Search Results (181)

Recurrent pregnancy loss (RPL) is defined as the occurrence of two or more pregnancy losses before 20 weeks of gestation. RPL is a common medical condition among reproductive-age women, with approximately 23 million cases reported annually worldwide. Up to 5% of pregnant women may experience two or more consecutive pregnancy losses. Previous studies have investigated risk factors for RPL, including maternal age, uterine pathology, genetic anomalies, infectious agents, endocrine disorders, thrombophilia, and immune dysfunction. However, RPL is a disease caused by a complex interaction of genetic factors, environmental factors (e.g., diet, lifestyle, and stress), epigenetic factors, and the immune system. In addition, due to the lack of research on genetics research related to RPL, the etiology remains unclear in up to 50% of cases. Platelets play a critical role in pregnancy maintenance. This study examined the associations of platelet receptor and ligand gene variants, including integrin subunit beta 3 (ITGB3) rs2317676 A > G, rs3809865 A > T; fibrinogen gamma chain (FGG) rs1049636 T > C, rs2066865 T > C; glycoprotein 1b subunit alpha (GP1BA) rs2243093 T > C, rs6065 C > T; platelet endothelial cell adhesion molecule 1 (PECAM1) rs2812 C > T; and platelet endothelial aggregation receptor 1 (PEAR1) rs822442 C > A, rs12137505 G > A, with RPL prevalence. In total, 389 RPL patients and 375 healthy controls (all Korean women) were enrolled. Genotyping of each single nucleotide polymorphism was performed using polymerase chain reaction–restriction fragment length polymorphism and the TaqMan genotyping assay. All samples were collected with approval from the Institutional Review Board at Bundang CHA Medical Center. The ITGB3 rs3809865 A > T genotype was strongly associated with RPL prevalence (pregnancy loss [PL] ≥ 2: adjusted odds ratio [AOR] = 2.505, 95% confidence interval [CI] = 1.262–4.969, p = 0.009; PL ≥ 3: AOR = 3.255, 95% CI = 1.551–6.830, p = 0.002; PL ≥ 4: AOR = 3.613, 95% CI = 1.403–9.307, p = 0.008). The FGG rs1049636 T > C polymorphism was associated with a decreased risk in women who had three or more pregnancy losses (PL ≥ 3: AOR = 0.673, 95% CI = 0.460–0.987, p = 0.043; PL ≥ 4: AOR = 0.556, 95% CI = 0.310–0.997, p = 0.049). These findings indicate significant associations of the ITGB3 rs3809865 A > T and FGG rs1049636 T > C polymorphisms with RPL, suggesting that platelet function influences RPL in Korean women. Full article

Background/Objectives: An initial COVID-19 candidate vaccine containing a purified ancestral SARS-CoV-2 spike antigen was characterized with an ELISA using recombinant monoclonal antibodies (mAbs) generated against this variant. Upon the emergence of a new Beta (B.1.351) spike variant early in the pandemic, the assessment of a bivalent vaccine containing ancestral and Beta spike antigens began. Due to accelerated project timelines, mAbs generated specifically against the Beta spike antigen were not available at the time to address assay development and vaccine testing requirements. Methods: Using only the initial mAb panel raised against the ancestral spike antigen, an epitope-blocking ELISA strategy was developed to independently measure Beta spike antigen in bivalent vaccine formulations. To facilitate this, epitope profiling of spike antigens from both ancestral and Beta variants was performed with biolayer interferometry and hydrogen–deuterium exchange mass spectrometry using the original panel of mAbs. Results: The resulting blocking ELISA was precise and specific for the Beta spike antigen and detected the expected amount of this antigen in bivalent vaccine formulations. The specific amount of ancestral spike protein in the bivalent vaccine was also confirmed using the original ELISA developed at the onset of the pandemic. Conclusions: This epitope-blocking strategy helped to overcome key reagent availability issues and could be applied to other projects involving related proteins. Full article

Background/Objectives: For viral entry into host cells, the spike (S) protein of coronavirus (CoV) uses its S1 domain to bind to the host receptor and S2 domain to mediate the fusion between virion and cellular membranes. The S1 domain acquired multiple mutations as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolved to give rise to Variant of Concerns (VOCs) but the S2 domain has limited changes. In particular, the stem helix in S2 did not change significantly and it is fairly well-conserved across multiple beta-CoVs. In this study, we generated a murine mAb 7B2 binding to the stem helix of SARS-CoV-2. Methods: MAb 7B2 was isolated from immunized mouse and its neutralization activity was evaluated using microneutralization, plaque reduction and cell–cell fusion assays. Bio-layer interferometry was used to measure binding affinity and AlphaFold3 was used to model the antibody–antigen interface. Results: MAb 7B2 has lower virus neutralizing and membrane block activities when compared to a previously reported stem helix-binding human mAb S2P6. Alanine scanning and AlphaFold3 modeling reveals that residues K1149 and D1153 in S form a network of polar interactions with the heavy chain of 7B2. Conversely, S2P6 binding to S is not affected by alanine substitution at K1149 and D1153 as indicated by the high ipTM scores in the predicted S2P6-stem helix structure. Conclusions: Our detailed characterization of the mechanism of inhibition of 7B2 reveals its distinctive binding model from S2P6 and yields insights on multiple neutralizing and highly conserved epitopes in the S2 domain which could be key components for pan-CoV vaccine development. Full article

Background/Objectives: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus Disease 2019 (COVID-19), has rapidly evolved, giving rise to multiple Variants of Concern—including Alpha, Beta, Gamma, Delta, and Omicron—which emerged independently across different regions. Licensed COVID-19 vaccines primarily target the highly mutable spike protein, resulting in reduced efficacy due to immune escape by emerging variants. Previously, we developed a live attenuated Francisella tularensis LVS ΔcapB single-vector platform COVID-19 vaccine, rLVS ΔcapB/MN, expressing the conserved membrane (M) and nucleocapsid (N) proteins from the early SARS-CoV-2 WA-01/2020 strain. In this study, we evaluate the efficacy of rLVS ΔcapB/MN and an enhanced version, rLVS ΔcapB::RdRp/MN, which additionally expresses the conserved RNA-dependent RNA polymerase (RdRp) protein from the same strain, in a hamster model. Methods: Both vaccine candidates were administered orally or intranasally to golden Syrian hamsters (equal numbers of males and females) and evaluated against intranasal challenge with SARS-CoV-2 Delta (B.1.617.2-AY.1) and Omicron (BA.5) variants. Results: Vaccinated animals developed robust, TH1-biased IgG responses specific to the nucleocapsid protein. Following SARS-CoV-2 challenge, immunized hamsters exhibited reduced weight loss, lower oropharyngeal and lung viral titers, and improved lung pathology scores compared with unvaccinated controls. Conclusion: These findings support the potential of this universal vaccine to provide broad protection against current and future SARS-CoV-2 variants, with minimal need for updating. Full article

The existence of s-DAPK-1, an alternatively spliced variant of DAPK-1, adds complexity to our understanding of the proteins involved in the regulation of cell survival, apoptosis, and autophagy. DAPK-1 has been implicated in the regulation of these processes; however, it remains unclear whether s-DAPK-1 also plays a similar role or a separate function; thus, determining its involvement in these processes is challenging due to the limited understanding of its regulation, interacting partners, function, and three-dimensional (3D) structure. Hence, this study was aimed at (1) understanding the regulation of s-DAPK-1 by predicting its microRNA targets, (2) predicting the 3D structure of s-DAPK-1, (3) its physicochemical and thermodynamic properties, (4) its interacting partners, and (5) molecular functions using computational methods. To achieve this aim, various bioinformatics tools and in silico webservers, such as ProteinPrompt, ProtParam, ProtScale, ScooP, Hawkdock, Phyre2, I-TASSER, PSIPRED, SAVES, and PROCHECK, along with user-friendly databases, such as NCBI, TarBase, and Protein Data Bank (PDB), were employed. For miRNA prediction, we used TarBase, and identified the specific microRNAs targeting s-DAPK-1. Furthermore, the Phyre2 database demonstrated that s-DAPK-1 possesses 40% alpha helices and 4% beta strands, forming a stable 3D structure. Additionally, s-DAPK-1 demonstrated stability to withstand high temperatures, suggesting that it is a thermostable protein. Moreover, s-DAPK-1 was found to interact with a variety of proteins involved in tumor progression and gene regulation, including a prion protein and histone H2B type 2-E (H2B2E). This suggests that s-DAPK-1 may perform diverse molecular functions such as regulation of metabolic processes, nucleic acid binding, and mRNA splicing by interacting with different proteins. Full article

Flagellates of the genus Giardia are intestinal parasites with a broad host range. Several Giardia duodenalis variants (assemblages) recently elevated to species rank—G. duodenalis (assemblage A1), G. intestinalis (A2) and Giardia enterica (B) are human pathogens. Giardia enterica has been reported in some hystricomorph rodents such as wild crested porcupines (Hystrix cristata), but no data were previously available from Brazilian porcupines (Coendou prehensilis) and naked mole rats (Heterocephalus glaber). The aim of this study is to genetically identify the Giardia isolates from these three rodent species, all housed in a zoological institution. Fecal samples were processed using the Bailenger concentration method, and DNA was extracted from the sediments using commercial kits. Partial PCR amplification and sequencing of the glutamate dehydrogenase, beta-giardin, and triose-phosphate isomerase genes revealed that all isolates belonged to G. enterica, showing 99–100% identity with sequences available in GenBank. Prevalences could not be reliably estimated due to small group sizes and the resulting proportions may be biased. To our knowledge, this is the first report identifying Giardia (G. enterica) in C. prehensilis and H. glaber, thus expanding the known host range of this parasite species and reinforcing the importance of surveillance in captive wild hosts. Full article

(1) Background: The currently circulating variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exhibits resistance to antibodies induced by vaccines. The World Health Organization recommended the use of monovalent XBB.1 sublineages (e.g., XBB.1.5) as an antigenic component in 2023. (2) Objective: In this study, we aimed to develop vaccines based on the XBB.1.5 receptor-binding domain (RBD) to combat the recently emerged SARS-CoV-2 XBB and JN.1 variants, as well as previously circulating variants. (3) Methods: Glycoengineered Pichia pastoris was utilized to produce a recombinant XBB.1.5 RBD protein with mammalian-like and fucose-free N-glycosylation. The XBB.1.5 RBD was mixed with Al(OH)₃:CpG adjuvants to prepare monovalent vaccines. Thereafter, the XBB.1.5 RBD was mixed with the Beta (B.1.351), Delta (B.1.617.2), or Omicron (BA.2) RBDs (1:1 ratio), along with Al(OH)₃:CpG, to prepare bivalent vaccines. BALB/c mice were immunized with the monovalent and bivalent vaccines. Neutralizing antibody titers were assessed via pseudovirus and authentic virus assays; humoral immune responses were analyzed by RBD-binding IgG subtypes. (4) Results: The monovalent vaccine induced higher neutralizing antibody titers against Delta, BA.2, XBB.1.5, and JN.1 compared to those in mice immunized solely with Al(OH)₃:CpG, as demonstrated by pseudovirus virus assays. The XBB.1.5/Delta RBD and XBB.1.5/Beta RBD-based bivalent vaccines provided potent protection against the BA.2, XBB.1.5, JN.1, and KP.2 variants, as well as the previously circulating Delta and Beta variants. All monovalent and bivalent vaccines induced high levels of RBD-binding IgG (IgG1, IgG2a, IgG2b, and IgG3) antibodies in mice, suggesting that they elicited robust humoral immune responses. The serum samples from mice immunized with the XBB.1.5 RBD-based and XBB.1.5/Delta RBD-based vaccines could neutralize the authentic XBB.1.16 virus. (5) Conclusions: The XBB.1.5/Beta and XBB.1.5/Delta RBD-based bivalent vaccines are considered as potential candidates for broad-spectrum vaccines against SARS-CoV-2 variants. Full article

Mutation is one of the most important drivers of viral evolution and genome variability, allowing viruses to potentially evade host immune responses and develop drug resistance. In the context of COVID-19, local genomic surveillance of circulating virus populations is therefore critical. The goals of this study were to describe the distribution of different SARS-CoV-2 lineages, assess their genomic differences, and infer virus importation events in Bangladesh. We individually aligned 1965 SARS-CoV-2 genome sequences obtained between April 2020 and June 2021 to the Wuhan-1 sequence and used the resulting multiple sequence alignment as input to infer a maximum likelihood phylogenetic tree. Sequences were assigned to lineages as described by the hierarchical Pangolin nomenclature scheme. We built a phylogeographic model using the virus population genome sequence variation to infer the number of virus importation events. We observed thirty-four lineages and sub-lineages in Bangladesh, with B.1.1.25 and its sub-lineages D.* (979 sequences) dominating, as well as the Beta variant of concern (VOC) B.1.351 and its sub-lineages B.1.351.* (403 sequences). The earliest B.1.1.25/D.* lineages likely resulted from multiple introductions, some of which led to larger outbreak clusters. There were 570 missense mutations, 426 synonymous mutations, 18 frameshift mutations, 7 deletions, 2 insertions, 10 changes at start/stop codons, and 64 mutations in intergenic or untranslated regions. According to phylogeographic modeling, there were 31 importation events into Bangladesh (95% CI: 27–36). Like elsewhere, Bangladesh has experienced distinct waves of dominant lineages during the COVID-19 pandemic; this study focuses on the emergence and displacement of the first wave-dominated lineage, which contains mutations seen in several VOCs and may have had a transmission advantage over the extant lineages. Full article

The global impact of the COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), persists in part due to the emergence of new variants. Understanding variant-specific infection dynamics and pathogenesis in murine models is crucial for identifying phenotypic changes and guiding the development of countermeasures. To address the limitations of earlier studies that investigated only a few variants or used small sample sizes, we evaluated clinical disease, infection kinetics, viral titers, cellular localization, and histopathologic changes in the lungs and brains of transgenic B6.Cg-Tg(K18-ACE2)2Prlmn/J (“K18”) and corresponding genetic control (C57BL/6J) mice expressing human angiotensin-converting enzyme 2 (hACE2). Six SARS-CoV-2 variants were assessed: B.1 (WA1-like), alpha, beta, delta, omicron, and omicron XBB.1.5, using cohorts of ≥18 mice. Following intranasal inoculation with B.1, alpha, beta, or delta variants, K18 mice experienced rapid weight loss and reached euthanasia criteria by 5–6 days post-inoculation (dpi). In contrast, K18 mice inoculated with both omicron variants recovered to their starting weight within 4–6 dpi. Infectious SARS-CoV-2 was detected in the oropharynx at 1 and2 dpi, in the lungs at 2, 4, and 6 dpi, and in the brain at 4 and 6 dpi for all variants except omicron. SARS-CoV-2 nucleoprotein was detected, and interstitial pneumonia of varying severity was observed in K18 mice infected with all variants. Brain lesions were identified in mice infected with the B.1, beta, and delta variants 6 dpi. As K18 mice express hACE2 in the brain—a feature not present in humans—we also compared infection dynamics of three variants to those of a mouse-adapted WA1 strain in C57BL/6J mice lacking the human ACE2 gene. C57BL/6J mice did not experience lethal disease, exhibited milder pneumonia, and had no evidence of neuroinvasion despite similar infection kinetics to K18 mice. These findings demonstrate contrasting phenotypes across the two models and reduced tropism and pathology of omicron compared to earlier variants in both models. This comprehensive analysis of SARS-CoV-2 variants in two mouse models provides valuable insights for model and variant selection for future studies. Full article

Wastewater surveillance has become a valuable tool to monitor the emergence of SARS-CoV-2 variants of concern (VOCs) at the community level. In this study, we aimed to evaluate the presence of Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1617.2), and Omicron (B.1.1.529) VOCs in samples from the inlet of a wastewater treatment plant (WWTP) as well as from two different sewer interceptors (SI-1 and SI-2) from the urban sewage system in Santiago de Compostela (Galicia, NW of Spain) throughout 2021 and January 2022. For this purpose, detection and quantification of the four VOCs was performed using four duplex SARS-CoV-2 allelic discrimination RT-qPCR assays, targeting the S-gene. An N1 RT-qPCR gene assay was used as a reference for the presence of SARS-CoV-2 RNA in wastewater samples. All VOCs were detected in wastewater samples. Alpha, Beta, Delta, and Omicron VOCs were detected in 45.7%, 7.5%, 66.7%, and 72.7% of all samples, respectively. Alpha VOC was dominant during the first part of the study, whereas Delta and Omicron detection peaks were observed in May–June and December 2021, respectively. Some differences were observed among the results obtained for the two city sectors studied, which could be explained by the differences in the characteristics of the population between them. Wastewater-based epidemiology allowed us to track the early circulation and emergence of SARS-CoV-2 variants at a local level, and our results are temporally concordant with clinical data and epidemiological findings reported by the health authorities. Full article

SARS-CoV-2 virus and its variants remain a global health threat, due to their capacity for rapid evolution. Variants throughout the COVID-19 pandemic exhibited variations in virulence, impacting vaccine protection and disease severity. Investigating nonstructural protein variants is critical to understanding viral evolution and manipulation of host protein interactions. We focus on nonstructural protein 3 (nsp3), with multiple domains with different activities, including viral polyprotein cleavage, host deubiquitylation, de-ISGylation, and double-membrane vesicle formation. Using affinity purification–mass spectrometry (AP-MS), we identify differential protein interactions in nsp3 caused by mutations found in variants identified between 2019 and 2024: Alpha 20I, Beta 20H, Delta 21I, Delta 21J, Gamma 20J, Kappa 21B, Lambda 21G, Omicron 21K, and Omicron 21L. A small set of amino acid substitutions in the N-terminal region of nsp3 (nsp3.1) could be traced to increased interactions with RNA-binding proteins, which are vital in viral replication. Meanwhile, variants of the central region of nsp3 (nsp3.2) were found to share interactions with protein quality control machinery, including ER-associated degradation. In this construct, shared trends in interactor enrichment are observed between Omicron 21K and Delta 21I. These results underscore how minor mutations reshape host interactions, emphasizing the evolutionary arms race between the host and virus. We provide a roadmap to track the interaction changes driven by SARS-CoV-2 variant evolution. Full article

Type 2 diabetes (T2D) is a prevalent chronic disease in the Korean population, influenced by lifestyle, dietary habits, and genetics. This study aimed to identify the effects of food intake and genetic factors on T2D progression in Korean adults using a multi-state illness-death model. We analyzed three transition models: normal glucose tolerance (NGT) to prediabetes (PD), NGT to T2D, and PD to T2D. We first identified dietary patterns significantly associated with each transition, using multivariate Cox proportional hazards models. Then, we assessed the impact of single-nucleotide polymorphisms (SNPs) on each transition, incorporating these dietary patterns as covariates. Our analysis revealed significant associations between the identified dietary patterns and the risk of PD and T2D incidence among individuals with NGT. We also identified novel genetic variants associated with disease progression: two SNPs (rs4607517 in Glucokinase [GCK] and rs758982 in Calcium/Calmodulin-Dependent Protein Kinase II Beta [CAMK2B]) in the NGT to PD model, and eight SNPs in the NGT to T2D model, including variants in the Zinc Finger Protein 106 (ZNF106), PTOV1 Extended AT-Hook Containing Adaptor Protein (PTOV1), Proprotein Convertase Subtilisin/Kexin Type 2 (PCSK2), Forkhead Box D2 (FOXD2), Solute Carrier Family 38 Member 7 (SLC38A7), and Neuronal Growth Regulator 1 (NEGR1) genes. Functional annotation analysis using ANNOVAR revealed that rs4607517 (GCK) and rs59595912 (PTOV1) exhibited high Combined Annotation-Dependent Depletion (CADD) and Deleterious Annotation of Genetic Variants using Neural Networks (DANN) scores, suggesting potential pathogenicity and providing a functional basis for their association with T2D progression. Integrating dietary and genetic factors with a multi-state model, this comprehensive approach offers valuable insights into T2D development and highlights potential targets for prevention and personalized interventions. Full article

Since the onset of the COVID-19 pandemic, various severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants have emerged. Although the primary site of SARS-CoV-2 infection is the lungs, it can also affect the brain and induce neurological symptoms. However, the specific effects of different variants on the brain remain unclear. In this study, a whole-transcriptome analysis was conducted using the brain tissues of K18-hACE2 mice infected with the ancestral B.1 (Wuhan) variant and with major SARS-CoV-2 variants of concern, including B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta) and B.1.529 (Omicron). After sequencing, differential gene expression, gene ontology (GO) and genome pathway enrichment analyses were performed. An Immune Cell Abundance Identifier (ImmuCellAI) was used to identify the abundance of different cell populations. Additionally, RT-qPCR was used to validate the RNA-seq data. The viral load and hierarchical clustering analyses divided the samples into two different clusters with notable differences in gene expression at day 6 post-infection for all variants compared to the control group. GO and the Kyoto Encyclopedia of genes and genomes enrichment analyses revealed similar patterns of pathway enrichment for different variants. ImmuCellAI revealed the changes in immune cell populations, including the decrease in CD4⁺ T and B cell proportions and the increase in CD8⁺ T and dendritic cell proportions. A co-expression network analysis revealed that some genes, such as STAT1, interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α), were dysregulated in all variants. A RT-qPCR analysis for IL-6, CXCL10 and IRF7 further validated the RNA-seq analysis. In conclusion, this study provides, for the first time, an extensive transcriptome analysis of a K18-hACE2 mouse brain after infection with major SARS-CoV-2 variants. Full article

The Severe Acute Respiratory Syndrome-related Coronavirus 2 (SARS-CoV-2), a causative agent of the COVID-19 disease, has been constantly evolving since its first identification. Mutations that are embedded in the viral genomic RNA affect the properties of the virus and lead to the emergence of new variants. During the COVID-19 pandemic, the World Health Organization has identified more than ten variants of the SARS-CoV-2 virus. Five of these—Alpha, Beta, Gamma, Delta, and Omicron—were classified as variants of concern (VOCs), as they caused significant outbreaks of the disease. Additionally, two progeny variants of Omicron, designated JN.1 and KS.1, are still causing new waves of infections. Due to the emergence of various SARS-CoV-2 variants, in some cases, it has become important to identify a particular variant in a sample. Here, we have developed a multiplexed probe-based real-time PCR system for the identification of SARS-CoV-2 VOCs (Alpha, Beta, Gamma, Delta, Omicron B.1.1.529/BA.1, and Omicron BA.2), as well as modern Omicron variants JN.1 and KS.1. The sensitivity and specificity of the PCR system have been tested using isolated viral genomes and RNA preparations from human nasopharyngeal swabs. The system allows for rapid identification of coronavirus variants in the cryopreserved and fresh samples. Full article

The use of dairy slurries as organic fertilizer amendments is a common practice in agriculture as a cost-saving measure, as well as a residue management strategy. However, concerns related to the increase in antibiotic resistance in the environment under the scope of the One Health strategy are increasing. In this study, we aimed to assess resistome enrichment driven by dairy slurry application in four southern Chile dairy farms. Slurry pits, rhizospheres of Lolium perenne amended with those slurries, and bulk soils were sampled. Thirteen antibiotic-resistance genes (ARGs, tetA, tetG, tetM, tetQ, tetW, tetX, sul1, sul2, blaCTX_M, bla_OXA-1, blaTEM, ermB, and dfrA1) for five antibiotic classes (tetracyclines, sulfonamides, beta-lactams, macrolides, and trimethoprim–sulfamethoxazole), two related integrases (intl1 and intl2), and total bacteria (16S rRNA) abundance was measured by quantitative PCR (qPCR). Then, the abundance profiles of two enzyme-inactivated ARGs (tetX and blaTEM) were determined. The differences between the bacterial communities inhabiting the different sample types were explored with 16S rRNA metabarcoding. In general, all measured ARGs were detected in slurries. A decreasing trend in ARG copy numbers was observed with increasing soil depth, with the exception of tetX, whose abundance increased in the bulk soil at specific farms. The tetX and blaTEM communities revealed no differences in the relative abundance of variants in any of the samples. Finally, taxonomic and structural differences were found among all sample types. Thus, the enrichment of the sampled farm soil resistomes was driven by the application of the raw slurries as fertilizer. Full article