Variation in structural motifs within SARS-related coronavirus spike proteins.

SARS-CoV-2 is the third known coronavirus (CoV) that has crossed the animal-human barrier in the last two decades. However, little structural information exists related to the close genetic species within the SARS-related coronaviruses. Here, we present three novel SARS-related CoV spike protein structures solved by single particle cryo-electron microscopy analysis derived from bat (bat SL-CoV WIV1) and civet (cCoV-SZ3, cCoV-007) hosts. We report complex glycan trees that decorate the glycoproteins and density for water molecules which facilitated modeling of the water molecule coordination networks within structurally important regions. We note structural conservation of the fatty acid binding pocket and presence of a linoleic acid molecule which are associated with stabilization of the receptor binding domains in the "down" conformation. Additionally, the N-terminal biliverdin binding pocket is occupied by a density in all the structures. Finally, we analyzed structural differences in a loop of the receptor binding motif between coronaviruses known to infect humans and the animal coronaviruses described in this study, which regulate binding to the human angiotensin converting enzyme 2 receptor. This study offers a structural framework to evaluate the close relatives of SARS-CoV-2, the ability to inform pandemic prevention, and aid in the development of pan-neutralizing treatments.

Carrier screening for present disease prevalence and recessive genetic disorder in Taiwanese population.

Carrier screening is important to people have a higher prevalence of severe recessive or X-linked genetic conditions. This study is aimed that the frequency and uncertain nature of genetic variants was identified in Taiwanese population, providing individuals with information at risk of inherited diseases and their heritability to newborns. A total of 480 subjects receiving genetic counseling with no family history of inherited disorders were recruited into a cohort from 2018 to 2022. Next-generation sequencing (NGS) panel for autosomal dominant (AD), autosomal recessive (AR) and X-linked diseases was sequenced to assess disease prevalence and carrier frequency for the targeted diseases. Publicly available NGS datasets were analyzed following a tier-based system and ACMG recommendation. 5.3% of subjects showed the presence of variants for genetic disorder, and 2.3% of them were determined with AD. 14 of subjects with pathogenic variants were carriers for AR. The inherited genes were LDLR for AD disorders and AR disorders included GAA and ATP7B. 21.6% of subjects had highest carrier frequency of GJB2 gene. 0.5% of subjects had highest frequency of GJB6 for AR condition. In conclusions, the variants in LDLR, GAA and ATP7B genes were identified in Taiwanese population, indicating individuals had higher risk of Pompe disease, Wilson's disease and familial hypercholesterolemia. Taiwanese individuals carrying GJB2 and GJB6 had the considerable risk of hearing loss passing to their offspring.

Development of Broad-Spectrum Nanobodies for the Therapy and Diagnosis of SARS-CoV-2 and Its Multiple Variants.

The continuous evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evaded the efficacy of previously developed antibodies and vaccines, thus remaining a significant global public health threat. Therefore, it is imperative to develop additional antibodies that are capable of neutralizing emerging variants. Nanobodies, as the smallest functional single-domain antibodies, exhibit enhanced stability and penetration ability, enabling them to recognize numerous concealed epitopes that are inaccessible to conventional antibodies. Herein, we constructed an immune library based on the immunization of alpaca with the S1 subunit of the SARS-CoV-2 spike protein, from which two nanobodies, Nb1 and Nb2, were selected using phage display technology for further characterization. Both nanobodies, with the binding residues residing within the receptor-binding domain (RBD) region of the spike, exhibited high affinity toward the S1 subunit. Moreover, they displayed cross-neutralizing activity against both wild-type SARS-CoV-2 and 10 ο variants, including BA.1, BA.2, BA.3, BA.5, BA.2.75, BF.7, BQ.1, EG.5.1, XBB.1.5, and JN.1. Molecular modeling and dynamics simulations predicted that both nanobodies interacted with the viral RBD through their complementarity determining region 1 (CDR1) and CDR2. These two nanobodies are novel tools for the development of therapeutic and diagnostic countermeasures targeting SARS-CoV-2 variants and potentially emerging coronaviruses.

[Application of microwave vacuum drying with process management in extract drying of personalized traditional Chinese medicine preparations].

Extracts are important intermediates in the production of traditional Chinese medicines preparations. The drying effect of extracts will directly affect the subsequent production process and the quality of the preparation. To meet the requirements of high drug loading, short time consumption, and simple production process of personalized traditional Chinese medicine preparations, this study explored the application of multi-program microwave vacuum drying process in the extract drying of personalized traditional Chinese medicine preparations. The influencing factors of microwave vacuum drying process were investigated for 5 excipients and 40 prescriptions. Taking the feasibility of drying, drying rate, drying time, and dried extract status as indicators, this study investigated the feeding requirements of microwave vacuum drying. With the dried extract status as the evaluation indicator, the three drying programs(A, B, and C) were compared to obtain the optimal drying condition. The experimental results showed that the optimal feeding conditions for microwave vacuum drying were material layer thickness of 2 cm and C program(a total of 7 drying processes), which solved the problem of easy scorching in microwave drying with process management. Furthermore, the preset moisture content of the dried extract in microwave drying should be 4%-5%, so that the dried extract of traditional Chinese medicine preparation had uniform quality, complete drying, and no scorching. This study lays a foundation for the application of microwave drying in the production of traditional Chinese medicine preparations, promoting the high-quality development of personalized traditional Chinese medicine preparations.

The roles and regulatory mechanisms of TGF-ß and BMP signaling in bone and cartilage development, homeostasis and disease.

Transforming growth factor-ßs (TGF-ßs) and bone morphometric proteins (BMPs) belong to the TGF-ß superfamily and perform essential functions during osteoblast and chondrocyte lineage commitment and differentiation, skeletal development, and homeostasis. TGF-ßs and BMPs transduce signals through SMAD-dependent and -independent pathways; specifically, they recruit different receptor heterotetramers and R-Smad complexes, resulting in unique biological readouts. BMPs promote osteogenesis, osteoclastogenesis, and chondrogenesis at all differentiation stages, while TGF-ßs play different roles in a stage-dependent manner. BMPs and TGF-ß have opposite functions in articular cartilage homeostasis. Moreover, TGF-ß has a specific role in maintaining the osteocyte network. The precise activation of BMP and TGF-ß signaling requires regulatory machinery at multiple levels, including latency control in the matrix, extracellular antagonists, ubiquitination and phosphorylation in the cytoplasm, nucleus-cytoplasm transportation, and transcriptional co-regulation in the nuclei. This review weaves the background information with the latest advances in the signaling facilitated by TGF-ßs and BMPs, and the advanced understanding of their diverse physiological functions and regulations. This review also summarizes the human diseases and mouse models associated with disordered TGF-ß and BMP signaling. A more precise understanding of the BMP and TGF-ß signaling could facilitate the development of bona fide clinical applications in treating bone and cartilage disorders.

Cell signaling and transcriptional regulation of osteoblast lineage commitment, differentiation, bone formation, and homeostasis.

The initiation of osteogenesis primarily occurs as mesenchymal stem cells undergo differentiation into osteoblasts. This differentiation process plays a crucial role in bone formation and homeostasis and is regulated by two intricate processes: cell signal transduction and transcriptional gene expression. Various essential cell signaling pathways, including Wnt, BMP, TGF-ß, Hedgehog, PTH, FGF, Ephrin, Notch, Hippo, and Piezo1/2, play a critical role in facilitating osteoblast differentiation, bone formation, and bone homeostasis. Key transcriptional factors in this differentiation process include Runx2, Cbfß, Runx1, Osterix, ATF4, SATB2, and TAZ/YAP. Furthermore, a diverse array of epigenetic factors also plays critical roles in osteoblast differentiation, bone formation, and homeostasis at the transcriptional level. This review provides an overview of the latest developments and current comprehension concerning the pathways of cell signaling, regulation of hormones, and transcriptional regulation of genes involved in the commitment and differentiation of osteoblast lineage, as well as in bone formation and maintenance of homeostasis. The paper also reviews epigenetic regulation of osteoblast differentiation via mechanisms, such as histone and DNA modifications. Additionally, we summarize the latest developments in osteoblast biology spurred by recent advancements in various modern technologies and bioinformatics. By synthesizing these insights into a comprehensive understanding of osteoblast differentiation, this review provides further clarification of the mechanisms underlying osteoblast lineage commitment, differentiation, and bone formation, and highlights potential new therapeutic applications for the treatment of bone diseases.

Wnt/ß-catenin signaling components and mechanisms in bone formation, homeostasis, and disease.

Wnts are secreted, lipid-modified proteins that bind to different receptors on the cell surface to activate canonical or non-canonical Wnt signaling pathways, which control various biological processes throughout embryonic development and adult life. Aberrant Wnt signaling pathway underlies a wide range of human disease pathogeneses. In this review, we provide an update of Wnt/ß-catenin signaling components and mechanisms in bone formation, homeostasis, and diseases. The Wnt proteins, receptors, activators, inhibitors, and the crosstalk of Wnt signaling pathways with other signaling pathways are summarized and discussed. We mainly review Wnt signaling functions in bone formation, homeostasis, and related diseases, and summarize mouse models carrying genetic modifications of Wnt signaling components. Moreover, the therapeutic strategies for treating bone diseases by targeting Wnt signaling, including the extracellular molecules, cytosol components, and nuclear components of Wnt signaling are reviewed. In summary, this paper reviews our current understanding of the mechanisms by which Wnt signaling regulates bone formation, homeostasis, and the efforts targeting Wnt signaling for treating bone diseases. Finally, the paper evaluates the important questions in Wnt signaling to be further explored based on the progress of new biological analytical technologies.

Deficiency of Cbfß in articular cartilage leads to osteoarthritis-like phenotype through Hippo/Yap, TGFß, and Wnt/ß-catenin signaling pathways.

Osteoarthritis (OA) is the most prevalent degenerative joint disorder, causing physical impairments among the elderly. Core binding factor subunit ß (Cbfß) has a critical role in bone homeostasis and cartilage development. However, the function and mechanism of Cbfß in articular cartilage and OA remains unclear. We found that Cbfßf/fAggrecan-CreERT mice with Cbfß-deficiency in articular cartilage developed a spontaneous osteoarthritis-like phenotype with articular cartilage degradation. Immunofluorescence staining showed that Cbfßf/fAggrecan-CreERT mice exhibited a significant increase in the expression of articular cartilage degradation markers and inflammatory markers in the knee joints. RNA-sequencing analysis demonstrated that Cbfß orchestrated Hippo/Yap, TGFß/Smad, and Wnt/ß-catenin signaling pathways in articular cartilage, and Cbfß deficiency resulted in the abnormal expression of downstream genes involved in maintaining articular cartilage homeostasis. Immunofluorescence staining results showed Cbfß deficiency significantly increased active ß-catenin and TCF4 expression while reducing Yap, TGFß1, and p-Smad 2/3 expression. Western blot and qPCR validated gene expression changes in hip articular cartilage of Cbfß-deficient mice. Our results demonstrate that deficiency of Cbfß in articular cartilage leads to an OA-like phenotype via affecting Hippo/Yap, TGFß, and Wnt/ß-catenin signaling pathways, disrupting articular cartilage homeostasis and leading to the pathological process of OA in mice. Our results indicate that targeting Cbfß may be a potential therapeutic target for the design of novel and effective treatments for OA.

The proteomic analysis uncovers the cellular responses to the African swine fever virus membrane proteins p54, p17, and pB117L.

African swine fever virus (ASFV) infection causes African swine fever (ASF), a highly contagious and fatal disease that poses severe threat to swine production. To gain insights into the host responses to ASFV, we generated recombinant adenovirus Ad5 expressing viral membrane proteins p54, p17, and pB117L individually and infected an alveolar cell line, 3D4/21, with these recombinant viruses. Then, the cell lysates were analyzed using label-free quantification proteomic analysis method. A total of 2158 differentially expressed proteins (DEPs) were identified, of which 817, 466, and 875 proteins were from Ad5-p54-, Ad5-p17-, Ad5-pB117L-infected 3D4/21 cells, respectively. Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed distinct yet interconnecting patterns of protein interaction networks. Specifically, the Ad5-p54 virus infection enriched the DEPs primarily involved in the metabolic pathways, endocytosis, adherens junction, and SNARE interactions in vesicular transport. The Ad5-p17 virus infection enriched the DEPs in endocytosis, ubiquitin-mediated proteolysis, N-Glycan biosynthesis, and apoptosis, while the Ad5-pB117L virus infection enriched the DEPs in metabolic pathways, endocytosis, oxidative phosphorylation, and focal adhesion. In summary, these results provide a comprehensive proteinomics analysis of the cellular responses to three ASFV membrane proteins, thus facilitating our understanding of ASFV pathogenesis.

Correction: RNAi-Mediated Silencing of Atp6i and Atp6i Haploinsufficiency Prevents Both Bone Loss and Inflammation in a Mouse Model of Periodontal Disease.

[This corrects the article DOI: 10.1371/journal.pone.0058599.].

Cbfß regulates Wnt/ß-catenin, Hippo/Yap, and Tgfß signaling pathways in articular cartilage homeostasis and protects from ACLT surgery-induced osteoarthritis.

As the most common degenerative joint disease, osteoarthritis (OA) contributes significantly to pain and disability during aging. Several genes of interest involved in articular cartilage damage in OA have been identified. However, the direct causes of OA are poorly understood. Evaluating the public human RNA-seq dataset showed that CBFB (subunit of a heterodimeric Cbfß/Runx1, Runx2, or Runx3 complex) expression is decreased in the cartilage of patients with OA. Here, we found that the chondrocyte-specific deletion of Cbfb in tamoxifen-induced Cbfbf/f;Col2a1-CreERT mice caused a spontaneous OA phenotype, worn articular cartilage, increased inflammation, and osteophytes. RNA-sequencing analysis showed that Cbfß deficiency in articular cartilage resulted in reduced cartilage regeneration, increased canonical Wnt signaling and inflammatory response, and decreased Hippo/Yap signaling and Tgfß signaling. Immunostaining and western blot validated these RNA-seq analysis results. ACLT surgery-induced OA decreased Cbfß and Yap expression and increased active ß-catenin expression in articular cartilage, while local AAV-mediated Cbfb overexpression promoted Yap expression and diminished active ß-catenin expression in OA lesions. Remarkably, AAV-mediated Cbfb overexpression in knee joints of mice with OA showed the significant protective effect of Cbfß on articular cartilage in the ACLT OA mouse model. Overall, this study, using loss-of-function and gain-of-function approaches, uncovered that low expression of Cbfß may be the cause of OA. Moreover, Local admission of Cbfb may rescue and protect OA through decreasing Wnt/ß-catenin signaling, and increasing Hippo/Yap signaling and Tgfß/Smad2/3 signaling in OA articular cartilage, indicating that local Cbfb overexpression could be an effective strategy for treatment of OA.

Cbfß regulates Wnt/ß-catenin, Hippo/Yap, and TGFß signaling pathways in articular cartilage homeostasis and protects from ACLT surgery-induced osteoarthritis.

As the most common degenerative joint disease, osteoarthritis (OA) contributes significantly to pain and disability during aging. Several genes of interest involved in articular cartilage damage in OA have been identified. However, the direct causes of OA are poorly understood. Evaluating the public human RNA-seq dataset showed that Cbfß, (subunit of a heterodimeric Cbfß/Runx1,Runx2, or Runx3 complex) expression is decreased in the cartilage of patients with OA. Here, we found that the chondrocyte-specific deletion of Cbfß in tamoxifen-induced Cbfßf/fCol2α1-CreERT mice caused a spontaneous OA phenotype, worn articular cartilage, increased inflammation, and osteophytes. RNA-sequencing analysis showed that Cbfß deficiency in articular cartilage resulted in reduced cartilage regeneration, increased canonical Wnt signaling and inflammatory response, and decreased Hippo/YAP signaling and TGF-ß signaling. Immunostaining and western blot validated these RNA-seq analysis results. ACLT surgery-induced OA decreased Cbfß and Yap expression and increased active ß-catenin expression in articular cartilage, while local AAV-mediated Cbfß overexpression promoted Yap expression and diminished active ß-catenin expression in OA lesions. Remarkably, AAV-mediated Cbfß overexpression in knee joints of mice with OA showed the significant protective effect of Cbfß on articular cartilage in the ACLT OA mouse model. Overall, this study, using loss-of-function and gain-of-function approaches, uncovered that low expression of Cbfß may be the cause of OA. Moreover, Local admission of Cbfß may rescue and protect OA through decreasing Wnt/ß-catenin signaling, and increasing Hippo/Yap signaling and TGFß/Smad2/3 signaling in OA articular cartilage, indicating that local Cbfß overexpression could be an effective strategy for treatment of OA.

SARS-CoV-2 spike-reactive naïve B cells and pre-existing memory B cells contribute to antibody responses in unexposed individuals after vaccination.

Introduction: Since December 2019, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) has presented considerable public health challenges. Multiple vaccines have been used to induce neutralizing antibodies (nAbs) and memory B-cell responses against the viral spike (S) glycoprotein, and many essential epitopes have been defined. Previous reports have identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike-reactive naïve B cells and preexisting memory B cells in unexposed individuals. However, the role of these spike-reactive B cells in vaccine-induced immunity remains unknown. Methods: To elucidate the characteristics of preexisting SARS-CoV-2 S-reactive B cells as well as their maturation after antigen encounter, we assessed the relationship of spike-reactive B cells before and after vaccination in unexposed human individuals. We further characterized the sequence identity, targeting domain, broad-spectrum binding activity and neutralizing activity of these SARS-CoV-2 S-reactive B cells by isolating monoclonal antibodies (mAbs) from these B cells. Results: The frequencies of both spike-reactive naïve B cells and preexisting memory B cells before vaccination correlated with the frequencies of spike-reactive memory B cells after vaccination. Isolated mAbs from spike-reactive naïve B cells before vaccination had fewer somatic hypermutations (SHMs) than mAbs isolated from spike-reactive memory B cells before and after vaccination, but bound SARS-CoV-2 spike in vitro. Intriguingly, these germline-like mAbs possessed broad binding profiles for SARS-CoV-2 and its variants, although with low or no neutralizing capacity. According to tracking of the evolution of IGHV4-4/IGKV3-20 lineage antibodies from a single donor, the lineage underwent SHMs and developed increased binding activity after vaccination. Discussion: Our findings suggest that spike-reactive naïve B cells can be expanded and matured by vaccination and cocontribute to vaccine-elicited antibody responses with preexisting memory B cells. Selectively and precisely targeting spike-reactive B cells by rational antigen design may provide a novel strategy for next-generation SARS-CoV-2 vaccine development.

The crosstalk between macrophages and cancer cells potentiates pancreatic cancer cachexia.

With limited treatment options, cachexia remains a major challenge for patients with cancer. Characterizing the interplay between tumor cells and the immune microenvironment may help identify potential therapeutic targets for cancer cachexia. Herein, we investigate the critical role of macrophages in potentiating pancreatic cancer induced muscle wasting via promoting TWEAK (TNF-like weak inducer of apoptosis) secretion from the tumor. Specifically, depletion of macrophages reverses muscle degradation induced by tumor cells. Macrophages induce non-autonomous secretion of TWEAK through CCL5/TRAF6/NF-κB pathway. TWEAK promotes muscle atrophy by activating MuRF1 initiated muscle remodeling. Notably, tumor cells recruit and reprogram macrophages via the CCL2/CCR2 axis and disrupting the interplay between macrophages and tumor cells attenuates muscle wasting. Collectively, this study identifies a feedforward loop between pancreatic cancer cells and macrophages, underlying the non-autonomous activation of TWEAK secretion from tumor cells thereby providing promising therapeutic targets for pancreatic cancer cachexia.