Phage-Bacterial Interaction Alters Phenotypes Associated with Virulence in Acinetobacter baumannii.

Bacteriophages exert strong selection on their bacterial hosts to evolve resistance. At the same time, the fitness costs on bacteria following phage resistance may change their virulence, which may affect the therapeutic outcomes of phage therapy. In this study, we set out to assess the costs of phage resistance on the in vitro virulence of priority 1 nosocomial pathogenic bacterium, Acinetobacter baumannii. By subjecting phage-resistant variant Ev5-WHG of A. baumannii WHG40004 to several in vitro virulence profiles, we found that its resistance to phage is associated with reduced fitness in host microenvironments. Also, the mutant exhibited impaired adhesion and invasion to mammalian cells, as well as increased susceptibility to macrophage phagocytosis. Furthermore, the whole-genome sequencing of the mutant revealed that there exist multiple mutations which may play a role in phage resistance and altered virulence. Altogether, this study demonstrates that resistance to phage can significantly alter phenotypes associated with virulence in Acinetobacter baumannii.

PHDtools: A platform for pathogen detection and multi-dimensional genetic signatures decoding to realize pathogen genomics data analyses online.

OBJECTIVES: Facing the emerging diseases, rapid identification of the pathogen and multi-dimensional characterization of the genomic features at both isolate-level and population-level through high-throughput sequencing data can provide invaluable information to guide the development of antiviral agents and strategies. However, a user-friendly program is in urgent need for clinical laboratories without bioinformatics background to decode the complex big genomics data. METHODS: In this study, we developed an interactive online platform named PHDtools with a total of 15 functions to analyze metagenomics data to identify the potential pathogen and decode multi-dimensional genetic signatures including intra-/inter-host variations and lineage-level variations. The platform was applied to analyze the meta-genomic data of the samples collected from the 172 imported COVID-19 cases. RESULTS: According to the analytical results of mNGS, 27 patients were found to have the co-infections of SARS-CoV-2 with either influenza virus (n = 9) or human picobirnavirus (n = 19). Enough coverages of all the assembled SARS-CoV-2 genomes provided the sub-lineages of Omicron variant, and the number of mutations in the non-structural genes and M gene was increased, as well as the intra-host variations occurred in E and M gene were under positive selection (Ka/Ks > 1). These findings of increased or changed mutations in the SARS-CoV-2 genome characterized the current adaptive evolution patterns of Omicron sub-lineages, and revealed the evolution speed of these sub-lineages might increase. CONCLUSIONS: Consequently, the application of PHDtools has proved that this platform is accurate, user-friendly and convenient for clinical users who are deficient in bioinformatics, and the clinical monitor of SARS-CoV-2 genomes by PHDtools also highlighted the potential evolution features of current SARS-CoV-2 and indicated that the development of anti-SARS-CoV-2 agents and new-designed vaccines should incorporate the gene variations other than S gene.

Long Non-Coding RNA BCYRN1 Drives Cellular Processes to Promote Asthma Development.

INTRODUCTION: Asthma is a common chronic inflammatory respiratory disease worldwide. The long non-coding RNA (lncRNA) BCYRN1 has been shown to function in the inhibition of smooth muscle cell differentiation and vascular development, but its function and potential molecular mechanisms of lncRNA BCYRN1 in bronchial smooth muscle cells (BSMCs) in asthma remain unknown. METHODS: Quantitative real-time polymerase chain reaction (RT-qPCR) was performed to detect the expression level of lncRNA BCYRN1 in blood and sputum of asthma patients. The effects of lncRNA BCYRN1 on the proliferation and migration of BSMCs were explored by cell counting kit-8, Transwell, colony formation, and flow cytometry analysis. Differentially expressed genes between lncRNA BCYRN1 overexpression and knockdown cells were identified using RNA-seq and verified using RT-qPCR. RESULTS: LncRNA BCYRN1 was upregulated in asthma patients. Overexpression of lncRNA BCYRN1 significantly promoted the proliferation and migration of BSMCs, inhibited cell apoptosis and affected cell cycle arrest, promoting DNA replication. These effects were reversed after lncRNA BCYRN1 inhibition. RNA-seq identified 434 common differentially expressed genes in the lncRNA BCYRN1 overexpression and knockdown groups and verified their expression levels by RT-qPCR. Gene ontology, Kyoto Encyclopedia of Genes and Genomes and Gene set enrichment analysis indicated that these genes were mainly involved in external stimulus, cell cycle, growth factor activity, cytokine interactions, and inflammatory response. CONCLUSION: The identification of the highly expressed lncRNA BCYRN1 in patients with asthma, combined with functional experiments and transcriptional data, suggests that lncRNA BCYRN1 can mediate the development of asthma and can be used as a promising diagnostic and prognostic biomarker for asthma.

Addition of terlipressin to norepinephrine in septic shock and effect of renal perfusion: a pilot study.

PURPOSE: Terlipressin improves renal function in patients with septic shock. However, the mechanism remains unclear. Here, we aimed to evaluate the effects of terlipressin on renal perfusion in patients with septic shock. MATERIALS AND METHODS: This pilot study enrolled patients with septic shock in the intensive care unit of the tertiary hospital from September 2019 to May 2020. We randomly assigned patients to terlipressin and usual care groups using a 1:1 ratio. Terlipressin was intravenously pumped at a rate of 1.3 µg/kg/hour for 24 h. We monitored renal perfusion using renal contrast-enhanced ultrasound (CEUS). The primary outcome was peak sonographic signal intensity (a renal perfusion parameter monitored by CEUS) at 24 h after enrollment. RESULTS: 22 patients were enrolled in this study with 10 in the terlipressin group and 12 in the usual care group. The baseline characteristics of patients between the two groups were comparable. The peak sonographic signal intensity at 24 h after enrollment in the terlipressin group (60.5 ± 8.6 dB) was significantly higher than that in the usual care group (52.4 ± 7.0 dB; mean difference, 7.1 dB; 95% CI, 0.4-13.9; adjusted p = .04). Patients in the terlipressin group had a lower time to peak, heart rates, norepinephrine dose, and a higher stroke volume at 24 h after enrollment. No significant difference in the urine output within 24 h and incidence of acute kidney injury within 28 days was found between the two groups. CONCLUSIONS: Terlipressin improves renal perfusion, increases stroke volume, and decreases norepinephrine dose and heart rates in patients with septic shock.

Steep Decline in Binding Capability of SARS-CoV-2 Omicron Variant (B.1.1.529) RBD to the Antibodies in Early COVID-19 Convalescent Sera and Inactivated Vaccine Sera.

A new SARS-CoV-2 variant B.1.1.529 was named by the WHO as Omicron and classified as a Variant of Concern (VOC) on 26 November 2021. Because this variant has more than 50 mutations, including 30 mutations on the spike, it has generated a lot of concerns on the potential impacts of the VOC on COVID-19. Here through ELISA assays using the recombinant RBD proteins with sequences the same to that of SARS-CoV-2 WIV04 (lineage B.1), the Delta variant and the Omicron variant as the coating antigens, the binding capabilities between the RBDs and the antibodies in COVID-19 convalescent sera and vaccine sera after two doses of the inactivated vaccine produced by Sinopharm WIBP are compared with each other. The results showed that the Omicron variant may evade antibodies induced by the ancestral strain and by the inactivated vaccine, with significant reduction in the binding capability of its RBD much greater than that of the Delta variant.

Genetic Signatures from Adaptation of Bacteria to Lytic Phage Identify Potential Agents To Aid Phage Killing of Multidrug-Resistant Acinetobacter baumannii.

With the increasing morbidity and mortality rates associated with multidrug-resistant bacteria, interest in bacteriophage therapy has been revived. However, bacterial resistance to phage infection threatens the usefulness of phage therapy, especially its inclusion in modern medicine. Multidrug-resistant Acinetobacter baumannii is a top-priority pathogen requiring urgent intervention and new therapeutic approaches, such as phage therapy. Here, we experimentally adapted A. baumannii WHG40004 to its lytic phage P21 and thereafter isolated a phage-resistant bacterial mutant, named Ev5-WHG. We then aimed to identify potential agents to aid phage killing of Ev5-WHG by analyzing its genome and that of the wild-type strain. The enriched Gene Ontology (GO) analysis based on genetic alterations in minor alleles and mutations showed that pathways such as zinc ion transport and cell membrane synthesis could play certain roles in phage resistance. Remarkably, the combination of zinc acetate and P21 showed increased bactericidal effect on Ev5-WHG. Significantly also, we showed that P21 completely prevented the growth of wild-type WHG40004 in the presence of antibiotics (meropenem and imipenem). The results from this study indicate that the analysis of phage resistance signatures during adaptation of bacteria to a lytic phage can inform the choice of agents to work cooperatively with phage to limit and/or reverse resistance. This approach could be important for guiding future successful phage therapy. IMPORTANCE Bacteriophages have proven very useful as alternative therapeutic agents in combating multidrug-resistant bacterial infections; however, bacterial resistance to phages threatens their use. In this study, we showed a new strategy of leveraging genetic signatures that accompany phage resistance in bacteria to predict agents that can be used with lytic phages to combat multidrug-resistant Acinetobacter baumannii. Significantly, this approach was helpful in suggesting the use of zinc acetate to reduce resistance in phage-resistant bacteria, as well as the use of phage with antibiotics meropenem and imipenem to prevent resistance in a wild-type strain of multidrug-resistant A. baumannii. The approach of this study will be helpful for improving the outcome of phage therapy and in overcoming antimicrobial resistance.

Developing an Amplification Refractory Mutation System-Quantitative Reverse Transcription-PCR Assay for Rapid and Sensitive Screening of SARS-CoV-2 Variants of Concern.

With the emergence and wide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs), such as the Delta variant (B.1.617.2 lineage and AY sublineage), it is important to track VOCs for sourcing of transmission. Currently, whole-genome sequencing is commonly used for detecting VOCs, but this is limited by the high costs of reagents and sophisticated sequencers. In this study, common mutations in the genomes of SARS-CoV-2 VOCs were identified by analyzing more than 1 million SARS-CoV-2 genomes from public data. Among them, mutations C1709A (a change of C to A at position 1709) and C56G, respectively, were found in more than 99% of the genomes of Alpha and Delta variants and were specific to them. Then, a method using the amplification refractory mutation system combined with quantitative reverse transcription-PCR (ARMS-RT-qPCR) based on the two mutations was developed for identifying both VOCs. The assay can detect as little as 1 copy/µL of the VOCs, and the results for identifying Alpha and Delta variants in clinical samples by the ARMS-RT-qPCR assay showed 100% agreement with the results using sequencing-based methods. The whole assay can be completed in 2.5 h using commercial fluorescent PCR instruments. Therefore, the ARMS-RT-qPCR assay could be used for screening the two highly concerning variants Alpha and Delta by normal PCR laboratories in airports and in hospitals and other health-related organizations. Additionally, based on the unique mutations identified by the genomic analysis, similar molecular assays can be developed for rapid identification of other VOCs. IMPORTANCE The current stage of the pandemic, led by SARS-CoV-2 variants of concern (VOCs), underscores the necessity to develop a cost-effective and rapid molecular diagnosis assay to differentiate the VOCs. In this study, over 1 million SARS-CoV-2 genomic sequences of high quality from GISAID were analyzed and a network of the common mutations of the lineages was constructed. The conserved unique mutations specific for SARS-CoV-2 VOCs were found. Then, ARMS-RT-qPCR assays based on the two unique mutations of the Alpha and Delta variants were developed for the detection of the two VOCs. Application of the assay in clinical samples demonstrated that the current method is a convenient, cost-effective, and rapid way to screen the target SARS-CoV-2 VOCs.

RNA-binding protein CELF6 is cell cycle regulated and controls cancer cell proliferation by stabilizing p21.

CELF6, a member of the CELF family of RNA-binding proteins, regulates muscle-specific alternative splicing and contributes to the pathogenesis of myotonic dystrophy (DM), however the role of CELF6 in cancer cell proliferation is less appreciated. Here, we show that the expression of CELF6 is cell cycle regulated. The cell cycle-dependent expression of CELF6 is mediated through the ubiquitin-proteasome pathway, SCF-ß-TrCP recognizes a nonphospho motif in CELF6 and regulates its proteasomal degradation. Overexpression or depletion of CELF6 modulates p21 gene expression. CELF6 binds to the 3'UTR of p21 transcript and increases its mRNA stability. Depletion of CELF6 promotes cell cycle progression, cell proliferation and colony formation whereas overexpression of CELF6 induces G1 phase arrest. The effect of CELF6 on cell proliferation is p53 and/or p21 dependent. Collectively, these data demonstrate that CELF6 might be a potential tumor suppressor, CELF6 regulates cell proliferation and cell cycle progression via modulating p21 stability.

Aloin Suppresses Lipopolysaccharide-Induced Inflammatory Response and Apoptosis by Inhibiting the Activation of NF-κB.

Numerous herbal-derived natural products are excellent anti-inflammatory agents. Several studies have reported that aloin, the major anthraquinone glycoside obtained from the Aloe species, exhibits anti-inflammatory activity. However, the molecular mechanism of this activity is not well understood. In this report, we found that aloin suppresses lipopolysaccharide-induced pro-inflammatory cytokine secretion and nitric oxide production, and downregulates the expression of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). Aloin inhibits the phosphorylation and acetylation of the NF-κB p65 subunit by suppressing the upstream kinases p38 and Msk1, preventing LPS-induced p65 translocation to the nucleus. We have also shown that aloin inhibits LPS-induced caspase-3 activation and apoptotic cell death. Collectively, these findings suggest that aloin effectively suppresses the inflammatory response, primarily through the inhibition of NF-κB signaling.

Maternal dietary exposure to selenium nanoparticle led to malformation in offspring.

Selenium (Se) is an essential element and its biological activity is related to its speciation. It is also well-known that in excess it can cause teratogenesis in fish and birds. In this study we compared dietary toxicity of elemental selenium nanoparticles (SeNPs) with selenite and selenomethionine (Se-Met). Japanese medaka (Oryzias latipes) was used as a laboratory model to determine Se effects on adults and their offspring. Adult females were individually exposed using a dry diet fortified with 0, 10 or 20 µg/g of the three Se species for 7 days and then allowed to breed for 3 days. Fertilization rate and the proportion of malformed offspring were examined. The three Se diets led to significant increase in maternal tissue Se concentration in the order of Se-Met >>selenite > SeNP. However, in terms of proportion of malformed offspring, the effect of Se-Met = selenite > SeNP. The malformations included pericardial edema and craniofacial changes, which were typical for Se toxicity. The mismatch of maternal ovary Se concentration and proportion of malformed offspring suggested total Se concentration is a poor predictor of toxicity and teratogenesis. Comparing expression of four genes related to oxidative stress in maternal tissue also showed that there were significant differences in expression patterns between three Se diets in the order of selenite = SeNP > Se-Met. Our results showed that SeNPs cause similar toxicity as other Se species but require further study to elucidate the underlying mechanism.