Development of an Inactivated Vaccine Candidate, BBIBP-CorV, with Potent Protection against SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens global public health. The development of a vaccine is urgently needed for the prevention and control of COVID-19. Here, we report the pilot-scale production of an inactivated SARS-CoV-2 vaccine candidate (BBIBP-CorV) that induces high levels of neutralizing antibodies titers in mice, rats, guinea pigs, rabbits, and nonhuman primates (cynomolgus monkeys and rhesus macaques) to provide protection against SARS-CoV-2. Two-dose immunizations using 2 µg/dose of BBIBP-CorV provided highly efficient protection against SARS-CoV-2 intratracheal challenge in rhesus macaques, without detectable antibody-dependent enhancement of infection. In addition, BBIBP-CorV exhibits efficient productivity and good genetic stability for vaccine manufacture. These results support the further evaluation of BBIBP-CorV in a clinical trial.

Micropeptide PACMP inhibition elicits synthetic lethal effects by decreasing CtIP and poly(ADP-ribosyl)ation.

Synthetic lethality through combinatorial targeting DNA damage response (DDR) pathways provides exciting anticancer therapeutic benefit. Currently, the long noncoding RNAs (lncRNAs) have been implicated in tumor drug resistance; however, their potential significance in DDR is still largely unknown. Here, we report that a human lncRNA, CTD-2256P15.2, encodes a micropeptide, named PAR-amplifying and CtIP-maintaining micropeptide (PACMP), with a dual function to maintain CtIP abundance and promote poly(ADP-ribosyl)ation. PACMP not only prevents CtIP from ubiquitination through inhibiting the CtIP-KLHL15 association but also directly binds DNA damage-induced poly(ADP-ribose) chains to enhance PARP1-dependent poly(ADP-ribosyl)ation. Targeting PACMP alone inhibits tumor growth by causing a synthetic lethal interaction between CtIP and PARP inhibitions and confers sensitivity to PARP/ATR/CDK4/6 inhibitors, ionizing radiation, epirubicin, and camptothecin. Our findings reveal that a lncRNA-derived micropeptide regulates cancer progression and drug resistance by modulating DDR, whose inhibition could be employed to augment the existing anticancer therapeutic strategies.

Hemolymph exosomes inhibit Spiroplasma eriocheiris infection by promoting Tetraspanin-mediated hemocyte phagocytosis in crab.

Exosomes released from infected cells are thought to play an important role in the dissemination of pathogens, as well as in host-derived immune molecules during infection. As an intracellular pathogen, Spiroplasma eriocheiris is harmful to multiple crustaceans. However, the immune mechanism of exosomes during Spiroplasma infection has not been investigated. Here, we found exosomes derived from S. eriocheiris-infected crabs could facilitate phagocytosis and apoptosis of hemocytes, resulting in increased crab survival and suppression of Spiroplasma intracellular replication. Proteomic analysis revealed the altered abundance of EsTetraspanin may confer resistance to S. eriocheiris, possibly by mediating hemocyte phagocytosis in Eriocheir sinensis. Specifically, knockdown of EsTetraspanin in E. sinensis increased susceptibility to S. eriocheiris infection and displayed compromised phagocytic ability, whereas overexpression of EsTetraspanin in Drosophila S2 cells inhibited S. eriocheiris infection. Further, it was confirmed that intramuscular injection of recombinant LEL domain of EsTetraspanin reduced the mortality of S. eriocheiris-infected crabs. Blockade with anti-EsTetraspanin serum could exacerbate S. eriocheiris invasion of hemocytes and impair hemocyte phagocytic activity. Taken together, our findings prove for the first time that exosomes modulate phagocytosis to resist pathogenic infection in invertebrates, which is proposed to be mediated by exosomal Tetraspanin, supporting the development of preventative strategies against Spiroplasma infection.

The novel TERF2::PDGFRB fusion gene enhances tumorigenesis via PDGFRB/STAT5 signalling pathways and sensitivity to TKI in ph-like ALL.

Patients with Philadelphia chromosome-like acute lymphoblastic leukaemia (Ph-like ALL) often face a grim prognosis, with PDGFRB gene fusions being commonly detected in this subgroup. Our study has unveiled a newfound fusion gene, TERF2::PDGFRB, and we have found that patients carrying this fusion gene exhibit sensitivity to dasatinib. Ba/F3 cells harbouring the TERF2::PDGFRB fusion display IL-3-independent cell proliferation through activation of the p-PDGFRB and p-STAT5 signalling pathways. These cells exhibit reduced apoptosis and demonstrate sensitivity to imatinib in vitro. When transfused into mice, Ba/F3 cells with the TERF2::PDGFRB fusion gene induce tumorigenesis and a shortened lifespan in cell-derived graft models, but this outcome can be improved with imatinib treatment. In summary, we have identified the novel TERF2::PDGFRB fusion gene, which exhibits oncogenic potential both in vitro and in vivo, making it a potential therapeutic target for tyrosine kinase inhibitors (TKIs).

Structural and molecular basis for urea recognition by Prochlorococcus.

Nitrogen (N) is an essential element for microbial growth and metabolism. The growth and reproduction of microorganisms in more than 75% of areas of the ocean are limited by N. Prochlorococcus is numerically the most abundant photosynthetic organism on the planet. Urea is an important and efficient N source for Prochlorococcus. However, how Prochlorococcus recognizes and absorbs urea still remains unclear. Prochlorococcus marinus MIT 9313, a typical Cyanobacteria, contains an ABC-type transporter, UrtABCDE, which may account for the transport of urea. Here, we heterologously expressed and purified UrtA, the substrate-binding protein of UrtABCDE, detected its binding affinity toward urea, and further determined the crystal structure of the UrtA/urea complex. Molecular dynamics simulations indicated that UrtA can alternate between "open" and "closed" states for urea binding. Based on structural and biochemical analyses, the molecular mechanism for urea recognition and binding was proposed. When a urea molecule is bound, UrtA undergoes a state change from open to closed surrounding the urea molecule, and the urea molecule is further stabilized by the hydrogen bonds supported by the conserved residues around it. Moreover, bioinformatics analysis showed that ABC-type urea transporters are widespread in bacteria and probably share similar urea recognition and binding mechanisms as UrtA from P. marinus MIT 9313. Our study provides a better understanding of urea absorption and utilization in marine bacteria.

Landscape of T cell epitopes displays hot mutations of SARS-CoV-2 variant spikes evading cellular immunity.

The continuous evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been accompanied by the emergence of viral mutations that pose a great challenge to existing vaccine strategies. It is not fully understood with regard to the role of mutations on the SARS-CoV-2 spike protein from emerging viral variants in T cell immunity. In the current study, recombinant eukaryotic plasmids were constructed as DNA vaccines to express the spike protein from multiple SARS-CoV-2 strains. These DNA vaccines were used to immunize BALB/c mice, and cross-T cell responses to the spike protein from these viral strains were quantitated using interferon-γ (IFN-γ) Elispot. Peptides covering the full-length spike protein from different viral strains were used to detect epitope-specific IFN-γ+ CD4+ and CD8+ T cell responses by fluorescence-activated cell sorting. SARS-CoV-2 Delta and Omicron BA.1 strains were found to have broad T cell cross-reactivity, followed by the Beta strain. The landscapes of T cell epitopes on the spike protein demonstrated that at least 30 mutations emerging from Alpha to Omicron BA.5 can mediate the escape of T cell immunity. Omicron and its sublineages have 19 out of these 30 mutations, most of which are new, and a few are inherited from ancient circulating variants of concerns. The cross-T cell immunity between SARS-CoV-2 prototype strain and Omicron strains can be attributed to the T cell epitopes located in the N-terminal domain (181-246 aa [amino acids], 271-318 aa) and C-terminal domain (1171-1273 aa) of the spike protein. These findings provide in vivo evidence for optimizing vaccine manufacturing and immunization strategies for current or future viral variants.

Mesonia profundi sp. nov., isolated from deep-sea sediment of the Mariana Trench.

A Gram-stain-negative, aerobic, rod-shaped, non-flagellated, non-gliding bacterial strain, designated MT50T, was isolated from a deep-sea sediment sample collected from the Mariana Trench. Optimal growth of strain MT50T was observed at 25 °C, pH 7.0-7.5 and in the presence of 3-5 % (w/v) NaCl. The strain was positive for oxidase and catalase. Phylogenetic analysis of 16S rRNA gene sequences revealed that strain MT50T is affiliated with the genus Mesonia, showing the highest sequence similarity (98.5 %) to the type strain of Mesonia ostreae. The digital DNA-DNA hybridization and average nucleotide identity values between strain MT50T and four closely related type strains of known Mesonia species (14.1-54.8 % and 72.7-86.8 %, respectively) were all below the threshold values to discriminate bacterial species, indicating that strain MT50T is affiliated with a novel species within the genus. The genomic G+C content deduced from the genome of strain MT50T was 36.2 mol%. The major fatty acids of strain MT50T were iso-C15 : 0, iso-C17 : 0 3-OH and anteiso-C15 : 0. The predominant respiratory quinone of the strain was MK-6. The polar lipids of strain MT50T included phosphatidylethanolamine and two unidentified lipids. Based on the polyphasic data presented in this study, strain MT50T represents a novel species of the genus Mesonia, for which the name Mesonia profundi sp. nov. is proposed. The type strain is MT50T (=MCCC 1K07833T=KCTC 92380T).

Identification and characterization of an L-type lectin from obscure puffer Takifugu obscurus in response to bacterial infection.

L-type lectins (LTLs) contain a carbohydrate recognition domain homologous to leguminous lectins, and have functions in selective protein trafficking, sorting and targeting in the secretory pathway of animals. In this study, a novel LTL, designated as ToERGIC-53, was cloned and identified from obscure puffer Takifugu obscurus. The open reading frame of ToERGIC-53 contained 1554 nucleotides encoding 517 amino acid residues. The deduced ToERGIC-53 protein consisted of a signal peptide, a leguminous lectin domain (LTLD), a coiled-coil region, and a transmembrane region. Quantitative real-time PCR showed that ToERGIC-53 was expressed in all examined tissues, with the highest expression level in the liver. The expression of ToERGIC-53 was significantly upregulated after infection with Vibrio harveyi and Staphylococcus aureus. Recombinant ToERGIC-53-LTLD (rToERGIC-53-LTLD) protein could not only agglutinate and bind to one Gram-positive bacterium (S. aureus) and three Gram-negative bacteria (V. harveyi, V. parahaemolyticus and Aeromonas hydrophila), but also bind to glycoconjugates on the surface of bacteria such as lipopolysaccharide, peptidoglycan, mannose and galactose. In addition, rToERGIC-53-LTLD inhibited the growth of bacteria in vitro. All these results suggested that ToERGIC-53 might be a pattern recognition receptor involved in antibacterial immune response of T. obscurus.

Mediating Role of Liver Dysfunction in the Association between Arsenic Exposure and Diabetes in Chinese Adults: A Nationwide Cross-Sectional Study of China National Human Biomonitoring (CNHBM) 2017-2018.

Inconsistent results have been reported regarding the association between low-to-moderate arsenic (As) exposure and diabetes. The effect of liver dysfunction on As-induced diabetes remains unclear. The cross-sectional study included 10,574 adults from 2017-2018 China National Human Biomonitoring. Urinary total As (TAs) levels were analyzed as markers of As exposure. Generalized linear mixed models and restricted cubic splines models were used to examine the relationships among TAs levels, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentrations, and diabetes prevalence. Mediating analysis was performed to assess whether liver dysfunction mediated the association between TAs and diabetes. Overall, the OR (95% CI) of diabetes in participants in the second, third, and fourth quartiles of TAs were 1.08 (0.88, 1.33), 1.17 (0.94, 1.45), and 1.52 (1.22, 1.90), respectively, in the fully adjusted models compared with those in the lowest quartile. Serum ALT was positively associated with TAs and diabetes. Additionally, mediation analyses showed that ALT mediated 4.32% of the association between TAs and diabetes in the overall population and 8.86% in the population without alcohol consumption in the past year. This study suggested that alleviating the hepatotoxicity of As could have implications for both diabetes and liver disease.

Effect of sacubitril valsartan on heart failure with mid-range or preserved ejection fraction in patients on maintenance hemodialysis: real-world experience in a single-center, prospective study.

BACKGROUND: This study aimed to evaluate the effect of sacubitril valsartan (SV) on heart failure (HF) hospitalization and cardiovascular mortality in patients on hemodialysis with HF with preserved ejection fraction (EF; HFpEF). METHODS: This single-center, prospective study enrolled 155 stable hemodialysis patients with EF > 40% who were followed up for 12 months. Fifty-nine patients were treated with SV; the others were matched for EF (57.89 ± 9.35 vs. 58.00 ± 11.82, P = 0.9) at a ratio of 1:1 and included as controls. The target dosage of SV was 200 mg/day. RESULTS: Twenty-three (23/155; 14.84%) had HF with mid-range EF (HFmrEF), while 132 (85.16%) had HFpEF. After SV treatment, the peak early diastolic transmitral flow velocity/peak early diastolic mitral annular tissue velocity(E/e') improved from 17.19 ± 8.74 to 12.80 ± 5.52 (P = 0.006), the left ventricular (LV) end-diastolic diameter decreased from 53.14 ± 7.67 mm to 51.56 ± 7.44 mm (P = 0.03), and the LV mass index decreased from 165.7 ± 44.6 g/m2 to 154.8 ± 24.0 g/m2 (P = 0.02). LVEF (P = 0.08) and LV global longitudinal strain (P = 0.7) did not change significantly. The composite outcome of first and recurrent HF hospitalization or cardiovascular death showed no difference between group. However, the Acute Dialysis Quality Initiative Workgroup (ADQI) HF class improved in 39 and 15 patients and worsened in 1 and 11 patients in the SV and control groups, respectively (P < 0.001). Age, diabetes mellitus, and pulmonary arterial pressure were independent risk factors for HF hospitalization and cardiovascular mortality in patients with HFpEF. CONCLUSIONS: SV improved LV hypertrophy, diastolic function, and the ADQI class for HF; however, it failed to reduce the composite endpoints of HF hospitalization and cardiovascular disease-related mortality over 12 months of follow-up in patients on maintenance hemodialysis with EF of > 40%.

Impact of COVID-19 pandemic responses on tuberculosis incidence: insights from Shantou, China.

BACKGROUND: Various measures taken against the COVID-19 pandemic are not only effective in reducing the spread of the disease, but also lead to some unexpected results. This article regarded these measures as an intervention and explored their impact on the incidence of tuberculosis in Shantou, China. METHODS: The incidence rate and the surveillance data of tuberculosis from January 1st, 2018 to December 31st, 2021 were provided by the Shantou Tuberculosis Prevention and Control Institute. Data were divided into pre-pandemic period (January 1st, 2018 - December 31st, 2019) and pandemic periods (January 1st, 2020 - December 31st, 2021). The Interrupted Time Series (ITS) was used to analyze the trend of tuberculosis incidence prior to and during the COVID-19 epidemic. RESULTS: The results showed that the incidence of tuberculosis cases in Shantou decreased significantly (p < 0.05) during the pandemic as compared to that prior to the pandemic. Among them, the 45-64 age group and the 65 + age group have statistically significant declines. When patients were stratified by occupation, the unemployed and those working in agriculture reduced the most. CONCLUSIONS: In response to the pandemic, measures like lockdowns and quarantines seem to have reduced tuberculosis incidence. However, this does not imply a true decrease. Underlying causes for the reduced true incidence need further scrutiny. Findings offer a preliminary exploration of interventions designed for one disease but functioning as unexpected results for another.

Percutaneous Intra-arterial Hyaluronidase Injection for Hyaluronic Acid Filler Embolism Threatening Skin Barrier Integrity: Implementation of a Stepwise Treatment Protocol.

BACKGROUND: Hyaluronic acid (HA) filler-induced vascular embolism that threatens skin integrity is an urgent situation. There is increasing evidence that percutaneous intra-arterial hyaluronidase injection is an effective therapeutic technique for it. However, until now, there is a lack of a unifying protocol about the technique. OBJECTIVES: This study aims to provide a conclusion of percutaneous intra-arterial hyaluronidase injection along with adjunctive measures on the treatment of occlusions precipitated by HA-based filler and develop a stepwise treatment protocol. METHODS: We searched PubMed for peer-reviewed studies, consensus statements, case series, and case reports using a variety of keywords. RESULTS: High-dose, pulsed hyaluronidase is the mainstay for the treatment of HA filler-induced embolism, but percutaneous intra-arterial hyaluronidase injection is a more effective technique. Until now, hyaluronidase is injected into three arteries percutaneously, including facial artery, supratrochlear artery, and superficial temporal artery. Furthermore, the adjunctive measures that may optimize clearance of an occlusion and/or skin barrier repair such as the use of image guidance and CGF should be considered. CONCLUSION: Vascular occlusions that threaten skin integrity are an urgent matter which requires accurate diagnosis and effective intervention. Percutaneous intra-arterial hyaluronidase injection along with adjunctive measures performed in a stepwise manner is key to an optimal outcome. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Dapansutrile Ameliorates Atrial Inflammation and Vulnerability to Atrial Fibrillation in HFpEF Rats.

BACKGROUND: Numerous studies have demonstrated that NLRP3 inflammasomes are key players in the progression of atrial fibrillation (AF) in heart failure with preserved ejection fraction (HFpEF). This study aimed to analyse the effect of pharmacological inhibition of NLRP3 inflammasomes using dapansutrile (DAPA), an oral NLRP3-specific inhibitor. METHODS: Dahl salt-sensitive rats were fed a high-salt diet (HSD, 8% NaCl) to induce HFpEF. Either DAPA (200 mg/kg/day) or saline was administered daily via gavage for 4 weeks. Electrophysiological studies were performed to assess the AF inducibility. Confocal fluorescence microscopy and western blot analysis were used to study calcium handling. RESULTS: The DAPA-treated HFpEF rats were less prone to AF induction by programmed electrical stimulation. Atrial fibrosis and inflammation were attenuated in DAPA-treated HFpEF hearts. Dapansutrile treatment showed an increase in the Ca2+ transient sarcoplasmic reticulum-Ca2+ load, and protein expression of SERCA2; NCX1 and phosphorylation of PLB at Thr17 were decreased following DAPA treatment. The increased frequency of spontaneous Ca2+ spark in the HFpEF rats was related to the hyperphosphorylation of RyR2 at Ser2814, which was blunted in DAPA treatment. Dapansutrile treatment also decreased the phosphorylation of CaMKII expression in the HFpEF rats. Mechanistically, DAPA exerts an anti-arrhythmic effect, mainly by inhibiting activation of the NLRP3 inflammasome. CONCLUSION: These data provide evidence that the beneficial cardiac effects of DAPA are associated with reduced atrial inflammation and improved CaMKII-dependent Ca2+-handling abnormalities via blunting activation of the NLRP3 inflammasome, and DAPA may be beneficial in a rat model of HFpEF-induced AF.

Targeting NAD Metabolism: Rational Design, Synthesis and In Vitro Evaluation of NAMPT/PARP1 Dual-Target Inhibitors as Anti-Breast Cancer Agents.

The malignancy of breast cancer poses a global challenge, with existing treatments often falling short of desired efficacy. Extensive research has underscored the effectiveness of targeting the metabolism of nicotinamide adenine dinucleotide (NAD), a pivotal molecule crucial for cancer cell survival and growth, as a promising anticancer strategy. Within mammalian cells, sustaining optimal NAD concentrations relies on two key enzymes, namely nicotinamide phosphoribosyltransferase (NAMPT) and poly(ADP-ribose) polymer 1 (PARP1). Recent studies have accentuated the potential benefits of combining NAMPT inhibitors and PARP1 inhibitors to enhance therapeutic outcomes, particularly in breast cancer. In this study, we designed and synthesized eleven novel NAMPT/PARP1 dual-target inhibitors. Among them, compound DDY02 exhibited acceptable inhibitory activities against both NAMPT and PARP1, with IC50 values of 0.01 and 0.05 µM, respectively. Moreover, in vitro evaluations revealed that treatment with DDY02 resulted in proliferation inhibition, NAD depletion, DNA damage, apoptosis, and migration inhibition in MDA-MB-468 cells. These results posit DDY02, by targeting NAD metabolism through inhibiting both NAMPT and PARP1, as a promising lead compound for the development of breast cancer therapy.

Hyperbaric oxygen therapy promotes the browning of white fat and contributes to the healing of diabetic wounds.

Non-healing wounds are one of the chronic complications of diabetes and have remained a worldwide challenge as one of the major health problems. Hyperbaric oxygen (HBO) therapy is proven to be very successful for diabetic wound treatment, for which the molecular basis is not understood. Adipocytes regulate multiple aspects of repair and may be therapeutic for inflammatory diseases and defective wound healing associated with aging and diabetes. Endothelial cell-derived extracellular vesicles could promote wound healing in diabetes. To study the mechanism by which HBO promotes wound healing in diabetes, we investigated the effect of HBO on fat cells in diabetic mice. A diabetic wound mouse model was established and treated with HBO. Haematoxylin and eosin (H&E) staining and immunofluorescence were used for the analysis of wound healing. To further explore the mechanism, we performed whole-genome sequencing on extracellular vesicles (EVs). Furthermore, we conducted in vitro experiments. Specifically, exosomes were collected from human umbilical vein endothelial cell (HUVEC) cells after HBO treatment, and then these exosomes were co-incubated with adipose tissue. The wound healing rate in diabetic mice treated with HBO was significantly higher. HBO therapy promotes the proliferation of adipose precursor cells. HUVEC-derived exosomes treated with HBO significantly promoted fat cell browning. These data clarify that HBO therapy may promote vascular endothelial cell proliferation and migration, and promote browning of fat cells through vascular endothelial cells derived exosomes, thereby promoting diabetic wound healing. This provides new ideas for the application of HBO therapy in the treatment of diabetic trauma.

Performance of the MeltPro TB assay as initial test for diagnosis of pulmonary tuberculosis with drug-resistance detection.

BACKGROUND: The MeltPro TB assay (MeltPro) is a molecular rapid diagnostic test designed for detecting resistance to antituberculosis drugs. However, the performance of MeltPro as an initial diagnostic test for simultaneously detecting the presence of Mycobacterium tuberculosis (MTB) and drug resistance has not been evaluated. This study aims to assess the performance of MeltPro as initial diagnostic test for simultaneous detection of MTB and drug resistance in clinical samples from patients with presumptive pulmonary tuberculosis (PTB). METHODS: A retrospective analysis was conducted on 1283 patients with presumptive PTB from two clinical centers, out of which 875 were diagnosed with PTB. The diagnostic accuracy of MeltPro, Xpert MTB/RIF (Xpert), and MGIT 960 for PTB detection was evaluated. Rifampicin (RIF), isoniazid (INH), ethambutol (EMB), streptomycin (STR), and fluoroquinolone (FQ) resistance were detected using MeltPro, with Xpert and/or the broth microdilution plate method (MYCOTB) results as references. RESULTS: For the diagnosis of PTB, MeltPro showed a sensitivity of 69.0%, which was similar to Xpert (72.7%; P > 0.05) and higher than MGIT (58.1%; P < 0.001). The specificity of MeltPro was 97.1%, similar to Xpert (98.0%; P > 0.05). In smear-negative patients, MeltPro's sensitivity was 50.9%, similar to Xpert (56.5%; P > 0.05), and higher than MGIT (33.1%; P < 0.001). Based on Xpert and/or MYCOTB results, MeltPro exhibited a sensitivity and specificity of 98.3% and 99.2%, respectively, for detecting RIF resistance. Based on MYCOTB results, MeltPro's sensitivity for detecting resistance to INH, EMB, STR, and FQ was 96.4%, 89.1%, 97.5%, and 90.3%, respectively, with specificities of 96.0%, 96.0%, 95.2%, and 99.4%, respectively. CONCLUSION: The MeltPro TB assay could potentially be an effective alternative as the initial test for rapid diagnosis of PTB with drug-resistance detection in clinical practice.

Biofilm formation stabilizes metabolism in a Roseobacteraceae bacterium under temperature increase.

Ocean warming profoundly impacts microbes in marine environments; yet, how lifestyle (e.g., free living versus biofilm associated) affects the bacterial response to rising temperature is not clear. Here, we compared transcriptional, enzymatic, and physiological responses of free-living and biofilm-associated Leisingera aquaemixtae M597, a member of the Roseobacteraceae family isolated from marine biofilms, to the increase in temperature from 25℃ to 31℃. Complete genome sequencing and metagenomics revealed the prevalence of M597 in global ocean biofilms. Transcriptomics suggested a significant effect on the expression of genes related to carbohydrate metabolism, nitrogen and sulfur metabolism, and phosphorus utilization of free-living M597 cells due to temperature increase, but such drastic alterations were not observed in its biofilms. In the free-living state, the transcription of the key enzyme participating in the Embden-Meyerhof-Parnas pathway was significantly increased due to the increase in temperature, accompanied by a substantial decrease in the Entner-Doudoroff pathway, but transcripts of these glycolytic enzymes in biofilm-forming strains were independent of the temperature variation. The correlation between the growth condition and the shift in glycolytic pathways under temperature change was confirmed by enzymatic activity assays. Furthermore, the rising temperature affected the growth rate and the production of intracellular reactive oxygen species when M597 cells were free living rather than in biofilms. Thus, biofilm formation stabilizes metabolism in M597 when grown under high temperature and this homeostasis is probably related to the glycolytic pathways.IMPORTANCEBiofilm formation is one of the most successful strategies employed by microbes against environmental fluctuations. In this study, using a marine Roseobacteraceae bacterium, we studied how biofilm formation affects the response of marine bacteria to the increase in temperature. This study enhances our understanding of the function of bacterial biofilms and the microbe-environment interactions in the framework of global climate change.

SARS-CoV-2 evolves to reduce but not abolish neutralizing action.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) have prolonged coronavirus disease 2019 (COVID-19) pandemic by escaping pre-existing immunity acquired by natural infection or vaccination. Elucidation of VOCs' mutation trends and evasion of neutralization is required to update current control measures. Mutations and the prevalence of VOCs were analyzed in the global immunization coverage rate context. Lentivirus-based pseudovirus neutralization analysis platforms for SARS-CoV-2 prototype strain (PS) and VOCs, containing Alpha, Beta, Gamma, Delta, and Omicron, were constructed based on the spike protein of each variant and HEK 293T cell line expressing the human angiotensin-converting enzyme 2 (hACE2) receptor on the surface, and an enhanced green fluorescent protein reporter. Serum samples from 65 convalescent individuals and 20 WIBP-CorV vaccine recipients and four therapeutic monoclonal antibodies (mAbs) namely imdevimab, casirivimab, bamlanivimab, and etesevimab were used to evaluate the neutralization potency against the variants. Pseudovirus-based neutralization assay platforms for PS and VOCs were established, and multiplicity of infection (MOI) was the key factor influencing the assay result. Compared to PS, VOCs may enhance the infectivity of hACE2-293T cells. Except for Alpha, other VOCs escaped neutralization to varying degrees. Attributed to favorable and emerging mutations, the current pandemic Omicron variant of all VOCs demonstrated the most significant neutralization-escaping ability to the sera and mAbs. Compared with the PS pseudovirus, Omicron had 15.7- and 3.71-fold decreases in the NT50 value (the highest serum dilution corresponding to a neutralization rate of 50%); and correspondingly, 90% and 43% of immunization or convalescent serum samples lost their neutralizing activity against the Omicron variant, respectively. Therefore, SARS-CoV-2 has evolved persistently with a strong ability to escape neutralization and prevailing against the established immune barrier. Our findings provide important clues to controlling the COVID-19 pandemic caused by new variants.

DNA-free CRISPR-Cas9 gene editing of wild tetraploid tomato Solanum peruvianum using protoplast regeneration.

Wild tomatoes (Solanum peruvianum) are important genomic resources for tomato research and breeding. Development of a foreign DNA-free clustered regularly interspaced short palindromic repeat (CRISPR)-Cas delivery system has potential to mitigate public concern about genetically modified organisms. Here, we established a DNA-free CRISPR-Cas9 genome editing system based on an optimized protoplast regeneration protocol of S. peruvianum, an important resource for tomato introgression breeding. We generated mutants for genes involved in small interfering RNAs biogenesis, RNA-DEPENDENT RNA POLYMERASE 6 (SpRDR6), and SUPPRESSOR OF GENE SILENCING 3 (SpSGS3); pathogen-related peptide precursors, PATHOGENESIS-RELATED PROTEIN-1 (SpPR-1) and PROSYSTEMIN (SpProSys); and fungal resistance (MILDEW RESISTANT LOCUS O, SpMlo1) using diploid or tetraploid protoplasts derived from in vitro-grown shoots. The ploidy level of these regenerants was not affected by PEG-Ca2+-mediated transfection, CRISPR reagents, or the target genes. By karyotyping and whole genome sequencing analysis, we confirmed that CRISPR-Cas9 editing did not introduce chromosomal changes or unintended genome editing sites. All mutated genes in both diploid and tetraploid regenerants were heritable in the next generation. spsgs3 null T0 regenerants and sprdr6 null T1 progeny had wiry, sterile phenotypes in both diploid and tetraploid lines. The sterility of the spsgs3 null mutant was partially rescued, and fruits were obtained by grafting to wild-type (WT) stock and pollination with WT pollen. The resulting seeds contained the mutated alleles. Tomato yellow leaf curl virus proliferated at higher levels in spsgs3 and sprdr6 mutants than in the WT. Therefore, this protoplast regeneration technique should greatly facilitate tomato polyploidization and enable the use of CRISPR-Cas for S. peruvianum domestication and tomato breeding.

Effect of proximal artery restriction on flow reduction and cardiac function in hemodialysis patients with high-flow arteriovenous fistulas.

OBJECTIVE: Arteriovenous fistula is the preferred vascular access for hemodialysis patients. High-flow arteriovenous fistula may cause high-output heart failure. Various procedures are used to reduce high-flow arteriovenous fistula. This study aimed to assess the efficacy of proximal artery restriction combined with distal artery ligation on flow reduction for high-flow arteriovenous fistula and on cardiac function and echocardiographic changes in patients undergoing hemodialysis. METHODS: A retrospective analysis was performed on data collected from the medical records of patients undergoing hemodialysis with heart failure and high-flow arteriovenous fistula between May 2018 and May 2021. Thirty-one patients were treated with proximal artery restriction (banding juxta-anastomosis of the proximal artery) combined with distal artery ligation (anastomosis distal artery ligation). Changes in the Acute Dialysis Quality Initiative Workgroup cardiac function class, blood pressure, and echocardiography before and 6 months after flow restriction were compared, and post-intervention primary patency was followed-up. RESULTS: The technical success rate of the surgery was 100%, and no surgery-related adverse events occurred. Blood flow and blood flow/cardiac output decreased significantly after flow restriction. Blood flow decreased from 2047.21 ± 398.08 mL/min to 1001.36 ± 240.42 mL/min, and blood flow/cardiac output decreased from 40.18% ± 6.76% to 22.34% ± 7.21% (P < .001). Post-intervention primary patency of arteriovenous fistula at 6, 12, and 24 months was 96.8%, 93.5%, and 75.2%, respectively. The Acute Dialysis Quality Initiative Workgroup cardiac function class improved significantly after 6 months of flow restriction (P < .001). The systolic and diastolic left heart function improved, as evidenced by a significant decrease in left atrial volume index, left ventricular end-diastolic/end-systolic diameters, left ventricular end-diastolic volume, left ventricular mass index, cardiac output, and cardiac index and an increase in lateral peak velocity of longitudinal contraction, average septal-lateral s', and lateral early diastolic peak velocity after flow restriction (P < .05). Systolic pulmonary artery pressure decreased from 32.36 ± 8.56 mmHg to 27.57 ± 8.98 mmHg (P < .05), indicating an improvement in right heart function. CONCLUSIONS: Proximal artery restriction combined with distal artery ligation effectively reduced the blood flow of high-flow arteriovenous fistula and improved cardiac function.