Serrated lesions (SLs), including sessile serrated lesions (SSL) and traditional serrated adenomas (TSA) have become subject of increased interest for their role as CRC precursors.Study aim was to evaluate the risk to develop total metachronous advanced neoplasia (T-MAN) at follow-up in patients with index SL compared to a matched cohort without SL.Patients 45-74y with SLs were identified through pathology database search. SL patients were matched 2:1 by sex; age; synchronous polyps ; timing of index, to patients without SL. Primary outcome was risk of T-MAN (advanced adenoma or high-risk SL) at follow-up. Secondary outcomes included risk of T-MAN stratified by synchronous polyps and SL characteristics.1425 patients were included . The SL group had greater risk of T-MAN compared to the non-SL group [Hazard-ratio (HR)=6.12 3.91-9.58)]. Patients with SL+HRA had higher risk of T-MAN compared to HRA alone [HR=2.62 (95%CI 1.45-4.71)], as well as patients with SL+LRA compared to LRA alone [HR=7.03 (95%CI 2.78-18.44)], and SL without adenoma compared to no-adenoma [HR=14.87 (95%CI 6.51-33.95)]. Presence of proximal SSL (HR=9.30), large SSL (HR=17.87) and proximal large SSL (HR=24.99), but not distal SSL, was associated with greater risk for T-MAN.Patients with SLs are at greater risk for developing T-MAN regardless of synchronous adenomas. Patients with SL and HRA, and those with large or proximal SSLs appear to be at greatest risk for T-MAN.OtherACGR. Djinbachian Grant / Research support from: Grant from the American College of Gastroenterology for the conduction of this project, M.-L. Lafontaine: None Declared, J. Anderson: None Declared, H. Pohl: None Declared, T. Dufault: None Declared, M. Boivin: None Declared, M. Bouin: None Declared, D. von Renteln Grant / Research support from: Daniel von Renteln is supported by a “Fonds de Recherche du Québec Santé” (FRQS) career development award and has received research funding from ERBE, Ventage, Pendopharm, Fujifilm, and Pentax., Consultant of: Boston Scientific and Pendopharm,


Correction to: BMC Infectious Diseases (2022) 22, 897. https://doi.org/10.1186/s12879-022-07887-1Error 1:Following publication of the original article , the autError 2:Following publication of the original article , the aut


Candida auris is a globally spreading yeast pathogen causing bloodstream infections with high mortality in critically ill patients. The inherent antifungal drug resistance of most C. auris isolates and threat of multidrug-resistant strains create a need for adjunct immunotherapeutic strategies. While C. albicans candidemia was shown to induce immune paralysis and activation of inhibitory immune checkpoints, in vivo data on host responses to C. auris bloodstream infection are lacking as is an immunocompetent murine infection model to study the immunopathology and immunotherapy of C. auris sepsis. Therefore, herein, we developed an immunocompetent C. auris sepsis model by intravenously infecting C57BL/6 mice with 1.5 × 108 to 8 × 108 yeast cells of aggregate-forming (AR-0384) and nonaggregative (AR-0381) C. auris reference isolates. Both isolates caused reproducible, inoculum-dependent increasing morbidity, mortality, and fungal burden in kidney tissue. Notably, morbidity and mortality outcomes were partially decoupled from fungal burden, suggesting a role of additional modulators of disease severity such as host immune responses. Flow cytometric analyses of splenic immune cells revealed significant upregulation of the programmed cell death protein 1 (PD-1) on T cells and its ligand PD-L1 on macrophages from mice infected with C. auris AR-0384 compared to uninfected mice. PD-L1 expression on macrophages from AR-0384-infected mice strongly correlated with fungal tissue burden (Spearman’s rank correlation coefficient [ρ] = 0.95). Altogether, our findings suggest that C. auris sepsis promotes a suppressive immune phenotype through PD-1/PD-L1 induction, supporting further exploration of PD-1/PD-L1 blockade as an immunotherapeutic strategy to mitigate C. auris candidiasis.IMPORTANCE Health authorities consider Candida auris to be one of the most serious emerging nosocomial pathogens due to its transmissibility, resistance to disinfection procedures, and frequent antifungal drug resistance. The frequency of multidrug-resistant C. auris isolates necessitates the development of novel therapeutic platforms, including immunotherapy. However, in vivo data on host interactions with C. auris are scarce, compounded by the lack of reliable immunocompetent mammalian models of C. auris candidemia. Herein, we describe a C. auris sepsis model in immunocompetent C57BL/6 mice and demonstrate reproducible and inoculum-dependent acute infection with both aggregate-forming and nonaggregative reference isolates from different clades. Furthermore, we show that C. auris sepsis induces upregulation of the PD-1/PD-L1 immune checkpoint pathway in infected mice, raising the potential of a therapeutic benefit of immune checkpoint blockade. Our immunocompetent model of C. auris sepsis could provide a facile preclinical platform to thoroughly investigate immune checkpoint blockade and combination therapy with antifungals. Candida auris  isolate bank   , which rre (MSS)  was quanre (MSS) –14, with10.1128/mSphere.00817-21.1TEXT S1Candida auris inoculums and infection of mice, determination of fungal burden in kidney tissue, isolation of murine splenocytes, and fluorescent labelling of splenocytes. Download Text S1, DOCX file, 0.03 MB.Detailed descriptions of experimental procedures for preparation of Copyright © 2022 Wurster et al.2022Wurster et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 8 AR-0381 cells remained sublethal, and the mice developed only mild signs of distress  (8 and 8 × 108) of AR-0381 cells caused increasing morbidity  and 7-day mortality (33% and 44%)   and B. Iand 44%)  and B. Tand 44%) . Likewisectively .8 yeast cells of the aggregate-forming isolate AR-0384 caused robust morbidity  and 17% 7-day mortality     and B. Irs, 2.0)  and B. Hrs, 2.0) . This trrs, 2.0) , suggestC. auris infection (in vitro and in vivo studies yielded divergent (strain- and inoculum-dependent) results regarding the comparative pathogenicity and immunopathology of aggregate-forming and nonaggregative C. auris strains  and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) on T cells, natural killer (NK) cells, and natural killer T (NKT) cells was determined by flow cytometry as described in C. auris-infected mice harbored significantly higher frequencies of PD-1-positive T cells than uninfected mice  (To test whether = 0.015)  and C. I= 0.015) .C. auris-infected mice than in controls  (P = 0.01)   and C. I = 0.01) . Collect in mice  and patiThe main limitations of this pilot study include testing of only one isolate per aggregation phenotype and a limited number of animals tested per condition. Furthermore, our study focused solely on the induction of coinhibitory immune checkpoint molecules and did not dynamically capture the net state of pro- and anti-inflammatory immune signals in the bloodstream and infected tissues.C. auris infection model in immunocompetent C57BL/6 mice and demonstrate the induction of coinhibitory immune checkpoint signals after C. auris bloodstream infection. These data provide a theoretical framework for PD-1/PD-L1 blockade as a potential immunotherapeutic strategy to mitigate C. auris candidiasis. We and others previously demonstrated a therapeutic benefit of the PD-1/PD-L1 pathway blockade in murine models of invasive mold infections (C. albicans sepsis (C. auris infection model in cost-efficient C57BL/6 mice could serve as a facile preclinical platform to study checkpoint inhibitors as an investigational therapy for C. auris sepsis. Furthermore, our immunocompetent model could complement the published cyclophosphamide-immunosuppressed and/or neutrophil elastase-deficient murine C. auris sepsis models (C. auris in different host backgrounds.Despite these limitations, we herein describe a simple and reproducible fections –22 and Cs sepsis , 23, 24.s models  and allo10.1128/mSphere.00817-21.2TABLE S1Table S1, DOCX file, 0.02 MB.Antibodies used for flow cytometry. Antibody solutions were prepared in 100 μL of flow cytometry buffer per sample. Download Copyright © 2022 Wurster et al.2022Wurster et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


Correction to: Eur J Nucl Med Mol Imaging (2022) 49:632–651https://doi.org/10.1007/s00259-021-05603-wThe authors regret a typo in the units assessing hyperglycaemia, which should be defined as > 8.9 mmol/L, and not as erroneously mentioned in the published version as > 8.9 nmol/L.


Gram-negative pathogens with multidrug resistance (MDR) or difficult-to-treat resistance (DTR) are increasingly common, resulting in limited treatment options. These resistant pathogens are often isolated from the respiratory tract or bloodstream and can result in significant patient morbidity and mortality. Imipenem/relebactam is a combination of imipenem with relebactam, a β-lactamase inhibitor of class A and C β-lactamases. We evaluated the activity of imipenem/relebactam and comparators  against gram-negative MDR and DTR isolates that were collected from patients with lower respiratory tract (RTI) or bloodstream infections (BSI) in the United States (US).In 2018-2020, 24 clinical labs participated in the global SMART surveillance program in the US, and each collected up to 100 consecutive aerobic or facultative gram-negative pathogens per year from patients with RTI and 50 from BSI. MICs were determined using CLSI broth microdilution and interpreted with CLSI breakpoints.P. aeruginosa (n=2018), 11.9% of E. coli (n=1952), 13.1% of K. pneumoniae (n=1080), 24.1% of E. cloacae complex (n=460), 19.7% of K. aerogenes (n=304), and 6.1% of S. marcescens isolates (n=304); DTR isolates were found among 7.1%, 0.1%, 2.4%, 1.5%, 0%, and 0.8%, respectively. Imipenem/relebactam was active against 61% of MDR P. aeruginosa, 43-53 percentage points higher than the studied comparator β-lactams, and against 49% of DTR P. aeruginosa, which were nonsusceptible to all studied commonly used β-lactams and levofloxacin (Table). Imipenem/relebactam was also active against 87-100% of MDR Enterobacterales, including against 97% of MDR K. pneumoniae, 18-96 percentage points higher than the studied comparator β-lactams, and against 85% of DTR K. pneumoniae.MDR isolates were found among 13.5% of collected in vitro data, imipenem/relebactam represents a promising treatment option in the US for patients with RTI or BSI caused by highly resistant gram-negative pathogens that pose substantial treatment challenges.Based on these Fakhar Siddiqui, MD, MBA, Merck & Co., Inc.: employee|Merck & Co., Inc.: Stocks/Bonds Karri A Bauer, PharmD, Merck & Co., Inc. Merck Research Laboratories: Stocks/Bonds Charles A. DeRyke, PharmD, Merck & Co., Inc. Merck Research Laboratories: Stocks/Bonds Katherine Young, M.S., Merck & Co., Inc.: Stocks/Bonds.


Chronic inflammation, a hallmark of type 2 diabetes (T2D) and adiposity, increases the risk for age-related co-morbidities, including physical frailty. This study aimed to examine the effects of a mobile technology-enhanced behavioral lifestyle educational intervention on frailty and associated clinical, inflammatory, and laboratory outcomes in overweight older adults with T2D. Twenty participants  with T2D were recruited to complete a single-arm 6-month lifestyle intervention modified from the Action for Health in Diabetes (Look AHEAD) study, enhanced with technology for self-monitoring of diet and physical activity. Clinical assessments were collected at baseline and end of the study and analyzed with paired t-tests. Inflammatory cytokines  were quantified using a commercially available multiplex kit . Clinical lab analysis was performed by Quest Diagnostics . Eighteen participants completed the study . At baseline, 13 participants were pre-frail, 4 were frail based on Fried frailty criteria. Inflammatory cytokines and liver enzymes values were within normal limits. At follow-up, the following outcomes significantly improved: frailty score -44% (p=0.01), BMI -3% (P< 0.00), alanine transaminase -18% (p=0.03), aspartate aminotransferase -13% (p< 0.00), IL2 -33% (p=0.01), IL4 -31% (p=0.05), IFNγ -36% (p=0.03), GM-CSF -43% (p=0.03). No other significant differences were observed. The results suggest the efficacy of a technology-enhanced lifestyle intervention on frailty and associated clinical, inflammatory, and laboratory outcomes in overweight older adults with T2D.


Ecdyonurusaurasiussp. nov., a micro-endemic species reported from several streams within the Aurès Mountains (north-eastern Algeria), is described and illustrated at nymphal, subimaginal and imaginal stages of both sexes. Critical morphological diagnostic characters distinguishing the new species are presented, together with molecular affinities as well as notes on the biology and distribution of the species. Ecdyonurus Eaton, 1868 belongs to the Ecdyonurinae Ulmer, 1920, a subfamily with rather challenging and controversial taxonomy as genera delineation and phylogeny are still partially unsolved or in process  and Helvetoraeticus Bauernfeind & Soldán, 2012 (15 species), according to the arrangement of setae on the superlingua, the number of bristles on the ventral side of the labrum and the number of comb-shaped bristles on the maxilla in nymphs, as well as the shape of the apical sclerite of the male genitalia.Ecdyonurusrothschildi Navás, 1929 and Ecdyonurusifranensis Vitte & Thomas, 1988, whereas one remains doubtful: Ecdyonurusvenosusvar.constantinicus Lestage, 1925, and the presence of Ecdyonurusvenosus  mentioned by Ecdyonurus.Currently, four taxa of this genus are reported from North Africa . Two of Ecdyonurusrothschildi from an oasis in Biskra Province, north-eastern Algeria, based on a male imago. The species was redescribed by E.aurantiacus  species group. Later, E.rothschildi, E.dispar  and E.aurantiacus nymphs. The species is now known from all Maghreb countries and is one of the most widespread species . We collected and reared fresh material at all stages. After critical observations and comparison with other Ecdyonurus species, we have clearly distinguished a new Algerian endemic species.The present study aims to examine BNP) and the Western Aurès Massif   substitution model . Two independent analyses of four MCMC chains run for one million generations with trees sampled every 1000 generations were implemented, and 100 000 generations were discarded as a burnin after visually verifying run stationarity and convergence in Tracer ver. 1.7.2  and included 25% of parsimony informative sites. The COI gene tree grouped the five sequences of Ecdyonurusaurasius sp. nov. into a well-supported monophyletic clade, and was supported as a distinct species in the ASAP analysis  to 20.1% (mean distance to E.aurantiacus), with a minimum distance of 7.1% between GBIFCH01119302 / GBIFCH00673192 and EC-CH0 sequences.The Ecdyonurinae Ulmer, 1920Taxon classificationAnimaliaEphemeropteraHeptageniidaeDambri, Benhadji & Sartorisp. nov.11A25021-4118-5EC5-B4CF-3CFE86311118https://zoobank.org/0A552D79-3329-4CCA-9724-D01492F82D7BHolotype. Algeria • male imago in ethanol, with its corresponding nymphal and subimaginal exuviae, Wilaya de Batna, Charchar, 35°24'22"N, 6°23'21"E, 1340 m. a.s.l., 09 Nov. 2021, B. Dambri coll. (GBIFCH01128855) [MZL] • Paratypes. 1 male imago, with its nymphal and subimaginal exuviae (GBIFCH01128846), 1 female imago, with its nymphal and subimaginal exuviae (GBIFCH01128858), [MZL]; 6 female imagos [IB-US], same data as holotype; 1 male imago, 1 male subimago [IB-US], 2 female imagos, 7 male subimagos [FEEL-UB2], 06 Nov. 2021; 1 male imago [IB-US], 1 male imago [FEEL-UB2], 20 Oct. 2021; 1 male imago, with its nymphal and subimaginal exuviae (GBIFCH01119304), 1 female imago, with its nymphal and subimaginal exuviae (GBIFCH01128861) [MZL], 17 Oct. 2021; 1 female imago with its subimaginal exuvia, 1 female subimago (GBIFCH01128849) [MZL], 15 Oct. 2021; 1 female subimago, 1 male subimago [IB-US], 1 male subimago (GBIFCH01128853) [MZL], 2 nymphs [FEEL-UB2], 10 Oct. 2021; 3 nymphs [IB-US], 2 nymphs (GBIFCH01128857), 1 nymph on slide (GBIFCH01119301) [MZL], 09 Oct. 2021; 7 nymphs [IB-US], 15 nymphs [FEEL-UB2], 18 Jun. 2020; 15 nymphs [IB-US], 18 nymphs [FEEL-UB2], 5 nymphs (GBIFCH01128850) [MZL], 3 Mar. 2020; same locality, B. Dambri coll; 10 nymphs (GBIFCH00832138), 2 nymphs on slide (GBIFCH00673191-GBIFCH00673192), 1 male imago (GBIFCH00673193), 1 male imago, 1 female imago, 2 female subimagos (GBIFCH00832125), 23 Jun. 2019, same locality, L. Kechemir coll. et leg. [MZL]Other paratypes. Algeria • Wilaya de Batna, Berbaga, 35°24'01N, 6°24'31"E, 1445 m. a.s.l., 1 male imago, with its nymphal and subimaginal exuviae (GBIFCH01119302), 1 female subimago with its nymphal exuvia (GBIFCH01128848) [MZL], 5 Nov. 2021; 1 male imago (GBIFCH01128852), 2 nymphs (GBIFCH01128847), 1 nymph on slide (GBIFCH01119303) [MZL], 13 nymphs [IB-US], 5 nymphs [FEEL-UB2], 4 Nov. 2021; 1 male imago [IB-US], 12 nymphs [FEEL-UB2], 30 Nov. 2020; 1 nymph [IB-US], 16 nymphs [FEEL-UB2], 03 May 2020; 1 male imago [IB-US], 10 nymphs [FEEL-UB2], 02 Mar. 2020, B. Dambri coll. Algeria • Wilaya de Khenchela, Yabous, 35°21'11"N, 6°38'35"E, 1420 m. a.s.l., 2 female imagos [IB-US], 2 female subimagos, 3 nymphs [FEEL-UB2], 22 Oct. 2021; 1 female subimago [IB-US], 2 nymphs [FEEL-UB2], 1 female imago with its subimaginal exuvia, 1 female subimago (GBIFCH01128854) [MZL], 13-14 Oct. 2021; 1 male imago with its subimaginal exuvia GBIFCH01128851), 1 female imago with is subimaginal exuvia (GBIFCH01128845) [MZL], 12 Oct. 2021; 5 nymphs [IB-US], 1 female imago, 2 male subimagos, 6 nymphs [FEEL-UB2], 09 Oct. 2021; 4 nymphs [IB-US], 2 nymphs [FEEL-UB2], 1 nymph (GBIFCH01128859) [MZL], 20 Jul. 2020; 2 nymphs [IB-US], 19 nymphs [FEEL-UB2], 1 nymph (GBIFCH01128856) [MZL], 02 Jun. 2020; 1 female subimago with its nymphal exuvia [IB-US], 8 nymphs [FEEL-UB2], 09 May 2020; 1 female subimago [IB-US], 15 nymphs [FEEL-UB2], 08 Mar. 2020; 1 female subimago with its nymphal exuvia [IB-US], 3 nymphs [FEEL-UB2], 23 Feb. 2020, B. Dambri coll. Algeria • Wilaya de Batna, Inoughissen, 35°16'42"N, 6°32'34"E, 1670 m. a.s.l., 1 nymph (GBIFCH01128863) [MZL], 07 Jul. 2020; 1 nymph (GBIFCH01128865) [MZL], 18 Apr. 2020, B. Dambri coll.35°33'03"N, 6°00'22"E, 1262 m. a.s.l.,1 nymph [IB-US], 17 Jun. 2020; 3 nymphs [IB-US], 10 nymphs [FEEL-UB2], 1 nymph (GBIFCH01128860) [MZL], 20 Apr. 2020, B. Dambri coll. Algeria • Wilaya de Batna, Bouailef, 35°37'01"N, 6°11'17"E, 1060 m, 1 nymph [IB-US], 08 Mar. 2020, B. Dambri coll.Algeria • Wilaya de Batna, oued Chaâba, Aurasiusmons; aurasius is a noun in apposition.Aurès mountains were coined by the Berber people as Awras, meaning tawny; translated by the Romans as Male imago Size: body length: 9.0–9.8 mm; forewing length 9.1–10.9 mm; cerci broken. General body color distinctly brown to reddish-brown . The new species presents more affinities with the two other North African endemics but can be distinguished from E.rothschildi by the much longer pronotal projections, the shape of the stout setae on the dorsal surface of femora (pointed in the latter), the shape of the gills  and the shape of the glossae ((inner margin rounded and convex in E.rothschildi). Ecdyonurusaurasius sp. nov. differs from E.ifranensis by the shape of the labrum (less broad in E.ifranensis), the shape of the stout setae on the dorsal surface of femora (pointed in E.ifranensis), and the shape of the glossae similar to E.rothschildi. In males, E.aurasius sp. nov. differs from E.rothschildi, E.dispar and E.aurantiacus by the compound eyes separated and not touching (character not stated in E.ifranensis description), from E.aurantiacus and E.dispar by the posterior margin of the basal sclerite smooth, and from E.ifranensis by the first transversal vein in the costal field surrounded by a dark brown maculation (the same in E.rothschildi), and by the shape of the posterior margin of the basal sclerite rounded (straight in E.ifranensis). It is also worth noting that E.aurasius sp. nov. differs from the two other North African species by the nervous ganglia tinted in purple in female imagos, whereas they are colorless in E.rothschildi and E.ifranensis.By the shape of the penis lobes and the posterolateral projections of the abdomen, E.dispar . The nymEcdyonurusaurasius sp. nov., as known so far, is restricted to the Aurès region. The species has been recorded from only six localities in the Western Aurès area; most habitats are located in the highest part of the streams, within altitudes ranging from 1010 to 1800 m a.s.l. These sites are represented by small mountain watercourses with gravel substrate  and the lowest one was observed at the Bouailef site .ate Fig. . The aveEphemeroptera species sporadically occurring in the same sites were Caenisluctuosa , Baetischelif Soldan, Godunko & Thomas, 2005 and Baetissinespinosus Soldán & Thomas, 1983.The mature nymphs and subimagos (together with early-instar nymphs) were observed in May/June and another generation observed in September/October, thus suggesting a bivoltine life cycle. The other


Brain metabolic-sensory targets for modulatory glucose-sensitive endocrine and neurochemical signals remain unidentified. A hypothalamic astrocyte primary culture model was here used to investigate whether glucocorticoid receptor (GR) and noradrenergic signals regulate astrocyte glucose  and/or energy (5′-AMP-activated protein kinase [AMPK]) sensor reactivity to glucoprivation by sex. Glucose-supplied astrocytes of each sex showed increased GLUT2 expression after incubation with the GR agonist dexamethasone (DEX) or norepinephrine (NE); DEX plus NE (DEX/NE) augmented GLUT2 in the female, but not in male. Glucoprivation did not alter GLUT2 expression, but eliminated NE regulation of this protein in both sexes. Male and female astrocyte glucokinase profiles were refractory to all drug treatments, but were down-regulated by glucoprivation. Glucoprivation altered AMPK expression in male only, and caused divergent sex-specific changes in activated, i.e., phosphoAMPK (pAMPK) levels. DEX or DEX/NE inhibited  or stimulated  AMPK and pAMPK proteins in both glucose-supplied and -deprived astrocytes. In male, NE coincidently up-regulated AMPK and inhibited pAMPK profiles in glucose-supplied astrocytes; these effects were abolished by glucoprivation. In female, AMPK profiles were unaffected by NE irrespective of glucose status, whereas pAMPK expression was up-regulated by NE only during glucoprivation. Present outcomes document, for each sex, effects of glucose status on hypothalamic astrocyte glucokinase, AMPK, and pAMPK protein expression and on noradrenergic control of these profiles. Data also show that DEX and NE regulation of GLUT2 is sex-monomorphic, but both stimuli impose divergent sex-specific effects on AMPK and pAMPK. Further effort is warranted to characterize mechanisms responsible for sex-dimorphic GR and noradrenergic governance of hypothalamic astrocyte energy sensory function. AMPK5′-AMP-activated protein kinaseCaMKKβcalcium/calmodulin-dependent protein kinase kinase-betaDEXdexamethasoneGCKglucokinaseGLUT2glucose transporter-2IIHinsulin-induced hypoglycemiaNEnorepinephrinepAMPKphosphoAMPKPP1protein phosphorylase-11The glucose-regulatory neural circuitry monitors cellular metabolic status and systemic energy substrate storage to exert appropriate control of central and peripheral effector motor functions –5. Many l-lactate for trafficking to neurons  and F = 19.86, p < 0.001; glucose status main effect: F = 16.63, p = 0.001; treatment main effect: F = 34.84, p < 0.001; glucose status/treatment interaction: F = 5.97, p = 0.006]) and GCK  = 15.22, p < 0.001; glucose status main effect: F = 95.43, p < 0.001; treatment main effect: F = 2.49, p = 0.097; glucose status/treatment interaction: F = 1.20, p = 0.342] and F = 6.70, p = 0.001; glucose status main effect: F = 36.47, p < 0.001; treatment main effect: F = 3.25, p = 0.050; glucose status/treatment interaction: F = 0.220, p = 0.881]) protein expression in each sex. For each sex, treatment groups including astrocytes supplied with 5.5 mM glucose (G5.5), at left, are illustrated by gray bars, while glucose-deprived (G0) treatment groups, at right, are shown in white. Data indicate that DEX or NE alone each significantly increased GLUT2 levels in glucose-supplied male and female astrocytes . DEX and NE co-incubation (cross-hatched gray bars) reduced  or elevated  GLUT2 content relative to vehicle controls. In each sex, GLUT2 protein profiles were unaffected by glucoprivation (G0/V [solid white bars] versus G5.5/V). In glucose-deprived male and female astrocytes, GLUT2 profiles were elevated by DEX , but were refractory to NE ; DEX plus NE treatment (cross-hatched white bars) increased GLUT2 expression to levels measured after incubation with DEX alone. In each sex, hypothalamic astrocyte GCK content was decreased in response to glucoprivation, but was altered by any drug treatment when glucose was present or absent.Research outcomes described below were obtained using a characterized hypothalamic primary astrocyte culture model to address the question of whether hypothalamic astrocyte primary cultures established from male and/or female rats express glucose-sensitive plasma membrane and glycolytic pathway glucose sensors, and these nutrient gauges are regulated by glucocorticoid or noradrenergic input alone or by interaction of these regulatory stimuli. F = 37.09, p < 0.001; glucose status main effect: F = 38.86, p < 0.001; treatment main effect: F = 54.84, p < 0.001; glucose status/treatment interaction: F = 18.78, p < 0.001] and F = 7.32, p = 0.001; glucose status main effect: F = 15.07, p = 0.001; treatment main effect: F = 11.24, p < 0.001; glucose status/treatment interaction: F = 0.82, p = 0.499]) and pAMPK  = 19.16, p < 0.001; glucose status main effect: F = 13.28, p = 0.002; treatment main effect: F = 29.35, p < 0.001; glucose status/treatment interaction: F = 10.95, p < 0.001] and F = 21.65, p < 0.001; glucose status main effect: F = 3.68, p = 0.067; treatment main effect: F = 42.89, p < 0.001; glucose status/treatment interaction: F = 6.39, p = 0.002]) expression. Results show that glucose-supplied male astrocytes exhibited reductions in AMPK expression in response to DEX or NE alone; this negative response was exacerbated by DEX plus NE treatment. In the female, AMPK levels were elevated by DEX given alone or together with NE, but this protein was unaffected by NE alone. Glucose withdrawal suppressed AMPK content in male, but not female astrocytes. DEX alone or DEX plus NE decreased or stimulated AMPK expression in male versus female glucose-deprived astrocytes, respectively. Astrocyte pAMPK levels were diminished  or augmented  by DEX, according to sex. NE stimulated or had no effect on this protein profile in male versus female, respectively. In each sex, pAMPK content was affected similarly by DEX alone versus DEX plus NE treatment. Glucoprivation correspondingly up-  or down-  regulated pAMPK expression in male and female astrocytes. In glucose-deprived male glial cells, pAMPK profiles were refractory to DEX or NE, but were inhibited by DEX plus NE. Glucose-deprived female astrocytes, on the other hand, showed elevated pAMPK expression in response to all three treatments with the greatest magnitude of increase caused by DEX alone.The ultra-sensitive energy gauge AMPK monitors the cellular AMP/ATP ratio. Current work examined the premise that hypothalamic astrocyte total AMPK protein and/or activated, e.g., phosphorylated AMPK (pAMPK) protein profiles are affected by glucose withdrawal in a sex-specific manner, and the related question that AMPK activation may be differentially controlled by DEX or NE when glucose is present versus absent. Data presented in F = 7.26, p = 0.001; glucose status main effect: F = 6.05, p = 0.026; treatment main effect: F = 8.61, p = 0.001; glucose status/treatment interaction: F = 6.32, p = 0.005) or female  = 11.13, p < 0.001; glucose status main effect: F = 8.34, p = 0.011; treatment main effect: F = 19.51, p < 0.001; glucose status/treatment interaction: F = 3.67, p = 0.035) astrocyte CaMMKβ protein profiles. Data reveal that CaMMKβ expression in glucose-supplied astrocytes of either sex was insensitive to DEX or NE treatment alone or together. NE inhibited this protein in glucose-deprived male cells; this decline was amplified by combinatory NE plus DE treatment. In female astrocytes incubated without glucose, CaMMKβ content was increased by exposure to either DEX or DEX plus NE. F = 9.63, p < 0.001; glucose status main effect: F = 32.24, p < 0.001; treatment main effect: F = 2.05, p = 0.147; glucose status/treatment interaction: F = 8.34, p = 0.005) or female  = 6.82, p = 0.001; glucose status main effect: F = 37.94, p < 0.001; treatment main effect: F = 3.09, p = 0.057; glucose status/treatment interaction: F = 0.19, p = 0.903) astrocyte PP1 protein levels. NE alone had divergent effects on PP1 expression in male glucose-supplied (increased) versus glucose-deprived (decreased) astrocytes. NE plus DEX had no effect or suppressed PP1 profiles in male astrocytes when glucose was present or absent, respectively. Female astrocyte PP1 content was diminished during glucoprivation, but was insensitive to any drug treatment.AMPK activity is regulated in part by the upstream stimulatory kinase enzyme CaMMKβ as well as the inhibitory phosphatase PP1. It was of interest here to investigate whether action of DEX alone or in coordination with NE controls expression of these enzyme proteins in hypothalamic astrocytes. F = 17.04, p < 0.001; glucose status main effect: F = 38.47, p < 0.001; treatment main effect: F = 25.59, p < 0.001; glucose status/treatment interaction: F = 1.35, p = 0.295) or female  = 5.14, p = 0.001; glucose status main effect: F = 6.70, p = 0.016; treatment main effect: F = 5.44, p = 0.005; glucose status/treatment interaction: F = 4.31, p = 0.014) astrocyte GR protein expression. Data show that GR protein was decreased in G5.5 male astrocytes after incubation with DEX, NE, or DEX plus NE, and that the magnitude of this reduction was greatest in response to the latter combinatory dosing. Meanwhile, GR expression in female G5.5 astrocytes was unaffected by any of these treatments. Glucoprivation did not modify GR expression in astrocytes of either sex. Treatment with either DEX or DEX plus NE resulted in diminished GR protein profiles in G0 male and female astrocytes.Research here addressed the issue of whether male and/or female rat hypothalamic astrocyte primary cultures are directly receptive to glucocorticoid control by means of GR expression, and if so, whether this receptor protein profile is sensitive to glucose availability and regulated by glucocorticoid and noradrenergic stimuli in the presence versus absence of glucose. F = 10.83, p < 0.001; glucose status main effect: F = 22.84, p < 0.001; treatment main effect: F = 9.43, p = 0.001; glucose status/treatment interaction: F = 8.23, p = 0.002] and F = 23.78, p < 0.001; glucose status main effect: F = 0.27, p = 0.608; treatment main effect: F = 52.99, p = <0.001; glucose status/treatment interaction: F = 2.40, p = 0.106]), α2-AR  = 20.78, p < 0.001; glucose status main effect: F = 77.61, p < 0.001; treatment main effect: F = 14.40, p < 0.001; glucose status/treatment interaction: F = 8.23, p = 0.002] and F = 14.31, p < 0.001; glucose status main effect: F = 5.52, p = 0.032; treatment main effect: F = 15.74, p < .001; glucose status/treatment interaction: F = 15.80, p < 0.001]), and β1-AR  = 3.09, p = 0.029; glucose status main effect: F = 7.39, p = 0.015; treatment main effect: F = 2.02, p = 0.152; glucose status/treatment interaction: F = 2.74, p = 0.077] and F = 7.45, p < 0.001; glucose status main effect: F = 5.86, p = 0.023; treatment main effect: F = 7.13, p = 0.001; glucose status/treatment interaction: F = 8.36, p = 0.001]) protein expression. Data show that DEX alone inhibited α1-AR protein, but did not regulate α2-AR or β1-AR levels in glucose-supplied male astrocytes. However, DEX alone stimulated each of these AR variant proteins in the female. Glucoprivation did not alter α1-AR, α2-AR, or β1-AR expression profiles in the male, but up-regulated α2-AR and β1-AR levels in female astrocytes. In glucose-deprived male astrocytes, DEX decreased α2-AR content, while NE suppressed α1-AR and α2-AR proteins. DEX plus NE had an equivalent effect to DEX alone on α1-AR expression, but had greater inhibitory effects on α2-AR levels compared to DEX or NE alone. Female glucose-deprived astrocytes showed augmentation of α1-AR and α2-AR, but not by β1-AR by DEX. DEX plus NE increased α1-AR levels to an extent similar to DEX alone, but did not alter α2-AR expression. Both NE alone and NE plus DEX inhibited β1-AR profiles in female astrocytes deprived of glucose.A critical objective of current work was to examine whether glucose status controls AR variant protein expression in male and female hypothalamic astrocytes, and to determine if DEX and NE act alone or synergistically to regulate these proteins during glucose availability versus deprivation. 4in vivo regarding neural control of glucose homeostasis in each sex.The current project used a hypothalamic astrocyte primary culture model to address the premise that these glia express glucoprivic-responsive glucose and energy sensing molecular biomarkers, and that glucose-sensitive hormone (glucocorticoid) and/or neurotransmitter (noradrenergic) stimuli govern sensor reactivity to glucose deficiency in a sex-specific manner. Outcomes show that in astrocytes of each sex, membrane glucose sensor GLUT2 and glycolytic pathway sensor GCK protein profiles are controlled by GR and AR input or by glucose, respectively. Yet, expression and activity of the energy sensor AMPK in these cells is subject to sex-dimorphic regulation by DEX, NE, and glucose. Results provide novel evidence that glucose modulates noradrenergic control of astrocyte GLUT2 (both sexes), AMPK , pAMPK (both sexes), and α1- and α2-AR  proteins. Evidence that co-administration of NE and DEX to male, but not female astrocytes attenuates (GLUT2) or exacerbates , effects of DEX alone infers that intersection of GR and AR signaling may be sex-specific. Ongoing work seeks to elucidate mechanisms that underlie differential male versus female astrocyte responses to GR or NE stimulation. There is also a need for understanding of functional implications of glucocorticoid and noradrenergic regulation of hypothalamic astrocyte metabolic sensory functions Present outcomes document expression of the characterized glucose sensors GLUT2 and GCK by hypothalamic astrocytes, and show that GR and AR input regulates the former, but not the latter protein in each sex. Evidence here that DEX treatment augments GLUT2 profiles in astrocytes of either sex, despite dissimilar regulatory effects of DEX on male (decreased) versus female (unchanged) astrocyte GR expression, infers that glucocorticoids may control GR input volume as well as post-receptor signaling in a sex-specific manner. GLUT2 ostensibly employs distinctive structural features to perform glucose transport versus monitoring functions . Data heThe characterized DEX dosage used here down-regulated astrocyte AMPK and pAMPK in male, but stimulated these proteins in the female, revealing bi-directional, sex-specific glucocorticoid regulation of AMPK expression and activity state in this brain cell type. This divergent hormone action may involve, in part, dissimilar GR signal volume in the two sexes as GR protein expression declined in DEX-treated male astrocytes, but was unaffected by this treatment in the female. DEX may also impose sex-contingent control of downstream post-receptor signaling, transcriptional, and/or post-translational events that govern astrocyte AMPK and pAMPK protein profiles. As the current study focused on a single DEX dose, there would be benefit from future investigation of whether varying GR ligand concentrations elicit proportionate modifications in AMPK and pAMPK levels over all or a segment of a comprehensive, physiological-like dosage range. There also remains a need to assess whether suppressive versus amplifying stimulatory effects of DEX on AMPK and pAMPK expression are associated with specific hormone concentration(s). Our findings that glucoprivation attenuates DEX-induced augmentation of male astrocyte pAMPK content infer that metabolic modulation of GR-mediated action on AMPK activation state may occur in this sex only. Noradrenergic control of this astrocyte energy sensor is evidently also sex-specific as NE incubation suppressed total AMPK protein and stimulated pAMPK levels in male astrocytes, yet had no effect either on protein profile in the female. Notably, glucoprivation was observed to elicit a directional shift in NE control  on male astrocyte AMPK, and to promote a loss or gain of NE regulation of pAMPK profiles in male versus female, respectively. These data point to sex-dependent metabolic modulation of noradrenergic governance of hypothalamic astrocyte AMPK activity.Current results show that neither DEX nor NE regulates the upstream stimulatory kinase CaMKKβ in the presence of glucose, yet during glucoprivation DEX elevates this protein profile in the female, while NE suppresses CaMKKβ expression in the male. PP1 levels in glucose-supplied astrocytes of either sex are similarly refractory to DEX, whereas NE enhances this protein in the male only. Notably, the direction of noradrenergic control of PP1 expression in male astrocytes is altered, e.g., switches from stimulatory to inhibitory when glucose is withdrawn. These outcomes support the novel concept that astrocyte energy status may determine whether glucocorticoid and noradrenergic signals are able to regulate astrocyte AMPK activity via upstream enzymes that govern phosphorylation, e.g., during glucose deficiency. DEX treatment of glucose-deprived female astrocytes stimulated CaMKKβ expression without affecting PP1 profiles, coinciding with up-regulated pAMPK. Thus, in this sex, CaMKKβ is likely required for glucocorticoid augmentation of AMPK activity. Interestingly, NE did not modify either CaMKKβ or PP1 expression, but also increased pAMPK profiles, apparently by kinase/phosphatase-independent mechanisms. Glucose-deprived male astrocyte CaMKKβ and PP1 profiles were resistant or inhibited by DEX, respectively, yet pAMPK expression was unaffected by either stimulus, inferring that potential augmentation of pAMPK levels by noradrenergic diminution of PP1 may be counter-balanced by signals that blunt AMPK activation.It is intriguing to observe that glucoprivic-associated diminution of astrocyte GCK expression coincided with up- or down-regulated pAMPK protein in male versus female, respectively. These findings infer that in the latter sex, augmenting effects of diminished glucose catabolism on sensor activity are apparently offset by regulatory signals that attenuate AMPK phosphorylation and/or decreased allosteric activation of AMPK due to energy production from non-glucose substrates.Present data also show that NE did not alter any astrocyte AR variant protein profile in either sex, but α1-AR (both sexes), α2-AR , and β1-AR  proteins are responsive to DEX. The observed efficacy of DEX to up-regulate all three AR variant proteins in the female may contribute to unique effects of combinatory DEX plus NE treatment on specific astrocyte target proteins in that sex. Glucose stability evidently modulates DEX regulation of specific AR proteins in each sex, as glucoprivation abolishes DEX inhibition of α1-AR profiles and allows DEX to suppress α2-AR levels in the male, and in the female eliminates DEX stimulation of β1-AR expression. Glucoprivic governance of noradrenergic regulation of AR protein profiles is, on the other hand, sex-specific as this metabolic stress allows inhibitory noradrenergic control of α1-AR and α2-AR proteins in male hypothalamic astrocytes only.in vivo models of glucose-sensitive glucocorticoid and noradrenergic signaling to the hypothalamus to understand, for each sex, hormonal and neurotransmitter regulation of astrocyte sensing and communication of cellular energetic stability or imbalance.In summary, current research presents unique proof that hypothalamic astrocytes express membrane and glycolytic pathway glucose-sensory biomarkers in addition to the energy sensor AMPK . Results


Existing chemotherapy treatments for breast cancer patients are high on toxicity. There are very limited options available for triple-positive breast cancer (TPBC) patients, and there have not been any major breakthrough for targeted therapy for triple-negative breast cancer (TNBC) patients. Therefore, there is a need to identify common therapeutic targets for breast cancer patients. In this manuscript, we compared the sphingolipid profiles of cancer cell lines representing TPBC and TNBC, and correlated these profiles with the proliferation and migration properties the of cell types. We then associated the sphingolipid profiles for each subtype specific cell line with transcriptional and translational expression of corresponding metabolizing enzymes. Our results suggested that ceramide kinase (CERK) that catalyzes the synthesis of ceramide-1-phosphates from ceramides is dysregulated in both cell types. We also showed that the targeting of CERK at transcriptional level by siRNA therapeutics or inhibiting the CERK activity by hydrogel-mediated delivery of chemical inhibitors can be an effective strategy to slow down the tumor progression. Therefore, CERK emerges as a potential therapeutic target that can be explored further for cancer therapy.Sphingolipids are key signaling biomolecules that play a distinct role in cell proliferation, migration, invasion, drug resistance, metastasis, and apoptosis. Triple-negative (ER−PR−HER2−) and triple-positive (ER+PR+HER2+) breast cancer  subtypes reveal distinct phenotypic characteristics and responses to therapy. Here, we present the sphingolipid profiles of BT-474 and MDA-MB-231 breast cancer cell lines representing the TPBC and TNBC subtypes. We correlated the level of different classes of sphingolipids and the expression of their corresponding metabolizing enzymes with the cell proliferation and cell migration properties of BT-474 and MDA-MB-231 cells. Our results showed that each cell type exhibits a unique sphingolipid profile, and common enzymes such as ceramide kinase  are deregulated in these cell types. We showed that siRNA/small molecule-mediated inhibition of CERK can alleviate cell proliferation in BT-474 and MDA-MB-231 cells, and cell migration in MDA-MB-231 cells. We further demonstrated that nanoparticle-mediated delivery of CERK siRNA and hydrogel-mediated sustained delivery of CERK inhibitor to the tumor site can inhibit tumor progression in BT-474 and MDA-MB-231 tumor models. In summary, distinct sphingolipid profiles of TPBC and TNBC representing cell lines provide potential therapeutic targets such as CERK, and nanoparticle/hydrogel mediated pharmacological manipulations of such targets can be explored for future cancer therapeutics. Lipid reprogramming has emerged as a key hallmark for cancer pathogenesis ,2. SphinCeramide, the central player in the sphingolipid pathway, induces apoptosis and cell senescence in response to chemotherapy and radiation therapy A 9,10].,10.9,10]+ tumors), and are categorized as triple-positive (TPBC) phenotypes . A. A21]. Aenotypes . Therefoenotypes . In contenotypes . Chemothenotypes ,28.Each breast cancer subtype has a unique molecular portrait that influences its response to therapy, prognosis, and outcome. Earlier studies have reported the sphingolipid profile in luminal (ER+PR+) and TNBC (ER−PR−HER2−) subtypes ,30. Howe®, Saint Louis, MO, USA. Fetal bovine serum (Cat#10270) was purchased from, Waltham, MA, USA. Matrigel (Cat#354234) was purchased from Corning Inc., Bedford, MA, USA. Penicillin-Streptomycin solution (SV30079) was purchased from Cytiva HyCloneTM, South Logan, OH, USA. Trypsin (Cat#TCL007), MEM Medium (Cat#AL081) and sodium bicarbonate (GRM849) were purchased from HiMedia Laboratories, Mumbai, India.Materials. Human breast cancer cell lines BT-474 and MDA-MB-231 were purchased from American Type Culture Collection . DMEM (Cat#D5648), DPBS (Cat#D5652), Crystal Violet Dye (Cat#C0775) were purchased from Sigma–AldrichTM DNase (Cat#AM2238) was from Invitrogen, Vilnius, Lithuania. iScriptTM cDNA synthesis kit (Cat#1708891) and iTaq™ universal SYBR®Green Supermix (Cat#1725124) were purchased from Bio-Rad Laboratories, Hercules, CA, USA. RNase Inhibitor (Cat#AM2694), and PierceTM BCA protein assay kit (Cat#23227) were purchased from Thermo Scientific. Complete™, Rockford, IL, USA. Protease Inhibitor Cocktail (P8340-1ML) was purchased from Sigma–Aldrich®, Saint Louis, MO, USA. Sigma. CerK siRNA (Cat#L-004061-00-0005) and scrambled siRNA (Cat# D-001810-10-05) were purchased from Dharmacon Inc., Cambridge, UK. Ceramide Kinase Inhibitor, NVP231 (Cat#3960) was purchased from Tocris Bioscience, Bristol, UK.Lipofectamine 2000 (Cat#11668019) and TURBO® Polar Lipids, Inc., Alabaster, OK, USA. LASS1 (CERS1) (Cat#H00010715-A01), LASS2 (CERS2) (Cat#H00029956-M01A), LASS4 (CERS4) (Cat#H00079603-M01), LASS6 (CERS6) (Cat#H00253782-M01) were purchased from Abnova, Taipei, Taiwan. LASS5 (CERS5) (Cat#ab73289), CerK (Cat#ab155061), SPHK1 (Cat#ab109522), SPHK2 (Cat#ab264042), A-SMase (SMPD1) (Cat#ab83354), N-SMase1 (SMPD2) (Cat#ab131330), N-SMase2 (SMPD3) (Cat#ab199399), N-SMase3 (SMPD4) (Cat#ab133935), UGCG (Cat#ab124296), SMS1 (Cat#ab135365), SMS2 (Cat#ab237681), GBA1 (Cat#ab88300), GLB1 (Cat#ab96239), B4GALT6 (Cat#ab200639), Goat anti-mouse IgG (L+H) (Cat#ab6789) were purchased from Abcam, Cambridge, UK. ASAH1 (Cat#HPA005468), ASAH2 (Cat#ABS457), β-actin (Cat#A5541) were purchased from Sigma–Aldrich®, Saint Louis, MO, USA. Goat anti-rabbit IgG-HRP was purchased from (Cat#sc-2004) Santa Cruz Biotechnology, Inc., Dallas, TX, USA. Ceramide/Sphingoid Internal Standard Mixture II (Cat#LM6005-1EA) was purchased from AvantiN,N′-methylene bisacrylamide (Cat#M7279), glycine (Cat#G8898), MTT (Cat#M5655), triethyl ammonium bicarbonate buffer (Cat#T70408), and iodoacetamide (144-48-9) were purchased from Sigma–Aldrich®, Saint Louis, MO, USA. Tris (Cat#MB029), NaCl (Cat#GRM853), polyoxyethylenesorbitan monolaurate (Tween® 20) (Cat#P7949), glycerol (Cat#GRM1027), bovine serum albumin fraction-V (Cat#GRM105) were purchased from HiMedia Laboratories, Mumbai, India. Paraformaldehyde (PFA) (Cat 81847) was purchased from Thomas Baker, Mumbai, India. Ammonium hydroxide (Cat#16227) and hydrochloric acid (Cat#29505) was purchased from Thermo Fisher Scientific, Waltham, MA, USA. MOPS free acid (Cat#MB0360) and acrylamide (Cat#AB1032) were purchased from Bio Basics Inc., Markham, ON, Canada. Sodium hydroxide (Cat#13913) and potassium hydroxide (Cat#84749) was purchased from Sisco Research Laboratories Pvt. Ltd., Maharashtra, India. Developer (Cat#4908216), Fixer (Cat#4908232) and XBT X-Ray film (Cat#6568307) were purchased from Carestream, Rochester, NY USA. Transwell Migration Plate (Cat#3464) was purchased from Corning Inc., Bedford, MA, USA. Nitrocellulose (Cat#HATF00010), Immobilon Western Chemiluminescent HRP substrate (Cat#WBKLS0500), ethanol (Cat#100983), glacial acetic acid (Cat#193402), chymotrypsin (11418467001), DMSO (Cat#276855), ethidium bromide (Cat#E8751), agarose (Cat#A9539), magnesium chloride (Cat#208337), sodium dodecyl sulfate (Cat#L3771), ammonium persulfate (Cat#A3678), Methanol (Cat#34966), chloroform (Cat#25669-1L), 2-propanol (Cat#34965), acetonitrile (Cat#34967), ammonium acetate (Cat#14267-25G), formic acid (Cat#56302-50ML), water (Cat#39253-4L) and ammonium formate (Cat#14266-25G) were purchased from Honeywell International Inc., Charlotte, NC, USA. ACQUITY UPLC BEH Shield RP18 column (Cat#186002854) was purchased from Waters™ Ltd., Milford, MA, USA.2 in humidified incubator. Cell culture. Human breast cancer cell lines BT-474 and MDA-MB-231 were cultured in DMEM high glucose media with 10% Fetal bovine serum, 100 units/mL penicillin, and 100 µg/mL streptomycin. Cells were grown at 37 °C with 5% CO5 per well) were seeded in six-well plate in DMEM media with 10% FBS and 10% penicillin and streptomycin, and incubated at 37 °C in CO2 incubator for 24 h. At 80–85% confluency, siRNA-lipofectamine complexes in 1:3 ratio were incubated for 25 min in MEM media, and cells were transfected with these complexes. After 6 h of transfection, media was removed, and cells were incubated with antibiotic-free media containing 10% FBS for 36 h. CERK silencing was confirmed by Western blotting.siRNA transfections in cell culture. BT-474 and MDA-MB-231 cells were transfected with siRNA (targeting CERK or scrambled) using Lipofectamine 2000. BT-474 and MDA-MB-231 cells  (5000 cells/well) were used, and cell proliferation assay was performed following previously described method [For transwell migration assay, BT-474 and MDA-MB-231 cells or siRNA transfected cells (scrambled or CERK siRNA) transfected cells  were seeded in transwell inserts (4 µm pore size). We then placed the inserts in 24-well cell culture plates containing DMEM with 10% FBS and incubated for 24 h at 37 °C. We fixed the migrated cells in 4% paraformaldehyde (PFA) (~5 min), and permeabilized with methanol (20 min). Cells were then stained with 2% crystal violet dye (15 min) followed by PBS washing to remove the extra stain on the cell surface. Cells were counted manually and imaged using a Nikon microscope. Pellet collection for RNA and protein isolation. For RNA isolation, cells were grown in 100 mm cell culture plates for 90% confluency. For pellet collection, media was aspirated from the plates and washed two times with DPBS. Trizol (1 mL) was added to the plate, and plates were incubated for 5 min. After incubation, cells were scraped and transferred into 1.5 mL centrifuge tubes, and were used immediately or stored at −80 °C. For protein isolation, cells were similarly washed, scraped out in DPBS, centrifuged at 5000 rpm for 5 min, and used immediately or stored at −80 °C.Quantitative Real-Time PCR. Total RNA extraction and qRT-PCR studies were performed using previously described method . All priWestern Blotting. Protein expression analysis was performed by Western blotting as per previously described method . ProteinIsolation and quantification of sphingolipids using LC-MS/MS. Collection of cell pellets, lipid isolation, LC-MS/MS analysis, and absolute quantitation of sphingolipids was performed as per our previously published method .Animal studies. Animal experimental protocols were reviewed and approved by the Institutional Animal Ethics Committee of Regional Centre for Biotechnology (RCB/IAEC/2021/96). The experiments were carried out as per the guidelines issued by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Govt. of India.6 cells were injected subcutaneously in the flank. Once the tumor was palpable (20–30 mm3), tumor volume and weight of the mice were recorded after every two days. Tumor volume was calculated as per the formula, L × B2/2 where L is the length of tumor and B is breadth of tumor. At the end of the experiment, tumors were excised, washed with ice-cold 1X PBS, and stored in Allprotect tissue reagent at −80 °C for further analysis. All tumor growth kinetic experiments were performed in female NOD SCID CB.17 mice. Prior to the cell injection, hair was shaved from flank of the mice. For comparative tumor kinetics experiments, respective cell lines (BT-474 and MDA-MB-231) were resuspended in FBS: Matrigel , and 3 × 103) were randomized into different groups with each group having 4–6 mice. Group 1 mice were left untreated, group 2 mice were injected intratumorally with scrambled siRNA, and group 3 mice were treated with target siRNA. The siRNA was injected in a volume of 40 µL with 300 ng of siRNA complexed with TAC6 polymer  at the tumor site [For siRNA experiments, mice with palpable tumor  were randomized into different groups with each group having 4–6 mice. Group 1 mice were left untreated, and group 2 mice were implanted subcutaneously with inhibitor (NVP-231)-loaded hydrogel [For inhibitor experiments, mice with palpable tumor .Visualization of sphingolipid metabolic pathways. Expression of genes involved in sphingolipid metabolism were quantified using RT-PCR. Protein levels were probed by Western blots and quantified using ImageJ, followed by normalization against actin. Lipidomics experiments were performed to quantify the metabolite (sphingolipid) levels. Log2 (fold changes) were calculated for MDA-MB-231 against BT-474 cells. The visualization was made using drawio, a cross-platform JavaScript based open source drawing application available on GitHub at We selected BT-474 and MDA-MB-231 breast cancer cell lines representing TPBC and TNBC subtypes. To quantify the levels of sphingolipid species in these two cell lines, we collected cell pellets, and isolated the total lipids enriched in sphingolipids. We performed qualitative and quantitative sphingolipidomics with absolute quantitation of 27 sphingolipid species including ceramides, glucosylceramides, lactosylceramides, sphingomyelins, ceramide-1-phosphates (C1P), sphingosine, and sphingosine-1-phosphate (S1P) using LC-MS/MS [p < 0.01), C18:0 , C22:0 , C24:0 , and C24:1  were elevated in MDA-MB-231 cells in comparison to BT-474 cells (p < 0.05), C18:0 , C20:0 , C22:0 , C24:0 , and C24:1 , are downregulated in MDA-MB-231 cells as compared to BT-474 cells (p < 0.05) (p < 0.05) species as compared to BT-474 cells (p < 0.05), C18:0 , C20:0 , C22:0 , and C24:0 , are less abundant in MDA-MB-231 cells as compared to BT-474 cells (p < 0.05), C18:0 , C20:0 , C22:0 , C24:0  and C24:1 , are elevated in MDA-MB-231 cells (p < 0.01) increase in levels of S1P   and C24:74 cells . We also74 cells C. It is 31 cells D. We alss of S1P  in MDA-Ms of S1P . TherefoSPT1) , SPT2 , delta 4-desaturase, sphingolipid 1 and 2 (DEGS1) , DEGS2 , and 3-ketodihydrosphingosine reductase (KDSR)  as compared to BT-474 cells , CERS2 , CERS3 , CERS4 , CERS5 , and CERS6  in MDA-MB-231 cells as compared to BT-474 cells , ASAH2 , alkaline ceramidase 1 and 3 ACER1 , and ACER3  are significantly upregulated in MDA-MB-231 cells as compared to BT-474 cells (p < 0.0005) in the expression of sphingosine kinase 1 (SPHK1) and a ~3.3-fold (p < 0.0005) increase in SPHK2 as compared to BT-474 cells that, again, positively correlates to higher S1P levels  in MDA-MB-231 cells (p < 0.01) decrease in the expression of S1P lyase (S1PL) in MDA-MB-231 cells over BT-474 cells.Among salvage pathway genes, expression of 74 cells . This inP levels B. Furthe31 cells B that coSMPD1) (p < 0.0001), SMPD2 , SMPD3 , and SMPD4  in MDA-MB-231 cells as compared to BT-474 cells (SMS1) (p < 0.0001), and downregulation of SMS2 (p < 0.0001) in MDA-MB-231 cells  in glucosylceramide synthase (UGCG) and a >50-fold increase (p < 0.01) in the expression of B4GALT6 in MDA-MB-231 cells in comparison to BT-474 cells  in glucosylceramidase 1 (GBA1) expression, responsible for breakdown of glucosylceramides, but we could not detect galactosidase (GLB1) expression due to low transcript levels. High expression of B4GALT6, thereby, support high lactosylceramide levels in MDA-MB-231 cells as compared to BT-474 cells in spite of high GBA1 expression (We observed a >100.0-fold increase (74 cells D. In spipression D.p < 0.05), CERS4 , CERS5 , but there was >2.0-fold (p < 0.05) decrease in the expression of CERS6 in comparison to BT-474 cells  gene expression in MDA-MB-231 cells as compared to BT-474 cells but it is not translated to protein expression, that might be due to different post-transcriptional and post-translational regulatory changes (p < 0.001) in MDA-MB-231 as compared to BT-474 cells is very well correlated with gene expression data and lower levels of sphingomyelins in MDA-MB-231 cells (p < 0.05), SMS2  and lower levels of SMPD4  as compared to BT-474 cells  increase in the expression of ASAH1 as expected from gene expression data, whereas there was no significant change in ASAH2 protein expression (p < 0.05), SPHK1 , and SPHK2  proteins in MDA-MB-231 cells as compared to BT-474 cells (p < 0.01) increase in UGCG expression in MDA-MB-231 cells in comparison to BT-474 cells, but there was a >7.5-fold decrease (p < 0.05) in the expression of B4GALT6 (p < 0.05) and low expression of GLB1  in MDA-MB-231 cells as compared to BT-474 cells  proliferation at 72 h as compared to MDA-MB-231 cells (p < 0.001) increase in migration of MDA-MB-231 cells as compared to BT-474 cells  were plated in 96-well plate, and proliferation index was determined by MTT assay. We observed that BT-474 cells exhibit >2.5-fold higher  of BT-474 cells after 72 h (p < 0.05) decrease in the number of colonies in BT-474 cells (p < 0.001) in cell proliferation (p < 0.001) (p < 0.0001) on knockdown of CERK by siRNA in MDA-MB-231 cells  E, and ce31 cells F.p < 0.05) decrease in cell proliferation (p < 0.01) decrease in number of colonies over untreated cells (p < 0.0001) decrease in cell proliferation of MDA-MB-231 cells on treatment with 1.0 µM of NVP-231 (p < 0.0001) decrease in number of colonies (p < 0.0001) decrease in number of migrated cells  decrease in tumor volume on final day (p < 0.0001) decrease in tumor volume on final day  decrease in tumor volume with NVP-231-Gel treatment on the final day as compared to untreated tumors (p < 0.05) decrease in volume of MDA-MB-231 tumors on final day , and worst prognosis, shorter survival and higher recurrence rate were positively associated to higher CERK expression among ER-negative cancers ,43. CERKPolymeric and lipid nanoparticles have emerged as effective delivery vehicles for gene therapeutics . In our CERK appears to be playing different roles in more aggressive metastatic breast cancer cell lines versus non-metastatic cell lines targeting cell migration and cell proliferation in a context-dependent manner. Hence, CERK can be a potential target for multiple breast cancer subtypes. However, more studies elucidating the molecular mechanism by which CERK controls these phenotypes and the extent of overlap in the pathways regulating them needs to be performed.


Infections caused by multidrug-resistant (MDR) bacteria are an increasingly common public health threat associated with worse outcomes in immunocompromised patients. Eravacycline (ERV) has potent in-vitro activity against MDR Gram-negative and Gram-positive bacteria and has demonstrated non-inferiority to meropenem in the phase III IGNITE4 trial; however, the trial excluded immunocompromised patients. We aimed to evaluate clinical and safety endpoints of immunocompromised patients receiving ERV as definitive therapy.Multicenter, retrospective, observational study conducted from October 2018 to April 2022. Adult hospitalized immunocompromised patients treated with ERV for ≥72 hours were included. Immunocompromised patients were defined as having any of the following: chemo or radiation therapy < 30 days of hospital admission, HIV/AIDS with CD4 < 200, chronic steroids  age was 62 (53-70) and 61.6% were male. Hospital length of stay was 28 (13-42) days and 67% were admitted to the intensive care unit. SOFA and APACHE II scores were 3.5 (1-7) and 16 (11-20), respectively. Common infection sources were intra-abdominal (26%) and lower respiratory tract (18%); 24% were bacteremic. Most patients had cultured Enterobacterales (58.7%) and Enterococci (37%) spp. infections. Of those, 21.3% were CRE and 19% were VRE. Infectious diseases consult was obtained in 91.8% of cases. Time elapsed from index culture collection to ERV initiation was 4 (2-8) days and duration of ERV therapy was 7 (4-12) days. In total, 81.3% of immunocompromised patients achieved 30-day survival and 90.7% did not have 30-day infection recurrence. Probable drug-related adverse events occurred in 5.3% of patients .A majority of immunocompromised patients receiving ERV as definitive therapy achieved 30-day survival and did not experience infection recurrence. ERV use in immunocompromised subpopulations will benefit from studies tailored to their specific characteristics.Kimberly C. Claeys, PharmD, BioFire Diagnostics: Honoraria Bruce M. Jones, Pharm.D., FIDSA, BCPS, AbbVie: Advisor/Consultant|AbbVie: Honoraria|La Jolla: Honoraria|Melinta: Advisor/Consultant|Paratek: Honoraria|Regeneron: Honoraria.


Respiratory syncytial virus (RSV) is a public health burden; no vaccine is currently available. An mRNA-based RSV vaccine (mRNA-1345) encoding the RSV prefusion stabilized F (preF) glycoprotein is under clinical investigation.A phase 1, randomized, observer-blind, placebo-controlled, dose-ranging study assessed safety and immunogenicity of mRNA-1345 in younger adults  and older adults  (NCT04528719). YA and OA were randomized to receive 1 dose of mRNA-1345  or placebo.In all, 74 YA participants  and 202 OA participants  received study injections. mRNA-1345 was well-tolerated in both groups, with lower reactogenicity observed in OA vs YA at higher doses. Injection site pain was the most frequent local solicited adverse reaction . Erythema and swelling were less frequent . Overall, 57.9-100% (YA) and 53.2-78.7% (OA) of mRNA-1345 and 40.0% (YA) and 45.5% (OA) of placebo groups reported ≥ 1 systemic SAR, most commonly headache, fatigue, myalgia, and arthralgia. As expected, neutralizing antibodies (nAbs) were present at baseline ; mRNA-1345 significantly boosted antibody titers through month (M) 1 in YA and OA, with comparable immunogenicity observed across age groups. M1 geometric mean fold rise (GMFR) for RSV-A nAbs were 20.0-22.3 (YA) and 12.1-16.6 (OA) and for RSV-B, nAbs were 11.7-14.4 (YA) and 8.7-12.6 (OA). M1 PreF binding antibody (bAb) GMFRs were 16.1-21.7 (YA) and 8.4-12.1 . Peak antibody titers declined through M6, but levels remained ≥ 4.1-fold above BL with minimal dose response. M6 GMFR for RSV-A nAbs were 7.0-9.6 (YA) and 4.1-5.8 (OA) and for RSV-B, nAbs were 5.0-8.9 (YA) and 4.5-5.5 (OA). M6 PreF bAbs GMFR were 5.9-7.0 (YA) and 4.1-4.7 (OA). Antibody decline over time was comparable in YA and OA cohorts.mRNA-1345 is well-tolerated in YA and OA. Antibody levels were boosted substantially above BL through M6 in both cohorts. These data support the continued development of mRNA-1345 as an RSV vaccine.Grace L. Chen, MD, MPH, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Runa Mithani, PharmD, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Archana Kapoor, PhD, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Sophia Lu, PhD, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Laila El Asmar, PhD, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Catherine A. Panozzo, PhD, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Christine A. Shaw, PhD, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Sonia K. Stoszek, PhD, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds Allison August, MD, Moderna, Inc.: Salary|Moderna, Inc.: Stocks/Bonds.


Correction: Environ Health 20, 44 (2021)https://doi.org/10.1186/s12940-021-00726-xFollowing the publication of the original article , the autCV70  ultrasound imaging system using a linear array 7.5 MHz transducer .In brief, high resolution B-mode ultrasound carotid artery images were acquired using a Siemens Acuson However, the correct information should state:X 300  ultrasound imaging system using a linear arrayVF 10-5 (8.0 MHz) transducer .In brief, high resolution B-mode ultrasound carotid artery images were acquired using a Siemens Acuson


Brassica species, and they play important roles in defense due to their anti-nutritive and toxic properties. Here, we conducted a genome-wide association study of six glucosinolate metabolites (mGWAS) in rapeseed, including three aliphatic glucosinolates , one aromatic glucosinolate (m157 gluconasturtiin) and two indole glucosinolates (m165 indolylmethyl glucosinolate and m172 4-hydroxyglucobrassicin), respectively. We identified 113 candidate intervals significantly associated with these six glucosinolate metabolites. In the genomic regions linked to the mGWAS peaks, 187 candidate genes involved in glucosinolate biosynthesis  and novel genes  were predicted based on the mGWAS, combined with analysis of differentially expressed genes. Our results provide insight into the genetic basis of glucosinolate biosynthesis in rapeseed and should facilitate marker-based breeding for improved seed quality in Brassica species.Glucosinolates (GSLs) are secondary plant metabolites that are enriched in rapeseed and related  Brassica species, providing these plants with their pungent odor [Glucosinolates (GSLs) are secondary metabolites comprising sulfur and nitrogen that are specially produced in ent odor ,2,3. GSLent odor ,5. Howevent odor ,7,8,9,10β-D-thiosaccharide and (Z)-n-hydroxamic sulfate, but have variable R-side chain groups due to the precursor amino acids [γ-glutamyl peptide (GGP1) and C-S lyase (Super root 1 (SUR1)), and then to the GSL core structure by glucosyltransferases (UGT74s) and sulfotransferases (SOTs) [O-methyltransferases (IGMTs) for indole GSLs [Generally, GSLs share the same basic structure, including no acids . The lenno acids ,13,14. Tno acids ,16, whicno acids ,17,18,19s (SOTs) ,22,23,24s (SOTs) . The corole GSLs ,27,28.Arabidopsis, the glucosinolate biosynthesis pathway is one of the best characterized specialized metabolite pathways [AtTSB1, AtCYP79B2, AtCYP79B3, AtCYP83B1 and AtST5a and so on), leading to the accumulation of GSLs [MYB51 and the GSL biosynthesis gene CYP83B1 to regulate the de novo biosynthesis of indole GSLs, but also regulates the expression of genes involved in side chain modification , thereby increasing the biosynthesis of indole GSLs [In pathways . Progrespathways ,32,33,34 of GSLs . The ali of GSLs ,37, whil of GSLs ,39. Seve of GSLs . Further of GSLs ,41. A reole GSLs .Brassica napus L.) is used worldwide as an oil crop and source of edible vegetable oil and feed meal. Therefore, breeding rapeseed varieties with low GSL levels in seeds is an important breeding goal. To date, many rapeseed resources with low seed GSL contents have been developed in polyploid rapeseed [B. napus seeds has been challenging. In this study, we performed a genome-wide association study for six glucosinolate metabolites in 143 rapeseed accessions using 239,945 SNP markers obtained by resequencing [B. napus with improved quality.Rapeseed , whereas the level of m172 showed a higher correlation with those of three aliphatic GSLs  than with that of m165 (Based on ultrahigh-performance liquid chromatography-heated electrospray ionization-tandem mass spectrometry (UPLC-HESI-MS/MS) analysis, we obtained six glucosinolate metabolites with high content in rapeseed at 35 days after flowering (DAF), including three aliphatic GSLs , one aromatic GSL (m157 gluconasturtiin) and two indole GSLs (m165 indolylmethyl-glucosinolate and m172 4-hydroxyglucobrassicin) a,b. Corr of m165 , indicat of m165 . The coe of m165 . Further of m165 , indicat of m165 . Therefo of m165 a, but al of m165 b.p-value < 4.17 × 10−6. The results of mGWAS for the six glucosinolate metabolites in two years (2017cq and 2018cq) and BLUP (best linear unbiased prediction) values  values  are summB. napus genome  in two years and BLUP values. In addition, qGSL-A01-4, qGSL-A02-2, qGSL-A03-3, qGSL-A05-2, qGSL-A06-1, qGSL-A06-3, qGSL-A09-6, qGSL-A09-7, qGSL-C04-2 and qGSL-C08-5 were repeatedly detected in at least one year or BLUP value. In these repeatedly detected interval regions, most of them were simultaneously associated with three glucosinolate metabolites  or any two of these  [Based on the physical positions of the significant SNPs, we mapped the intervals to the corresponding chromosomes of the reference genome h (v4.1)  and searBased on the known GSL biosynthesis pathway, we identified 64 candidate genes in the GSL biosynthesis pathway (including genes encoding enzymes in this pathway and their homologs) among the intervals, which showed differential expression during seed development in BnHG vs. BnLG . γ-glutamyl peptidase 1 (GGP1), S-alkyl thiohydrogen oxidase lyase (SUR1), sulfotransferases , flavin-monooxygenase glucosinolate S-oxygenase 5 (FMOGS-OX5), oxoglutarate-dependent dioxygenase (AOP3) and indole-glucoside O-methyltransferases , were identified on the following chromosomes: A01 , A02 , A03 , A04 , A05 , A06 , A07 , A08 , A09 , C02 , C04 , C06  and C07 , respectively , methylthioalkyl malate synthase (MAM1), bile acid transporter (BAT5), cytochrome P450s , UDP-glucosyl transferases , glutathione S-transferases , ectively , indicatMYB34, MYB51, MYB122 and WRKY33, which are involved in GSL biosynthesis [B. napus.Previously studies have identified MYB transcription factors that regulate GSL biosynthesis in various plants ,38,50,69ynthesis ,70, wereβ-glucosidases (BGLUs), one ABC transporter family protein (PEN3) and six glucosidolate glucohydrolases (TGGs), respectively , three nitrile specifier proteins (NSPs), one glutathione gamma-glutamylcysteine transferase (PCS1), fifteen ectively . Correspectively . Among tectively .IMD1, MAM1, IGMTs, MYB34, MYB51, MYB122 and so on) and novel genes  were identified within the confidence intervals of GSLs. Our findings not only demonstrate the reliability of the association genetics approach, but also provide new insight into elucidating the biosynthesis of these GSLs in B. napus seeds.Altogether, numerous key homologous genes, including known genes  and BnaIMD1 (BnaA02g02020D), which are located in candidate intervals qGSL-A02-1 and qGSL-C02-11, associated with three aliphogenic GSLs and one aromatic GSL  and BanGS-OH (BnaA04g17900D), which participate in the side chain modification process of aliphatic GSL biosynthesis and showed higher expression profiles in BnHG vs. BnLG (CYP79 genes [CYP83 genes [GGP1 [SUR1 [UGT74 genes [SOT genes [BnaCYP79F1 (BnaA06g11010D), BnaCYP79B2 (BnaA08g16100D), BnaCYP83A1 (BnaA04g24160D and BnaC04g47910D), BnaGGP1 , BnaSUR1 (BnaA09g10030D), BnaUGT74B1 (BnaA09g29790D), BnaUGT74C1 (BnaC04g42530D), BnaSOT16 (BnaA07g31260D), BnaSOT17 (BnaA06g12720D) and BnaSOT18  , NSP genes (3), CAD1 (1), TGG genes (6) and NIT genes (3) (BnaSOT12 (BnaA02g27300D) is homologous to AtSOT12, which functions in flavonoid, brassinosteroid and salicylic acid activity, and is involved in plant responses to salt, osmotic stress and phytohormones [BnCYP81D11 (BnaA02g29380D and BnaA02g29390D), BnaCYP81D7 (BnaA03g23030D), BnaCYP81G1 , BnaCYP81F3 (BnaA08g15650D), BnaCYP81F4 (BnaA08g15660D) and BnaCYP81K1 (BnaC02g36740D), are located in the interval regions associated with glucosinolate metabolites and showed differential expression in BnHG vs. BnLG seeds  . In addienes (3) , which aBnaA08g16D, BnaCYP BnaA06g10D, BnaCYLG seeds . Among tynthesis ,103, butLG seeds . SimilarCYP81F2, IGMT1 and IGMT2 [A. thaliana [B. napus  biosynthesis, specifically the production of 4-methoxyindole-3-ylmethyl glucosinolate (4MI3G), by directly activating the expression of nd IGMT2 , while tnd IGMT2 ,70,105. thaliana . In thisB. napus . Our finB. napus seeds , 46 medium-glucosinolate content (45~100 μmol/g) and 27 high-glucosinolate content (>100 μmol/g), respectively  and low  seed glucosinolate content were selected from 143 B. napus accessions for transcriptome sequencing (RNA-Seq), respectively. The seeds of 20, 30 and 40 DAF were also collected and pooled from five or more individuals in BnHG and BnLG plants. All samples were stored at −80 °C until further analysis.In total, 143 ectively . All accThe total RNA was extracted from the seeds of 20, 30 and 40 DAF using an EZ-10 DNAaway RNA Mini-Preps kit  following the manufacturer instructions. Then, the qualified RNA samples were used for libraries construction and sequenced on Illumina Hiseq 2000 platform with 150 bp paired-end reads . The gene expression profiles were evaluated using FPKM (fragments per kilo base of exon model per million) values. Genes were considered as differentially expressed genes (DEGs) with a minimum 2-fold difference in expression (|log2FC| ≥ 1). g at 4 °C for 10 min. Then, the residues were repeatedly extracted. Eventually, the mixed liquid supernatants were used for UPLC-HESI-MS/MS analysis after being filtered by a 0.22 μm nylon filter. All experiments were performed in at least three replicates for each accession.The raw metabolites were extracted from fresh seeds described in our previous research , with miThe UPLC-HESI-MS/MS was performed using Dionex UltiMateTM 3000 UHPLC system  coupled to a Thermo Scientifific Q-Exactive System equipped with an S-Lens ionizer source , including the precolumn  and Acquity UPLCBEH C18 column . The parameters were as follows: the mobile phases A (0.1% formic acid) and B (0.1% acetonitrile aqueous solution), 37 °C column temperature, 0.3 mL/min flow rate and 10 μL injection volume, respectively. The mobile phase gradients are 0–2 min, 5% B–10% B; 2–10 min, 10–25% B; 10–13 min, 25–95% B; 13–16 min, 95% B; 16–16.5 min, 95–5% B; 16.5–21 min, 5% B. The mass spectrometry was detected in negative ion mode with a scanning range of 100 to 1200 (m/z), 3.5 kV ion source voltage, 350 °C capillary temperature, 35 sheath gas, 10 auxiliary gas and 0 backblow air, respectively.http://prime.psc.riken.jp/compms/msdial/main.htmL#MSP, accessed on 2 April 2022), and automatically converted by ABF  converter  [−1 . Raw data of UPLC-HESI-MS/MS were firstly treated with MS-DAIL ver4.1 MSP negative database (ch 2020) . Correspch 2020) ,110. Furch 2020) , and thehttp://www.eXtension.org/pages/61006, accessed on 14 October 2020), respectively. The content of six glucosinolate metabolites in the 2017 and 2018 growing seasons and resulting values of BLUP were used as phenotypes for GWAS, respectively. The heritability was calculated by using the multi-year repeated model. In addition, the quantitative data of glucosinolate metabolites were divided into 10 grades, and Shannon–Wiener Diversity Index was used to calculate metabolites [t-test) were performed [The glucosinolate metabolites were detected in two consecutive years (2017 and 2018) with replications; we further obtained the best linear unbiased prediction (BLUP) of six glucosinolate metabolites per accession using a linear model using an R script  and GATK (version 3.2) to process local realignment and base quality detection for the alignment results, sequentially. Further, we then used AMtools mpileup (version 0.1.19–44428cd) and GATK to perform SNP calling. In total, 239,945 high-quality SNPs with a minor allele frequency (MAF) <5% were used for further analysis. The six models, including naïve, Q, K, PCA, K + Q and K + PCA model, were applied to determine the statistical associations between phenotypes and genotypes. Quantile–quantile (QQ) plots were used for false-positive correction for association analyses. In this study, genome-wide association analysis for six glucosinolate metabolites was carried out using the GLM with Q model and MLM with Q + K model by TASSEL 5.2.1 software. The population structure Q matrix was completed by admixture_linux-1.3.0 software [P  <  P  =  1/N , and the threshold of significance was set to p < 4.17 × 10−6.Detailed methods used for SNP genotyping and mapping were previously described ,47,48. I-bzh’  using thsoftware . The sigbzh reference genome  [The significant interval regions were mapped to the Darmor-er 2022) , which wer 2022) . Finally


Correction: J Exp Clin Cancer Res 38, 435 (2019)https://doi.org/10.1186/s13046-019-1439-xFigure Figure Figure Figure Following publication of the original article , errors The corrections do not have any effect on the final conclusions of the paper.Additional file 1: Figure S1. JMJD2C promoted the metastasis of CRC LoVo cells. a-c Real time PCR and western blotting were performed to confirm the gene silencing and overexpressing efficiency for JMJD2C. LoVo was transiently transfected with shRNA/NT vector, shRNA/JMJD2C vector, empty overexpression vector, or JMJD2C overexpression vector. d Migration assays of LoVo cells transfected with shRNA/NT, shRNA/ JMJD2C, empty vector, or JMJD2C overexpression vector, respectively. e Numbers of migrated cells are shown as mean ± SD; n = 3. *, P < 0.05; **, P < 0.01 (t test).


Platythyreaclypeata species group is reviewed and three species, including one new species, P.homasawinisp. nov., are recognized. This species group is distinguished from the P.parallela species group by the reddish-brown body, the elliptical shape of the propodeal spiracle, the elongate antennal scape, and the distinctly narrowed posteriad space between frontal carinae. Platythyreahomasawinisp. nov., from Thailand and China, is described based on the worker caste. The type series of the new species was collected on the forest floor from dead wood in an advanced stage of decomposition. A key to the Oriental species of the genus Platythyrea based on the worker caste is provided.The  Platythyrea Roger, 1863  is a rarely collected group with recent reviews listing 39 species ; P.quadridenta Donisthorpe, 1941; P.tricuspidata Emery, 1900 and P.janyai Phengsi, Jaitrong, Ruangsittichai & Khachonpisitsak, 2018). Among them, five species are reported from Thailand, and they belong to two species groups (sensu Brown 1975): the P.clypeata group (P.clypeata and P.janyai) and P.parallela group   . Our recTHNHM). The specimens were compared with high-resolution images of holotypes and paratypes of closely related species  available on P.janyai were also examined. The holotype and paratypes of P.homasawini sp. nov. are deposited in THNHM.This study is mainly based on the material deposited in the Natural History Museum of the National Science Museum, Thailand  process vertically to a line intersecting the dorsal most point of the node.PL Petiole length: length of petiole measured in lateral view from the anterior articulation to the posterior articulation of petiole.PW Pronotal width: maximum width of pronotum measured in dorsal view.SL Scape length: straight-line length of the antennal scape, excluding the basal constriction or neck.SI Scape index = SL/HW ×100.TL Total length: total outstretched length of the individual, from the mandibular apex to the gastral apex.Abbreviations of the type depositories are as follows:BMNHThe Natural History Museum, London, U.K.MHNGMuséum d’Histoire Naturelle, Geneva, Switzerland.THNHM Natural History Museum of the National Science Museum, Thailand.Platythyrea species were made at the Microscopic Center, Faculty of Science, Burapha University with a LEO 1450 VP scanning electron microscope on gold coated specimens.Scanning electron microscope images of The species group can be characterized by the following characteristics: 1) body reddish brown; 2) space between frontal carinae distinctly narrowed posteriad , deposited in MHNG . Holotype of P.thwaitesi: alate queen from Sri Lanka, deposited in BMNH .THNHM-I-24983) and 1 dealate queen (THNHM-I-24982), Laos, Vientiane, Pak Ngum District, Ban Phang Dang, ca 300 m alt., 14.VI.2010, W. Jaitrong leg., Colony no. WJT-LAO-143 (THNHM-I-24981) • 1 worker (THNHM-I-24984), Laos, Vientiane, Pak Ngum District, Ban Phang Dang, 14.VI.2010, Sk. Yamane leg., Colony no. LA10-SKY-128 • 1 worker (THNHM-I-24981) Laos, Vientiane, Pak Ngum District, Ban Phang Dang, ca 300 m alt., 12.VI.2010, W. Jaitrong leg. – Thailand • 9 workers (THNHM-I-02423 to THNHM-I-02431), eastern Thailand, Chachoengsao Province, Tha Takiab District, Khao Ang Reu Nai Wildlife Sanctuary, 27.IX.2002, W. Jaitrong leg., Colony no. WJT270902-01 • 3 workers (THNHM-I-02432 to THNHM-I-02434), same locality, date and collector, Colony no. WJT270902-1 • 6 workers (THNHM-I-02435 to THNHM-I-02440), eastern Thailand, Sa Kaeo Province, Khao Ang Reu Nei Wildlife Sanctuary, 26.VI.2003, W. Jaitrong leg., Colony no. WJT03-TH-228; 22 workers (THNHM-I-02441 to THNHM-I-02452) and 1 male (THNHM-I-02453), eastern Thailand, Chanthaburi Province, Soi Dao District, 14.V.2008, W. Jaitrong leg., Colony no. WJT08-E065 • 14 workers  and 1 dealate queen, central Thailand, Nakhon Nayok Prov., Muang Dist., Nang Rong Temple, in rotting wood, 29.VIII.2018, W. Jaitrong leg., Colony no. WJT290819-09.Laos • 10 workers (n = 10). and inhabits primary dry evergreen forest and disturbed forests. All colonies of this species were collected from dead wood on the forest floor in an advanced stage of decomposition.Sri Lanka, Vietnam, Laos (Vientiane), and Thailand  Fig. .Platythyreaclypeata is similar to P.gracillima, P.janyai, and P.prizo. However, P.clypeata can be easily separated from P.gracillima by the following characteristics (characters of P.gracillima in parentheses unless otherwise stated): 1) body size smaller ; 2) eye smaller and flat ; 3) clypeus narrow and rather convex (clypeus broad and rather flat); 4) declivity of propodeum concave ; 5) seen from back propodeal declivity rounded above ; 6) petiole laterally swollen ; 7) head finely punctate (head rather smooth).Platythyreaclypeata can be distinguished from P.prizo by the following characteristics (characters of P.prizo in parentheses unless otherwise stated): 1) head shorter ; 2) eye flat and small ; 3) eye without erect pubescence (eye covered with extremely fine short erect pubescence); 4) propodeum junction obtusely angulated (propodeum junction armed with a pair of short teeth or tubercles); 5) in profile, posterodorsal corner of petiole forming an acute angle (roundly convex).P.janyai (characters of P.janyai in parentheses unless otherwise stated) by 1) head relatively longer ; 2) eye clearly smaller ; 3) eye flat (eye convex); 4) head finely punctate with dense shallow foveae ; 5) in profile petiole slightly longer than high and in dorsal view its node slightly narrower posteriorly ; 6) ventral outline of petiole feebly concave (weakly convex) , northern Thailand, Chiang Mai Prov., Doi Saket Dist., Ban Mae Pong, 24.XI.2021, K. Homasawin leg., Colony No. WJT241121-01. Paratypes: 29 workers , same data as holotype.THNHM-I-02463), northern Thailand, Chiang Mai Prov., Muang Dist., restored forest, 8.V.2002, S. Sonthichai leg. • 1 worker (THNHM-I-02454), western Thailand, Kanchanaburi Prov., Thong Pha Phum N.P., Natural Forest, 8.III.2005, W. Sakchooeong leg.China • 1 worker, Yunnan Prov., Menghai County, Meng’a Town, Papo Village, 1280 m, No. A97-2318, collected from secondary monsoon evergreen broadleaf forest, 10.IX.1997, Zhenghui Xu leg. – Thailand • 1 worker . TL 7.72–7.80, HL 1.56–1.64, HW 1.08–1.12, SL 1.64–1.68, EL 0.16–0.20, PW 0.96–0.99, ML 2.80–2.84, PL 1.08–1.12, PH 0.60–0.64, DPW 0.40–0.44, CI 68–69, EI 15–19, SI 150–159.holotype and paratypes). Head and Thailand (Chiang Mai and Kanchanaburi provinces) Fig. .The specific name is dedicated to Mr Kaisihanat Homasawin, who donated the type series.P.janyai but it can be separated from P.janyai by the following characteristics (characters of P.janyai in parentheses unless otherwise stated): 1) head weakly widening anteriorly (head not widening anteriorly); 2) dorsal outline of petiole weakly convex ; 3) posterior margin of petiolar node without a concavity in the middle (posterior margin of petiolar node with a concavity in the middle): 4) body surface with thin pubescence (body surface with thick pubescence); 5) head longer ; 6) antennal scape relatively long, 1/3 of its length extending beyond posterolateral corner of head ; 7) eye smaller and flat ; 8) seen from back propodeal declivity rounded above ; 9) dorsal outline of petiole weakly convex .The new species is similar to Platythyreahomasawini can be easily separated from P.clypeata by the following characteristics (characters of P.clypeata in parentheses unless otherwise stated): 1) head in full-face view, posterior margin weakly concave ; 2) antennal scape relatively long, 1/3 of its length extending beyond posterolateral corner of head ; 3) clypeus broad (clypeus narrow); 4) eye larger ; 5) mesosoma relative longer ; 6) mesopleuron not demarcated from mesonotum ; 7) in profile view, propodeal junction roundly convex ; 8) in dorsal view, posterior margin of petiole clearly convex ; 9) lateral face of head entirely micropunctate ; 10) gaster superficially shagreened (finely reticulate).Platythyreahomasawini can be distinguished from P.gracillima by the following characteristics (characters of P.gracillima in parentheses unless otherwise stated): 1) clypeus roundly convex (rather flat in P.gracillima); 2) petiole laterally convex; 3) seen from above longer than high ; 4) seen from above a little more than twice as long as broad); 5) mandible finely micropunctate (mandible finely and densely punctate); 6) head entirely finely micropunctate (rather smooth in P.gracillima).Taxon classificationAnimaliaHymenopteraFormicidaePhengsi, Jaitrong, Ruangsittichai & Khachonpisitsak, 201880F1B0B2-8F43-5645-B08A-D5F34E0D516DPlatythyreajanyaiHolotype (THNHM-I-02392) and three paratypes (THNHM-I-02393 to THNHM-I-02395) workers, southern Thailand, Phatthalung Province, Si Banphot District, Riang Thong Waterfall, Khao Pu Khao Ya National Park, 28.IX.2007, W. Jaitrong leg., Colony no. WJT07-TH-2060 (THNHM-I-02392), deposited in THNHM (examined).THNHM-I-02465) – Thailand • 2 workers, southern Thailand, Trang Province, Na Yong District, Khao Chong Botanical Garden, 7.XI.2014, W. Jaitrong leg., Colony No WJT071114-2 (THNHM-I-02421 to THNHM-I-02422) • 5 workers, same locality and collector, 26.XII.2018, Colony no. WJT261218-1 (THNHM).Malaysia • 1 worker, western Malaysia, Selangor, Ulu Gombak, 22.III.2013, F. Ito leg. ((n = 4).TL 6.63–6.96, HL 1.42–1.45, HW 1.06, SL 1.39–1.42, EL 0.20, ML 2.21–2.31, PL 0.73–0.79, PH 0.53, DPW 0.40, CI 72–74, EI 18, SI 131–134.holotype and paratypes). Head and Malaysia  Fig. .Platythyreajanyai is similar to P.clypeata, P.homasawini and P.gracillima. However, P.janyai can be easily separated from P.gracillima by the following characteristics (characters of P.gracillima in parentheses unless otherwise stated): 1) body size smaller ; 2) eye relatively smaller ; 3) seen from back, propodeal declivity tapering above ; 4) petiole laterally convex, seen from above longer than broad . For differentiation of P.janyai and P.clypeata, see “Comparative notes” under P.clypeata, and of P.janyai and P.homasawini, see under P.homasawini.Platythyrea are now known around the world. Among them, six species are found in Thailand, and they belong to two species groups : P.clypeata group  and P.parallela group .With this study, 40 valid species of the genus The shape of the frontal lobe, mandibular shape and dentition, and shape of mesosoma and petiolar node were characters used by Brown (1975) to distinguish the two species groups mentioned here. These morphological characters were confirmed and used by several authors who described new species after Brown (1975) . The preP.clypeata group can be distinguished from other congeners mainly by the following: body reddish brown; space between frontal carinae distinctly narrowed posteriad; propodeal spiracle opening elliptical, petiole longer than high, and posterior margin concave. The worker caste of Platythyrea species is generally monomorphic with little variation in size within species. Thus, worker body size can be used for separating large and small species. In this study, worker body size has been used to separate P.homasawini sp. nov., P.janyai, and P.clypeata.The Platythyreaclypeata is distinctly allopatric with P.janyai in geographic subregion in continental Asia . However, P.clypeata and P.janyai both were found in lowland (< 300 m a.s.l.). Platythyreaclypeata inhabits various habitats including plantations, secondary forests, and primary dry evergreen forests. Platythyreajanyai is confined to primary evergreen forests. Platythyreahomasawini sp. nov. is restricted to highland hill evergreen forests.tal Asia . It has


Cystobasidium ongulense has been reported from East Ongul Island near Syowa Station, East Antarctica, as a new basidiomycetous yeast species. This species has cold active lipases and cellulases that are active even at subzero temperatures. We report draft genome sequences of five Cystobasidium ongulense strains isolated from East Antarctica. Cystobasidium ongulense was isolated from East Ongul Island, East Antarctica, and reported as a new species of basidiomycete yeast  isolated from East Ongul Island and three strains  isolated from Inhovde, near Syowa Station.g for 5 min at 4°C. The cell pellets were washed with sterile distilled water and precipitated by centrifugation again. The cell pellets were dried using a vacuum freeze dryer. The lyophilized cells were powdered in a mortar and used for genome extraction. The genomic DNA of five Cystobasidium ongulense strains was extracted and purified using a NucleoBond AXG100 column with a Nucleo buffer set III (TaKaRa Bio) according to the manufacturer’s protocol. The concentration and purification of the genomic DNA were determined using a NanoDrop spectrophotometer (Thermo Scientific) and the Qubit double-stranded DNA (dsDNA) broad-range (BR) assay kit (Thermo Scientific). Then, the genomic DNA was digested using the microTUBE (Covaris), and the genomic library was constructed using the Illumina TruSeq DNA PCR-free library preparation kit (Illumina). The quality of the library was confirmed using the Quant-iT PicoGreen dsDNA assay kit, and the library size was checked using the Agilent 2200 TapeStation system . The paired-end sequence reaction was performed on a HiSeq 2500 instrument (Illumina). Adapters and low-quality bases were trimmed using fastp ver. 0.12.4 (–ab initio gene predictions were carried out using AUGUSTUS ver. 3.4.0 (Each strain was cultured in yeast extract-peptone-dextrose (YPD) liquid medium (Difco) at 15°C for 5 days. The cells from cultures were collected by centrifugation at 3,500 × . 0.12.4 , annotat. 0.12.4 –11 automr. 3.4.0 . The resr. 3.4.0  and AGATr. 3.4.0 , Phobiusr. 3.4.0 , antiSMAr. 3.4.0 , and Eggr. 3.4.0  against r. 3.4.0 .C. ongulense strains is shown in A summary of the genomic analysis results of the five C. ongulense will help us understand how Antarctic fungi adapt to the extreme environment.We believe that the genomic information of PRJDB6568 with accession numbers BQUO00000000.1 (9A-2), BQUP00000000.1 (9A-5), BQUQ00000000.1 (051-20-1), BQUR00000000.1 (056-1-20Y-3), and BQUS00000000.1 (056-20-8). The raw reads are available under SRA accession numbers DRR118811, DRR118812 (9A-2), DRR118813, DRR118814 (9A-5), DRR118815, DRR118816 (051-20-1), DRR118817, DRR118818 (056-1-20Y-3), and DRR118819, DRR118820 (056-20-8).This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under BioProject accession number