Mutant Proteins Research Articles

Computational Mutagenesis of GPx7 and GPx8: Structural and Stability Insights into Rare Genetic and Somatic Missense Mutations and Their Implications for Cancer Development.

Background/Objectives: Somatic and genetic mutations in glutathione peroxidases (GPxs), including GPx7 and GPx8, have been linked to intellectual disability, microcephaly, and various tumors. GPx7 and GPx8 evolved the latest among the GPx enzymes and are present in the endoplasmic reticulum. Although lacking a glutathione binding domain, GPx7 and GPx8 possess peroxidase activity that helps the body respond to cellular stress. However, the protein mutations in these peroxidases remain relatively understudied. Methods: By elucidating the structural and stability consequences of missense mutations, this study aims to provide insights into the pathogenic mechanisms involved in different cancers, thereby aiding clinical diagnosis, treatment strategies, and the development of targeted therapies. We performed saturated computational mutagenesis to analyze 2926 and 3971 missense mutations of GPx7 and GPx8, respectively. Results: The results indicate that G153H and G153F in GPx7 are highly destabilizing, while E93M and W142F are stabilizing. In GPx8, N74W and G173W caused the most instability while S70I and S119P increased stability. Our analysis shows that highly destabilizing somatic and genetic mutations are more likely pathogenic compared to stabilizing mutations. Conclusions: This comprehensive analysis of missense mutations in GPx7 and GPx8 provides critical insights into their impact on protein structure and stability, contributing to a deeper understanding of the roles of somatic mutations in cancer development and progression. These findings can inform more precise clinical diagnostics and targeted treatment approaches for cancers.

Pharmacokinetic predictions and molecular docking of phytocompounds derived from Carissa edulis on mutant p53 protein in breast cancer

Globally, breast cancer is a leading cause of death, and the current chemotherapeutic agents are not devoid of drawbacks. Nevertheless, the potential of medicinal plants for cancer treatment and prevention remains immense. The current study aimed at elucidating the interactions between phytocompounds identified in Carissa edulis (C. edulis) and mutant p53, drug-likeness and potential toxicity. Molecular docking analysis was employed to analyse the interaction of compounds and the target protein. Lipinski’s Rule of Five was used to analyse drug-likeness properties. Liquid chromatography-mass spectroscopy (LC-MS) and gas chromatography-mass spectroscopy (GC-MS) were employed to detected presence of phytocompounds in the extracts. Quantitative analyses revealed presence of phytocompounds which have been previously reported to have antiproliferative effects. The docked compounds had a binding efficiency ranging from -4.00 to -8.44 kcal/mol. Most of the studied phytocompounds were found to be within the "Rule of 5" without any violation. Regarding toxicity, rutin, catechin, betulin, tocopherol, sitosterol, beta-amyrin, menthol, and epicatechin exhibited no inhibitory effect on CYP450 enzymes. Moreover, most compounds had high intestinal absorption, whereas few compounds cross blood brain barrier. A bioavailability score of 0.55 was exhibited by most bioactive compounds analysed. These results underscore the therapeutic potential of C. edulis for future anticancer drug development.

Structural Analysis of the SCN1A R1648H Mutation in Epilepsy Using Alpha Fold

Epilepsy is one of the most common chronic, noncommunicable neurological conditions in the world, affecting around 50 million people worldwide. The condition is characterized by repeated seizures caused by unusual electrical activity in the brain. The major causes of the condition include brain injury or infection, metabolic problems, neurotransmitter imbalance, and genetic factors. Besides the major causes listed above, genetic mutations also play an important role in certain forms. SCN1A is one of the key genes involved and is linked with Dravet syndrome—a severe developmental and epileptic condition—and a list of epileptic conditions. The SCN1A gene produces a specific protein, part of the sodium channel (NaV1.1), in nerve cells. This protein can regulate sodium ions across the cell membranes, which is critical for how the electrical signals move and can be controlled. However, the mutations in SCN1A can disrupt such electrical signal movement processes, leading to nerve transmission problems and possibly increasing the chance of seizures. This study employs AlphaFold, an artificial intelligence-based protein structure prediction tool, to explore the structural changes caused by the R1648H mutation. By comparing the wild-type and mutant SCN1A proteins, we aim to identify specific alterations contributing to sodium channel malfunctioning in epileptic conditions. Understanding these molecular consequences can bridge the gap between genetic mutations and clinical outcomes, potentially informing the development of targeted therapies and personalized medicine approaches for SCN1A-related epilepsies.

Pathological PNPase variants with altered RNA binding and degradation activity affect the phenotype of bacterial and human cell models

Abstract Human PNPase (hPNPase) is an essential RNA exonuclease located in mitochondria, where it contributes to RNA import from the cytoplasm, degradation of mitochondrial RNA and R-loop homeostasis. Biallelic mutations in the hPNPase PNPT1 gene cause different genetic diseases, ranging from hereditary hearing loss to Leigh syndrome. In this work, we used an Escherichia coli model to test the effects of four pathological PNPT1 mutations associated with diseases of different severity. Moreover, we generated a new human cell model by introducing PNPT1 mutations into 293T cells via CRISPR-Cas editing. Notably, the bacterial cells expressing the different mutant alleles exhibited similar phenotypes consistent with hPNPase loss of function. In contrast, the human cell model responded differently to the two mutations tested, with responses correlating with the severity of the respective pathologies. We interpreted the data derived from both models in the light of the in vitro RNA binding and degradation activity of the wild-type and mutated hPNPase variants. We found that all pathogenic mutations tested caused defects in protein assembly and affected the degradation and RNA binding efficiency to varying degrees. However, the severity of the conditions caused by different mutations did not correlate with the catalytic activity of the mutant proteins.

Bioinformatic analysis of TFR2, HJV, and HFE nonsense mutations in hereditary hemochromatosis: Insights into the pathogenesis of diabetic mellitus and heart failure

Sequence variations in TFR2, HJV, and HFE proteins are causative contributors to a wide range of disorders of iron metabolism, which could result in hereditary hemochromatosis, leading to heart failure and diabetes. Known nonsense variants were retrieved from ClinVar, identifying common variants in diabetes mellitus and heart failure, followed by protein-protein interaction analysis through STRING and Cytoscape for hub genes identification, and then structural modeling through AlphaFold and mutation incorporation through DESMOND Maestro to assess their potential impact on protein function. These mutations have not been previously bioinformatically examined, and thus this research presents the foundational studies for further investigations. The modeling and comparative superimposition analysis of wild and mutant proteins showed that nonsense mutations in TFR2 result in truncated proteins that disrupt crucial domains for iron uptake and transport. Similarly, HJV nonsense mutants, including R63* and C321*, truncate the protein and prevent hepcidin regulation. Nonsense mutations in HFE disrupt conserved regions within MHC class I-like antigen recognition and immunoglobulin C1-set domains, potentially leading to abnormal iron buildup. These findings enhance our understanding of the pathogenic mechanisms of genes with these mutations and highlight the importance of genotyping for predicting the severity of iron overload disorders, aiding effective clinical interventions. HFE mutations primarily affect the MHC class I-like antigen recognition domain, while HJV mutations disrupt hepcidin expression and BMP signaling, contributing to juvenile hemochromatosis. Additionally, previously unreported truncating mutations, such as Q12* and R105*, significantly impair function, exacerbating iron overload and disease progression.

Role of Mutual Information Profile Shifts in Assessing the Pathogenicity of Mutations on Protein Functions: The Case of Pyrin Variants Associated With Familial Mediterranean Fever.

This study presents a novel method to assess the pathogenicity of pyrin protein mutations by using mutual information (MI) as a measure to quantify the correlation between residue motions or fluctuations and associated changes affecting the phenotype. The concept of MI profile shift is presented to quantify changes in MI upon mutation, revealing insights into residue-residue interactions at critical positions. We apply this method to the pyrin protein variants, which are associated with an autosomal recessively inherited disease called familial Mediterranean fever (FMF) since the available tools do not help predict the pathogenicity of the most penetrant variants. We demonstrate the utility of MI profile shifts in assessing the effects of mutations on protein stability, function, and disease phenotype. The importance of MI shifts, particularly the negative shifts observed in the pyrin example, as indicators of severe functional effects is emphasized. Additionally, the exploration of potential compensatory mechanisms suggested by positive MI shifts, which are otherwise random and inconsequential, is highlighted. The study also discusses challenges in relating MI profile changes to disease severity and advocates for comprehensive analysis considering genetic, environmental, and stochastic factors. Overall, this study provides insights into the molecular mechanisms underlying the pathogenesis of FMF and offers a framework for identifying potential therapeutic targets based on MI profile changes induced by mutations.

Kelch-like ECH-associated Protein 1/Nuclear Factor Erythroid 2-related Factor 2 Pathway and Its Interplay with Oncogenes in Lung Tumorigenesis.

Nuclear factor erythroid 2-related factor 2 (NRF2), a transcription factor regulating cellular redox homeostasis, exhibits a complex role in cancer biology. Genetic mutations in the Kelch-like ECH-associated protein 1 (KEAP1)/NRF2 system, which lead to NRF2 hyperactivation, are found in 20% to 30% of lung cancer cases. This review explores the intricate interplay between NRF2 and key oncogenic pathways in lung cancer, focusing on the interaction of KEAP1/NRF2 system with Kirsten rat sarcoma virus (KRAS), tumor protein P53 (TP53), epidermal growth factor receptor (EGFR), and phosphatidylinositol 3-kinases (PI3K)/AKT signaling. While NRF2 activation alone is insufficient to initiate tumorigenesis, it can significantly impact tumor initiation and progression when combined with oncogenic drivers such as KRAS. The review highlights the context-dependent effects of NRF2, from its protective role against chemical carcinogen-induced tumor initiation to its potential promotion of tumor growth in established cancers. These findings suggest the need for nuanced, stage-specific approaches to targeting the NRF2 pathway in cancer therapy.

Non-Structural Protein-13 Mutations in European Isolates of SARS-CoV-2 Changed Protein Stability

Objective: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) became one of the most important health problems of the 21st century. Non-structural protein-13 (nsp13/helicase) plays an important role in the replication of the viral genome and the viral life cycle. The SARS-CoV-2 genome has undergone thousands of mutations since the disease first appeared. Mutations pose a threat to the validity of therapeutics due to changes in protein structure. Modeling alterations caused by mutations in the viral proteome contributes to the development of effective antivirals. The changes in protein structure and stability caused by mutations seen in European isolates of SARS-CoV-2 were analyzed in the study with the aim of contributing to studies on the development of new anti-virals and the validity of existing therapeutics. Methods: The changes in protein structure after mutation were modeled with deep learning algorithms. The alterations in protein stability were analyzed by SDM2, mCSM, DUET and DynaMut2. Results: The mutation analysis revealed four (Pro77Leu, Gly170Ser, Tyr324Cys, and Arg392Cys) missense mutations in the nsp13 protein in European isolates of SARS-CoV-2. Mutations caused changes in protein structure (rmsd 0.294 Å) and stability (-.58 ≤ ΔΔG ≤ .003 kcal.mol-1). The atomic interactions formed by the mutant residues in the three-dimensional conformation of the protein have changed. Conclusion: The mutations seen in European isolates for nsp13 of SARS-CoV-2 may lead to the emergence of different phenotypes in terms of viral activity. For this reason, the study may contribute to the success of the fight against the virus with different treatment approaches in different regions.

Bioinformatics Analysis of a 4bp Homozygous Deletion Mutation of EDAR Gene Identified as an Important Cause of Hypohidrotic Ectodermal Dysplasia in Pakistan

Hypohidrotic Ectodermal Dysplasia (HED) is a rare congenital disorder characterized by reduced hair, the absence of sweat glands, dental anomalies, and craniofacial malformations. This condition can be inherited through X-linked, autosomal recessive, or autosomal dominant modes of inheritance. Mutations in four specific genes (EDAR, EDA, WNT10A, and EDARADD) are known to cause HED. In a study to identify a pathogenic mutation in a consanguineous Pakistani family with the autosomal recessive form of HED, microsatellite markers were utilized to genotype the known loci associated with the disease. The condition in this family was mapped to the EDAR gene locus located on chromosome 2q11-q13. Upon screening the EDAR gene, a novel homozygous 4bp deletion (718delAAGA) in exon 8 was identified, which segregated with the disease phenotype. This 4bp deletion in the EDAR gene results in a frameshift and early termination of translation, producing a truncated protein of 245 amino acids instead of the normal length of 539 amino acids. Various bioinformatics tools were employed to analyze the pathogenic mutation linked to a significant number of HED cases in Pakistan. I-TASSER was used to model the protein structure, CASTp facilitated the identification of various pockets, and STiTCH 3.1 determined EDA to be the ligand for EDAR. Docking analysis were conducted for both the mutant and wild-type EDAR proteins with EDA, revealing notable differences in the interaction sites between the docked complexes of the normal and mutant forms of EDAR with the ligand EDA. These analyses provided insights into the protein's structural features, active sites, interactions, and the overall impact of the mutation.

Association between the EHBP1 SNPs and dyslipidemia in the end-stage renal disease patients with dialysis in Chinese Han population

BackgroundLipid metabolism is influenced by mutations in the EH domain binding protein 1 gene (EHBP1). This study investigated the link between the EHBP1 single-nucleotide polymorphisms (SNPs) and dyslipidemia risks in maintenance dialysis patients with end-stage renal disease in Chinese Han population.MethodsA total of 539 patients were divided into dyslipidemia (379) and control (160) groups. The patients with dyslipidemia were divided into four subgroups: high low-density lipoprotein cholesterol, low high-density lipoprotein cholesterol (HDLC), high triglyceride (TG) and high total cholesterol groups. The genotype distributions of three EHBP1 SNPs (rs2710642, rs10496099 and rs1168816) were determined by high-throughput sequencing technology and were analyzed via generalized multifactor dimension reduction and binary logistic regression analysis.ResultsThe high-TG and control groups differed in terms of the genotype frequency of the rs2710642. One haplotype was detected in both the dyslipidemia and high-TG groups. The risk of dyslipidemia was 2.72-fold higher in participants with rs2710642GG compared with those of rs2710642AA and 2.62-fold higher compared with those with rs2710642AA + GA. Subjects who carried rs2710642GG had a 2.94 times greater risk of high TG levels than those who carried rs2710642AA and a 2.89 times greater risk than those who carried rs2710642AA + GA. Compared with those who carried rs2710642AA + GA, those who carried rs2710642GG were 2.53 times more likely to have low HDLC levels. The rs2710642–body mass index (BMI) (≥ 24 kg/m2) and rs11688816A–rs2710642G haplotype interactions increased the risk of dyslipidemia, and the rs2710642–BMI (≥ 24 kg/m2) interaction increased the risk of high TG levels. The rs10496099–rs2710642 and rs10496099–rs2710642–rs11688816 interactions increased the risk of low HDLC levels.ConclusionsThese results suggest that the EHBP1 rs2710642G and rs2710642GG and interactions with rs11688816A or BMI (≥ 24 kg/m2) were linked to higher dyslipidemia risks in end-stage renal disease patients in Chinese Han population.

Immune Stress-induced Tumor Mutation Burden and Neoantigen Expression in 4T1 Mammary Cancer Cells: A Potential Mechanism for Long-term Survival in Patients Treated With Immune Checkpoint Inhibitors.

The Kaplan-Meier curves for patients treated with immune checkpoint inhibitors (ICIs) display a small group of potentially-cured patients with long-term survival, creating a 'kangaroo-tail' shape of the survival curve. However, the mechanistic basis of this phenomenon and what occurs in patients whose cancer is resistant to ICIs remain unclear. The present study aimed to answer these questions. We analyzed mutations in mouse 4T1 mammary-gland-derived cancer cells expressing the hemagglutinin antigen (4T1-HA), which were grown in either wild-type mice or cytotoxic T-lymphocyte (CTL)-loaded immunocompromised mice (RAG-/- + ACT) under immune stress. These mutations were compared to those in 4T1-HA cells grown in RAG-/- mice without immune stress as a control. The number of gene mutations, the tumor mutation burden (TMB) and microsatellite instability (MSI) scores were increased in the cancer cells under immune stress. The mutations in the antigen protein were such that the protein retained its immunogenicity and could still function as a neoantigen. Repeated immune recognition of additional neoantigens may lead to the kangaroo-tail survival phenomenon. The common genetic mutations of the analyzed 4T1-HA cells under immune stress included genes related to immune response. Analysis of alternative splicing of genes showed that are accumulated gene alterations under immune stress related to cancer-cell proliferation. Copy-number variation (CNV) analysis indicated that normal-antigen presentation and immune responses may be impaired under immune stress. Cancer cells, under immune stress, may acquire both immune escape capabilities and increased immunogenicity. This dual effect could lead to either resistance or response to ICIs, respectively.

Huntingtin interactome reveals huntingtin role in regulation of double strand break DNA damage response (DSB/DDR), chromatin remodeling and RNA processing pathways.

Huntington's Disease (HD), a progressive neurodegenerative disorder with no disease-modifying therapies, is caused by a CAG repeat expansion in the HD gene encoding polyglutamine-expanded huntingtin (HTT) protein. Mechanisms of HD cellular pathogenesis and cellular functions of the normal and mutant HTT proteins are still not completely understood. HTT protein has numerous interaction partners, and it likely provides a scaffold for assembly of multiprotein complexes many of which may be altered in HD. Previous studies have implicated DNA damage response in HD pathogenesis. Gene transcription and RNA processing has also emerged as molecular mechanisms associated with HD. Here we used multiple approaches to identify HTT interactors in the context of DNA damage stress. Our results indicate that HTT interacts with many proteins involved in the regulation of interconnected DNA repair/remodeling and RNA processing pathways. We present evidence for a role for HTT in double strand break repair mechanism. We demonstrate HTT functional interaction with a major DNA damage response kinase DNA-PKcs and association of both proteins with nuclear speckles. We show that S1181 phosphorylation of HTT is regulated by DSB, and can be carried out (at least in vitro) by DNA-PK. Furthermore, we show HTT interactions with RNA binding proteins associated with nuclear speckles, including two proteins encoded by genes at HD modifier loci, TCERG1 and MED15, and with chromatin remodeling complex BAF. These interactions of HTT may position it as an important scaffolding intermediary providing integrated regulation of gene expression and RNA processing in the context of DNA repair mechanisms.

Unique target binding by the C-terminal region of FHR1 provides a new perception of aHUS pathology

IntroductionFHR1 is a multifunctional human plasma protein with three C-terminal domains, namely short consensus repeats (SCR) 3–5, showing 98% sequence-identity with the complement inhibitor Factor H. We show that FHR1 uses all three C-terminal SCR to make surface contact. The conserved C-terminal regions of FHR1 and Factor H are altered in patients with atypical-hemolytic-uremic-syndrome. Therefore, we compared FHR1 isoforms with sequence-variations in SCR3, and pathogenic mutants with sequence variations in SCR5.MethodsFHR1 binding to apoptotic cells was evaluated EM and fluorescent microscopy and in kidney biopsies. FHR1 and Factor H variants and mutants were generated and expressed. The variants and mutant proteins were tested in binding studies to C3b , C3d and heparin, in hemolytic assays and for the induction of inflammatory cytokines. The action profiles of FHR1 and Factor H were calculated and compared.ResultsFunctional data revealed that residues YVQ vs HLE in SCR3 and LA vs SV in SCR5 altered ligand binding and surface interaction, influenced target recognition and complement control. Amino-acid-sequence variations in SCR3 influenced FHR1 contact with surface constituents, such as glycosaminoglycans. By contrast, SCR5, the most C-terminal domain, was more relevant for C3b/C3d contact. Notably, wild-type FHR1LA selected C3d, while pathogenic aHUS-associated alterations FHR1SV selected C3b. In consequence mutant FHR1SV altered fined-tuned FHR1-directed effector functions while pathogenic Factor HLA modified C3-convertase control.DiscussionThis influences timing of complement control and inflammatory effector actions at modified self-surfaces. Pathogenic FHR1SV, directed to C3b-decorated targets, adds inflammatory activity at a time when C3-convertase control is appropriate and conversely, mutant Factor HLA adds C3-convertase control at C3d-coated surfaces when inflammatory effector functions are favorable. Further, our computational modeling approach confirms such distinct effects of FHR1 monomers and dimers as compared to flexible Factor H. These effects may explain inappropriate timing of complement regulation and inflammation of the aHUS-derived mutant proteins FHR1SV and Factor HLA.

Evidence of an emerging triple-reassortant H3N3 avian influenza virus in China

The H3 subtype of avian influenza virus (AIV) stands out as one of the most prevalent subtypes, posing a significant threat to public health. In this study, a novel triple-reassortant H3N3 AIV designated A/chicken/China/16/2023 (H3N3), was isolated from a sick chicken in northern China. The complete genome of the isolate was determined using next-generation sequencing, and the AIV-like particles were confirmed via transmission electron microscopy. Subsequent phylogenetic analyses revealed that HA and NA genes of the H3N3 isolate clustered within the Eurasian lineage of AIVs, exhibiting the closest genetic relationship with other H3N3 AIVs identified in China during 2023. Interestingly, the HA and NA genes of the nove H3N3 isolate were originated from H3N8 and H10N3 AIVs, respectively, and the six internal genes originated from prevalent H9N2 AIVs. These findings indicated the novel H3N3 isolate possesses a complex genetic constellation, likely arising from multiple reassortment events involving H3N8, H9N2, and H10N3 subtype influenza viruses. Additionally, the presence of Q226 and T228 in the HA protein suggests the H3N3 virus preferentially binds to α-2,3-linked sialic acid receptors. The HA cleavage site motif (PEKQTR/GIF) and the absence of E627K and D701N mutations in PB2 protein classify the virus as a characteristic low pathogenicity AIV. However, several mutations in internal genes raise concerns about potential increases in viral resistance, virulence, and transmission in mammalian hosts. Overall, this study provides valuable insights into the molecular and genetic characterization of the emerging triple-reassortant H3N3 AIVs, and continued surveillance of domestic poultry is essential for monitoring the H3N3 subtype evolution and potential spread.

Molecular and clinical characterization of two independent Chinese families with protein C deficiency.

This study aims to investigate the clinical characterization and molecular pathogenic basis of hereditary protein C (PC) deficiency in two independent Chinese families, and conduct in vitro expression studies on the newly discovered p.Trp444Arg mutation. The PC activity (PC: A) was tested using the chromogenic substrate, and PC antigen (PC: Ag) was detected via enzyme-linked immunosorbent assay (ELISA). To identify the mutation sites, nine exons of the PROC gene were amplified by PCR, and the products were directly sequenced. The conservation and pathogenicity of the mutations, as well as changes in the spatial structure of PC proteins before and after mutations, were analyzed using ClustalX-2.1-win, online bioinformatics software, and PyMOL. The function of the mutant proteins was detected using the thrombin generation assay (TGA). Recombinant PC was ectopically expressed in HEK293T cells, with mRNA levels quantified by RT-qPCR. The recombinant protein was further characterized using Western blotting, ELISA, and immunofluorescence microscopy. Proband A and B, aged 39 and 63 respectively, are both diagnosed with deep vein thrombosis (DVT) in both lower limbs and pulmonary embolism (PE). Two missense mutations, p.Arg440Cys and p.Trp444Arg, were identified in the probands. Bioinformatics and protein modeling analyses revealed that the two mutations probably affected the normal function of PC. The thrombin generation assay revealed impaired thrombin generation capacity in both probands, with proband B showing more severe impairment. In vitro expression experiments demonstrated that p.Trp444Arg do not significantly affect mRNA expression levels of PC protein compared to wild-type, but result in lower PC: Ag content and protein expression in the supernatant and higher levels in the lysate. These two mutations may be the causes of reduced PC in two independent Chinese families. Notably, this is the first reported instance of the p.Trp444Arg mutation.

Improving Cellular Protein Content of Saccharomyces cerevisiae Based on Adaptive Evolution and Flow Cytometry-Aided High Throughput Screening.

Enhancing the protein content and production efficiency of Saccharomyces cerevisiae is crucial as an alternative protein source. This study screened nongenetically modified yeast strains with high protein content for food ingredient production and explored the underlying mechanisms. Yeast protein levels were found to correlate with RNA, leading to a high-throughput screening method using RNA fluorescence and flow cytometry. Four mutant libraries (∼200,000 cells) were generated through adaptive laboratory evolution in protein synthesis inhibitors, resulting in the high protein mutant content B1, with a protein content of 65.8 g/100 g dry cell weight in shake flasks. In a 45 L bioreactor using fed-batch fermentation with ethanol below 1.5 g/L, B1's protein content increased to 70.3 g/100 g dry cell weight, an 18.5% rise. Mannan and β-glucan levels in the cell wall decreased by 21.7 and 30.5%, potentially enhancing protein extraction for food production. Transcriptome analysis revealed that increased protein content results from down-regulating the cell cycle and meiosis-related genes. Validation of differentially expressed genes demonstrated that up-regulating SUT1 and down-regulating CNM67 are key for enhancing protein synthesis and accumulation. This study proposes a nongenetic screening method for high protein content S. cerevisiae strains, achieving the highest reported protein content.

Loss of CHOP Prevents Joint Degeneration and Pain in a Mouse Model of Pseudoachondroplasia

Pseudoachondroplasia (PSACH), a severe dwarfing condition characterized by impaired skeletal growth and early joint degeneration, results from mutations in cartilage oligomeric matrix protein (COMP). These mutations disrupt normal protein folding, leading to the accumulation of misfolded COMP in chondrocytes. The MT-COMP mouse is a murine model of PSACH that expresses D469del human COMP in response to doxycycline and replicates the PSACH chondrocyte and clinical pathology. The basis for the mutant-COMP pathology involves endoplasmic reticulum (ER) stress signaling through the PERK/eIF2α/CHOP pathway. C/EBP homologous protein (CHOP), in conjunction with a TNFα inflammatory process, upregulates mTORC1, hindering autophagy clearance of mutant COMP protein. Life-long joint pain/degeneration diminishes quality of life, and treatments other than joint replacements are urgently needed. To assess whether molecules that reduce CHOP activity should be considered as a potential treatment for PSACH, we evaluated MT-COMP mice with 50% CHOP (MT-COMP/CHOP+/−), antisense oligonucleotide (ASO)-mediated CHOP knockdown, and complete CHOP ablation (MT-COMP/CHOP−/−). While earlier studies demonstrated that loss of CHOP in MT-COMP mice reduced intracellular retention, inflammation, and growth plate chondrocyte death, we now show that it did not normalize limb growth. ASO treatment reduced CHOP mRNA by approximately 60%, as measured by RT-qPCR, but did not improve limb length similar to MT-COMP/CHOP+/−. Interestingly, both 50% genetic reduction and complete loss of CHOP alleviated pain, while total ablation of CHOP in MT-COMP mice was necessary to preserve joint health. These results indicate that (1) CHOP reduction therapy is not an effective strategy for improving limb length and (2) pain and chondrocyte pathology are more responsive to intervention than the prevention of joint damage.

Evolutionary variation of the monkeypox virus detected for the first time in Nantong, Jiangsu.

Monkeypox (MPOX) is a zoonotic disease caused by Orthopoxvirus monkeypox (MPXV), belonging to the Orthopoxvirus genus, and exhibits symptoms similar to smallpox. In 2024, the monkeypox outbreak in the Democratic Republic of the Congo continued to develop, raising widespread global public health concerns. In September 2023, the first local monkeypox outbreak was reported in Nantong, Jiangsu Province, China. Whole-genome sequencing of samples from seven confirmed patients identified a new lineage, C.1.1, which may be related to imported cases from Japan. Evolutionary analysis of MPXV showed fewer mutations mediated by Apolipoprotein B mRNA Editing Catalytic polypeptide-like 3 (APOBEC3). Additionally, mutations in the N2L protein disrupted transcription initiation, while changes in the Cytomegalovirus-encoded immunomodulatory protein B (CrmB) led to structural instability of the protein. It is hoped that these findings will provide insights for future research on the evolutionary mechanisms of the monkeypox virus and the development of vaccines.

Pan-KRAS inhibitors BI-2493 and BI-2865 display potent anti-tumor activity in tumors with KRAS wild-type allele amplification.

KRASG12C selective inhibitors, such as sotorasib and adagrasib, have raised hopes of targeting other KRAS mutant alleles in cancer patients. We report that KRAS wild-type amplified tumor models are sensitive to treatment with the small molecule KRAS inhibitors BI-2493 and BI-2865. These pan-KRAS inhibitors directly target the "OFF" state of KRAS and result in potent anti-tumor activity in pre-clinical models of cancers driven by KRAS mutant proteins. Here, we used the high-throughput cellular viability PRISM assay to assess the anti-proliferative activity of BI-2493 in a 900+ cancer cell line panel, expanding on our previous work. KRAS wild-type amplified cancer cell lines, with a copy number >7, were identified as the most sensitive, across cell lines with any KRAS alterations, to our pan-KRAS inhibitors. Importantly, our data suggest that a KRAS "OFF" inhibitor is better suited to treat KRAS wild-type amplified tumors than a KRAS "ON" inhibitor. KRAS wild-type amplification is common in patients with gastroesophageal cancers where it has been shown to act as a unique cancer driver with little overlap to other actionable mutations. The pan-KRAS inhibitors BI-2493 and BI-2865 show potent anti-tumor activity in vitro and in vivo in KRAS wild-type amplified cell lines from this and other tumor types. In conclusion, this is the first study to demonstrate that direct pharmacological inhibition of KRAS shows anti-tumor activity in preclinical models of cancer with KRAS wild-type amplification, suggesting a novel therapeutic concept for patients with cancers bearing this KRAS alteration.

Influenza Virus-Associated Acute Necrotizing Encephalopathy in Two Young Children: Case Report

Background and Clinical Significance: Acute necrotizing encephalopathy (ANE) represents a severe complication, mainly described in children, of influenza virus infection. We report the cases of two young girls with ANE associated with influenza virus infection who were diagnosed by MRI cerebral scan. Case Presentation: A 7-year-old girl with a history of a previous episode of ANE presented with a worsening drowsy state and seizures. In the second case, an otherwise healthy 5-year-old girl presented with fever, seizures, and marked neurological deterioration. In both cases, nasopharyngeal swab testing was positive for influenza virus A, while cerebral MRI indicated ANE. Despite aggressive treatment with high-dose corticosteroids and a five-day course ofimmunoglobulins, the ultimate prognosis was poor in both patients. ANE is a serious complication of viral infections in children, with a high mortality rate and a broad spectrum of neurological sequelae. To date, the pathophysiology and management of influenza virus-induced ANE remain uncertain. Although ANE is usually sporadic, familial and recurrent cases have been reported, and anRAN-binding protein (RANBP2) mutation has occasionally been associated with its occurrence.Conclusions: Rapid recognition of neurological symptoms and suspicion of a viral trigger, especially in influenza-like illnesses, are both essential for the timely administration of effective therapy. Further research is needed to clarify the pathophysiology of ANE and establish the best therapeutic strategies to fight such a deadly disease.