Gene Deletion Research Articles

Nephrogenic diabetes insipidus results from a novel in-frame deletion of AVPR2 gene in monozygotic-twin boys and their mother and grandmother.

Mutations in the AVPR2 gene are the most common cause of nephrogenic diabetes insipidus(NDI). In-frame deletions of the AVPR2 gene are a rare variant that results in NDI. We report a novel variant of the p.H138del in an NDI family with twin male patients and three female carriers of different clinical phenotypes. The proband's blood genome was sequenced witha panel, and the variants were classified according to ACMG/AMP (2015) guidelines. X chromosome inactivation (XCI) was analyzed in the peripheral blood of his mother, grandmother, and maternal aunt, respectively. The haplotypes of the X chromosome were determined using their STR loci. A novel in-frame deletion in the AVPR2 gene was detected in monozygotic-twin boys, and his mother, grandmother, and maternal aunt were heterozygous carriers. The two boys showed typical NDI, and their mother and grandmother presented polydipsia, polydipsia, and polyuria, but the maternal aunt did not have similar symptoms. The blood XCI results of the mother, grandmother, and maternal aunt showed random inactivation (36.18 , 48.37, and 49.30 %, respectively). The X haplotype indicated that the variant of the mother and grandmother was on their activated X chromosomes(Xa), while the maternal aunt's variant was on her inactivated X chromosome(Xi). In-frame deletion of the AVPR2 gene within its functional domain can significantly affect protein function, which is one of the vital causes of NDI. The clinical variability of female carriers of AVPR2 is associated with underlying environmental and epigenetic factors or complex recombination of the X chromosomes.

Strategies for genetic manipulation of the halotolerant black yeast Hortaea werneckii: ectopic DNA integration and marker-free CRISPR/Cas9 transformation.

Hortaea werneckii is a halotolerant black yeast commonly found in hypersaline environments. This yeast is also the causative agent of tinea nigra, a superficial mycosis of the palm of the hand and soles of the feet of humans. In addition to their remarkable halotolerance, this black yeast exhibits an unconventional cell division cycle, alternating between fission and budding cell division. Cell density and the salt concentration in their environment regulate which cell division cycle H. werneckii uses. Although H. werneckii have been extensively studied due to their unique physiology and cell biology, deciphering the underlying mechanisms behind these remarkable phenotypes has been limited due to the lack of genetic tools available. Here, we report a new ectopic integration protocol for H. werneckii using polyethylene glycol-CaCl2 mediated protoplast transformation. This approach relies on a drug (hygromycin B) resistance gene to select for successful integration of the genetic construct. The same construct was used to express cytosolic green fluorescent protein. Finally, we developed a marker-free CRISPR/Cas9 protocol for targeted gene deletion using the melanin synthesis pathway as a visual reporter of successful transformation. These transformation strategies will allow testing hypotheses related to H. werneckii cell biology and physiology.IMPORTANCEHortaea werneckii is a remarkable yeast capable of growing in high salt concentration, and its cell division cycle alternates between fission-like and budding. For these unique attributes, H. werneckii has gathered interest in research programs studying extremophile fungi and cell division. Most of our understanding of H. werneckii biology comes from genomic analyses, the usage of drugs to target a particular pathway, or the heterologous expression of its genes in S. cerevisiae. Nonetheless, H. werneckii has remained genetically intractable. Here, we report on two strategies to transform H. werneckii: ectopic integration of a plasmid and gene deletion using CRISPR/Cas9. These approaches will be fundamental to expanding the experimental techniques available to study H. werneckii, including live-cell imaging of cellular processes and reverse genetic approaches.

Клиническое наблюдение гипоталамического ожирения у ребенка с нейрофиброматозом I типа и глиомой хиазмально-селлярной области

Neurofibromatosis (NF) is a genetic disorder characterized by the development of various types of benign or malignant tumors affecting ether the central or the peripheral nervous systems and manifested also on the skin in the form of “café au lait” spots as well as other signs sometimes. Thus, this disorder requires multidisciplinary approach. The clinical case observed in the Article is no exception.: a patient with coffee-colored spots all over the body from an early age was described with similar familial symptoms in the patient’s father, two brothers. At the age of 4 months old, after the first DTaP vaccination, nystagmus appeared, after the second vaccination - progressive deterioration of vision, and at the age of 3 to 4 years old the hearing loss joined in. For the 6 years the patient was observed by pediatrician, ophthalmologist, otolaryngologist and neurologist and received courses of retrobulbar injections of vascular-metabolic drugs, hardware treatment and physiotherapy with slightly positive effect. The first MR imaging of the brain was performed at the age of 7 y/o when a glioma was detected in the chiasmatic-sellar region. Starting the age of 7 y/o the patient gradually developed and increased hypothalamic obesity with subsequent metabolic complications in the forms of impaired glucose tolerance, hyperinsulinism and insulin resistance, arterial hypertension and non-alcoholic fatty liver disease. At the age of 12 a heterozygous mutation in the NF1 gene (deletion of 4 nucleotides) in exon 5 was detected: c.495_498delTGTT:p.T165fs, which made it possible to establish a diagnosis of NF type 1. This case reflects the multifaceted nature of the disease, the importance of a multidisciplinary approach and its early diagnosis.

MamF-like proteins are distant Tic20 homologs involved in organelle assembly in bacteria

Organelle-specific protein translocation systems are essential for organelle biogenesis and maintenance in eukaryotes but thought to be absent from prokaryotic organelles. Here, we demonstrate that MamF-like proteins are crucial for the formation and functionality of bacterial magnetosome organelles. Deletion of mamF-like genes in the Alphaproteobacterium Magnetospirillum gryphiswaldense results in severe defects in organelle positioning, biomineralization, and magnetic navigation. These phenotypic defects result from the disrupted targeting of a subset of magnetosomal proteins that contain C-terminal glycine-rich integral membrane domains. Phylogenetic analyses reveal an ancient evolutionary link between MamF-like proteins and plastidial Tic20. Our findings redefine the molecular roles of MamF-like proteins and suggest that organelle-specific protein targeting systems also play a role in bacterial organelle formation.

A Gene Cassette Vd276-280 in Verticillium dahliae Contains Two Genes that Affect Melanized Microsclerotium Formation and Virulence.

Verticillium dahliae is a soilborne phytopathogenic fungus causing Verticillium wilt on hundreds of plant species. Several sequenced genomes of V. dahliae are available, but functional characterization of most genes has just begun. Based on our previous comparison of the transcriptome from the wild-type and ΔVdCf2 strains, a significant upregulation of the gene cassette, Vd276-280, in the ΔVdCf2 strain was observed. In this study, the functional characterization of the Vd276-280 gene cassette was performed. Agrobacterium-mediated knockout of this gene cassette in V. dahliae significantly inhibited conidiation, melanized microsclerotium formation in the mutant strains, and their virulence toward cotton. Furthermore, deletion of individual genes in the Vd276-280 gene cassette identified that the disruption of VDAG_07276 and VDAG_07280 delayed microsclerotium formation, inhibited conidiation, and reduced virulence toward cotton. Our data suggest that VDAG_07276 and VDAG_07280 in the Vd276-280 gene cassette mainly act as positive regulators of development and virulence in V. dahliae.

Dynamic regulation of proximal tubular autophagy from injury to repair after ischemic kidney damage

BackgroundThe role of proximal tubular autophagy in repairing kidney injury following ischemia remains unclear.MethodsIn this study, we utilized mice with conditional deletion of the Atg5 gene in proximal tubules and monitored the long-term dynamic regulation of autophagy following ischemic acute kidney injury (AKI).ResultsThe results showed that Atg5-deficient proximal tubule epithelial cells exhibited damaged mitochondria, concentric membranes, and lysosomal accumulation 24 h after ischemia/reperfusion. However, 28 days after ischemia/reperfusion, concentric membrane bodies remained, but lysosomal accumulation was no longer observed. Notably, the absence of Atg5 in renal tubular epithelial cells impaired renal function and led to increased tubular cell proliferation and oxidative stress in the early stage of injury. However, during the repair period following AKI, Atg5 deficiency exhibited no significant difference in the expression of proliferating cell nuclear antigen (PCNA) and 4-hydoxynonenal (4HNE), suggesting that the improvement in renal fibrosis associated with Atg5 deficiency is unlikely to result from its effect on cell proliferation or reactive oxygen species levels. Additionally, Atg5 deficiency inhibits the secretion of profibrotic factor fibroblast growth factor 2 (FGF2) from the early stage of renal injury to the recovery stage of AKI, indicating that autophagy-specific regulation of FGF2 secretion is a dynamic process overlapping with other stages of injury. Furthermore, increased co-localization of ATG5 with 4HNE and FGF2 was observed in patient samples.ConclusionIn summary, our results suggest that the dynamic regulation of autophagy on key molecules involved in kidney injury and repair varies with the stage of kidney injury.Graphical

Co-occurrence of direct and indirect extracellular electron transfer mechanisms during electroactive respiration in a dissimilatory sulfate reducing bacterium.

Understanding the extracellular electron transfer mechanisms of electroactive bacteria could help determine their potential in microbial fuel cells (MFCs) and their microbial syntrophy with redox-active minerals in natural environments. However, the mechanisms of extracellular electron transfer to electrodes by sulfate-reducing bacteria (SRB) remain underexplored. Here, we utilized double-chamber MFCs with carbon cloth electrodes to investigate the extracellular electron transfer mechanisms of Desulfovibrio vulgaris Hildenborough (DvH), a model SRB, under varying lactate and sulfate concentrations using different DvH mutants. Our MFC setup indicated that DvH can harvest electrons from lactate at the anode and transfer them to cathode, where DvH could further utilize these electrons. Patterns in current production compared with variations of electron donor/acceptor ratios in the anode and cathode suggested that attachment of DvH to the electrode and biofilm density were critical for effective electricity generation. Electron microscopy analysis of DvH biofilms indicated DvH utilized filaments that resemble pili to attach to electrodes and facilitate extracellular electron transfer from cell to cell and to the electrode. Proteomics profiling indicated that DvH adapted to electroactive respiration by presenting more pili- and flagellar-related proteins. The mutant with a deletion of the major pilus-producing gene yielded less voltage and far less attachment to both anodic and catholic electrodes, suggesting the importance of pili in extracellular electron transfer. The mutant with a deficiency in biofilm formation, however, did not eliminate current production indicating the existence of indirect extracellular electron transfer. Untargeted metabolomics profiling showed flavin-based metabolites, potential electron shuttles.IMPORTANCEWe explored the application of Desulfovibrio vulgaris Hildenborough in microbial fuel cells (MFCs) and investigated its potential extracellular electron transfer (EET) mechanism. We also conducted untargeted proteomics and metabolomics profiling, offering insights into how DvH adapts metabolically to different electron donors and acceptors. An understanding of the EET mechanism and metabolic flexibility of DvH holds promise for future uses including bioremediation or enhancing efficacy in MFCs for wastewater treatment applications.

Arrayed CRISPR libraries for the genome-wide activation, deletion and silencing of human protein-coding genes.

Arrayed CRISPR libraries extend the scope of gene-perturbation screens to non-selectable cell phenotypes. However, library generation requires assembling thousands of vectors expressing single-guide RNAs (sgRNAs). Here, by leveraging massively parallel plasmid-cloning methodology, we show that arrayed libraries can be constructed for the genome-wide ablation (19,936 plasmids) of human protein-coding genes and for their activation and epigenetic silencing (22,442 plasmids), with each plasmid encoding an array of four non-overlapping sgRNAs designed to tolerate most human DNA polymorphisms. The quadruple-sgRNA libraries yielded high perturbation efficacies in deletion (75-99%) and silencing (76-92%) experiments and substantial fold changes in activation experiments. Moreover, an arrayed activation screen of 1,634 human transcription factors uncovered 11 novel regulators of the cellular prion protein PrPC, screening with a pooled version of the ablation library led to the identification of 5 novel modifiers of autophagy that otherwise went undetected, and 'post-pooling' individually produced lentiviruses eliminated template-switching artefacts and enhanced the performance of pooled screens for epigenetic silencing. Quadruple-sgRNA arrayed libraries are a powerful and versatile resource for targeted genome-wide perturbations.

Capture of Totipotency in Mouse Embryonic Stem Cells in the Absence of Pdzk1.

Totipotent cells can differentiate into three lineages: the epiblast, primitive endoderm, and trophectoderm. Naturally, only early fertilized embryos possess totipotency, and they lose this ability as they develop. The expansion of stem cell differentiation potential has been a hot topic in developmental biology for years, particularly with respect to the generation totipotent-like stem cells. Here, the study describes the establishment of totipotency in embryonic stem cells (ESCs) via the deletion of a single gene, Pdzk1. Pdzk1-knockout (KO) ESCs substantially contribute to the fetus, placenta, and yolk sac in chimera assays but can also self-organize to form standard blastocyst-like structures containing the three lineages efficiently; thus, they exhibit full developmental potential as early blastomeres. Single-cell transcriptome and bulk RNA-seq comprehensive analyses revealed that Pdzk1-KO activates several lineage inducers (C1qa, C1qb, Fgf5, and Cdx2) to break down barriers between embryonic and extraembryonic tissues, making these lineages switch smoothly and resulting in a totipotent-like state. This versatile and scalable system provides a robust experimental model for differentiation potency and cell fate studies.

High-resolution genetic analysis of whole APC gene deletions: a report of two cases and patient characteristics

Familial adenomatous polyposis (FAP) is an autosomal dominant syndrome caused by germline variants in the APC gene, leading to the development of numerous colorectal polyps and significantly increases the risk of colorectal cancer. A diagnosis is typically made using colonoscopy, and genetic testing can assist in patient surveillance and carrier identification. Recent advances include the use of whole-genome array comparative genomic hybridization (a-CGH), which provides better resolution of genetic imbalances. We aimed to explore the specific features of FAP patients with whole APC gene deletions using high-resolution a-CGH and to compare patient characteristics. Two polyposis patients with whole APC deletions were identified, and the lost genetic sizes ranged from 0.3–1.1 Mb. Nervous abnormalities were a characteristic symptom in a patient with a 1.1 Mb loss. A patient with an approximately 0.3 Mb loss, which included the entire APC gene, presented a polyposis phenotype without intellectual disability. The comparison of genetic losses, with or without intellectual disability, revealed 7 genetic changes. Consequently, EPB41L4A is a candidate gene associated with the neurogenic phenotype.

Heterologous Biosynthesis of Taxifolin in Yarrowia lipolytica: Metabolic Engineering and Genome-Scale Metabolic Modeling.

Taxifolin, also known as dihydroquercetin (DHQ), is a flavonoid recognized for its potent antioxidant properties and a wide range of biological activities, including anti-tumor, antiviral, and immunomodulatory effects. Conventional extraction and chemical synthesis methods for taxifolin are often limited by low yields and associated environmental concerns. In this study, we investigated the heterologous biosynthesis of taxifolin in Yarrowia lipolytica through a combination of metabolic engineering and genome-scale metabolic modeling (GSM), complemented by flux balance analysis (FBA). We engineered Yarrowia lipolytica by introducing key biosynthetic genes and successfully synthesized taxifolin using naringenin (NAR) as a substrate, chosen for its low cost. Fermentation experiments demonstrated an optimal taxifolin yield of 10% at a substrate concentration of 200mg/L naringenin, with a maximum yield of 26.4mg/L taxifolin at 1g/L naringenin. To further enhance production, we applied a marker-free Cre-loxP-based gene integration method, allowing stable genomic integration of key genes, which increased taxifolin yield to 34.9mg/L at 1g/L naringenin. Additionally, intermediate metabolites eriodictyol (ERI) and dihydrokaempferol (DHK) accumulated to concentrations of 89.2mg/L and 21.7mg/L, respectively. Furthermore, we integrated metabolic data into a GSM and applied FBA to optimize the taxifolin biosynthetic pathway. Through Pareto frontier analysis, sensitivity analysis, flux variability analysis, and single gene deletion simulations, we identified key genetic modifications that significantly enhanced taxifolin yield. Overexpression of GND1 and IDP2 increased yields by 94% and 155%, respectively, while knockout of LIP2 led to a 46% increase. Using tri-baffled shake flasks to improve oxygen supply resulted in a 120% yield increase, whereas YPG medium decreased yield by 59%, validating our model's accuracy. To ensure stable and efficient gene expression, we integrated multi-copy constructs into the ribosomal DNA (rDNA) locus of Yarrowia lipolytica, doubling taxifolin production. These results demonstrate the effectiveness of GSM and FBA in addressing bottlenecks in microbial taxifolin biosynthesis and provide a basis for future optimization and large-scale production.

A Child with Non-Growth Hormone-Deficient Short Stature with Chromosome 2 Deletion of 2q35-36.1: A Case Report and Literature Review of Haploinsufficiency of Epha4 Gene

AbstractNon-growth hormone-deficient short stature is a group of growth developmental disorders with complex pathophysiological mechanisms involving multiple pathways, including abnormalities in the growth hormone/insulin-like growth factor 1 signal pathway. We report a 4-year-old girl with severe growth and developmental delay: her height was 91 cm (<–3 standard deviation [SD]), and her growth rate was 3 to 4 cm/year (<–2 SD). Growth hormone stimulation tests indicated a normal peak growth hormone level (11.1 μg/L), while insulin-like growth factor-1 levels were reduced (7.98 nmol/L, <–1SD). Whole exome sequencing and copy number variation analysis revealed a 3.19 Mb deletion at 2q35-q36.1 on chromosome 2, encompassing all coding regions of the Epha4 gene. This study suggests an association between Epha4 gene deletion and non-growth hormone-deficient short stature, highlighting the importance of further research on this gene and its related signaling pathways to understand the molecular mechanisms of the disease and potential therapeutic approaches.

Genotype First Approach as a Diagnostic Strategy for Precision Medicine in Moroccan Families with Nephronophthisis

AbstractNephronophthisis is a group of autosomal recessive kidney diseases characterized by chronic tubulointerstitial abnormalities leading to kidney failure. The incidence of nephronophthisis varies globally, and its genetic heterogeneity presents significant difficulties in diagnosis. Herein, we present a molecular diagnosis of four unrelated Moroccan families fulfilling the clinical criteria of nephronophthisis. As first diagnosis step, screening for a homozygous NPHP1 gene deletion, the most common mutation, was performed by using multiplex polymerase chain reaction. Additionally, clinical exome sequencing was carried out on patients in whom no NPHP1 deletion was identified. Three novel pathogenic variants in NPHP1, INVS, and NPHP4 genes were identified and confirmed by Sanger sequencing in the proband and other family members. These variants are absent from genetic databases of patients and controls of Moroccan origin. The bioinformatics analysis classified these variants as pathogenic. Our findings expand the genotypic spectrum of nephronophthisis and highlight the importance of genetic testing in patients from low- and middle-income countries to obtain a precise diagnosis of nephronophthisis. This helps facilitate an appropriate and personalized genetic counseling and improves clinical outcomes for patients with this condition.

Biosynthesis of Tetramate Derivatives in Penicillium crustosum Reveals the Involvement of ortho-Quinone Methide in Crosstalk of Multiple Pathways.

Genome mining and gene deletion experiments in Penicillium crustosum proved the involvement of the PKS-NRPS PemA and the trans-enoyl reductase PemB in the formation of three enantiomeric clavatol-containing tetramate pairs. Overexpression of a transcription factor significantly improved the product yields. Feeding experiments provided evidence for their formation via 1,4-Michael addition of hydroxyclavatol to two tetramates from the Pem pathway. This study provides another example of the involvement of reactive intermediates in multiple biosynthetic pathways.

Structure-optimized sgRNA selection with PlatinumCRISPr for efficient Cas9 generation of knockouts.

A single guide RNA (sgRNA) directs Cas9 nuclease for gene-specific scission of double-stranded DNA. High Cas9 activity is essential for efficient gene editing to generate gene deletions and gene replacements by homologous recombination. However, cleavage efficiency is below 50% for more than half of randomly selected sgRNA sequences in human cell culture screens or model organisms. We used in vitro assays to determine intrinsic molecular parameters for maximal sgRNA activity including correct folding of sgRNAs and Cas9 structural information. From the comparison of over 10 data sets, we find major constraints in sgRNA design originating from defective secondary structure of the sgRNA, sequence context of the seed region, GC context, and detrimental motifs, but we also find considerable variation among different prediction tools when applied to different data sets. To aid selection of efficient sgRNAs, we developed web-based PlatinumCRISPr, an sgRNA design tool to evaluate base-pairing and sequence composition parameters for optimal design of highly efficient sgRNAs for Cas9 genome editing. We applied this tool to select sgRNAs to efficiently generate gene deletions in Drosophila Ythdc1 and Ythdf, that bind to N 6 methylated adenosines (m6A) in mRNA. However, we discovered that generating small deletions with sgRNAs and Cas9 leads to ectopic reinsertion of the deleted DNA fragment elsewhere in the genome. These insertions can be removed by standard genetic recombination and chromosome exchange. These new insights into sgRNA design and the mechanisms of CRISPR-Cas9 genome editing advance the efficient use of this technique for safer applications in humans.

Could the transcription factor AtnN coordinating the aspercryptin secondary metabolite gene cluster in Aspergillus nidulans be a global regulator?

Products of dormant secondary metabolite gene clusters of fungal genomes can be exploited for medical purposes as bioactive agents. These clusters can be switched on under oxidative stress and may endow fungi with a versatile chemical armory in a competitive niche. In Aspergillus nidulans, the aspercryptin gene cluster, including the synthase [atnA (AN7884)] and its transcription factor (atnN), was activated under menadione sodium bisulfite (MSB) treatment. In this study, we generated and phenotypically examined the gene deletion and overexpression mutants of atnN and studied the secondary metabolite production of the mutants. Overexpression of atnN significantly reduced the colony growth of surface cultures compared to the control. The ΔatnN gene deletion strain showed higher sensitivity to tert-butyl hydroperoxide (tBOOH), while the atnNOE strain was more resistant to MSB, Congo Red, and sorbitol. Interestingly, deletion of atnN decreased cleistothecia formation of A. nidulans. Manipulation of atnN affected the synthesis of several secondary metabolites, for example, the siderophore production of A. nidulans. The extracellular triacetylfusarinine C (TAFC) production decreased, while the intracellular ferricrocin (FC) concentration of the cultures increased in the atnNOE mutant cultivating A. nidulans in a complex medium containing 1% mycological peptone and 2% maltose. In Czapek-Dox Broth medium, increased asperthecin production was observed in the ΔatnN mutant. The mycotoxin sterigmatocystin synthesis elevated in the ΔatnN mutant, while reduced in the atnNOE mutant on minimal medium. Our study supports previous observations that secondary metabolite production is coordinated in a complex way, and the linkage of stress response, sexual reproduction, and secondary metabolite production can be governed by several transcription factors.

Functional impact of a deletion in Mycobacterium bovis BCG Moreau celA1 gene

Several mycobacterial species are known to cause human diseases, such as tuberculosis and leprosy. In addition to these pathogenic species, there are also saprophytic representatives, which occasionally cause opportunistic infections. It is well established that numerous mycobacteria produce biofilms containing cellulose, and their genomes frequently harbor genes involved in cellulose degradation, such as celA1. Notably, the BCG Moreau vaccine strain carries a specific deletion of two-base pairs, resulting in a predicted protein with fewer than 100 amino acids in the catalytic portion at the C-terminal end. We investigated the functional consequences of this polymorphism and observed that recombinant enzyme from the Moreau strain lack catalytic activity. Furthermore, compared to the Pasteur strain, Moreau is unable to utilize carboxymethylcellulose (CMC) as the sole carbon source. These findings suggest an absence of cellulolytic activity in this strain, which may influence the bacterium virulence.

Molecular identification and characterization of the superoxide dismutase (SOD) gene family in Tetranychus urticae (Acari: Tetranychidae) and the role of TuSOD2 gene under short-term heat stress

Superoxide dismutases (SODs) are key enzymes for scavenging insects' excess reactive oxygen species (ROS) and play a crucial role in resisting a variety of stresses. The deletion of SOD genes can alter the response of insects to abiotic stresses. In this study, four genes of the superoxide dismutase family in Tetranychus urticae were identified and analyzed, among which three copper/zinc SODs and a manganese SOD. The result of quantitative real-time PCR showed that TuSOD2 expression level was up-regulated significantly under short-term heat stress, but TuSOD1, TuSOD2, and TuSOD4 were down-regulated or no significant difference, which indicated that T. urticae responded to oxidative stress induced by heat stress by raising the expression level of superoxide dismutase gene. Among them, TuSOD2 gene plays a key role in response to heat stress in T. urticae, and other SOD genes may be responsible for other oxidative stresses. RNA interference experiments showed that the knocking down of TuSOD2 reduced the activities of various antioxidant enzymes, resulting in a decline in the tolerance to high temperature and an increase in the mortality of T. urticae under short-term heat stress. To summarize, SOD, especially TuSOD2 plays an important role in treating short-term heat stress in T. urticae.

Association Between NK Cell Genetic Variants and the Development of Long COVID Associated- and Prepandemic Small Fiber Neuropathy.

Long coronavirus disease 2019 (COVID) (LC) symptoms including pain and autonomic dysfunction are in some patients associated with small-fiber neuropathy (SFN). The pathomechanisms underlying SFN are mostly unclear. Natural killer (NK) cells play a crucial role in immune regulation, viral clearance and nerve metabolism. The aim of this study was to identify associations between development of small-fiber dysfunction dependent and independent of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and human genetic markers associated with specific NK cell functions. The genetic markers assessed in all cohorts included: FCGR3A, IGHG1, HLA-E, NKG2C, and rs9916629. Genotyping was performed using TaqMan assays, Sanger sequencing and touchdown polymerase chain reaction. We assessed human cytomegalovirus (HCMV) IgG serostatus in all participants, and screened for anti-neuronal, anti-glial and anti-ganglioside autoantibodies in both patient cohorts. We included 50 LC patients with newly-emerged symptoms of small-fiber dysfunction after SARS-CoV-2 infection, 27 prepandemic SFN patients and 320 control persons. Markers associated with low NKG2C response, that is, deletion of the NKG2C gene and lack of prior HCMV infection (IgG seronegativity), occurred significantly more frequently in prepandemic SFN patients compared to LC patients and controls (p = 0.0109 and 0.0005, respectively). In conclusion, markers of impaired NKG2C pathways are associated with prepandemic SFN, but not with Long COVID-associated small-fiber dysfunction.

Two NPC1 homologous proteins are involved in asexual reproduction, pathogenicity, and lipid trafficking in Phytophthora sojae

Niemann-Pick type C (NPC) disease is characterized by lysosomal lipid storage disorders and defects in lipid trafficking, primarily due to mutations in the NPC1 protein. Two NPC1 homologous proteins are present in the genome of Phytophthora sojae, named as PsNPC1-1 and PsNPC1-2. Both proteins exhibit high sequence identity, consistent conserved functional domains, similar gene expression patterns, and comparable subcellular localization. Deletion of a single PsNPC1 gene did not result in significant phenotypic changes. However, simultaneous deletion of both PsNPC1 genes led to reduced mycelial growth, decreased sporangial production, impaired pathogenicity, and an inability to release normal zoospores in P. sojae. Furthermore, dysfunction of PsNPC1s did not completely block the absorption and utilization of exogenous sterols by P. sojae. While lipidome analysis revealed that the relative contents of fatty acyls, sphingolipids and saccharolipids were significantly elevated in the double-gene deletion mutant, alongside obvious alterations in glycerophospholipid and glycerolipid metabolism. Additionally, we observed a significant down-regulation of PsCDP-AP protein along with its interactions with both PsNPC1s. Deletion of PsCDP-AP also impaired asexual reproduction and virulence of P. sojae. These findings demonstrate that both PsNPC1 proteins may collaborate with other key regulators to modulate asexual reproduction, pathogenicity and lipid trafficking in P. sojae.