Gain Of Function Research Articles

Adaptive Prescribed‐Time Control for Marine Surface Vehicles: A Time‐Varying Gain Approach

ABSTRACTThis study investigates an adaptive prescribed‐time control scheme for marine surface vehicles (MSVs). Based on the introduced time‐varying gain function, this article provides a sufficient condition for practical prescribed‐time stability. The aim is to provide a theoretical basis for the prescribed‐time controller design. The upper bound of the settling time can be accurately preset by adjusting the design parameters. Furthermore, adaptive parameter technique is utilized to handle the unknown lumped disturbances. Theory confirms that the tracking error of the MSV satisfies the conditions for practical prescribed‐time stability. The superiority and effectiveness of the scheme are verified through simulation analysis.

Bag2 protects against doxorubicin-induced cardiotoxicity by maintaining Pink1-mediated mitophagy

The clinical application of Doxorubicin (DOX) is limited due to its cardiotoxicity. Mitophagy dysfunction is the primary cause of DOX-induced cardiotoxicity (DIC). However, the precise mechanism by which DOX regulates mitophagy remains elusive. Bag2 (BCL2-associated athanogene 2) is a cochaperone implicated in multiple pathological states. The aim of this study was to investigate the potential cardio-protective effects of Bag2 in DIC. C57BL/6 mice and AC16 cells were used to establish DIC model. The expression of Bag2 were measured by western blotting and immunohistochemical. The effects of Bag2 on DIC were assessed through functional gain and loss experiments. Through in vitro and in vivo experiments, we found that Bag2 expression was significantly reduced after DOX treatment. Both Bag2 knockdown and DOX administration resulted in apoptosis, mitochondrial dysfunction, and impaired mitophagy. Conversely, Bag2 overexpression exerted protective effects against these phenotypes induced by DOX stimulation. Mechanistically, Bag2 maintained mitophagy activation by binding to Pink1 and protecting it from proteasome-dependent degradation, thereby preserving mitochondrial function and protecting against myocardial lesions. Our findings suggest that Bag2 may serve as a promising therapeutic target for the treatment of DIC.

Ruxolitinib Treatment Ameliorates Clinical, Immunological, and Transcriptomic Aberrations in Patients with STAT3 Gain-of-Function Disease

BackgroundSTAT3 gain-of-function (GOF) disease presents with lymphoproliferation, autoimmunity, and failure to thrive. While JAK inhibitors have alleviated symptoms, their effects on disease pathogenesis remain unclear. ObjectiveWe prospectively investigated the clinical, immunological, and transcriptomic responses of four STAT3 GOF patients under long-term ruxolitinib treatment. MethodsWe conducted clinical and immunological evaluations at baseline and after ruxolitinib treatment at 3, 8, 12, and over 12 months. Our assessments included circulating T follicular helper cells (cTFH), regulatory T (Treg) cells, cytokine levels, and proliferation assays. Furthermore, we investigated the transcriptomic changes with treatment and conducted T-cell receptor sequencing. ResultsRuxolitinib achieved substantial control over the clinical manifestations. Post-treatment evaluations demonstrated a notable increase in naive CD4+ and CD8+ T cell populations, alongside a significant reduction in effector memory T cells. Additionally, there was a decrease in cTFH cells and double-negative T cells. Treg cell percentages and their canonical markers, already reduced before treatment, further declined with ruxolitinib. The treatment did not alter IFN-γ, IL-17A, IL-10, IL-4 cytokine productions of CD4+ T cells. Importantly, ruxolitinib effectively normalized the previously dysregulated transcriptome profile in peripheral blood mononuclear cells, bringing it closer to that of healthy controls. This normalization was most striking in the downregulation of STAT3-targeted genes, interferon-related genes, myeloid cell activation, and cytotoxic effector CD8+ T-cell genes, with effects persisting for up to 12 months. Self-reactive T-cell indices based on TCR repertoire analysis revealed potential auto-reactive cell clones in the patient samples. ConclusionRuxolitinib reversed cellular and transcriptomic signatures, enhancing our understanding of the disease's pathophysiology and highlighting essential immunological markers for precise monitoring.

Idiopathic Distal Sensory Polyneuropathy and Fibromyalgia Syndrome: A Comparative Phenotyping Study.

Painful idiopathic distal sensory polyneuropathy (IDSP) and fibromyalgia syndrome (FMS) are cryptogenic chronic pain syndromes. The contribution of small fibre pathology (SFP) in FMS remains controversial. This study aims to quantify small nerve pathology in participants with IDSP and FMS and identify relationships of SFP with sensory phenotypes. In this study, 73 individuals (FMS: 25, IDSP: 23, healthy volunteers: 25) underwent comprehensive assessment, including neurological exams, questionnaires, sensory tests, and corneal confocal microscopy. IDSP participants displayed lower wind-up ratio (WUR) relative to FMS (p<0.001), loss of function to thermal and mechanical stimuli and elevated neuropathy disability scores compared to FMS and healthy volunteers (all p<0.001). FMS participants demonstrated gain of function to heat and blunt pressure pain responses relative to IDSP, and healthy volunteers (heat: p=0.002 and p=0.003; pressure: both p<0.001) and WUR (both p<0.001). FMS participants exhibited reduced corneal nerve fibre density (p=0.02), while IDSP participants had lower global corneal nerve measures (density, branch density, and length) relative to healthy volunteers (all p<0.001). Utilising corneal nerve fibre length, SFP was demonstrated in 66.6% of participants (FMS: 13/25; IDSP: 22/23). Participants with SFP,in both FMS and IDSP, reported symptoms indicative of small nerve fibre disease. Although distinctions in pain distributions are evident between individuals with FMS and IDSP, over 50% of participants between the two conditions displayed both a loss and gain of thermal and mechanical function suggestive of shared mechanisms. However, sensory phenotypes were associated with the presence of SFP in IDSP but not in FMS.

Mutation-based mechanism and evolution of the potent multidrug efflux pump RE-CmeABC in Campylobacter

The resistance-nodulation-cell division (RND) superfamily of multidrug efflux systems are important players in mediating antibiotic resistance in gram-negative pathogens. Campylobacter jejuni, a major enteric pathogen, utilizes an RND-type transporter system, CmeABC, as the primary mechanism for extrusion of various antibiotics. Recently, a functionally potent variant of CmeABC (named RE-CmeABC) emerged in clinical Campylobacter isolates, conferring enhanced resistance to multiple antibiotic classes. Despite the clinical importance of RE-CmeABC, the molecular mechanisms for its functional gain and its evolutionary trajectory remain unknown. Here, we demonstrated that amino acid substitutions in RE-CmeB (inner membrane transporter), but not in RE-CmeA (periplasmic protein) and RE-CmeC (outer membrane protein), in conjunction with a nucleotide mutation in the promoter region of the efflux operon, are responsible for the functional gain of the multidrug efflux system. We also showed that RE-cmeABC is emerging globally and distributed in genetically diverse C. jejuni strains, suggesting its possible spread by horizontal gene transfer. Notably, many of RE-cmeABC harboring isolates were associated with the human host including strains from large disease outbreaks, indicating the clinical relevance and significance of RE-CmeABC. Evolutionary analysis indicated that RE-cmeB likely originated from Campylobacter coli, but its expansion mainly occurred in C. jejuni, possibly driven by antibiotic selection pressure. Additionally, RE-cmeB, but not RE-cmeA and RE-cmeC, experienced a selective sweep and was progressing to be fixed during evolution. Together, these results identify a mutation-based mechanism for functional gain in RE-CmeABC and reveal the key role of RE-CmeB in facilitating Campylobacter adaptation to antibiotic selection.

Inositol 1,4,5-Trisphosphate Receptor Mutations Associated with Human Disease: Insights into Receptor Function and Dysfunction.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are ubiquitous intracellular Ca2+ release channels. Their activation, subcellular localization, abundance, and regulation play major roles in defining the spatiotemporal characteristics of intracellular Ca2+ signals, which are in turn fundamental to the appropriate activation of effectors that control a myriad of cellular events. Over the past decade, ∼100 mutations in ITPRs associated with human diseases have been documented. Mutations have been detailed in all three IP3R subtypes and all functional domains of the protein, resulting in both gain and loss of receptor function. IP3R mutations are associated with a diverse array of pathology including spinocerebellar ataxia, peripheral neuropathy, immunopathy, anhidrosis, hyperparathyroidism, and squamous cell carcinoma. This review focuses on how studying the altered activity of these mutations provides information relating to IP3R structure and function, the physiology underpinned by specific IP3R subtypes, and the pathological consequences of dysregulated Ca2+ signaling in human disease.

Convex set reliability-based optimal attitude control for space solar power station with bounded and correlated uncertainties

Considering the bounded and correlated uncertainties in the inherently nonlinear attitude dynamics, this paper proposes an uncertain optimal attitude control for space solar power stations (SSPS), subjected to the convex set-based reliability constraint. To accurately and cost-effectively quantify the uncertainty in the SSPS attitude control system, the uncertain attitude dynamics are proposed based on nominal SSPS with convex set-based uncertainties. Given the known bounds and correlations of the random variables, the convex set-based attitude dynamics of SSPS can be conveniently constructed and transformed into corresponding uncertain state-space equations. Uncertain state and output can be solved using the extended-order matrix and uncertainty propagation method. A convex set-based Riccati equation is: developed using the convex set theory, traditional algebraic Riccati equation (ARE), and Lyapunov equation, which can obtain all the uncertain components of feedback gain, input torque, and cost function, respectively. Convex set-based time-dependent reliability (CSTR) is applied to assess the safety of the attitude control system by setting critical values and considering the time-dependent effect, which is regarded as an important constraint in the optimal attitude control issue with uncertainty. A numerical example of SSPS is provided to verify the proposed uncertain controller within the reliability-constrained optimization framework, demonstrating its effectiveness in managing nonlinear system dynamics under uncertainty.

GoFCards: an integrated database and analytic platform for gain of function variants in humans.

Gain-of-function (GOF) variants, which introduce new or amplify protein functions, are essential for understanding disease mechanisms. Despite advances in genomics and functional research, identifying and analyzing pathogenic GOF variants remains challenging owing to fragmented data and database limitations, underscoring the difficulty in accessing critical genetic information. To address this challenge, we manually reviewed the literature, pinpointing 3089 single-nucleotide variants and 72 insertions and deletions in 579 genes associated with 1299 diseases from 2069 studies, and integrated these with the 3.5 million predicted GOF variants. Our approach is complemented by a proprietary scoring system that prioritizes GOF variants on the basis of the evidence supporting their GOF effects and provides predictive scores for variants that lack existing documentation. We then developed a database named GoFCards for general geneticists and clinicians to easily obtain GOF variants in humans (http://www.genemed.tech/gofcards). This database also contains data from>150 sources and offers comprehensive variant-level and gene-level annotations, with the aim of providing users with convenient access to detailed and relevant genetic information. Furthermore, GoFCards empowers users with limited bioinformatic skills to analyze and annotate genetic data, and prioritize GOF variants. GoFCards offers an efficient platform for interpreting GOF variants and thereby advancing genetic research.

A Novel Heterozygous NFKB2 Variant in a Multiplex Family with Common Variable Immune Deficiency and Autoantibodies Against Type I IFNs.

We studied a family with three male individuals across two generations affected by common variable immune deficiency (CVID). We identified a novel missense heterozygous variant (c.2602T>A:p.Y868N) of NFKB2 in all patients and not in healthy relatives. Functional studies of the mutant allele in an overexpression system and of the patients' cells confirmed the deleteriousness of the NFKB2 variant and genotype, respectively, on the activation of the non-canonical NF-κB signaling pathway. Impaired processing of p100 into p52 underlies p100 accumulation, which results in gain-of-function (GOF) of IκBδ inhibitory activity and loss-of-function (LOF) of p52 transcriptional activity. The three patients' plasma contained autoantibodies that neutralized IFN-α2 and/or IFN-ω, accounting for the severe or recurrent viral diseases of the patients, including influenza pneumonia in one sibling, and severe COVID-19 and recurrent herpes labialis in another. Our results confirm that NFKB2 alleles that are IκBδ GOF and p52 LOF can underlie CVID and drive the production of autoantibodies neutralizing type I IFNs, thereby predisposing to severe viral diseases.

White box specification of intervention policies for prescriptive process monitoring

Prescriptive process monitoring methods seek to enhance business process performance by triggering real-time interventions, such as offering discounts to increase the likelihood of a positive outcome (e.g., a purchase). At the core of a prescriptive process monitoring method lies an intervention policy, which determines under which conditions and when to trigger an intervention. While state-of-the-art prescriptive process monitoring approaches rely on black-box intervention policies derived through reinforcement learning, algorithmic decision-making requirements sometimes dictate that the business stakeholders must be able to understand, justify, and adjust these intervention policies manually. To address this requirement, this article proposes WB-PrPM (White-Box Prescriptive Process Monitoring), a framework that enables stakeholders to define intervention policies in business processes. WB-PrPM is a rule-based system that helps decision-makers balance the demand for effective interventions with the imperatives of limited resource capacity. The framework incorporates an automated method for tuning the parameters of the intervention policies to optimize a total gain function. An evaluation is presented using real-life datasets to examine the tradeoffs among various parameters. The evaluation reveals that different variants of the proposed framework outperform existing baselines in terms of total gain, even when default parameter values are used. Additionally, the automated parameter optimization approach further enhances the total gain.

Initial experience with Double-vein Embolization in Hungary

IntroductionIn recent years several new techniques have emerged to induce hypertrophy of the future liver remnant prior to major hepatectomies. We aimed to summarize our initial experience with Double-vein Embolization as the first center in Hungary. MethodsBetween March 2023 and August 2024 a total of 16 Double-vein Embolization procedures were performed in Semmelweis University. Future liver remnant volume was calculated based on computed tomography scans obtained within 4 weeks prior and 2–3 weeks after the procedure. Tc-99m mebrofenin hepatobiliary scintigraphy results were available for 12/16 patients. ResultsTechnical success rate was 100 %. No major complication was observed. Successful resection rate was 93.8 %. One patient died due to post-hepatectomy liver failure. Future liver remnant volume and ratio increased significantly after the procedure compared to baseline (433.1 ± 163.8 cm3 vs. 603.5 ± 201.8 cm3, p < 0.0001 and 27.2 ± 6.5 % vs. 37 ± 8.8 %, p < 0.0001, respectively). Future liver remnant clearance improved significantly 1 and 2 weeks after the procedure (1.68 ± 0.58 %/min/m2 vs. 2.44 ± 0.64 %/min/m2 and 2.39 ± 0.31 %/min/m2, respectively). Mean function gain was 50.6 % after one week and 60.1% after two weeks, respectively. DiscussionVolumetric and functional outcomes in the present study are comparable with results reported in the literature. Our findings provide further evidence that Double-vein Embolization is a safe procedure that offers sufficient volumetric and functional gain in most candidates for liver resection. However, further studies are needed to define the exact place of this new technique in clinical practice.

KRT80 in hepatocellular carcinoma plays oncogenic role via epithelial-mesenchymal transition and PI3K/AKT pathway.

Hepatocellular carcinoma (HCC), a globally prevalent form of cancer, is featured by aggressive growth and early metastasis. Elucidating the underlying mechanism and identifying the effective therapy are critical for advanced HCC patients. In the study, we detect that KRT80 was upregulated in HCC samples. HCC patients with higher KRT80 are associated with worse overall survival after surgery. Gain-of and loss-of function studies show that KRT80 enhanced HCC cells proliferation, migration, invasion, and angiogenesis, whereas its silencing abolishes the effects in vivo and in vitro. Mechanistic investigation shows that KRT80 may function as an independent prognostic risk factor and act as an oncogene by influencing EMT and modulating the PI3K/AKT signaling pathway. Together, these findings suggest that KRT80 may be a potential oncogene and a good indicator in predicting prognosis. Targeting KRT80 can offer new insights into the prevention and treatment of HCC.

Brain Function Status may be Related to Pelvic Floor Function and Pregnancy Weight Gain in Postpartum Women with Pelvic Floor Dysfunction

Background: Postpartum women often show depression and anxiety, abnormal memory and cognitive function, and pelvic floor function problems. Brain function test is also a new research direction generated combining life science and computer science. This study aimed to observe the brain function status of postpartum women and analyze the correlation between brain function and pelvic floor function and gestational weight. Methods: A cross-sectional study of women with medical record in our hospital. A total of 88 outpatient postpartum women with pelvic floor dysfunction including 48 cases of cesarean section and 40 cases of vaginal delivery who underwent brain function tests from August 2022 to September 2023 and met the inclusion criteria were included. Basic demographic information, brain function tests, pelvic floor examination, and pregnancy-related data were extracted. The brain function status of women after vaginal delivery and cesarean section was analyzed statistically, and analysis of the relationship between pelvic floor assessment and brain function. Results: Of the 88 postpartum women, &gt;50% showed abnormal findings in the hypoxia index, emotional resistance, sleep index, outside focus, brain fatigue, brain emptying, and reaction speed. Statistical differences in brain fatigue (p &lt; 0.001) and brain emptying (p = 0.002) were observed between postpartum women with vaginal delivery and cesarean section. Correlation analysis results revealed that prolapse was correlated with brain emptying (p &lt; 0.001), and weakly correlated anxiety tendency (p = 0.05), sleep index (p = 0.02), introverted brain (p = 0.05), brain fatigue (p = 0.02). Pelvic floor muscle strength was associated with emotional resistance (p = 0.03), brain inhibition (p = 0.04), and brain stability (p = 0.03) weakly. Weight gain during pregnancy was related to inner focus (p = 0.001), and weakly related to oxygen deficiency index (p = 0.04), brain stability (p = 0.03), brain coordination (p = 0.01), brain emptying (p = 0.04), and reaction speed (p = 0.01). Conclusions: This study observed that most postpartum women have abnormal brain function, including mood, sleep, fatigue, cerebral oxygen metabolism, concentration, and information processing ability, and showed that postpartum brain function problems might correlated with pelvic floor function and pregnancy weight gain.

POU3F4 up-regulates Gli1 expression and promotes neuronal differentiation and synaptic development of hippocampal neural stem cells

BackgroundNeural stem cells (NSCs) are considered to be the most promising cell type for cell replacement therapy in neurodegenerative diseases. However, their low neuronal differentiation ratio impedes their application in such conditions. Elucidating the molecular mechanism of NSC differentiation may provide the necessary experimental basis for expanding their application. Previous studies have indicated that POU3F4 can induce neuronal differentiation of NSCs, this study aims to underly the possible exact mechanism of POU3F4 on the NSC differentiation and development.MethodsNSCs were isolated and cultured from the hippocampus of neonatal mice. The frozen hippocampal sections were prepared for immunohistochemical staining. Synaptic development was assessed using electron microscopy. High-throughput sequencing was employed to analyze the gene expression profile following the overexpression of Brn4. Gene expression levels were determined through Western blotting and qRT-PCR. Cell cycle and differentiation were evaluated using flow cytometry and immunofluorescent staining.ResultsIt was found that POU3F4 promoted the neuronal differentiation of hippocampal NSCs and synapse development, and inhibited NSC proliferation. POU3F4-deficient mice exhibited impairments in learning and memory. RNA sequencing and ChIP assays confirmed that Gli1 was downstream of POU3F4. Loss and gain function experiments indicated that Gli1 mediated POU3F4 promoting neuronal differentiation and synapse development. Forced expression of Gli1 in hippocampus improved learning and memory function of animal models.ConclusionsThe results suggest that POU3F4 and Gli1 promote neuronal differentiation and synaptic development of NSCs, and that Gli1 partially mediates the effects of POU3F4.Graphical abstract

RAS signaling pathway is essential in regulating PIEZO1-mediated hepatic iron overload in dehydrated hereditary stomatocytosis.

PIEZO1 encodes a mechanoreceptor, a cation channel activated by mechanical stimuli. Gain-of-function (GoF) variants in PIEZO1 cause dehydrated hereditary stomatocytosis (DHS), or xerocytosis, a pleiotropic syndrome characterized by anemia and iron overload. DHS patients develop hepatic iron overload independent of the degree of anemia and transfusion regimen. PIEZO1-GoF variants suppress hepcidin expression in both hepatic cellular model and constitutive/macrophage-specific Piezo1-GoF mice model. Therefore, PIEZO1-GoF variants regulate hepcidin expression by a crosstalk between hepatocytes (HCs) and macrophages with a still unknown mechanism. Transcriptomic and proteomics analysis in the human hepatic Hep3B cells engineered for the PIEZO1-R2456H variant (PIEZO1-KI) revealed alterations in the actin cytoskeleton regulation, MAPK cascade, and RAS signaling. These changes mainly occur through a novel key regulator, RRAS, whose protein and mRNA levels are regulated by PIEZO1 activation and inhibition. This regulation was further confirmed in C57BL/6 mouse primary HCs treated with Yoda-1 and/or GsMTx-4. Indeed, PIEZO1-KI cells exhibited hyper-activated RAS-GTPase activity that is rescued by PIEZO1 inhibition, restoring expression of the hepcidin gene HAMP. A negative correlation between RAS signaling and HAMP regulation was confirmed by inhibiting RAS-GTPase and MEK1-2 activity. Conversely, rescued HAMP gene expression requires downregulation of RRAS, confirming negative feedback between RAS-MAPK and BMP/SMADs pathways in HAMP regulation. We demonstrated that PIEZO1-GoF variants influence the actin cytoskeleton organization by activating the hepatic RAS signaling system. Understanding the role of RAS signaling in regulating iron metabolism could pave the way for new therapeutic strategies in DHS and other conditions characterized by iron overload.

Overexpression of autophagy enhancer PACER/RUBCNL in neurons accelerates disease in the SOD1G93A ALS mouse model

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal paralytic disorder associated with motor neuron death. Mutant superoxide dismutase 1 (SOD1) misfolding and aggregation have been linked to familial ALS, with the accumulation of abnormal wild-type SOD1 species being also observed in postmortem tissue of sporadic ALS cases. Both wild-type and mutated SOD1 are reported to contribute to motoneuron cell death. The autophagic pathway has been shown to be dysregulated in ALS. Recent evidence suggests a dual time-dependent role of autophagy in the progression of the disease. PACER, also called RUBCNL (Rubicon-like), is an enhancer of autophagy and has been found diminished in its levels during ALS pathology in mice and humans. Pacer loss of function disturbs the autophagy process and leads to the accumulation of SOD1 aggregates, as well as sensitizes neurons to death. Therefore, here we investigated if constitutive overexpression of PACER in neurons since early development is beneficial in an in vivo model of ALS. We generated a transgenic mouse model overexpressing human PACER in neurons, which then was crossbred with the mutant SOD1G93A ALS mouse model. Unexpectedly, PACER/SOD1G93A double transgenic mice exhibited an earlier disease onset and shorter lifespan than did littermate SOD1G93A mice. The overexpression of PACER in neurons in vivo and in vitro increased the accumulation of SOD1 aggregates, possibly due to impaired autophagy. These results suggest that similar to Pacer loss-of function, Pacer gain-of function is detrimental to autophagy, increases SOD1 aggregation and worsens ALS pathogenesis. In a wider context, our results indicate the requirement to maintain a fine balance of PACER protein levels to sustain proteostasis.

Technical challenges of intracellular flow cytometry-based assays as a functional complement to diagnosis of signaling defects of inborn errors of immunity: PI3K pathway as a case of study.

The use of next-generation sequencing in inborn errors of immunity (IEI) has considerably increased the identification of novel gene variants, many of which are identified in patients without the described clinical phenotype or with variants of uncertain pathogenic significance in previously described genes. Properly designed functional and cellular assays, many necessarily accomplished by research-based laboratories, reveal the pathogenic consequences of the gene variants and contribute to diagnosis. Activated PI3Kδ syndrome (APDS) is a rare disease that can be divided into APDS1, caused by gain of function (GOF) mutations in PIK3CD gene, and APDS2, with loss of function (LOF) variants in the PIK3R1 gene. Both entities present hyperactivation of the PI3K pathway, which can be analyzed through Akt and S6 phosphorylation status. Our objective was to perform an accurate, robust, and reproducible functional assay to analyze the phosphorylation status of proteins in the PI3K-Akt-S6 pathway by flow cytometry, to contribute to diagnosis, to monitor treatments, and to establish intra-assay standardization. We illustrate the robustness and reproducibility of our experimental procedure in patients with APDS who had high Akt and/or S6 phosphorylation levels at baseline, and after anti-IgM stimulation in B cells. We show the relevance of an appropriate cohort of samples from healthy donors, processed within the same conditions as the suspected samples, in particular the time frame for sample processing once blood is collected. We highlight the importance of B cell stimulation through B cell receptor signaling, which is highly recommended, especially for samples that would be processed more than 24 hours after blood extraction. Also, having a defined experimental procedure is important, including the cytometer setup, which allows cytometer reproducibility for a period of time, enabling the comparison of a sample at different times.

Elucidating the pathobiology of Cerebellar Ataxia with Neuropathy and Vestibular Areflexia Syndrome (CANVAS) with its expanded RNA structure formation and proteinopathy

Numerous neurological disorders are linked to sequences rich in guanine repeats found in introns, exons, and regulatory regions of genes. These sequences have been observed to form stable G-quadruplex (GQ) structures both in vitro and in vitro. Cerebellar Ataxia with Neuropathy and Vestibular Areflexia Syndrome (CANVAS), a slowly progressive neurodegenerative disorder, is associated with the biallelic expansion of (AAGGG)n pathogenic repeats in the second intron of the RFC1 gene. Though these G-rich pathogenic repeats in other neurological diseases are associated with protein loss of function, RNA gain of function, and/or protein gain of function, not much is known about the pathological mechanism associated with CANVAS. Herein, we report the formation of stable GQ conformations in the CANVAS-associated repeats i.e., r(AAGGG)n, where ‘r’ stands for RNA. These GQs are critical regulators in neurological disorders leading to RNA foci formation and RNA binding protein sequestration. They also alter other causative processes like intron retention, which leads us to hypothesize a toxic Proteinopathy mechanism in CANVAS. Various biophysical and biomolecular assays characterized the interactions of three aggregation-prone RNA-binding proteins (RBPs): heterogeneous nuclear ribonucleoprotein H1/F (hnRNP H1/F), and DGCR8 with different pathogenic repeats [(AAGGG)9] in vitro, further affirming the hypothesis. The biophysical observations are further supported by molecular dynamics analysis and cell-based studies, putting us a step closer to elucidating the pathological mechanism(s) in CANVAS neuropathy, paving the way for the development of innovative therapeutic interventions.

Discovery of Potent and Selective Blockers Targeting the Epilepsy-Associated KNa1.1 Channel.

Gain-of-function (GOF) mutations of the sodium-activated potassium channel KNa1.1 (Slack, Slo2.2, or KCa4.1) induce severe, drug-resistant forms of epilepsy in infants and children. Although quinidine has shown promise in treating KCNT1-related epilepsies compared to other drugs, its limited efficacy and substantial side effects necessitate the development of new KNa1.1 channel inhibitors. In this study, we developed a novel class of KNa1.1 inhibitors using combined silico approaches and structural optimization. Among these inhibitors, compound Z05 was identified as a selective potential KNa1.1 inhibitor, especially against the hERG channel. Moreover, its binding site and potential counteraction to a GOF mutant Y796H were identified by the mutation studies. Our data also showed that Z05 had significant pharmacological profiles, including high brain penetration and moderate oral bioavailability, offering a valuable in vitro tool compound for further drug development in treating KCNT1-related epilepsies.

Deciphering the tissue-specific functional effect of Alzheimer risk SNPs with deep genome annotation

Alzheimer’s disease (AD) is a highly heritable brain dementia, along with substantial failure of cognitive function. Large-scale genome-wide association studies (GWASs) have led to a set of SNPs significantly associated with AD and related traits. GWAS hits usually emerge as clusters where a lead SNP with the highest significance is surrounded by other less significant neighboring SNPs. Although functionality is not guaranteed even with the strongest associations in GWASs, lead SNPs have historically been the focus of the field, with the remaining associations inferred to be redundant. Recent deep genome annotation tools enable the prediction of function from a segment of a DNA sequence with significantly improved precision, which allows in-silico mutagenesis to interrogate the functional effect of SNP alleles. In this project, we explored the impact of top AD GWAS hits around APOE region on chromatin functions and whether it will be altered by the genetic context (i.e., alleles of neighboring SNPs). Our results showed that highly correlated SNPs in the same LD block could have distinct impacts on downstream functions. Although some GWAS lead SNPs showed dominant functional effects regardless of the neighborhood SNP alleles, several other SNPs did exhibit enhanced loss or gain of function under certain genetic contexts, suggesting potential additional information hidden in the LD blocks.