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1.
Mol Pharm ; 2024 Jun 26.
Article in English | MEDLINE | ID: mdl-38920116

ABSTRACT

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.

2.
Elife ; 132024 May 28.
Article in English | MEDLINE | ID: mdl-38805545

ABSTRACT

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.


Subject(s)
Cartilage, Articular , Hippo Signaling Pathway , Homeostasis , Osteoarthritis , Transforming Growth Factor beta , YAP-Signaling Proteins , Animals , Cartilage, Articular/metabolism , Mice , Osteoarthritis/genetics , Osteoarthritis/metabolism , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta/genetics , YAP-Signaling Proteins/metabolism , YAP-Signaling Proteins/genetics , Wnt Signaling Pathway , beta Catenin/metabolism , beta Catenin/genetics , Signal Transduction , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Humans , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/genetics
3.
Microbes Infect ; : 105348, 2024 Apr 30.
Article in English | MEDLINE | ID: mdl-38697277

ABSTRACT

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.

4.
PLoS Pathog ; 20(5): e1012158, 2024 May 28.
Article in English | MEDLINE | ID: mdl-38805567

ABSTRACT

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.

5.
Int J Biol Sci ; 20(6): 1965-1977, 2024.
Article in English | MEDLINE | ID: mdl-38617544

ABSTRACT

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.


Subject(s)
Cartilage, Articular , Osteoarthritis , Animals , Mice , Aggrecans , beta Catenin/genetics , Osteoarthritis/genetics , Phenotype , Transforming Growth Factor beta , Wnt Signaling Pathway/genetics
6.
Zhongguo Zhong Yao Za Zhi ; 49(3): 625-633, 2024 Feb.
Article in Chinese | MEDLINE | ID: mdl-38621866

ABSTRACT

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.


Subject(s)
Medicine, Chinese Traditional , Microwaves , Vacuum , Desiccation/methods , Plant Extracts
7.
Cancer Cell ; 42(5): 885-903.e4, 2024 May 13.
Article in English | MEDLINE | ID: mdl-38608702

ABSTRACT

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.


Subject(s)
Cachexia , Cytokine TWEAK , Macrophages , Pancreatic Neoplasms , Cachexia/metabolism , Cachexia/etiology , Cachexia/pathology , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/complications , Cytokine TWEAK/metabolism , Animals , Humans , Macrophages/metabolism , Mice , NF-kappa B/metabolism , Cell Line, Tumor , Tumor Microenvironment , Muscular Atrophy/metabolism , Muscular Atrophy/etiology , Muscular Atrophy/pathology , Chemokine CCL5/metabolism , Signal Transduction , TNF Receptor-Associated Factor 6/metabolism , Tumor Necrosis Factors/metabolism , Receptors, CCR2/metabolism , Chemokine CCL2/metabolism , Mice, Inbred C57BL
9.
Front Immunol ; 15: 1355949, 2024.
Article in English | MEDLINE | ID: mdl-38420128

ABSTRACT

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.


Subject(s)
COVID-19 , Memory B Cells , Humans , SARS-CoV-2 , Antibody Formation , COVID-19 Vaccines , COVID-19/prevention & control , Vaccination , Antibodies, Monoclonal
10.
bioRxiv ; 2024 Jan 16.
Article in English | MEDLINE | ID: mdl-38293189

ABSTRACT

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.

11.
Cell Res ; 34(2): 101-123, 2024 02.
Article in English | MEDLINE | ID: mdl-38267638

ABSTRACT

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.


Subject(s)
Bone Morphogenetic Proteins , Chondrogenesis , Transforming Growth Factor beta , Animals , Humans , Mice , Bone and Bones/metabolism , Cartilage/metabolism , Homeostasis , Transforming Growth Factor beta/metabolism , Bone Morphogenetic Proteins/metabolism
13.
J Hum Genet ; 69(3-4): 115-118, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38135707

ABSTRACT

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.


Subject(s)
Hepatolenticular Degeneration , Infant, Newborn , Humans , Prevalence , Hepatolenticular Degeneration/genetics , Genetic Counseling , Genes, Recessive , Mutation
14.
EMBO Rep ; 24(11): e56614, 2023 11 06.
Article in English | MEDLINE | ID: mdl-37789674

ABSTRACT

ATPase family AAA domain-containing protein 1 (ATAD1) maintains mitochondrial homeostasis by removing mislocalized tail-anchored (TA) proteins from the mitochondrial outer membrane (MOM). Hepatitis C virus (HCV) infection induces mitochondrial fragmentation, and viral NS5B protein is a TA protein. Here, we investigate whether ATAD1 plays a role in regulating HCV infection. We find that HCV infection has no effect on ATAD1 expression, but knockout of ATAD1 significantly enhances HCV infection; this enhancement is suppressed by ATAD1 complementation. NS5B partially localizes to mitochondria, dependent on its transmembrane domain (TMD), and induces mitochondrial fragmentation, which is further enhanced by ATAD1 knockout. ATAD1 interacts with NS5B, dependent on its three internal domains (TMD, pore-loop 1, and pore-loop 2), and induces the proteasomal degradation of NS5B. In addition, we provide evidence that ATAD1 augments the antiviral function of MAVS upon HCV infection. Taken together, we show that the mitochondrial quality control exerted by ATAD1 can be extended to a novel antiviral function through the extraction of the viral TA-protein NS5B from the mitochondrial outer membrane.


Subject(s)
Hepacivirus , Hepatitis C , Humans , Hepacivirus/metabolism , Viral Proteins/metabolism , Hepatitis C/metabolism , Mitochondria/metabolism , Antiviral Agents , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
15.
Signal Transduct Target Ther ; 8(1): 393, 2023 10 06.
Article in English | MEDLINE | ID: mdl-37802996

ABSTRACT

Long-term humoral immunity to SARS-CoV-2 is essential for preventing reinfection. The production of neutralizing antibody (nAb) and B cell differentiation are tightly regulated by T follicular help (TFH) cells. However, the longevity and functional role of TFH cell subsets in COVID-19 convalescents and vaccine recipients remain poorly defined. Here, we show that SARS-CoV-2 infection and inactivated vaccine elicited both spike-specific CXCR3+ TFH cell and CXCR3- TFH cell responses, which showed distinct response patterns. Spike-specific CXCR3+ TFH cells exhibit a dominant and more durable response than CXCR3- TFH cells that positively correlated with antibody responses. A third booster dose preferentially expands the spike-specific CXCR3+ TFH cell subset induced by two doses of inactivated vaccine, contributing to antibody maturation and potency. Functionally, spike-specific CXCR3+ TFH cells have a greater ability to induce spike-specific antibody secreting cells (ASCs) differentiation compared to spike-specific CXCR3- TFH cells. In conclusion, the persistent and functional role of spike-specific CXCR3+ TFH cells following SARS-CoV-2 infection and vaccination may play an important role in antibody maintenance and recall response, thereby conferring long-term protection. The findings from this study will inform the development of SARS-CoV-2 vaccines aiming to induce long-term protective immune memory.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , COVID-19 Vaccines , Antibodies, Neutralizing , Vaccines, Inactivated
16.
Sci Total Environ ; 904: 166811, 2023 Dec 15.
Article in English | MEDLINE | ID: mdl-37673249

ABSTRACT

A novel wastewater treatment plant process was constructed to overcome the challenge of simultaneous nitrate removal and phosphorus (P) recovery. The results revealed that the P and nitrate removal efficiency rose from 39.0 % and 48.4 % to 92.8 % and 93.6 % after 136 days of operation, and the total P content in the biofilm (TPbiofilm) rose from 15.8 mg/g SS to 57.8 mg/g SS. Moreover, the increase of TPbiofilm changed the metabolic mode of denitrifying polyphosphate accumulating organisms (DPAOs), increasing the P concentration of the enriched stream to 172.5 mg/L. Furthermore, the acid/alkaline fermentation led to the rupture of the cell membrane, which released poly-phosphate and ortho-phosphate of cell/EPS in DPAOs and released metal­phosphorus (CaP and MgP). In addition, high-throughput sequencing analysis demonstrated that the relative abundance of DPAOs involved in P storage increased, wherein the abundance of Acinetobacter and Saprospiraceae rose from 8.0 % and 4.1 % to 16.1 % and 14.0 %. What's more, the highest P recovery efficiency (98.3 ± 1.1 %) could be obtained at optimal conditions for struvite precipitation (pH = 7.56 and P: N: Mg = 1.87:3.66:1) through the response surface method (RSM) simulation, and the precipitates test analysis indicated that P recovery from biofilm sludge was potentially operable. This research was of great essentiality for exploring the recovery of P from biofilm sludge.


Subject(s)
Phosphorus , Sewage , Phosphorus/metabolism , Nitrates/metabolism , Denitrification , Anaerobiosis , Bioreactors , Polyphosphates , Biofilms , Waste Disposal, Fluid/methods , Nitrogen
17.
Int J Oral Sci ; 15(1): 35, 2023 08 21.
Article in English | MEDLINE | ID: mdl-37599332

ABSTRACT

The biomolecular mechanisms that regulate tooth root development and odontoblast differentiation are poorly understood. We found that Atp6i deficient mice (Atp6i-/-) arrested tooth root formation, indicated by truncated Hertwig's epithelial root sheath (HERS) progression. Furthermore, Atp6i deficiency significantly reduced the proliferation and differentiation of radicular odontogenic cells responsible for root formation. Atp6i-/- mice had largely decreased expression of odontoblast differentiation marker gene expression profiles (Col1a1, Nfic, Dspp, and Osx) in the alveolar bone. Atp6i-/- mice sample RNA-seq analysis results showed decreased expression levels of odontoblast markers. Additionally, there was a significant reduction in Smad2/3 activation, inhibiting transforming growth factor-ß (TGF-ß) signaling in Atp6i-/- odontoblasts. Through treating pulp precursor cells with Atp6i-/- or wild-type OC bone resorption-conditioned medium, we found the latter medium to promote odontoblast differentiation, as shown by increased odontoblast differentiation marker genes expression (Nfic, Dspp, Osx, and Runx2). This increased expression was significantly blocked by anti-TGF-ß1 antibody neutralization, whereas odontoblast differentiation and Smad2/3 activation were significantly attenuated by Atp6i-/- OC conditioned medium. Importantly, ectopic TGF-ß1 partially rescued root development and root dentin deposition of Atp6i-/- mice tooth germs were transplanted under mouse kidney capsules. Collectively, our novel data shows that the prevention of TGF-ß1 release from the alveolar bone matrix due to OC dysfunction may lead to osteopetrosis-associated root formation via impaired radicular odontoblast differentiation. As such, this study uncovers TGF-ß1 /Smad2/3 as a key signaling pathway regulating odontoblast differentiation and tooth root formation and may contribute to future therapeutic approaches to tooth root regeneration.


Subject(s)
Odontoblasts , Transforming Growth Factor beta1 , Female , Animals , Mice , Culture Media, Conditioned , Cell Differentiation , Signal Transduction , Disease Models, Animal , Tooth Root
18.
Microbiol Spectr ; 11(4): e0042323, 2023 08 17.
Article in English | MEDLINE | ID: mdl-37378517

ABSTRACT

Dengue virus (DENV) is the most widespread arbovirus, causing symptoms ranging from dengue fever to severe dengue, including hemorrhagic fever and shock syndrome. Four serotypes of DENV (DENV-1 to -4) can infect humans; however, no anti-DENV drug is available. To facilitate the study of antivirals and viral pathogenesis, here we developed an infectious clone and a subgenomic replicon of DENV-3 strains for anti-DENV drug discovery by screening a synthetic compound library. The viral cDNA was amplified from a serum sample from a DENV-3-infected individual during the 2019 epidemic; however, fragments containing the prM-E-partial NS1 region could not be cloned until a DENV-3 consensus sequence with 19 synonymous substitutions was introduced to reduce putative Escherichia coli promoter activity. Transfection of the resulting cDNA clone, plasmid DV3syn, released an infectious virus titer of 2.2 × 102 focus-forming units (FFU)/mL. Through serial passages, four adaptive mutations (4M) were identified, and addition of 4M generated recombinant DV3syn_4M, which produced viral titers ranging from 1.5 × 104 to 6.7 × 104 FFU/mL and remained genetically stable in transformant bacteria. Additionally, we constructed a DENV-3 subgenomic replicon and screened an arylnaphthalene lignan library, from which C169-P1 was identified as exhibiting inhibitory effects on viral replicon. A time-of-drug addition assay revealed that C169-P1 also impeded the internalization process of cell entry. Furthermore, we demonstrated that C169-P1 inhibited the infectivity of DV3syn_4M, as well as DENV-1, DENV-2, and DENV-4, in a dose-dependent manner. This study provides an infectious clone and a replicon for the study of DENV-3 and a candidate compound for future development against DENV-1 to -4 infections. IMPORTANCE Dengue virus (DENV) is the most prevalent mosquito-transmitted virus, and there is no an anti-dengue drug. Reverse genetic systems representative of different serotype viruses are invaluable tools for the study of viral pathogenesis and antiviral drugs. Here, we developed an efficient infectious clone of a clinical DENV-3 genotype III isolate. We successfully overcame the instability of flavivirus genome-length cDNA in transformant bacteria, an unsolved issue for construction of cDNA clones of flaviviruses, and adapted this clone to efficiently produce infectious viruses following plasmid transfection of cell culture. Moreover, we constructed a DENV-3 subgenomic replicon and screened a compound library. An arylnaphthalene lignan, C169-P1, was identified as an inhibitor of virus replication and cell entry. Finally, we demonstrated that C169-P1 exhibited a broad-spectrum antiviral effect against the infections with DENV-1 to -4. The reverse genetic systems and the compound candidate described here facilitate the study of DENV and related RNA viruses.


Subject(s)
Dengue Virus , Flavivirus , Animals , Humans , Dengue Virus/genetics , Serogroup , DNA, Complementary/pharmacology , Antiviral Agents/pharmacology , Flavivirus/genetics , Replicon , Clone Cells
19.
Bioresour Technol ; 384: 129284, 2023 Sep.
Article in English | MEDLINE | ID: mdl-37302767

ABSTRACT

A novel process was proposed for simultaneous denitrification and phosphorus (P) recovery. The increased nitrate concentration facilitated the activity of denitrifying P removal (DPR) in P enrichment, which stimulated P uptake and storage, making P more readily accessible for release into the recirculated stream. The total P content in the biofilm (TPbiofilm) rose to 54.6 ± 3.5 mg/g SS as the nitrate concentration increased from 15.0 to 25.0 mg/L, while the P concentration of the enriched stream reached 172.5 ± 3.5 mg/L. Moreover, the abundance of denitrifying polyphosphate accumulating organisms (DPAOs) increased from 5.6% to 28.0%, and the increased nitrate concentration facilitated the process of carbon, nitrogen, and P metabolism due to the rise in the genes involved in critical functions of metabolism. Acid/alkaline fermentation analysis indicated that the EPS release was the primary P-release pathway. Additionally, pure struvite crystals were obtained from the enriched stream and fermentation supernatant.


Subject(s)
Sewage , Wastewater , Phosphorus/metabolism , Nitrates , Denitrification , Bioreactors , Organic Chemicals , Nitrogen , Waste Disposal, Fluid
20.
Biomedicines ; 11(6)2023 May 27.
Article in English | MEDLINE | ID: mdl-37371652

ABSTRACT

In the early 1960s, heat shock proteins (HSPs) were first identified as vital intracellular proteinaceous components that help in stress physiology and reprogram the cellular responses to enable the organism's survival. By the early 1990s, HSPs were detected in extracellular spaces and found to activate gamma-delta T-lymphocytes. Subsequent investigations identified their association with varied disease conditions, including autoimmune disorders, diabetes, cancer, hepatic, pancreatic, and renal disorders, and cachexia. In cardiology, extracellular HSPs play a definite, but still unclear, role in atherosclerosis, acute coronary syndromes, and heart failure. The possibility of HSP-targeted novel molecular therapeutics has generated much interest and hope in recent years. In this review, we discuss the role of Extracellular Heat Shock Proteins (Ec-HSPs) in various disease states, with a particular focus on cardiovascular diseases.

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