Mycobacterium tuberculosis (Mtb) and Salmonella enterica serovar Typhimurium (Stm) and localizes to Mtb in macrophages. To determine the combined contribution of membrane damage-sensing galectins to immunity, we generated Gal-3, -8, and -9 triple knockout (TKO) mice. Mtb infection of primary macrophages from TKO mice resulted in defective autophagic flux but normal bacterial replication. Surprisingly, these mice had no discernable defect in resistance to acute infection with Mtb, Stm or Listeria monocytogenes, and had only modest impairments in bacterial growth restriction and CD4 T cell activation during chronic Mtb infection. Collectively, these findings indicate that while Gal-3, -8, and -9 respond to an array of intracellular pathogens, together these membrane damage-sensing galectins play a limited role in host resistance to bacterial infection.Macrophages employ an array of pattern recognition receptors to detect and eliminate intracellular pathogens that access the cytosol. The cytosolic carbohydrate sensors Galectin-3, -8, and -9  recognize damaged pathogen-containing phagosomes, and Gal-3 and Gal-8 are reported to restrict bacterial growth via autophagy in cultured cells. However, the contribution of these galectins to host resistance during bacterial infection in vivo remains unclear. We found that Gal-9 binds directly to  Listeria monocytogenes, Salmonella enterica serovar Typhimurium, and Mycobacterium tuberculosis (Mtb), and were only modestly susceptible to chronic Mtb infection. Our data suggest that Gal-3, -8 and -9 are not critical for innate immune responses during acute infection and may play a more prominent role in the adaptive immune response. These results broaden our understanding of the role of membrane damage-sensing pathways in host defense against bacterial infection.Intracellular bacterial pathogens cause many of the world’s most deadly infectious diseases. A common requirement for nearly all intracellular pathogens is the ability to damage the endomembrane compartments in which they reside, which allows pathogens access to the nutrient-rich cytosol of the host. However, membrane damage also creates a “pattern of pathogenesis” that triggers antimicrobial immune responses. Galectin-3, -8, and -9  act as a surveillance system for membrane damage and Gal-3 and Gal-8 inhibit bacterial growth by activating autophagy, a cellular pathway that can capture cytosolic bacteria and degrade them in lysosomes. Membrane damage-sensing galectins were hypothesized to promote bacterial killing during acute infection yet their role in the immune response of an infected animal remains unclear. Here, we show that mice deficient for Gal-3, -8, and -9 had no defects in resistance to acute infection with the pathogens  Infectious diseases caused by intracellular bacterial pathogens are among the leading causes of mortality worldwide . BacteriMycobacterium tuberculosis (Mtb), Salmonella enterica serovar Typhimurium (Stm), Listeria monocytogenes (Lm), Shigella flexneri, and Legionella pneumophila (Lp), inflict phagosomal membrane damage that leads to the recruitment of Gal-3, -8, and -9  at 30°C. Cells were harvested mid-log phase for in vitro binding studies.Primary murine bone marrow-derived macrophages (BMMs) were isolated and cultured in BMM media  as previously described . For all-/- and Gal-3-/- cells, RAW 264.7 macrophages were transduced with lentiviral particles containing lentiCRISPR v2 (Addgene #52961) expressing Cas9 and sgRNAs. The following sgRNAs were used: ACGGACAGTGGGGTCCTG , CCCCGGCCCAACTGCCCC . Cells were genotyped and frameshift mutations were confirmed by Sanger sequencing. To generate Gal-9 shRNA knockdown THP-1 cells, shRNAs were designed to target human Gal-9 mRNA and cloned into the pLKO.1 vector (Addgene #10878). The sense shRNA sequences are CCTGGTGCAGAGCTCAGATTT  and GCTCTGTGCAGCTGTCCTA . Lentiviral particles were produced in Lenti-X cells (Clontech) per manufacturer’s instructions. Early passage THP-1 cells were transduced with lentiviral particles containing the shRNA expression vector. The cells were selected in 0.5 μg/mL puromycin to obtain a polyclonal population. Knockdown efficiency was assessed via qPCR using the following primers: 5’-CCGAAAAATGCCCTTCGTCC-3’ (forward), 5’- ACCTTGAGGCAGTGAGCTTC-3’ (reverse) and determined to be 95% and 91% knockdown for shGal-9 #1 and shGal-9 #2, respectively.To generate Gal-9-3xFLAG cells, RAW 264.7 macrophages were transduced with lentiviral particles containing Gal-9-3xFLAG expressed from pLenti-puro (Addgene #39481). To generate Gal-9Mtb-infected BMMs were treated with vehicle control (DMSO) or bafilomycin A1 (Sigma) at the indicated concentrations for 3 hours prior to fixation.For lysosomal damage experiments, BMMs were incubated with LLOMe (Sigma) at the indicated concentrations for 2 hours prior to fixation. For the autophagic flux experiments, Mtb-LUX experiments, cells were infected by removing media from wells, overlaying cell monolayers with bacterial suspensions, and spinfecting for 10 minutes at 300 rcf.To ensure bacterial replication or autophagy targeting was not impacted by differences in macrophage concentration, WT BMMs were plated in 96-well plates at the target cell density, while TKO BMMs were plated on a cell density gradient in order to ensure equivalent plating numbers of each genotype. After plating, cells were allowed to adhere and equilibrate for 2 to 3 days prior to infection. On day 0 of infection, one plate was fixed in PBS with 4% PFA, washed, DAPI stained, and nuclei were counted at 5X magnification on an Opera Phenix microscope, thus allowing determination of cell density-matched wells. During analysis, wells of different genotypes were only compared if cell counts were matched within <5% of each other. For microscopy, CFU, and Mtb-LUX infections, cells were plated at 4 x 104 cells per well (target density) in Nunc 96-well white walled TC treated plates (Thermo Fisher). For IFN-γ treatment, cells were incubated with 1.5 ng/mL IFN-γ the day before infection. BMMs were infected at a MOI of 1 or 5. Luminescence of Mtb-LUX-infected BMMs in cell density-matched wells was measured as previously described [For escribed .Mtb microscopy experiments, WT Mtb-GFP or ΔeccC Mtb-GFP were used. BMMs were plated in either 24-well glass-bottom plates (Cellvis) at 2 x 105 cells per well, or 96-well PerkinElmer Cell Carrier Ultra plates at 2.5 x 104 cells per well (target density). All infections for microscopy used an MOI of 2. At indicated time points, microscopy plates were washed in warm PBS, fixed in PBS with 4% PFA, washed three times with PBS, and stored in PBS at 4°C before immunostaining. For lysotracker experiments, cells were stained with 50 nM Lysotracker Red DND-99 in BMM media for 3 hours prior to fixation and imaged immediately after washing out the fixative.For all Mtb CFU experiments, BMMs were plated in 24-well TC treated plates at 2 x 105 cells per well, infected with WT Mtb Erdman strain using an MOI of 2, and Mtb CFU enumeration was performed as previously described [For escribed .Mtb infection of THP-1 cells, THP-1 cells were differentiated for 3 days in DMEM with 2 mM L-glutamine, 1 mM sodium pyruvate, and 10% heat-inactivated fetal calf serum with 10 ng/mL PMA and 0.1 ng/mL Vitamin D. On day 0 of differentiation, THP-1 cells were plated at 4 x 104 cells per well in a 96-well plate. The media was exchanged with fresh differentiation media daily. Bacterial suspension was prepared as described above and THP-1 cells were spinfected for 5 minutes. Cells were incubated at 37°C for 30 minutes. THP-1 cells were washed 3 times with PBS with 1% heat-inactivated horse serum. Differentiation media lacking phenol red with PMA and Vitamin D was replaced after the washes. Luminescence of Mtb-LUX-infected THP-1 cells was measured as previously described [For escribed . Half of5 BMMs were plated on 24-well plates 24 hours prior to infection. For IFN-γ treatment, cells were incubated with 0.01 μg/mL IFN-γ the night before infection. Bacteria grown to mid-log phase as described above were pelleted and resuspended in BMM media. Suspension was incubated at 37°C for 30 minutes for opsonization. Bacteria were then pelleted and resuspended to 1 x 109 CFU/mL prior to spinfection with BMMs for 5 minutes at 250 rcf followed by 37°C incubation for 30 minutes for phagocytosis. Cells were washed with PBS and incubated with BMM media with 100 μg/mL gentamicin at 37°C incubation for 30 minutes, then washed with PBS and incubated with BMM media with 5 μg/mL gentamicin. For CFU enumeration, cells were washed with PBS and lysed in 500 uL water for 10 minutes prior to lysate serial dilution and plating on LB agar plates.1 x 106 BMMs were plated on 14 coverslips in 60 mm non-TC treated dishes and infected the next day at an MOI of 0.25. Lm CFU enumeration was performed as previously described [A total of 3 x 10escribed .5 WT or TKO BMMs were plated on 96-well plates and incubated overnight. Medium was replaced with bacterial suspension in BMM media at an MOI of 0.01. At indicated time points, luminescence emission was measured at 470 nm with a Spectra-L plate reader (Molecular Devices).Bacterial replication was measured using a luminescence-based replication assay as previously described . BrieflyFollowing fixation, washing, and storage in PBS at 4°C, cells were blocked and permeabilized in PBS with 5% FBS or 2% BSA and 0.1% Triton X-100 (blocking/permeabilization buffer) for 30 minutes at room temperature. For LC3 immunostaining, cells were incubated with ice cold methanol for 10 minutes on ice, then washed three times with PBS prior to the blocking and permeabilization step above. Cells were then stained in blocking/permeabilization buffer overnight at 4°C with the following primary antibodies at the indicated dilutions: anti-Gal-9 , anti-Gal-3 , anti-polyubiquitin , anti-ubiquitin K63-specific , anti-ubiquitin K48-specific , anti-p62 , anti-Tax1bp1 , anti-Optineurin , and anti-LC3 . Cells were then washed three times with PBS for 5 minutes each. DAPI and secondary antibody staining was performed at room temperature for 30–60 minutes using anti-mouse or anti-rabbit Alexa Fluor 647 or 488 (Invitrogen) at the above indicated dilutions. Cells were washed three times with PBS for 5 minutes then imaged.All microscopy experiments were performed with an Opera Phenix High-Content Screening System confocal microscope (PerkinElmer). Microscopy plates were imaged at 10X magnification for nuclei counting and cell density matching, 40X or 63X magnification for colocalization or puncta quantification, or 63X magnification for representative images. Automated analysis for colocalization, nuclei counting, and puncta quantification was performed with PerkinElmer Harmony software package. For colocalization analysis, a maximum intensity projection was generated from multiple z-stacks and autophagy markers and bacteria were identified using a spot finder algorithm. Autophagy marker spots were then filtered based on size and only spots >25–40 pixels in area were selected for analysis to limit nonspecific colocalization of small puncta with bacteria. Bacteria with at least one pixel of overlap with size filtered autophagy marker spots were counted as a colocalization event, and % colocalization was defined as the total number of colocalization events divided by the total number of bacteria.Mtb at an MOI of 1 for 6 hours. Cells were then detached using 1 mM EDTA in PBS and fixed for 10 minutes at room temperature using freshly prepared periodate-lysine-paraformaldehyde stain . Following fixation, cells were pelleted and resuspended in PBS without washing and stored at 4° until embedding was performed. Samples were embedded in 10% gelatin and infiltrated overnight with 2.3 M sucrose/20% polyvinyl pyrrolidone in PIPES/MgCl2 at 4°C. Samples were trimmed, frozen in liquid nitrogen, and sectioned with a Leica Ultracut UCT7 cryo-ultramicrotome (Leica Microsystems). Ultrathin sections of 50 nm were blocked with 5% FBS/5% NGS for 30 minutes and subsequently incubated with mouse anti-FLAG (Sigma F3165) for 1 hour at room temperature. Following washes in block buffer, sections were incubated with goat anti-mouse IgG + IgM 18 nm colloidal gold conjugated secondary antibody (Jackson ImmunoResearch Laboratories) for 1 hour. Sections were stained with 0.3% uranyl acetate/2% methyl cellulose and viewed on a JEOL 1200 EX transmission electron microscope (JEOL USA) equipped with an AMT 8 megapixel digital camera and AMT Image Capture Engine V602 software (Advanced Microscopy Techniques). All labeling experiments were conducted in parallel with controls omitting the primary antibody.RAW 264.7 cells expressing mouse Gal-9-3xFLAG were infected with Mtb, Lm, and Lp experiments. TKO strain 2  was used for the Stm macrophage and mouse experiments.All mice used were specific pathogen free, maintained in 12 hour light-dark cycle, and given standard chow diet ad libitum. 8 to 12-week-old male and female mice were used. WT C57BL/6J mice were obtained from Jackson Laboratories (JAX). CRISPR/Cas9 targeting was performed by electroporation of Gal-3, Gal-8, and Gal-9-targeting Cas9-sgRNA RNP complexes into fertilized zygotes from C57BL/6J female mice  . TKO micMtb Erdman strain frozen stock was diluted in sterile water and sonicated at 90% amplitude three times for 30 seconds. 8 to 12-week-old male and female mice were infected with approximately ~75 CFUs  or >400 CFUs (high dose) of prepared inoculum using a Glas-Col inhalation exposure system. At indicated time points, lungs and spleens were isolated, homogenized, and plated on 7H10 plates with Polymyxin B , Carbenicillin (50 mg/L), Trimethoprim lactate (20 mg/L) and Amphotericin B (5 mg/L). Plates were incubated at 37°C for 25–28 days before counting colonies. For survival experiment, mouse weight was measured weekly and mice were sacrificed after losing 15% of maximum body weight.To prepare infection inoculum, Stm strain IR715 was cultured as described above. Mice received 100 μl of sterile PBS or 1000 CFUs diluted in 100 μl PBS by intraperitoneal injection. Mouse weights were monitored daily during infection. Systemic bacterial levels were characterized at day 1, 2, or 3 post-infection by harvesting liver and spleen and enumerating CFU. Liver and spleen were collected in PBS, weighed, and homogenized. Homogenates were serially diluted and plated on LB agar plates. CFU per gram tissue was calculated after overnight growth at 37°C.Lm was grown at 37°C with shaking at 200 rpm to mid-log phase. Bacteria were washed in PBS and resuspended at 5 x 105 colony-forming units (CFU) per 1 mL of sterile PBS. Mice were then injected with 1 x 105 CFU via the tail vein. 48 hours post-infection, livers and spleens were collected, homogenized, and plated on LB streptomycin 200 μg/mL to determine the number of CFU per organ.Spleens were isolated and pressed through a 70 μm cell strainer to generate a single cell suspension. Lung cells were isolated and digested in RPMI with Liberase TM (70 μg/ml) and DNase I (30 μg/ml) in gentleMACS C-tubes then placed at 37°C for 30 minutes, followed by gentleMACS tissue homogenization for single cell suspension. Cells were pelleted and RBCs were lysed using ACK lysing buffer. Cells were washed, pelleted, resuspended in X-VIVO TM 15 (Lonza) and counted on a hemocytometer or using fluorescent count beads on an SH800 Sony Sorter. Cells were plated, stimulated overnight with indicated peptides (2 μg/ml) or Concanavalin A , developed, and analyzed as previously described . ConA-stAnalysis of statistical significance was performed with Prism 9 (GraphPad).S1 FigA) Cryosections of RAW 264.7 cells stably expressing Gal-9-FLAG infected with WT Mtb (MOI = 1) 6 hours post-infection. Gal-9 localization to extra-phagosomal membranes (black arrowheads), phagosomes (black arrows), and Mtb (white arrowheads); b, bacteria. Four representative micrographs are shown from a dataset of 26 total images. Scale bar = 0.5 μm. (B) Quantification of (A) for Gal-9-FLAG localization to indicated structures; EP membrane, extra-phagosomal membranes. Values are a percent of total Gal-9-FLAG puncta in the dataset.((TIFF)Click here for additional data file.S2 FigA) Immunoblots of bone marrow-derived macrophage (BMM) lysates from WT and TKO mice (strain 2) probed for indicated proteins. (B) Confocal microscopy of WT or TKO BMMs treated with LLOMe at the indicated concentrations for 2 hours and immunostained for LC3. (C) Quantification of (B) for LC3 puncta per cell. Figures are representative of three independent experiments . An average of 400 cells were analyzed per technical replicate (C). Error bars represent SD from four technical replicates, and **p<0.01, ***p<0.001 by unpaired t-test.((TIFF)Click here for additional data file.S3 FigA) Confocal microscopy of WT or TKO BMMs infected with Mtb-GFP (MOI = 2) 8 hours post-infection and immunostained for K48- or K63-linked polyubiquitin, or p62. (B) Quantification of (A) for Mtb-GFP colocalization with indicated markers. (C) Confocal microscopy of WT or TKO BMMs infected with Mtb-GFP (MOI = 2) 8 hours post-infection and immunostained for p62, Tax1bp1, or Optineurin. (D) Quantification of (C) for Mtb-GFP colocalization with indicated markers. (E) Quantification of polyubiquitin puncta 8 hours post-infection in mock- or Mtb-GFP-infected (MOI = 2) WT or TKO BMMs. (F) Quantification of Lysotracker puncta 8 hours post-infection in mock- or Mtb-GFP-infected (MOI = 2) WT or TKO BMMs. (G) Quantification of LC3 colocalization with WT or ΔeccC Mtb (MOI = 2) at indicated time points. Figures are representative of two (A-D) independent experiments or represent one (G) or two  independent experiments. An average of 747 , 926 , 1464 , and 429 (G) cells were analyzed per technical replicate. Error bars represent SD from three  or four  technical replicates, and **p<0.01, ****p<0.0001 by unpaired t-test.((TIFF)Click here for additional data file.S4 FigA) control or Gal-9 knockdown shRNA THP-1 cells infected with Mtb-LUX (MOI = 1), (B) control, Gal-3-/-, or Gal-9-/- RAW 264.7 cells infected with Mtb-LUX (MOI = 1), (C) WT or Gal-9-/- BMMs infected with Mtb-LUX (MOI = 2), or (D) Gal-8+/- or Gal-8-/- BMMs infected with Mtb-LUX (MOI = 1). Figures are representative of two  or three (C) independent experiments, or represent four independent experiments (A). Error bars represent SD from three technical replicates, and **p<0.01, ***p<0.001 by unpaired t-test.RLU-fold change of ((TIFF)Click here for additional data file.S5 FigA) ~18 CFUs or (B) ~136 CFUs of Mtb Erdman and bacterial loads in lungs were enumerated by plating for CFU at indicated time points. n = 3–5 mice per genotype. Figures represent one experiment. Bars represent the mean, error bars represent SEM, and p-values were determined by unpaired t-test.WT and TKO female mice were aerosol infected with ((TIFF)Click here for additional data file.S6 FigA) WT and TKO male mice were aerosol infected with ~450 CFUs of Mtb Erdman and bacterial loads in lungs were enumerated by plating for CFU at indicated time points. n = 5 mice per genotype. (B) Same as in (A) but bacterial loads in spleens. Figures represent one experiment. Bars represent the mean, error bars represent SEM, and p-values were determined by unpaired t-test.((TIFF)Click here for additional data file.S7 FigA) WT and TKO mice were aerosol infected with ~100 CFUs of Mtb Erdman and splenocytes were isolated for IFN-γ ELISpot to enumerate Mtb-specific T cells. Mtb-specific spot-forming cells (SFC) responding to re-stimulation in vitro with Mtb peptides are shown for each condition relative to Concanavalin A (ConA) total T cells and WT. n = 10–11 mice per genotype. (B) Same as in (A) but ELISpot performed with lung single cell suspension. n = 4 mice per genotype. (C) ELISpot on splenocytes following high-dose aerosol inoculation (~800 CFUs). n = 5 mice per genotype. Figures represent data from three (A) independent experiments or one  independent experiment. Error bars represent SD, and ****p<0.0001 by unpaired t-test.((TIFF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Antimicrobial Resistance & Infection Control (2020) 9:13610.1186/s13756-020-00802-wThe original article [1] contained an error in author, Gedefaw Diress Alen ’s name which has since been amended.


Increased breakthrough infections among nursing home residents (NHR) are clinically consequential despite good vaccine coverage and substantial antibody titers. While vaccination blunted severe outcomes, breakthrough infections may arise from lower vaccine-induced antibody quality. We aimed to assess vaccine-induced antibody quality among NHR and younger healthcare workers (HCW) using antibody avidity.We longitudinally sampled 50 NHR and 30 HCWs, with or without prior history of COVID-19, who received the BNT162b2 mRNA vaccine at 0-14 days before, 2 weeks, and 4-6 months after each vaccine dose up to the bivalent (BV) boosters. Primary outcomes included anti-spike, anti-Receptor Binding Domain (RBD), and avidity levels to the ancestral Wuhan, Delta, and Omicron BA.1 strains determined by bead-multiplex immunoassay and ability to bind after 6M urea vs. saline incubation.After the primary series, anti-spike and RBD titers increased significantly to all 3 strains in both NHR and HCW. Avidity levels progressively increased across all time points up to the 3rd dose. Interestingly, this increase continued 6-8 months after vaccination suggesting that affinity maturation continues to occur for many months after the primary vaccination series. NHRs without prior infection have the poorest avidity levels to Delta and Omicron, especially before the 3rd dose. The 3rd dose raised avidity levels substantially in all groups and for all strains. Avidity levels after boosting, across the 3rd and 4th doses, demonstrate evidence of durability. Preliminary findings after BV boost show a continuation of avidity maturation with a larger impact on prior infected individuals.Wuhan Spike AvidityAvidity index of the Wuhan spike antibody across different time points among naive and prior infected individuals. Prevax- before primary series; Postvax- 2 weeks after primary series; M6- 6 months after primary series; Postboost- 2 weeks after 1st monovalent booster; M3M6postboost- 3-6months post booster; Postboost 2- 2 weeks after 2nd monovalent booster. NH- Nursing home; HCW- Healthcare WorkersOmicron BA.1 spike AvidityAvidity index of the BA.1 spike antibody across different time points among naive and prior infected individuals. Prevax- before primary series; Postvax- 2 weeks after primary series; M6- 6 months after primary series; Postboost- 2 weeks after 1st monovalent booster; M3M6postboost- 3-6months post booster; Postboost 2- 2 weeks after 2nd monovalent booster. NH- Nursing home; HCW- Healthcare WorkersThis study underscores the importance of booster vaccination among NHR and HCWs. The booster dose increases avidity, adding to vaccine-induced antibody functional ability. Higher avidity antibodies have higher cross-reactivity to other SARS-CoV-2 strains, as Wuhan boosting improves Delta and Omicron avidity, and should improve protection from ever-evolving strains. Higher avidities may help explain how the vaccine’s protective effects persist even while antibody titers fade between vaccine doses.Stefan Gravenstein, MD, MPH, CDC: Grant/Research Support|Genentech: Advisor/Consultant|Genentech: Grant/Research Support|GSK: Advisor/Consultant|GSK: Honoraria|Janssen: Advisor/Consultant|Janssen: Honoraria|NIH: Grant/Research Support|Pfizer: Grant/Research Support|Pfizer: Honoraria|Sanofi: Advisor/Consultant|Sanofi: Grant/Research Support|Sanofi: Honoraria|Seqirus: Grant/Research Support|Seqirus: Honoraria David Canaday, MD, Pfizer: Grant/Research Support


Multisystem inflammatory syndrome in children (MIS-C) is a rare but severe complication of SARS-CoV-2. Identifying clinical and demographic characteristics associated with MIS-C mortality and description of MIS-C decedents over time may help identify children at increased risk for poor outcomes and potentially inform treatment decisions.This investigation included all MIS-C cases with known outcome reported to CDC MIS-C national surveillance from February 2020─December 2022. Clinical and demographic characteristics were compared between survivors and decedents. Decedents were described over time and classified by date of MIS-C illness onset into five pandemic waves corresponding to five peaks of MIS-C activity : (1) 2/19/2020─6/28/2020, (2) 6/29/2020─10/17/2020, (3) 10/18/2020─7/8/2021, (4) 7/9/2021─12/7/ 2021, and (5) 12/8/2021─12/31/2022. Waves 4 and 5 were periods of predominant Delta and Omicron SARS-CoV-2 variant circulation, respectively.Seventy-six decedents and 8,691 survivors were reported (Table). A higher proportion of decedents compared with survivors were aged 16-20 years , from the South  and had MIS-C illness onset in wave 1 . Half (51%) of decedents had an underlying medical condition compared with 24% of survivors (p< 0.01). A higher proportion of decedents than survivors had severe cardiovascular (95% vs 76%), respiratory (99% vs 36%), renal (59% vs 19%), and neurologic (30% vs 4%) involvement, and fewer had mucocutaneous (33% vs 75%). Deaths in the 16-20 age group were predominant in waves 1-4 but comprised only 15% of deaths in wave 5, when 57% of decedents were aged 0-4 years . Race and ethnicity of decedents varied over time. Severe cardiovascular and respiratory involvement was high ( >85-100%) over time in decedents; other organ manifestations varied by MIS-C wave.Older age (16-20 years), residing in the South, illness early in the pandemic, underlying medical conditions, and severe organ involvement were all significantly associated with death among children with MIS-C. MIS-C decedent characteristics are dynamic and may continue to evolve with changes in underlying population immunity, SARS-CoV-2 transmission, and variant emergence.All Authors: No reported disclosures


Correction: Harm Reduction Journal (2023) 20:30 10.1186/s12954-023-00761-6\Following publication of the original article [1], the affiliation for the author Stephanie Tzanetis has been updated as “CanTEST , Canberra, Australia.”The original article has been corrected.


Purpose: This study aimed to evaluate the prognosis of glioma patients with different molecular subtypes of who treated with intensity-modulated radiation therapy (IMRT).Methods: We collected 45 glioma patients treated in our hospital between January 2017 and December 2020. All enrolled patients received postoperative IMRT and were divided into two groups based on the Isocitrate dehydrogenase (IDH status). Overall survival (OS) and progression-free survival (PFS) were estimated retrospectively.p = 0.16). While the 1-year PFS of IDH-mut group was significantly higher than that in ΙDH-wild group . Subgroup analysis revealed that the 1-year PFS of IDH-mut/1p/19q codeletion group and IDH-mut/1p/19q noncodeletion group was significantly higher than in IDH-wild type patients. For patients with IDH-mut/MGMT-methylation, the outcome was no significant difference in OS, but PFS was longer than other subtypes.Results: The median follow-up was 22 months (range 2–108.5 months). The 1-year OS of IDH-mut group and ΙDH-wild group was similar (77.3% vs. 81.5%, Conclusion: This retrospective study showed that 1-year PFS of patients with IDH mutated was better than IDH-wild type patients. In subgroups analysis, the outcomes were shown that patients with IDH-mut/ 1p/19q codeletion and patients with IDH-mut/1p/19q noncodeletion had longer 1-year PFS than IDH-wild type patients, but the OS was similar between the subgroups. Patients with IDH-mut/MGMT-methylation had the best prognosis in the whole subgroups. However, these results still need further confirmation of large sample size, prospectively, randomized controlled trails. Glioma is one of the most common malignant central nervous system tumor. According to central brain tumor registry of the United States (CBTRUS), glioma account for 27% of all CNS and approximately 80% of malignant tumors . The treWe retrospectively collected glioma patients diagnose in the Second Affiliated Hospital of Guangxi Medical University from January 2017 to December 2020. The inclusion criteria were as follows: (1) Age between 16 and 70 years old; (2) histopathology confirmed astrocytoma, oligodendroglioma, glioblastoma, anaplastic astrocytoma, anaplastic oligodendroglioma; (3) Assessable IDH1,IDH2,MGMT,1p/19q status; (4) CT or MRI before surgery; (5) Didn’t receive radiotherapy, chemotherapy or other treatments before; (6) Double cancer were excluded; (7) ECOG score ≤2; (8) All the inclusion patients received post-radiotherapy; The exclusion criteria were as follows: (1) Patients who received tumor treated as before; (2) More than two types malignant tumors; (3) Incomplete follow-up data.Clinical data on patients’ age, gender, pathological type, WHO grade, preoperative tumor size, surgical resection method, radiotherapy dose, recurrence site from the medical history system of The Second Affiliated Hospital of Guangxi Medical University. All the patients received radiotherapy 4–8 weeks after surgery. According to the status of IDH, we divided enrolled patients into two groups: IDH mutate group (IDH-mut group) and IDH wild group (IDH-wild group), regardless their histopathology status. Patient characteristics were summarized in in situ hybridization (FISH).DNA was extracted from tumor tissues by standard methods, and all the samples were examined by experienced pathologists to ensure every sample had 80% or more tumor cell content. The techniques to determine MGMT promoter methylation by one-stage methylation-specific PCR  (methylaAll the enrolled patients were received 6 MV-X ray liner accelerator of intensity modulated radiation therapy (IMRT). Patients were immobilized with an individual head-neck-shoulder thermoplastic mask in the supine position to ensure reproducibility of patient positioning during planning CT and the following radiation. The slice thickness was 3 mm of the CT scan.The target volumes were administered according to the protocol of the EORTC and RTOG. Target delineation as follows:The gross tumor volume (GTV) included the contrast-enhancing regions which present on T1-weighted preoperative MRI scans and all the surgical areas. The clinical target volume (CTV) was determined by the GTV with a margin up to 2 cm. The planning target volume (PTV) was created by extending a 3 to 5 mm margin of the corresponding CTV.The gross tumor volume-1 (GTV-1) as the EORTC described before, but GTV-1 included the perifocal edema regions which was visible on T2 or FLAIR sequence. The clinical target-1 (CTV-1) was based on the GTV with a margin of up to 2 cm . The planning target volume-1 (PTV-1) was the CTV1 extended 3 to 5 mm margin. The gross tumor volume-2 (GTV-2) included the surgical regions and all the contrast-enhancing regions detected on T1-weighted MRI. CTV-2 defined as the GTV-2 extended 2 cm margin. The planning target volume-2 (PTV-2) was 3 to 5 mm CTV2 margin expansion.The prescription dose were as follows: the patients who diagnosed with low-grade gliomas treated with 54 Gy, but if patients had visible residual tumor on MRI, the dose boosted to 60 Gy, the PCTV dose was 45 to 54 Gy. For high-grade gliomas, the prescription dose was PGTV 60 Gy and PCTV 54 Gy.Concurrent chemotherapy was administered with temozolomide: temozolomide 75 mg/m2, daily during radiotherapy.Adjuvant chemotherapy: temozolomide 150–200 mg/ m2/d1-d5, every 28 days a cycle, total 6 cycles.http://www.r-project.org/))). The differences between curves were assessed using the log-rank test . OS was calculated from the time of histological confirmation to the date of death or loss/last time to follow-up. PFS was defined as the time interval between initiation of radiation therapy and the date of the CT or ΜRI examination that confirmed progression according to the RANO criteria [P < 0.05 (2-sided) was considered to indicate statistical significance.The clinical characteristics of enrolled patients were used χ2 test with SPSS v22.0 . The Kaplan-Meier estimator analyses were calculated the Overall Survival (ΟS) and progression-free survival (PFS) . The median age was 46 . Of the 45 samples, 22 (48.9%) of the patients with IDH mutation, including 7 patients with astrocytoma (WHO grade 2), 6 with anaplastic astrocytoma (WHO grade 3), 2 with anaplastic oligodendroglioma (WHO grade 3) and 7 with glioblastoma (WHO grade 4). And 23 of 46 (51.1%) patients were ΙDH wild-type, including 4 astrocytoma patients (WHO grade 2), 6 anaplastic astrocytoma patients (WHO grade 3) and 13 glioblastoma patients (WHO grade 4). In IDH-mut group, there were 14 patients had MGMT methylation, 8 were MGMT-nonmethylation. For IDH-wild group, 8 of the 23 patients had MGMT methylation, while 15 patients were MGMT-nonmethylation. All patients underwent postoperative IMRT. A total of 77.3% of the patients (17 of 22) were treated with radiotherapy concurrent temozolomide in IDH-mut group, and 36.4% of the patients (8 of 22) received RT followed by chemotherapy using temozolomide. Meanwhile, all the patients received temozolomide as the concurrent chemotherapy regimen in IDH-wild group, and 11of 23 (47.8%) patients received adjuvant chemotherapy by using temozolomide after RT. The baseline characteristics of patients, stratified by IDH status, were shown in p = 0.16) were similar in IDH-mut group and ΙDH-wild group. While the rates of 1-year PFS  were significantly higher in IDH-mut group than the ΙDH-wild group (There were 24 deaths (10 of 22 (45.5%) in IDH-mut group and 14 of 23 (60.9%) in the ΙDH-wild group) and 24 patients’ recurrences (9 of 22 (40.9%) in IDH-mut group and 15 of 23 (65.2%) in the ΙDH-wild group). All patients were recurrent in radiation field. The rates of 1-year OS  had IDH-mut/1p/19q codeletion, 10 (22.2%) had IDH-mut/1p/19q noncodeletion and 23 (51.1%) were IDH-wild type. The 1-year OS rate was 83.3% for IDH-mut/1p/19q codeletion group, 70.0% for IDH-mut/1p/19q noncodeletion group, and 81.5% for IDH-wild type group  had IDH-wild/MGMT-unmethylation, 8 (17.8%) were IDH-wild/MGMT-methylation, 8 (17.8%) were IDH-mut/MGMT-unmethylation and 14 (31.1%) were IDH-mut/MGMT-methylation. The 1-year OS rate was 72.0% for IDH-wild/MGMT-unmethylation group, 71.4% for IDH-wild/MGMT-methylation group, 75.0% for IDH-mut/MGMT-unmethylation group and 78.6% for IDH-mut/MGMT-methylation. The 4 molecular subgroups were no significantly associated with OS (Multivariate analysis of 45 valid cases showed no significant prognostic factor in OS and PFS. Univariate analysis revealed that only IDH status was significant prognostic factor of PFS .The WHO classification of brain tumor in version 1,2016  proposedIn conclusion, this retrospective study showed that patients with IDH mutation had better PFS than those patients with IDH-wild type. In terms of IDH-mut/1p/19q codeletion and IDH-mut/1p/19q noncodeletion of glioma patients, the outcomes were shown longer PFS than patients with IDH-wild type, but no difference in OS among the subgroups. Patients with IDH-mut/MGMT-methylation had the best prognosis in the subgroups. However, these results still needs further confirmation of large sample size, prospectively, randomized controlled trails.


Emerging data suggest that plasma with an anti-SARS-CoV-2 level of ≥2 × 104 binding antibody units/mL (BAU/mL) protects against SARS-CoV-2 Omicron BA.1 infection. Specimens were collected using a cross-sectional random sampling approach. For PRNT50 studies, 63 previously analyzed specimens by PRNT50 versus SARS-CoV-2 wild-type, Alpha, Beta, Gamma, and Delta were analyzed by PRNT50 versus Omicron BA.1. The 63 specimens plus 4,390 specimens  were also tested using the Abbott SARS-CoV-2 IgG II Quant assay . In the vaccinated group, the percentages of specimens with any measurable PRNT50 versus wild-type or VOC were wild type (21/25 [84%]), Alpha (19/25 [76%]), Beta (18/25 [72%]), Gamma (13/25 [52%]), Delta (19/25 [76%]), and Omicron BA.1 (9/25 [36%]). In the unvaccinated group, the percentages of specimens with any measurable PRNT50 versus wild type or VOC were wild-type SARS-CoV-2 (16/39 [41%]), Alpha (16/39 [41%]), Beta (10/39 [26%]), Gamma (9/39 [23%]), Delta (16/39 [41%]), and Omicron BA.1 (0/39) . None of the 4,453 specimens tested by the Abbott Quant assay had a binding capacity of ≥2 × 104 BAU/mL. Vaccinated donors were more likely than unvaccinated donors to neutralize Omicron when assessed by a PRNT50 assay.The 50% plaque reduction neutralization assay (PRNTIMPORTANCE SARS-CoV-2 Omicron emergence occurred in Canada during the period from November 2021 to January 2022. This study assessed the ability of donor plasma collected earlier (January to March 2021) to generate any neutralizing capacity against Omicron BA.1 SARS-CoV-2. Vaccinated individuals, regardless of infection status, were more likely to neutralize Omicron BA.1 than unvaccinated individuals. This study then used a semiquantitative binding antibody assay to screen a larger number of specimens  for individual specimens that might have high-titer neutralizing capacity against Omicron BA.1. None of the 4,453 specimens tested by the semiquantitative SARS-CoV-2 assay had a binding capacity suggestive of a high-titer neutralizing capacity against Omicron BA.1. These data do not imply that Canadians lacked immunity to Omicron BA.1 during the study period. Immunity to SARS-CoV-2 is complex, and there is still no wide consensus on correlation of protection to SARS-CoV-2. Since 6–12–) assays . Other, ) assays , 19.50. This assay generates semiquantitative results which can be converted into binding antibody units [BAU] per milliliter  is a high-throughput assay that is simpler to operationalize than PRNTlliliter . A priorRS-CoV-2 . High-thRS-CoV-2 .4 BAU/mL may identify high-titer neutralizing plasma against Omicron BA.1 that could then be used in convalescent plasma clinical trials  (50 had evidence of an anti-S or anti-receptor binding domain (RBD) signal (with or without anti-N)  are presented in Study specimens were subsamples of a larger repeated cross-sectional design with random cross-sectional sampling. Previously, 65 specimens were analyzed by PRNTS-CoV-2)  as well S-CoV-2) . All spe anti-N) . Sixty-t50 results were not compared directly. Instead, the numbers of specimens producing any neutralizing antibodies  were compared within vaccinated and unvaccinated groups.Since different cell lines were used to understand the neutralizing capacity of donor plasma against Omicron SARS-CoV-2, median PRNTSmall numbers of specimens for individuals with a vaccine history and an anti-N signal (possible evidence of a past SARS-CoV-2 infection) led to the combination of data from donors vaccinated with an anti-N signal  and dono50, plasma from vaccinated donors was more likely than plasma from unvaccinated donors to neutralize VOCs (including Omicron BA.1) and wild type. For wild-type neutralization, the proportions were vaccinated (21/25 [84%]) versus unvaccinated (16/39 [41%]) . For Alpha neutralization, the proportions were vaccinated (19/25 [76%]) versus unvaccinated (16/39 [41%]) . For Beta neutralization, the proportions were vaccinated (18/25 [72%]) versus unvaccinated (10/39 [26%]) . For Gamma neutralization, the proportions were vaccinated (13/25 [52%]) versus unvaccinated (9/39 [23%]) . For Delta neutralization, the proportions were vaccinated (19/25 [76%]) versus unvaccinated (16/39 [41%]) . For Omicron (BA.1) neutralization, the proportions were vaccinated (9/25 [36%]) versus unvaccinated (0/39) .When neutralizing capacity was measured by PRNT50 for Omicron and the Abbott Quant Assay, 4,390 randomly sampled specimens were tested by the Abbott Quant assay . The monthly distribution of these 4,453 specimens collected in 2021 was 1,499 in January, 1,465 in February, and 1,489 in March. The BAU per milliliter values of these 4,453 specimens are presented in 4 BAU/mL.In addition to the 63 specimens tested by PRNT50 against wild type, Alpha, Beta, Gamma, Delta, and Omicron BA.1) than plasma from unvaccinated blood donors. In the unvaccinated group, none of the plasma specimens had measurable PRNT50 titers versus Omicron BA.1. As previously noted, specimens were collected when seroprevalence to SARS-CoV-2 was <10% and when most Canadians with a history of SARS-CoV-2 infection would have been infected with wild-type or Alpha SARS-CoV-2  had lower levels of pseudovirus neutralization against BA.1 than convalescent-phase serum from BA.1-infected patients (n = 31) (n = 64] and 6 months postinfection [n = 36]) collected prior to the emergence of BA.1 exhibited decreased neutralization against BA.1 than wild-type SARS-CoV-2 when measured with a 50% fluorescent focus reduction neutralization titer (FFRNT50) assay (n = 24) or 1 dose of vaccine (n = 20) also exhibited reduced neutralization against BA.1 compared to wild type using a pseudovirus assay (n = 10]) had reduced neutralization of BA.1, using a focus-forming neutralization assay  . In anot0) assay . Convaleus assay . A smallon assay .4 BAU/mL, which has been previously associated with high-titer plasma against Omicron BA.1  first identified median BAU/mL levels of ≥2 × 10nfection . This stnfection –53. The fection –, and vacfection –.50  or completely vaccine hesitant (relying on immunity from an earlier infection with wild-type or Alpha SARS-CoV-2). Those individuals may have impaired humoral protection against Omicron BA.1 SARS-CoV-2 infection. Therefore, even in populations with high rates of SARS-CoV-2 infection, vaccination  is an important strategy in reducing the burden of severe disease and death , 59. ThiInstitutional ethics board clearance for this project was received from the University of Alberta and the following institutions: Canadian Blood Services and Sinai Health, Toronto .n = 1,500/month) for a 12-month period from January, February, and March of 2021  , February , and March  for testing with the Abbott Quant assay.Canadian Blood Services collects retention EDTA plasma  specimens as previously described , 32, 60.50 neutralization assays  were previously selected using a published tiered testing approach , 13.Serological evidence of anti-N positivity was defined as the presence of an anti-N signal by at least one of the Abbott Architect anti-N SARS-CoV-2 IgG assay or the Sinai Health anti-N assay (see previous publication ).50 experiments. Vero cell cultures were used for Wuhan wild type  and variant of concern strains . Culture conditions for wild type, Alpha, Beta, Gamma, and Delta followed the experimental conditions previously described , plates were air-dried, and plaques were counted on a lightbox . The OmWe tested 4,453 randomly selected retention specimens by using the Abbott Quant assay  as per the manufacturer’s guidelines and as previously described . These s50 values were assessed for the presence (yes or no) of any measurable neutralizing response against wild type and variant  SARS-CoV-2.A study identification number was assigned by the information technology team at Canadian Blood Services. All samples were labeled with a study identification number, and all data were stored with this number. Researchers did not have access to the donor-identifying data. Data were stored using a password-protected Microsoft Excel  spreadsheet. Descriptive data , Fisher's exact test (two-sided), odds ratios, and 95% CIs were calculated with GraphPad Prism  was used to analyze data. PRNT


E. coli, K. pneumoniae, E. faecalis, P. vulgaris) with clinical efficacy against recurrent urinary tract infections (UTIs). Here, MV140 was evaluated in a murine model of acute uropathogenic E. coli (UPEC)-induced UTI using the UTI89 strain. MV140 vaccination resulted in UPEC clearance, concomitant with increased influx of myeloid cells in urine, CD4+ T cells in the bladder, and a systemic adaptive immune response to both MV140-containing E. coli and UTI89.MV140 is a mucosal vaccine of inactivated whole bacteria ( Escherichia coli -UPEC- being the most common causative agent), mostly affect women and acute infections are commonly treated with antibiotics (Urinary tract infections (UTIs) are common and recur frequently. They are primarily of bacterial origin  that has shown long-term efficacy (up to a year of follow-up) in the prevention of recurrent UTI in women  and psoriasin S100a7a) levels which have been shown to be activated during a UTI 0a7a leve were anaE. coli strains, as well as against the whole MV140 bacterial mixture, compared to unvaccinated but infected control mice. This antibody response was already present early upon infection (24h p.i.) and slightly increased two weeks later (day 14 p.i.)  14 p.i.) .n vitro than control mice following stimulation with either MV140 or heat-killed UTI89 at comparable rates  cells  cells , 6, 15. Although the induced antibody response might be involved in MV140 protection, the key role of T cells, particularly Th17, in mucosal infections due to extracellular bacteria has been highlighted elsewhere . Activatin vitro the T cell-polarizing properties of human monocyte-derived DCs primed with MV140  , 15, 23.To summarize, our data indicate that MV140 induces strong systemic and local adaptive immunity in the bladder against a clinical UPEC isolate, which correlates with early bacterial clearance after infection.Female C57BL/6 Wild-type mice (8- to 10-week-old) from Jackson laboratory were used. Mice were maintained under specified pathogen-free conditions in a barrier facility under a 12 h light-dark cycle. All experimental procedures were approved by the animal studies committee of Washington University in St. Louis School of Medicine .Escherichia coli, V113 Klebsiella pneumoniae, V125 Enterococcus faecalis, V127 Proteus vulgaris  at 300 Formazin Turbidity Units , or vaccine excipients (control) diluted in phosphate-buffered saline , 3 times a week for 2 weeks, and rested for an additional week.As immunization protocol, mice were intranasally (i.n.) administered with 50 μL of MV140, a suspension of equal amounts of whole-cell heat-inactivated bacteria: V121 7 colony forming units (CFUs) of UTI89 in PBS, as published elsewhere  were plated in 96-well plates  and in vitro stimulated for 72h with heat-killed UTI89, MV140  or control, and cytokine production  measured in cell-free culture supernatants by Multiplex cytokine assay , following manufacturer’s instructions.Spleens were used to prepare single cell suspensions following conventional protocols. Cells  from serum samples obtained at indicated timepoints. Briefly, 96-well non-tissue culture-treated plates (Greiner Bio-One) were pretreated with poly-L-lysine (Sigma-Aldrich) for 1h under UV light. Then, plates were coated with the heat-inactivated whole cell bacteria mixture MV140, or heat-killed V121 E. coli or UTI89  overnight at 4°C, and, subsequently, incubated with mouse serum for 2h at room temperature (RT). Specific Igs were detected using biotin rat anti-mouse IgG, peroxidase and o-phenylenediamine dihydrochloride . Plates were read at 492 nm .Specific IgG for MV140, MV140-containing strain Bladders were digested at 37°C for 30 minutes in RPMI-1640 with 10mM HEPES, collagenase D, and DNAse , forced through a 70 μm cell strainer (Corning), and washed with 5% fetal bovine serum  in PBS. Urines were diluted in fluorescence-activated single cell sorting (FACS) Buffer (PBS supplemented with 5 mM EDTA and 3% FBS) for 30 minutes , prior to staining. Single cell suspensions resuspended on ice-cold FACS Buffer were stained with anti-CD45 eFluor450 (eBioscience), anti-CD3-A700 (eBioscience), anti-CD4-PE/Cy7 (BioLegend), anti-CD8-BrilliantViolet605 (BioLegend), anti-CD11b-PE/Cy5 (eBioscience), anti-Ly6C-APC/Cy7 (BioLegend), anti-Ly6G-FITC (BioLegend), anti-F4/80-PE (eBioscience), anti-CD64-APC (BioLegend) and 7-AAD (BioLegend). Purified anti-FcɣRIII/II (BioLegend) was used to block Fc-receptors. Data was acquired on LSR II flow cytometer (BD) and analyzed with FlowJo software v10.0 (BD).°C. Sections were washed with PBS-T and further incubated with respective secondary Alexa Fluor– conjugated antibody (Invitrogen) in 1:600 for 1.5h at RT, followed by staining with DAPI for 15 min, washed and mounted in Fluoromount G (Southern Biotech). Slides were analyzed with a Zeiss 700 confocal microscope and fluorescence intensity quantified with the ImageJ software.For immunofluorescence analysis, bladders were fixed in methacarn  and embedded in paraffin. Bladder sections were deparaffinized using neoclear and rehydrated, pretreated with 0.3% Triton X-100 in PBS (PBS-T), and boiled in citrate buffer, 1 mM EDTA, 10 mM TRIS, 0.05% Tween 20 (pH 9). Sections were rinsed with PBS-T, treated with FX Signal Enhancer (Invitrogen) at RT for 30 min and blocked for 60 mins with sera. Further, anti–psoriasin  and anti-UPIIIa  were incubated overnight at 4For RT-qPCR, bladders were flash frozen, and RNA extracted using TRIzol reagent (Invitrogen) according to manufacturer protocol followed by gDNA digestion with TURBO DNA-free kit (Invitrogen). cDNA was generated using Superscript II Reverse Transcriptase (Invitrogen). qPCR was performed with standard SYBR green (Applied Byosistems) on a Rotor-Gene PCR cycle (Corbett Life Science). Gene specific primers Cramp , S100a7a  and Actb  were used. Fold-changes were calculated using Ct method and normalized internally to respective control.The statistical analysis was performed using Prism (GraphPad Software). Statistical significance for comparison between treatment groups was determined using Mann-Whitney or unpaired Student’s t tests, according to normal distribution evaluated by Shapiro-Wilk test. Outliers were identified by means of Tukey’s range test. For antibody analysis, arbitrary units were calculated from the optical density (OD) values according to standard curves, after subtracting the OD values of blank and adjusting by the serum dilutions performed. Differences were considered significant at p < 0.05. Except when specified, only significant differences are shown.1


Saccharum spp.) production and improving the water use efficiency (WUE) is a critical trait for the sustainability of this bioenergy crop. The molecular mechanism underlying WUE remains underexplored in sugarcane. Here, we investigated the drought-triggered physiological and transcriptional responses of two sugarcane cultivars contrasting for drought tolerance, ‘IACSP97-7065’ (sensitive) and ‘IACSP94-2094’ (tolerant). After 21 days without irrigation (DWI), only ‘IACSP94-2094’ exhibited superior WUE and instantaneous carboxylation efficiency, with the net CO2 assimilation being less impacted when compared with ‘IACSP97-7065’. RNA-seq of sugarcane leaves at 21 DWI revealed a total of 1,585 differentially expressed genes (DEGs) for both genotypes, among which ‘IACSP94-2094’ showed 617 (38.9%) exclusive transcripts (212 up- and 405 down-regulated). Functional enrichment analyses of these unique DEGs revealed several relevant biological processes, such as photosynthesis, transcription factors, signal transduction, solute transport, and redox homeostasis. The better drought-responsiveness of ‘IACSP94-2094’ suggested signaling cascades that foster transcriptional regulation of genes implicated in the Calvin cycle and transport of water and carbon dioxide, which are expected to support the high WUE and carboxylation efficiency observed for this genotype under water deficit. Moreover, the robust antioxidant system of the drought-tolerant genotype might serve as a molecular shield against the drought-associated overproduction of reactive oxygen species. This study provides relevant data that may be used to develop novel strategies for sugarcane breeding programs and to understand the genetic basis of drought tolerance and WUE improvement of sugarcane.Drought is a major constraint to sugarcane ( Saccharum spp.), a main feedstock for sugar and biofuel production, is strongly impacted during dry summer spells  and water consumption . Importantly, WUE is often seen as a parameter associated with the level of drought tolerance in plants (Water use efficiency (WUE) is a complex trait connected to plant biomass yield , describn plants . Howevern plants . While gHere we conducted a thorough investigation of the drought-triggered responses in two sugarcane cultivars contrasting for drought tolerance: ‘IACSP97-7065’ (drought-sensitive) and ‘IACSP94-2094’ (drought-tolerant). Plants were subjected to controlled water deficit regimes under greenhouse conditions, and physiological parameters (including WUE) were estimated after 21 days without irrigation (DWI) and after the rehydration stage (recovery). Massive leaf transcriptome analyses were carried out at the maximum drought stress (21 DWI) using the RNA-seq (Illumina platform), and gene expression data were validated by quantitative reversed transcript amplification (RT-qPCR). Based on functional analyses, we report several biological pathways that were remarkably responsive in the drought-tolerant genotype. Our results provide new clues to unveil the intrinsic drought coping strategies in sugarcane, which may be useful for biotechnological applications and plant breeding programs.Saccharum spp.) cultivars contrasting for drought tolerance, ‘IACSP97-7065’ (drought-sensitive) and ‘IACSP94-2094’ (drought-tolerant), which were developed by the “Sugarcane Program” . The cultivars displayed differential growth and yield in drought-prone areas of Cerrado (the Brazilian “savanna”) (3) filled with soil previously analyzed  according to We analyzed two sugarcane (avanna”) . The plan = 3). Each biological replicate was composed of a bulk of three fully expanded and light-exposed (+1) leaves.For the transcriptome analysis, +1 leaves (the first fully expanded leaf) from both genotypes were collected at 21 DWI for RNA extraction. The experiments were conducted in biological triplicates (2 assimilation (PN), instantaneous carboxylation efficiency , stomatal conductance (gs), transpiration (E), and water use efficiency . These estimates were performed on +1 leaves under photosynthetic photon flux density of 2000 µmol m-2 s-1 and 385 ± 6 ppm CO2 concentration, considering natural air temperature  and humidity  between 09:00 and 11:00 AM. Some additional photochemical parameters were estimated, such as the maximum (Fv/Fm) and effective (ΦPSII) quantum efficiency of photosystem II (PSII) and the non-photochemical quenching (NPQ). Fv/Fm is based on fluorescence signals from dark-adapted leaves, whereas ΦPSII is based on fluorescence signals from light-adapted leaves, following the pulse saturation method  (vs. rehydration), and condition factors (irrigated vs. drought), followed by Tukey’s test . The physiological raw data are shown in The physiological responses of both sugarcane cultivars to water deficit were estimated at 21 DWI and rehydration stage (seven days after rewatering) compared to irrigated controls. An infrared gas analyzer  was used to measure chlorophyll fluorescence and leaf gas-exchange parameters, such as CO, 0.8 s) . The leavia 1.2% agarose electrophoresis. Libraries were assembled according to the recommendations of TruSeq RNA Sample Prep kit . Then, four cDNA libraries were paired-end sequenced by a HiSeq 2000 Illumina sequencer. CLC Genomics Workbench  software was used for quality check, filtering, and trimming  procedures. The raw transcriptome data were deposited in the NCBI Sequence Read Archive (SRA) database (BioProject ID: PRJNA882367).Total RNA was extracted using a Lithium Chloride (LiCl) method  and purihttps://ics.hutton.ac.uk/tablet/) was used to identify mismatches between reads and reference. Reads per kilobase of exon per million mapped reads (RPKM) were calculated, and differentially expressed genes (DEGs) were identified by CLC Genomics Workbench software contrasting drought versus irrigated treatments at p-value ≤ 0.05 and Benjamini and Hochberg adjusted FDR  < 0.05. Gene expression values were then normalized in log2Fold-change (FC). In this study, only DEGs with |log2FC| greater or equal to 1 were considered for further analyses.Transcriptome mapping, gene count, and gene expression analysis were conducted by CLC Genomics Workbench software under default settings (length fraction = 0.5 and similarity fraction 0.8). Cleaned reads were mapped to Sugarcane Assembled Sequences (SAS) from Sugarcane EST Project (SUCEST) database  as refere-value < 10-5) in OmicsBox v.1.1.164 (via the WEGO tool (https://biodb.swu.edu.cn/cgi-bin/wego/index.pl), which determined the significance level  between the gene numbers (related to GO terms) in the two sugarcane data sets (drought-tolerant versus drought-sensitive). Kyoto Encyclopedia of Genes and Genomes (KEGG) . To visualize the deregulated biological pathways, DEGs were first mapped to plant protein categories (BIN ontologies) from Mercator4 v5.0 .The search for orthologs of the DEGs was executed by the BLASTX tool (.1.1.164  against s (KEGG)  pathway s (KEGG)  against or4 v5.0 , then suor4 v5.0 , which g25S rRNA1 and 25S rRNA2 were used as reference genes. RT-qPCR expression data was retrieved by Bio-Rad CFX Manager software, and statistical analysis (drought versus irrigated) was performed using the software REST 2009  using the gene expression values from the RNA-Seq and RT-qPCR experiments. This correlation analysis was performed using GraphPad Prism v. 8.0.0 for Windows .Fifteen DEGs detected by RNA-Seq were randomly selected for gene expression validation by reversed transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. Primer pairs are shown in EST 2009  with a pQ method , followe2 assimilation was observed for both the drought-tolerant (70%) and -sensitive (91%) cultivars .Changes in physiological parameters of both cultivars were investigated at the maximum water deficit (21 DWI) and after rehydration Figure 1All the gas-exchange and photochemical parameters were recovered after rehydration of the plants from both cultivars and all plants showed similar values, but at lower levels than those observed for the irrigated control plants. Notably, the WUE of the rehydrated ‘IACSP94-2094’ plants remained at higher levels in comparison to the control  and watering regime treatments, which rendered a total of 113,815,386 (85%) high-quality paired-end reads after quality control . From these, ‘IACSP97-7065’ displayed 308 particular DEGs (183 up- and 125 down-regulated), whereas ‘IACSP94-2094’ exhibited 617 particular DEGs (212 up- and 405 down-regulated). Moreover, the sugarcane genotypes shared numerous similarly regulated genes (435 up- and 223 down-regulated) and only 2 with opposite transcriptional profiles (Saccharum spp. sequences (6.5%) and to close grass species, such as Miscanthus lutarioriparius (42.8%), Sorghum bicolor (21.8%), Zea mays (8.1%), and others (11.2%). The top BLAST hits for each annotated DEG are listed in This study revealed a total of 1,585 (3.6%) differentially expressed genes  showing no differential expression. The reproducibility and accuracy of the expression levels of the 15 DEGs were supported by a strong correlation  between the RNA-seq and RT-qPCR data , a protein involved in tolerance to extreme desiccation (serine/threonine-protein kinase (SAPK3) was 2.3-fold more expressed in plants of the drought-sensitive genotype as compared to those from ‘IACSP94-2094’. The fact that some genes showed differential expression for only one of the genotypes is noteworthy. The NAC21 transcription factor (log2FC = 4.7) and universal stress protein  were up-regulated, particularly in ‘IACSP97-7065’, whereas the chloroplast ferredoxin-5 was down-regulated (log2FC = -1.53). On the other hand, the ‘IACSP94-2094’ genotype exhibited exclusive differential expression for ATP-dependent RNA helicase A-like , auxin-like 1 , and tau class glutathione S-transferase 6 .Considering the RT-qPCR data (iccation  was showThe Gene Ontology (GO) enrichment analysis highlighted many GO terms for the transcriptomic analysis of the plants from both cultivars under drought stress Figure 3KEGG pathway enrichment analysis indicated additional ‘IACSP94-2094’ and ‘IACSP97-7065’ DEGs involved in plant hormone signal transduction, MAPK signaling pathway, starch and sucrose metabolism, biosynthesis of amino acids, carbon fixation in photosynthetic organisms, carbon metabolism, glycerophospholipid metabolism, among others Figure 4AP2-EREBP, bHLH, and NAC)  transcription factors, (ii) photosynthesis, (iii) redox homeostasis, and (iv) solute transport Figure 6NF-Y (SCRLLR1109E12.g), DELLA (SCQSST1037G07.g), WRKY1 (SCQGLR2025A07.g), ORR23 (SCJFSB1011E04.g), DOF2 (SCRLAD1136C12.g), SCL6 (SCBFRZ2016F05.g), TGAL6-like (SCRURT2012D03.g), ILR3-like (SCCCRT1003E06.g), CDF1-like (SCRLAD1136C12.g), and CBP60G-1 (SCEQRT2025A05.g) as down-regulated, and HOX29-like (SCCCAM1C09G11.g), bZIP23 (SCVPLB1018G11.g), DREB1C (SCQGLR1041E11.g), ZFP1-like (SCMCLR1122C01.g), bHLH035 (SCEZRZ1016C11.g), RAP2-10-like (SCRLAD1043B06.g), ERF060 (SCRLLV1050E06.g), and bZIP/RISBZ5 (SCCCCL4005C09.g) as up-regulated (PPDK1 (SCRLCL6031H05.g) and RSH1 (SCEZRZ3051A09.g) as down-regulated and CAB4 (SCCCST1002F06.g), CP29.1 (SCUTST3131A12.g), CPN10 (SCUTLR1058D06.G), CPN20 (SCJLLR1106F03.G), CPN60 (SCCCCL4005E12.G), CURT (SCMCSD1061A04.G), PPIL1 (SCEQFL5044B07.G), psaF (SCACSB1039A11.G), psaO (SCJFAD1013H02.G), psb27 (SCUTSB1076D12.G), psbX (SCRFLB1055B09.G), and rbcL (SCJFLR1073E09.g) as up-regulated (TRX-H (SCAGLR2011H07.G), RBOHB (SCJLRT1023G05.G), and GSTT (SCBGLR1002F10.G) (GAT1 (SCEZRZ3015D04.G), PHT1 (SCCCLR1001G10.G), NHX2 (SCRLFL4028D02.G), SWEET2b-like (SCBGLR1120E06.G), ZIF/TOM (SCCCCL7002B06.G), MOT1 (SCJLRT2052C07.G), MCU2 (SCQSLB1049D10.G), G3P1 (SCCCST2002F04.G), TauE/SafE4 (SCCCLR1C03H03.G), and NIPA4 (SCJLST1020D04.G) as down-regulated, and NRT1/PTR6.4 (SCACRZ3035B05.G), OEP23 (SCCCST3C03A11.G), PIP2-1 (SCRFLR1012A08.G), KT10 (SCCCST2002G11.G), MCU (SCBFRT1064C07.G), ERD4 (SCJLRZ1024A10.G), and PIP2-5 (SCJFRT1059C11.G) as up-regulated. Within these four sets , the drought-tolerant genotype highlights its greater drought-responsiveness with 52 exclusive DEGs, whereas the drought-sensitive showed only 18.Focusing on the drought-tolerant genotype, the set of transcription factors comprised 18 DEGs (10 down- and eight up-regulated) not found in ‘IACSP97-7065’, including egulated Figure 6e.g., metabolic impairment) limitations, as well as decreases in transpiration rate  were reduced for both cultivars at 21 DWI (i) was also maintained in ‘IACSP94-2094’ plants at 21 DWI, whereas a substantial decrease was found in ‘IACSP97-7065’, which suggests a biochemical impairment of photosynthesis in the drought-sensitive genotype. Upon rehydration, WUE was significantly higher in ‘IACSP94-2094’ compared to its irrigated control. The improvement of WUE in ‘IACSP94-2094’ may be explained by the apparent slow recovery of stomatal conductance , as severe drought stress could accumulate high concentrations of ABA that might take longer to be degraded  and secondary  transport (PIP2-1 and PIP2-5), K+ transporter (KT10), chloroplast outer envelope protein (OEP23), mitochondrial calcium uptake channel (MCU), and early-responsive to dehydration 4 (ERD4). In hexaploid wheat, OEP16-2 is responsive to heat stress and confers drought tolerance when overexpressed in Arabidopsis thaliana transgenic lines  isoforms, MAP3K17 (SCJLRT1006B11.g) and MAP3K17-like (SCVPRZ3029A08.g), which are activated by ABA signaling and osmotic stresses in A. thaliana (2+) – a universal secondary messenger - in water-constrained conditions induces signal transduction by Ca2+ binding proteins, such as calmodulin-like (CML) proteins, which interact with other dehydration-responsive molecules , particularly in the drought-tolerant genotype. Functional studies overexpressing CML genes have demonstrated improved tolerance to multiple abiotic stresses in plants  cascade to elicit a physiological response . Accordie plants . Here, tthaliana . Increasolecules . In factolecules . In ricet stress . Our expn plants ; however2 availability, which impairs photosynthesis (psaF and psaO) and PSII (psb27 and psbX) genes, our results unveiled up-regulated genes involved in the Calvin-Benson cycle, including the large subunit of the ribulose-bisphosphate carboxylase [Rubisco (rbcL)], its co-chaperones  and chaperonin (CPN60), and a beta-type carbonic anhydrase (βCA - SCEQLB1063G04.G). In C4 plants, β-CA converts CO2 to bicarbonate , CAB4, and CP29.1. These proteins are responsible for the assembly of antenna complexes, which is essential for plants adapting to ever-changing environmental conditions  families have been reported to be involved in response to abiotic stresses in sugarcane . As we ra leaves . Cuticul– PeSHN1 . Moreoveic lines . Althouge plants , and bZIt stress .RBOHB) in ‘IACSP94-2094’, which is a ROS-generating enzyme induced through ABA signaling in drought-stressed plants (TRX-H) and theta glutathione S-transferase (GSTT). Additionally, our RT-qPCR analysis disclosed that GSTU6 transcript accumulates especially in the drought-tolerant genotype. Plant GSTs are well-studied detoxifying enzymes associated with tolerance to various abiotic stresses, including drought, in several crops  in plants, which operate in signal transduction pathways toward stress responses . The resd plants . Previoud plants  and greed plants . Consistal crops . Neverthr stress . Thioredr stress , of whicd stress . TherefoMiscanthus lutarioriparius, which were assigned to photosynthesis, stomatal regulation, and responses to abiotic stresses. Among these genes, our transcriptome analysis of ‘IACSP94-2094’ showed three possible orthologs under drought conditions: thioredoxin-like CXXS1 (TRX-H), pyruvate orthophosphate dikinase1 (SCRLCL6031H05.g), and lysine-specific histone demethylase 1 (SCBGLR1023B11.g), whereas in ‘IACSP97-7065’, we found cysteine-rich receptor-like protein kinase 10 (SCSGAD1009D03.g) and soluble starch synthase II-2 (SCSGSB1009B08.b). In sorghum, Panicum miliaceum L.) genotype, in which drought-triggered transcriptome was mainly enriched in signal transduction, MAPK signaling, and carbon metabolism pathways  of sugarcane genes underlying these biological pathways.In the absence of transcriptome studies focused on sugarcane WUE under water deficit, a few studies in close related grasses have contributed to elucidate some of the genetic components of the plant responses . Fan et pathways . As our In summary, the present study disclosed the physiological and transcriptional adaptations of contrasting sugarcane genotypes in coping with drought stress conditions. The drought-tolerant genotype ‘IACSP94-2094’ displays superior WUE under water deficit and rehydration, relying on less sensitivity on reducing carbon assimilation under drought and increasing water use after rehydration. Accordingly, this genotype also shows a stable carboxylation efficiency under water deficit, with higher levels than the drought-sensitive genotype. Similar to a previous microarray study , our RNAThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI Sequence Read Archive (SRA) database (BioProjectID: PRJNA882367).SC, AF, and RR conceived and supervised the project, designed the experiments, and acquired resources. ML developed the sugarcane genotypes. JN and RR performed the physiological experiments. RM provided expertise in RNA sequencing procedures. JN performed RNA sequencing and RT-qPCR analysis. TP and RR contribute with formal data analysis and text editing. DC curated the RNA-Seq data, conducted functional analyses, prepared all figures and tables, and wrote the entire manuscript. All authors read and approved the final manuscript. All authors contributed to the article.


The anti-viral activity of SAMHD1 is regulated by dephosphorylation of the residue T592. However, the impact of T592 phosphorylation on dNTPase activity is still under debate. Whether additional cellular functions of SAMHD1 impact anti-viral restriction is not completely understood. We report BLaER1 cells as a novel human macrophage HIV-1 infection model combined with CRISPR/Cas9 knock-in (KI) introducing specific mutations into the SAMHD1 locus to study mutations in a physiological context. Transdifferentiated BLaER1 cells harbor active dephosphorylated SAMHD1 that blocks HIV-1 reporter virus infection. As expected, homozygous T592E mutation, but not T592A, relieved a block to HIV-1 reverse transcription. Co-delivery of VLP-Vpx to SAMHD1 T592E KI mutant cells did not further enhance HIV-1 infection indicating the absence of additional SAMHD1-mediated anti-viral activity independent of T592 dephosphorylation. T592E KI cells retained dNTP levels similar to WT cells indicating uncoupling of anti-viral and dNTPase activity of SAMHD1. The integrity of the catalytic site in SAMHD1 was critical for anti-viral activity, yet a poor correlation between HIV-1 restriction and global cellular dNTP levels was observed in cells harboring catalytic core mutations. Together, we emphasize the complexity of the relationship between HIV-1 restriction, SAMHD1 enzymatic function, and T592 phospho-regulation and provide novel tools for investigation in an endogenous and physiological context.Sterile α motif and HD domain-containing protein 1 (SAMHD1) is a dNTP triphosphate triphosphohydrolase (dNTPase) and a potent restriction factor for immunodeficiency virus 1 (HIV-1), active in myeloid and resting CD4We introduce BLaER1 cells as an alternative myeloid cell model in combination with CRISPR/Cas9-mediated gene editing to study the influence of sterile α motif and HD domain-containing protein 1 (SAMHD1) T592 phosphorylation on anti-viral restriction and the control of cellular dNTP levels in an endogenous, physiologically relevant context. A proper understanding of the mechanism of the anti-viral function of SAMHD1 will provide attractive strategies aiming at selectively manipulating SAMHD1 without affecting other cellular functions. Even more, our toolkit may inspire further genetic analysis and investigation of restriction factors inhibiting retroviruses and their cellular function and regulation, leading to a deeper understanding of intrinsic anti-viral immunity. Additionally, allele-specific sequencing as described for SAMHD1 KO was performed, if required. Absence of large deletions in the region between amplification primers was tested by PCR and analytic gel electrophoresis. Presence of both alleles was confirmed by quantitative genomic PCR , exon 6 , or exon 8  specific PrimeTime qPCR Assay (IDT), human TERT TaqMan Copy Number Reference (Thermo Fisher) and PrimeTime Gene Expression Master Mix (IDT) on a CFX384 machine.For CRISPR/Cas9-mediated SAMHD1 knock-out (KO), 200 pmol Edit-R Modified Synthetic crRNA targeting escribed . RibonucpH 7.5)]  and screomic PCR , perform− R−luc (− R+ (HIV-1-mCherry) were produced, as detailed previously (− R−luc (a kind gift of Nathaniel Landau) or pNL4.3 IRES mCherry E− R+ (a kind gift of Frank Kirchhoff) were co-transfected together with pCMV-VSV-G into 293T/17 cells using 18 mM polyethylenimine (Sigma-Aldrich). Filtered (0.45 µm) supernatants were treated with 1 U/mL DNAse I  and purified through a 20% sucrose cushion . Viral stocks were titrated for β-galactosidase activity on TZM-bl cells. Virus-like particles containing Vpx (VLP-Vpx) were produced in an analog manner using pSIV3+ . For more recent experiments (+ cell population. Infection was quantified after 24 h by FACS or qPCR (for HIV-1-mCherry), or alternatively by adding 50 µL/well britelite plus reagent (PerkinElmer) and measurement on a Pherastar FS (BMG) (for HIV-1-luc). To show VLP-Vpx-mediated SAMHD1 degradation, 4.4 × 105 transdifferentiated BLaER1 cells were treated in a 12-well tissue culture plate in the same conditions and concentrations as stated above.VSV-G pseudotyped HIV-1 reporter viruses pNL4.3 E− R−luc   and stained with CD11b-APC , CD19-PE , CD14-PacBlue , CD163-PE , CD206-APC , CD11c-VioBlue , CD4-APC , CXCR4-PE , CCR5-PE  or respective isotype controls  and Fixable Viability Dye eFluor 780 (Thermo Fisher) in presence of FC Block . Stained cells were washed twice in FACS buffer and fixed in 2% paraformaldehyde , before analyzing on a LSR II instrument (BD). For readout of HIV-1-mCherry infection, up to six wells of a 96-well plate were pooled and stained with CD11b-APC and Fixable Viability Dye eFluor 780 as detailed above. For intracellular SAMHD1 staining, cells were fixed in Cytofix Buffer , subsequent to cell surface staining, and permeabilization with Perm Buffer III , before staining with anti-SAMHD1  or an isotype control  and anti-rabbit IgG-DyLight 405 . Infected cells were analyzed on a BD LSRFortessa.For flow cytometric analysis of BLaER1 transdifferentiation and surface marker expression, 1 × 10g, 4°C) and protein content was measured by Bradford assay using Protein Assay Dye Reagent Concentrate (BioRad). Twenty micrograms of total protein was denaturated  in NuPAGE LDS Sample Buffer and Reducing Reagent (Thermo Fisher) and separated on a NuPAGE 4–12% Bis-Tris gradient gel (Thermo Fisher) in MOPS running buffer . Transfer was performed in an XCell II Blot Module in NuPAGE Transfer Buffer (Thermo Fisher) onto a Hybond P 0.45 PVDF membrane . After blocking in 5% BSA or milk powder (Carl Roth) in TBST , primary antibodies anti-GAPDH , anti-Cyclin B1 , anti-Cyclin A2 , anti-SAMHD1 , anti-SAMHD1 , and anti-SAMHD1-pT592  diluted in 5% BSA or milk powder in TBST were applied overnight at 4°C. Subsequent to washing in TBST, anti-rabbit IgG, horseradish peroxidase (HRP)-linked antibody (CST) was applied  and the membrane was washed again before detection on a FUSION FX7 (VilberLourmat) using ECL Prime reagent (GE). If required, membranes were stripped of bound antibody in stripping buffer , 100 mM β-mercaptoethanol; 1 h, 65°C). Band densities were determined with FUSION software (Vilber Lourmat).For immunoblot, cells were washed in PBS, lysed in radioimmunoprecipitation buffer  supplemented with proteinase and phosphatase inhibitor (Roche) for 30 min on ice. Lysate was cleared  on a CFX384.HIV-1-mCherry copy number was quantified by qPCR as detailed previously . In brie6 transdifferentiated BLaER1 cells were washed in PBS and subjected to methanol extraction of dNTPs, followed by quantification of all four dNTPs by single nucleotide incorporation assay, as described previously (g), cells were fixed , permeabilized , and stained using HCS CellMask Deep Red Stain . Z-Stack of stained cells was acquired using confocal imaging platform Operetta (Perkin Elmer) and volume was calculated as a sum of cell areas in all relevant Z-stacks using Harmony software (Perkin Elmer).For measurement of cellular dNTP levels, 2 × 10eviously . CD19 det-test, as well as non-parametric Kruskal–Wallis test or parametric one-way ANOVA, corrected against multiple testing using Dunn’s or Dunnet correction, respectively.Statistical analysis was performed using GraphPad Prism (V8). Mean and standard deviations are shown. Statistical significance was assessed using unpaired two-tailed


The rapid and wide spread of carbapenemase-producing Enterobacterales (CPE) has become a global concern. Currently, β-lactams with β-lactamase inhibitors  are used for treatment of CPE infection as one of the effective agents. However, some of combinations do not have activity against MBLs. Thus, we believe that the discriminating between MBL and serine-CPE is important. The eCIM has been published by Clinical & Laboratory Standards Institute (CLSI) to differentiate to MBL from serine-CPE. The eCIM is required EDTA, that is not clinical diagnostic agent. In this study, we examined simplifying the eCIM procedure by using the EDTA blood collection tube (BCT-eCIM).(Table 1). The eCIM were performed and interpreted by following CLSI procedures. For BCT-eCIM, whole quantity of 2-mL TSB was inoculated into a BD Vacutainer EDTA Tube (2-mL) (Becton-Dickinson) (Vacutainer-eCIM) or an EDTA-2K Insepack II-D 2-mL tube  (Insepack-eCIM); the tubes conform to International Organization for Standardization 6710, hence the 2-mL TSB inoculated tubes obtain final concentration of 4.1 to 6.8 mM EDTA. The BCT-eCIM used these tubes as a substitute for 0.5 mM EDTA TSB tube of eCIM, and any other procedures followed the eCIM procedure. Additionally, we performed the 4 mM- and 7 mM-eCIM to verify the effect of EDTA concentration (variable region of BCT-eCIM).A total of 53 CPE (39 MBL and 14 Serin-CPE) were tested. The test population included 39 clinical isolates, and 14 reference strains from NCTC or ATCC Table 1Bacterial strainsTable 2 and Figure 1 show the performance data of each eCIMs and the positive and negative Quality control results, respectively. The sensitivity of 4 mM-eCIM, 5 mM-eCIM, 7 mM-eCIM, Vacutainer-eCIM and Insepack-eCIM were 37/40 (93%), 36/40 (90%), 36/40 (90%), 39/40 (98%) and 39/40 (98%), respectively. The specificity of 4 mM-eCIM, 5 mM-eCIM, 7 mM-eCIM, Vacutainer-eCIM and Insepack-eCIM were 11/13 (85%), 13/13 (100%), 13/13 (100%), 12/13 (92%) and 12/13 (92%), respectively.Table 2Results obtained using the eCIM and BCT-eCIMOur methods (BCT-eCIMs) were superior to sensitivity compared to conventional eCIMs. Therefore, BCT-eCIM may be useful in clinical settings as the simplifying method.Hiroshige Mikamo, M.D, Ph.D, Asahi Kasei Pharma Corporation: Grant/Research Support|Merck Sharp & Dohme: Honoraria|Pfizer Inc.: Grant/Research Support|Pfizer R&D Japan: Honoraria|Sumitomo Pharma Co., Ltd.: Grant/Research Support|Sumitomo Pharma Co., Ltd.: Honoraria


Immunocompromised (IC) persons are at increased risk of severe COVID-19 and can have a suboptimal immune response to a primary 2-dose COVID-19 vaccine series; thus, vaccination recommendations for IC persons include additional COVID-19 vaccine doses. We describe outcomes and vaccination status among US IC children hospitalized with laboratory-confirmed COVID-19 during a period of SARS-CoV-2 Omicron circulation.Children with and without an IC condition hospitalized for COVID-19 were enrolled as case-patients into a vaccine effectiveness investigation as part of the Overcoming COVID-19 network of 31 hospitals across 23 states. Data on IC conditions abstracted from the medical record included the presence of an “active or prior oncologic disorder” or a “non-oncologic immunosuppressive disorder.” Results for IC case-patients with COVID-19 enrolled from 12/18/2022–4/3/2023 (Omicron period) were analyzed by age group and vaccination status.> 1 additional underlying medical condition (Table 1). Thirty (13%) IC case-patients were aged 8 months–4 years, 115 (48%) 5–11 years, and 94 (39%) 12–18 years. Life-threatening illness or death occurred in 1 (3%) IC case-patient aged 8 months–4 years, 8 (7%) aged 5–11 years , and 13 (14%) aged 12-18 years (including 2 deaths). Among 209 IC case-patients aged 5–18 years, 26 (23%) of those with oncologic and 27 (28%) with non-oncologic disorders had received 2 doses of a monovalent primary COVID-19 vaccine series; 6 (5%) and 14 (14%) had received > 3 doses, respectively . A significantly higher proportion of IC case-patients  had received at least 2 COVID-19 vaccine doses compared with non-IC patients .Of 239 IC case-patients, 127 (53%) had an oncologic disorder and 112 (47%) non-oncologic; 120 (50%) had > 5 years and > 99% survived. Vaccination coverage was low among all case-patients. A higher proportion of IC children who received >2 vaccine doses were among those case-patients hospitalized for COVID-19 compared with non-IC children. Additional doses to improve protection in IC children are recommended.During Omicron, immunocompromised children hospitalized for COVID-19 were more frequently aged Regina Simeone, PhD, Pfizer: Stocks/Bonds Natasha B. Halasa, MD, MPH, Merck: Grant/Research Support|Quidell: Grant/Research Support|Quidell: donation of kits|Sanofi: Grant/Research Support|Sanofi: vaccine support


The avidity of the T-cell receptor (TCR) for antigenic peptides presented by the MHC (pMHC) on cells is an essential parameter for efficient T cell-mediated immunity. Yet, whether the TCR-ligand avidity can drive the clonal evolution of virus antigen-specific CD8 T cells, and how this process is determined in latent Cytomegalovirus (CMV)- against Epstein-Barr virus (EBV)-mediated infection remains largely unknown.To address these issues, we quantified monomeric TCR-pMHC dissociation rates on CMV- and EBV-specific individual TCRαβ clonotypes and polyclonal CD8 T cell populations in healthy donors over a follow-up time of 15-18 years. The parameters involved during the long-term persistence of virus-specific T cell clonotypes were further evaluated by gene expression profiling, phenotype and functional analyses.Within CMV/pp65-specific T cell repertoires, a progressive contraction of clonotypes with high TCR-pMHC avidity and low CD8 binding dependency was observed, leading to an overall avidity decline during long-term antigen exposure. We identified a unique transcriptional signature preferentially expressed by high-avidity CMV/pp65-specific T cell clonotypes, including the inhibitory receptor LILRB1. Interestingly, T cell clonotypes of high-avidity showed higher LILRB1 expression than the low-avidity ones and LILRB1 blockade moderately increased T cell proliferation. Similar findings were made for CD8 T cell repertoires specific for the CMV/IE-1 epitope. There was a gradual in vivo loss of high-avidity T cells with time for both CMV specificities, corresponding to virus-specific CD8 T cells expressing enhanced LILRB1 levels. In sharp contrast, the EBV/BMFL1-specific T cell clonal composition and distribution, once established, displayed an exceptional stability, unrelated to TCR-pMHC binding avidity or LILRB1 expression.These findings reveal an overall long-term avidity decline of CMV- but not EBV-specific T cell clonal repertoires, highlighting the differing role played by TCR-ligand avidity over the course of these two latent herpesvirus infections. Our data further suggest that the inhibitor receptor LILRB1 potentially restricts the clonal expansion of high-avidity CMV-specific T cell clonotypes during latent infection. We propose that the mechanisms regulating the long-term outcome of CMV- and EBV-specific memory CD8 T cell clonotypes in humans are distinct. Primary Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infection is associated with a robust T cell response, followed by the establishment of a lifelong latency, while the T cell response persists long-term. In healthy individuals, herpesviruses are generally well controlled, through a fine balance between viral determinants and host immune surveillance. Yet, CMV and EBV cause considerable morbidity and mortality, particularly in immunocompromised individuals , 2. The Extensive research has been undertaken to determine which main drivers are underlying the selection and long-term maintenance of the large populations of CMV-specific memory CD8 T cells . Competi495-503 and HLA-A2/IE-1316-324) versus EBV-specific (HLA-A2/BMFL1280-288) CD8 T cells over time. Our findings reveal a critical role played by TCR avidity-driven repertoire evolution in the long-term outcome (i.e. 15 to 18 years) of CMV-specific but not of EBV-specific CD8 T cell responses in healthy individuals. LILRB1 inhibitor checkpoint was also preferentially expressed in high-avidity CMV-specific T cell clonotypes and may potentially limit their life expectancy during persistent infection.We had the unique opportunity to study the parameters driving TCRαβ clonal evolution between CMV and EBV chronic infection from six HLA-A2-positive healthy individuals in a longitudinal study, during an observational period of up to 18 years. Specifically, we assessed the structural TCR-pMHC avidity by monomeric TCR-ligand off-rate measurements , 34 combThis study protocol was reviewed and approved according to the relevant regulatory standards from the Ethics Committee for Clinical Research of the University of Lausanne  in accordance with the Declaration of Helsinki. All healthy donors have provided written informed consent for the use of blood samples in medical research.n) as described previously (n+15y and Tn+18y). No clinical data were available on their acute infection in the past, but all individuals remained in excellent health during this long-term follow-up. Peripheral blood mononuclear cells (PBMCs) centrifuged in Ficoll-Hypaque (Pharmacia) were cryopreserved in 10% DMSO and stored in liquid nitrogen until further use.Leukapheresis were collected at Interregional Blood Transfusion SRC from six HLA-A2-positive healthy individuals latently infected with the CMV and/or EBV viruses  in 2002 (i.e. time-point Teviously . All don495-503 (NLVPMVATV) or EBV/BMFL1280-288 (GLCTLVAML) peptide  at 4°C for 45 minutes, followed by cell surface marker APC-A750 anti-CD8 (Beckman Coulter) at 4°C for 30 minutes. Virus-specific CD8 T cells (CD8+multimer+) were sorted on a FACS Aria (BD Biosciences) flow cytometer as 300-1000 cells, before further cloning by limiting dilution and expansion in RPMI 1640 medium (Gibco) supplemented with 8% human serum, 150 U/ml human recombinant IL-2 (a gift from GlaxoSmithKline), 1 μg/ml PHA (Sodiag), and 1×106/ml 30 Gy-irradiated allogeneic PBMCs as feeder cells. T cell clones (2x104 cells) were directly processed through direct cell lysis and cDNA synthesis. cDNA sample was then subjected to individual PCR using a set of previously validated forward primers specific for the different known TRBV or TRAV gene subfamilies and two reverse primers specific for the corresponding C-beta or C-alpha gene segments  and described according to the IMmunoGeneTics (IMGT) nomenclature (Thawed PBMCs were positively enriched using anti-CD8-coated magnetic microbeads (Miltenyi Biotec), stained in PBS, 0.2% BSA, and 5 mM EDTA with PE-labeled HLA-A*0201 multimers loaded with native CMV/pp65segments . PCR pronclature .ex vivo TRBV family identified by flow cytometry from donors BCL4 and BCL6. Briefly, virus-specific CD8 T cells, which stained positive for a given TRBV family were sorted, in vitro cloned and TRBV-CDR3 sequenced as described above.CD8-enriched T cells from PBMCs were initially stained at 4°C for 45 minutes with CMV/pp65-specific or EBV/BMFL1-specific multimers, followed by cell surface marker APC-A750, FITC or APC anti-CD8 (Beckman Coulter) and antibodies against the different identified TRBV families , CMV/pp65495-503 (NLVPMVATV) or CMV/IE-1316-324 (VLEETSVML) peptides. NTAmers prepared with CD8 binding-deficient HLA-A*0201 monomers (i.e. CD8-null NTA) bearing the D227K/T228A mutations in the HLA α3 domain  and modeled  using Prism software .NTAmers were synthetized by the Peptide and Tetramer Core Facility, Ludwig Cancer Research, UNIL CHUV  as described previously . Dually 3 domain  were useex vivo sorted cells (between 300 and 8000 cells) was extracted using the RNeasy Micro Kit (Qiagen) according to the manufacturer’s protocol. RNA-sequencing libraries were prepared using the Clontech SMART Seq v4 Ultra Low RNA kit  and sequencing was performed on the Illumina HiSeq 2500. Library preparation, sequencing and analyses were performed by the Lausanne Genomic Technologies Facility  as described in detail in the Supplemental Data section. Gene expression data have been deposited in the NCBI Gene Expression Omnibus (GSE246111).CD8-enriched from PBMCs were stained with CMV/pp65-specific multimers followed by cell surface marker APC-A750, FITC or APC anti-CD8 (Beckman Coulter) and antibodies against the different identified TRBV families  staining (20 minutes at 4°C in PBS) was used to discriminate live/dead cells. LSRII (BD Biosciences) and Cytoflex (Beckman Coulter) flow cytometers were used to acquire the data. The percentage of positive cells or the level of expression of each marker (geometric mean fluorescence intensity [gMFI]) were analyzed using FlowJo software . For the dimensional reduction analyses, samples at each time-point were downsized using the downsample V3 plugin  before concatenation and analysis with both the t-SNE built-in module and the FlowSOM plugin  were isolated from thawed healthy donors PBMCs and stained in PBS, 0.2% BSA and 5 mM EDTA with AF700 anti-CD8 (Biolegend), APC-efluor780 anti-CD28 (eBioscience), ECD anti-CD45RA (Beckman Coulter), APC or SB600 anti-CD85j  and BV421 anti-CD57 (BD Biosciences) for 30 minutes at room temperature. When required CD8M plugin . FlowSOM495-503 peptide (10-8M), washed, and incubated with CFSE-labelled (Invitrogen) CMV/pp65-specific CD8 T cell clones of high or low avidity at an E/T ratio of 1:1 in RPMI 1640 medium (Gibco) supplemented with 8% human serum and 50 U/ml human recombinant IL-2 (GlaxoSmithKline) in the presence of monoclonal IgG2B mouse anti-human LILRB1  blocking antibody or an IgG2B isotype control used at 5 μg/ml (R&D Systems). After 3 days, cells were washed, stained in PBS with Near-IR Vivid (Invitrogen) at 4°C for 30 minutes before acquisition on a LSRII (BD Biosciences) cytometer. The percentage of divided cells, as well as the expansion and replication indexes were determined using the proliferation analysis tools available in FlowJo .30-Gy-irradiated HLA-A*0201-positive PBMCs were pulsed 1 hour at 37°C with the native CMV/pp65www.graphpad.com), Statistical tests were used as indicated throughout the manuscript.Data were analyzed using the GraphPad Prism software -1316-324- and EBV/BMFL1280-288-specific populations within total CD8 T cells over time was monitored by ex vivo fluorescent multimer staining  antibodies combined with epitope-specific multimers for the ex vivo staining of CMV/pp65- or EBV/BMFL1-specific CD8 T cells at the TRBV level  is a reliable parameter, independent of the activation state of the T cell   . Each spoff and low CD8 binding dependency as shown by their capacity to bind CD8-null NTAmers), as depicted by clono 1 and 2 (BCL4), clono 5 and 6 (BCL6) and clono 8 (BCL1). Conversely, the second subgroup was characterized by TCRs of low binding avidity (i.e. fast off-rates/low koff and high CD8 binding dependency), such as clono 3 and 4 (BCL4), clono 7 (BCL6) and clono 9 (BCL1). Moreover, the relative prevalence of most high avidity clonotypes showed a decline over time, contrasting to the increase in relative frequency of the low avidity ones  and CD8 binding capacity (off) and were largely CD8 binding-dependent compared to TRBV20 clonotypes. Both high and low avidity EBV/BMFL1-specific TCR clonotypes displayed a great stability in terms of relative frequency during the period of 15 years  and CD8 binding-deficient (i.e. CD8-null NTA) NTAmers. Similar to the CMV/pp65 clonal repertoires  were observed on CMV/pp65-specific bulk T cells at the later time-point (i.e. Tn+15y), revealing a significant decrease in the overall TCR binding avidity over time in three out of four donors , but not EBV (i.e. BMFL1280-288 epitope) latency.Compared to the CMV/pp65 model, the overall off-rates of EBV/BMFL1-specific ex vivo sorted CMV-pp65/TRBV-specific sub-populations at Tn and Tn+15y , associated in CD8 T cells with aging and CMV latent infection  of high avidity but not that of low avidity T cell clones  of CMV/pp65-specific T cell clonotypes from BCL4 upon direct LILRB1 blockade and o LILRB1  only indion data Figure 5ion data  during tl clones .ex vivo phenotype and off-rate measurements alongside. Studies were further performed on the two immune-dominant CMV/A2 CD8 T cell epitopes (i.e. CMV/pp65495-503 versus CMV/immediate-early (IE)-1316-324) only detectable in donor BCL6  and fraction B (i.e. slower off-rates)  during long-term antigen exposure. This was not the case for EBV/BMFL1-specific CD8 T cell repertoires, in which the clonal composition and distribution  are kept highly stable for at least 15 years. Together, these findings indicate that TCR-pMHC binding avidity is a determining factor driving the clonal evolution of long-lasting CMV- but not EBV-specific memory CD8 T cell responses in humans. In addition, LILRB1 checkpoint receptor was preferentially expressed in high-avidity CMV-specific TCRαβ clonotypes and correlated in those cells to their gradual frequency decline over time. The main weakness of the study is the relative low number of available volunteers (n = 6), due to the exceptionally long period of follow-up and the fact that chronic CMV/pp65-specific T cell responses are typically composed of highly restricted co-dominant clonotypes (n = 15). Consequently, this somehow limits the strength of our statistical-based conclusions.Here, we investigated whether TCR-ligand avidity can directly drive the long-term maintenance of particular herpesvirus-specific CD8 TCRαβ clonotypes and searched for factors that regulate clonal repertoires in latent human CMV versus EBV infection, over a follow-up time of 15-18 years. Our data revealed the progressive Differences in the nature of these β-herpesviruses during latent infection could in part explain the gradual contraction of high avidity CMV/pp65- and CMV/IE-1-specific T cell clonotypes observed over time, contrasting with the striking stability of EBV/BMFL1-specific CD8 T cell clonal repertoires. In the context of EBV infection, some infected B cells become long-lived, namely the latently infected memory B cells . EBV rea495-503 and HLA-A2/IE-1316-324). As shown here in humans, longer observation periods (i.e. >15-20 years) are required to reveal the contraction of high-avidity CMV/epitope-specific clones in the latency infection phase, as T cell repertoires can be maintained relatively stable during at least several years . Since HLA-A2/BMFL1280-288 is an epitope from the early protein BMFL1 essentially expressed during the acute phase of infection, the stability of the observed clonotype repertoires over extended periods of time could potentially be explained by the relative absence of epitope expression during the latent phase. Nonetheless, TCR clonotype composition and distribution against HLA-A2/BMFL1280-288 were also highly preserved during transient immunological perturbations after non-myeloablative chemotherapy  is frequently found on the surface of human CD8 T cells . This isity loss . Interesity loss  recentlyex vivo assessment of TCR-pMHC binding kinetics at the surface of live T cells have proven technically challenging until recently  values were found for all studied representatives of a given CMV- or EBV-specific clonotype over time (Tn versus Tn+15y), indicating that TCR-pMHC dissociation rate represents a stable intrinsic biometric at the TCRαβ clonotype level. Our findings nicely fit with another recent report performed on MCMV-specific TCR repertoires during the course of infection using TCR-pMHC koff-rate measurements with reversible Streptamers . The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.BC: Conceptualization, Data curation, Formal Analysis, Methodology, Writing – original draft. BD: Formal Analysis, Methodology, Writing – review & editing. LC-I: Formal Analysis, Methodology, Writing – review & editing. MA: Formal Analysis, Methodology, Writing – review & editing. SP: Formal Analysis, Methodology, Writing – review & editing. MH: Conceptualization, Formal Analysis, Investigation, Methodology, Supervision, Validation, Writing – review & editing. NR: Conceptualization, Formal Analysis, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing.