Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has infected >600 million people in the ongoing global pandemic. Several variants of the SARS-CoV-2 have emerged in the last >2 years, challenging the continued efficacy of current COVID vaccines. Therefore, there is a crucial need to investigate a highly cross-protective vaccine effective against variants of SARS-CoV-2. In this study, we examined seven lipopeptides derived from highly conserved, immunodominant epitopes from the S, N, and M proteins of SARS-CoV-2, that are predicted to contain epitopes for clinically protective B cells, helper T cells (TH) and cytotoxic T cells (CTL). Intranasal immunization of mice with most of the lipopeptides led to significantly higher splenocyte proliferation and cytokine production, mucosal and systemic antibody responses, and induction of effector B and T lymphocytes in both lungs and spleen, compared to immunizations with the corresponding peptides without lipid. Immunizations with Spike-derived lipopeptides led to cross-reactive IgG, IgM and IgA responses against Alpha, Beta, Delta, and Omicron Spike proteins as well as neutralizing antibodies. These studies support their potential for development as components of a cross-protective SARS-CoV-2 vaccine. Recent reports suggest that nAb responses, elicited by vaccines and/or natural infections, is an accurate predictor of protection against SARS-CoV-2 –5. Gener+/CD8+ T cells have shown to induce cross-reactive immunity that is resistant to mutations acquired by SARS-CoV-2 variants of concerns (VOCs) and effectively preventing VOC escape  and membrane (M) proteins, in addition to the spike protein . FurtherC escape . For a bin vitro and in vivo assays. We found that lipopeptides existed as larger micelle-like particles compared to their peptide counterpart, and stimulated APCs by the upregulation CD40, CD86 and HLA-DR molecules without the activation of TLR-2 and TLR-4 receptors. Intranasal immunizations with individual lipopeptides in mice generated stronger antigen-specific cellular, and mucosal as well as systemic humoral immune responses, compared to their native peptide immunizations. In addition, we provide evidence of the induction of cross-protective immunity upon mucosal immunization with the designed lipopeptides.In this study, we identified seven highly conserved, cross-reactive, immunodominant peptides from the S, N, and M proteins of SARS-CoV-2, MERS-CoV, SARS-CoV and other common cold coronaviruses that are predicted to contain epitopes for clinically protective B cells, helper T cells (TH) and cytotoxic T cells (CTL), and which also bind to multiple MHC class I and II molecules (covering >98% of human population) Table 1 1, P3, P5, P7, P9, P11, and P13, ranged from 134.2-229.8 nm, 356.2-2792 nm, 79.32-309.9 nm, 1256- 4033 nm, 125.6- 1510 nm, 246.2-1856 nm, and 361-3554 nm, respectively , P4 (41.7%), P5 (7.1%), P6 (5.7%), P7 (9.6%), and P8 (17.3%) (3 (174.0%), P5 (145.5%), P6 (9.4%), P7 (15.9%), P8 (23.5%), P9 (13.9%), P10 (26.2%), P12 (4.1%), and P13 (2.4%) (5 (110.0%), P7 (7.9%), P8 (244.7%), P9 (400.7%), P10 (386.9%), P11 (332.1%), P12 (267.6%), P13 (340.0%), and P14 (289.7%) (1 (p=0.0028), P3 (p=0.0291), P5 (0.0053), P9 (p<0.0001), P11 (p=0.0015), and P13 (p=0.0005), and native peptide stimulation with P8 (p=0.002), P10 (p<0.0001), P12 (p=0.0056), and P14 (p=0.0029). THP-1 cells stimulated with lipopeptides, or peptides did not produce detectable levels of proinflammatory cytokines  or stimulate human TLR-2 and -4 receptors . We found that Pnd immunization, spleens were examined for antigen-specific proliferation responses using a colorimetric BrdU incorporation assay. Splenocytes from all immunized groups were also stimulated with ConA (T cell mitogen), which was used as a positive control  and P2   twice, 14 days apart. Eight days after the 21-P14, that were restimulated ex vivo with their respective native peptides (1 μg/ml). We found that splenocytes from lipopeptide immunizations, P1, P3, P9, and P13, produce higher levels of IFN-γ, TNF-α, IL-2, and IL-4, compared to their respective peptide immunization groups , TNF-α , and IL-2  significantly correlated with antigen-specific splenocyte proliferation responses , and serum samples collected from P5 (p<0.0001), P6 (p<0.0001), P7 (p=0.0041), P9 (p<0.0001), and P10 (p=0.0004) immunizations elicited a significantly higher anti-(S-N fusion)-IgA antibody response, compared to unimmunized controls (7 (p=0.0011) and P9 (p=0.0242) induced significantly higher anti-(S-N fusion)-IgA antibody titers, compared to their respective peptide immunizations. P5 induced a significantly higher anti-(S-N fusion)-IgA titer at a 1:2 dilution, compared to the P6 (p<0.0001) (5-P10) led to a significant anti-(N)-IgA antibody titer, compared to unimmunized controls -IgA titers compared to their respective peptide, with p<0.0001 and p=0.0347, respectively. Nucleocapsid-derived lipopeptide immunizations showed a strong mucosal anti-(S-N fusion)-IgA and anti-(N)-IgA antibody response compared to their respective nucleocapsid-derived peptide immunization. In contrast, membrane-derived lipopeptide and peptide (P11-P14) immunizations did not elicit anti-(M)-IgA antibodies (BAL fluid from P1-P14 immunized mice (1-P4) and nucleocapsid-derived (P5-P10) lipopeptide and peptide immunizations produced significant amounts of anti-(S-N fusion)-IgM antibodies (5-P10 immunized mice induced significantly higher anti-(N)-IgM antibodies, compared to the unimmunized controls (7 immunization induced a significantly higher anti-(S-N fusion)-IgM and anti-(N)-IgM antibody response, compared to its native peptide immunization . Also, P9 immunization induced a significantly higher anti-(N)-IgM antibody titer, compared to P10 (p<0.0001). Membrane-derived lipopeptide and peptide immunizations with P11 and P12 produced significantly higher anti-(M)-IgM antibody titers, compared to unimmunized controls (p<0.0001) -IgG antibody titres, compared to unimmunized controls (p<0.0001) (1 was significantly higher compared to its native peptide immunization (p<0.0001). Notably, P3 and P4 immunizations produced the highest anti-(S-N fusion)-IgG antibody titer compared to all other immunization groups. All nucleocapsid-derived lipopeptide and peptide immunizations have significantly higher anti-(N)-IgG antibody titres, compared to unimmunized controls (p<0.0001) (5 and P9 immunizations had significantly higher anti-(N)-IgG antibody titres, compared to their respective native peptide immunizations, with p=0.0036 and p=0.0089, respectively. Furthermore, we found that anti-(M)-IgG antibodies were only produced by P11 immunizations (1-P4), and nucleocapsid- (P5-P10) derived lipopeptide and peptide immunizations to their respective antigens.Lastly, we measured systemic IgG antibody responses against the S-N fusion, N, and M proteins of SARS-CoV-2. Immunizations with spike-derived and nucleocapsid-derived lipopeptides and peptides induced strong IgG antibody responses to their respective antigens. P1-P4) lipopeptides and peptides against SARS-CoV-2 VOCs: Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617), and Omicron (B.1.1.529). P1 immunization showed significantly higher mucosal IgA antibody titers, compared to unimmunized controls and its respective peptide immunization, against all SARS-CoV-2 VOCs  , B.1.351 (p=0.0049), B.1.617 (p=0.0188), and B.1.1.529 (p<0.0001) variants of SARS-CoV-2   and B.1.617 (p=0.0088) variants , B.1.351 (p<0.0001), and B.1.1.529 (p=0.0059). Similarly, P3 immunizations induced significantly higher IgG antibody titers compared to P4, against the B.1.1.7, B.1.351, B.1.617, and B.1.1.529 variants (p<0.0001). Evidently, the spike-derived lipopeptide immunizations induced a more robust cross-variant IgG antibody response, compared to their respective peptide immunizations.From serum samples collected from P1-P4 immunizations were tested for their ability to block/neutralize viral binding to target host ACE2 receptor, using a cPass SARS-CoV-2 Neutralization kit. Serum from a non-spike lipopeptide immunization, P5, was used as a negative control. Serum antibody responses elicited by P1 and P3 immunizations showed a percent inhibition of 23.5% and 22.0%, respectively  had significantly higher neutralizing capabilities compared to their respective peptides . Next, we found that BAL antibody responses from P1 (23.7%) and P2 (20.3%) immunization showed significantly higher neutralizing capabilities compared to P5 , whereas P3 (12.1%) and P4 (12.2%) immunizations showed similar neutralization capability.Antibody responses in BAL and serum samples collected from Pectively Figure 6+CD3-), helper T cells , and cytotoxic T cells  to identify functional changes among the immunization groups. Using clustering analysis, we identified cell clusters (Pop#) within B cells, TH and CTLs , TH cells with B cell and CTL helper function (3.0% vs. 0.0%), CTL helper function (43.9% vs. 12.7%), and B cell helper function (34.3% vs. 0.8%), and CTLs with effector (25.1% vs. 20.5%) and pleiotropic (46.2% vs. 33.6%) properties, compared to peptide immunizations , and higher percentage of regulatory B cells (45.0% vs. 31.1%), compared to peptide immunizations  activated APCs more efficiently, compared to their respective peptides, enabling the integration of the innate and adaptive systems without the need of an adjuvant. Furthermore, we found that lipopeptides did not stimulate TLR-2 and -4 receptors or produce detectable amounts of proinflammatory cytokines (The upregulation of antigen-presenting (HLA-DR) and costimulatory (CD40/CD86) molecules demonstrate enhanced capacity to present antigen on APCs and prime T cells. Interestingly, we found that lipopeptides derived from SARS-CoV-2 antigens led to higher upregulation of HLA-DR, CD40, and CD86 in comparison to the corresponding peptides and medium-alone control , 36. Thie system . Our resytokines . These p1, P3, P5, P9, and P13) induced significantly higher recall responses, upon ex vivo re-stimulation by corresponding lipopeptides and native peptides, compared to peptide immunizations  may drive T cells into anergic states. Anergic conditions are not desirable for a vaccine as it demonstrates the failure of our immune system to mount a response against the targeted antigen  and TH2 (IL-4) cytokines have been suggested to modulate immune responses towards cell-mediated or humoral immunity. IFN-γ functions to educate immune cells to recognize and eliminate pathogens, and associates with protective immunity against intracellular pathogens  marked the presence of long-lasting, antigen-specific cellular responses  and cellular (cytotoxic T cell clusters) immune responses  demonstrated a cross-reactive mucosal IgA and systemic IgM/IgG responses against four different SARS-CoV-2 VOCs   responses, compared to the corresponding peptide groups Figure 4he blood . The indIn conclusion, our results clearly demonstrate that intranasal immunization with lipopeptides, even without any added adjuvant, lead to significant induction of mucosal antibody responses and systemic cellular and humoral immune responses. This study describes an important and innovative finding that opens new avenues for developing a broadly protective vaccine for SARS-CoV-2 virus and its variants, and potentially other heterologous coronaviruses.1, P3, P5, P7, P9, P11, and P13) and their corresponding native peptides , derived from highly conserved and functional regions of the S-, N- and M-proteins of SARS-CoV-2, were custom synthesized by Genscript Inc.  with >96% purity , and a standardized Sizing and Zeta report was generated by Zetasizer XPLORER software v1.10 to provide mean particle size for each sample.6 cells/ml) were seeded with P1-P14, at 1 μg/ml. The cultures were incubated in 5% CO2, at 37°C for 24 hours. The activation of THP-1 monocytes was determined by flow cytometry analysis. A total of 2×105 THP-1 cells from each culture was stained with co-stimulatory markers  using established procedures from Thermofisher , and subsequently, performed a percent of control calculation (Percent of Control =100 × iMFIExp./iMFIControl) for graphing (THP-1 monocytes (ATCC TIB-202) were grown in medium containing RPMI-1640 (Gibco), 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (P/S), and 1% L-glutamine, and maintained at a confluency between 70-80%. THP-1 cells  cell lines are stably transfected with human TLR2 and TLR4 genes. Upon TLR signalling, a secreted embryonic alkaline phosphatase (SEAP) reporter gene is induced, and SEAP is secreted, which is detected by a QUANTI-Blue™ detection assay.5 cells/ml) and HEK-Blue™ hTLR4 (2.5×105 cells/ml) cells were seeded with P1-P14, at 1 μg/ml and 10 μg/ml, in triplicates. The cultures were incubated in 5% CO2, at 37°C for 24 hours, and supernatants were collected to run QUANTI-Blue™ detection according to the assay protocol. Using a DTX 880 Plate Reader (Beckman Coulter), optical density (OD) readings were taken of QUANTI-Blue™ detection assays, at 620nm. Data was expressed as mean ± SEM of triplicate cultures.HEK-Blue™ hTLR2 and HEK-Blue™ hTLR4 cell lines were grown according to their respective handling procedures. HEK-Blue™ hTLR2  for Health Sciences and were conducted according to the guidelines of the Canadian Council of Animal Care (CCAC). Four to six-week-old male C57BL/6 mice were purchased from Charles River Laboratory and housed in a pathogen-free animal facility (HSLAS) at the University of Alberta. Mice were immunized twice, 14 days apart with individual lipopeptides or peptides (10 μg/mouse), intranasally in a total volume of 30 μL (15 μL in each nostril). Mice were euthanized 8 days after second immunization and bronchoalveolar lavages (BAL), sera, lungs, and spleens were collected. Unimmunized mice were used as controls.5 cells splenocytes from immunized mice and 4×105 antigen presenting cells  were incubated with corresponding lipopeptides and peptides at various concentrations described in the figure legends  and isolated lung lymphocytes (1×105 cells) from immunized mice were stained with extracellular  and intracellular markers  using established protocols from Thermofisher  1X for 4h at 37°C and subsequently stained with extracellular and intracellular markers as mentioned above. Samples were run on Attune NxT flow cytometer  and analyzed by FlowJo v10.8 Software. Lung and spleen samples from immunized groups P1-P14 were concatenated to provide consistencies in clustering methods and creating tSNE (t-distributed stochastic neighbor embedding) plots. Using Phenograph and FlowSOM clustering algorithms, lung lymphocytes and splenocytes were classified into different clusters based on phenotypic markers that were expressed, and overlayed on tSNE plots. Violin Box was used to acquire MFI values of phenotypic markers that each cluster expressed, which was represented by heatmaps, created on Morpheus . The U-PLEX Biomarker Group 1 (mouse) assay was used to analyze cytokines profiles of IFNγ, IL-10, IL-2, IL-4, and TNFα. The MSD plates were read on MESO QuickPlex SQ120 and analyzed on DISCOVERY WORKBENCH 4.0 Analysis Software.Cytokine concentrations were measured from supernatants collected from antigen-specific splenocyte proliferation cultures of PSerum and BAL samples were pooled from three immunized mice and ran in duplicates for each dilution, on 96-well plates. The plates were coated with SARS-CoV-2 nucleocapsid, membrane, a spike-nucleocapsid fusion protein, and the spike proteins of Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617), and Omicron (B.1.1.529) variants . For detection of IgG and IgM in serum, and IgA in BAL, the antibody ELISA procedure described earlier was followed . The abs1-P4 immunized groups were tested for neutralizing antibodies. Triplicate samples were run using the cPass SARS-CoV-2 Neutralization Antibody Detection Kit according to the manufacturer’s instructions. Serum and BAL samples were run at 1:9 dilutions and undiluted, respectively, and read using a DTX 880 Plate Reader (Beckman Coulter). Data are expressed as mean ± SEM of triplicates.Serum and BAL samples from PP ≤ 0.05 was used to indicated significance.Data were analyzed, and graphed using GraphPad Prism Software 9.4.1. Data was presented as mean ± SEM of 2-3 replicate values of 3 pooled mice and statistical differences were analyzed by one-way or two-way ANOVAs, adjusted for multiple comparisons. In addition, Spearman’s test was performed to determine the correlations between cytokines and proliferation values. A The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by University of Alberta’s Animal Care and Use Committee (ACUC) for Health Sciences (AUP00000212) and according to the guidelines of the Canadian Council of Animal Care (CCAC).RP, design, planning, execution of experiments, data analyses, writing first draft and editing. BA, conception, funding, design, planning, supervision, data analyses, writing and editing. All authors contributed to the article and approved the submitted version.


The minimum inhibitory concentrations (MICs) were tested using a Trek Diagnostic System (Thermo Fisher Scientific). Susceptibility was determined by CLSI broth microdilution and interpreted with CLSI M100 (2021) breakpoints.From 2017-2021, 554 Gram-negative isolates were collected from 16 pediatric departments across China. GNB isolates from pediatric departments were mainly derived from bloodstream (n = 135), intraperitoneal (n = 129), lower respiratory tract (n = 198) and urinary tract (n = 92) infections (E. coli (34.30%) and K. pneumoniae (27.62%) were the most common pathogens, followed by P. aeruginosa (15.34%). Susceptibility of species with more than 20 isolates were showed in Table 1. The susceptibility of P. aeruginosa to C/T was 89.41%, which was the highest among beta-lactams and was second only to amikacin. The susceptibilities of E. coli and K. pneumoniae isolates to C/T were 92.63% and 58.17%, respectively. When exclude carbapenem-resistant E. coli and K. pneumoniae, the susceptibility to C/T increased to 96.17% and 86.14%. C/T showed similar activities to E. coli and K. pneumoniae isolated from pediatric patients < 1 year (Table 2). Thirteen P. aeruginosa isolates were collected from patients < 1 year and 13/13 were susceptible to C/T (Table 3).P. aeruginosa and E. coli obtained from pediatric patients in China showed a high susceptibility to C/T. When excluding carbapenem resistant isolates, C/T also showed good activities against K. pneumoniae.Pengcheng Li, M.D., MSD China: Employee of MSD China


Polybrominated diphenyl ethers (PBDEs) are commercially used flame retardants that bioaccumulate in human tissues, including breast milk. PBDEs produce endocrine and metabolic disruption in experimental animals and have been associated with diabetes and metabolic syndrome (MetS) in humans, however, their sex-specific diabetogenic effects are not completely understood. Our past works show glucolipid dysregulation resulting from perinatal exposure to the commercial penta-mixture of PBDEs, DE-71, in C57BL/6 female mice.As a comparison, in the current study, the effects of DE-71 on glucose homeostasis in male offspring was examined. C57BL/6N dams were exposed to DE-71 at 0.1 mg/kg/d (L-DE-71), 0.4 mg/kg/d (H-DE-71), or received corn oil vehicle (VEH/CON) for a total of 10 wks, including gestation and lactation and their male offspring were examined in adulthood.In vivo glucose challenge showed marked glucose intolerance (H-DE-71) and incomplete clearance (L- and H-DE-71). Moreover, L-DE-71-exposed mice showed altered glucose responses to exogenous insulin, including incomplete glucose clearance and/or utilization. In addition, L-DE-71 produced elevated levels of plasma glucagon and the incretin, active glucagon-like peptide-1 (7-36) amide (GLP-1) but no changes were detected in insulin. These alterations, which represent criteria used clinically to diagnose diabetes in humans, were accompanied with reduced hepatic glutamate dehydrogenase enzymatic activity, elevated adrenal epinephrine and decreased thermogenic brown adipose tissue (BAT) mass, indicating involvement of several organ system targets of PBDEs. Liver levels of several endocannabinoid species were not altered.Compared to VEH/CON, DE-71 exposure produced hypoglycemia after a 11 h fast (H-DE-71). An increased fast duration from 9 to 11 h resulted in lower blood glucose in both DE-71 exposure groups. Our findings demonstrate that chronic, low-level exposure to PBDEs in dams can dysregulate glucose homeostasis and glucoregulatory hormones in their male offspring. Previous findings using female siblings show altered glucose homeostasis that aligned with a contrasting diabetogenic phenotype, while their mothers displayed more subtle glucoregulatory alterations, suggesting that developing organisms are more susceptible to DE-71. We summarize the results of the current work, generated in males, considering previous findings in females. Collectively, these findings offer a comprehensive account of differential effects of environmentally relevant PBDEs on glucose homeostasis and glucoregulatory endocrine dysregulation of developmentally exposed male and female mice. Considerable evidence points to a potential contribution of endocrine- (EDCs) and metabolism-disrupting chemicals (MDCs) to the rapid increase in the incidence of these metabolic diseases . One claetration –14. Thervia exposed dams and accumulate in male and female offspring liver and brain  is informative in understanding the diabetic phenotype of T2D. The incretin GLP-1 is secreted by enteroendocrine L-cells and acts on pancreatic β-cells to promote postprandial insulin secretion. In T2D, this insulinotropic effect is either impaired or absent leading to hyperglycemia . ActivatHaving observed sexually dimorphic risk and susceptibility of perinatally exposed females to MetS , 29, thead libitum in glass water bottles. Procedures on the care and treatment of animals were performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the University of California, Riverside, Institutional Animal Care and Use Committee (AUP#20170026 and 20200018).C57BL/6N mice were obtained from both Charles River Laboratories  and Taconic Biosciences . Mice were group housed 2-4 per cage and maintained in a non-specific pathogen free vivarium on a 12 h light/dark cycle at an ambient temperature of 20.6-23.9 °C and relative humidity of 20-70%. Mice were provided rodent chow  and municipal tap water Dosing solutions were prepared as described previously , 32. In via the dam was accomplished as described previously  and ex vivo analysis of hepatic, adipose, adrenal, and endocrine parameters (Cohort 2). The DE-71 exposure and testing paradigm is shown in Perinatal PBDE exposure eviously . In brieeviously Figure 1eviously , 20, 38,ex ratio . Male ofITT, was calculated using the formula (0.693 x 100) x t1/2-1 to determine in vivo insulin sensitivity from 0-30 min post insulin injection. Fasting blood glucose was determined from baseline values (t=0) obtained in IPGTT (11h) and ITT (9h). GTT and ITT sample sizes were different because ITT effect size was expected to be less (as estimated by our previous work (Mice were fasted overnight (ON) for 11h and then injected with glucose . Glucose was measured directly from tail blood (~1 uL) at time 0, 15, 30, 60, and 120 min post-glucose challenge. A calibrated glucometer and test strips  were used to measure plasma glucose concentrations. Seven days following IPGTT, an insulin tolerance test (ITT) was performed with Humulin R (Eli Lilly) bolus  on mice fasted ON for 9h. Tail blood was collected, and glucose was sampled in the same manner as IPGTT . For area calculations, the area under (AUC) or above the glycemia curve (inverse AUC) from 0-120 min post injection was used. The percent blood glucose reduction rate after insulin administration, Kous work ) and, thDuring sacrifice, under terminal isoflurane anesthesia, cardiac blood (0.3-1 mL) was collected and centrifuged at 16,000 x g for 20 min at 4 °C. After the addition of a cocktail of protease inhibitors and EDTA, the plasma samples were stored at -80°C until further use. The following organs were excised and weighed: liver, pancreas, spleen, adrenal glands and interscapular brown adipose tissue (BAT). Plasma, liver and adrenal samples were snap-frozen over dry ice and stored at -80 °C for later analysis of plasma hormones, adrenal epinephrine, liver endocannabinoids and enzymatic activity.via cardiac puncture (ad libitum fed state) was analyzed for metabolic hormones using commercially available kits according to manufacturer’s instructions. Plasma insulin was measured using commercial ELISA kits  as previously described , 10 pmol d4-oleoylethanolamide , and 500 pmol d5-2-arachidonoyl-sn-glycerol . Following homogenization of samples, 2 mL of chloroform and 1 mL of water were added, followed by centrifugation at 2000 x g for 15 min at 4°C. The organic phase was extracted and the pooled lower phases were dried under N2 gas followed by resuspension in 0.1 mL methanol:chloroform (9:1). Analysis of endocannabinoids (EC) was performed via ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC/MS/MS) as previously described 6. The mixture was incubated at 0°C for 20 min and the oxidation reaction was stopped by the addition of 60 µL of a 9 N NaOH solution containing 4 mg/ml ascorbic acid . Fluorescence emission was determined at 520 nm (excitation wavelength at 420 nm) using a fluorescence plate reader (Promega). Epinephrine concentration  was converted from the mean fluorescence intensity units of each sample using calibration standards and polynomial curve fitting.Epinephrine content in adrenal glands was measured using a modification of the trihydroxyindole method as described previously . Brieflypost hoc testing for multiple group comparisons. For parametric ANOVAs, Tukey’s post hoc test was used when both sample sizes and variance were equal, and a Dunnett’s T3 test was used when sample size and variance were not equal. For non-parametric ANOVA, a Dunn’s post hoc test was performed. For Two-way ANOVA, Tukey’s, Sidak’s and Holm-Sidak’s post hoc tests were used. Differences were deemed significant at p<0.05. Data are expressed as mean ± standard error of the mean (s.e.m) unless indicated otherwise.Statistical analyses were performed using GraphPad Prism v.9.4.1. A one-way analysis of variance (ANOVA) was used to test the main effect of one factor. When normality assumption failed, as determined using a Shapiro-Wilk test, a Kruskal-Wallis H ANOVA was used. A Brown-Forsythe ANOVA was used if the group variances were significantly different. Data for fasting glycemia were analyzed by two-way ANOVA for main effects of exposure and fasting duration. ITT and GTT experiments were analyzed by repeated measures two-way or mixed model ANOVA to determine main effects of exposure and time. ANOVA was followed by Exposure effect F=9.34, p<0.001, Tukey’s post-hoc VEH/CON vs L-DE-71, p=0.03 and lower in L-DE-71 vs H-DE-71, p=0.0003). Relative liver weight was 8% greater in H-DE-71 relative to L-DE-71 =2.824, p=0.0675, Tukey’s post-hoc L-DE-71 vs H-DE-71 p<.05). The absolute and relative weights of pancreas and spleen were similar across groups.Exposure effect F= 6.65, p<0.01; Time effect F= 24.6, p<0.0001; Exposure x Time F= 0.76 ns; Sidak’s post hoc test, 11 h: VEH/CON vs H-DE-71, p<0.01; 9 h vs 11 h, L-DE-71 p<0.01, H-DE-71, p<0.05 (We examined fasting blood glucose (FBG) after 9 and 11 h fasting using glycemia values from basal time points obtained in ITT and GTT experiments, respectively. , p<0.05 Figure 2=4.767, p<0.05; Time effect F=330.2, p<0.0001; Time x Exposure F =3.112, p<0.01)  and 60 min post injection (p<0.05) and in L-DE-71 vs H-DE-71 at t=30 (p<0.01) and 60 min post injection (p<0.01).To investigate the effects of DE-71 on glucose tolerance, glycemia was measured during GTT over the 120 min post-injection time course =278.6, p<0.0001; Exposure effect F=9.39 p<0.001; Time x Exposure F=5.94, p<0.0001). Mean glycemia values for H-DE-71 were significantly different from VEH/CON at t=15 , 30 (p<0.01) and 60 min post injection (p<0.01) and from L-DE-71 at t=15 (p<0.01), t=30 (p<0.01) and 60 min post injection (p<0.001).Since FBG after an 11 h fast was lower in H-DE-71 relative to VEH/CON =5.21, p<0.05, Tukey’s post-hoc VEH/CON vs H-DE-71, p<0.029, L-DE-71 vs H-DE-71, p<0.013; Percent basal glycemia AUC, Brown-Forsythe ANOVA: Exposure effect F=10.1, p<0.001, Dunnett’s T3 post-hoc VEH/CON vs H-DE-71, p<0.004, L-DE-71 vs H-DE-71 p<0.0003 .The differences in magnitude and duration of glycemia are integrated using the area under the glucose curve, AUCTime effect F=56.2, p<0.0001; Exposure effect F=2.40, ns; Time x Exposure F=1.90, p<0.05) . When glycemia is expressed as a percent of baseline, L-DE-71 exposed mice displayed incomplete recovery or glucose clearance/utilization at 90 and 120 min after insulin injection =33.7, p<0.0001; Exposure effect F=8.014, p<0.001; Time x Exposure F=1.23, ns)  and 120 min (p<0.001). This is represented as a greater mean latency to reach the minimum insulin-induced hypoglycemia in L-DE-71 relative to VEH/CON =9.07, p<0.01; Dunn’s post hoc VEH/CON vs L-DE-71, p<0.01)  was plotted using absolute =2.024, ns, Sidak’s post-hoc VEH/CON vs L-DE-71, p<0.05) (ITTinsulin measured over the first 30 min post-injection (The inverse area under the glucose response curve (inverse AUCad libitum fed state) =3.684, p=0.0409; Dunnet’s post-hoc VEH/CON vs L-DE-71 p=0.0473; Glucagon, One-way ANOVA: Exposure effect F=5.61, p=0.02; Dunnett’s T3 post-hoc VEH/CON vs L-DE-71 p=0.0449) =16.06, p<.0001; Tukey’s post-hoc VEH/CON vs L-DE-71 p=0.0001, VEH/CON vs H-DE-71 p=0.002). Adrenal weights were not different across experimental groups =6.82, p=0.061; Tukey’s post-hoc VEH/CON vs L-DE-71 p=0.01, L-DE-71 vs H-DE-71 p=0.01) =19.51, p<.0001; Dunnett’s post-hoc VEH/CON vs L-DE-71 p=0.002, VEH/CON vs H-DE-71 p<0.0001) (p=0.03).Exposure to DE-71 decreases glucose tolerance which may be due to enhanced hepatic glucose production. To examine this possibility, we tested the hypothesis that PBDEs increase the activity of GDH a hepatic gluconeogenic enzyme. We found that exposure to L- and H-DE-71 significantly <0.0001) Figure 7Male F1 mice exposed to either dose of DE-71 displayed normal liver levels of the endocannabinoid (EC), anandamide , and related fatty acid ethanolamides, docosahexaenoyl ethanolamide (DHEA) and oleoylethanolamide (OEA), when compared to VEH/CON . The combination of DE-71 effects - exaggerated fasting hypoglycemia, glucose intolerance and incomplete glucose clearance/utilization in response to insulin challenge demonstrates the complex actions of PBDEs on glucose homeostasis in males. Previously, we have observed abnormal glucose metabolism and other parameters in their DE-71-exposed female siblings  activity increases energy expenditure by burning fat and increasing metabolic rate and can utilize glucose especially when stimulated by insulin . TherefoIt is important to note that developmental exposure to DE-71 ends at weaning, but that dysregulated glucoregulatory phenotypes in exposed male and female offspring are observed in adulthood . We specin vivo and ex vivo parameters measured in DE-71-exposed male offspring indicate broad, complex effects of developmental exposure to PBDEs on glucose homeostasis and glucoregulatory parameters involving various organ systems. Our results support human studies reporting the positive association between body burdens of PBDEs and T2D and MetS  (AUP# 20170026 and 20200018).Conceptualization, MC-C, EK. Methodology, MC-C, EK, ND, JK, BC. Validation, MC-C, EK, BC, PP, ND. Formal Analysis, EK, MC-C, PP, ND. Investigation, EK, BC, PP, JK, AB, MC-C, MD. Writing – Original Draft, EK, MC-C, AB. Writing – Review & Editing, MC-C, EK, AB. Visualization, EK. Resources, MC-C., ND. Data Curation, EK, MC-C. Supervision, MC-C, EK, ND. Project Administration, MC-C, EK. Funding Acquisition, MC-C, EK, ND. All authors contributed to the article and approved the submitted version.


The COVID-19 pandemic has led to a considerable increase in Central-Line Associated Bloodstream Infections (CLABSI) and Catheter Associated Urinary Tract Infections (CAUTI). However, it remains unclear how this affected different population groups.This retrospective observational cohort study included CLABSI and CAUTI in a tertiary care facility. Information was collected on patient demographics, hospitalization, comorbidities, and COVID-19 status. Chi-square and Wilcoxon rank-sum tests were used for categorical and continuous variable comparisons. GEE models compared pre- and pandemic periods by interrupted time series analysis.From 1/1/2018 to 5/31/2022 98,791 patients had 151,550 hospital admissions. Of those, 17,796 patients had 29,483 central lines placed and 45,180 patients had 65,422 Foleys. 314 patients developed 338 CLABSI and 216 patients had 217 CAUTI. 1,552 patients tested positive for COVID-19 with 22 developing CLABSI and 14 CAUTI. The pre-pandemic downward trend in CLABSI and CAUTI was reversed during COVID-19 (p< 0.05).Black patients and those with other/unknown race had higher CLABSI per patient with central lines (p< 0.02) and higher device days (p< 0.0001) compared to white patients. Hispanic/Latino patients acquired more CLABSI per patient compared to non-Hispanic/non-Latino patients (p=0.04). During COVID-19 the already higher device days in Hispanic/Latino patients further increased compared to non-Hispanic/non-Latino patients (p< 0.0001).Black patients had higher CAUTI infection rates per patient with Foleys compared to white and other/unknown patients (p< 0.05). CAUTI per device days were also higher in black patients compared to white patients (p=0.02). No difference in CAUTI burden was detected for ethnic groups throughout the study period. Black and non-Hispanic/non-Latino patients had higher Foley usage days than other patients (p< 0.0001). Foley days decreased during COVID-19 (p=0.01).We detected health outcome disparities affecting black (CLABSI and CAUTI) and Hispanic/Latino (CLABSI) patients. Infections increased during the pandemic without altering race/ethnicity differences. Device utilization was significantly higher in affected groups.All Authors: No reported disclosures


In TANGO, switching to dolutegravir/lamivudine (DTG/3TC) was noninferior compared to continuing a tenofovir alafenamide (TAF)-based regimen, however switching from bictegravir (BIC)/emtricitabine (F)/TAF was not evaluated. Here, we present efficacy and safety of switching to DTG/3TC compared with continuing B/F/TAF in virologically suppressed adults through 24 weeks.DYAD (NCT04585737) is an ongoing, open-label clinical trial that randomized adults with HIV-1 RNA< 50 copies/mL and no prior virologic failure (2:1) to switch to once-daily fixed-dose DTG/3TC or remain on B/F/TAF. The primary endpoint is the proportion with HIV-1 RNA ≥ 50 c/mL at Week (W) 48 . A planned W24 interim analysis assessed noninferiority with a 6% margin.Overall, 222 adults  were randomized. At W24, 3 (2%) participants on DTG/3TC and 3 (4%) on B/F/TAF had HIV-1 RNA ≥ 50 c/mL  meeting noninferiority criteria. Through W24, 4 on DTG/3TC and 3 on B/F/TAF met confirmed virologic withdrawal (CVW) criteria and underwent resistance testing. 2/7 had treatment-emergent resistance. One B/F/TAF CVW developed M184M/I and G140G/S at W12; at study discontinuation (DC), HIV-1 RNA< 50 c/mL and the participant remained on B/F/TAF. One DTG/3TC CVW had no resistance on study genotype but underwent repeat genotypic testing outside the study (ordered by clinic provider) and had M184V at W12; at study DC, HIV-1 RNA< 50 c/mL on DTG/3TC and the participant was subsequently transitioned to DTG + darunavir/cobicistat (DRV/c). One non-CVW DTG/3TC participant developed M184V and K65R at W12 (genotype inadvertently collected at first episode of unconfirmed viremia); at study DC, HIV-1 RNA< 50 c/mL on DTG/3TC and the participant was subsequently transitioned to DTG+DRV/c. Drug-related adverse events (AEs) and withdrawals due to AEs occurred in 31 (21%) and 6 (4%) participants with DTG/3TC and 1 (1%) and 0 participants with B/F/TAF, respectively.DYAD demonstrated noninferior efficacy of switching to DTG/3TC vs. continuing B/F/TAF at W24. Higher AE rates in the DTG/3TC arm are likely consistent with the open-label nature of this switch study.Charlotte-Paige M. Rolle, MD, MPH, Gilead: Advisor/Consultant|Gilead: Grant/Research Support|Gilead: Honoraria|Janssen: Advisor/Consultant|ViiV: Advisor/Consultant|ViiV: Grant/Research Support|ViiV: Honoraria Federico Hinestrosa, MD, AbbVie: Honoraria|Gilead Sciences: Advisor/Consultant|Gilead Sciences: Honoraria|MSD: Honoraria|ViiV Healthcare: Advisor/Consultant|ViiV Healthcare: Honoraria Edwin DeJesus, MD, Gilead Sciences, Inc: Advisor/Consultant|Theratechnology: Advisor/Consultant|Theratechnology: Honoraria


Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis are frequent etiologic agents of CABP. Ceftaroline fosamil is a parenteral cephem approved treatment of patients with CABP caused by S. pneumoniae (including cases with concurrent bacteremia), methicillin-susceptible Staphylococcus aureus (MSSA), H. influenzae, and some species of Enterobacterales. In this study we report the in vitro activity of ceftaroline and comparators against isolates from community-acquired respiratory tract infections (CARTI) collected through a global surveillance program.Community-acquired bacterial pneumonia (CABP) is a frequent cause of patient morbidity and mortality. S. pneumoniae, H. influenzae, M. catarrhalis, MSSA, and methicillin-resistant S. aureus (MRSA) were tested. The isolates  originated from Asia/South Pacific ; Europe ; Latin America (671/8.5%); Middle East/Africa (659/8.4%); and North America (Canada only) (380/4.8%). Ceftaroline and comparator agent MICs were determined by CLSI M07 broth microdilution methodology. MICs were interpreted using 2023 CLSI M100 MIC breakpoints.Clinically relevant, non-duplicate, isolates cultured from respiratory specimens by clinical laboratories in 54 countries in 2018-2021 were collected by the ATLAS Surveillance Program central laboratory . Community-acquired infections were defined as those from patients < 48 hours in hospital. In total, 7,886 isolates of in vitro activity of ceftaroline and comparator agents is summarized in the table. Greater than 99% of S. pneumoniae and 100% of MSSA were susceptible to ceftaroline, including penicillin-nonsusceptible S. pneumoniae based on a dosage of 600 mg every 12h. Sixty-two (9.6%) MRSA were ceftaroline-susceptible-dose-dependent (MIC 2-4 µg/mL) based on a dosage of 600 mg every 8h administered over 2h, with the majority from (n) Thailand (9), S. Korea (7), and China (6). Four isolates, from S. Korea (2), China (1), and Ukraine (1) were resistant to CPT (MIC of ≥8 µg/mL). 99.6% of H. influenzae were susceptible to ceftaroline.The in vitro activity against current pathogens associated with CABP from a global collection.Ceftaroline demonstrated potent Meredith Hackel, PhD, Pfizer Inc.: Honoraria|Venatorx: Paid fees for conducting the study and abstract preparation Gregory Stone, PhD, Pfizer: Stocks/Bonds Daniel F. Sahm, PhD, Merck & Co., Inc.: Honoraria|Pfizer Inc.: Honoraria|Venatorx: Paid fees for conducting the study and abstract preparation


Dialysis, renal transplant (RTX) and autoimmune kidney disease (AKD) patients are at higher risk of COVD-19 and suboptimal humoral responses post primary SARS-CoV-2 vaccination. As such, these groups have been prioritised for further SARS-CoV-2 vaccine doses. Limited data are available on their immunogenicity.nd, 3rd and 4th SARS-CoV-2 vaccines. Endpoints were median anti-S IgG titres and seroconversion (anti-S IgG titre >1896 MFI).Patients on dialysis or with a RTX or AKD who received primary SARS-CoV-2 vaccination were prospectively recruited from three UK sites. SARS-CoV-2 IgG spike antibody (anti-S IgG) and nucleocapsid titres were measured with a Luminex assay post 2rd dose , with 27/58 (47%) of RTX and 17/46 (37%) of AKD patient non-responders seroconverting. Seroconversion among RTX and AKD patients overall improved to 78% (114/146) and 79% (122/155) respectively (p< 0.05), but not in dialysis patients post 3rd dose. 212/400 (53%) had serology post 4th dose , with 10/22 (46%) and 3/25 (12%) of RTX and AKD patient non-responders seroconverting. Anti-S IgG titre and seroconversion were similar within each group post 4th dose .628 patients  comprising 240 dialysis, 194 RTX, and 194 AKD patients were recruited, with seroconversion of 96%, 61% and 70% respectively post two vaccines, and median anti-S IgG titres (IQR) of 30604 (24393–31826), 5829 (455–28296) and 18711 (1233–30226). 400/627 (64%) had anti-S IgG titres post 3rd dose in a multivariable model were rituximab use within six months, mycophenolate, prednisolone, chronic kidney disease and AKD , and post 4th dose, rituximab alone (p< 0.001).Predictors of non-seroconversion post 3rd but not 4th dose improved anti-S IgG titres and seroconversion in renal patients. Immunosuppressive use, particularly rituximab, was associated with reduced serologic responses . Patients on immunosuppressives should therefore be prioritised for consideration of additional pre-exposure prophylactic agents.After primary SARS-CoV-2 vaccination, a 3Michael Chen-Xu, MBChB, MRCP, MPH, GSK: Grant/Research Support Rachel Jones, MD, GSK: Advisor/Consultant|GSK: Grant/Research Support|Roche: Grant/Research Support|Vifor Pharma: Advisor/Consultant Rona M. Smith, MD MRCP, GSK: Grant/Research Support|Union Therapeutics: Grant/Research Support


Remdesivir (RDV), a nucleotide analog prodrug that targets the viral RNA-dependent RNA polymerase Nsp12, is approved to treat COVID-19 in hospitalized and nonhospitalized patients. Obeldesivir (ODV), an oral mono-5’-isobutyryl ester prodrug that is metabolized into the same active triphosphate as RDV, is being evaluated in Phase 3 clinical trials. The antiviral activity of RDV and ODV against previous Omicron subvariants (BA.1 to BQ.1.1) was maintained with respect to the ancestral WA1 strain. We assessed RDV and ODV antiviral activity against recent Omicron subvariants BF.7, BQ.1, XBB.1.5, CH.1.1, and XBF using clinical isolates and/or site-directed mutants (SDMs).Global Initiative on Sharing Avian Influenza Data (GISAID) EpiCoV database sequences. Structural analysis of identified substitutions was conducted on a prior cryo-electron microscopy-based model of the replication-transcription complex. Antiviral activity of RDV and ODV against subvariant clinical isolates was assessed by nucleoprotein ELISA in A549-hACE2-TMPRSS2 cells and by SDMs in the replicon systemThe prevalence of Nsp12 substitutions in Omicron subvariants was evaluated by analysis of 50 vs WA1). Evaluation of CH.1.1 and XBF by introducing variant-defining mutations into the replicon showed no change in in vitro susceptibility (< 1.8-fold change). Phenotyping of clinical isolates of BF.7, BQ.1, XBB.1.5, and CH.1.1 indicated no loss of RDV or ODV in vitro antiviral activity (< 1.3-fold change).Genomic analysis of > 2 million Omicron subvariant sequences revealed unique substitutions in Nsp12 vs WA1. No new defining substitutions in Nsp12 were found compared with earlier Omicron variants. The defining substitutions (≥ 75% of sequences) included P323L , Y273H (BQ.1), and G671S . Less prevalent substitutions were observed with frequencies from 1-3.5%: L247F, T248I, V257F, N507I, A529V, F694Y; none had direct interaction with the incoming RDV nucleotide triphosphate or the viral RNA, except N507I. Phenotyping of individual substitutions using SDMs showed no loss of RDV or ODV susceptibility (< 1.2-fold change in ECRDV and ODV retained potent in vitro antiviral activity against all tested Omicron subvariants with potencies comparable to WA1.Lauren Rodriguez, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Jiani Li, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Dong Han, MS, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Ross Martin, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Jasmine Moshiri, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Nadine Peinovich, MPH, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds John P. Bilello, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Jason K. Perry, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds Charlotte Hedskog, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds


Agrobacterium radiobacter strain MD22b was isolated from infected fruit from Vatan Farm, a dekhkan farm in Yangibog . The 5.7-Mbp draft genome sequence presented here shares homology with chromosomes 1 and 2, as well as with the Ti plasmid from agrobacteria. Agrobacterium radiobacter (Beijerinck and van Delden 1902) Conn 1942 (formerly Agrobacterium tumefaciens) . The fruit was rinsed three times in sterile distilled water and homogenized. Aliquots were spread onto Roy-Sasser agar (Agrobacterium-like colony was picked and identified as A. radiobacter by its API 20 NE (bioMérieux) profile (1-4-6-7-7-4-4). DNA was extracted from cells cultivated in LB for 24 h at 30°C with shaking using the GenElute bacterial genomic DNA kit (Sigma-Aldrich). Sequencing of the 16S rRNA gene using the primer combination 5′-AGRGTTTGATYHTGGCTCAG-3′ (27f_mod) and 5′-TASGGHTACCTTGTTACGACTT-3′ (1492r_mod) (A. radiobacter strain ATCC 23308 (GenBank accession number MT534521.1) using BLASTn v. 2.13.0 searches against the GenBank v. 252 nonredundant nucleotide database (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). For genome sequencing by Eurofins Genomics, a NEBNext Ultra II DNA preparation kit was used, and Illumina NovaSeq 6000 S2 paired-end genomic sequencing was performed with a read length of 2 × 151 bp, resulting in 10,125,972 reads with a Phred score of ≥28 and a total of 1,518,897,000 sequenced bases. Additional quality control was performed to remove any remaining adapter sequences using the Trim Reads tool in the CLC Genomics Workbench v. 20.1. Assembly was performed using the CLC de novo assembly tool, resulting in a total sequence length of 5,736,602 bp and a total gapped length of 5,736,406 bp, distributed in 38 contigs, with an N50 value of 267,803 bp, coverage of 265×, and GC content of 59.44%. Unless otherwise noted, default parameters were used for all software. Annotation of the draft genome was done using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v. 6.1 (https://www.ncbi.nlm.nih.gov/genome/annotation_prok). The genome completeness was estimated at 100% using CheckM v. 1.0.18 (Agrobacterium with pairwise average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values of 97.97% and 83.8%, respectively, against the type strain A. radiobacter NCPPB 3001 using the ANI calculator (http://enve-omics.ce.gatech.edu) (https://ggdc.dsmz.de/ggdc.php#) (A. radiobacter K84 was performed, revealing significant homology with both chromosomes of strain K84 (repA and repB, and the plasmid Ti replication initiator gene, repC. The reported data will be useful for future understanding of the genetic diversity and virulence potential of A. radiobacter in Central Asia.ser agar  plates afile 1-4--7-7-4-4.rain K84 . A complA. radiobacter strain MD22b have been deposited in DDBJ/ENA/GenBank under accession numbers OP364099 and JANDHV000000000, respectively. The associated BioProject, SRA, and BioSample accession numbers are PRJNA856860, SRR21562299, and SAMN29594852, respectively.The partial 16S rRNA gene and whole-genome shotgun sequences of


Pantoea agglomerans has been isolated from various habitats including disease plants. Here, we present the genome of P. agglomerans strain NBBC-01 obtained from rotten potatoes that were infected by Ditylenchus desstructor. The genome information will prove advantageous in elucidating its ecological role.The Gram-negative bacterium  Pantoea agglomerans has been shown to promote plant growth  in Wuhan, China, which was infected with a plant-parasitic nematode Ditylenchus destructor  following the manufacturer’s protocols. The DNA purity, concentration, and integrity were assessed with NanoDrop One spectrophotometer , Qubit v.4.0 Fluorometer , and 0.75% agarose gel electrophoresis, respectively. Size selection (20–30 kb) was carried out for long-read sequencing utilizing the PippinHT system . The DNA library was generated without DNA shearing utilizing a ligation sequencing kit ), Oxford, UK] and sequenced with a Nanopore PromethION sequencer instrument (ONT) on a R9.4.1 flow cell FLO-MIN106). Guppy v.4.4.2 (. Guppy vP. agglomerans DSM3493T (GenBank accession: GCA_900185875). The values of digital DNA–DNA hybridization (P. agglomerans NBRC 102470 (GenBank accession: GCA_001598475) is 88.7%. Thus, NBEC-018 was identified as a P. agglomerans isolate.The high-quality short- and long-read sequences were assembled into a whole chromosome and plasmids using Unicycler v.0.4.9 . All sofdization , 13 betw


There is growing evidence that people with tuberculosis (TB) are at higher risk of mortality even after treatment completion. However, there are limited data on post-TB treatment mortality rates and causes of death in the United States (U.S.).Cohort study of all adults (age ≥ 18 years) diagnosed with TB in Georgia (U.S.) who finished treatment between January 1, 2008, and December 31, 2019. We obtained post-TB treatment mortality data from the Centers for Disease Control and Prevention National Death Index and general Georgia population death rates and causes of death for the same period from public databases. We calculated age- and sex-standardized mortality rates (SMRs) for the TB cohort and general Georgia population and the SMR ratio between these two groups .Among 3,183 patients alive at the time TB treatment was stopped, 2,391 (75.1%) were culture-confirmed, 625 (19.6%) were extrapulmonary, and 15 (< 1%) were rifampin and isoniazid resistant. The median age at TB diagnosis was 44 years (interquartile range (IQR): 32-57), 2,094 (65.8%) were male, and 1,557 (48.9%) were U.S.-born. 328 (10.3%) were co-infected with HIV. Most patients  completed treatment. Reasons for treatment non-completion included adverse events (n=11), patient declined treatment (n=13), and lost to follow-up (n=186). The median post-TB treatment follow-up was 6.4 years (IQR 3.4-9.0) and 233 (7.3%) patients died. Causes of death included cardiovascular disease and malignancy  and HIV . The age and sex adjusted SMR ratio of the TB cohort compared to the general Georgia population was 0.93 (95% CI: 0.78-1.09). The SMR ratio in a subgroup analysis restricted to U.S.-born persons was 1.6 (95% CI: 1.36-1.77).All data is shown as n (%) unless stated otherwise. Abbreviations: TB = tuberculosis, IQR = inter-quartile range 1. Other races = American Indian/Alaska Native , multiracial  2. Other risk factors = end-stage renal disease , post-organ transplantation , TNF-a antagonist therapy , immunosuppression (not HIV/AIDS) , incomplete LTBI , other ; missing data All rates are per 1000, . Abbreviations: TB = tuberculosis 1. Entire TB cohort: deaths n = 233, person-time = 18,440 person-years 2. Georgia population: deaths n = 900,874, person-time = 90,796,252 person-years 3. Race includes only non-Hispanic White and non-Hispanic Black categories: TB cohort (race standardization): deaths n = 212, person-time = 14,095 person-years GA population (race standardization): deaths n = 888,261, person-time = 85,630,800 person-years 4. Culture-confirmed TB cohort: deaths n = 182, person-time = 12,821 person-yearsOur data suggests the overall cohort of patients who finish TB treatment are not at higher risk of death compared to the general population. Analysis restricted to U.S.-born persons, however, did show a slightly higher risk of death post-TB treatment, which suggests the so-called "healthy immigrant effect" could have affected our post-TB treatment mortality estimates.All Authors: No reported disclosures


A simultaneous, high-throughput and sensitive method for analysing nine neonicotinoid pesticides (NEOs) and four metabolites (NEOms) in urine using a liquid chromatography-tandem mass spectrometry (LC–MSMS) was developed. The method detection limit (MDL) and lowest concentration minimum reporting limit (LCMRL) of the nine NEOs were 0.0013–0.048 ng/ml and 0.0050–0.17 ng/ml, respectively. The MDL and LCMRL of the four NEOms were 0.0052–0.52 ng/ml and 0.011–1.6 ng/ml, respectively. Intermediate precision for the nine NEOs and four NEOms was 7.5–12.5% and 7.4–10.9%, respectively. Accuracy for the nine NEOs and four NEOms was 3.83–5.60% and 3.01–29.2%, respectively. The developed method was applied to analyse urine samples collected from participants of a large-scale birth cohort study, namely, the Japan Environment and Children's Study (JECS).•The NEO and NEOm concentrations in 100 µl urine samples were analysed using a highly sensitive LC–MSMS.•Automated solid phase extraction in a 96-well plate was utilised to achieve high-throughput analysis.•Intermediate precision and accuracy were less than 12.5% and 94.8–99.1%, respectively.  Specifications tableNeonicotinoid pesticides (NEOs) are agonists of neuronal nicotinic acetylcholine receptors 13C6) (ACE-IS), thiacloprid (pyridymethyl-13C6) (TCP-IS), sulfoxaflor  (SUL-IS), flonicamid  (FLN-IS), thiamethoxam  (THX-IS), dinotefuran (furylmethyl-13C5) (DIN-IS), clothianidin  (CLO-IS), imidacloprid (pyridymethyl-13C6) (IMI-IS), nitenpyram  (NIT-IS), acetamiprid-N-desmethyl  (dm-ACE-IS), TCP-amid (pyridymethyl-13C6) (TCP-amid-IS), CLO-desmethyl  (dm-CLO-IS) and IMD-olefin  (IMI-OF-IS), were purchased from Cambridge Isotope Laboratories, Inc. . Acetonitrile (purity 99.8%), ammonium acetate (guaranteed reagent), formic acid (purity ≥98.0%), methanol (purity 99.8%) and ultrapure water  were used for this analysis.The sample preparation method was based on our previous study TM 6500 mass spectrometer  was used to detect and quantify target analytes. The MSMS system was operated using electrospray ionisation positive mode, and multiple reaction monitoring was performed. An ACQUITY UPLC HSS T3  was used as an analytical column. The LC and MSMS parameters are listed in The Nexera X2 system  was used for separation. A Triple Quad. Sulfoxaflor (SUL) was a diastereomer. The two isomers were quantified separately as SUL-A and SUL-B. All samples that fell outside the calibration range were reanalysed following further dilution.The calibration range is shown in Table 5A pooled urine sample was prepared using maternal samples collected from volunteers to create QC samples. Levels of analytes varied in actual samples, so we tried to create the QC sample to reflect such concentrations. The QC samples had the concentration of dinotefuran around 2 ng/ml. To double the concentration, NEOs and NEOms were fortified such that the concentrations of ACE, TCP, SUL-A, SUL-B, FLN, THX, DIN, CLO, IMI, NIT, dm-ACE, TCP-amid, dm-CLO and IMI-OF were 0.5, 0.5, 0.5, 0.5, 10, 2.0, 5.0, 5.0, 5.0, 5.0, 5.0, 1.0, 20 and 100 ng/ml, respectively. The QC samples were subjected to the same procedure as the unknown samples, with five replicates analysed in each analytical sequence. The lowest concentration minimum reporting limit (LCMRL) was calculated following the U.S. Environmental Protection Agency's instructions 2) higher than 0.990. Repeatability and intermediate precision were determined based on ISO 5725:1994 and 27148:2010, with QC sample measurements (n = 100). QC for day-to-day analysis was determined using a Shewhart control chart  of 1.40 (0.27), 1.36 (0.31), 0.19 (0.06) and 1.34 (0.47) ng/ml for ACE, CLO, TCP and THX, respectively, were analysed in nine replicates. All measured concentrations fell within the certified ranges  samples (NMIJ CRM 7408-a) with certified concentrations  at 25°C (1.0125 g/cmd ranges .Table 7RNot applicable.Yukiko Nishihama: Conceptualization, Formal analysis, Writing – original draft, Visualization. Shoji F. Nakayama: Methodology, Software, Writing – review & editing, Supervision, Project administration. Tomohiko Isobe: Methodology, Investigation, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Enterobacteriaceae bloodstream infections (CRE-BI) in children from Latin America and Caribbean (LAC ) is scarce.Systematic reviews about the burden and use of resource related to Carbapenem Resistant To analyze demographic, epidemiological, clinical, use of resource and microbiological data from CRE-BI in LACA systematic review following Cochrane methods, PRISMA/MOOSE statements was performed (PROSPERO 2022 CRD42022352504). We searched all databases  from 1/1/2012 to 9/30/2022. Independent selection, data extraction, and risk of bias assessment (RoB) were performed with COVIDENCE. Types of study included; observational/surveillance/experimental/quasi-experimental. Population: Inpatients < 19 years old, with CRE-BI. Outcomes: age, underlying conditions, previous . treatment and use of resources. Statistical analysis: proportion meta-analyses applying an arc-sine transformation and RoBTo, We conducted proportion meta-analyses and applied arc-sine. R studio was used25/858 screened studies included for full text assessment with 243 patients were included. The most represented countries were Brazil (30%), Colombia (26%) and Argentina (22%). Median age was 34.5 months (IRQ 14-85). Predominant resistance mechanisms: KPC (96%), NDM (58%), OXA (27%). Pool proportion were: Male 55%. Underlying conditions 100% (immunocompromised host 84%), Previous Colonization with CRE was 42% (CI95% 13-78) and broad spectrum antibiotic used was 88% (CI95% 46-98%), most commonly carbapenems 84% (CI95% 72-91%) Bacteremia secondary to intra-abdominal infection was 80%. Combination antimicrobial treatment was 96% (CI95% 22-100%). Median length of stay 22.5 (IQR 22-30 days). Overall case fatality ratio was 48% (IC95% 27-69%).CRE-BI cause morbidity and mortality mainly in pediatric population with underlying disease in LAC region. Considering its high impact in public health, it's mandatory to establish adequate policies and programs to improve antimicrobial stewardship.All Authors: No reported disclosures


Fig 1). Stimulation of mucosal antibodies and T cells, immune effectors also associated with protection against influenza, are anticipated to enhance VE.Adults over age 65 years exhibit increased susceptibility to influenza viruses, accounting for 70-85% of annual influenza-related US fatalities. Licensed vaccines mainly induce serum humoral immunity and remain the primary method of influenza prevention, but vaccine effectiveness (VE) remains particularly low in older individuals (Table 1). Demographics were comparable across cohorts (Table 2).A randomized, double-blind, double-dummy, placebo-controlled Phase 1b study (NCT05163847) was conducted at 6 US study sites with the investigational intranasal M2SR (M2 deficient Single Replication) and Fluzone High Dose (HD) vaccine that contained HA & NA from A/Cambodia/e0826360/2020 (Cam2020). Adults aged 65-85 years (n = 305) received either one administration of Cam2020 M2SR alone, Cam2020 M2SR along with HD, HD alone, or placebo . Moreover, M2SR coadministered with HD induced HAI and NAI antibodies in significantly higher proportion of subjects than HD alone . M2SR alone and M2SR coadministered with HD elicited significant mucosal secretory IgA (sIgA) antibodies against vaccine antigen, Cam2020, and drifted H3N2 Darwin2021 strain . Similarly, T cells positive for IFNg and GrzB secretion were significantly induced by M2SR only or by M2SR coadministered with HD . HD alone did not induce mucosal antibodies or T cell responses .Study vaccination was well-tolerated. Intranasal M2SR generated significantly increased serum HAI and NAI antibodies compared with placebo . These broad immune responses support the potential for the safe coadministration of M2SR with HD to enhance protection against influenza infection in this highly vulnerable age group.Coadministration of intranasal M2SR with intramuscular Fluzone HD resulted in a multi-faceted immune response in older adults aged 65-85 years old with a higher proportion of responders across all immune measurements compared with either vaccine alone. Coadministration of the two vaccines resulted in significant rises in serum HAI and NAI titers compared to HD alone (Joseph Eiden, MD, PhD, FluGen, Inc.: Advisor/Consultant Bryan Murray, M.D., FluGen, Inc.: Advisor/Consultant Renee Herber, B.S., FluGen, Inc.: Salary Roger Aitchison, ScM, FluGen, Inc.: Advisor/Consultant David Marshall, B.S., FluGen, Inc.: salary Michael Moser, Ph.D., FluGen, Inc.: salary Pamuk Bilsel, Ph.D., FluGen, Inc.: salary


Fusarium verticillioides is a filamentous fungus that causes plant diseases and harms human health through cancer-inducing mycotoxin and life-threatening Fusariosis. Given its threat to agriculture and public health, genome assembly of this fungus is critical to our understanding of its pathobiology and developing antifungal drugs. Here, we report a gap-free genome assembly of F. verticillioides using PacBio HiFi data and high-throughput chromosome capture (Hi-C) sequencing data. The assembled 42.0 Mb sequence contains eleven gapless chromosomes capturing all centromeres and 19 of all 22 telomeres. This assembly represents a significant improvement over previous version on contiguity (contig N50: 4.3 Mb), completeness (BUSCO score: 99.0%) and correctness (QV: 88.8). A total of 15,230 protein-coding genes were predicted, 6.2% of which are newly annotated genes. In addition, we identified three-dimension chromatin structures such as TADs-like structures and chromatin loops based on Hi-C data of ultra-high coverage. This gap-free genome of F. verticillioides is an excellent resource for further panoramic understanding mechanisms of fungal genome evolution, mycotoxin production and pathogenesis on plant and human host. Fusarium verticillioides, a filamentous fungus belonging to Fusarium fujikuroi species complex, causes Fusarium ear rot of maize, a major crop worldwide. Besides yield loss, various mycotoxins are produced during fungal infection of maize, reducing the quality of corn products. The best characterized F. verticillioides mycotoxins are fumonisins, a group of polyketide mycotoxins associated with esophageal cancer and neural tube birth defects in human populations consuming the contaminated maize products1. Although F. veticillioides is considered nonpathogenic to healthy human being, it can become a serious threat to individuals with compromised immune system such as those infected by undergoing organ transplants2. Human infection by F. verticillioides commonly known as Fusariosis has been a surging life threat to the immunocompromised patients due to limited options of antifungal drugs for treatment and emergence of multi-drug resistance3. Therefore, elucidation of molecular mechanisms underlying fungal pathogenesis and antifungal resistance in F. verticillioides is crucial to both agricultural safety and public health.F. verticillioides strain 7600 was released in 20104 with a contig N50 of 392.3 kb. Recently, several updated versions of F. verticillioides genome assemblies are available in NCBI  genome database. Although these genome assemblies have since facilitated the genetic studies of fungal biological processes, they are highly fragmented with several hundreds to thousands of contigs. The fact that F. verticillioides has 11 chromosomes suggests the presence of gaps in these assembly versions. Furthermore, no telomere and centromere sequences have been reported in any F. verticillioides genome assembly available, leaving these essential and complex genomic regions unexplored. A complete genome sequence for F. verticillioides would enable accurate characterization of the fungal genome function, regulation and evolution, shedding light on mechanisms of growth, development, pathogenicity and mycotoxin production.Despite the importance of this fungus, its complete genome sequence has not been assembled and thoroughly analyzed, impeding dissection of molecular and evolutionary mechanisms underlying its pathogenesis, secondary metabolism and drug resistance. The first genome assembly of F. verticillioides, and update the genome annotations based on the improved genome assembly. We sequenced the genome of F. verticillioides strain 7600 to produce high-fidelity (HiFi) long reads of PacBio  single-molecule real-time (SMRT) sequencing, and Hi-C (high throughput chromatin conformation capture) data using Illumina pair-end sequencing. In total, we generated 4.1 Gb (~96.7X coverage) PacBio HiFi raw reads with a N50 of 10.0 kb. and 53.8 Gb Hi-C data   and Flye7 to obtain draft genome assemblies which were individually polished using Nextpolish (v.1.4.0)8 followed by assembly merge using quickmerge (https://github.com/mahulchak/quickmerge)  contains 11 gap-free chromosomes  compared to the previous assembly Table . CompariF. verticillioides contained all centromeres on 11 chromosomes  Hi-C data containing 95.8% valid interaction pairs after initial quality control  slant and stored in −80 °C freezer. F. verticillioides 7600 mycelia and spores harvested from two-day old PDB (potato dextrose broth) culture in 150 rpm shaker at 25 °C were used to isolate high molecular weight DNA using CTAB (cetyltrimethylammonium bromide) method11. A total of 15 µg purified genomic DNA were used to construct a standard PacBio SMRTbell library using PacBio SMRT Express Template Prep Kit 2.0 . The sequencing was performed using a PacBio Sequel II instrument at Biomarker Technologies Corporation .F. verticillioides was prepared from cross-linked chromatins of fungal mycelia using a standard Hi-C protocol12. The constructed Hi-C sequencing library was sequenced by a test run and examined for valid interaction read pair ratios using HiCPro (v.3.1.0)13 before going through high coverage sequencing. The library was sequenced by Illumina NovaSeq. 6000 to yield 10.5 Gb (249.7 coverage) paired-end reads. The valid interaction pairs of Hi-C sequencing reads were used to anchor all contigs using Juicer (v.1.5)9, followed by using a 3D-DNA correction pipeline10 and manually correction with Juicebox (v.1.11.08)14. compartment A/B were analyzed using HiTC (v.1.40.1)15 and Cworld-dekker (https://github.com/dekkerlab/cworld-dekker), TADs-like structures and chromosome loop were identified by Juicer (v.1.5)9. Three-dimensional structure visualization of the whole genome using pyGenomeTracks (v.3.7)16.Hi-C library construction of 5, HiCanu (v.1.4)6 (parameters: -assemble -pacbio-hifi oeaErrorRate = 0.001), Flye (v.2.9)7 and NextDenovo (v.2.5.0) , to assemble, respectively, and then sorted the number of contigs of different assemblers in ascending order. Based on four assemblies we used quickmerge (v.0.3) (https://github.com/mahulchak/quickmerge) to produce a merged genome assembly, and finally used Juicebox14 to manually adjust misassemblies.To optimize the genome assembly strategy and take into account the differences of assembly algorithm between software, we used Hifiasm (v.0.16.1)F. verticillioides using Trizol  agents following manufacturer recommendation protocol. The RNA Nano 6000 Assay Kit of Agilent Bioanalyzer 2100 system  was used to evaluate the total RNA integrity. The total RNA used for library preparation first enriched the mRNA with polyA tail through Oligo (dT) magnetic beads. The mRNA was then subjected to sequencing library construction using Illumina True-seq transcriptome kit  with an insert size of 370bp–420bp, and sequenced by an Illumina Novaseq. 6000 platform at Biomarker Technologies Corporation  to generate 150 bp paired-end reads. RNA-seq data was checked for quality using fastp (v.0.23.2)17, mapped to F. verticillioides genome assembly using hisat2 (v.2.1.0)18, followed by calculating mapping ratios by samtools (v.1.15)19.Total RNA was extracted from the mycelia of de novo prediction and similarity aligmment to annotate it via RepeatModeler (v. 1.0.11)20 (parameters: -database -engine ncbi -pa) and softmasked genome by RepeatMasker (v. 4.1.2.p1)21. RepeatMasker’s perl script (rmOutToGFF3.pl) converts various types of repeat sequence annotation results into a common generic feature format (GFF) version 3. Gene model prediction combined with the following three aspects of evidence: (a) ab initio prediction, (b) homologous protein, (c)RNA-seq evidence. During the ab initio prediction, we firstly trained the GeneMark-ET model for five rounds using BRAKER2 (v.2.1.6)22 (parameters:–species = Fv–fungus–softmasking–genome–bam–prot_seq–prg = gth–gff3–rounds = 5), whose process employed GeneMark-ET23, NCBI BLAST24, DIAMOND25 and GenomeThreader26. We then trained the SNAP27 semi-HMM model for two rounds using MAKER28 . AUGUSTUS29 used the built-in Fusarium genome feature model. To provide homologous protein evidences for gene prediction, we downloaded the protein data of this species (anchored chromosomes) from the public database, including 7600 (NCBI Assembly ID: GCA_ 000149555.1), BRIP53590 (NCBI Assembly ID: GCA_003316995.2), BRIP53590 (NCBI Assembly ID: GCA_003317015.2) and BRIP14953 (NCBI Assembly ID: GCA_003316975.2). For transcriptome data, RNA-seq reads from the vegetative phase were firstly aligned to our genome assembly through hisat218 for BRAKER2 (v.2.1.6)22. Then, we performed reference-based assembly and de novo assembly of transcriptomes by Scallop (v0.10.5)30 and Trinity (v.2.8.4)31 , respectively. Transcripts obtained by two methods are de-redundant with CD-HIT (v.4.6)32 (parameters:-I -c 0.99 -T 50 -M 100000 -o). The above three evidences are integrated by MAKER (v.3.01.03)28 to predict the final gene model. Rfam/Infernal (v.1.1.4)33 (parameters: cmscan–cut_ga–rfam–nohmmonly–fmt 2 –clanin –tblout) and tRNAscan-SE (v. 2.0.9)34 (parameters: -E -X 20 –f -m -b -j–detail) are used to infer genome-wide non-coding RNAs. To compare the previous (NCBI: GCA_000149555.1) and our genome assembly, we performed the genome annotation on two genome assemblies by using the same software, parameters, and protein data.For repetitive sequences, we firstly use 35 with accession number of SRR2100352136, SRR2100352037, SRR2100351938, respectively. The gap-free genome assembly is deposited under the same BioProject at NCBI (GCA_027571605.1) and also in Genome Warehouse of National Genomics Data Center (https://ngdc.cncb.ac.cn/) at China National Center for Bioinformation under the accession number of GWHBQEB0000000039. Genome annotations including protein-coding regions, repeat sequence and ncRNA annotation files have been submitted to the online open access repository Figshare40.The raw PacBio HiFi sequencing data, Hi-C data and RNA-seq data have been deposited in the National Center for Biotechnology Information (NCBI) under the BioProject (PRJNA868307)14. We then aligned the species’ mitochondrial genome to our assembly by megaBLAST24, which found no errors. Finally, we also used megaBLAST24 to aligned our genome assembly against a common database (ftp://ftp.ncbi.nlm.nih.gov/pub/kitts/contam_in_euks.fa.gz) to identify potentially contaminated sequences sequencing adaptor sequence (ftp://ftp.ncbi.nlm.nih.gov/pub/kitts/adaptors_for_screening_euks.fa) and nucleotide sequence database (remote mode), which again found no contamination.To get a nearly complete and error-free nuclear genome, we first manually corrected the assembly using Hi-C read alignment within the Juicebox41 (parameters: -W repetitive_k15.txt -t 104 -ax map-pb) and the quality value (QV) was assessed using Merqury (v.1.3)42 (parameters: k = 18 count). Second, the BUSCO 43 analysis was conducted to reflect the completeness of genome assembly. The final F. verticillioies gap-free genome assembly has a QV of 88.8, completeness of 99.7% and BUSCO score of 99%, suggesting the high accuracy and completeness of the assembly, respectively 44. This assembly has reduced the length of gaps from 90,816 in previous version to 0, and captured eleven centromeres and nineteen telomeres (TTAGGG) except missing three telomeres via trf (v. 4.09.1)45 (parameters: 2 7 7 80 10 90 2000 -d -m -l 2) from assemblies and raw sequences, one each at the end of Chr2 and Chr4 47 (parameters: intersect -wa -wb -f 1.0), which account for 99.5% of the old version genome and 93.6% of this study genome. Compared to previous version (NCBI: GCA_000149555.1), our assembly has corrected three major inversions (Fig. 48 plot.The resolved fungal telomere and centromere regions have been well covered by PacBio HiFi reads that span these complex regions Fig.  by IGV (hr4 Fig. . There iools v.2.0.047 (paSupplementary Table 1


Correction: BMC Public Health 23, 960 (2023)https://doi.org/10.1186/s12889-023-15934-yFollowing publication of the original article , the autFig. 2 correct version of Fig. 1


Little is known about the first line induction chemotherapy cycles for HIV-associated diffuse large B-cell lymphoma (DLBCL) as these are less common than HIV-negative lymphoma. Currently, the optimal treatment cycles option remains undefined. Therefore, we performed a multi-center study to analyze the clinical characteristics and outcomes of HIV-associated DLBCL patients in different treatment modes in China.Totally 273 newly diagnosed HIV-associated DLBCL patients at eleven large academic centers from October 2008 to October 2021, were analyzed.p<0.001). Cox multivariate analysis showed that age ≥60 , high IPI score (3-5) , B symptoms , elevated LDH  and received less than 4 cycles chemotherapy  were independent risk factor for adverse prognosis based on overall survival (OS).In the entire cohort, the median age was 47 years  at lymphoma diagnosis, and 223 patients were male (81.7%). One hundred and ninety-four (71.1%) patients were germinal center B-cell-like lymphoma (GCB) subtype. Most patients  had elevated lactate dehydrogenase (LDH), and advanced Ann Arbor stage (78.9% 213/273) at diagnosis. High international prognostic index (IPI) score (3-5) at diagnosis was found in 65.2% (178/273) of patients. One hundred and fifty-five patients (56.8%) had extranodal involvement. The median CD4 cell count was 168/μl , of whom 174 (63.7%) had a CD4 cell count below 200/μl. The median follow‐up of our cohort was 10.1 (0.1-160) months. The overall 2-year OS rates 58.0%. Median OS times in the 0, 1-3, 4-6, and >6 cycles chemotherapy cohort were 7.1 months, 20.0 months, not reached, and not reached, respectively (Hazard Ratio (HR)=0.549, 95% Confidence interval (CI) 0.451-0.667; These results demonstrated that 4-6 cycles chemotherapy were significantly associated with improved outcomes in HIV-associated DLBCL patients. However, >6 cycles chemotherapy did not further improve the survival of patients. Human immunodeficiency virus (HIV)-associated lymphoma is a rare aggressive non-Hodgkin lymphoma. The incidence rate reported in 2017 was 100/100, 000 to 300/100,000 in population infected with HIV, becoming the highest incidence rate among HIV-associated cancers in the United States , regardless of chemotherapy regimen or dose intensity for HIV-associated DLBCL patients. This is because patients are easily complicated by opportunistic infections, resulting in an increase in mortality , 5. Withp<0.001)  received no anti-lymphoma chemotherapy because of fear of discrimination and poor financial situation. Median survival times in the patients who no received any anti-lymphoma chemotherapy was only 3.5 months, which was significantly lower than that of patients receiving chemotherapy  classification criteria.cART included two nucleoside reverse transcriptase inhibitors and one nonnucleoside reverse transcriptase inhibitor. The chemotherapy cohort included patients who received any of the following regimens: etoposide, doxorubicin, vincristine, cyclophosphamide and prednisone (EPOCH); rituximab, etoposide, doxorubicin, vincristine, cyclophosphamide and prednisone (R-EPOCH).18F-fluorodexyglucose positron emission tomography/computed tomography (PET/CT) or computed tomography (CT) were performed for radiological evaluation. Responses were classified according to standard criteria per each institution, typically aligning with Lugano criteria, which including complete response (CR), partial response (PR), stable disease (SD) and progressive disease (PD).Progression-free survival (PFS) was defined as the time from lymphoma diagnosis to disease progression, relapse or death from any cause. Overall survival (OS) was defined as the time from lymphoma diagnosis to last follow-up or death from any cause. Survival was estimated using Kaplan–Meier curves and compared by the log-rank test. The Cox proportional hazards regression model was used in multivariate analysis to determine prognostic factors. All statistical tests were two‐sided, and p ≤ 0.05 was considered statistically significant. All statistical data were analyzed with GraphPad Prism 9.2-microglobulin (β2-MG), and advanced Ann Arbor stage (78.9% 213/273) at diagnosis. High international prognostic index (IPI) score (3-5) at diagnosis was found in 65.2% (178/273) of patients. One hundred and fifty-five patients (56.8%) had extranodal involvement. Totally 53(19.4%) patients had bone marrow involvement, 24(8.8%) patients had central nervous system (CNS) involvement, 64 (23.4%) had poor Eastern Cooperative Oncology Group performance status (ECOG PS 2-4), and 150 (54.9%) had B symptoms at diagnosis. The median CD4 cell count at diagnosis was 168/μl , of whom 174 (63.7%) had a CD4 cell count below 200/μl at diagnosis. Among the Epstein-Barr virus (EBV) status, EBV load was elevated (5×103 copies/ml) in 104 (38.1%) patients. Of all patients, 37(13.6%) had positive HBsAg and 7(2.6%) were positive for anti-Hepatitis C virus (HCV) antibody.Two hundred and seventy-three newly diagnosed HIV-associated DLBCL were included in this study. The median age was 47 years  at lymphoma diagnosis, and 223 patients were male (81.7%). One hundred and ninety-four (71.1%) patients were germinal center B-cell-like lymphoma (GCB) subtype. Most patients  had elevated lactate dehydrogenase (LDH),  elevated serum β2-MG, Ann Arbor stage, IPI score, extra-nodal involvement, bone marrow involvement, CNS involvement, CD4 cell count, and chemotherapy regimen. The 0 cycle chemotherapy cohort featured more cases of GCB . Baseline clinical characteristics are summarized in Patients were stratified based on the induction chemotherapy, where 38/273 (13.9%), 71/273 (26.0%), 73/273 (26.7%), and 91/273 (33.3%) were in the 0, 1-3, 4-6, and >6 cycles chemotherapy cohort, respectively. In the study, some patients received no anti-lymphoma therapy because of fear of discrimination and poor financial situation, some patients had less than four cycles of anti-lymphoma therapy also due to poor financial situation. This is because Central and Western China AIDS Lymphoma League (CALL) is located in southwest China, and the economic level of the patients is low. Patient characteristics were similar between the four cohorts, including gender, age, residence, educational level, HIV transmission route, cART therapy, years of HIV infection before lymphoma, ECOG-PS, the presence of B symptoms, elevated serum LDH, elevated serum β.In this cohort study, the median chemotherapy cycles were 4 , 38 (13.9%) received no anti-lymphoma therapy, 71 (26.0%) had less than four cycles of chemotherapy, 73 (26.7%) patients received four to six cycles of chemotherapy, and 91 (33.3%) patients received more than six cycles of chemotherapy. Of the 235 patients who received chemotherapy, 80 (34.0%) received EPOCH regimen and 155 (66.0%) received R-EPOCH regimen. 239 (87.5%) were administered cART. Totally 209 patients were evaluated for best treatment response at the end of treatment, including 65 patients who received EPOCH and 144 patients who received R-EPOCH. The results indicated an overall response rate (ORR) of 63.1% and 80.6%, respectively.CR rate were 23.1% and 42.4%, respectively  . Median PFS times in the 0, 1-3, 4-6, and >6 cycles chemotherapy cohort were 7.1 months, 10.1 months, 36.5 months, and 26.4 months, respectively (Hazard Ratio (HR)=0.713, 95% Confidence interval (CI) 0.600-0.847; p<0.001) . Median OS times were 7.1 months, 20.0 months, not reached, and not reached, respectively  . These results demonstrated that 4-6 cycles chemotherapy were significantly associated with improved outcomes in HIV-associated DLBCL patients. However, >6 cycles chemotherapy did not further improve the survival of patients.The median follow‐up of our cohort was 10.1 (0.1-160) months. Median PFS and OS were 17 months and 38.1 months respectively. The overall 2-year PFS and OS rates were 46.8% and 58.0%, respectively  . Median . Medianp=0.049) . Median OS times were 27.7 months and not reached. The overall 2-year OS rates were 52.6% and 68.5%, respectively (p=0.048) . These data suggested that rituximab administration was significantly associated with improved outcomes.Median PFS times in the no received rituximab and received rituximab groups were 16 months and not reached. The overall 2-year PFS rates were 41.0% and 53.0%, respectively (p=0.049) . Median p=0.006), high IPI score (3-5) , B symptoms , poor ECOG-PS , elevated LDH , Extra-nodal involvement  and received less than 4 cycles chemotherapy  were predictive of worse PFS. Age ≥60 , high IPI score (3-5) , B symptoms , elevated LDH  and received less than 4 cycles chemotherapy  were predictive of worse OS.Univariate analysis showed that advanced stage , B symptoms  and received less than 4 cycles chemotherapy  were independent risk factors for adverse prognosis based on PFS. Age ≥60 , high IPI score (3-5) , B symptoms , elevated LDH  and received less than 4 cycles chemotherapy  were independent risk factor for adverse prognosis based on OS .Cox multivariate analysis showed that high IPI score (3-5)  in May 2021. The CALL is a public welfare organization dedicated to the diagnosis and treatment of HIV-associated lymphoma patients. This is the first multi‐center retrospective study with HIV-associated DLBCL in eleven China academic centers from CALL from October 2008 to October 2021. The present study included 273 HIV-associated DLBCL cases, which is the largest cohort reported in China to date. To the best of our knowledge, this is also the first systematic study to explore the effect of chemotherapy cycles on prognosis in a large series of HIV-associated DLBCL.HIV-associated malignancies are divided into acquired immune deficiency syndrome-defining and non-acquired immune deficiency syndrome-defining cancers based on the coincidence rate. Kaposi’s sarcoma, Non-Hodgkin’s lymphoma and invasive cervical cancer are considered acquired immune deficiency syndrome- defining cancers. Other cancers, which including Hodgkin’s lymphoma, hepatocellular carcinoma, oral and pharyngeal cancers, lung cancer, anal cancer, vulvar cancer and penile cancer, are considered non-acquired immune deficiency syndrome-defining cancers , 2. The The present cohort study showed that the median age was 47 years  at lymphoma diagnosis, and 223 patients were male (81.7%), in agreement with the overall male-to-female HIV incidence rate in China . CompareCurrently, there are no standard guidelines for treatment of HIV-associated DLBCL. In the pre-cART era, median survival times only 5 to 8 months for HIV-associated DLBCL patients. This is because patients are easily complicated by opportunistic infections, resulting in an increase in mortality , 5. In tp=0.03) and PFS  compared with HIV-negative patients (p=0.006) and median OS  compared with no receiving rituximab (p=0.049). The overall 2-year OS rates were 52.6% and 68.5%, respectively (p=0.048). The current data also suggested that rituximab administration was significantly associated with improved outcomes in HIV-associated DLBCL. In AMC034, rituximab combined with EPOCH was assessed, and the results were compared with R-CHOP in AMC010. The results showed that a higher CR rate (69%) was obtained for R-EPOCH compared with R-CHOP (47%)  . In the % . In thOP (47%) .p<0.001), and CR rate  were significantly higher in patients who received chemotherapy (p<0.001) were independent risk factor for adverse prognosis.The optimal treatment cycles option of first line induction chemotherapy for HIV-associated DLBCL remains undefined. Compared with HIV-negative DLBCL patients, HIV-associated DLBCL patients are difficult to accept long-term hospitalization chemotherapy, whether there is an appropriate reduction in the number of chemotherapy cycles, the survival of patients will not be affected? Wu D retrospectively analyzed the clinical features of 100 HIV-associated lymphoma patients, which including 66 HIV-associated DLBCL. 15 patients (15%) did not received chemotherapy. Compared to those who did not received chemotherapy, the 2-year OS rate (41.0% vs. 0%, otherapy . In our otherapy . Once thotherapy , 20–24. In summary, our multi-center study suggested that received less than 4 cycles chemotherapy predicted poor OS and PFS in HIV-associated DLBCL patients. Therefore, we recommend that patients receive at least 4 cycles of systemic chemotherapy. More high-quality randomized controlled studies were needed to test our findings.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.CW, YW, JL, and HM conceived and designed the study, analyzed the data, and drafted and revised the paper. HZ, YC, and YL conceptualized and designed the study. All authors provided critical comments to the manuscript. All authors contributed to the article and approved the submitted version.