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The spike (S) protein is a leading vaccine candidate against SARS-CoV-2 infection. The S1 domain of S protein, which contains a critical receptor-binding domain (RBD) antigen, potentially induces protective immunoreactivities against SARS-CoV-2. In this study, we presented preclinical evaluations of a novel insect cell-derived SARS-CoV-2 recombinant S1 (rS1) protein as a potent COVID-19 vaccine candidate. The native antigenicity of rS1 was characterized by enzyme-linked immunosorbent assay with a neutralizing monoclonal antibody targeting the RBD antigen. To improve its immunogenicity, rS1-adjuvanted with fucoidan/trimethylchitosan nanoparticles (FUC-TMC NPs) and cytosine-phosphate-guanosine-oligodeoxynucleotides (CpG-ODNs) were investigated using a mouse model. The S1-specific immunoglobulin G (IgG) titers, FluoroSpot assay, pseudovirus- and prototype SARS-CoV-2-based neutralization assays were assessed. The results showed that the rS1/CpG/ FUC-TMC NPs (rS1/CpG/NPs) formulation induced a broad-spectrum IgG response with potent, long-lasting, and cross-protective neutralizing activity against the emerging SARS-CoV-2 variant of concern, along with a Th1-biased cellular response. Thus, the rS1/CpG/NPs formulation presents a promising vaccination approach against COVID-19.

BackgroundDrug repurposing is a fast and effective way to develop drugs for an emerging disease such as COVID-19. The main challenges of effective drug repurposing are the discoveries of the right therapeutic targets and the right drugs for combating the disease.MethodsHere, we present a systematic repurposing approach, combining Homopharma and hierarchal systems biology networks (HiSBiN), to predict 327 therapeutic targets and 21,233 drug-target interactions of 1,592 FDA drugs for COVID-19. Among these multi-target drugs, eight candidates (along with pimozide and valsartan) were tested and methotrexate was identified to affect 14 therapeutic targets suppressing SARS-CoV-2 entry, viral replication, and COVID-19 pathologies. Through the use of in vitro (EC50 = 0.4 μM) and in vivo models, we show that methotrexate is able to inhibit COVID-19 via multiple mechanisms.ResultsOur in vitro studies illustrate that methotrexate can suppress SARS-CoV-2 entry and replication by targeting furin and DHFR of the host, respectively. Additionally, methotrexate inhibits all four SARS-CoV-2 variants of concern. In a Syrian hamster model for COVID-19, methotrexate reduced virus replication, inflammation in the infected lungs. By analysis of transcriptomic analysis of collected samples from hamster lung, we uncovered that neutrophil infiltration and the pathways of innate immune response, adaptive immune response and thrombosis are modulated in the treated animals.ConclusionsWe demonstrate that this systematic repurposing approach is potentially useful to identify pharmaceutical targets, multi-target drugs and regulated pathways for a complex disease. Our findings indicate that methotrexate is established as a promising drug against SARS-CoV-2 variants and can be used to treat lung damage and inflammation in COVID-19, warranting future evaluation in clinical trials.

The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants has altered the trajectory of the COVID-19 pandemic and raised some uncertainty on the long-term efficiency of vaccine strategy. The development of new therapeutics against a wide range of SARS-CoV-2 variants is imperative. We, here, have designed an inhalable siRNA, C6G25S, which covers 99.8% of current SARS-CoV-2 variants and is capable of inhibiting dominant strains, including Alpha, Delta, Gamma, and Epsilon, at picomolar ranges of IC

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes severe “flu-like” symptoms that can progress to acute respiratory distress syndrome (ARDS), pneumonia, renal failure, and death. From the therapeutic perspective, 3-chymotrypsin-like protein (3CLpro) is a plausible target for direct-acting antiviral agents because of its indispensable role in viral replication. The flavonoid ugonin J (UJ) has been reported to have antioxidative and anti-inflammatory activities. However, the potential of UJ as an antiviral agent remains unexplored. In this study, we investigated the therapeutic activity of UJ against SARS-CoV-2 infection. Importantly, UJ has a distinct inhibitory activity against SARS-CoV-2 3CLpro, compared to luteolin, kaempferol, and isokaempferide. Specifically, UJ blocks the active site of SARS-CoV-2 3CLpro by forming hydrogen bonding and van der Waals interactions with H163, M165 and E166, G143 and C145, Q189, and P168 in subsites S1, S1′, S2, and S4, respectively. In addition, UJ forms strong, stable interactions with core pharmacophore anchors of SARS-CoV-2 3CLpro in a computational model. UJ shows consistent anti-inflammatory activity in inflamed human alveolar basal epithelial A549 cells. Furthermore, UJ has a 50% cytotoxic concentration (CC50) and a 50% effective concentration (EC50) values of about 783 and 2.38 µM, respectively, with a selectivity index (SI) value of 329, in SARS-CoV-2-infected Vero E6 cells. Taken together, UJ is a direct-acting antiviral that obstructs the activity of a fundamental protease of SARS-CoV-2, offering the therapeutic potential for SARS-CoV-2 infection.

Carbapenem-resistant Acinetobacter nosocomialis (CRAn) is a significant public health concern. Tigecycline non-susceptible CRAn (Tn-CRAn) isolates have emerged worldwide. Tigecycline resistance is mainly related to the overexpression of AdeABC efflux pump controlled by AdeRS two-component system (TCS). Two novel tetracycline derivatives, omadacycline and eravacycline, may present a treatment option for CRAn. This study investigated the in vitro antimicrobial activity of tigecycline, omadacycline and eravacycline against clinical CRAn isolates and the contribution of efflux pumps in their resistance. Eighty-nine clinical CRAn isolates, including 57 Tn-CRAn isolates were evaluated for minimum inhibitory concentrations (MICs) by the broth microdilution. The relationship between the antimicrobial resistance and efflux pump expression was assessed by their responses to the efflux pump inhibitor 1-(1-naphthylmethyl)-piperazine (NMP). The contribution of the AdeABC efflux pump in their resistance was determined by the complementation of the AdeRS two-component system in wild-type, adeRS operon and adeB gene knockout strains. Among the 89 isolates, omadacycline and eravacycline MICs were correlated closely with those of tigecycline. They demonstrated improved potency, based on MIC90 values, by showing a 4 to 8-fold greater potency than tigecycline. The synergetic effects of tigecycline, omadacycline and eravacycline with NMP were observed in 57 (100%), 13 (22.8%), and 51 (89.5%) of Tn-CRAn isolates, respectively. Further analysis showed that the laboratory strain carrying the Type 1 adeRS operon increased the tigecycline, omadacycline and eravacycline MICs by 4–8-folds, respectively. Eravacycline demonstrated improved potency over tigecycline against populations of CRAn, including Tn-CRAn isolates. The over-expression of AdeABC efflux pumps was directly activated by the AdeRS two-component system and simultaneously reduced the susceptibilities of tigecycline, eravacycline, and omadacycline. Omadacycline and eravacycline MICs were correlated closely with those of eravacycline.

Herein, positively surface-charged silver nanoparticles (AgNPs) capped with trimethylchitosan nitrate (TMCN) were synthesized using an environmentally friendly method. Nano-sized TMCN-AgNPs (~80 nm) with high zeta potential (>30 mV) provide sufficient static repulsion to stabilize colloid AgNPs in aqueous solutions without aggregation for >3 months. In in vitro cell cycle assays, TMCN-AgNPs showed low cytotoxicity towards L929 cells. A microdilution inhibition assay demonstrated the antifungal potential of TMCN-AgNPs, with a minimum inhibitory concentration of 0.06 mM against Candida tropicalis ATCC 750, and 0.46 mM against both Candida albicans ATCC 76615 and Candida glabrata ATCC 15545. Moreover, the addition of TMCN-AgNPs at 0.23 mM significantly reduced biofilm formation in 96-well plates with C. albicans and C. tropicalis. Importantly, when zebrafish eggs were infected with Candida cells, 0.23 mM TMCN-AgNPs greatly diminished the amount of biofilm on eggs and rescued the survival of embryos by up to 70%.

Elizabethkingia genus is emerging in hospitals and resistant to multiple antibiotics. The intrinsic imipenem resistance of Elizabethkingia genus is related to two chromosome-encoded metallo-beta-lactamases (MBLs), BlaB and GOB. This study was aimed to investigate the in vitro activity of imipenem, vancomycin, and rifampicin in clinical Elizabethkingia species. The distribution and heterogeneity of MBLs responsible for imipenem resistance were also evaluated. A total of 167 Elizabethkingia isolates from different patients were collected, including E. anophelis (142), E. meningoseptica (11), and E. miricola (14). All isolates were evaluated by the broth microdilution assay, ethylenediaminetetraacetic acid (EDTA) combination disk test, and EDTA-based microdilution test. The characteristics of BlaB and GOB were evaluated in phylogenetic analysis and heterologous expression experiments. Most of the isolates were susceptible to rifampin (94%), whereas none of the isolates were susceptible to imipenem. Vancomycin showed intermediate effectiveness. EDTA could reduce 4 folds or more minimum inhibitory concentrations (MICs) of imipenem in 105 isolates (62.9%). Of the isolates, the amino acid sequences of BlaB and GOB were divided into 22 and 25 different types, respectively. Phylogenetic analysis showed BlaB and GOB are species-specific proteins. Furthermore, GOB and BlaB from E. anophelis showed higher imipenem hydrolysis efficiency than those from the other two species. Rifampicin remained the most active agent in the current study. The mechanism of Elizabethkingia resistance to imipenem primarily stemmed from MBLs but other mechanisms could also exist, which requires further investigation.

Herein, we describe an improved procedure for the green synthesis of chondroitin sulfate stabilized silver nanoparticles (ChS-AgNPs). Glucose was used as a reducing agent under alkaline conditions to obtain a small particle size (10 nm), and the reduction was complete within one hour at room temperature. The concentration of NaOH affected the reaction rate, formation yield, and particle size of ChS-AgNPs. The formation of AgNPs was confirmed using UV-vis, TEM, XRD, and XPS. ChS-AgNPs showed excellent catalytic activities in the reduction of 4-nitrophenol by NaBH

Tigecycline (TGC)-resistant extensively drug-resistant Acinetobacter baumannii (XDRAB) is an increasing threat in regard to nosocomial infections. The resistance-nodulation-cell division (RND) efflux pump has played an important role in TGC resistance. In this study, total 81 TGC-resistant XDRAB isolates were analyzed for their responses to the efflux pump inhibitor 1-(1-naphthylmethyl)-piperazine (NMP). We found that NMP could reduce by 4-fold or greater than 4-fold the minimum inhibitory concentration (MIC) of TGC in 45 isolates (55.6 %). After typing with pulsed-field gel electrophoresis (PFGE), group A appeared to be the major cluster with good synergistic response to NMP. Transcripts of the AdeABC efflux pump gene were consistently more correlated with TGC resistance than transcripts of the AdeFGJ or AdeIJK efflux pump genes in these isolates. Of the 81 isolates, the amino acid sequences of AdeR and AdeS were further classified and combined into 31 different codes. Although the dissemination of TGC-resistant XDRAB isolates was genetically diverse in our hospital, their responses to NMP conversion were still strain-dependent. We found that AdeRS combination codes were better than PFGE typing in separating groups of isolates with different sensitivity to NMP conversion.

The chikungunya virus (CHIKV), an arthritogenic alphavirus, has caused explosive epidemics involving millions of cases. Globally expanding pandemics involving CHIKV and post-CHIKV rheumatic disorders are increasing public health concerns. However, no antiviral interventions or vaccines to control CHIKV infection have yet been approved. Although suramin has been possess anti-CHIKV activity in vitro , whether suramin has anti-CHIKV activity in vivo remains unknown. This study aimed to determine whether suramin treatment could ameliorate CHIKV-induced arthritis in a C57BL / 6 mice model. C57BL / 6 mice were infected with CHIKVs to evaluate anti-CHIKV activities of suramin in terms of histopathology, viral burden and disease score. Not only did suramin treatment substantially decrease viral loads, but it also significantly ameliorated acute foot lesions in mice. In addition, suramin treatment markedly restores cartilage integrity and reduces the number of IHC positive chondrocyte in mice infected with CHIKV strains 0810bTw and 0706aTw. This in vivo study highlights the potential ability of suramin to treat CHIKV infection in clinical settings.

Overexpression of the efflux pump AdeABC is associated with tigecycline resistance of multi-drug resistant Acinetobacter baumannii (MDRAB). A two-component regulatory system, sensor AdeS and regulator AdeR proteins regulate the pump. However, the detailed mechanism of the AdeR protein to enhance the expression of adeABC operon is not well defined. We illustrated the biological characteristics of AdeR proteins by comparing a mutant AdeR protein of a tigecycline resistant MDRAB to the wild AdeR protein. By analyzing a series of deletion constructs, a minimal gene cassette of the intercistronic spacer DNA fragment specifically bound with the adeR protein and resulted in band shifting in electrophoresis mobility shifting assays (EMSA). A conserve direct repeat motif was observed in the intercistronic spacer DNA. We demonstrated the AdeR protein was a direct-repeat-binding protein. Two common residue mutations on the AdeR proteins of tigecycline resistant MDRAB isolates could reduce their binding affinity with the intercistronic spacer. The free intercistronic spacer may then more efficiently support the read-through of the adeABC operon during the co-transcriptional translation in tigecycline resistant MDRAB isolates.

Thirty-two vanB2 Enterococcus faecium isolates were found to harbor Tn 5382 . Twenty-four isolates had a 1,419-bp sequence inserted within the open reading frame (ORF) C of Tn 5382 . This 1,419-bp sequence contained a 638-bp ORF with a 72% amino acid sequence homology with the transposase gene of IS 150 . Thirty isolates had the pbp5 gene linked to Tn 5382 .

Thirteen clinical isolates of m ultidrug- r esistant Acinetobacter baumannii r esistant to c arbapenems (MRAB-C) with tigecycline nonsusceptibility were collected from individual patients in this study. None of the 13 isolates shared the same strain characteristics in molecular typing. All of them showed increased adeB transcription, as predicted. However, none of these tigecycline-nonsusceptible MRAB-C isolates were found to possess previously known adeRS mutations. Upregulation of adeB transcription may result from cross stimulation by other mechanisms.

Meticillin-resistant and vancomycin-intermediate Staphylococcus aureus (VISA) has emerged worldwide. However, clonal dissemination of VISA in hospitals has rarely been reported. We investigated 43 isolates of meticillin-resistant VISA [vancomycin minimum inhibitory concentrations (MICs) of 4 microg/mL in 35 isolates and 8 microg/mL in 8 isolates) recovered from 21 hospitalised patients. A glycopeptide was given prior to isolation of VISA in 14 of the patients. Five patients (23.8%) died despite vancomycin therapy. All isolates were inhibited by tigecycline at 0.5 microg/mL, linezolid at 1 microg/mL and ceftobiprole at 2 microg/mL. Five isolates (11.6%) had reduced susceptibility to daptomycin (MICs of 1-2 microg/mL). In addition, 6 of the 43 VISA isolates had decreased susceptibility to autolysis by 0.05% Triton X-100. All 43 VISA isolates carried staphylococcal chromosome cassette mec (SCCmec) type III and accessory gene regulator (agr) group I but none carried the Panton-Valentine leukocidin gene (lukS-lukF). None of the enterococcal van genes were detected in the 43 VISA isolates. Molecular typing generated by pulsed-field gel electrophoresis revealed that all isolates belonged to one pulsotype, indicating clonal dissemination of VISA isolates in the hospital. The high rate of non-susceptibility to daptomycin amongst these VISA isolates is alarming and indicates the limitation of this agent for the treatment of infections due to VISA.

Sternal osteomyelitis is an uncommon complication of Calmette-Guerin bacillus vaccination. Herein we describe a 4-month-old Taiwanese infant with a growing parasternal mass resulting from sternal osteomyelitis. By using DNA sequencing analysis, we identified the etiology as Calmette-Guerin bacillus vaccination.

Over-expression of AdeABC efflux pump stimulated continuously by the mutated AdeRS two component system has been found to result in antimicrobial resistance, even tigecycline (TGC) resistance, in multidrug-resistant Acinetobacter baumannii (MRAB). Although the insertion sequence, ISAba1, contributes to one of the AdeRS mutations, the detail mechanism remains unclear. In the present study we collected 130 TGC-resistant isolates from 317 carbapenem resistant MRAB (MRAB-C) isolates, and 38 of them were characterized with ISAba1 insertion in the adeS gene. The relationship between the expression of AdeABC efflux pump and TGC resistant was verified indirectly by successfully reducing TGC resistance with NMP, an efflux pump inhibitor. Further analysis showed that the remaining gene following the ISAba1 insertion was still transcribed to generate a truncated AdeS protein by the Pout promoter on ISAba1 instead of frame shift or pre-termination. Through introducing a series of recombinant adeRS constructs into a adeRS knockout strain, we demonstrated the truncated AdeS protein was constitutively produced and stimulating the expression of AdeABC efflux pump via interaction with AdeR. Our findings suggest a mechanism of antimicrobial resistance induced by an aberrant cytoplasmic sensor derived from an insertion element.