Wild-type Variants Research Articles

Discovery of a Potential Allosteric Site in the SARS-CoV-2 Spike Protein and Targeting Allosteric Inhibitor to Stabilize the RBD Down State using a Computational Approach.

The novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a worldwide public health crisis. At present, the development of effective drugs and/or related therapeutics is still the most urgent and important task for combating the virus. The viral entry and associated infectivity mainly rely on its envelope spike protein to recognize and bind to the host cell receptor angiotensin-converting enzyme 2 (ACE2) through a conformational switch of the spike receptor binding domain (RBD) from inactive to active state. Thus, it is of great significance to design an allosteric inhibitor targeting spike to lock it in the inactive and ACE2-inaccessible state. This study aims to discover the potential broad-spectrum allosteric inhibitors capable of binding and stabilizing the diverse spike variants, including the wild type, Delta, and Omicron, in the inactive RBD down state. In this work, we first detected a potential allosteric pocket within the SARS-CoV-2 spike protein. Then, we performed large-scale structure-based virtual screening by targeting the putative allosteric pocket to identify allosteric inhibitors that could stabilize the spike inactive state. Molecular dynamics simulations were further carried out to evaluate the effects of compound binding on the stability of spike RBD. Finally, we identified three potential allosteric inhibitors, CPD3, CPD5, and CPD6, against diverse SARS-CoV-2 variants, including Wild-type, Delta, and Omicron variants. Our simulation results showed that the three compounds could stably bind the predicted allosteric site and effectively stabilize the spike in the inactive state. The three compounds provide novel chemical structures for rational drug design targeting spike protein, which is expected to greatly assist in the development of new drugs against SARS-CoV-2.

EXTH-46. TESTING PRECISION GENOMICS-GUIDED THERAPY IN A MODEL OF ANAPLASTIC PLEOMORPHIC XANTHOASTROCYTOMA: DUAL INHIBITION OF CDK4/6 AND MEK

Abstract Typical survival for pediatric high-grade gliomas remains less than 18 months despite recent improved understanding of the molecular drivers of these tumors. Hyperactivating MAPK and CDK4/6 pathway mutations are common and targetable alterations implicated in tumorigenesis and malignant transformation in pediatric glioma. We have established and characterized a novel patient-derived xenograft (PDX) model, RHT128, from a pediatric patient diagnosed with the high-grade glioma anaplastic pleomorphic xanthoastrocytoma (APXA). The molecular landscape of PDX RHT128 exhibits molecular fidelity to the patient’s tumor. Clinical precision genomics analysis of the tumor revealed a novel BRAF chromosomal rearrangement and CDKN2A/B deletion. Based on this molecular signature, the patient was treated with MEK inhibitor trametinib as a monotherapy and, following progression of disease, with CDK4/6 inhibitor ribociclib. However, the tumor continued to progress. In this study our objective is to simultaneously target the CDK4/6 and MAPK pathways in RHT128 and determine to what extent this combination therapy minimizes emergence of therapeutic resistance. Single-agent efficacy assessments in a subcutaneous RHT128 model, showing significant dose-dependent reduction in tumor volume after treatment with abemaciclib (p<0.05), palbociclib (p<0.01), and the blood-brain barrier-permeable MEK inhibitor mirdametinib (p<0.0001). Analysis of the global kinome in CDK4/6 inhibitor-treated PDX tissues compared to vehicle treatment using multiplexed-inhibitor bead chromatography–mass spectrometry demonstrated effective inhibition of CDK4/6. Moreover, this analysis revealed dose-dependent alterations in the MAPK and PI3K pathways and modulation of critical neurotransmitter pathways in treated tissues. Future studies will explore efficacy of these MEK and CDK4/6 inhibitors in combination and in our BRAF wild-type and BRAF V600E variant APXA models to gain further mechanistic insights. The pervasiveness of alterations to the MAPK and CDK4/6 pathways in pediatric gliomas make this approach promising for further study in a broader range of these deadly tumors.

Impact of Genetic Polymorphisms and Drug-Drug Interactions Mediated by Carboxylesterase1 on Remimazolam Deactivation.

Remimazolam (Byfavo®), a recent FDA-approved ester-linked benzodiazepine, offers advantages in sedation, such as rapid onset and predictable duration, making it suitable for broad anesthesia applications. Its favorable pharmacological profile is primarily attributed to rapid hydrolysis, the primary metabolism pathway for its deactivation. Thus, understanding remimazolam hydrolysis determinants is essential for optimizing its clinical use. This study aimed to identify the enzyme(s) and tissue(s) responsible for remimazolam hydrolysis and to evaluate the influence of genetic polymorphisms and drug-drug interactions (DDIs) on its hydrolysis in the human liver. An initial incubation study with remimazolam and phosphate buffer saline (PBS), human serum, and the S9 fractions of human liver and intestine demonstrated that remimazolam was exclusively hydrolyzed by human liver S9 fractions. Subsequent incubation studies utilizing a Carboxylesterase inhibitor (Bis-para-nitrophenylphosphate, BNPP), recombinant human Carboxylesterase1 (CES1) and Carboxylesterase 2 (CES2) confirmed that remimazolam is specifically hydrolyzed by CES1 in human liver. Furthermore, in vitro studies with wild-type CES1 and CES1 variants transfected cells revealed that certain genetic polymorphisms significantly impair remimazolam deactivation. Notably, the impact of CES1 G143E was verified using individual human liver samples. Moreover, our evaluation of the DDIs between remimazolam and several other substrates/inhibitors of CES1-including simvastatin, enalapril, clopidogrel and sacubitril- found that clopidogrel significantly inhibited remimazolam hydrolysis at clinically relevant concentrations, with CES1 genetic variants potentially influencing the interactions. In summary, CES1 genetic variants and its interacting drugs are crucial factors contributing to interindividual variability in remimazolam hepatic hydrolysis, holding the potential to serve as biomarkers for optimizing remimazolam use. Significance Statement This investigation demonstrates that remimazolam is deactivated by CES1 in the human liver, with CES1 genetic variants and DDIs significantly influencing its metabolism. These findings emphasize the need to consider CES1 genetic variability and potential DDIs in remimazolam use, especially in personalized pharmacotherapy to achieve optimal anesthetic outcomes.

Transthyretin Cardiac Amyloidosis: Insights into Wild-Type Variants and Familial Amyloid Cardiomyopathy

Transthyretin (TTR) cardiac amyloidosis is a progressive condition characterized by amyloid fibril deposition in the heart, leading to heart failure. This review focuses on insights into wild-type (ATTRwt) and familial (ATTRm) variants of TTR amyloidosis. The pathophysiology involves TTR tetramer destabilization, monomer misfolding, and amyloid fibril formation. Therapeutic advances, including TTR stabilizers like tafamidis and Diflunisal, and gene-silencing agents such as patisiran and inotersen, show promise in managing the disease. Despite these advancements, early diagnosis remains challenging, and side effects of current therapies necessitate the development of safer, more effective treatments. Future research should focus on novel therapies, improved diagnostic methods, and personalized treatment strategies to enhance patient outcomes. This review underscores the importance of continued innovation and collaboration in tackling TTR cardiac amyloidosis.

Design of the conserved epitope peptide of SARS-CoV-2 spike protein as the broad-spectrum COVID-19 vaccine

Our previous study has found that monoclonal antibodies targeting a conserved epitope peptide spanning from residues 1144 to 1156 of SARS-CoV-2 spike (S) protein, namely S(1144–1156), can broadly neutralize all of the prevalent SARS-CoV-2 strains, including the wild type, Alpha, Epsilon, Delta, and Gamma variants. In the study, S(1144–1156) was conjugated with bovine serum albumin (BSA) and formulated with Montanide ISA 51 adjuvant for inoculation in BALB/c mice to study its potential as a vaccine candidate. Results showed that the titers of S protein-specific IgGs and the neutralizing antibodies in mouse sera against various SARS-CoV-2 variants, including the Omicron sublineages, were largely induced along with three doses of immunization. The significant release of IFN-γ and IL-2 was also observed by ELISpot assays through stimulating vaccinated mouse splenocytes with the S(1144–1156) peptide. Furthermore, the vaccination of the S(1143–1157)- and S(1142–1158)-EGFP fusion proteins can elicit more SARS-CoV-2 neutralizing antibodies in mouse sera than the S(1144–1156)-EGFP fusion protein. Interestingly, the antisera collected from mice inoculated with the S(1144–1156) peptide vaccine exhibited better efficacy for neutralizing Omicron BA.2.86 and JN.1 subvariants than Omicron BA.1, BA.2, and XBB subvariants. Since the amino acid sequences of the S(1144–1156) are highly conserved among various SARS-CoV-2 variants, the immunogen containing the S(1144–1156) core epitope can be designed as a broadly effective COVID-19 vaccine.Key points• Inoculation of mice with the S(1144–1156) peptide vaccine can induce bnAbs against various SARS-CoV-2 variants.• The S(1144–1156) peptide stimulated significant release of IFN-γ and IL-2 in vaccinated mouse splenocytes.• The S(1143–1157) and S(1142–1158) peptide vaccines can elicit more SARS-CoV-2 nAbs in mice.

Tirzepatide, GIP(1-42) and GIP(1-30) display unique signaling profiles at two common GIP receptor variants, E354 and Q354.

Type 2 diabetes (T2D) and obesity are prevalent metabolic disorders affecting millions of individuals worldwide. A new effective therapeutic drug called tirzepatide for the treatment of obesity and T2D is a dual agonist of the GIP receptor and GLP-1 receptor. Tirzepatide is clinically more effective than GLP-1 receptor agonists but the reasons why are not well understood. Tirzepatide reportedly stimulates the GIP receptor more potently than the GLP-1 receptor. However, tirzepatide signaling has not been thoroughly investigated at the E354 (wildtype) or Q354 (E354Q) GIP receptor variants. The E354Q variant is associated increased risk of T2D and lower body mass index. To better understand GIP receptor signaling we characterized the activity of endogenous agonists and tirzepatide at both GIP receptor variants. Using Cos7 cells we examined wildtype and E354Q GIP receptor signaling, analyzing cAMP and IP1 accumulation as well as AKT, ERK1/2 and CREB phosphorylation. GIP(1-42) and GIP(1-30)NH2 displayed equipotent effects on these pathways excluding CREB phosphorylation where GIP(1-30)NH2 was more potent than GIP(1-42) at the E354Q GIP receptor. Tirzepatide favored cAMP signaling at both variants. These findings indicate that tirzepatide is a biased agonist towards Gαs signaling and suggests it equally activates the wildtype and E354Q GIP receptor variants. We also observed differences between the pharmacology of the GIP receptor variants with endogenous peptides, which may help to explain differences in phenotype. These findings contribute to a comprehensive understanding of GIP receptor signaling, and will aid development of therapies combating T2D and obesity.

Clinical report and genetic analysis of a Chinese family with retinitis pigmentosa 79 caused by a novel loss-of-function HK1 variant.

Retinitis pigmentosa (RP) is a genetically heterogeneous disease. RP 79 has been associated with heterozygous variants of hexokinase 1 (HK1). Only two missense HK1 variants have been reported in 11 families. To discover the molecular pathogenic mechanism of RP and validate the biological harm of HK1 through in vitro experiments. We conducted a genetic analysis of a 3-year-old female patient with RP and her family. We also evaluated the ocular phenotypes caused by HK1 (the identified variant). Peripheral blood samples were collected from the patient, her parents, and her brother, and trio whole-exome sequencing was performed. A protein structure analysis was performed to assess the functional impact of the variant, and a mutant plasmid was constructed for the quantitative polymerase chain reaction (qPCR) and western blot (WB) analysis of the effects of the variant on transcription and protein translation. The patient harbored the NM_000188.3: c.613del (p.Ala205Leufs*3) variant, which is a heterozygous variant of HK1. Sanger sequencing confirmed the presence of this variant in the patient; however, the patient's parents and brother had the wild-type variant. The protein structure analysis indicated that the variant resulted in a truncated protein caused by premature termination of amino acid coding. The qPCR results indicated that the variant may not have affected the transcription process. However, the WB analysis demonstrated that the mutant HK-1 protein was not expressed and that the wild-type group exhibited normal expression. Our patient had a loss-of-function (LoF) variant of HK1, which may be the genetic cause of typical features of RP that are observed at an early age. These findings expand the spectrum of HK1 variants and phenotypes and suggest that LoF variants of HK1 may represent a specific pathogenic mechanism of RP.

Anti-SARS-CoV-2 IgM Antibody Levels Measured by an In-House ELISA in a Convalescent Latin Population Persist over Time and Exhibit Neutralizing Capacity to Several Variants of Concern.

The coronavirus, SARS-CoV-2, is the causative agent for COVID-19, first registered in Wuhan, China and responsible for more than 6 million deaths worldwide. Currently, RT-PCR is the gold-standard method for diagnosing COVID-19. However, serological tests are needed for screening acute disease diagnosis and screening large populations during the COVID-19 outbreak. Herein, we described the development and validation of an in-house enzyme-linked immunosorbent assay (ELISA) for detecting the levels of anti-spike-1-RBD IgM antibody (CovIgM-ELISA) in well-defined serum/plasma panel for screening and identifying subjects infected with SARS-CoV-2 in a Latin population. In-house CovIgM-ELISA has the format of an indirect ELISA. It was optimized by checkerboard titration using recombinant SARS-CoV-2 spike-S1-RBD protein as an antigen. We found that, compared to the RT-PCR as the standard method, the in-house CovIgM-ELISA displayed sensitivities of 96.15% and 93.22% for samples collected up to 30 or 60 days after infection, respectively, as well as 95.59% specificity with 97.3% accuracy. The agreement kappa value (κ) of our CovIgM-ELISA was substantial when compared to RT-PCR (κ = 0.873) and the anti-SARS-CoV-2 IgM ELISA (InBios Int) (κ = 0.684). The IgM levels detected in the population positively correlated with the neutralizing activity against the wild-type, Alpha and Delta variants of concern, but failed to neutralize Omicron. These data indicate that our in-house CovIgM-ELISA is a compatible performing assay for the detection of SARS-CoV-2 infection.

Distinct roles for the domains of the mitochondrial aspartate/glutamate carrier citrin in organellar localization and substrate transport

ObjectiveCitrin, the mitochondrial aspartate/glutamate carrier isoform 2, is structurally and mechanistically the most complex SLC25 family member, because it consists of three-domains and forms a homodimer. Each protomer has an N-terminal calcium-binding domain with EF-hands, followed by a substrate-transporting carrier domain and a C-terminal domain with an amphipathic helix. The absence or dysfunction of citrin leads to citrin deficiency, a highly prevalent pan-ethnic mitochondrial disease. Here, we aim to understand the role of different citrin domains and how they contribute to pathogenic mechanisms in citrin deficiency. MethodsWe have employed structural modelling and functional reconstitution of purified proteins in proteoliposomes to assess the transport activity and calcium regulation of wild-type citrin and pathogenic variants associated with citrin deficiency. We have also developed a double knock-out of citrin and aralar (AGC1), the two paralogs of the mitochondrial aspartate/glutamate carrier, in HAP1 cells to perform mitochondrial imaging and to investigate mitochondrial localisation. ResultsUsing 33 pathogenic variants of citrin we clarify determinants of sub-cellular localization and transport mechanism. We identify crucial elements of the carrier domain that are required for transport, including those involved in substrate binding, network formation and dynamics. We show that the N-terminal domain is not involved in calcium regulation of transport, as previously thought, but when mutated causes a mitochondrial import defect. ConclusionsOur work introduces a new role for the N-terminal domain of citrin and demonstrates that dysfunction of the different domains contributes to distinct pathogenic mechanisms in citrin deficiency.

Longitudinal assessment of SNPs rs72552763 and rs622342 in SLC22A1 over HbA1c control among Mexican-Mestizo diabetic type 2 patients.

Background: In Mexico, 75% of diabetes mellitus type 2 (DMT2) patients are not in glycaemic control criteria (HbA1c<7%); this entails a significantly variable drug response. Amongst the factors influencing such variability, are genetics, more specifically, single nucleotide polymorphisms (SNPs). Three genes implied in metformin pharmacokinetics are SLC22A1, SLC22A2, and SLC22A3, which are polymorphic. While there have been cross-sectional studies on their SNPs impact over drug response, a longitudinal study would contribute valuable information on their effect over time. Methods: SNPs of SLC22A1 (rs72552763, rs622342, rs12208357, rs2282143, rs594709, rs628031, and rs683369), SLC22A2 (rs316019), and SLC22A3 (rs2076828), were determined through PCR-TR. The clinical records of 69 patients undergoing metformin monotherapy were retrospectively assessed. Metformin is the first line treatment against DMT2. A level of HbA1c <7% (time 0) was considered as an inescapable inclusion criterion. The study's cases were those patients who reported HbA1c ≥ 7% (time1) after time 0 (t0). Kaplan-Meier curves including a Log-Rank test and a Cox multivariate analysis of proportional risks were performed. Aim: Determining clinical, biochemical, and genetic variables which may affect non-control (HbA1c ≥ 7%) survival time spans amongst DMT2 Mexican-Mestizo patients undergoing metformin monotherapy at Hospital Regional de Alta Especialidad de Ixtapaluca (HRAEI) between October 2013 and December 2023. Results: All 69 patients were monitored over a median period of 642 days (273-1,134). A comparison between time 0 and time 1 (t1) revealed differences in weight (p = 0.036), metformin dose mg/kg/day (p = 0.003), plasmatic glucose mg/dL (p = 0.048), and HbA1c (p < 0.001). The median non-control survival rate was different across the 3 genotypes of rs62552763 in SLC22A1 (p = 0.0034) and the dominant genotypic model GAT/GAT vs. GAT/del + del/del (p = 0.009). There were differences between rs622342 genotypes as well (p = 0.041). In GAT/GAT the Cox model found HR = 0.407 (IC95%: 0.202-0.818, p = 0.011) in the univariate analysis and HR = 0.418 (IC95%: 0.204-0.856, p = 0.034) in the multivariate analysis, adjusted by initial metformin dose (mg/kg/day), initial weight (kg), and final metformin dose (mg/kg/day). Genotype A/A of rs622342 in SLC22A1, reported HR = 0.392 (IC95%: 0.169-0.910, p = 0.029) in the multivariate analysis as well. Conclusion: Among DMT2 Mexican-Mestizo patients undergoing metformin monotherapy the minor allele del in rs72552763 and the minor allele C in rs622342 reported a significantly shorter survival median respect to the wild type variant. Patients carrying del in rs72552763 or C in rs622342, both in SLC22A1, will reach non-control in less time with respect to other patients. Therefore these genotypes may constitute a therapeutic response biomarker for this population.

The comparison of pathogenicity among SARS-CoV-2 variants in domestic cats

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been detected or isolated from domestic cats. It is unclear whether cats play an important role in the SARS-CoV-2 transmission cycle. In this study, we examined the susceptibility of cats to SARS-CoV-2, including wild type and variants, by animal experiments. Cats inoculated with wild type, gamma, and delta variants secreted a large amount of SARS-CoV-2 for 1 week after the inoculation from nasal, oropharyngeal, and rectal routes. Only 100 TCID50 of virus could infect cats and replicate well without severe clinical symptoms. In addition, one cat inoculated with wild type showed persistent virus secretion in feces for over 28 days post-inoculation (dpi). The titer of virus-neutralizing (VN) antibodies against SARS-CoV-2 increased from 11 dpi, reaching a peak at 14 dpi. However, the omicron variant could not replicate well in cat tissues and induced a lower titer of VN antibodies. It is concluded that cats were highly susceptible to SARS-CoV-2 infection, but not to the Omicron Variant, which caused the attenuated pathogenicity.

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 thromboembolism (TE), commonly manifesting in pediatric patients. This study aimed to elucidate the pathogenic mechanisms of two novel PC mutations, C238G and R189W, identified in Thai children with both venous and arterial TE. The effects of wild-type (WT), C238G, and R189W PC variants were investigated through transient transfection of HEK293T cells. PC secretion levels were measured, and immunofluorescence analysis was performed to assess intracellular localization. ER stress-related gene expression and UPR activation were evaluated. Structural analysis was conducted to explore the significance of the C238 and R189W residue in PC functionality. The C238G mutation led to a severe 95% reduction in PC secretion, while R189W showed a 30% decrease compared with WT. Immunofluorescence revealed that C238G-PC was predominantly retained in the ER, indicating protein misfolding. C238G-expressing cells exhibited significant upregulation of ER stress-related genes and UPR activation. In contrast, R189W resulted in only a modest increase in UPR gene expression, suggesting a less pronounced impact on protein folding and secretion. Structural analysis demonstrated the critical role of the C238 residue in maintaining PC's disulfide bond and overall conformation. This study reveals distinct molecular mechanisms by which the C238G and R189W mutations contribute to PC deficiency and increased thrombotic risk. The findings emphasize the essential role of the C238 residue in preserving PC structure and secretion, enhancing the understanding of PC deficiency-associated TE in pediatric patients.

Abstract IA-04: Targeting the oncogenic state of RAS with tri-complex inhibitors

Abstract KRAS oncogenic driver mutations occur in approximately 30% of all human cancers, and in particular, in over 90% of patients with pancreatic ductal adenocarcinoma (PDAC). Small molecule inhibitors that selectively target the inactive, GDP-bound state of KRAS G12C mutant proteins have demonstrated clinical efficacy in non-small cell lung cancer (NSCLC) as monotherapies, and in the small subset of PDAC patients that harbor this mutation. However, there are no currently approved RAS-targeted therapy options for the majority of patients with non-G12C RAS mutant PDAC. We have designed a series of tri-complex inhibitors that specifically target the GTP-bound, active state of RAS (RAS(ON)). These small molecule inhibitors bind non-covalently to an abundant intracellular protein, cyclophilin A (CypA). The resulting binary complex selectively engages RAS(ON), forming a tri-complex that sterically inhibits RAS interaction with downstream effectors. We have generated mutant-selective inhibitors that covalently engage RAS(ON) G12C (RMC-6291) or RAS(ON) G12D (RMC-9805). In addition, RMC-6236 is a RAS(ON) multi-selective inhibitor that non-covalently inhibits the active, GTP-bound state of mutant and wild-type variants of the canonical RAS isoforms (KRAS, NRAS, and HRAS). Here we will describe the mechanism of action of these investigational agents and the preclinical profiles that support their clinical evaluation in patients with PDAC as monotherapies and in combination therapy regimens with standard-of-care agents or as novel RAS(ON) inhibitor doublets. Citation Format: Mallika Singh. Targeting the oncogenic state of RAS with tri-complex inhibitors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr IA-04.

A - 117 Double Trouble: a Case Study on Cardiac Amyloidosis and Alzheimer’s Disease

Abstract Objective Transthyretin amyloidosis (ATTR) is an increasingly recognized cause of heart failure amongst older adults. The median age upon diagnosis is 75 with 90% of patients reported as men. The amyloidogenic protein is transthyretin (TTR), which is primarily produced in the liver, but is also produced in the choroid plexus and neurons under distress. There are two types of ATTR: an autosomal dominant and a wild-type (wt) variant. ATTRwt is the amyloid precursor in the cardiac depositions found in older adults. We present the first neuropsychological evaluation of a patient with ATTRwt. Method An 87-year-old right-handed White woman reported a 2 year history of slowly progressive short term memory decline. Her family noted rapid cognitive decline and paranoia approximately 6 months ago. Medical history was notable for heart failure, along with lumbar stenosis, carpal tunnel syndrome, arthritis, scoliosis, and hyperlipidemia. ATTRwt diagnosis was made during cardiac clearance for lumbar surgery. Neuroimaging documented age-related cerebral atrophy and chronic microvascular ischemic disease. Results Inspection of data revealed an amnestic presentation, with impaired confrontational naming, spatial judgment, and processing speed performances. She denied depressogenic and anxiolytic symptoms while family endorsed symptoms of apathy and executive dysfunction. Conclusions The relationship between cardiac and nervous systems has garnished increasing attention in the literature and in clinical planning. Overall, the relationship between cardiac TTR amyloid and brain amyloid beta deposition in humans is unclear. Neuropsychologists should be aware of this heart condition that is increasingly recognized in older adults and the intriguing findings implicated around the cardiac-brain axis.

Blockade of endothelin receptors mitigates SARS-CoV-2-induced osteoarthritis.

Joint pain and osteoarthritis can occur as coronavirus disease 2019 (COVID-19) sequelae after infection. However, little is known about the damage to articular cartilage. Here we illustrate knee joint damage after wild-type, Delta and Omicron variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in vivo. Rapid joint injury with cystic lesions at the osteochondral junction was observed in two patients with post-COVID osteoarthritis and recapitulated in a golden Syrian hamster model. SARS-CoV-2-activated endothelin-1 signalling increased vascular permeability and caused viral spike proteins leakage into the subchondral bone. Osteoclast activation, chondrocyte dropout and cyst formation were confirmed histologically. The US Food and Drug Administration-approved endothelin receptor antagonist, macitentan, mitigated cystic lesions and preserved chondrocyte number in the acute phase of viral infection in hamsters. Delayed macitentan treatment at post-acute infection phase alleviated chondrocyte senescence and restored subchondral bone loss. It is worth noting that it could also attenuate viral spike-induced joint pain. Our work suggests endothelin receptor blockade as a novel therapeutic strategy for post-COVID arthritis.

Exploring SCN5A variants associated with atrial fibrillation in atrial cardiomyocytes derived from human induced pluripotent stem cells: A characterization study

BackgroundAtrial fibrillation (AF) poses a major risk for heart failure, myocardial infarction, and stroke. Several studies have linked SCN5A variants to AF, but their precise mechanistic contribution remains unclear. Human induced pluripotent stem cells (hiPSCs) provide a promising platform for modeling AF-linked SCN5A variants and their functional alterations. ObjectiveThe purpose of this study was to assess the electrophysiological impact of 3 AF-linked SCN5A variants (K1493R, M1875T, N1986K) identified in 3 unrelated individuals. MethodsCRISPR-Cas9 was used to generate a new hiPSC line in which NaV1.5 was knocked out. Following differentiation into specific atrial cardiomyocyte by using retinoic acid, the adult wild-type (WT) and 3 AF variants were introduced into the NaV1.5 knockout (KO) line through transfection. Subsequent analysis including molecular biology, optical mapping, and electrophysiology were performed. ResultsThe absence of NaV1.5 channels altered the expression of key cardiac genes. NaV1.5 KO atrial-like cardiomyocytes derived from human induced pluripotent stem cells displayed slower conduction velocities, altered action potential (AP) parameters, and impaired calcium transient propagation. The transfection of the WT channel restored sodium current density, AP characteristics and the expression of several cardiac genes. Among the AF variants, 1 induced a loss of function (N1986K) while the other 2 induced a gain of function in NaV1.5 channel activity. Cellular excitability alterations and early afterdepolarizations were observed in AF variants. ConclusionOur findings suggest that distinct alterations in NaV1.5 channel properties may trigger altered atrial excitability and arrhythmogenic activity in AF. Our KO model offers an innovative approach for investigating SCN5A variants in an adult human cardiac environment.

Left Atrial Strain in Omicron-Type COVID-19 Patients

BackgroundInformation about left atrial (LA) 2-dimensional (2D) strain parameters in patients with the Omicron variant of COVID-19 is limited. The aim of this study is to evaluate LA strain (LAS) in COVID-19 patients with the Omicron variant and compare it to that of propensity-matched patients with the wild-type (WT) variant. MethodsA total of 148 consecutive patients who were hospitalized with Omicron COVID-19 underwent an echocardiographic evaluation within the first day after hospital admission and were compared to propensity-matched patients (1:1) with the WT variant. LA 2D speckle tracking echocardiography parameters included the following: LAS, reservoir (LASr); LAS, conduit (LAScd); LAS, contraction (LASct); and LASr to E/e' ratio. The values for the parameters that occurred during acute Omicron-type infection were compared with those found on historic examinations in 36 patients. ResultsCompared to the matched WT cohort, patients with acute Omicron-type infection had similar LASr (31.3% ± 13.3% vs 33.0% ± 14.2%), LAScd (–18.7% ± 9.8% vs –18.6% ± 10.8%), and LASct (–12.5% ± 8.6% vs –13.6% ± 8.2%) values (P > 0.2 for all), but a higher E/e' ratio (11.8 ± 6 vs 10.1 ± 7; P = 0.03). Surprisingly, LASr (31.9% ± 13.7% vs 22.6% ± 13.9%; P = 0.04) and LAScd (–18.7% ± 9.7% vs –10.7% ± 6.6%; P < 0.001) improved in patients during acute infection. LASr, LAScd, and LASr/(E/e') were significantly associated with an increased risk of either in-hospital mortality, need for mechanical ventilation, or the combined event. ConclusionsIn hospitalized patients with an Omicron COVID-19 infection, LAS parameters are similar to those of matched patients with WT variant and are associated with mortality and respiratory deterioration. These abnormalities were recorded previously in the 36 patients with historical echocardiograms, suggesting that they are related to background cardiac disease.

Variant-specific antibody profiling for tracking SARS-CoV-2 variant infections in children and adolescents.

Monitoring the seroprevalence of SARS-CoV-2 in children and adolescents can provide valuable information for effective SARS-CoV-2 surveillance, and thus guide vaccination strategies. In this study, we quantified antibodies against the spike S1 domains of several SARS-CoV-2 variants (wild-type, Alpha, Delta, and Omicron variants) as well as endemic human coronaviruses (HCoVs) in 1,309 children and adolescents screened between December 2020 and March 2023. Their antibody binding profiles were compared with those of 22 pre-pandemic samples from children and adolescents using an in-house Luminex®-based Corona Array (CA). The primary objectives of this study were to (i) monitor SARS-CoV-2-specific antibodies in children and adolescents, (ii) evaluate whether the S1-specific antibody response can identify the infecting variant of concern (VoC), (iii) estimate the prevalence of silent infections, and (iv) test whether vaccination or infection with SARS-CoV-2 induce HCoV cross-reactive antibodies. Both SARS-CoV-2 infection and vaccination induced a robust antibody response against the S1 domain of WT and VoCs in children and adolescents. Antibodies specific for the S1 domain were able to distinguish between SARS-CoV-2 VoCs in infected children. The serologically identified VoC was typically the predominant VoC at the time of infection. Furthermore, our highly sensitive CA identified more silent SARS-CoV-2 infections than a commercial ELISA (12.1% vs. 6.3%, respectively), and provided insights into the infecting VoC. Seroconversion to endemic HCoVs occurred in early childhood, and vaccination or infection with SARS-CoV-2 did not induce HCoV S1 cross-reactive antibodies. In conclusion, the antibody response to the S1 domain of the spike protein of SARS-CoV-2 is highly specific, providing information about the infecting VoC and revealing clinically silent infections.

Nuclear Localization of Human SOD1 in Motor Neurons in Mouse Model and Patient Amyotrophic Lateral Sclerosis: Possible Links to Cholinergic Phenotype, NADPH Oxidase, Oxidative Stress, and DNA Damage.

Amyotrophic lateral sclerosis (ALS) is a fatal disease that causes degeneration of motor neurons (MNs) and paralysis. ALS can be caused by mutations in the gene that encodes copper/zinc superoxide dismutase (SOD1). SOD1 is known mostly as a cytosolic antioxidant protein, but SOD1 is also in the nucleus of non-transgenic (tg) and human SOD1 (hSOD1) tg mouse MNs. SOD1's nuclear presence in different cell types and subnuclear compartmentations are unknown, as are the nuclear functions of SOD1. We examined hSOD1 nuclear localization and DNA damage in tg mice expressing mutated and wildtype variants of hSOD1 (hSOD1-G93A and hSOD1-wildtype). We also studied ALS patient-derived induced pluripotent stem (iPS) cells to determine the nuclear presence of SOD1 in undifferentiated and differentiated MNs. In hSOD1-G93A and hSOD1-wildtype tg mice, choline acetyltransferase (ChAT)-positive MNs had nuclear hSOD1, but while hSOD1-wildtype mouse MNs also had nuclear ChAT, hSOD1-G93A mouse MNs showed symptom-related loss of nuclear ChAT. The interneurons had preserved parvalbumin nuclear positivity in hSOD1-G93A mice. hSOD1-G93A was seen less commonly in spinal cord astrocytes and, notably, oligodendrocytes, but as the disease emerged, the oligodendrocytes had increased mutant hSOD1 nuclear presence. Brain and spinal cord subcellular fractionation identified mutant hSOD1 in soluble nuclear extracts of the brain and spinal cord, but mutant hSOD1 was concentrated in the chromatin nuclear extract only in the spinal cord. Nuclear extracts from mutant hSOD1 tg mouse spinal cords had altered protein nitration, footprinting peroxynitrite presence, and the intact nuclear extracts had strongly increased superoxide production as well as the active NADPH oxidase marker, p47phox. The comet assay showed that MNs from hSOD1-G93A mice progressively (6-14 weeks of age) accumulated DNA single-strand breaks. Ablation of the NCF1 gene, encoding p47phox, and pharmacological inhibition of NADPH oxidase with systemic treatment of apocynin (10 mg/kg, ip) extended the mean lifespan of hSOD1-G93A mice by about 25% and mitigated genomic DNA damage progression. In human postmortem CNS, SOD1 was found in the nucleus of neurons and glia; nuclear SOD1 was increased in degenerating neurons in ALS cases and formed inclusions. Human iPS cells had nuclear SOD1 during directed differentiation to MNs, but mutant SOD1-expressing cells failed to establish wildtype MN nuclear SOD1 levels. We conclude that SOD1 has a prominent nuclear presence in the central nervous system, perhaps adopting aberrant contexts to participate in ALS pathobiology.

Evaluation of the binding interactions between Plasmodium falciparum Kelch-13 mutant recombinant proteins with artemisinin.

Malaria, an ancient mosquito-borne illness caused by Plasmodium parasites, is mostly treated with Artemisinin Combination Therapy (ACT). However, Single Nucleotide Polymorphisms (SNPs) mutations in the P. falciparum Kelch 13 (PfK13) protein have been associated with artemisinin resistance (ART-R). Therefore, this study aims to generate PfK13 recombinant proteins incorporating of two specific SNPs mutations, PfK13-V494I and PfK13-N537I, and subsequently analyze their binding interactions with artemisinin (ART). The recombinant proteins of PfK13 mutations and the Wild Type (WT) variant were expressed utilizing a standard protein expression protocol with modifications and subsequently purified via IMAC and confirmed with SDS-PAGE analysis and Orbitrap tandem mass spectrometry. The binding interactions between PfK13-V494I and PfK13-N537I propeller domain proteins ART were assessed through Isothermal Titration Calorimetry (ITC) and subsequently validated using fluorescence spectrometry. The protein concentrations obtained were 0.3 mg/ml for PfK13-WT, 0.18 mg/ml for PfK13-V494I, and 0.28 mg/ml for PfK13-N537I. Results obtained for binding interaction revealed an increased fluorescence intensity in the mutants PfK13-N537I (83 a.u.) and PfK13-V494I (143 a.u.) compared to PfK13-WT (33 a.u.), indicating increased exposure of surface proteins because of the looser binding between PfK13 protein mutants with ART. This shows that the PfK13 mutations may induce alterations in the binding interaction with ART, potentially leading to reduced effectiveness of ART and ultimately contributing to ART-R. However, this study only elucidated one facet of the contributing factors that could serve as potential indicators for ART-R and further investigation should be pursued in the future to comprehensively explore this complex mechanism of ART-R.