Pathogenic Mechanisms Research Articles

A Homozygous Variant in NAA60 Is Associated with Primary Familial Brain Calcification.

Primary familial brain calcification (PFBC) is a monogenic disorder characterized by bilateral calcifications in the brain. The genetic basis remains unknown in over half of the PFBC patients, indicating the existence of additional novel causative genes. NAA60 was a recently reported novel causative gene for PFBC. The aim was to identify the probable novel causative gene in an autosomal recessive inherited PFBC family. We performed a comprehensive genetic study on a consanguineous Chinese family with 3 siblings diagnosed with PFBC. We evaluated the effect of the variant in a probable novel causative gene on the protein level using Western blot, immunofluorescence, and coimmunoprecipitation. Possible downstream pathogenic mechanisms were further explored in gene knockout (KO) cell lines and animal models. We identified a PFBC co-segregated homozygous variant of c.460_461del (p.D154Lfs*113) in NAA60. Functional assays showed that this variant disrupts NAA60 protein localization to Golgi and accelerated protein degradation. The mutant NAA60 protein alters its interaction with the PFBC-related proteins PiT2 and XPR1, affecting intracellular phosphate homeostasis. Further mass spectrometry analysis in NAA60 KO cell lines revealed decreased expression of multiple brain calcification-associated proteins, including reduced folate carrier (RFC), a folate metabolism-related protein. Our study replicated the identification of NAA60 as a novel causative gene for autosomal recessive PFBC, demonstrating our causative variant leads to NAA60 loss of function. The NAA60 loss of function disrupts not only PFBC-related proteins (eg, PiT2 and XPR1) but also a wide range of other brain calcification-associated membrane protein substrates (eg, RFC), and provided a novel probable pathogenic mechanism for PFBC. © 2024 International Parkinson and Movement Disorder Society.

The roles and mechanisms of endoplasmic reticulum stress-mediated autophagy in animal viral infections.

The endoplasmic reticulum (ER) is a unique organelle responsible for protein synthesis and processing, lipid synthesis in eukaryotic cells, and the replication of many animal viruses isclosely related to ER. A considerable number of viral proteins are synthesised during viral infection, resulting in the accumulation of unfolded and misfolded proteins in ER, which in turn induces endoplasmic reticulum stress (ERS). ERS further drives three signalling pathways (PERK, IRE1, and ATF6) of the cellular unfolded protein response (UPR) to respond to the ERS. In numerous studies, ERS has been shown to mediate autophagy, a highly conserved cellular degradation mechanism to maintain cellular homeostasis in eukaryotic cells, through the UPR to restore ER homeostasis. ERS-mediated autophagy is closely linked to the occurrence and development of numerous viral diseases in animals. Host cells can inhibit viral replication by regulating ERS-mediated autophagy, restoring the ER's normal physiological process. Conversely, many viruses have evolved strategies to exploit ERS-mediated autophagy to achieve immune escape. These strategies include the regulation of PERK-eIF2α-Beclin1, PERK-eIF2α-ATF4-ATG12, IRE1α-JNK-Beclin1, and other signalling pathways, which provide favourable conditions for the replication of animal viruses in host cells. The ERS-mediated autophagy pathway has become a hot topic in animal virological research. This article reviews the most recent research regarding the regulatory functions of ERS-mediated autophagy pathways in animal viral infections, emphasising the underlying mechanisms in the context of different viral infections. Furthermore, it considers the future direction and challenges in the development of ERS-mediated autophagy targeting strategies for combating animal viral diseases, which will contribute to unveiling their pathogenic mechanism from a new perspective and provide a scientific reference for the discovery and development of new antiviral drugs and preventive strategies.

Unraveling the Molecular Pathogenesis of Protein C Deficiency-Associated VTE: Insights from Protein C Mutations C238G and R189W in Thai Patients.

Protein C (PC) deficiency is a well-established risk factor for venous thromboembolism (VTE), often manifesting in pediatric patients. This study aimed to elucidate the pathogenic mechanisms underlying two novel PC mutations, C238G and R189W, identified in Thai children with VTE. Through transient transfection of HEK293T cells, the effects of the wild-type (WT), C238G, and R189W PC variants were investigated. The C238G mutation demonstrated a severe (95%) reduction in PC secretion, while the R189W showed a 30% decrease compared to WT. Immunofluorescence analysis revealed that the C238G-PC was predominantly localized in the endoplasmic reticulum (ER), suggesting intracellular retention due to protein misfolding. This was accompanied by a significant upregulation of ER stress-related genes in C238G-expressing cells, indicating the activation of the unfolded protein response (UPR). In contrast, the R189W mutation led to a modest upregulation of UPR genes, implying a less severe impact on protein folding and secretion. Structural analysis highlighted the critical role of the highly conserved C238 residue in maintaining the disulfide bond and 3D conformation essential for PC functionality. These findings provide insights into the distinct molecular pathogenic mechanisms by which the C238G and R189W mutations result in PC deficiency and increased thrombotic risk in affected individuals. This current study emphasizes the importance of the C238 residue in preserving PC structural integrity and secretion, contributing to our understanding of PC deficiency-associated VTE.

Oral inoculation of fusobacterium nucleatum exacerbates ulcerative colitis via the secretion of virulence adhesin FadA.

Fusobacterium nucleatum (F. nucleatum), an anaerobic resident of the oral cavity, is increasingly recognized as a contributing factor to ulcerative colitis (UC). The adhesive properties of F. nucleatum are mediated by its key virulence protein, FadA adhesin. However, further investigations are needed to understand the pathogenic mechanisms of this oral pathogen in UC. The present study aimed to explore the role of the FadA adhesin in the colonization and invasion of oral F. nucleatum in dextran sulphate sodium (DSS)-induced colitis mice via molecular techniques. In this study, we found that oral inoculation of F. nucleatum strain carrying the FadA adhesin further exacerbated DSS-induced colitis, leading to elevated alveolar bone loss, disease severity, and mortality. Additionally, CDH1 gene knockout mice treated with DSS presented increases in body weight and alveolar bone density, as well as a reduction in disease severity. Furthermore, FadA adhesin adhered to its mucosal receptor E-cadherin, leading to the phosphorylation of β-catenin and the degradation of IκBα, the activation of the NF-κB signalling pathway and the upregulation of downstream cytokines. In conclusion, this research revealed that oral inoculation with F. nucleatum facilitates experimental colitis via the secretion of the virulence adhesin FadA. Targeting the oral pathogen F. nucleatum and its virulence factor FadA may represent a promising therapeutic approach for a portion of UC patients.

Exploring Fatty Acid β-Oxidation Pathways in Bacteria: From General Mechanisms to DSF Signaling and Pathogenicity in Xanthomonas.

Fatty acids (FAs) participate in extensive physiological activities such as energy metabolism, transcriptional control, and cell signaling. In bacteria, FAs are degraded and utilized through various metabolic pathways, including β-oxidation. Over the past ten years, significant progress has been made in studying FA oxidation in bacteria, particularly in E. coli, where the processes and roles of FA β-oxidation have been comprehensively elucidated. Here, we provide an update on the new research achievements in FAs β-oxidation in bacteria. Using Xanthomonas as an example, we introduce the oxidation process and regulation mechanism of the DSF-family quorum sensing signal. Based on current findings, we propose the specific enzymes required for β-oxidation of several specific FAs. Finally, we discuss the future outlook on scientific issues that remain to be addressed. This paper supplies theoretical guidance for further study of the FA β-oxidation pathway with particular emphasis on its connection to the pathogenicity mechanisms of bacteria.

Evidence on Hidradenitis Suppurativa as an Autoinflammatory Skin Disease

Hidradenitis suppurativa (HS) is a chronic and debilitating inflammatory skin disease that often exhibits heterogeneity in its clinical presentation, especially in the context of its rare syndromic forms. The pathogenesis of HS results from a complex interplay of genetic predisposition, innate and adaptive immunity dysregulation, smoking, obesity and environmental factors. In the early phase of the disease, the innate immune system is hyperactivated, contributing to tissue damage and triggering the activation and amplification of the adaptive immune response, which plays a pivotal role in the chronic stages of the disease. Recent studies focused on elucidating the importance of innate immunity impairment and autoinflammation in HS and increasing evidence has emerged on the occurrence of the disease in the context of well-known monogenic and polygenic autoinflammatory syndromes (AIDs). This review provides a comprehensive examination of the current scientific background supporting the contribution of autoinflammation to HS etiology, including genetic data, molecular studies and clinical evidence, as well as the association between HS and AIDs. However, further research is needed to shed light on the pathogenic mechanism of this challenging condition and to identify potential perspectives for future therapeutic approaches.

Identification of ribosome biogenesis genes and subgroups in ischaemic stroke

BackgroundIschaemic stroke is a leading cause of death and severe disability worldwide. Given the importance of protein synthesis in the inflammatory response and neuronal repair and regeneration after stroke, and that proteins are acquired by ribosomal translation of mRNA, it has been theorised that ribosome biogenesis may have an impact on promoting and facilitating recovery after stroke. However, the relationship between stroke and ribosome biogenesis has not been investigated.MethodsIn the present study, a ribosome biogenesis gene signature (RSG) was developed using Cox and least absolute shrinkage and selection operator (LASSO) analysis. We classified ischaemic stroke patients into high-risk and low-risk groups using the obtained relevant genes, and further elucidated the immune infiltration of the disease using ssGSEA, which clarified the close relationship between ischaemic stroke and immune subgroups. The concentration of related proteins in the serum of stroke patients was determined by ELISA, and the patients were divided into groups to evaluate the effect of the ribosome biogenesis gene on patients. Through bioinformatics analysis, we identified potential IS-RSGs and explored future therapeutic targets, thereby facilitating the development of more effective therapeutic strategies and novel drugs against potential therapeutic targets in ischaemic stroke.ResultsWe obtained a set of 12 ribosome biogenesis-related genes (EXOSC5, MRPS11, MRPS7, RNASEL, RPF1, RPS28, C1QBP, GAR1, GRWD1, PELP1, UTP, ERI3), which play a key role in assessing the prognostic risk of ischaemic stroke. Importantly, risk grouping using ribosome biogenesis-related genes was also closely associated with important signaling pathways in stroke. ELISA detected the expression of C1QBP, RPS28 and RNASEL proteins in stroke patients, and the proportion of neutrophils was significantly increased in the high-risk group.ConclusionsThe present study demonstrates the involvement of ribosomal biogenesis genes in the pathogenesis of ischaemic stroke, providing novel insights into the underlying pathogenic mechanisms and potential therapeutic strategies for ischaemic stroke.

Congenital myopathies: pathophysiological mechanisms and promising therapies.

Congenital myopathies (CMs) are a kind of non-progressive or slow-progressive muscle diseases caused by genetic mutations, which are currently defined and categorized mainly according to their clinicopathological features. CMs exhibit pleiotropy and genetic heterogeneity. Currently, supportive treatment and pharmacological remission are the mainstay of treatment, with no cure available. Some adeno-associated viruses show promising prospects in the treatment of MTM1 and BIN1-associated myopathies; however, such gene-level therapeutic interventions target only specific mutation types and are not generalizable. Thus, it is particularly crucial to identify the specific causative genes. Here, we outline the pathogenic mechanisms based on the classification of causative genes: excitation-contraction coupling and triadic assembly (RYR1, MTM1, DNM2, BIN1), actin-myosin interaction and production of myofibril forces (NEB, ACTA1, TNNT1, TPM2, TPM3), as well as other biological processes. Furthermore, we provide a comprehensive overview of recent therapeutic advancements and potential treatment modalities of CMs. Despite ongoing research endeavors, targeted strategies and collaboration are imperative to address diagnostic uncertainties and explore potential treatments.

The causal effects of inflammatory and autoimmune skin diseases on thyroid diseases: evidence from Mendelian randomization study

BackgroundTo clarify the controversy between inflammatory or autoimmune skin diseases and thyroid diseases, we performed two-sample Mendelian randomization (MR) analyses.ParticipantsGenetic data on factors associated with atopic dermatitis (AD, n=40,835), seborrheic dermatitis (SD, n=339,277), acne (n=363,927), rosacea (n=299,421), urticaria (n=374,758), psoriasis (n=373,338), psoriasis vulgaris (n=369,830), systemic lupus erythematosus (SLE, n=14,267), vitiligo (n=353,348), alopecia areata (AA, n=361,822), pemphigus (n=375,929), bullous pemphigoid (BP, n=376,274), systemic sclerosis (SSc, n=376,864), localized scleroderma (LS, n=353,449), hypothyroidism (n=314,995 or n=337,159), and hyperthyroidism (n=281,683 or n=337,159) were derived from genome-wide association summary statistics of European ancestry.Main measuresThe inverse variance weighted method was employed to obtain the causal estimates of inflammatory or autoimmune skin diseases on the risk of thyroid diseases, complemented by MR-Egger, weighted median, and MR-pleiotropy residual sum and outlier (MR-PRESSO).Key resultsAD, SLE, SD, and psoriasis vulgaris were associated with an increased risk of hypothyroidism, whereas BP was associated with a lower risk of hypothyroidism (all with p < 0.05). The multivariable MR analyses showed that AD (OR = 1.053; 95%CI: 1.015-1.092; p = 0.006), SLE (OR = 1.093; 95%CI: 1.059-1.127; p < 0.001), and SD (OR = 1.006; 95%CI: 1.002-1.010; p = 0.006) independently and predominately contributed to the genetic causal effect on hypothyroidism after adjusting for smoking. The results showed no causal effects of inflammatory or autoimmune skin diseases on hyperthyroidism.ConclusionThe findings showed a causal effect of AD, SLE, SD on hypothyroidism, but further investigations should be conducted to explore the pathogenic mechanisms underlying these relationships.

Blockade of STING activation alleviates microglial dysfunction and a broad spectrum of Alzheimer's disease pathologies.

Abnormal glial activation promotes neurodegeneration in Alzheimer's disease (AD), the most common cause of dementia. Stimulation of the cGAS-STING pathway induces microglial dysfunction and sterile inflammation, which exacerbates AD. We showed that inhibiting STING activation can control microglia and ameliorate a wide spectrum of AD symptoms. The cGAS-STING pathway is required for the detection of ectopic DNA and the subsequent immune response. Amyloid-β (Aβ) and tau induce mitochondrial stress, which causes DNA to be released into the cytoplasm of microglia. cGAS and STING are highly expressed in Aβ plaque-associated microglia, and neuronal STING is upregulated in the brains of AD model animals. The presence of the APOE ε4 allele, an AD risk factor, also upregulated both proteins. STING activation was necessary for microglial NLRP3 activation, proinflammatory responses, and type-I-interferon responses. Pharmacological STING inhibition reduced a wide range of AD pathogenic features in AppNL-G-F/hTau double-knock-in mice. An unanticipated transcriptome shift in microglia reduced gliosis and cerebral inflammation. Significant reductions in the Aβ load, tau phosphorylation, and microglial synapse engulfment prevented memory loss. To summarize, our study describes the pathogenic mechanism of STING activation as well as its potential as a therapeutic target in AD.

Evaluating renal injury characteristics in different rat models of hyperuricemia and elucidating pathological molecular mechanisms via serum metabolomics

Hyperuricemia has emerged as a significant global health concern, closely associated with various metabolic disorders. The adverse effects frequently observed with current pharmacological treatments for hyperuricemia highlight the urgent need for reliable animal models to elucidate the disease’s pathophysiological mechanisms, thereby facilitating the development of safer and more effective therapies. In this study, we established three rat models of hyperuricemia using potassium oxonate, either alone or in combination with fructose and adenine. Each model exhibited distinct pathological changes, with the combination of potassium oxonate, fructose, and adenine causing significantly more severe damage to liver and kidney functions than potassium oxonate alone. Serum metabolomics analyses revealed profound dysregulation in the metabolic pathways of purine, pyrimidines, and glutathione, underscoring the pivotal role of oxidative stress in the progression of hyperuricemia. We identified key biomarkers such as orotidine, ureidosuccinic acid, uracil, and pseudouridine, which are associated with uric acid-induced damage to hepatic and renal systems. MetOrigin tracing analysis further revealed that differential metabolites related to hyperuricemia are primarily involved in host-microbiome co-metabolic pathways, particularly in purine metabolism, with bacterial phyla such as Pseudomonadota, Actinomycetota, and Ascomycota being closely linked to the critical metabolic processes of uric acid production. These findings not only enhance our understanding of the pathogenic mechanisms underlying hyperuricemia but also provide a robust experimental model foundation for the development of innovative treatment strategies.

Identification and validation of potential prognostic biomarkers in glioblastoma via the mesenchymal stem cell infiltration level

AimsMesenchymal stem cells (MSCs) are key components in promoting glioblastoma (GBM) progression. This study aimed to explore new therapeutic targets and related pathogenic mechanisms based on different MSCs infiltration levels in GBM patients.MethodsWe estimated the relationship between cell infiltration and prognosis of GBM. Subsequently, key risk genes were identified and prognostic models were constructed by LASSO-Cox analysis. The risk genes were validated by five independent external cohorts, single-cell RNA analysis, and immunohistochemistry of human GBM tissues. TIDE analysis predicted responsiveness to immune checkpoint inhibitors in different risk groups.ResultsThe MSCs infiltration level was negatively associated with survival in GBM patients. LOXL1, LOXL4, and GUCA1A are key risk genes that promote GBM progression and may act through complex intercellular communication.ConclusionThis research has provided a comprehensive study for exploring the MSCs infiltration environment on GBM progression, which could shed light on novel biomarkers and mechanisms involved in GBM progression.

Long Non-coding RNA DNM3OS: Pathogenic Roles and Molecular Mechanisms in Pathophysiological Processes.

Long non-coding RNA (lncRNA) is a class of single-stranded RNA biomolecules involving over 200 nucleotides and does not encode proteins. Research on lncRNA has become a hot spot for the past few years. DNM3OS (Dynamin 3 Opposite Strand), which has been clearly identified as a regulatory lncRNA, exerts an integral role in the pathophysiology of multiple human diseases. The current review study summarizes the pathogenic mechanism of DNM3OS in various pathophysiological processes, aiming to reveal its important value as a therapeutic drug target for related human diseases and provide a new way for targeted therapy. Through systematic retrieval and in-depth study of relevant articles in PubMed, this article analyzes and summarizes the pathogenic roles and molecular mechanisms in pathophysiological processes of long non-coding RNA DNM3OS. DNM3OS exerts an important regulatory role in the occurrence and development of bone diseases, neoplastic diseases, fibrotic diseases, inflammatory diseases, and many other diseases. DNM3OS is a potential new biomarker and therapeutic target for the treatment of a series of diseases, consisting of bone diseases, neoplastic diseases, fibrotic diseases, and inflammatory diseases.

Expert consensus on the diagnosis and therapy of endo-periodontal lesions.

Endo-periodontal lesions (EPLs) involve both the periodontium and pulp tissue and have complicated etiologies and pathogenic mechanisms, including unique anatomical and microbiological characteristics and multiple contributing factors. This etiological complexity leads to difficulties in determining patient prognosis, posing great challenges in clinical practice. Furthermore, EPL-affected teeth require multidisciplinary therapy, including periodontal therapy, endodontic therapy and others, but there is still much debate about the appropriate timing of periodontal therapy and root canal therapy. By compiling the most recent findings on the etiology, pathogenesis, clinical characteristics, diagnosis, therapy, and prognosis of EPL-affected teeth, this consensus sought to support clinicians in making the best possible treatment decisions based on both biological and clinical evidence.

Advanced Insights into Competitive Endogenous RNAs (ceRNAs) Regulated Pathogenic Mechanisms in Metastatic Triple-Negative Breast Cancer (mTNBC)

Triple-negative breast cancer is aggressive and challenging to treat because of a lack of targets and heterogeneity among tumors. A paramount factor in the mortality from breast cancer is metastasis, which is driven by genetic and phenotypic alterations that drive epithelial–mesenchymal transition, stemness, survival, migration and invasion. Many genetic and epigenetic mechanisms have been identified in triple-negative breast cancer that drive these metastatic phenotypes; however, this knowledge has not yet led to the development of effective drugs for metastatic triple-negative breast cancer (mTNBC). One that may not have received enough attention in the literature is post-translational regulation of broad sets of cancer-related genes through inhibitory microRNAs and the complex competitive endogenous RNA (ceRNA) regulatory networks they are influenced by. This field of study and the resulting knowledge regarding alterations in these networks is coming of age, enabling translation into clinical benefit for patients. Herein, we review metastatic triple-negative breast cancer (mTNBC), the role of ceRNA network regulation in metastasis (and therefore clinical outcomes), potential approaches for therapeutic exploitation of these alterations, knowledge gaps and future directions in the field.

Selenium deficiency and T-2 toxin trigger ferroptosis in cartilage from Kashin-Beck diseases

Kashin-Beck disease (KBD) is a prevalent, endemic, and degenerative cartilage injury disorder, characterized by high rates of teratogenicity and disability. The etiology and pathogenesis of KBD are not fully understood, although research suggests that selenium deficiency and exposure T-2 toxin are significant environmental risk factors. The initial pathological changes of KBD manifest as necrosis of deep chondrocytes, dedifferentiation of chondrocytes, excessive apoptosis of chondrocytes, and subsequent disruption of extracellular matrix metabolism. However, the precise pathogenic mechanisms of chondrocyte damage in KBD remain incompletely understood. Ferroptosis is auniqueform of programmed cell death triggered byiron-dependent lipid peroxide accumulation. It has been shown to contribute to cartilage damage and chondrocyte death in various osteoarticular conditions, particularly osteoarthritis (OA). Notably, KBD not only exhibits clinical and pathologicalsimilarities with OA, but also indicates a potential association with ferroptosis in morphological and molecular similarities. Additionally, the environmental risk factors T-2 toxin exposure and selenium deficiency are also significant contributors toferroptosis. Consequently, it is plausible to postulate that environmental risk factors may trigger ferroptosis, leading to the initiation of cartilage damage in KBD. Our hypothesis can be verified through both in vitro and in vivo experiments. Chondrocyte injury induced by ferroptosis may be a novel finding in KBD, which is important for clarifying its etiology and developing effective therapeutic strategies.

Disability independent of cerebral white matter demyelination in progressive multiple sclerosis.

The pathogenic mechanisms contributing to neurological disability in progressive multiple sclerosis (PMS) are poorly understood. Cortical neuronal loss independent of cerebral white matter (WM) demyelination in myelocortical MS (MCMS) and identification of MS patients with widespread cortical atrophy and disability progression independent of relapse activity (PIRA) support pathogenic mechanisms other than cerebral WM demyelination. The three-dimensional distribution and underlying pathology of myelinated T2 lesions were investigated in postmortem MCMS brains. Postmortem brain slices from previously characterized MCMS (10 cases) and typical MS (TMS) cases (12 cases) were co-registered with in situ postmortem T2 hyperintensities and T1 hypointensities. T1 intensity thresholds were used to establish a classifier that differentiates MCMS from TMS. The classifier was validated in 36 uncharacterized postmortem brains and applied to baseline MRIs from 255 living PMS participants enrolled in SPRINT-MS. Myelinated T2 hyperintensities in postmortem MCMS brains have a contiguous periventricular distribution that expands at the occipital poles of the lateral ventricles where a surface-in gradient of myelinated axonal degeneration was observed. The MRI classifier distinguished pathologically confirmed postmortem MCMS and TMS cases with an accuracy of 94%. For SPRINT-MS patients, the MRI classifier identified 78% as TMS, 10% as MCMS, and 12% with a paucity of cerebral T1 and T2 intensities. In SPRINT-MS, expanded disability status scale and brain atrophy measures were similar in MCMS and TMS cohorts. A paucity of cerebral WM demyelination in 22% of living PMS patients raises questions regarding a primary role for cerebral WM demyelination in disability progression in all MS patients and has implications for clinical management of MS patients and clinical trial outcomes in PMS. Periventricular myelinated fiber degeneration provides additional support for surface-in gradients of neurodegeneration in MS.

An efficient gene targeting system using Δku80 and functional analysis of Cyp51A in Trichophyton rubrum.

Trichophyton rubrum is one of the most frequently isolated fungi in patients with dermatophytosis. Despite its clinical significance, the molecular mechanisms of drug resistance and pathogenicity of T. rubrum remain to be elucidated because of the lack of genetic tools, such as efficient gene targeting systems. In this study, we generated a T. rubrum strain that lacks the nonhomologous end-joining-related gene ku80 (Δku80) and then developed a highly efficient genetic recombination system with gene targeting efficiency that was 46 times higher than that using the wild-type strain. Cyp51A and Cyp51B are 14-α-lanosterol demethylase isozymes in T. rubrum that promote ergosterol biosynthesis and are the targets of azole antifungal drugs. The expression of cyp51A mRNA was induced by the addition of the azole antifungal drug efinaconazole, whereas no such induction was detected for cyp51B, suggesting that Cyp51A functions as an azole-responsive Cyp51 isozyme. To explore the contribution of Cyp51A to susceptibility to azole drugs, the neomycin phosphotransferase (nptII) gene cassette was inserted into the cyp51A 3'-untranslated region of Δku80 to destabilize the mRNA of cyp51A. In this mutant, the induction of cyp51A mRNA expression by efinaconazole was diminished. The minimum inhibitory concentration for several azole drugs of this strain was reduced, suggesting that dermatophyte Cyp51A contributes to the tolerance for azole drugs. These findings suggest that an efficient gene targeting system using Δku80 in T. rubrum is applicable for analyzing genes encoding drug targets.

Myosin ATPase inhibition fails to rescue the metabolically dysregulated proteome of nebulin-deficient muscle.

Nemaline myopathy (NM) is a genetic muscle disease, primarily caused by mutations in the NEB gene (NEB-NM) and with muscle myosin dysfunction as a major molecular pathogenic mechanism. Recently, we have observed that the myosin biochemical super-relaxed state was significantly impaired in NEB-NM, inducing an aberrant increase in ATP consumption and remodelling of the energy proteome in diseased muscle fibres. Because the small-molecule Mavacamten is known to promote the myosin super-relaxed state and reduce the ATP demand, we tested its potency in the context of NEB-NM. We first conducted in vitro experiments in isolated single myofibres from patients and found that Mavacamten successfully reversed the myosin ATP overconsumption. Following this, we assessed its short-term in vivo effects using the conditional nebulin knockout (cNeb KO) mouse model and subsequently performing global proteomics profiling in dissected soleus myofibres. After a 4week treatment period, we observed a remodelling of a large number of proteins in both cNeb KO mice and their wild-type siblings. Nevertheless, these changes were not related to the energy proteome, indicating that short-term Mavacamten treatment is not sufficient to properly counterbalance the metabolically dysregulated proteome of cNeb KO mice. Taken together, our findings emphasize Mavacamten potency in vitro but challenge its short-term efficacy in vivo. KEY POINTS: No cure exists for nemaline myopathy, a type of genetic skeletal muscle disease mainly derived from mutations in genes encoding myofilament proteins. Applying Mavacamten, a small molecule directly targeting the myofilaments, to isolated membrane-permeabilized muscle fibres from human patients restored myosin energetic disturbances. Treating a mouse model of nemaline myopathy in vivo with Mavacamten for 4weeks, remodelled the skeletal muscle fibre proteome without any noticeable effects on energetic proteins. Short-term Mavacamten treatment may not be sufficient to reverse the muscle phenotype in nemaline myopathy.

Next-generation sequencing in pediatric-onset epilepsies: Analysis with target panels and personalized therapeutic approach.

The objective of this study is to report the results of the genetic analysis in a large and well-characterized population with pediatric-onset epilepsies and to identify those who could benefit from precision medicine treatments. In this retrospective observational study, we consecutively recruited patients with pediatric-onset epilepsy observed at a tertiary neurological center over a time span of 7 years, collecting clinical and laboratory findings. Following in-depth diagnostic process to exclude possible structural and metabolic causes of the disease, patients with a suspected genetically determined etiology underwent next-generation sequencing (NGS) screening with panels for the analysis of target genes causative of epilepsy. We detected likely pathogenic or pathogenic variants (classes IV and V) in 24% of the 562 patients who underwent genetic investigations. By the evaluation of patients' data, we observed that some features (onset of epilepsy before one year old, presence of neurological deficits, psychomotor delay/cognitive disability, and malformative aspects at brain MRI) were significantly associated with class IV or V variants. Moreover, statistical analysis showed that the diagnostic yield resulted higher for patients affected by Progressive Myoclonic Epilepsy (PME) and with early onset developmental and epileptic encephalopathies (DEE), compared with focal epilepsies, genetic generalized epilepsies, DEE with onset at/after 1 y.o., and unclassified epileptic syndromes. According to the results of the genetic screening, up to 33% of patients carrying class IV or V variants resulted potentially eligible for precision medicine treatments. The large-scale application of NGS multigene panels of analysis is a useful tool for the molecular diagnosis of patients with pediatric-onset epilepsies, allowing the identification of those who could benefit from a personalized therapeutic approach. The analysis of patients with pediatric-onset epilepsy using advanced technologies for the screening of all the implicated genes allows the identification of the cause of diseases in an ever-increasing number of cases. Understanding the pathogenic mechanisms could, in some cases, guide the selection and optimization of appropriate treatment approaches for patients.