current study, we studied EZH2-mediated epigenetic regulation of renal gluconeogenesis in three different mouse models of renal fibrosis. Method Mouse models of renal fibrosis were established by unilateral ureteral obstruction (UUO) operation, unilateral ischemia reperfusion injury or folic acid (FA) injection. Human proximal tubular cell line (HK2), primary tubular cells or primary tubules was used as an in vitro or ex vivo model. EZH2 was conditional knockout or inhibited by 3-DZNeP in mice. Inhibition of EZH2 by 3-DZNeP or overexpressed by adenovirus was performed in vitro. RNA sequencing and cleavage under targets and tagmentation (CUT&Tag) sequencing analysis were performed and followed by quantitative PCR, CUT&Tag and Western blotting analysis. Glucogenic metabolites were measured and systemic glucose metabolism was assessed by tolerance tests. Results By deletion of Ezh2 gene in mice through a ubiquitous expressed Cre/Esr1 system, we confirmed the pro-fibrotic effect of EZH2 in unilateral ureteral obstruction (UUO) kidneys. Through RNA sequencing and CUT&Tag sequencing analysis, we found that the phosphoenolpyruvate carboxykinase 1 (PCK1), a critical enzyme in gluconeogenesis, is negatively regulated by the methyltransferase EZH2 in fibrotic kidneys, which was further confirmed by quantitative PCR, CUT&Tag and Western blotting analysis in renal cells and UUO or FA kidneys. We further showed that pharmaceutical inhibition of EZH2 by 3-DZNeP enhances PCK1 expression in fibrotic kidneys of three mouse models. Moreover, the concentration of lactate, a glucogenic metabolic intermediate, was increased in mouse fibrotic kidneys, which was reduced by EZH2 inhibition. A further experiment showed that systemic glucose metabolism was impaired in UUO mice and restored by EZH2 inhibition as assessed by insulin or glucose tolerance test. Upregulated EZH2 in UUO kidneys is colocalized with the proximal tubular marker as shown by immunofluorescent staining. The direct anti-gluconeogenesis effect of EZH2 on renal proximal epithelial cells and primary renal tubules was shown by analysis of gluconeogenic gene expression and production of glucose or lactate in human HK2 cells treated with EZH2 inhibitor or exogeneous expression of EZH2 by adenovirus transfection. Importantly, we further confirmed the direct effect of tubular EZH2 on gluconeogenesis in tubular specific deleted EZH2 mice (Ezh2fl/fl;Cdh16-Cre). Moreover, inhibition of PCK1 by 3-mercaptopropionic acid abrogated the pro-gluconeogenic effect and anti-fibrotic effect of EZH2 inhibitor in renal cells and UUO kidneys. Finally, RNA sequencing data from kidney allograft biopsies showed that EZH2 is progressively increased in CKD patients. Conclusion We conclude that EZH2 promotes renal tubulointerstitial fibrosis through epigenetic inhibition of PCK1. Our study suggests that therapeutic restoration of the impaired renal gluconeogenesis is beneficial to CKD.

Deciphering the genetic landscape of lumpy skin disease: Unraveling variable virulence through comprehensive genome sequence analysis in India

Lumpy Skin Disease (LSD), a poxvirus disease affecting cattle, emerged in India in 2019 and intensified in 2022, resulting in significant economic losses for dairy farmers. There was unusual shift in mortality and morbidity patterns during the second wave. A comprehensive genetic study conducted, analyzing samples from 2019 to 2022 revealed circulation of two distinct subclades (subclade 1.2a and 1.2b) in India, with the latter showing a different pattern in morbidity and mortality. Notably, the Ankyrin repeats gene-based analysis could differentiate animals with varying clinical scores. Genetic variations were significant, with unique deletions identified, including a 12-nucleotide deletion in the GPCR gene in virus isolates collected during 2022 outbreaks, not reported earlier in Indian LSDV strains. A crucial finding was a significant 95-nucleotide deletion in the Functional Resolution Sequence (FRS) repeats of LSDV genomes from 2022 outbreaks, absent in 2019 samples. These deletions may have influenced the virus's virulence in India.

Exploration of Gene Therapy for Alport Syndrome.

Alport syndrome is a hereditary disease caused by mutations in the genes encoding the alpha 3, alpha 4, and alpha 5 chains of type IV collagen. It is characterized by hematuria, proteinuria, progressive renal dysfunction, hearing loss, and ocular abnormalities. The main network of type IV collagen in the glomerular basement membrane is composed of α3α4α5 heterotrimer. Mutations in these genes can lead to the replacement of this network by an immature network composed of the α1α1α2 heterotrimer. Unfortunately, this immature network is unable to provide normal physical support, resulting in hematuria, proteinuria, and progressive renal dysfunction. Current treatment options for Alport syndrome include angiotensin-converting enzyme inhibitors and angiotensin receptor blockers, which aim to alleviate glomerular filtration pressure, reduce renal injury, and delay the progression of renal dysfunction. However, the effectiveness of these treatments is limited, highlighting the need for novel therapeutic strategies and medications to improve patient outcomes. Gene therapy, which involves the use of genetic material to prevent or treat diseases, holds promise for the treatment of Alport syndrome. This approach may involve the insertion or deletion of whole genes or gene fragments to restore or disrupt gene function or the editing of endogenous genes to correct genetic mutations and restore functional protein synthesis. Recombinant adeno-associated virus (rAAV) vectors have shown significant progress in kidney gene therapy, with several gene therapy drugs based on these vectors reaching clinical application. Despite the challenges posed by the structural characteristics of the kidney, the development of kidney gene therapy using rAAV vectors is making continuous progress. This article provides a review of the current achievements in gene therapy for Alport syndrome and discusses future research directions in this field.

Tools for Cre-Mediated Conditional Deletion of Floxed Alleles from Developing Cerebellar Purkinje Cells.

The Cre-lox system is an indispensable tool in neuroscience research for targeting gene deletions to specific cellular populations. Here we assess the utility of several transgenic Cre lines, along with a viral approach, for targeting cerebellar Purkinje cells (PCs) in mice. Using a combination of a fluorescent reporter line (Ai14) to indicate Cre-mediated recombination and a floxed Dystroglycan line (Dag1flox ), we show that reporter expression does not always align precisely with loss of protein. The commonly used Pcp2Cre line exhibits a gradual mosaic pattern of Cre recombination in PCs from Postnatal Day 7 (P7) to P14, while loss of Dag1 protein is not complete until P30. Ptf1aCre drives recombination in precursor cells that give rise to GABAergic neurons in the embryonic cerebellum, including PCs and molecular layer interneurons. However, due to its transient expression in precursors, Ptf1aCre results in stochastic loss of Dag1 protein in these neurons. NestinCre , which is often described as a "pan-neuronal" Cre line for the central nervous system, does not drive Cre-mediated recombination in PCs. We identify a Calb1Cre line that drives efficient and complete recombination in embryonic PCs, resulting in loss of Dag1 protein before the period of synaptogenesis. AAV8-mediated delivery of Cre at P0 results in gradual transduction of PCs during the second postnatal week, with loss of Dag1 protein not reaching appreciable levels until P35. These results characterize several tools for targeting conditional deletions in cerebellar PCs at different developmental stages and illustrate the importance of validating the loss of protein following recombination.

Cymoxanil disrupts RNA synthesis through inhibiting the activity of dihydrofolate reductase.

The agricultural fungicide cymoxanil (CMX) is commonly used in the treatment of plant pathogens, such as Phytophthora infestans. Although the use of CMX is widespread throughout the agricultural industry and internationally, the exact mechanism of action behind this fungicide remains unclear. Therefore, we sought to elucidate the biocidal mechanism underlying CMX. This was accomplished by first performing a large-scale chemical-genomic screen comprising the 4000 haploid non-essential gene deletion array of the yeast Saccharomyces cerevisiae. We found that gene families related to de novo purine biosynthesis and ribonucleoside synthesis were enriched in the presence of CMX. These results were confirmed through additional spot-test and colony counting assays. We next examined whether CMX affects RNA biosynthesis. Using qRT-PCR and expression assays, we found that CMX appears to target RNA biosynthesis possibly through the yeast dihydrofolate reductase (DHFR) enzyme Dfr1. To determine whether DHFR is a target of CMX, we performed an in-silico molecular docking assay between CMX and yeast, human, and P. infestans DHFR. The results suggest that CMX directly interacts with the active site of all tested forms of DHFR using conserved residues. Using an in vitro DHFR activity assay we observed that CMX inhibits DHFR activity in a dose-dependent relationship.

The Ugd, a capsular polysaccharide synthesis protein, regulates the bacterial motility in Vibrio alginolyticus

Vibrio alginolyticus is one of the most common opportunistic pathogens in marine animals and humans. In this study, A transposon mutation library of the V. alginolyticus E110 was used to identify motility-related genes, and we found three flagellar and one capsular polysaccharide (CPS) synthesis-related genes were linked to swarming motility. Then, gene deletion and complementation further confirmed that CPS synthesis-related gene ugd is involved in the swarming motility of V. alginolyticus. Phenotype assays showed that the Δugd mutant reduced CPS production, decreased biofilm formation, impaired swimming ability, and increased cytotoxicity compared to the wild-type strain. Transcriptome analysis showed that 655 genes (15%) were upregulated and 914 genes (21%) were downregulated in the Δugd strain. KEGG pathway and heatmap analysis revealed that genes involved in two-component systems (TCSs), chemotaxis, and flagella assembly pathways were downregulated in the Δugd mutant. On the other hand, genes involved in pathways of human diseases, biosynthesis ABC transporters, and metabolism were upregulated in the Δugd mutant. The RT-qPCR further validated that ugd-regulated genes are associated with motility, biofilm formation, virulence, and TCSs. These findings imply that ugd may be an important player in the control of some physiological processes in V. alginolyticus, highlighting its potential as a target for future research and potential therapeutic interventions.

SntB Affects Growth to Regulate Infecting Potential in Penicillium italicum.

Penicillium italicum, a major postharvest pathogen, causes blue mold rot in citrus fruits through the deployment of various virulence factors. Recent studies highlight the role of the epigenetic reader, SntB, in modulating the pathogenicity of phytopathogenic fungi. Our research revealed that the deletion of the SntB gene in P. italicum led to significant phenotypic alterations, including delayed mycelial growth, reduced spore production, and decreased utilization of sucrose. Additionally, the mutant strain exhibited increased sensitivity to pH fluctuations and elevated iron and calcium ion stress, culminating in reduced virulence on Gannan Novel oranges. Ultrastructural analyses disclosed notable disruptions in cell membrane integrity, disorganization within the cellular matrix, and signs of autophagy. Transcriptomic data further indicated a pronounced upregulation of hydrolytic enzymes, oxidoreductases, and transport proteins, suggesting a heightened energy demand. The observed phenomena were consistent with a carbon starvation response potentially triggering apoptotic pathways, including iron-dependent cell death. These findings collectively underscored the pivotal role of SntB in maintaining the pathogenic traits of P. italicum, proposing that targeting PiSntB could offer a new avenue for controlling citrus fungal infections and subsequent fruit decay.

Functional characterization of three novel dense granule proteins in Neospora caninum using the CRISPR-Cas9 system

Neospora caninum is an obligate intracellular parasite that infects a wide range of mammalian species, and particularly causes abortions in cattle and nervous system dysfunction in dogs. Dense granule proteins (GRAs) are thought to play an important role in the mediation of host-parasite interactions and facilitating parasitism. However, a large number of potential GRAs remain uncharacterized, and the functions of most of the identified GRAs have not been elucidated. Previously, we screened a large number GRAs including NcGRA27 and NcGRA61 using the proximity-dependent biotin identification (BioID) technique. Here, we identified a novel GRA protein NcGRA85 and used C-terminal endogenous gene tagging to determine its localization at the parasitophorous vacuole (PV) in the tachyzoite. We successfully disrupted three gra genes (NcGRA27, NcGRA61 and NcGRA85) of N. caninum NC1 strain using CRISPR-Cas9-mediated homologous recombination and phenotyped the single knockout strain. The NcGRA61 and NcGRA85 genes were not essential for parasite replication and growth in vitro and for virulence during infection of mice, as observed by replication assays, plaque assays and in vitro virulence assays in mice. Deletion of the NcGRA27 gene in the NC1 strain reduced the in vitro replication and growth of the parasite, as well as the pathogenicity of the NC1 strain in mice. In summary, our findings provide a basis for in-depth studies of N. caninum pathogenesis and demonstrate the importance of NcGRA27 in parasite growth and virulence, most likely a new virulence factor of N. caninum.

Platform establishment of the Cre-loxP recombination system for genetic manipulation of the Lumpy skin disease virus

Lumpy skin disease virus (LSDV) is a rapidly emerging pathogen in Asia, including China. Genetic manipulation of the LSDV is essential for the elucidation of the pathogenic mechanism and biological function of the LSDV-encoded protein. In this study, we established a platform for the Cre-loxP recombination system under a modified early-late H5 promoter of the VACV for quick construction of the recombinant LSDV virus. The recombinant virus, LSDV-EGFP-ΔTK, was purified and obtained using serial limited dilution and picking the single cells methods. Using the lentiviral package system, a Cre recombinase enzyme stable expression MDBK cell line was established to supply the Cre recombinase for the reporter gene excision. A genetically stable, safe TK gene-deleted LSDV (LSDV-ΔTK) was constructed using homologous recombination and the Cre-loxP system. It was purified using limited dilution in the MDBK-Cre cell line. Establishing the Cre-loxP recombination system will enable sequential deletion of the interested genes from the LSDV genome and genetic manipulation of the LSDV genome, providing technical support and a platform for developing the attenuated LSDV vaccine.