Characterization of erenumab and rimegepant on calcitonin gene-related peptide induced responses in Xenopus Laevis oocytes expressing the calcitonin gene-related peptide receptor and the amylin-1 receptor

BackgroundThe clinical use of calcitonin gene-related peptide receptor (CGRP-R) antagonists and monoclonal antibodies against CGRP and CGRP-R has offered new treatment possibilities for migraine patients. CGRP activates both the CGRP-R and structurally related amylin 1 receptor (AMY₁-R). The relative effect of erenumab and the small-molecule CGRP-R antagonist, rimegepant, towards the CGRP-R and AMY-R needs to be further characterized.MethodsThe effect of CGRP and two CGRP-R antagonists were examined in Xenopus laevis oocytes expressing human CGRP-R, human AMY₁-R and their subunits.ResultsCGRP administered to receptor expressing oocytes induced a concentration-dependent increase in current with the order of potency CGRP-R> > AMY₁-R > calcitonin receptor (CTR). There was no effect on single components of the CGRP-R; calcitonin receptor-like receptor and receptor activity-modifying protein 1. Amylin was only effective on AMY₁-R and CTR. Inhibition potencies (pIC₅₀ values) for erenumab on CGRP induced currents were 10.86 and 9.35 for CGRP-R and AMY₁-R, respectively. Rimegepant inhibited CGRP induced currents with pIC₅₀ values of 11.30 and 9.91 for CGRP-R and AMY₁-R, respectively.ConclusionOur results demonstrate that erenumab and rimegepant are potent antagonists of CGRP-R and AMY₁-R with 32- and 25-times preference for the CGRP-R over the AMY₁-R, respectively. It is discussed if this difference in affinity between the two receptors is the likely reason why constipation is a common and serious adverse effect during CGRP-R antagonism but less so with CGRP binding antibodies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s10194-022-01425-9.     

Catechins as a Potential Dietary Supplementation in Prevention of Comorbidities Linked with Down Syndrome

Plant-derived polyphenols flavonoids are increasingly being recognized for their medicinal potential. These bioactive compounds derived from plants are gaining more interest in ameliorating adverse health risks because of their low toxicity and few side effects. Among them, therapeutic approaches demonstrated the efficacy of catechins, a major group of flavonoids, in reverting several aspects of Down syndrome, the most common genomic disorder that causes intellectual disability. Down syndrome is characterized by increased incidence of developing Alzheimer’s disease, obesity, and subsequent metabolic disorders. In this focused review, we examine the main effects of catechins on comorbidities linked with Down syndrome. We also provide evidence of catechin effects on DYRK1A, a dosage-sensitive gene encoding a protein kinase involved in brain defects and metabolic disease associated with Down syndrome.     

Evaluation of the Immunogenicity in Mice Orally Immunized with Recombinant Lactobacillus casei Expressing Porcine Epidemic Diarrhea Virus S1 Protein

Porcine epidemic diarrhea (PED), characterized by diarrhea, vomiting, and dehydration, is an acute enteric infectious disease of pigs. The disease is caused by porcine epidemic diarrhea virus (PEDV), which infects the intestinal mucosal surface. Therefore, mucosal immunization through the oral route is an effective method of immunization. Lactic acid bacteria, which are acid resistant and bile-salt resistant and improve mucosal immunity, are ideal carriers for oral vaccines. The S1 glycoprotein of PEDV mediates binding of the virus with cell receptors and induces neutralizing antibodies against the virus. Therefore, we reversely screened the recombinant strain pPG-SD-S1/Δupp ATCC 393 expressing PEDV S1 glycoprotein by Lactobacillus casei deficient in upp genotype (Δupp ATCC 393). Mice were orally immunized three times with the recombinant bacteria that had been identified for expression, and the changes of anti-PEDV IgG and secreted immunoglobulin A levels were observed over 70 days. The results indicated that the antibody levels notably increased after oral administration of recombinant bacteria. The detection of extracellular cytokines on the 42nd day after immunization indicated high levels of humoral and cellular immune responses in mice. The above results demonstrate that pPG-SD-S1/Δupp ATCC 393 has great potential as an oral vaccine against PEDV.     

A Unique Robust Dual-Promoter-Driven and Dual-Reporter-Expressing SARS-CoV-2 Replicon: Construction and Characterization

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2, SARS2) remains a great global health threat and demands identification of more effective and SARS2-targeted antiviral drugs, even with successful development of anti-SARS2 vaccines. Viral replicons have proven to be a rapid, safe, and readily scalable platform for high-throughput screening, identification, and evaluation of antiviral drugs against positive-stranded RNA viruses. In the study, we report a unique robust HIV long terminal repeat (LTR)/T7 dual-promoter-driven and dual-reporter firefly luciferase (fLuc) and green fluorescent protein (GFP)-expressing SARS2 replicon. The genomic organization of the replicon was designed with quite a few features that were to ensure the replication fidelity of the replicon, to maximize the expression of the full-length replicon, and to offer the monitoring flexibility of the replicon replication. We showed the success of the construction of the replicon and expression of reporter genes fLuc and GFP and SARS structural N from the replicon DNA or the RNA that was in vitro transcribed from the replicon DNA. We also showed detection of the negative-stranded genomic RNA (gRNA) and subgenomic RNA (sgRNA) intermediates, a hallmark of replication of positive-stranded RNA viruses from the replicon. Lastly, we showed that expression of the reporter genes, N gene, gRNA, and sgRNA from the replicon was sensitive to inhibition by Remdesivir. Taken together, our results support use of the replicon for identification of anti-SARS2 drugs and development of new anti-SARS strategies targeted at the step of virus replication.     

Understanding the Driving Forces That Trigger Mutations in SARS-CoV-2: Mutational Energetics and the Role of Arginine Blockers in COVID-19 Therapy

SARS-CoV-2 is a global challenge due to its ability to mutate into variants that spread more rapidly than the wild-type virus. Because the molecular biology of this virus has been studied in such great detail, it represents an archetypal paradigm for research into new antiviral drug therapies. The rapid evolution of SARS-CoV-2 in the human population is driven, in part, by mutations in the receptor-binding domain (RBD) of the spike (S-) protein, some of which enable tighter binding to angiotensin-converting enzyme (ACE2). More stable RBD-ACE2 association is coupled with accelerated hydrolysis of furin and 3CLpro cleavage sites that augment infection. Non-RBD and non-interfacial mutations assist the S-protein in adopting thermodynamically favorable conformations for stronger binding. The driving forces of key mutations for Alpha, Beta, Gamma, Delta, Kappa, Lambda and Omicron variants, which stabilize the RBD-ACE2 complex, are investigated by free-energy computational approaches, as well as equilibrium and steered molecular dynamic simulations. Considered also are the structural hydropathy traits of the residues in the interface between SARS-CoV-2 RBD and ACE2 protein. Salt bridges and π-π interactions are critical forces that create stronger complexes between the RBD and ACE2. The trend of mutations is the replacement of non-polar hydrophobic interactions with polar hydrophilic interactions, which enhance binding of RBD with ACE2. However, this is not always the case, as conformational landscapes also contribute to a stronger binding. Arginine, the most polar and hydrophilic among the natural amino acids, is the most aggressive mutant amino acid for stronger binding. Arginine blockers, such as traditional sartans that bear anionic tetrazoles and carboxylates, may be ideal candidate drugs for retarding viral infection by weakening S-protein RBD binding to ACE2 and discouraging hydrolysis of cleavage sites. Based on our computational results it is suggested that a new generation of “supersartans”, called “bisartans”, bearing two anionic biphenyl-tetrazole pharmacophores, are superior to carboxylates in terms of their interactions with viral targets, suggesting their potential as drugs in the treatment of COVID-19. In Brief: This in silico study reviews our understanding of molecular driving forces that trigger mutations in the SARS-CoV-2 virus. It also reports further studies on a new class of “supersartans” referred to herein as “bisartans”, bearing two anionic biphenyltetrazole moieties that show potential in models for blocking critical amino acids of mutants, such as arginine, in the Delta variant. Bisartans may also act at other targets essential for viral infection and replication (i.e., ACE2, furin cleavage site and 3CLpro), rendering them potential new drugs for additional experimentation and translation to human clinical trials.     

Quercetin improves contrast-induced acute kidney injury through the HIF-1α/lncRNA NEAT1/HMGB1 pathway

ContextThe risk of contrast-induced acute kidney injury (CI-AKI) is increasing and the harm is great. Quercetin is the main active component in Abelmoschus manihot (L.) Medik (Malvaceae) and was reported to reduce the expression of HIF-1α.ObjectiveWe investigate whether quercetin improves the CI-AKI through the HIF-1α/lncRNA NEAT1/HMGB1 pathway.Materials and methodsHK-2 cells were treated with iohexol (200 mg/mL) for 6 h to establish a CI-AKI model. Quercetin (20 μM) was administered to CI-AKI cells cultured in dishes for 24 h. Cell morphology was observed by a fluorescence microscope. MTT and TUNEL assays were used to detect cell survival rate and apoptosis. Relative mRNA levels were measured by qRT-PCR. Protein levels were detected using western blotting. IL-6 and TNF-α protein levels were tested by Elisa assay. Targeting binding sites of HIF-1α and lncRNA NEAT1 were detected by luciferase assay.ResultsThe IC₅₀ value of quercetin was 163.25 μM. The expression levels of HIF-1α, lncRNA NEAT1 and HMGB1 were upregulated in the CI-AKI cell model. Quercetin diminished cell injury and apoptosis via inhibiting HIF-1α. Silencing of HIF-1α targeting lncRNA MEAT1 diminished cell injury and apoptosis. Silencing lncRNA NEAT1 has the same effect via suppressing HMGB1 expression. Collectively, quercetin diminished cell injury and apoptosis in CI-AKI cell model via the inhibition of HIF-1α on lncRNA NEAT1/HMGB1 signalling pathway.Discussion and conclusionsQuercetin diminished cell injury and apoptosis in CI-AKI cell mode via the inhibition of HIF-1α on the lncRNA NEAT1/HMGB1 signalling pathway, offering a potential novel therapeutic target for CI-AKI therapy.     

New‐onset fulminant type 1 diabetes after severe acute respiratory syndrome coronavirus 2 vaccination: A case report

Fulminant type 1 diabetes is characterized by a rapid progression of insulin deficiency triggered by viral infection. Here, we report a case of a 45‐year‐old Japanese woman with fulminant type 1 diabetes that developed 8 days after receiving messenger ribonucleic acid vaccine against severe acute respiratory syndrome coronavirus 2. She had been healthy and had no symptoms suggestive of viral infection before the vaccination. Laboratory tests showed exhaustion of insulin secretion and negative results for islet autoantibodies. Human leukocyte antigen genotype analysis showed the DRB1*04:05 and DQB1*04:01 alleles. This is the first case report of new‐onset fulminant type 1 diabetes after severe acute respiratory syndrome coronavirus 2 vaccination, and suggests that a severe acute respiratory syndrome coronavirus 2 vaccine might trigger the onset of fulminant type 1 diabetes in susceptible individuals. However, a causal relationship remains to be identified, and further studies are required to determine the incidence of such cases.     

维药胡桐泪的多糖含量测定及其糖组成分析

目的:测定市场上实际流通的商品胡桐泪药材中多糖的含量并对其单糖组成进行分析,以多糖为指标评价药材质量。方法:采用苯酚-硫酸法对总多糖进行含量测定;应用核磁共振氢谱(1H-NMR)分析单糖组成;运用高效凝胶渗透色谱(HPGPC)测定总多糖的分子量分布。结果:以葡萄糖计,4批新式胡桐泪总多糖含量平均值为(7.36±1.74)%,6批老式胡桐泪总多糖含量平均值为(1.53±0.99)%;新式与老式胡桐泪总多糖的单糖组成相似,主要由阿拉伯糖和半乳糖组成,还含有木糖、甘露糖、鼠李糖等,部分样品尚含有较高含量的葡萄糖;新旧类型样品总多糖在分子量分布上存在较大差异。结论:新式胡桐泪的总多糖含量平均值较老式高,以多糖含量为评价指标时,新式样品总体质量较老式更好;应用核磁共振氢谱可以快速、准确地鉴定单糖组成。     

Circulating tumor DNA predicts efficacy of a dual AKT/p70S6K inhibitor (LY2780301) plus paclitaxel in metastatic breast cancer: plasma analysis of the TAKTIC phase IB/II study

The phosphatidylinositol‐3‐kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway is frequently activated in HER2‐negative breast cancer and may play a role in taxane resistance. The phase IB/II TAKTIC trial ({"type":"clinical-trial","attrs":{"text":"NCT01980277","term_id":"NCT01980277"}}NCT01980277) has shown that combining a dual AKT and p70 ribosomal protein S6 kinase (p70S6K) inhibitor (LY2780301) taken orally with weekly paclitaxel in HER2‐negative advanced breast cancer is feasible, with preliminary evidence of efficacy. We wanted to explore whether circulating tumor DNA (ctDNA) may be a surrogate marker of treatment efficacy in this setting. Serial plasma samples were collected and cell‐free DNA was sequenced using low‐coverage whole‐genome sequencing, and analysis was completed with droplet digital polymerase chain reaction (PCR) for some patients with driver mutations. Baseline tumor fraction (TF) and TF after 7 weeks on treatment were compared to progression‐free survival (PFS) and the overall response rate. We also explored circulating copy number alterations associated with treatment failure. Of the 51 patients enrolled in the TAKTIC trial, at least one plasma sample was available for 44 cases (96 timepoints). All patients with tumor TP53, PI3KCA, or AKT1 mutations harbored at least one of these alterations in plasma. TF at inclusion was correlated with PFS (6m‐PFS was 92% for ctDNAneg patients vs 68% for ctDNApos cases; hazard ratio [HR] = 3.45, 95% confidence interval [CI] [1.34–8.90], P = 0.007). ctDNA status at week 7 was not correlated with prognosis. Even though most circulating copy number alterations were conserved at disease progression, some genomic regions of interest were altered in post‐progression samples. In conclusion, ctDNA detection at baseline was associated with shorter PFS in patients included in the TAKTIC trial. Plasma‐based copy number analysis may help to identify alterations involved in resistance to treatment.