Behavioral tests were conducted on day 15 and 30 postnicotine injection, while memory tests, brain neurotransmitters, antioxidants, and histopathological examination were examined at day 30 only. As a result, nicotine impaired rats' activity (hypoactivity and hyperactivity) and memory, induced anxiolytic and anxiogenic effects on rats, and altered neurotransmitters , and redox markers  levels in brain homogenates. Thickening and congestion of the meninges and degeneration of the cerebral neurons and glia cells were observed. Cosupplementation with NNG, vitamin E, and their combination with nicotine was beneficial in the alleviation of activity impairments and improved short memory and cognition defects and exploratory behaviors. Our results indicate the antioxidant potential of NNG and vitamin E by modulating redox markers and neurotransmitters in the brain. Thus, data suggest that the prophylactic use of NNG, vitamin E, and/or their combination for (45 days) may have a successful amelioration of the disrupted behavior and cognition and biochemical and histopathological alterations induced by nicotine.Nicotine is the major alkaloid present in cigarettes that induces various biochemical and behavioral changes. Nanonaringenin (NNG) and vitamin E are antioxidants that are reported to mitigate serious impairments caused by some toxins and oxidants. Thus, we aimed to investigate the efficacy of NNG, vitamin E, and their combinations to ameliorate behavioral, biochemical, and histological alterations induced by nicotine in rats. Adult male albino rats were randomly grouped into six equal groups (10 rats/group): control, N (nicotine 1 mg/kg b.w./day S/C from 15 The nACs remain , 5. Thess remain . In addis remain , 7 whichs remain . Therefos remain . Recentls remain . Flavonos remain –17. Amons remain . Naringes remain , anti-ins remain . Hence, s remain –28. Mores remain , 30.The larger surface area of nanoparticles per mass unit and their smaller size can lead to faster drug delivery and higher bioactivity compared to bulky particles –33. ThusTherefore, we aimed to evaluate the prophylactic role of NNG, vitamin E, and their combination in amelioration of impaired activity, anxiety, and learning induced by nicotine as well as biochemical and histological alterations posed by subcutaneous nicotine administration in rats.10H14N2)  and naringenin (C15H12O5)  were purchased from Glentham Life Sciences Ltd., England Unit 5 Ingoldmells Court Edinburgh Way, Corsham Wiltshire SN13 9XN, the United Kingdom. Vitamin E (dl-Alpha Tocopheryl Acetate) (C29H50O2) with item number 1770 was purchased from Puritan's Pride, INC Ronkonkoma, NY 11779, USA. The commercial diagnostic kits used for assaying of the reduced glutathione (GSH), malondialdehyde (MDA), catalase, hydrogen peroxide (H2O2), and acetylcholinesterase (AchE) were obtained from Biodiagnostic Company for Research Kits, Egypt. Enzyme-linked immunosorbent assay (ELISA) kits for rat dopamine  and rat 5-hydroxytryptamine (5-HT)  were provided by R&D System, USA. All other chemicals were of analytical grade.Nicotine hydrogen tartrate salt  was used for the characterization of NNG at the National Research Center, Dokki, Giza, Egypt. A droplet of a freshly prepared suspension was poured onto copper grids and left to dry in the air, then observed by high-resolution TEM. The TEM indicated that NNG droplets were nearly spherical crystal shaped with homogeneous nanometric size spreading .The present study was carried out on 60 adult male albino rats ranging between 120 and 150 g body weight. They were obtained from Helwan Farm of Laboratory Animals, Cairo, Egypt. Rats were acclimatized for 2 weeks, then they were housed in groups in metal cages under good ventilation and illumination conditions at room temperature (24°C ± 2°C), humidity (68%) under 12 hours light-dark cycle during the period of the experiment. Rats had free access to water and diet ad libitum. All experimental measures were performed in a strict guideline according to the recommendations for the care and use of laboratory animals and approved by the Institutional Animal Care and Use Committee at Beni-Suef University.n = 10/group).The rats were randomized into six equal groups .th–45th day) 5 days a week [50 of nicotine is 50 mg/kg for rats [The rats were injected subcutaneously with freshly prepared nicotine dissolved in physiological saline (1 mg/kg b.w./day) for 30 days (15s a week . This dos a week . The LD5for rats .The rats received NNG (25 mg/kg b.w./day) orally with a gastric tube for 45 days , 39.th–30th day. Nicotine was given an hour before NNG administration.The rats were given NNG (25 mg/kg b.w./day) for 45 days orally with a gastric tube, interrupted by nicotine injected subcutaneously (1 mg/kg b.w.) at the 15th–30th day. Nicotine was given an hour before vitamin E administration.The rats were given vitamin E dissolved in corn oil orally with a gastric tube for 45 days (200 mg/kg b.w./day) , interruth–30th day.The rats were given NNG (25 mg/kg b.w./day) and vitamin E (200 mg/kg b.w./day) orally with a gastric tube for 45 days interrupted by nicotine injected subcutaneously (1 mg/kg b.w.) at the 15The following behavioral tests were performed to evaluate the effect of the nicotine, NNG and/or vitamin E with nicotine injection on rats' activity and memory. Five rats per group were used. The tests were performed two hours after the nicotine injection.This test was conducted on days 15 and 30 after injection of nicotine. This test measured locomotion, exploration, and anxiety, according to Gould et al. . The appFor each rat, locomotion anxiety like behaviors , freezing (immobility) time, exploration  were measured.(1) Y-Maze Test. This test was conducted on day 30 of the experiment. The test is used to measure spatial short-term working memory by recording the spontaneous alternative behavior in the maze arms [aze arms . Accordiaze arms , Rasooliaze arms , and Balaze arms , arms we(2) Novel Object Recognition (NOR). The aim of this test is to evaluate the short-term memory and was performed at the end of the experiment. Novel object recognition test is used to evaluate learning and memory deficits in rats and mice depending on the natural behavior of a rat in interacting with novel objects more than familiar objects, which are known as recognition memory [n memory  accordinn memory , Leger en memory , and Limn memory . NOR was2O2 levels, and AchE activities. The third portion of brain tissue was kept at −80°C for ELISA assays of dopamine and serotonin.Twenty-four hours following the last dose, the animals were killed by cervical dislocation. Brain tissues were collected and washed with physiological saline (NaCl 0.9%), then divided into three parts. The first portion was preserved in a neutral buffered formalin solution for histopathological examination. The second portion was homogenized in 10 volume phosphate buffer saline (pH: 7) using a homogenizer , then centrifuged at 20,000 ×g for 15 minutes at 4°C. The supernatant was kept at −80°C for further biochemical assays of GSH, MDA, Catalase, H2O2 levels, and catalase activity according to the methods described by Satoh [The brain homogenate was used for the measurements of MDA, GSH, Hby Satoh , Beutlerby Satoh , and Aebby Satoh , respectby Satoh . Levels by Satoh  using raμm were cut and mounted on clear and dry glass slides. The obtained slides were stained with Hematoxylin and Eosin (H & E) and Bielschowsky's silver stain for histopathological examination using LEICA  connected to the light microscope using 10, 20, 40 objective lenses [Brain tissues were dissected carefully and washed with physiological saline (NaCl 0.9%), then immersed in neutral buffered formalin solution 10%. Tissue specimens of the brain were dehydrated in ethyl alcohol, cleared in xylol, impregnated in soft paraffin, and embedded in hard paraffin. Sections of 4–6 e lenses .p values below 0.05 were accepted for significance.The results were statistically analyzed by using Graph Pad In stat software . One-way analysis of variance (ANOVA) test followed by Tukey's post hoc test for experimental group comparison was used. The p < 0.05) in N and N + NNG groups compared to the control one. At day 30 of nicotine injection, a significant (p < 0.05) decrease was observed in N and N + E in comparison to the control group. On the contrary, on day 15, rearing showed a significant (p < 0.05) increase in the N group while a significant (p < 0.05) decrease was observed in N + NNG, N + E and N + NNG + E compared to the control and nicotine groups. At day 30 of nicotine injection, rearing was significantly (p < 0.05) reduced in N, N + NNG, and N + E compared to control. However, rats in N + NNG + E group performed a locomotor activity near to that of control group. In additions, nicotine significantly (p < 0.05) decreased the freezing time and immobility at day 15 of nicotine injection. However, NNG, N + NNG, N + E and N + NNG + E significantly (p < 0.05) prolonged the freezing time of rats at day 15 of nicotine injection. Moreover, N + NNG significantly (p < 0.05) increased the frequency and duration of rat's entrance into central squares at days 15 and 30 while N + NNG + E increased them significantly (p < 0.05) at day 15 only. Thus, the administration of nicotine-treated rats with NNG either alone in N + NNG group or in combination with vitamin E in N + NNG + E group could improve rats, exploratory behaviors.p < 0.05) decreased in N, N + NNG, and N + E groups, while it was moderately improved in the combination (N + NNG + E) group. DI was significantly (p < 0.05) decreased in the N group. The prophylactic administration of vitamin E and/or combination treatment with NNG and vitamin E showed an ameliorative effect on cognition by the restoration of the values to near normalcy.p < 0.05) in brain tissue of nicotine-treated rats compared with those in the control group. However, the concentration of dopamine significantly reduced (p < 0.05) in the nicotine group in comparison to the control group. 2O2 significantly increased (p < 0.05) in brain tissue homogenates of nicotine-treated rats compared with those in the control group. On the other hand, the prophylactic treatment with NNG and/or vitamin E parallel with nicotine administration showed an ameliorative effect by the restoration of these biomarkers' values to near normalcy. The supplementation of the nicotine-treated rats with either NNG or vitamin E induced the same degree of protection. In addition, NNG supplementation to normal rats did not alter these values in comparison to those in the control group, indicating no adverse effect of NNG on the brain.The influence of different treatments on the nicotine-induced histopathological alterations in brain tissue is presented in Figures 3Nicotine stimulates the release of different neurotransmitters such as acetylcholine, dopamine, norepinephrine, and serotonin (5-HT) by nAChRs activation . NicotinMonoamines are an important group of neuromodulators that are released onto spinal cord circuits and are critical for the expression of locomotion . DopaminThe obtained results showed that the NNG and vitamin E did not ameliorate the prominent reduction in rat's activity caused by nicotine at 15 days of the experiment, while their combination successfully relieved the nicotine effect on locomotion at day 30. This indicated that the ameliorative effect of vitamin E , 69 and Interestingly, NNG, vitamin E, and their combination prolonged immobility time at 15 days of the experiment. Tobacco was recorded to decrease vitamins C and E , 69. In The time rats spent freezing in the open field maze is an indicator of animal anxiety , 45. In The observed behaviors declared that nicotine was anxiolytic on day 15 (decreased freezing time) and anxiogenic (decreased rearing) on day 30 of the experiment. These results are supported by early reports , 74 provOur data revealed that NNG could ameliorate the anxiogenic effect of nicotine. Similarly, NNG alleviated anxiety impairment induced by deltamethrin in rats . This maThe obtained results showed that the administration of NNG alone or in combination with vitamin E could successfully mitigate the nicotine effect on activity. Meanwhile, NNG improved the exploratory behavior of rats. Likewise, NNG treated rats exhibited a long exploration time . ExploraOur findings indicated that the tested dose of nicotine impaired the working memory and cognition of rats. In the early studies, nicotine caused impairments in learning and memory . In addiOur data suggested that vitamin E had an ameliorative effect on the impaired cognition induced by nicotine. Meanwhile, a combination of vitamin E and NNG improved the cognition capability of rats. NNG could improve learning in experimental animals , 91. VitThe reported changes in brain monoamines (decrease DA and increase 5-HT) levels are consistent with Perez et al. , who fouα4β2 nAChRs on GABAergic neurons , which in turn leads to an increase in their firing and decreases dopaminergic activity [We suggest six explanations for decreased brain DA in the present study. The first is that prolonged exposure to nicotine (LTN) leads to increase nAChR desensitization that may alter the response of DA terminals to distinct firing patterns . The secactivity . The altactivity . Fourth,activity . Fifth, activity . Consequactivity . Sixth, activity . In addiactivity .The prophylactic treatment with NNG and/or vitamin E or a combination of both for 45 days showed an ameliorative effect as the values returned nearly to control. The supplementation of nicotine-treated rats with either NNG or vitamin E induced the same degree of protection. Similarly, Rahigude et al.  and Khaj2O2 concentrations that, in turn, utilize GSH and catalase enzyme resulting in a significant decrease in GSH content and catalase activity in the brain tissue of nicotine-treated group compared to the control group , where they act as potent antioxidants and protect nerve cells against oxidative damage , 133. Nal attack .As an anticholinesterase, NNG enhances memory inefficiency in diabetes  and cognIn the current study, the histopathological examinations support the biochemical analysis where the nicotine caused marked pathological changes in the cerebral cortex indicated by structural alterations, congestion of meningeal and cerebral blood vessels, prevascular edema, and degeneration of the cortical nerve cells and glial tissue. Cotreatment with oral NNG and/or vitamin E attenuates these changes in cerebral structure. Structural observations have confirmed the cerebroprotection nature of NNG and/or vitamin E on nicotine-induced oxidative lesions.Our findings suggested that nicotine may lead to oxidative stress and impairment in behavioral, biochemical, and histological variables. However, supplementation of nanonaringenin (NNG), vitamin E, and their combination for 45 days might exert successful amelioration of theses impairments as well as brain AchE, monoamines and redox markers alterations. Thus, NNG and vitamin E should be included in the diet to afford a protective effect against nicotine-induced cytotoxicity. Further studies are required to evaluate the efficacy of different doses of NNG and vitamin E to mitigate impairments induced by nicotine at different routes of administration and durations.


Suicide is a leading cause of death worldwide, presenting a serious public health problem. We aimed to investigate the biological basis of suicide completion using proteomics on postmortem brain tissue. Thirty-six postmortem brain samples (23 suicide completers and 13 controls) were collected. We evaluated the proteomic profile in the prefrontal cortex  using tandem mass tag-based quantification with liquid chromatography–tandem mass spectrometry. Bioinformatics tools were used to elucidate the biological mechanisms related to suicide. Subgroup analysis was conducted to identify common differentially expressed proteins among clinically different groups. Of 9801 proteins identified, 295 were differentially expressed between groups. Suicide completion samples were mostly enriched in the endocannabinoid and apoptotic pathways . Among the differentially expressed proteins, GSTT1 was identified as a potential biomarker among suicide completers with psychiatric disorders. Our findings suggest that the previously under-recognized endocannabinoid system and apoptotic processes are highly involved in suicide. Suicide is a leading cause of death worldwide. According to the World Health Organization, >800,000 people die by suicide every year . Among tOne of the biggest obstacles to identifying the biological basis of suicidal behavior is that most previous studies were conducted in living high-risk suicide patients , whose characteristics may differ from those of suicide completers , 17. ComAmong postmortem studies of suicide completers, candidate gene approaches predominated so that only important proteins in major pathways were indicated as biomarkers for suicide (i.e. brain-derived neurotrophic factor or receptor tyrosine kinase B ). HoweveThe current study aimed to identify alterations in the proteomic profile of the prefrontal cortex of the postmortem brain of subjects with suicidal behavior  using anPostmortem brain samples were obtained from the Seoul National University Department of Forensic Medicine. Suicide completers were defined as individuals whose cause of death was a self-inflicted injury as confirmed by a forensic expert during autopsy. The control group comprised brain samples obtained from victims of sudden death, whose cause of death was mostly cardiac, as confirmed by a forensic expert. The postmortem interval was calculated based on the estimated time of death and time of autopsy. A total of 36 brain samples were collected: 23 suicide completers and 13 sudden death controls. Fresh brain tissue samples from the dorsolateral prefrontal cortex  were dissected and stored at 80 °C until further use. The DLPFC was chosen considering both proven morphological differences of the part between suicide completers and controls  and Clinical information, including diagnostic estimation, was obtained from anonymized records, which contained the declaration of family, close friends, and police. Toxicological data were used to confirm this declaration. Toxicological screening, including medications and alcohol concentration, was performed using peripheral blood obtained at the time of autopsy. All information was primarily evaluated by forensic experts who participated in autopsies, as well as a psychiatrist, and the diagnosis was reviewed and confirmed by an independent experienced psychiatrist. Only samples with consensus diagnosis based on the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) criteria were included.This study protocol was approved by the Seoul National University Hospital Institutional Review Board (IRB No. 2004-216-1119)pH is one of the most widely used parameters in tissue quality measurements . Each brA total of 36 postmortem brain tissue samples were resected and subjected to a previously described sample preparation method, with some modifications , 37–39. g, 15 min, 20 °C) to remove SDS. The washed samples were incubated in 200 µL of 50 mM iodoacetamide in UA buffer at room temperature for 30 min to alkylate the reduced cysteine. After the UA buffer was exchanged with 40 mM TEAB, samples were digested with 0.1 µg/µL trypsin/Lys-C at a ratio of 1:100  for 18 h at 37 °C. To estimate the amount of peptides, tissue digestions were measured by a tryptophan fluorescence assay at 350 nm using an excitation wavelength of 295 nm [Protein digestion was performed using a combined approach including acetone precipitation and the filter-aided sample preparation procedure , 37, 38.Because of the limited number of tandem mass tag (TMT) channels, we decided to distribute the 36 samples in four TMT experimental sets. After all samples were randomly distributed, each experimental set had nine individual samples and one pooled sample Fig. . Prior tTMT-labeled samples were re-dissolved in 0.1% trifluoroacetic acid and desalted using an HLB OASIS column according to the manufacturer’s instructions. Consequently, high-pH reversed-phase peptide fractionation was performed using an Agilent 1260 bioinert HPLC  equipped with a 300 Extended-C18 column . Peptide samples were separated with a 60 min linear gradient from 5% to 40% ACN in 15 mM ammonium hydroxide at a flow rate of 0.4 mL/min. The sample was fractionated into 96 fractions, concatenated into 24 fractions with varying hydrophobicities to optimize coverage and liquid chromatography–tandem mass spectrometry (LC–MS/MS) run time. The fractions were lyophilized in a vacuum centrifuge and stored at −80 °C until LC–MS/MS analysis.m/z 200 with a mass range of 350–1700m/z. The automatic gain control (AGC) target value was 3 × 106 and the isolation window for MS/MS was 1.2m/z. The 15 most abundant ions were fragmented by higher-energy collisional dissociation with a normalized collision energy of 33 at a resolution of 35,000 at m/z 200. The AGC target value for MS/MS was 5 × 105. The maximum ion injection times for the survey and MS/MS scans were 30 and 120 ms, respectively. Dynamic exclusion was set at 40 s to prevent repeated sequencing.Peptide samples were analyzed using a Q-Exactive mass spectrometer equipped with an EASY-Spray ion source  coupled with an Easy-nano LC 1000 , following our established protocol , 37, 38.Homo sapiens database , which included an additional protein, chicken ovalbumin. The database search was conducted with the following parameters: full enzyme digestion using trypsin (after KR/−) up to two missed cleavages; a precursor ion mass tolerance of 20 ppm (monoisotopic mass); a fragment ion mass tolerance of 0.02 Da (monoisotopic mass); static modifications of 229.163 Da on lysine residues and peptide N-termini for TMT labeling and 57.02 Da on cysteine residues for carbamidomethylation; and dynamic modifications of 42.01 Da for protein N-term acetylation and 15.99 Da for methionine oxidation. In accordance with the established target-decoy search strategy [http://proteomecentral.proteomexchange.org) via the PRIDE partner repository, under identifier PXD029845 [The raw MS files were processed using Proteome Discoverer version 2.2 . The tandem mass spectra search was conducted using the SEQUEST-HT algorithm against the UniProt strategy , the seaXD029845 .U test for continuous variables and Pearson’s chi-square or Fisher’s exact test for categorical variables. Statistical analyses of proteomic data were performed based on normalized protein abundance using Perseus [t-test  was applied to identify significantly altered proteins. Normalized protein abundance was subjected to z-normalization followed by hierarchical clustering.The clinical variables of suicide completers and sudden death individuals were compared using the Mann–Whitney  Perseus . Initialp < 0.05, and FC > 1.2 were identified among subgroups of suicide attempters.To confirm whether the identified DEP was due to suicide itself or other related clinical factors, sub analyses were performed among suicide completers. Suicide completers were divided into groups according to history of psychiatric disorder, history of treatment with psychotropic medications, and alcohol intake. DEPs with p-values, which were then filtered at a cutoff value of 0.05. Ingenuity pathway analysis (IPA) was used for functional analysis . Fisher’s exact test (P < 0.05) was used in IPA to estimate the probability that a specific set of proteins was related to a certain pathway. The proteins included in each module were subjected to network analysis using IPA. Gene set enrichment analysis (GSEA) was performed using the GSEA software (GSEA version 4.1.0). The c5.go.bp.v7.4.symbols.gmt gene set was selected as the reference gene set. Weighted gene co-expression network analysis (WGCNA), a systems biology method to understand correlation patterns among genes across samples, was adopted to identify co-expressed proteins and modules associated with suicide and sudden death (Version 1.69 in R). The eigengene was then used to summarize modules, and the association between these modules and sample traits, such as clinical parameters, was investigated [Protein Gene Ontology (GO) was classified using the DAVID bioinformatics tool (version 6.8) . The GO stigated .p = 0.580). However, the sex ratio among suicide completers was nearly 1:1, while only men died of sudden death. The suicide method varied among suicide completers: 16 (69.6%) by drowning, 2 (8.7%) by jumping, 1 (4.3%) by crashing, and 4 (17.4%) by hanging. Among suicide completers, 12 (47.8%) had psychiatric disorders: 7 (30.4%) had schizophrenia, and 4 (17.4%) had major depressive disorder. The toxicological results showed that 13 (56.5%) subjects had not been taking psychotropic drugs, six were taking antipsychotics, and two were on antidepressants. Among suicide completers, 6 (26.1%) subjects drank alcohol at the time of suicide. The mean blood alcohol concentration of suicide victims was 0.092 (SD = 0.023), which did not differ from that of people who died of sudden death. There was no significant difference between groups in postmortem interval and measured pH  was performed between suicide completers and sudden death individuals. The results, including statistical significance, p-values, and fold-changes, are presented in Table Of the 9801 identified proteins, 8035 were quantifiable in individual brain tissue samples and were used for statistical analysis. To identify DEPs, Student’s According to the GO-enrichment analysis among 244 upregulated DEPs Fig. , regulatGO-enrichment analysis of the 51 downregulated DEPs resulted in similar pathways Fig. . CellulaSeveral highly enriched canonical pathways were identified with IPA, including the endocannabinoid developing neuron pathway, opioid signaling pathway, and endocannabinoid neuronal synapse pathway Fig. . Neurotrp-value are shown in Figs. In addition, the differentially expressed gene sets in suicide completers compared with controls detected by GSEA, their normalized enrichment score, and nominal β = 12, to construct the unsigned co-expression network (R2 = 0.7)  Fig. . A topol.7) Fig. . Subsequ.7) Fig. .Fig. 3WGThe dataset had two very large modules (blue and turquoise) and several smaller modules, indicating that the expression levels of numerous genes were strongly correlated Table . The bluThe top network using 393 proteins in the turquoise module having the largest correlation with the sudden death group was organismal injury and abnormalities; each protein in the network demonstrated high PS and MM values Fig. . The proIn subgroup analyses, certain proteins related to clinical characteristics  were identified among suicide completers Table . With reIn the current study, we identified protein profiles related to suicide and sudden death using postmortem brain tissue. Using bioinformatics tools, we discovered canonical pathways related to suicide completion, such as various neurotransmitter transmission signaling pathways, including endocannabinoid pathways. Through subgroup analyses, we found common proteins between suicide completion and other factors related to suicide such as psychiatric disorders, psychotropic use, and alcohol intake , 48.To confirm the reliability of our study results, comparative analysis with other studies was conducted. The most recent postmortem brain tissue study using a proteomic approach identified 5644 proteins using label-free proteomics acquisition through nano-flow liquid chromatography–electrospray tandem MS in five samples in the suicide and non-suicide group , while oAmong the various pathways identified, the endocannabinoid system has received increasing attention due to its role in the regulation of affective disorders. Previous studies have shown higher cannabinoid receptor type 1 (CB1) and CB1-mediated G-protein activation in the DLPFC of depressed suicidal subjects . MechaniInterestingly, the pathways reported in the current study included cellular degeneration, apoptosis, and related cytoskeleton structural changes. In GO analysis, the DEPs in suicidal subjects related to cellular component biogenesis and assembly were downregulated. This was also replicated in the IPA analysis where we found downregulation of the positive regulation of apoptotic signaling pathway and upregulation of the positive regulation of neuron death in the suicide group. Furthermore, the most significant proteins for suicide in the WGCNA analysis included CAPNS1 , which is suggested to play a neuroprotective role , and CSNThe major concern of the current study was whether the identified DEPs were solely related to suicide completion, which is a complex phenomenon, or related to other risk factors of suicide or the result of treatment. Therefore, we performed multiple subgroup analyses. First, we compared control vs. suicide without psychiatric disorder and control vs. suicide with psychiatric disorder to prove whether having a psychiatric disorder would have influenced our results Table , S11B. DGSTT1, which plays a role in coping with oxidative stress, was identified as a biomarker among suicide completers with psychiatric disorders. Since a GSTT1 polymorphism has already been noted as a key biomarker for schizophrenia, bipolar disorder, and other mood disorders –65, the S100A10 is a protein that modulates serotonergic 1B and glutamate receptor function and has been known to be related with suicide in many previous studies , 68. FurThere are several limitations to our study, such as the sample size and the evaluation procedure. First, the small sample size may have affected our analyses, particularly the subgroup analyses, with respect to statistical power. Moreover, the lack of standardized diagnostic evaluation after death raises a concern for suboptimal diagnosis. However, in 34.5–61.8% of suicide cases, there was no psychiatric diagnosis is East Asia, including China, Japan, and South Korea –73, whicSecond, there was a limitation in collecting control group samples given the male predominance for sudden death, already shown in previous epidemiologic studies , 76. To Third, there was a possibility of bias since we could use only the accessible postmortem brain gathered from autopsy. Most suicides in our samples were due to drowning; however, hanging accounts for >50% of suicides in South Korea . In addiFourth, significant confounders may have influenced our results. We tried to obtain all clinical information available from the police and the family of the deceased; however, the subject’s state right before suicide was still elusive. Although we performed several subgroup analyses to determine whether the differences in protein expression were affected by the given information , other unpredictable confounders such as undiscovered comorbidity might have affected the results . Furthermore, we were unable to obtain information on the chronic use of substances such as alcohol, nicotine, and illicit drugs. Considering that such information may influence the biological pathways we discovered (such as the endocannabinoid pathway) , they maNevertheless, our study has several strengths. The current study was the first to apply a proteomic approach to the postmortem brains of East Asians. A total of 36 postmortem brain samples were collected. Using high-throughput TMT-based quantification with high-resolution mass spectroscopy, 9802 proteins were simultaneously identified and quantified, the highest number so far reported in the literature. Moreover, we could comprehensively reveal potential biological pathways (i.e. endocannabinoid pathways and apoptotic pathways) related to suicide completion using multiple existing bioinformatics tools.In conclusion, our study uncovered the largest protein pool of East Asians related to suicide completion using various bioinformatics tools. Through multiple subgroup analyses, we found common biological markers between suicide completion and suicidal risk factors such as psychiatric disorders, which are one of the most important risk factors for suicide. The pathways revealed in this study are strong candidates to explain the biological basis of suicide.supplementary figuressupplementary tables


Subjects were grouped by %BML: lost mass (LM), gained mass (GM), or no change (NC). A one-way ANOVA compared groups on Ucol and FC. There were differences across groups for pre-practice Ucol (P < 0.01) and FC (P < 0.01). GM had a higher pre-practice Ucol than LM (P < 0.01) and NC (P < 0.05) and consumed more fluid than LM (P < 0.01) and NC (P < 0.05). A stepwise linear regression examined the extent that Ucol and FC were related to %BML. When predicting BML, FC accounted for 45% of variance (P < 0.01). The addition of pre-practice Ucol increased predicted variance explained . Subjects who gained mass during practice arrived with elevated urine color (Ucol 5 ± 2), while those who lost mass arrived with pale urine color (Ucol 3 ± 2). Findings indicate those who arrived with an elevated urine color attempted to improve hydration status by consuming more fluid and gaining body mass during exercise.Although studies use body mass changes or urine color to measure hydration status, the purpose of this study was to examine the relationship between pre-practice urine color and exercise body mass changes in female tackle football players. Twenty-six female American football players  volunteered. Fluid consumptions (FC) was measured during tackle football practices, while urine color (U An exercising individual's hydration status can impact a person's health, exercise capacity and athletic performance  and body mass changes, when compared to a 3-day baseline body mass measure  can use body mass loss and urine color measurements to educate the athlete on how they can ensure they are euhydrated at the start of exercise to broadly guide fluid consumption during exercise. Voluntary drinking sometimes results in over hydration but can also result in dehydration, even when athletes have access to fluids  at the start of exercise may gain more body mass by compensating with fluid consumption due to starting exercise in a hypohydrated state. Similarly, we hypothesized that participants whose urinary measures were indicative of being euhydrated prior to exercise may lose body mass from pre- to post-exercise.n = 26; Age: 29.9 ± 7.3 years, range: 22–48 years; Height: 165.2 ± 2.6 cm; Mass: 83.8 ± 24.4 kg; Body Mass Index: 30.8 ± 9.4; BSA: 1.9 ± 0.2). Exclusion criteria included any injury or condition that would preclude any football player from participating in football practices. This study was approved by the Institutional Review Board, and all participants read and signed an informed consent form. This was a convenience sample of participants from the same team.Subjects from this study were female professional football players  x100 . This is the start of the official season for women's tackle football. Data for each subject was recorded over the course of between 4 and 8 practices across two seasons. Because this study ranged across two seasons, not all participants were in attendance for every data collection day. Each football practice lasted 2 h, and participants were wearing full American football gear . When participants arrived at practice, they provided pre-practice urine samples for urine color in a standard, clear specimen cup, and semi-nude (shorts and sport bra) body mass measures  were then recorded  were calculated for all variables. Due to the within-subject repeated nature of the data, a multi-level model was used to examine the extent that the measure of hydration (pre-practice urine color) and fluid consumed were related to body mass loss. The grouping variable was individual subject, with BML as the dependent variable, and fluid consumed and urine color as co-variates.P < 0.05. SPSS 28 for Mac was used for all analyses.To further investigate the potential differences in hydration and fluid consumption when one gained, lost, or maintained body mass throughout an individual practice, data were grouped by body mass loss (LM), body mass gain (GM), and no body mass change (NC). An ANOVA was then used to compare the groups on fluid consumed, pre-practice urine color, and post-practice urine color. Given the within subject design that was not dependent on practices in this global assessment, all data were collapsed across days. While the ANOVA cannot account for the potential violation of independence due to collapsing across days, we wished to demonstrate in absolute numbers how BML and fluid consumed varied by pre-practice urine color on a given practice in way the linear multi-level modeling does not demonstrate. The significant α level was set at The model can be found in Overall Mean, SD, and range for BML and fluid consumed are listed in P < 0.01) and fluid consumed (P < 0.01). Specifically, the group that gained mass throughout practice (GM) had a higher pre-practice urine color than the group who lost mass, or LM (P < 0.01) and the no mass change (P < 0.05) groups (P < 0.01) and the NC (P < 0.05) groups (P < 0.01).There were differences across groups for pre-practice urine color  groups . The GM ) groups . The NC To our knowledge no study has specifically examined the relationship between pre-practice urinary hydration measures and body mass changes during female football practices in the field setting. Furthermore, the present study sought to examine whether those athletes that arrived in a hypohydrated state compensated for this during exercise by consuming more water. When looking at pre-practice urinary measures, we found that subjects who gained mass pre- to post-exercise arrived at practice in a hypohydrated state with an average urine color of 5 , 2. FurtThe direct relationships between hydration and BML revealed that for every liter of fluid consumed each participant was predicted to gain 0.1% body mass, and that consuming more water resulted in less predicted body mass loss. Additionally, greater pre-practice urine color predicted losing less body mass . Our fincol to better depict the hydration status of these athletes in a field setting. Athletes are often educated about body mass changes and encouraged to avoid both fluid losses >2% of their body mass as well as to prevent body mass gains during exercise.Using body mass to assess hydration can be one of the most common ways to measure hydration status, particularly in individuals that are euhydrated at the start of exercise; however, having a euhydrated pre-exercise body mass helps better quantify any changes in body mass during exercise. Body mass changes can be equated with losses of body water (1 kg = 1 L) with very minimal error during short duration exercise  that athletes often arrive to practice in a hypohydrated state  in order to provide accurate, individualized hydration recommendations to their patients.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Institutional Review Board of the University of South Florida. The patients/participants provided their written informed consent to participate in this study.RL, DL, and AT: conceptualization. RL and AT: methodology, formal analysis, data curation, visualization, and supervision. AT: software. RL, DL, AT, and VL: validation, writing—original draft preparation, and writing—review and editing. RL: investigation, resources, project administration, and funding acquisition. All authors have read and agreed to the published version of the manuscript.This research was funded in part by the University of South Florida New Investigator Grant, # 18325-0083616.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Staphylococcus aureus (MRSA) in NH roommates, compared it with expected prevalence, and determined specific body and environmental sites that may act as sources of roommate colonization. Roommate contamination was associated with index patient’s colonization (relative risk (RR): 2.57 (95% CI 1.04–6.37)) for MRSA, and index patient’s immediate environment contamination for VRE (RR: 3.60 (95% CI 1.59–8.12)). When specific index patient sites associated with roommate colonization were investigated, the side table  and the nurse call button (p = 0.001 and 0.052) stood out, together with patient hands in the case of VRE (p = 0.026). Future studies should be carried out to establish whether these sites should be a specific target of infection prevention campaigns in NHs with multiple occupancy rooms.Multiple room occupancy is common in Nursing Homes (NHs), and its role in transmission of antibiotic-resistant pathogens is unclear. We investigated prevalence of patient colonization and environmental contamination with vancomycin-resistant enterococci (VRE) and methicillin-resistant The online version contains supplementary material available at 10.1186/s13756-021-00972-1. Staphylococcus aureus (MRSA) are among the most commonly found antimicrobial-resistant pathogens in this vulnerable population, where they are found as often or more often than in acute care settings [Patient colonization and environmental contamination with multidrug-resistant organisms are common in Nursing Homes (NHs), where multiple room occupancy is a popular option. Vancomycin-resistant enterococci (VRE) and methicillin-resistant settings –3. Priorsettings ,4–6.In one intensive care unit study, it has been demonstrated that patients can be significantly more likely to acquire VRE and MRSA if the prior room occupant was colonized with the same organism Globally, caring for older adults in shared spaces is common. Since sharing rooms can lead to transmission of pathogens, we aimedto assess whether co-colonization and co-contamination with MRSA and VRE occurs more often than expected among roommates, to investigate the probability of colonization and contamination in roommates of a colonized patient, and to determine if and which specific source sites may be driving such burden.The present study is based on an in depth analysis of a subset of patients enrolled in a larger prospective cohort study of newly admitted patients in southeast Michigan NHs . PatientS. aureus isolates were screened for methicillin resistance using cefoxitin disc diffusion according to CLSI criteria. Growth suggestive of VRE on BEV6 was confirmed by pyrrolidonyl arylamidase testing .Samples were collected using Bactiswabs , and processed on the same day. Nares, oral, groin and perianal samples were streaked on TSA 5% sheep blood agar plates, MSA agar, and bile-esculin agar with 6 ug/ml vancomycin (BEV6) and incubated for up to 48 h at 36 °C. All other samples were enriched overnight in BHI broth, after which they were streaked and incubated as above. Growth suggestive of staphylococci on MSA was tested by catalase and coagulase test , and z test). The overall tested patient population included patients with roommates of unknown status and patients with no roommate.Colonization and contamination with MRSA and VRE were analyzed independently. Colonization and contamination rates were obtained for all enrolled patients and their immediate environment, respectively, at their first visit. To determine whether double room placement may result in increased likelihood of colonization and/or contamination, we compared actual frequencies of co-colonization in roommates to frequencies expected in the overall population of tested patients , using a simple proportion comparison test  of colonization of a roommate when the index patient sharing her/his room was colonized compared with when the index patient was not colonized. Similarly, we calculated the RR for environmental contamination of the roommate surrounding based on index patient’s surrounding contamination. and for the cross probabilities of body colonization and environmental contamination.Finally, to establish if and which body and/or environmental site/s may be specifically associated with an outsized risk of roommate colonization or contamination, we employed a site-specific strategy, by comparing rates for each sampling site in index patients and their immediate vicinity, according to their roommate’s overall colonization or contamination status (Fisher’s exact test).2 (2.6%) and 0.1712 (2.9%). Actual observations revealed 7.1% for both colonization (p = 0.028) and contamination (p = 0.054), pointing to a possible interdependency of MRSA status between roommates. For VRE, the prevalence of body colonization and room contamination among all patients was 33.2% in both cases, leading to an expected dual percentage of 11.0%. Actual data showed 21.4% contamination (p = 0.071), and 8.9% colonization. A relatively high prevalence of VRE was expected in such NH patients [Microbiological data was available on body and environmental MRSA and VRE burden for 1619 visits of 651 patients. In 56 cases, microbiological data was available for both patients sharing a double room. The rate of MRSA body colonization and room contamination upon admission for all patients with available microbiological data, and regardless of room occupancy status (single or double) was 0.161 (16.1%) and 0.171 (17.1%), respectively. Thus, the prevalence of dual colonization and contamination of both roommates, upon the assumption that their statuses are independent, would be expected to be 0.161In visits where matched microbiological data was available for both patients, index patient MRSA colonization translated to a higher risk of contamination of their roommate’s immediate environment , and a higher risk of roommate colonization, although not reaching statistical significance (RR 2.62 (0.74–9.26), p = 0.08) , p = 0.002) compared to a weaker evidence of cross contamination between the index patient and their roommate’s environment, (RR 1.87 (0.77–4.54), p = 0.17 and 1.67 (0.79–3.52), p = 0.18, respectively).Among specific sampling sites, the nurse call button appears to be more likely to be contaminated in index patients of MRSA-colonized roommates (p = 0.052), and the side table (top) in index patients of roommates with MRSA-contaminated surroundings (p = 0.047) Fig. . In the Among index patient body sites, the hands are the most strongly associated with roommate burden, including a statistically significant association when roommates’ immediate environment is contaminated with VRE (p = 0.026). Taken together, these observations indicate that not only patient colonization, but also environmental contamination may be associated with higher MRSA and VRE burden amongst patients sharing a room.Our original data suggests that MRSA and VRE colonization of a NH patient residing in a double room may be a risk factor for acquisition by her/his roommate, and points to patient hands as well as certain environmental sites as possible sources for transmission Interestingly, MRSA has been demonstrated to be capable of colonizing up to 12% of roommates of positive patients in double occupancy hospital rooms This study was not powered to prove transmission and its direction, thus the implication of index patient as a “source” must be taken with caution in the face of limited understanding of NH colonization and contamination dynamics. Still, it must be noted that the sub-population of patients from which our roommate pair visit data originated was not different from the general study population in terms of potential confounding risk factors such as age, sex, comorbidity and functional status (Additional file Additional file 1. Antimicrobial usage for each facility.


The current coronavirus disease 2019 (COVID-19) is a public health emergency. In this study, we aimed to evaluate the risk factors for mortality in severe and critical COVID-19 patients.We performed a retrospective study of patients diagnosed with severe and critical COVID-19 from four hospitals in Wuhan, China, by evaluating the clinical characteristics and laboratory results, and using Cox proportional hazards model to assess the risk factors involved in disease progression.9/L  that correlated with an increased mortality rate. The multivariable Cox proportional hazards model demonstrated that older age  and higher white blood cell count  were independent risk factors for determining COVID-19 associated mortality.In total, 446 patients with COVID-19 were enrolled. The study indicated a high mortality rate (20.2%) in severe and critical COVID-19 patients. At the time of admission, all patients required oxygen therapy, and 52 (12%) required invasive mechanical ventilation, of which 50 (96%) died. The univariate Cox proportional hazards model showed a white blood cell count of more than 10 × 10COVID-19 is associated with a significant risk of morbidity and mortality in the population. Older age and higher white blood cell count were found to be independent risk factors for mortality. In December 2019, a cluster of patients with pneumonia of unknown etiology was identified in Wuhan City, Hubei Province, China. Isolation of bronchoalveolar-lavage samples from patients with pneumonia in human airway epithelial cells and subsequent whole-genome sequencing revealed the presence of a novel beta-coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) . Severe To date, more than 2 million deaths of the pandemic and huge economic and social upheaval internationally . There iAll methods in this study were performed in accordance with the relevant guidelines and regulations. The study was approved by the Institutional Review Board (IRB) at the Hubei Provincial Hospital of Traditional Chinese Medicine (HBZY2020-C14–01) in Wuhan, P. R. China. Oral consent was obtained from patients or patients’ relatives. All cases came from Hubei Provincial Hospital of Traditional Chinese Medicine, Renmin Hospital of Wuhan University, Hubei Provincial Hospital of Integrated Chinese & Western Medicine, and JinYin-tan Hospital . The first patient was admitted on January 15, 2020 and the follow-up deadline for the last admitted patients was March 1, 2020. Also included in our study were 31 severe and critical patients in a previous publication .2)/oxygen concentration (FiO2) ≤ 300 mmHg, or ④ pulmonary imaging of blood showing significant progression of lesions > 50%. Critical patients must show one of the following: ① respiratory failure, requiring mechanical ventilation, ② shock, or ③combined failure of other organs requiring ICU monitoring and treatment  and death. The patient was discharged based on meeting the following four criteria: ① no fever for at least three days, ② significant improvement in both lungs on a chest computed tomography (CT) scan, ③ clinical relief from respiratory symptoms, and ④ two sequential SARS-CoV-2 RNA negative throat swab samples obtained at least 24 h apart , laboratory tests, and treatment outcomes of patients at the time of admission. The collected data were collated and reviewed by a team of professionally trained doctors. The collated experimental data included blood analysis , liver function (levels of glutamate transferase and aspartate transferase), renal function , and the levels of glucose, triglycerides, cholesterol, high-sensitivity cardiac troponin I, interleukin-6 (IL-6), procalcitonin, and C-reactive protein (CRP).Statistical analysis was performed using R version 4.0.2. Categorical variables were described by frequency and percentage, and continuous variables were described by median and interquartile range (IQR) values. Continuous variables were compared using independent group tests when the data were normally distributed; otherwise, the Mann-Whitney test was used.p < 0.05) in univariate analysis were included in the multivariable analysis. The Kaplan-Meier method was used to analyze the survival rate, and the log-rank test was used to compare the survival rates. We tested in the model interactions that were significant on a stratified analysis hazards ratio (HR) presented with 95% confidence intervals (CI). Testing for the proportional hazards assumption was conducted by checking the scaled Schoenfeld residuals using Survival package 3.2–3 in R version 4.0.2. The results showed that all variables satisfied the assumption. All statistical tests were mutually exclusive and statistical significance was set at p < 0.05.The Cox proportional hazards model was used to analyze baseline variables related to mortality. For each continuous variable , martingale residuals were utilized to assess each variable’s functional form. It turned out that the log transformed format is more appropriate to glucose and AST. Thus, the log transformed glucose and AST were included in the analyses. Variables that were found to be statistically significant (The median age of the 446 patients included in the study was 55 years (IQR 42–66); of which, 213 (47.76%) were male, 90 (20.2%) died during hospitalization, and 356 (79.8%) were discharged. The median length of hospital stay was 10 days (IQR 8–14) (Table 9/L), 52 (12%) with a leukocyte count above the normal range (white blood cell count more than 10 × 109/L), and about 50% had a decreased lymphocyte count (lymphocyte count less than 1.0 × 109/L). A total of 121 (27%) patients exhibited varying degrees of liver dysfunction, with alanine aminotransferase (ALT) or aspartate aminotransferase (AST) levels above the normal range. Renal dysfunction occurred in 32 (7%) patients, who showed elevated blood urea nitrogen or serum creatinine levels. There were 211 (47%) patients with abnormal blood glucose levels, and among them, 200 (45%) showed glucose levels above the normal range (glucose concentration more than 6.1 mmol/L), while 11 (2%) patients showed levels below the normal range (glucose concentration less than 3.9 mmol/L). Levels of CRP were increased in 67% of the patients (CRP more than 10 mg/L)  patients presented a leukocyte count below the normal range (white blood cell count less than 4 × 10% patientp < 0.001). Patients were divided into two groups based on age with a cut-off point of 60 years. The Kaplan-Meier survival curves are shown in Fig. p = 0.001], hypertension , and coronary heart disease . The rate of death increased 1.187-fold in patients with an increased leukocyte count. The leukocyte count cut-off was at the upper limit of the normal value range. The Kaplan-Meier survival curves are shown in Fig. 9/L while the probability of survival never fell below 50% for the other two groups. Other risk factors affecting death included lymphocyte count, and levels of creatinine, log transformed AST, log transformed glucose, as shown in Table We included 380 patients with complete data for Cox regression. The univariate Cox proportional hazards model showed that the rate of death increased 1.072- fold with an increase in age .The multivariable Cox proportional hazards model was used to identify independent risk factors for mortality. We found that older age  and higher white blood cell count  were highly significant independent risk factors  count was a highly significant independent risk factor for mortality in severe and critical COVID-19 patients. We further divided the WBC into 10 deciles and calculate the Pearson correlation between mortality and the WBC count within each decile. By doing this, we expected to find out the association between mortality and WBC count. The results showed that the highest decile of WBC count has a correlation of 0.39 with mortality while the association in the other deciles were much lower. An elevated white blood cell counts often indicates an ongoing inflammatory responses to bacterial infection or disease progression. Patients with severe virus infection were more likely to co-infected with bacteria due to low immune functions. In other coronavirus infections such as SARS-CoV and MERS-CoV, could found “bcytokine storms” and immunopathology. Excessive inflammation could occur while immune response was dysregulated. The inflammatory response could stimulate the production of inflammatory cells and speed up the apoptosis of lymphocytes . PatientSome studies based on the characteristics of patients , 19, 20,Although the lymphocyte count of most patients was low –28, it wPrior to the first outbreak of SARS, a limited number of coronaviruses, causing only mild illnesses such as the common cold, were known to infect in humans . Given tOur study has several limitations worth noting. Firstly, there was insufficient information on demographic characteristics, clinical symptoms, history of exposure, and personal history of the patients included in this study. Secondly, it was a retrospective study design and relied on data collected from case records. Information such as which patient comes from which hospital was not collected. There might be intracluster correlations among four hospitals, although we believe the impact on standard errors is minimal. Thirdly, the patients did not undergo all laboratory tests like neutrophil count, lactate dehydrogenase and serum ferritin tests. Therefore, their role might be underestimated in predicting death in hospitalized patients. Lastly, we studied a short-term prognosis of patients during hospitalization, however, SARS-CoV-2 is a newly discovered virus, and its onset, characteristics and prognosis are still being researched. So the validity of the risk factors for mortality derived from our cohort remains tentative, and we need a larger sample size and a longer follow-up period to validate our findings. In the later stages, follow-up of surviving patients must also be continued to better understand clinical course of the disease.The COVID-19 outbreak has been declared a global pandemic. In this study, we found that older age and higher white blood cell count were clinically useful independent risk factors for mortality of severe and critical COVID-19 patients in hospitalized. We hope this information will aid clinicians in the treatment of patients with COVID-19.


We retrospectively studied outcomes in patients treated with preoperative radiochemotherapy and surgery for esophageal squamous cell cancer. We put a special focus on the comparison of patients treated with 5-fluorouracil/cisplatin  or carboplatin/paclitaxel (‘CROSS’). First, the higher age and more comorbidities of ‘CROSS’ patients, along with a shorter intensive care/intermediate care unit stay, might reflect an improvement in supportive and surgical/perioperative procedures in the periods. Second, the ‘CROSS’ patients experienced more hematologic toxicity and were less likely to complete chemotherapy as per protocol. This indicates that efforts should be taken to guide patients through a toxic treatment regimen. Third, the negative prognostic impact of radiochemotherapy-related toxicities and the duration of the intensive care/intermediate care unit stay underlines that further optimization of treatment procedures remains an important goal. Toxicity profiles could be improved by tailoring the regimen to individual patients .We retrospectively studied outcomes in patients treated with preoperative radiochemotherapy and surgery for esophageal squamous cell cancer. We put special focus on the comparison of patients treated with 5-fluorouracil/cisplatin  or carboplatin/paclitaxel (‘CROSS’). We compared characteristics between patients treated according to ‘Walsh’ vs. ‘CROSS’. Cox regression was performed to test for an association of parameters with outcomes. Study eligibility was met by 90 patients. First, the higher age and more comorbidities of the ‘CROSS’ patients, along with a shorter intensive care/intermediate care stay, might reflect an improvement in supportive and surgical/perioperative procedures over the periods. Second, the ‘CROSS’ patients experienced more hematologic toxicity and were less likely to complete chemotherapy as per protocol. This indicates that efforts should be taken to guide patients through a toxic treatment regimen by supportive measures. Third, the negative prognostic impact of radiochemotherapy-related toxicities  and the duration of the intensive care/intermediate care unit stay underlines that further optimization of treatment procedures remains an important goal. We found no differences in tumor downstaging and survival between treatment regimen. Toxicity profiles could be improved by tailoring the regimen to individual patients . Esophageal cancer is often diagnosed at advanced stages  ,2. RecenIn the light of the aforementioned progress and developments, we retrospectively studied treatment results, toxicities, surgical outcomes, and prognostic factors in patients with squamous cell esophageal cancer treated with preoperative RCT and surgery at our institution. Here, to take into account the major shift in treatment strategies, we put a special focus on the differences in treatment according to ‘Walsh’ and ‘CROSS’.n = 3), and a combined partial resection of lung and liver (n = 1). Here, during pre-surgical staging examinations (n = 2 patients) or intraoperative examination (n = 1 patient), organ lesions were evident and were excised for further evaluation. In histopathological examination, these lesions were benign. In one patient, a partial lung resection was necessary due to adhesions after previous surgical interventions. In 15 patients (16.7%), an esophagectomy with cervical anastomosis was performed. An R0 resection was achieved in all of the patients. Please see A total of 90 patients were eligible for our study (n = 6), decrease in creatinine clearance (n = 3), infection (n = 1), deterioration of hearing (n = 1), and combined deterioration of hearing and decrease in creatinine clearance (n = 1). The reasons in patients treated with carboplatin/paclitaxel were: Leucopenia (n = 8), deterioration of clinical condition (n = 2), infection (n = 1), and elevated liver enzymes (n = 1). The rates of hematologic toxicity (≥grade 3) were higher in the carboplatin/paclitaxel group. Furthermore, we analyzed whether patients who experienced RCT-related toxicities were less likely to receive full chemotherapy. Indeed, the rates of patients with incomplete chemotherapy administration were significantly higher  received 100% of the planned chemotherapy cycles and dose, whereas significantly less patients in the carboplatin/paclitaxel group completed chemotherapy . The reaIn the whole study cohort, the 5-year locoregional control (LRC), progression-free survival (PFS), OS, and cancer-specific survival (CSS) were 84.1%, 46.2%, 49.7%, and 66.9%. In total, 40/90 patients (44.4%) died during follow-up. Among these, 21 patients died from esophageal cancer. In 19 patients, the cause of death was not further specified in the medical records. A total of 10/90 patients (11.1%) developed locoregional recurrence. Here, 4 patients had combined local and regional recurrences, 4 patients had isolated local recurrences, and 2 patients had isolated regional recurrences. In our study, 21/90 patients (23.3%) developed distant metastases. In the Cox regression analysis , there wThe outcome in patients with locally advanced esophageal cancer was significantly improved by the introduction of preoperative RCT ,10,18. TIn the present study, we compared different RCT regimens and, thus, have to consider the esophageal cancer incidence and local treatment practice in the periods 1999–2014  and 2014–2019 . Remarkably, we found patients treated with carboplatin/paclitaxel to be almost 10 years older (median 70.1 vs. 61.1 years) than patients treated with 5-fluorouracil/cisplatin. First, this might reflect the general tendency that the tumor incidence rises at the age of ≥60 years, whereas it declines in younger patients ,13,23,24Additionally, we found a shorter intensive care/intermediate care unit stay (median 4 vs. 7 days) in patients treated with carboplatin/paclitaxel. This finding is especially remarkable, since these patients were about 10 years older and had more comorbidities. This might reflect recent developments and efforts to improve surgical techniques and perioperative procedures . OverallFurthermore, we found that VMAT was used more frequently in patients treated with carboplatin/paclitaxel  than in patients treated with 5-fluorouracil/cisplatin (0/63 patients). This, again, represents technical developments and innovations in local therapies, whereas, with a limited number of patients, the results should not be overinterpreted. Here, the question may arise why we did not treat more patients with intensity-modulated radiotherapy (IMRT) or VMAT in the respective period. Based on previous reports of our clinic  and otheIn our study, we found higher rates of hematologic toxicities for the ‘CROSS’ regimen. In contrast, Münch et al. found the ‘Walsh’ regimen to be associated with higher rates of hematologic toxicities . These fAdditionally, we found that patients who experienced acute organ and/or hematologic toxicities ≥grade 3 were less likely to receive the complete chemotherapy and had worse outcomes (CSS) than patients who experienced toxicities <grade 3. This might be attributed to the reduced compliance to chemotherapy due to hematologic toxicities . HoweveFurthermore, we found worse outcomes (for CSS) in patients who experienced dysphagia ≥grade 3 during RCT. Previous authors reported that the tumor length is an independent prognostic factor in esophageal cancer . FurtherFinally, we found local treatment with either RCT regimen to be comparably effective. In the presented study, only 10/90 patients (11.1%) developed locoregional recurrence. In the whole study population, we found a pathological complete response in 35/90 patients (38.9%). In line with previous studies, our results underscore the prognostic relevance of pathological complete response . For thiWe studied the medical records of patients with esophageal cancer who were treated at our Department of Radiotherapy and Radiation Oncology. We included patients who received a preoperative RCT (5-fluorouracil/cisplatin or carboplatin/paclitaxel) and subsequent tumor resection for squamous cell esophageal cancer. Patients had adequate organ functions and performance status (ECOG 0-1) to undergo preoperative RCT. They were deemed operable by experienced surgeons at our University Medical Center during interdisciplinary tumor conferences. Additionally, the indication for preoperative RCT was set in the multidisciplinary tumor board. The staging procedures and the therapeutic management were based on the respective contemporary guidelines ,44,45. Tn = 3). For both 3D conformal radiotherapy and VMAT, the lung constraint was V (5 Gy) ≤ 30%. Before initiation of RCT, the isocentre and  the fields were simulated with a conventional x-ray simulator using oral contrast agent.Initially, in our clinic, the RCT regimen was based on the trial published by Walsh et al. . From 202 on day 7) or 5 weekly cycles of carboplatin (area under the curve of 2mg/mL*min on day 1) and paclitaxel (50 mg/m2 on day 1). The pretreatment examinations included blood cell counts and clinical chemistry analyses, an electrocardiogram, and the assessment of the 24-h urine creatinine clearance. The prophylaxis of nausea and vomiting was conducted according to the MASCC/ESMO guidelines [The chemotherapy regimen comprised 2 cycles (in the first and fifth week of RCT) of 5-fluorouracil (15 mg/kg/d over 5 days) and cisplatin  or death from any cause. The endpoint for CSS was patient death caused by esophageal cancer progression. The survival curves were created using Kaplan–Meier statistics. The comparisons of survival times for the different groups were performed using log-rank statistics and Cox regression analysis. The software Statistica (v. 13), SPSS (v. 26), and R (v. 4.0.2) with the plugin ‘KWWin’  were useFirst, we compared the characteristics of patients treated with carboplatin/paclitaxel and with 5-fluorouracil/cisplatin. The patients treated according to the ‘CROSS’ trial had a shorter intensive care/intermediate care unit stay. This might reflect an improvement in supportive and surgical/perioperative procedures in the different time periods. Second, patients treated with carboplatin/paclitaxel experienced more hematologic toxicities and were less likely to receive the complete chemotherapy. This indicates that efforts should be taken to carefully guide patients through a toxic treatment regimen by supportive measures . Additio


Asyndetus clavipes belongs to the subfamily Diaphorinae of Dolichopodidae. The mitogenome of A. clavipes was sequenced, the first representative complete mitogenome from this subfamily. This mitogenome is 15,385 bp in size, includes 13 protein-coding genes, 22 transfer RNAs, and two ribosomal RNAs. All genes have the same location and coding strand as in other published species of Dolichopodidae. Nucleotide composition is biased toward A and T, which together made up 78.6% of the entire genome. Bayesian inference strongly supported the monophyly of Empidoidea, Empididae and Dolichopodidae, with the phylogenetic relationships within Empidoidea: ((Dolichopodinae + Neurigoninae) + Diaphorinae) + ((Trichopezinae + (Empidinae + Oreogetoninae)) + Ocydromiinae).The long-legged fly  Asyndetus Loew, 1869 belongs to the subfamily Diaphorinae. It is a large genus of Dolichopodidae, worldwide distributed with 102 species  of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences (IPPCAAS) . Total genomic DNA was extracted from a whole-body (except the head) specimen using the QIAamp DNA Blood Mini Kit  and stored at −20 °C until needed. The mitogenome was sequenced in BeiRuiHeKang biotechnology company used NGS. 1 μg of genomic DNA was used to generate libraries with an average insert size of 350 bp, which were sequenced using the Illumina NovaSeq 6000 platform  with 150 bp paired-end reads on one sample per flow-cell lane. A total of 24,471,684 raw paired reads were generated. The quality of all sequences was checked using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Clean reads were assembled and annotated using the MitoZ v2.4 pipeline  and encodes 13 PCGs, 22 tRNA genes, and 2 rRNA genes. All genes have the same location and coding strand as in other published species of Dolichopodidae  initiate with ATT codons, and six PCGs  initiate with ATG codons. All PCGs (12 of 13) use the typical termination codons TAA while NAD4 uses TAG.The complete mitogenome of A. clavipes could provide important information for further studies of Dolichopodidae phylogeny. Further studies are needed to sequence more species from the subfamily Diaphorinae, as well as other subfamilies, which will enhance our understanding of molecular phylogeny in Dolichopodidae.Phylogenetic analysis was performed based on the nucleotide sequences of 13 PCGs from 12 Diptera species. Sequences were aligned using MAFFT v7.313 (Katoh and Standley


Haemophilus influenzae disease in Bellvitge University Hospital (2014–2019), reporting its evolution from a previous period (2008–2013) and analysing the non-typeable H. influenzae (NTHi) population structure using a clade-related classification. Clinical data, antimicrobial susceptibility and serotyping were studied and compared with those of the previous period. Population structure was assessed by multilocus sequence typing (MLST), SNP-based phylogenetic analysis and clade-related classification. The incidence of invasive H. influenzae disease remained constant between the two periods (average 2.07 cases per 100 000 population), while the 30 day mortality rate decreased . Immunosuppressive therapy (40 %) and malignancy (36 %) were the most frequent comorbidities. Ampicillin and fluoroquinolone resistance rates had increased between the two periods . NTHi was the main cause of invasive disease in both periods (84.3 and 85.3 %), followed by serotype f (12.9 and 8.8 %). NTHi displayed high genetic diversity. However, two clusters of 13 (n=20) and 5 sequence types (STs) (n=10) associated with clade V included NTHi strains of the most prevalent STs (ST3 and ST103), many of which showed increased frequency over time. Moreover, ST103 and ST160 from clade V were associated with β-lactam resistance. Invasive H. influenzae disease is uncommon, but can be severe, especially in the elderly with comorbidities. NTHi remains the main cause of invasive disease, with ST103 and ST160 (clade V) responsible for increasing β-lactam resistance over time.This study provides an update on invasive  Sequence reads were deposited in the European Nucleotide Archive (ENA) under the project accession number PRJEB42971 .Haemophilus influenzae is an opportunistic pathogen in the human respiratory tract that may cause community-acquired invasive infections with high mortality rates. Moreover, the rise in β-lactam resistance due to β-lactamase production is cause for concern. We provide an update on the current epidemiology of invasive H. influenzae disease, as well as an analysis of the heterogeneous non-typable H. influenzae (NTHi) population structure using a clade-related classification based on accessory genome analysis. Multilocus sequence typing (MLST) is useful for clustering capsulated H. influenzae isolates because they are clonal and have few sequence types (STs), but it is not the best method for studying the heterogeneous NTHi population. The use of a clade-related classification based on the presence or absence of some surface-associated proteins and virulence determinants is a valuable tool to address this issue. Interestingly, we have observed that despite the genetic diversity, more than half of the invasive NTHi isolates shared a common phylogenetic origin, clustering them in the same clade. Moreover, these isolates showed an increased frequency over time and were linked to β-lactamase production. This should raise our awareness of the possibility of clonal dissemination, and we should consider monitoring the evolution of these clones in invasive disease.Haemophilus influenzae is a Gram-negative coccobacillus that colonizes the human nasopharynx and throat in up to 80 % of children and 20–30 % of adults [H. influenzae (NTHi) [f adults . CapsulaH. influenzae is responsible for respiratory infections ranging from otitis media, sinusitis and conjunctivitis to deeper infections such as pneumonia and exacerbations of chronic obstructive pulmonary disease (COPD) and even invasive disease [H. influenzae type b conjugate vaccine, this serotype was the most common cause of invasive disease. Vaccine implementation has produced an epidemiological shift, with most invasive infections currently occurring in elderly patients with underlying conditions and caused mainly by NTHi followed by non-type-b serotypes [H. influenzae disease evolves, concerns are now focused on non-vaccine-preventable isolates and the growing proportion of β-lactam resistance by the acquisition of β-lactamases and alterations in penicillin-binding proteins (PBPs) [Despite the usual asymptomatic interaction with humans,  disease . Before erotypes . As the s (PBPs) .H. influenzae isolates, as they are highly clonal and associated with few sequence types (STs). By contrast, NTHi strains show high genetic heterogeneity [The structure of the bacterial population provides information on the evolution of a bacterial species, its genetic diversity and the dynamics of antimicrobial resistance determinants and pathogenic mechanisms . Classifogeneity . The claogeneity , could bH. influenzae clones, along with their antibiotic resistance profile and genetic variability, is essential to design useful prevention strategies. In this study, we provide an update on the epidemiology of invasive H. influenzae disease in Bellvitge University Hospital (2014–2019), monitoring changes from a previous period (2008–2013) [Epidemiological surveillance of invasive 08–2013) .H. influenzae disease between 2008 and 2019 , the same sample types considered in the previous study period (2008–2013) [H. influenzae isolates were identified by mass spectrometry using MALDI-TOF .A laboratory-based surveillance study on the epidemiology of invasive 008–2013 ; second 08–2013)  to facilH. influenzae disease were reviewed to obtain demographic and clinical data, including information on gender, age, source of infection, underlying conditions and 30 day mortality. The incidence of invasive H. influenzae disease was expressed as the number of cases per 100 000 population and was estimated using the adult population that received health coverage in Bellvitge University Hospital as the denominator, according to the Statistical Institute of Catalonia (https://www.idescat.cat).The medical records of patients with invasive Antimicrobial susceptibility was assessed by the microdilution method, using commercial panels  and following the recommendations for clinical breakpoints of the Clinical and Laboratory Standards Institute (CLSI) . β-lactaH. influenzae isolates identified during the second period (2014–2019) were subjected to whole-genome sequencing (WGS) for serotyping, MLST classification and the study of resistance-related mutations. Genomic DNA was extracted using the QIAamp DNA Mini Kit (Qiagen) and quantified by the QuantiFluor dsDNA System (Promega). Nextera XT was used to prepare the libraries, followed by paired-end sequencing on a MiSeq platform (Illumina). Sequences were assembled with the INNUca v4.2 pipeline (github.com/BUMMI/INNUca) using default parameters. MLST was performed using the mlst v2.4 software . New allele and ST numbers were registered in PubMLST (pubmlst.org). Full MST algorithm with PHYLOViZ [In silico serotyping was conducted using hicap (github.com/scwatts/hicap) [H. influenzae Rd KW20 (NC_000907) used as the reference. For phylogenetic analysis, core-SNPs were extracted with Snippy’s core module (snippy-core). Subsequently, a novel core-SNP phylogenetic tree, using RAxML-NG [All 68 invasive PHYLOViZ  was useds/hicap) . The mecd 2014–20 were subRAxML-NG . The Hi3RAxML-NG .et al. [et al. [github.com/micro-bellvitge/Phylogenetic-analysis-invasive-HINF).NTHi isolates were grouped into clades, as previously proposed . A totalet al. , 57 geno [et al. , and 58  [et al. . Phyloge [et al.  with def [et al.  . Of these, 150 (1.8 %) were responsible for invasive disease, most of which had been isolated from blood (88.0 %) and less frequently from cerebrospinal and pleural fluids . In the first period (2008–2013), 82 of the 3433 (2.4 %) H. influenzae isolates caused invasive disease, compared to 68 of the 4758 isolates (1.4 %) in the second period (2014–2019).A total of 8191H. influenzae disease in the adult population was 2.07 cases per 100 000 population, with no significant differences between the two periods . By age groups, the overall incidence was lower in young adults (≤64 years) than among those aged 65 or older  , as observed in each study period separately . The incidence in young adults decreased in the second period , while the incidence in older adults remained stable.The overall incidence of invasive n=27), which decreased between the periods  and did not differ between the age groups  . Nevertheless, 30 day mortality in young adults was associated with a higher CCI (5.2±3.8) compared to young adults who survived (3.0±2.7) , while no differences were observed in the elderly  (Table S2).The most common comorbidities were immunosuppressive therapy (40.0%) and solid organ malignancies (36.0%), which were strongly associated with 30 day mortality. Diabetes, heart disease and COPD were more common in the elderly, while HIV was more frequent in younger adults. The only difference observed between the two periods was an increase in the number of patients with heart disease. The overall 30 day mortality was 18.0 % (ctively) . The ChaSusceptibility rates for all the antibiotics tested were similar in the two periods . All theblaTEM-1 gene and one serotype f strain carried a blaROB-1 gene in a pB1000 plasmid (GU080063). The latter was also resistant to cefuroxime and cefepime. Amino acid substitutions in PBP3 were observed in 26 strains (38.2%) and were classified according to Dabernat et al. [During the second period (2014–2019), there was a slight increase in β-lactam resistance and an emergence of resistance to ciprofloxacin . Twelve t et al.  (Table 3n=59 in the first period and n=58 in the second period), 15 were caused by serotype f , four were due to serotype e , and two were due to serotype b strains   . The remn=3), while ST103 was the most prevalent in the second period (n=4), followed by ST3 and ST160 . Two of the most common STs, ST103 and ST160, produced a TEM-1 β-lactamase, and both of them showed increased prevalence from the previous period: ST103 from one to four isolates, while ST160, which was not detected in the first period, caused three cases of invasive disease in the second period. Despite the great variability among NTHi, PHYLOViZ revealed two clusters, which each consisted of clones that were linked to one another by sharing at least five MLST loci with another clone in the group . One of the clusters was related to ST3 and included 13 STs (n=20), while the other was associated with ST103 and included five STs (n=10). Moreover, there was an increase in the number of isolates belonging to these clusters, with 13 in the first period (ten associated with the ST3 cluster and three with the ST103 cluster) and 17 in the second period . On the other hand, capsulated strains showed high clonality, with a few related STs  identified for each serotype in both periods: ST124 (n=12) and ST2354, ST2361, and ST2366  for serotype f; ST18 and ST386  for serotype e; and ST6 and ST2364  for serotype b. No differences in ST distribution were observed in the capsulated strains between the periods.MLST and PHYLOViZ analysis showed high genetic heterogeneity, with 84 different STs identified among the 117 NTHi strains. The most common in the first period was ST57 , ST12, ST147 and ST1524 , and ST183, ST836, ST1591 and ST2350 . Moreover, nine of the 12 β-lactamase producing strains were included in this clade  compared to capsulated isolates  .In addition, each clade constituted a different monophyletic lineage, except for clades II, III and VI, in which the strains showed more diverse phylogenetic origins . The recH. influenzae disease is uncommon, but can be severe, especially in adults with comorbidities. H. influenzae is a matter of concern due to its community-acquired burden and the high prevalence of ampicillin resistance it presents [H. influenzae. In this study, we provide an update on the epidemiology of invasive H. influenzae disease over a 12 year period and analysed the population structure of NTHi using a clade-related classification.Invasive presents . For theBetween 2008 and 2019, the overall incidence of invasive disease in Bellvitge University Hospital was 2.07/100 000, which is consistent with the incidence rate found in Europe and the USA . InvasivH. influenzae infections caused by NTHi and non-type-b serotypes have gradually increased in Europe between 2000 and 2014 [et al. [Invasive and 2014 . Our stuand 2014 , while tand 2014  indicateand 2014 , serotypand 2014 , causingand 2014 . Langere [et al.  observedet al. [H. influenzae, which could explain the low prevalence of this β-lactamase type. Despite being low, fluoroquinolone resistance emerged in the second period. Fluoroquinolones are commonly used to treat respiratory infections, which may lead to resistance acquisition. Although the fluoroquinolone resistance rate is low worldwide [Antimicrobial resistance rates remained constant between the two periods of our study, with only a slight increase in β-lactam and fluoroquinolone resistance. TEM-1 β-lactamase was the most common cause of resistance to ampicillin, while one isolate carried a plasmid with the ROB-1 enzyme. San Millan et al.  demonstrorldwide , vigilanNTHi had higher genetic variability and a larger proportion of sites affected by recombination compared to capsulated isolates or other bacterial species . Most NTet al. [et al. [The second most common NTHi clade was VI followed by clade II, while clades I, III and IV were rarely observed. Isolates belonging to clades II and III showed high genetic variability, which could be solved by classification into subclades defined by their different monophyletic origins. On the other hand, clade VI isolates had different monophyletic origins, despite sharing the pattern of accessory genes described by Pinto et al. . The incet al. . However [et al.  classifiH. influenzae population can be missed if only MLST is performed, particularly if the sample size is small and the analysed clones do not belong to the most prevalent STs. For this reason, we suggest that the combination of phylogenetic analysis and clade-related classification based on the accessory genome is a good approach to monitor the evolution of invasive H. influenzae clones and to control the rise of clones associated with β-lactamase production. Further studies will elucidate clades with high phylogenetic variability.Although MLST is useful for classifying capsulated isolates and it did cluster some of the NTHi isolates, it is not the most appropriate technique to study the heterogeneous NTHi population. Details of the heterogeneity of the H. influenzae disease is mainly associated with NTHi and its incidence increases with age, especially in patients with underlying disease. Serotype f remains the most common among the capsulated strains, followed by serotype e, although both disappeared by the end of the study. Continued surveillance of invasive H. influenzae disease is necessary to control the emergence of NTHi and non-vaccine serotype isolates, as well as the rising resistance to β-lactams and fluoroquinolones. We propose that the NTHi population structure should be studied through phylogenetic analysis and clade-related classification, as it clusters the main STs associated with invasive disease. This is an alternative to overcome the limitations of MLST and to comprehend the genetic diversity and dynamics of NTHi.In conclusion, invasive Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.


Since the onset of the COVID-19 pandemic in December 2019, many countries around the world have imposed lockdown measures in order to reduce virus spread. Social isolation is known to have a significant psychological impact, potentially triggering alcohol misuse in adults. In our study, we aimed to investigate the effect of COVID-19 lockdown measures on alcohol consumption in adults in Bavaria.In this cross-sectional study, we enrolled 2067 participants, with 1961 young adults (mean age 23.3 ± 4.1) and 106 mature adults (mean age 66.7 ± 9.7). Participants were asked to complete a standardized questionnaire, semi-quantitatively evaluating the alcohol drinking behaviour before and during the pandemic lockdown.p <  0.001). Among young adults, 42% reported unchanged drinking behaviour compared to 76% in the mature adult group; 44% of young adults reported to drink less compared to only 7% of mature adults. An increase in alcohol consumption was only reported by 14% of young adults and 17% of mature adults. Interestingly, in the entire cohort, the change of alcohol intake was most pronounced among moderate drinkers (> 0 to < 5 drinks/week) in both age groups (p <  0.001). Ordinal logistic regression revealed female sex, low BMI and younger age to be associated with a decrease in number of self-reported drinks/week.After implementation of lockdown, the alteration of alcohol consumption was significantly different between young and mature adults  on April 24, 2020.The study was retrospectively registered at The online version contains supplementary material available at 10.1186/s12937-021-00699-0. Since the onset of the COVID-19 pandemic in December 2019, many countries around the world have imposed lockdown measures in order to reduce virus spread. In the German federal state of Bavaria, lockdown was implemented on the 21st of March by the local government . The survey invitation was emailed to students who consented to receive survey invitations at LMU Munich and 5 other Bavarian universities. Data for mature adults (older than 50 years) were collected in a similar online and print questionnaire, sent to randomly chosen mature adults from diverse educational and financial backgrounds in different regions of Bavaria. These included family members of hospital co-workers , who then passed on printouts to friends. A power calculation has not been performed prior to study conductance.In order to assess the change in alcohol consumption during lockdown, we conducted a large-scale survey among young adults and mature adults. Data for young adults (up to 50 years of age) were collected within the online cross-sectional COLA (COVID-19 Pandemic Lockdown in Young Adults) study, registered at Participants were asked to provide demographic data  and to compare their alcohol consumption during lockdown to before on a three-level scale . Also, they semi-quantified the number of drinks per week before, and after, lockdown implementation on an ordinal scale , with 1 drink corresponding to 500 ml of beer, 100 ml of wine, or 20 ml of liquors. See All data sets with information on age and results on semi-quantified number of drinks were included in the analysis. All data collection was performed in accordance with the Declaration of Helsinki. The study was approved by the ethics committee of the Ludwig-Maximilians-University (LMU) Munich, Germany . Participants were asked to report about their alcohol drinking behaviour before and during lockdown Fig. .2-Test (Pearson Chi-Square) for ordinal or nominal data. Three different univariate and multivariate logistic regression analyses were performed to identify the protective factors and risk factors for alterations in alcohol consumption during lockdown compared to before, i.e. for i) drinking more, ii) drinking less, or iii) drinking over 5 drinks per week. Additionally, an ordinal regression model was used with the drinks category being the dependent variable and age group , gender, BMI group , and a high education level (i.e. having acquired Abitur or university degree) serving as independent variables.Shapiro-Wilk test was applied for normality assessment. Differences between groups were evaluated using Kruskal-Wallis-Test for continuous data and Chip-value < 0.05 was regarded statistically significant for all tests. Continuous data are depicted as mean ± standard deviation or median [interquartile range (IQR)]. Statistical analysis was performed using R (RStudio version 1.2.5033).A n = 1385) vs. 58.1%  were female. Body mass index was significantly higher in the older group   and 106 mature adults (over 50 years) included in the analysis. Return rate for online and paper-based survey were 24% (1980 out of 8252) and 41% (77 out of 187), respectively. Mean age was 23.3 ± 4.1 and 66.7 ± 9.7, respectively, and 71.4%  of young adults reported to not have changed their drinking behaviour compared to 76% (n = 80) of mature adults. The fraction of participants stating to be drinking less was larger among the young adults  compared to mature adults , while only 14%  and 17%  consume more alcohol.The fraction of participants from the two age groups reporting to be consuming more, less, or an unchanged amount of alcohol since the implementation of lockdown were significantly different  (Supplementary Table n = 33), only 1 participant (3%) had a decreased alcohol consumption  from the young adults group decreased their alcohol consumption , age, abstinence (i.e. 0 drinks/week) and frequent drinking (more than 2 drinks/week) were indicators for drinking more or less during lockdown. However, in multivariate logistic regression models for drinking more or less during lockdown respectively, after adjustment for interactions, pre-lockdown abstinence (0 drinks/week) was the only factor protecting against drinking more , female vs. male gender , and BMI < 20 kg/m2 vs. BMI ≥ 20 and ≤ 25 kg/m2  were found to be associated with a decrease to a lower category for number of drinks per week. A BMI > 25 kg/m2 or a high education level had no statistically significant effects  or not alter (44%) their drinking habits. In contrast, only 7% of mature adults reduced their alcohol consumption, and in 17%, an increase was observed. Most mature adults (76%) did not change their drinking habits.The self-reported levels of alcohol consumption are comparable to the literature on alcohol consumption in young  or maturOur results on changes in drinking behaviour are in accordance with the proposed scenario of a decline in alcohol consumption during the early phase of the crisis . The effPerhaps, in mature adults, the lack of drinking in bars or restaurants was counter-balanced by home drinking . AlthougIn terms of prevention of chronic alcohol-related morbidities, the results are somewhat satisfactory for the moment, and a spike of drinking disorders or alcohol dependency does not seem likely. However, an economic crisis as a result of the current pandemic could, in the medium-term, lead to the proposed alternative scenario with high unemployment rates and an increased alcohol consumption due to more free time .This study has limitations and is subject to different types of bias. It is a cross-sectional online-based survey and results cannot be reassessed in the future. Data and conclusions drawn are based on self-reported levels of alcohol consumption, which may be affected by information bias. Data were collected at only one single time point, and people were asked to provide their self-assessed consumption amount before lockdown retrospectively. It was object to selection bias, with an unsystematic distribution of the questionnaire and low return rates, and instrument bias, as there were a paper-based and an online version of the questionnaire. Most factors evaluated in the models interacted with the age group since they were not evenly distributed between groups. This and the low number of participants in the mature adults group limit the generalizability of the results mainly to students in Bavaria.In conclusion, we show that alcohol consumption in Bavaria, Germany, during COVID-19 lockdown is altered in an age-dependent manner. Although an increase of drinking was not observed right now, further studies exploring regional differences and long-term effects are warranted.Additional file 1: Table S1. Results by BMI group. Table S2. Results by gender. Table S3. Alcoholic beverages per week in young adults. Table S4. Alcoholic beverages per week in mature adults. Table S5. Ordinal logistic regression model for drinking less during lockdown.Additional file 2. Online Questionnaire .