Drug-resistant tuberculosis (DR-TB), obesity, and malnutrition are growing public health problems in the world. However, little has discussed the impact of different BMI status on the emergence of TB drug resistance. We aimed to explore the drug-resistant profiles of DR-TB and its clinical predictors among underweight, overweight or obesity population.8957 newly diagnosed TB cases with drug susceptibility results and BMI data in Shandong China, from 2004 to 2019 were enrolled. Multivariable and univariable logistic regression models were applied to investigate the impact of BMI on different drug-resistance. Clinical predicators and drug-resistant profiles of DR-TB among obesity, underweight, normal TB group were also described.P < 0.05.Among 8957 TB cases, 6417 (71.64%) were normal weight, 2121 (23.68%) were underweight, 373 (4.16%) were overweight, and 46 (0.51%) were obese. The proportion of drug resistance and co-morbidity among normal weight, underweight, overweight, obese TB groups were 18.86%/18.25%/20.38%/23.91% (DR-TB), 11.19%/11.74%/9.65%/17.39% , 3.41%/3.06%/5.36%/0.00% , 4.21%/3.39%/5.36%/6.52% , 10.57%/8.44%/19.57%/23.91% (co-morbidity), respectively. Compared with normal weight group, underweight were associated with lower risk of streptomycin-related resistance , but contributed to a higher risk of MR-TB (isoniazid) (odds ratio (OR) 1.347, 95% CI 1.049–1.730; adjusted OR (aOR) 1.31, 95% CI 1.017–1.686), P < 0.05. In addition, overweight were positively associated with MDR-TB , isoniazid + rifampicin + streptomycin resistance : 1.061–3.577; aOR 2.113, 95% CI 1.141–3.912), Any isoniazid + streptomycin resistance , The higher risk of MDR-TB, isoniazid + rifampicin + streptomycin resistance, Any isoniazid + streptomycin resistance, and co-morbidity among overweight population implies that routine screening for drug sensitivity and more attention on co-morbidity among overweight TB cases may be necessary. In addition, underweight TB cases have a higher risk of isoniazid resistance. Our study suggests that an in-depth study of the interaction between host metabolic activity and infection of DR-TB may contribute more to novel treatment options or preventive measures, and accelerate the implementation of the STOP TB strategy. Mycobacterium tuberculosis (MTB), contributes to a largest number of deaths from infectious diseases (more than HIV/AIDS) [Tuberculosis (TB), is caused by IV/AIDS) , 2. AccoIV/AIDS) . AdditioIV/AIDS) . Nine peIV/AIDS) .Multidrug-resistant tuberculosis (MDR-TB) is defined as TB with resistance to at least isoniazid (INH) and rifampin (RFP), which is usually associated with longer hospitalization, more expensive treatment, and higher mortality . The bac2 was independent risk factor of latent tuberculosis infection  1.17, 95% CI 1.04–1.33) [The critical influences of diverse nutritional status on TB infection have been recognized for decades . Recent 04–1.33) . In summThis research intended to explore the association between BMI and primary DR-TB in the aspects as follows: (1) to describe the clinical characteristics of TB cases with four different BMI status ; (2) to illustrate the drug resistant profiles of TB cases subgroups stratified by BMI; (3) to analyze the relative risk of DR-TB including DR-TB , MDR-TB , mono-resistant tuberculosis , polydrug resistant tuberculosis , RFP-related resistance, INH-related resistance, streptomycin (SM)-related resistance, MR-TB (INH), INH + RFP + SM resistance (MDR3), INH + SM resistance (PDR2), Any INH + SM resistance among subgroups with different BMI; (4) to investigate the risk factors of DR-TB among TB cases subgroups stratified by BMI.The Ethics Committee of Shandong Provincial Hospital (SPH) and Shandong Provincial Chest Hospital (SPCH) approved for our study. Personal information of TB patients such as names were erased before data analysis and reporting. All methods were performed in accordance with relevant guidelines and regulations. Informed consent was obtained from all participants or, if participants are under 18, from a parent and/or legal guardian.2 [2) was 3.5% (95% CI 3.4–3.6) [Our study was conducted in Shandong, a coastal province in eastern China (36°24′N latitude and 118° 24′ E longitude), with 100 million inhabitants and an area of 157,100 km2 . A large2 . The rat2 . As estiNontuberculosis mycobacteria (NTM) infection; (3) BMI information missing; (4) extra-pulmonary cases , meanwhile their basic demography and clinical characteristics were collected. Information of BMI, age, sex , drinking (yes or no), smoking (yes or no), cavity (yes or no), and co-morbidity were collected through questionnaire. Nutritional status indicators except BMI were not routinely collected. Exclusion criteria: (1) cases with previous TB history; (2) 2 were in the underweight range, between 18.5 and 24.9 kg/m2 were in normal weight range, between 25 and 29.9 kg/m2 were in the overweight range, ≥ 30 kg/m2 were in the obese range [BMI below 18.5 kg/mse range . Mono-rese range . Multidrse range . Polydruse range . MR-TB  of SPCH. Initially, at least two sputum specimens of each eligible patient were collected. Then, isolates were cultured in Löwenstein-Jensen (L-J) medium according to the standard protocol, and then these growing colonies were used for strain identification and phenotypic DST . DST forP value < 0.05. All data analyses were conducted in SPSS software (version 20.0).Categorical baseline demographic and clinical features of 8957 TB cases including age , sex (men or women), smoker or non-smoker, drinker or non-drinker, cavity (yes/no), co-morbidity (yes/no), and drug resistant profiles in four subgroups with different BMI status were compared by Pearson Chi-square test or Fisher’s exact test. The age subgroups were divided according to previous publications , 22. BinAs shown in Table P < 0.05. Underweight TB cases had a higher proportion of asthma (0.85% vs 0.37%) and COPD (2.55% vs 1.78%) but with a lower rate of diabetes (3.58% vs 7.03%) than normal group, P < 0.05. There were no significant differences in sex, cavity, smoking and drinking between lower and normal BMI cases. Both these two groups have more males, but less drinkers, smokers, and cavity  or > 65 (34.64% vs 22.98%) age group, and they had a lower rate of co-morbidity (8.44% vs 10.57%), P < 0.05. In addition, overweight TB cases are less likely to be in 15–24 age group (6.18% vs 15.63%), P < 0.05. There were no significant differences in 0–14 age group, 65 + age group, sex, smoking and drinking between overweight and normal cases (Table The proportions of 25–44 age group (33.33% vs 15.63%), 45–64 age group (39.78% vs 34.21%), cavity (51% vs 43.66%), co-morbidity (19.57% vs 10.57%), COPD (3.22% vs 1.78%), diabetes (13.94% vs 7.03%), hypertension (3.75% vs 1.82%), and cancer (0.80% vs 0.16%) were higher in The rates of females (32.61% vs 17.19%), co-morbidity (23.91% vs 10.57%), diabetes (19.57% vs 7.03%) were higher in obese TB cases than in normal cases, P < 0.05. There were no significant differences in age, smoking, drinking and cavity between obese and normal cases  or MDR3 (INH + RFP + SM) (3.22% vs 1.68%) cases among overweight group than normal group, P < 0.05. There were no significant differences in other drug resistant sub-types between abnormal weight groups and normal group.As shown in Table P = 0.028), but contributed to a higher risk of MR-TB (INH) . In addition, overweight was positively associated with MDR-TB , INH + RFP + SM resistance , Any INH + SM resistance . However, there were no statistical differences on drug resistance between obese group and normal weight group. In univariable and multivariable multinomial logistic regression models, we also found that overweight may be a risk factor for MDR-TB   and overweight cases . In addition, co-morbidity except COPD, diabetes, hypertension  were positively associated with DR-TB in underweight cases. Among normal weight group, male  or cavitary  pulmonary tuberculosis were more likely to have anti-TB resistance   and overweight (BMI = 24–26.9 kg/m2)  contributed to a lower risk of TB infection [2 was observed to be independently associated with LTBI  [Obesity has become a global public health problem, and it’s reported that approximately 46% of adults and 15% of children China are overweight or obese . Plenty 04–1.33) . Interes04–1.33) , 26. Fur04–1.33) . Therefo04–1.33) , 30. The04–1.33) , and theOverweight TB cases had a higher possibility of suffering from co-morbidity including COPD, hypertension, diabetes, and cancer. Actually, previous studies have figured out that BMI was a reliable predictor of prevalent diabetes, hypertension, and COPD –33. A stPeople have found a significant association between underweight and higher risk of active TB, adverse TB treatment outcomes, tuberculosis-related mortality , 37–39. It has been observed that males were more likely to be affected with DR-TB among normal, underweight, or overweight population. However, there were inconsistent results in the previous literature about the influence of gender differences on vulnerability to MDR-TB –44. A stThis study had some strengths. Firstly, it’s the first study to investigate the effect of BMI on drug resistance among newly diagnosed pulmonary TB cases. Secondly, the relative risk of numerous drug-resistant sub-types including DR-TB , MDR-TB , MR-TB , PDR-TB , RFP-related resistance, INH-related resistance, SM-related resistance, IMR-TB (INH), INH + RFP + SM resistance (MDR3), INH + SM resistance (PDR2), Any INH + SM resistance among underweight, overweight, obese were analyzed, and could provide clinical reference for future pathologic and molecular studies on different resistant MTB strains. Thirdly, the large amount (DST results from a province of 100 million people) and long time span (from 2004 to 2019) of our data guaranteed the reliance of our findings.This study also had some limitations. Firstly, drug resistance of second-line anti-TB drugs were not routinely examined in China unless at the initiative requirements of patients. Thus, the association between BMI and second-line anti-TB resistance remains to be discovered in future. Secondly, this was a retrospective study, and may have information bias. Thirdly, some obese patients can also be malnourished, since BMI is only one of prognostic factors for assessing malnutrition , 29, 45.Our study has important implications on the global triple epidemics of underweight, obesity, and DR-TB. The positive effect of overweight on MDR-TB, INH + RFP + SM resistance, Any INH + SM resistance, and co-morbidity implies that routine screening for drug sensitivity and more attention on co-morbidity among overweight TB cases may be necessary. Although patients with normal weight had a lower proportion of drug-resistance than overweight or obese patients in Shandong, screening for drug resistance cannot be ignored either because patients with normal weight accounted for 74.64%. In addition, we found underweight TB cases have a higher risk of INH resistance, which provides a reference for clinical rational use of drugs. Our study suggests that an in-depth study of the interaction between host metabolic activity and infection of DR-TB may contribute more to novel treatment options or preventive measures, and accelerate the implementation of the STOP TB strategy.


In December 2020, B.1.1.7 lineage of SARS-CoV-2 was first detected in the United States and has since become the dominant lineage. Previous investigations involving B.1.1.7 suggested higher rates of transmission relative to non-B.1.1.7 lineages. We conducted a household transmission investigation to determine the secondary infection rates (SIR) of B.1.1.7 and non-B.1.1.7 SARS-CoV-2 lineages. From January–April 2021, we enrolled members of households in San Diego County, CA, and Denver, CO metropolitan area (Tri-County), with a confirmed SARS-CoV-2 infection in a household member with illness onset date in the previous 10 days. CDC investigators visited households at enrollment and 14 days later at closeout to obtain demographic and clinical data and nasopharyngeal (NP) samples on all consenting household members. Interim visits, with collection of NP swabs, occurred if a participant became symptomatic during follow-up. NP samples were tested for SARS-CoV-2 using TaqPath™ RT-PCR test, where failure to amplify the spike protein results in S-Gene target failure (SGTF) may indicate B.1.1.7 lineage. Demographic characteristics and SIR were compared among SGTF and non-SGTF households using two-sided p-values with chi-square tests; 95% confidence intervals (CI) were calculated with Wilson score intervals.552 persons from 151 households were enrolled. 91 (60%) households were classified as SGTF, 57 (38%) non-SGTF, and 3 (2%) indeterminant. SGTF and non-SGTF households had similar sex distribution  and age  and 31 years (IQR 15–45), respectively). Hispanic people accounted for 24% and 32% of enrolled members of SGTF and non-SGTF households, respectively (p=0.04). At least one secondary case occurred in 61% of SGTF and 58% of non-SGTF households (P=0.66). SIR was 52% (95%[CI] 46%-59%) for SGTF and 45% (95% CI 37%-53%) for non-SGTF households (P=0.18). SIRs were high in both SGTF and non-SGTF households; our findings did not support an increase in SIR for SGTF relative to non-SGTF households in this setting. Sequence confirmed SARS-CoV-2 samples will provide further information on lineage specific SIRs.All Authors: No reported disclosures


The elevated plus maze test was employed to evaluate anxiety-like behavior in rats, and in vivo microdialysis was used to measure the extracellular NE level in the BNST. In elevated plus maze tests, EtOHW rats but not EtOH-naive rats exhibited anxiety-like behavior when challenged with 7-minute mild restraint stress, which was, respectively, mitigated by prior intra-NTS infusion of the nitric oxide scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), nonselective NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME), or selective neuronal NOS (nNOS) inhibitor 7-nitroindazole (7-NI). Each of these agents also decreased the plasma corticosterone levels in EtOHW rats. In in vivo microdialysis, prior intra-NTS infusion of carboxy-PTIO, L-NAME, or 7-NI attenuated the mild stress-induced NE release in the BNST of EtOHW rats. Additionally, EtOHW rats showed increased solitary nNOS gene and protein expression. Moreover, the anxiolytic effect of intra-NTS administration of 7-NI was abolished by subsequent intra-NTS administration of sodium nitroprusside. These results suggest that elevation of solitary nitric oxide signaling derived from nNOS mediates stress-precipitated anxiety and norepinephrine release in the BNST during protracted EtOHW.Ethanol withdrawal (EtOHW) alters the pattern of neurohormonal and behavioral response toward internal and external stimuli, which mediates relapse to alcohol use even after a long period of abstinence. Increased noradrenergic signaling from the nucleus tractus solitarius (NTS) to the bed nucleus of the stria terminalis (BNST) during EtOHW underlies withdrawal-induced anxiety, while nitric oxide synthase (NOS) inhibitors injected into the periaqueductal area attenuate EtOHW-induced anxiety. Therefore, this study investigated the involvement of NOS within the NTS in anxiety and increased norepinephrine (NE) release in the BNST during protracted EtOHW in rats exposed to a mild stress. Rats were intraperitoneally administered 3 g/kg/day EtOH for 21 days followed by 28 days of withdrawal, and on the 28 Relapse is a major barrier in alcoholism therapy . EthanolElevated anxiety during EtOHW is the major negative emotional component for alcoholism relapse , and sevβ-adrenergic antagonists reduced the withdrawal-associated conditioned place aversion . However, as also seen in Figures into open arms: F = 9.22, p < 0.001, EtOH/vehicle/non-AMRS group (n = 8) versus EtOH/vehicle/AMRS group (n = 8), p < 0.001, EtOH/vehicle/AMRS group versus EtOH/carboxy-PTIO/AMRS group (n = 8), p < 0.01; EtOH/vehicle/AMRS group versus EtOH/L-NAME/AMRS group (n = 8), p < 0.01; EtOH/vehicle/AMRS group versus EtOH/7-NI/AMRS group (n = 8), p < 0.01; EtOH/vehicle/AMRS group versus EtOH/L-NIO/AMRS group (n = 8), p > 0.05; %timespent in open arms: F = 8.05, p < 0.001, EtOH/vehicle/non-AMRS group versus EtOH/vehicle/AMRS group, p < 0.001, EtOH/vehicle/AMRS group versus EtOH/carboxy-PTIO/AMRS group, p < 0.01; EtOH/vehicle/AMRS group versus EtOH/L-NAME/AMRS group, p < 0.01; EtOH/vehicle/AMRS group versus EtOH/7-NI/AMRS group, p < 0.01; EtOH/vehicle/AMRS group versus EtOH/L-NIO/AMRS group, p > 0.05]. Moreover, at the same dose, intra-NTS 7-NI alone did not produce any significant behavioral changes in rats (data not shown).As shown in Figures F(drug) = 19.92, p < 0.001, F(stress) = 32.25, p < 0.001, F(drug × stress) = 13.96, p < 0.01; saline/non-AMRS group (n = 6) versus EtOH/AMRS group (n = 6), p < 0.001; EtOH/non-AMRS group (n = 6) versus EtOH/AMRS group, p < 0.001; saline/AMRS group versus EtOH/AMRS group, p < 0.001]. However, as shown in F = 11.71, p < 0.001; EtOH/vehicle/non-AMRS group (n = 6) versus EtOH/vehicle/AMRS group (n = 6), p < 0.001, EtOH/vehicle/AMRS group versus EtOH/carboxy-PTIO/AMRS group (n = 6), p < 0.01], L-NAME , or 7-NI , but not L-NIO , inhibited the increased CORT secretion.Consistent with the results of the behavioral, as seen in n = 7): 4.25 ± 0.32 (pg/15 uL); saline/vehicle/AMRS group (n = 7): 4.75 ± 0.48; EtOH/vehicle/non-AMRS group (n = 7): 4.64 ± 0.41; EtOH/vehicle/AMRS group (n = 7): 5.19 ± 0.44; EtOH/carboxy-PTIO/AMRS group (n = 7): 4.38 ± 0.37; EtOH/L-NAME/AMRS group (n = 7): 4.23 ± 0.38; EtOH/7-NI/AMRS group (n = 7): 5.25 ± 0.37; EtOH/L-NIO/AMRS group (n = 7): 4.81 ± 0.37]. As seen in Figures F(treatment) = 0.07, p > 0.05, F(time) = 2.25, p > 0.05, F(treatment × time) = 0.70, p > 0.05; in F(treatment) = 16.79, p < 0.001; F(time) = 25.63, p < 0.001, F(treatment × time) = 9.55, p < 0.001; 15 min: saline/vehicle/AMRS group versus EtOH/vehicle/AMRS group, p < 0.001; 30 min saline/vehicle/AMRS group versus EtOH/vehicle/AMRS group, p < 0.05]. However, as shown in F(treatment) = 9.62, p < 0.001, F(time) = 87.92, p < 0.001, F(treatment × time) = 4.53, p < 0.001; 15 min: EtOH/vehicle/non-AMRS group EtOH/vehicle/AMRS group, p < 0.001, EtOH/vehicle/AMRS versus EtOH/carboxy-PTIO/AMRS group, p < 0.001; 30 min: EtOH/vehicle/non-AMRS group versus EtOH/vehicle/AMRS group, p < 0.01, EtOH/vehicle/AMRS group versus EtOH/carboxy-PTIO/AMRS group, p < 0.01], L-NAME , or 7-NI , but not L-NIO , prevented these increases. In addition, intra-NTS L-NIO alone did not significantly affect NE release in naive rats (data not shown).No significant differences in the basal extracellular NE level in the vBNST were found between the groups , whereas the AMRS did not alter nNOS protein expression . However, the AMRS markedly enhanced phospho-nNOS levels in EtOHW rats  but not in saline-treated controls . Additionally, neither EtOHW nor AMRS significantly affected eNOS or phospho-eNOS protein expressions .As shown in F(drug) = 84.40, p < 0.001, F(stress) = 0.04, p > 0.05, F(drug×stress) = 0.06, p > 0.05; saline/non-AMRS group (n = 6) versus EtOH/non-AMRS group (n = 6), p < 0.001; saline/AMRS group (n = 6) versus EtOH/AMRS group (n = 6), p < 0.001]. The AMRS did not alter nNOS mRNA expression .In agreement with the Western blot data, as seen in into open arms: F = 15.87, p < 0.001; saline/vehicle/AMRS/vehicle group (n = 6) versus EtOH/vehicle/AMRS/vehicle group (n = 6), p < 0.001; EtOH/vehicle/AMRS/vehicle group versus EtOH/7-NI/AMRS/vehicle group (n = 6), p < 0.001; EtOH/7-NI/AMRS/vehicle group versus EtOH/7-NI/AMRS/SNP group (n = 6), p < 0.01; saline/vehicle/AMRS/vehicle group versus EtOH/7-NI/AMRS/SNP group, p < 0.001; %timespent in open arms: F = 11.17, p < 0.001; saline/vehicle/AMRS/vehicle group versus EtOH/vehicle/AMRS/vehicle group, p < 0.001; EtOH/vehicle/AMRS/vehicle group versus EtOH/7-NI/AMRS/vehicle group, p < 0.01; EtOH/7-NI/AMRS/vehicle group versus EtOH/7-NI/AMRS/SNP group, p < 0.05; saline/vehicle/AMRS/vehicle group versus EtOH/7-NI/AMRS/SNP group, p < 0.01].To further determine the involvement of solitary nNOS in AMRS-induced anxiety during protracted EtOHW, another cohort of EtOHW rats was sequentially treated with 7-NI and SNP and then tested in the EPM . As showThe results of the present study showed that 7-minute AMRS provoked anxiety-like behaviors, enhanced plasma CORT secretion, and sensitized NE release in the vBNST in rats treated with EtOH but not saline, at 28 days after the final dose of EtOH or saline. However, all of these behavioral, hormonal, and neurochemical abnormalities were attenuated by prior intra-NTS infusion of carboxy-PTIO, L-NAME, or 7-NI, but not by L-NIO. EtOHW elevated nNOS, but not eNOS, protein expression in the NTS, concomitant with an increased nNOS mRNA level, and the AMRS increased the phosphorylation rate of nNOS in the NTS of EtOHW rats. Moreover, intra-NTS injection of SNP after 7-NI administration abolished the expected anxiolytic action of 7-NI. Taken together, these results suggest a critical role of solitary nNOS in anxiety and vBNST NE release induced by acute mild stress during protracted EtOHW.The susceptibility to stress during protracted EtOHW alters neurotransmission responses to certain stimuli that are normally innocuous to provoke pathophysiological consequences , 27. To Both anxiety and plasma CORT secretion are closely associated with increased noradrenergic transmission in the BNST , 33. In The NTS primarily integrates and transmits visceral and external information to the forebrain, forming the autonomic-affective functional basis for the body. The gaseous molecule NO serves as both a neurotransmitter and neuromodulator, and is synthesized by three isoforms of NOS, i.e., nNOS, iNOS, and eNOS. We reported previously that systemic nicotine administration increased hypothalamic NE release via activation of both nNOS and eNOS in the NTS . HoweverIn summary, prior intra-NTS infusion of carboxy-PTIO, L-NAME, or 7-NI, but not L-NIO, attenuated the anxiety and vBNST NE release induced by 7-minute AMRS during EtOHW. EtOHW enhanced both nNOS protein and mRNA expression in the NTS but did not affect the eNOS protein level. These observations suggest that nNOS activity is promoted in the NTS during protracted EtOHW, which sensitizes the NTS-BNST noradrenergic response to stress and results in anxiety-like behavior in rats .


Long term sequelae across multiple medical domains, including the respiratory, psychiatric, and neurocognitive have been reported after COVID-19. Studies evaluating the impact of this symptom burden, however, are lacking. We aimed to describe the self-reported occurrence of symptoms and their effect on patient functioning six months after their acute hospitalization for COVID-19. From a historical cohort study of patients hospitalized for COVID-19 between March 8, and June 14, 2020, we identified patients discharged home. The purpose of the study was explained, and they were asked to consent to a telephone questionnaire. We used a modified version of a previously validated general symptom questionnaire (GSQ-30) to assess multi-system symptom burden. The Patient Health Questionnaire-2 (PHQ-2) was used to screen for major depression. Of the original 565 patients, 258 patients were discharged home (45%). Of these, 57 (22%) patients were able to be contacted and agreed to participate in the survey. The mean (SD) age of the respondents was 55.1 (14.8) years, and 37 (64.9%) were female. The most common symptoms at follow-up were fatigue (60.0%), dyspnea (57.1%), feeling irritable, sad or decreased pleasure (56.4%), and memory difficulty (56.4%). Females had a significantly higher GSQ score (0.02) than males. Patients ages < 60 years tended to experience similar, if not greater, impaired functioning (p=0.07) compared with those ages ≥ 60 years (Table 1). Females were more likely to be irritable or sad (p=0.007), not feel rested on awakening (p=0.04), have shooting, stabbing and burning pain (p=0.02), have discomfort with normal light and sound (p=0.04), and have memory difficulty (p=0.04) than males (Table 2). Table 1. Self-Reported Post-Acute Sequelae of COVID syndrome in adults younger than 60 versus adults at or older than 60 Years. SD: Standard deviation, ICU: Intensive care unit, ED: Emergency department, GSQ - General symptom questionnaire, PHQ-2: Patient Health Questionnaire-2Table 2. Self-Reported Post-acute Sequelae of COVID syndrome in female versus male adults. SD: Standard deviation, ED: Emergency department, GSQ - General symptom questionnaire, PHQ-2: Patient Health Questionnaire-2Our study describes the clinical burden of post-acute sequelae of COVID-19 (PASC) in four core domains: fatigue, neurologic, neuro-psychiatric and viral-like symptoms. Over 45% of patients ages < 60 years suffered impaired functioning, compared with 21.1% of patient’s ages 60 years and above. Females had significantly higher GSQ scores than men which strongly corelates with the functional impairment among the females. Larger studies are needed to further validate our findings.All Authors: No reported disclosures


Caseins are major milk proteins that have an evolutionarily conserved role in nutrition. Sequence variations in thecasein genes affect milk composition in livestock species. Regulatory elements of the casein genes could be used to directthe expression of desired transgenes into the milk of transgenic animals. Dozens of casein alleles have been identified forgoats, cows, sheep, camels and horses, and these sequence variants are associated with altered gene expression and milkprotein content. Most of the known mutations affecting casein genes’ expression are located in the promoter and 3’-untranslated regions. We performed pronuclear microinjections with Cas9 mRNA and sgRNA against the first coding exon ofthe mouse Csn1s1 gene to introduce random mutations in the α-casein (Csn1s1) signal peptide sequence at the beginningof the mouse gene. Sanger sequencing of the founder mice identified 40 mutations. As expected, mutations clusteredaround the sgRNA cut site (3 bp from PAM). Most of the mutations represented small deletions (1–10 bp), but we detectedseveral larger deletions as well (100–300 bp). Functionally most mutations led to gene knockout due to a frameshift or astart codon loss. Some of the mutations represented in-frame indels in the first coding exon. Of these, we describe a novelhypomorphic Csn1s1 (Csn1s1c.4-5insTCC) allele. We measured Csn1s1 protein levels and confirmed that the mutation has anegative effect on milk composition, which shows a 50 % reduction in gene expression and a 40–80 % decrease in Csn1s1protein amount, compared to the wild-type allele. We assumed that mutation affected transcript stability or splicing by anunknown mechanism. This mutation can potentially serve as a genetic marker for low Csn1s1 expression. Caseins are major milk proteins that have an evolutionaryconserved role in nutrition . Casein locushas been studied for a long time to understand the principleof gene regulation and hormone-induced expression . At thesame time, regulatory elements of the casein genes could beused to direct the expression of desired transgenes into milkof transgenic animals .This strategy is frequently employed to create “enriched”milk with improved composition , or to achieve large scale productionof recombinant human proteins in mouse models  and in livestockspecies . Milk-specific signal peptidesare used in biotechnology to enhance recombinant proteinsecretion during lactation . During breeding, casein genes acquired many sequencevariations that can lead to altered gene expression and arecharacteristic of some goat and cow breeds . Many of thesesequence variants could be used as markers for breeding. Indairy industry casein composition is an important milk traitdirectly influencing quality of dairy products . Hypomorphic casein mutationare also associated with other traits such as litter size . For example, K. Wang with colleagues showedthat 11 bp del in the intron 8 of the goat Csn1s1 negativelyaffects the expression of the gene . Otherknown hypomorphic mutations are located in the promoterand 3′-untranslated regions (UTRs) of casein genes . Novel CRISPR methods greatly facilitate genome editing infarm animals , including targeted transgeneintegration  and mutation modeling . The latter approach has potential toexplain the molecular mechanism of how hypomorphic mutations affect milk proteins. In this report, we used CRISPR/Cas9to create a set of mutations within a signal peptide sequenceof the α-casein (Csn1s1) gene in mice. One of the mutantswas chosen to study effects of a small in-frame insertion onthe Csn1s1 expression during lactation.Generation and genotyping of the Csn1s1 mutant mice.In vitro transcription and purification of the gRNA wereperformed with MEGAshortscript™ T7 Transcription Kit(Thermo Fisher Scientific) and MEGAclear™ TranscriptionClean-Up Kit (Thermo Fisher Scientific) according to themanufacturer’s protocol. Cas9 mRNA (GeneArt™ CRISPRNuclease mRNA) was purchased from Thermo Fisher Scientific. 50 ng/μL Cas9 mRNA and 25 ng/μL gRNA (5′-GTGAGGATGAGGAGTTTCA-3′) were mixed in RNase-free water,backfilled into an injection needle with positive balancingpressure  and injected into thecytoplasm of zygotes (C57BL/6 × CBA background). Afterinjections, the embryos were cultured for 1 hour in drops ofM16 medium at 37 °C and an atmosphere of 5 % CO2. Theviable microinjected zygotes were transplanted the same day into oviducts of pseudopregnant CD-1 females (0.5 days aftercoitus). Isoflurane inhalation anesthesia was applied in theseexperiments.1.Primers for PCR were as follows: 5′-GCGCATAACTAAGCATCTTATGCT-3′ (forward primer), 5′-TGACTTGGAGTTTTAGATTTGGACA-3′ (reverse primer). Selected malemice founders were crossed with C57BL/6 females. Formutation c.4-5insTCC described in this paper, founder malewas crossed with two F1 heterozygous daughters. Offspringwas genotyped and two sibling females were selected foreach group  forfurther analysis.Mutations were detected using PCR and Sanger sequencingof the target region of the Csn1s1 exon 2 (Supplementary 1)http://www.bionet.nsc.ru/vogis/download/pict-2021-25/appx8.pdfSupplementary materials 1 and 2 are available at: All experiments were conducted at the Centre for Genetic Resources of Laboratory Animals at the Institute ofCytology and Genetics, SB RAS (RFMEFI61914X0005 andRFMEFI61914X0010). All experiments were performed inaccordance with protocols and guidelines approved by theAnimal Care and Use Committee Federal Research Centreof the Institute of Cytology and Genetics, SB RAS operatingunder standards set by regulations documents Federal HealthMinistry (2010/708n/RF), NRC and FELASA recommendations. Experimental protocols were approved by the BioethicsReview Committee of the Institute of Cytology and Genetics,SB RAS.Droplet digital PCR analysis. Total cellular RNA wasextracted from mouse mammary glands at day 8 of lactationusing TRI Reagent (Sigma-Aldrich). 1 μg of total RNA wasused to generate cDNA in a 20 μl reaction using RevertAidRT Kit (Thermo Fisher Scientific) with random hexamerprimers according to the manufacturer’s instructions. DropletDigital PCR (ddPCR) was performed using a QX100 system(Bio-Rad) with primers and probes specific for the Csn1s1and Csn2 mouse transcripts (Supplementary 2). The primersand probes sequences were as follows: 5′-TGTAGTGGATCAGGCACTGG-3′ (Csn1s1 forward primer), 5′-TCCTTGGAGACAATGGGCTT-3′ (Csn1s1 reverse primer), 5′-HEXCCAGTTCTCTGTTCAGCCCTTCCCACA-BHQ2–3′(Csn1s1 probe), 5′-AGGACTTGACAGCCATGAAGG-3′(Csn2 forward primer), 5′-ATGTTCAACAGATTCCTCACTGGA-3′ (Csn2 reverse primer), 5′-FAM-ATCCTCGCCTGCCTTGTGGCCCTTGC-BHQ1–3′ (Csn2 probe). ddPCRreactions were set in 20 μl volumes containing 1× ddPCR™Supermix for Probes (no dUTP), 900 nM primers and 250 nMprobes, and 1 μl of 5000-fold diluted cDNA. ddPCR reactionsfor each sample were performed in duplicates. PCR was conducted according to the following program: 95 °C for 10 min,then 40 cycles of 95 °C for 30 s and 61 °C for 1 min, witha ramp rate of 2 °C per second, and a final step at 98 °C for5 min. The results were analyzed using QuantaSoft software(Bio-Rad). Concentrations of cDNA copies of Csn1s1 andCsn2 were derived from ddPCR and relative expression ofCsn1s1 to Csn2 was calculated for each animal.Milk and mammary gland protein analysis. Milk wasobtained from narcotized female mice at day 8 of lactationafter oxytocin administration . Themilk was collected with a pipette attached to an aspiration device, transferred into a microcentrifuge tube and stored at–80 °C. Inguinal mammary glands (MGs) were extracted fromthe same (euthanized) mice and stored at –80 °C. For protein extraction MGs were minced in Dounce homogenizers,resuspended in RIPA buffer  with protease inhibitor cocktail (1x Complete ULTRA(Roche), 1x PhosSTOP (Roche), 5 mM NaF (Sigma)). Thelysates were incubated on ice for 30 min and then centrifugedat 4 °C for 10 min at 10 000 g. Supernatant was sonicatedand stored at –80 °C. Total protein concentrations for milkand MG lysates were determined with Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific), according to themanufacturer’s instructions. To prepare samples for SDSPAGE, milk or MG protein samples were mixed with RIPAand SDS-PAGE loading buffer (Bio-Rad) to a final concentration of 1 μg/μl and heated at 65 °C for 20 minutes. Thesamples (25 μg) were separated on a 12 % polyacrylamidegel and stained with Coomassie Blue G-250. ThermoFisherPageRuler™ Prestained Protein Ladder (10 to 180 kDa) wasused as a protein molecular weight marker. Csn1s1, albuminand total protein concentrations were evaluated using QuantityOne (Bio-Rad) and ImageJ software.Coomassie-stained gel was used for wet transfer of theproteins to PVDF membrane .The membrane was blocked with 5 % milk in TBST  for 2 hoursand incubated with primary mouse anti-Csn1s1 antibodies(1:1000)  overnight at4 °C. Next day membrane was repeatedly washed with TBSTand incubated with secondary mouse HRP-antibodies (1:1000) at 25 °C for 2 hours.Immunodetection was performed with ECL substrate solution, according tothe manufacturer’s instructions.Generation of the Csn1s1 mutant miceWe performed pronuclear microinjections with Cas9 mRNAand sgRNA against the first coding exon of the mouse Csn1s1gene . Cas9-induced mutations in this region couldpotentially affect signal peptide coding sequence and leadto altered milk composition. We screened founder mice by Sanger sequencing and identified multiple random mutations at the Cas9 cut site (presented in Supplementary 1).The sgRNA targeted the Csn1s1 site with high efficiencyas we detected 41 mutant alleles, 4–5 mosaic alleles  and only 4–5 wild-type allelesin 20 founder mice . Asexpected, mutations clustered around the sgRNA cut site(3 bp from PAM). Most of the mutations represented smalldeletions (1–10 bp), but we detected several larger deletionsas well (100–300 bp). Of note, some of the unique mutationvariants had increased incidence rate. For example, 12 bpdeletion (GAAACTCCTCAT) arose independently four timesand another 10 bp deletion (CCATGAAACT) – three times(see Supplementary 1). We suspect this bias towards somevariants is caused by microhomology-mediated end-joining(MMEJ), since these two mutations are flanked with 3 and 2similar nucleotides  (see Supplementary 1). Although mutations were mostly deleterious forthe gene expression and led to a Csn1s1 knockout (KO) byframeshift, several in-frame mutation variants resulted insubtle changes in signal peptide coding sequence withoutKO (see Supplementary 1). We selected one of the mutants,tagged Csn1s1c.4-5insTCC, which had a 3 bp insertion followingthe start codon . To study gene expression andmilk composition we chose 6 female siblings  from the Csn1s1c.4-5insTCCline for milk and mammary glands collection. Mutation c.4-5insTCC leads to reducedexpression of the Csn1s1 geneWe estimated the Csn1s1 gene expression in mammary glandsof wild-type and mutant mice at day 8 of lactation usingdroplet digital PCR (ddPCR). We selected Csn2 (β-casein) asa reference gene as it has a similar expression profile in mammary gland (see Supplementary 2). ddPCR analysis revealedthat Csn1s1:Csn2 ratio was roughly 1:3 (0.338) in wild-typesiblings , which is in agreement with publisheddata . Heterozygous and homozygousCsn1s1c.4-5insTCC siblings showed lower Csn1s1 expressionwith ratios around 1:4 (0.248) and 1:6 (0.168), respectively, compared to wild-type siblings. We also usedfemales from the parental strains C57BL/6 and CBA as controls for normal caseins level . In rare cases, Cas9activity can provoke rearrangements near the target locus.We sequenced mutation-flanking regions, including theCsn1s1 promoter and surrounding introns (2.3 kb+1.1 kb)from homozygous mice (data not shown). We also sequencedtop two off-targets for the Csn1s1 sgRNA in the F0 founder. No mutations werefound in the examined sequences. Thus, we could confirmthat the 3 bp in-frame insertion in the first coding exon led toa 30–50 % decrease in the Csn1s1 gene expression. Milk protein composition in c.4-5insTCC mice We measured Csn1s1 protein levels and confirmed that mutation has a negative effect on milk composition. We collectedmilk and mammary glands from lactating females at the day 8of lactation. Csn1s1 knockout mouse was taken from anotherexperiment (“KO”) as additional control for the Csn1s1 levels.Separation of milk proteins on 12 % SDS‐PAGE resulted ina typical band pattern for mouse milk . In wildtype mice, Csn1s1 represents a major protein fraction andcorresponds to approx. 30 % of total milk protein . In homozygous mutants, loss of Csn1s1 could beobserved both at the Coomassie-stained gel  andafter western blotting . Csn1s1 levels fell down to30 % in homozygotes, both for milk and for mammary glandlysates . However, exact ratios varied depending on the control protein band used for calculations. For instance, we performed the following calculations for the milkCsn1s1: Csn1s1 (gel) vs total protein (gel) – 40 % reduction; Csn1s1 (gel) vs albumin (gel) – 70 % reduction; Csn1s1(western blot membrane) vs total protein (gel) – 80 % reduction. This effect was even more pronounced in mammarygland lysates (intracellular casein levels): Csn1s1 (westernblot membrane) vs total gel – 80 % reduction.We report a novel in-frame Csn1s1 hypomorphic mutationthat leads to a 50 % gene expression decrease in mice. Inmost cases, mutation effect is tied to disruption of a regulatory element  . Frame-shifting indels in the coding sequencecould initiate transcript surveillance pathway called nonsense-mediated mRNA decay (NMD) . Alternatively, in-frame mutationscan lead to exon removal by alternative splicing . Essentially, exon skipping could be promoted by internal exon splicing enhancersand suppressors (ESEs and ESSs) which are hard to predict, unliketypical splice site mutations . In ourmutant mice, promoter had no alterations as the mutationhappened in the coding sequence, quite far from a transcription start site. We assumed that it affected transcript stabilityor splicing by unknown mechanism. It should also be notedthat mutated Csn1s1 protein was still secreted in milk, thusthe function of N-terminal signal peptide was not criticallyaffected by the mutation. 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The kidneys are among the organs most affected by leukemic involvement in chronic lymphocytic leukemia (CLL) patients in postmortem autopsy series 63%-90%) ,2. Howev%-90% ,2.9/L (lymphocytes: 60%); platelets, 247x109/L. The blood ﬁlm appearances were suggestive of CLL. The following biochemical tests were abnormal: blood urea nitrogen, 44 mg/dL ; serum creatinine (sCr), 6.16 mg/dL ; CKD-EPI estimated-glomerular filtration rate, 8 mL/min/1.73 m2 . His urine had no leukocytes or casts, with 3 erythrocytes/field and proteinuria in the non-nephrotic range (1779 mg/24 h). Serological tests for hepatitis B, hepatitis C, HIV, and autoimmune kidney disorders were negative. Serum-free light chain (sFLC) kappa was 317.5 mg/dL  and sFLC lambda was 22.2 mg/dL , but no monoclonal bands in serum or urine immunofixation were detected. Renal ultrasound showed normal-sized kidneys with no evidence of obstructive nephropathy. A renal biopsy was performed, which demonstrated heavy infiltration of diffuse monomorphic neoplastic lymphocytes in the interstitium (9/L). After a year of follow-up, his renal function had improved to sCr of 2.04 mg/dL, and a complete blood count revealed hemoglobin of 14.7 g/dL, leukocytes of 9.15x109/L, and platelets of 294x109/L.A 72-year-old white male without any renal disease presented with general malaise and weight loss. He had been diagnosed with stage B(1) B-CLL four years before and was treated with a bendamustine and rituximab protocol. Physical examination revealed general lymphadenopathy and hepatomegaly. Complete blood count findings were as follows: hemoglobin, 11.3 g/dL; leukocytes, 411.4x10rstitium . Six of rstitium  and 1C wBesides LI, renal impairment in CLL patients can be associated with prerenal azotemia, thrombotic microangiopathy, acute tubular necrosis, acute interstitial nephritis, uric acid nephropathy, light chain nephropathy, amyloidosis, obstructive nephropathy, glomerulonephritis, and cryoglobulinemia . LI of t


Correction to: J Foot Ankle Res 14, 15 (2021)https://doi.org/10.1186/s13047-020-00435-7Following publication of the original article , we wereThe original article has been corrected. Additional file 2 has been replaced.Additional file 2. Development of the systematic podiatry protocol.


A total of 50 neonates  participated in this study. LBW-VLBW neonates were further stratified as those with  and without  structural MRI (sMRI) abnormalities. TRUST and PC MRI studies were undertaken to determine OEF, CBF, and CMRO2. Ultimately, CMRO2 proved significantly lower (p = 0.01) in LBW-VLBW (vs term) neonates, both LBW-VLBW-a and LBW-VLBW-n subsets showing significantly greater physiologic deficits than term controls . CMRO2 and CBF in LBW-VLBW-a and LBW-VLBW-n subsets did not differ significantly (p > 0.05), although OEF showed a tendency to diverge (p = 0.15). However, OEF values in the LBW-VLBW-n subset differed significantly from those of term controls (p = 0.02). Compared with brain volume or body weight, these physiologic parameters yield higher area-under-the-curve (AUC) values for distinguishing neonates of the LBW-VLBW-a subset. The latter displayed distinct cerebral metabolic and hemodynamic, whereas changes were marginal in the LBW-VLBW-n subset  by comparison. Physiologic imaging may therefore be useful in identifying LBW-VLBW newborns at high risk of irreversible brain damage.Low birth-weight (LBW) and very low birth-weight (VLBW) newborns have increased risks of brain injuries, growth failure, motor difficulties, developmental coordination disorders or delay, and adult-onset vascular diseases. However, relatively little is known of the neurobiologic underpinnings. To clarify the pathophysiologic vulnerabilities of such neonates, we applied several advanced techniques for assessing brain physiology, namely T2-relaxation-under-spin-tagging (TRUST) magnetic resonance imaging (MRI) and phase-contrast (PC) MRI. This enabled quantification of oxygen extraction fraction (OEF), global cerebral blood flow (CBF), and cerebral metabolic rate of oxygen (CMRO Preterm newborns of low birth weight  or very low birth weight  present a substantial public health problem. In 2015, the worldwide prevalence was 14.6%, with 91% confined to low- and middle-income countries [primarily Southern Asia (24%) and sub-Saharan Africa (48%)]. More than 80% of perinatal deaths occur in LBW neonates .Although most LBW and VLBW preterm infants show catch-up gains in height and weight, they are still at increased risk of brain injuries , growth 133Xenon clearance  technologies have allowed noninvasive and quantitative measurements of critical neurophysiologic parameters, without need of contrast agents. The oxygen extraction fraction (OEF) of the brain is assessable using T2-relaxation-under-spin-tagging (TRUST) MRI technique , and glo2 using T2 prepared tissue relaxation inversion recovery (T2-TRIR) pulse sequences and ASL (2 and CBF in asphyxiated newborns with severe HIE (r = 0.88)  is another valuable means of perfusion MRI for this purpose. However, its signal-to-noise ratio (SNR) tends to be lower, the scan times lengthier, and there is a potential for confounding factors . In an earlier effort, infants with hypoxic ischemic encephalopathy (HIE) showed lower CBF and CMRO and ASL . Newborn = 0.88) . Our pre = 0.88) . Tortora = 0.88) . There h2 in a mixed group of LBW and VLBW newborns, comparing outcomes with those of term neonates.In the present study, we used TRUST MRI and PC MRI to determine OEF, CBF, and CMROn = 41) at birth weights >1,000 g but <2,500 g. Term neonates serving as controls (n = 9) had birth weights ≥2,500 g, were ≥37 weeks at birth, and showed no sMRI abnormalities. Population characteristics and results of blood gas analysis were listed in Our investigational protocol received approval from the Ethics Committee at Shengjing Hospital of China Medical University. Between June 2016 and December 2018, a total of 50 newborns accrued for study. The structural MRI pulse sequences performed included axial and sagittal T1-weighted imaging (TIWI), axial T2-weighted imaging (T2WI), and diffusion-weighted imaging (DWI). Congenital malformations, severe infections, or unusable MRI studies were grounds for exclusion. Subjects qualified as LBW-VLBW  obtained from all infants (LBW-VLBW and term) were viewed as Picture Archiving and Communication Systems (PACS) files by two neuroradiologists (YQ and XYS) with 10 years of experience, each blinded to group data. They independently scored MRI abnormalities for each infant, using a method similar to one already described  that focA 3T MRI system equipped with a phased-array head coil  was used for all MR scans. Prior to imaging procedures, a pediatrician sedated infants through nasal feedings of chloral hydrate (50 mg/kg). This is standard clinical practice at our institution. All newborns were well fed, visually monitored, and hearing protected during scans.2; and scan time, 30 s.Axial T1-weighted spin-echo images were acquired at the following settings: repetition time/echo time (TR/TE), 200 ms/2.3 ms; section thickness, 5 mm; field of view (FOV), 180 mm × 150 mm × 89 mm; matrix size, 224 × 162; and scan time, 34.4 s. For sagittal T1-weighted spin-echo images, the following settings were used: TR/TE, 250 ms/2.3 ms; section thickness, 5 mm; FOV, 230 mm × 230 mm × 107 mm; matrix size, 256 × 250; and scan time, 33.0 s. Axial T2WI used the following settings: TR/TE, 5,000 ms/80 ms; section thickness, 5 mm; FOV, 180 mm × 150 mm × 90 mm; matrix size, 112 × 112; and scan time, 40.9 s. Axial echo-planar imaging (EPI) DWI was performed as follows: TR/TE, 3,500 ms/63 ms; section thickness, 5 mm; FOV, 180 mm × 180 mm × 89 mm; b values, 0 and 1,000 s/mmOxygen extraction fraction was calculated as follows:where Ya and Yv signify arterial and venous oxygenation, respectively. Ya was measured peripherally, via pulse oximeter applied to neonatal toes. Yv was measured by TRUST MRI technique, given that blood T2 has a calibrationable relation with oxygenation level. This sequence utilizes a spin-labeling module to isolate pure venous blood signals, thereafter applying a series of T2-preparation pulses to modulate the MRI signal, the monoexponential fitting of which yields blood T2 . A T2-YvFor measuring CBF, PC MRI employed bipolar gradients to encode flow velocities of blood supplying major feeding arteries of the brain, specifically left and right internal carotid arteries (LICA and RICA) and left and right vertebral arteries (LVA and RVA), allowing quantitation of total blood flow to the brain. Flow values were then normalized to brain volume, expressing CBF in units of mL/100 g/min. Time-of-flight MR angiograms (TOF-MRAs) were first performed to allow visualization of above-referenced feeding arteries. Imaging slabs were positioned at center of epistropheus, with 60-mm saturation slabs placed above to suppress venous signals. Settings of TOF MRA were as follows: TR/TE, 20 ms/3.45 ms; flip angle, 18°; FOV, 90 mm × 90 mm × 20 mm; voxel size, 0.8 mm × 0.8 mm × 2 mm; and scan duration, 23.7 s.Based on MRA images, four PC MRI scans were performed by orienting imaging slices perpendicular to and centered on respective target arteries, as previously described . Scans oProcessing of PC MRI data again followed existing procedures . Manual 2 was calculated according to Fick’s principle  on scan age, sex, and diagnostic group (independent variables). In regression analyses, paired groups were encoded as follows: LBW-VLBW (0) vs term (1) neonates; LBW-VLBW-a (1) vs LBW-VLBW-n (0) subsets; LBW-VLBW-a neonates (1) vs term controls (0); and LBW-VLBW-n neonates (0) vs term controls (1). LBW-VLBW newborns underwent MRI scans once their vital signs had stabilized, and ventilators were no longer required (3 weeks old on average). Term infants had good vital signs and were amenable to scanning at any time (1 week old on average). There were distinct chronologic differences in scan ages of these pairings. Due to remarkable disparities , it was 1 and b2 were based on results of an age- and sex-adjusted regression model similarly reported  and term neonates (n = 9), LBW-VLBW-a (n = 24) and LBW-VLBW-n (n = 17) subsets, LBW-VLBW-a neonates (n = 24) and term controls (n = 24), and LBW-VLBW-n neonates (n = 17) and term controls (n = 24). We also analyzed birth weight, brain volume, and cerebral physiologic parameters independently, generating receiver operating characteristic (ROC) curves to distinguish the LBW-VLBW-a subset from all other neonates and from the LBW-VLBW group overall. Inter-rater variations in MRI scores determined by YQ and XYS were assessed via intraclass correlation coefficient (ICC), rated as follows: 1.0–0.81, excellent; 0.80–0.61, very good; 0.60–0.41, good; 0.40–0.21, reasonable; and 0.20–0.00, poor. All computations were driven by standard software , setting significance at p < 0.05.where coefficients breported . Correctp = 0.89); CMRO2, 29.9 ± 11.4 μmol/100 g/min vs 48.9 ± 12.4 μmol/100 g/min (p < 0.001); CBF, 13.6 ± 5.0 mL/100 g/min vs 19.5 ± 3.8 mL/100 g/min (p = 0.002); and brain volume, 265.2 ± 74.5 mL vs 354.1 ± 52.0 mL (p = 0.001). Only OEF proved similar, the other parameters differing significantly by group.In LBW-VLBW and term neonates, measured parameters were as follows: OEF, 31.3 ± 10.5% vs 30.8 ± 6.2% (n = 41), including both LBW-VLBW-a and LBW-VLBW-n subsets, are detailed in p > 0.05). Fifteen LBW-VLBW newborns had required mechanical ventilation.Population characteristics and diagnostic outcomes  of the LBW-VLBW group (2 was significantly lower (p = 0.01) in LBW-VLBW (vs term) newborns, CBF (p = 0.06) and OEF (p = 0.67) did not differ significantly. Furthermore, CMRO2 (p = 0.48) and CBF (p = 0.63) failed to show scan age-related increases during this period of early development; and despite significant differences in birth weight, LBW-VLBW and term neonates did not differ significantly in brain volume.We first compared physiologic measures of LBW-VLBW and term neonates . Althoug2 rates relative to controls . Contrary to expectations, no significant differences in CMRO2 (p = 0.30), CBF (p = 0.50), or OEF (p = 0.15) recordings were evident across subsets upon linear regression analysis. Relative to term neonates, OEF was significantly lower (p = 0.02) in the LBW-VLBW-n subset.Next, we compared the two subsets of LBW-VLBW neonates (LBW-VLBW-a and LBW-VLBW-n) with term newborns, both displaying lower CMRO1, scan age; b2, sex), with 95% confidence interval (CI) of coefficients and respective p-values. Once corrected for scan age and sex, group comparisons of birth weight, CMRO2, CBF, OEF and brain volume produced similar results , the LBW-VLBW-a subset vs term controls , and the LBW-VLBW-n subset vs term controls . This suggests that observed differences in physiologic parameters (CMRO2 and CBF) of the LBW-VLBW-a subset and term controls stemmed from brain injuries and did not reflect scan age or sex. Corrected OEF values in the LBW-VLBW-n subset did differ significantly (p < 0.001) from those of term controls. There were no significant differences in corrected values of LBW-VLBW-a and LBW-VLBW-n subsets (p > 0.05). results . Correctp = 0.25], OEF , CMRO2 , CBF , and brain volume  in distinguishing the LBW-VLBW-a subset from term and LBW-VLBW neonates are shown in 2 demonstrated fair diagnostic performance in this regard.Receiver operating characteristic curves for birth weight . Some term neonates were also discounted due to abnormal laboratory results or MRI findings. Presently, the potential for neurocognitive deficits in these infants and the relevance of CMRO2 reductions on neurocognitive deficits. We will also bolster subject recruitment, further stratifying by birth weight  and sex, and foster a multicenter approach to ascertain neurocognitive thresholds for reductions in CMRO2. Owing to brain immaturity, especially in VLBW infants, there is exceptional vulnerability to injuries undermining the microstructural integrity and tract connectivity of periventricular white matter (a vascular watershed territory)  . Knut harritory) . We must2 at brain level. Finally, the image quality in It is unclear at this point whether administering an anesthetic, such as chloral hydrate, has any cerebrovascular hemodynamic ramifications. There is no direct evidence in studies of humans and animals that it increases CBF or lowers glucose metabolism . However2, and higher OEF tended to show minimal structural abnormalities. Physiologic imaging may be a useful means of identifying those LBW-VLBW newborns at high risk of developing irreversible brain damage.In the present study, we have demonstrated that structural damage in LBW-VLBW-a neonates is associated with metabolic and hemodynamic deficits of the brain. LBW-VLBW-n neonates with lower CBF and CMROThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The study protocol had ethical approval from the Ethics Committee of Shengjing Hospital of China Medical University (IRB2015PS28K). Written informed consent from the participants’ legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.YQ contributed study concepts and design, and providing post-processing assistance. Both authors participated in analysis of experiment results, drafting and editing of the manuscript, and have read and approved the final manuscript for publication.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.


Clostridioides difficile infection (CDI) is the leading cause of life-threatening health care-related gastrointestinal illness worldwide. Phylogenetically appropriate closed reference genomes are essential for studies of C. difficile transmission and evolution. Here, we provide high-quality complete hybrid genome assemblies for the three most prevalent C. difficile strains causing CDI in Australia. Clostridioides difficile causes life-threatening diarrhea and health care-related gastrointestinal infections globally (C. difficile population distinct from that of the rest of the world (C. difficile strains causing C. difficile infection (CDI) in Australia, PCR ribotype 014 (RT014) , RT002 (11.8%), and RT056 (5.4%)  were selected from >1,500 isolates recovered from patients with symptomatic CDI, part of the ongoing nationwide longitudinal surveillance of CDI in Australia, the C. difficile Antimicrobial Resistance Surveillance (CDARS) study  for 48 h . ONT sequencing was performed on a MinION Mk1C device  using an R9 generation flow cell following a DNA by ligation protocol (SQK-LSK109). Filtlong v0.2.0 (https://github.com/rrwick/Filtlong) was used to filter the low-quality reads , resulting in 2.28 (S-0352), 2.59 (S-0253), and 6.66 (S-0942) Gb of sequence data, respectively. The hybrid assembly of ONT and Illumina reads was performed using Unicycler v0.4.8  and annotated using the NCBI Prokaryotic Genome Annotation Pipeline v5.2  and at the ENA under BioProject accession number PRJEB41588 (Illumina sequence data); see The genome data are available at GenBank under BioProject accession number


Scientific Reports 10.1038/s41598-021-81366-6, published online 19 January 2021Correction to: The original version of this Article contained extensive errors in the Reference list.Reference 26 was incorrectly split into Reference 26 and Reference 27.“Trainer, M. N. & Freud, P. J. High-Concentration Submicron Particle Size Distribution by Dynamic Light Scattering Power spectrum development with heterodyne technology advances biotechnology and nanotechnology measurements.”now reads:“Trainer, M. N. & Freud, P. J. High-Concentration Submicron Particle Size Distribution by Dynamic Light Scattering Power spectrum development with heterodyne technology advances biotechnology and nanotechnology measurements. Microtrac, Inc. Application Note SL-AN-05 Rev B. (2009).”As a result, References 27–47 were incorrectly listed as References 28–48.The original Article has been corrected.


The chemokine receptor 4 (CXCR4) and 7 (CXCR7) are G-protein-coupled receptors (GPCRs) activated through their shared ligand CXCL12 in multiple human cancers. They play a key role in the tumor/tumor microenvironment (TME) promoting tumor progression, targeting cell proliferation and migration, while orchestrating the recruitment of immune and stromal cells within the TME. CXCL12 excludes T cells from TME through a concentration gradient that inhibits immunoactive cells access and promotes tumor vascularization. Thus, dual CXCR4/CXCR7 inhibition will target different cancer components. CXCR4/CXCR7 antagonism should prevent the development of metastases by interfering with tumor cell growth, migration and chemotaxis and favoring the frequency of T cells in TME. Herein, we discuss the current understanding on the role of CXCL12/CXCR4/CXCR7 cross-talk in tumor progression and immune cells recruitment providing support for a combined CXCR4/CXCR7 targeting therapy. In addition, we consider emerging approaches that coordinately target both immune checkpoints and CXCL12/CXCR4/CXCR7 axis. Chemokines are small chemoattractant molecules that control cell migration, proliferation and survival in physiological and pathological processes including cancer . They ar−/− knock-out mice die before birth -dependent phosphorylation and interaction with β –arrestin  GPCRs encoded on chromosome 2.1 , 17. CXCre birth . CXCR4 −ogenesis . CXCL12 ogenesis . CXCL12-ogenesis . In addiogenesis . When CXarrestin . CXCR7 parrestin , angiogearrestin , neurogearrestin  and cardarrestin . Althougarrestin . Initialarrestin . Withoutarrestin . CXCR7, arrestin . CXCR7 carrestin . Studiesarrestin  or in hearrestin . CXCR4/Carrestin . Overallvia Epidermal Growth Factor Receptors (EGFRs) and Matrix Metallopeptidase 9 (MMP-9) (via angiogenesis (An active CXCL12/CXCR4 pathway is considered a feature of aggressive tumors  as it po (MMP-9) . Also CX (MMP-9) . CXCR7 a (MMP-9) . The pro (MMP-9) , 48. CXC (MMP-9) –51. In o (MMP-9)  while CX (MMP-9) . CXCR7 m (MMP-9) . In cerv (MMP-9) . In lung (MMP-9) . Accordi (MMP-9) . Convers (MMP-9) . CXCL12  (MMP-9) . In addi (MMP-9) . In neur (MMP-9) . In breaogenesis . Hence, ogenesis , EGFR , myeloid derived suppressor cells (MDSCs), and dendritic cells (DCs) to the tumor niche . CXCL12 via Raf-ERK pathway , CXCR4 is expressed in tumor endothelium sprouting tumor vessels  and CXCR pathway . CXCR7 e pathway  or IL-1b pathway , by lipo pathway  or durin pathway . CXCR7 i pathway  and surv pathway . It is s pathway , 84 and  pathway . CXCL12 and TECs . Thus, Celopment .DCs are the most potent antigen presenting cells (APCs) in the immune system . Immatur+CD25high FoxP3+) are CD4+ T cells with predominantly suppressive activity (5–10% of circulating CD4+ T cells in humans). Tregs impair immune effector cells function via cytokines, direct lysis, inhibitory receptors, metabolic disruption, IL-2 depletion or inducing an immunosuppressive microenvironment  potentiates migration toward vascular-associated CXCL12-positive cells in the BM. In lymphoma-bearing mice, TCXCR4 potentiates the effector function increasing tumor protection -γ respectively, particularly in macrophages (The only approved drug CXCR4 inhibitor is AMD3100 (known as Plerixafor or Mozobil)  while muin vivo . In prosin vivo . Other ain vivo  or CCX73in vivo . The antin vivo . Recentlin vivo . It bindrophages . Some anrophages . The cycrophages . A CXCR4rophages . Peptide+CD25–Foxp3+IL2+CD40L+ helper-like cells (-L1) small interfering RNA (siPD-L1). The nanocomplex promotes T cell infiltration, decreases alpha-smooth muscle actin (α-SMA) and collagen, reduces MDSCs and Tregs recruitment (Recently, CXCR4 antagonists have been coupled to ICIs with the intent to remodel TME improving ICIs efficacy . Since tke cells . Inhibitke cells  while take cells . A nanocruitment . Thus, cImmuno-resistance and vascularization are acquired tumor features that contribute to cancer growth and metastasis. Among the different signaling pathways, directly or indirectly involved in cancer immune-resistance and angiogenesis, CXCR4/CXCR7/CXCL12 is crucial for participating in cancer migration, angiogenesis and immunosuppressive cell recruitment. Thus, the inhibition of the CXCR4/CXCL12 or CXCR7/CXCL12 axis is attractive in cancers overexpressing both receptors such as colorectal cancer , renal cSSa, CI, and SSc contributed in conception and design of the study. AMT, AC, FA, and GG supervised the study. SSa, CI, and SSc wrote and edited the manuscript. SSa and CI equally contributed. All authors contributed to the article and approved the submitted version.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.


Staphylococcus aureus (CA-MRSA) has become a major cause of S. aureus infections globally. We report the complete genome sequences of three of the earliest CA-MRSA strains isolated from remote Australian Indigenous communities in the Kimberley region of Western Australia.Initially reported in Western Australia in the 1980s, community-associated methicillin-resistant  Staphylococcus aureus (MRSA) is a leading cause of health care-associated infections worldwide and has emerged as a major cause of infections in the community . Each genome was also sequenced with the Illumina NextSeq 500 platform using the Nextera XT DNA library preparation kit (150-bp paired-end chemistry) and the NextSeq 500/550 kit v2.5 . Nesoni clip (github.com/Victorian-Bioinformatics-Consortium/nesoni) was used to remove adaptor sequences and to quality filter the reads. Illumina reads were mapped to the genome assembly with Minimap2 v2.17-r941 . The ONT SQK-RAD004 kit was used to create DNA sequencing libraries, which were loaded onto an ONT FLO-MIN106 flow cell and sequenced using ONT MinKNOW v20.10.3 and MinKNOW Core v4.1.2. Base calling was performed with Guppy v4.4.1+1c81d62 (ONT), using the dna_r9.4.1_450bps_hac.cfg model. NanoFilt . Unfilte.17-r941  was used.17-r941 . Genome .17-r941 . DefaultPRJNA703734 and PRJNA703736 and Sequence Read Archive (SRA) accession numbers SRX11246052, SRX11246051, SRX11247745, SRX11247744, SRX11246056, and SRX11246055, as indicated in GenBank accession numbers for the genome assemblies are provided in


First-generation COVID-19 vaccines are matched to spike protein of the Wuhan-H1 (WT) strain. Convalescent and vaccinee samples show reduced neutralization of SARS-CoV-2 variants of concern (VOC). Next generation DNA vaccines could be matched to single variants or synthetically designed for broader coverage of multiple VOCs.The synthetic consensus (SynCon®) sequence for INO-4802 SARS-CoV-2 spike with focused RBD changes and dual proline mutations was codon-optimized . Sequences for wild-type (pWT) and B.1.351 (pB.1.351) were similarly optimized. Immunogenicity was evaluated in BALB/c mice. Pre-clinical efficacy was assessed in the Syrian Hamster model.Figure 1. Design Strategy for INO-4802INO-4802 induced potent neutralizing antibody responses against WT, B.1.1.7, P.1, and B.1.351 VOC in a murine model. pWT vaccinated animals showed a 3-fold reduction in mean neutralizing ID50 for the B.1.351 pseudotyped virus. INO-4802 immunized animals had significantly higher (p = 0.0408) neutralizing capacity (mean ID50 816.16). ID50 of pB.1.351 serum was reduced 7-fold for B.1.1.7 and significantly lower (p = 0.0068) than INO-4802 (317.44). INO-4802 neutralized WT (548.28) comparable to pWT. INO-4802 also neutralized P.1 (1026.6) . pWT, pB.1.351 or INO-4802 induced similar T-cell responses against all variants. INO-4802 skewed towards a TH1-response. All hamsters vaccinated with INO-4802 or pB.1.351 were protected from weight loss after B.1.351 live virus challenge. 4/6 pWT immunized hamsters were completely protected. pWT immunized hamsters neutralized WT (1090) but not B.1.351 (39.16). INO-4802 neutralized both WT (672.2) and B.1.351 (1121) . We observed higher increase of binding titers following heterologous boost with INO-4802 (3.6 – 4.4 log2-fold change) than homologous boost with pWT (2.0 – 2.4 log2 fold change) .Figure 2. INO-4802 Induces Functional Humoral Immune Response Against SARS-CoV-2 Variants of ConcernFigure 3. INO-4802 Protects Hamsters Against Challenge With B.1.351 Live VirusFigure 4. Heterologous Boost with INO-4802 Induces Humoral Immune Response Against SARS-CoV-2 VariantsVaccines matching single VOCs, like pB.1.351 and pWT, elicit responses against the matched antigen but have reduced cross-reactivity. Presenting a pan-SARS-CoV-2 approach, INO-4802 may offer substantial advantages in terms of cross-strain protection, reduced susceptibility to escape mutants and non-restricted geographical use.Katherine Schultheis, MSc, Inovio Pharmaceuticals (Employee) Charles C. Reed, PhD, Inovio Pharmaceuticals  Viviane M. Andrade, PhD, Inovio Pharmaceuticals Inc. (Employee) Richa Kalia, MS, Inovio Pharmaceuticals  Jared Tur, PhD, Inovio (Employee) Blake Schouest, PhD, Inovio Pharmaceuticals (Employee) Dustin Elwood, PhD, Inovio Pharmaceuticals (Employee) Arthur Doan, n/a, Inovio (Employee) Patrick Pezzoli, BS, Inovio (Employee) Dinah Amante, BS, Inovio (Employee) David Weiner, PhD, Inovio  J Joseph Kim, PhD, Inovio (Employee) Laurent Humeau, PhD, Inovio Pharmaceuticals (Employee) Stephanie Ramos, PhD, Inovio Pharmaceuticals (Employee) Trevor R. F. Smith, PhD, Inovio  Kate Broderick, PhD, Inovio (Employee).


S. aureus (MRSA), vancomycin-resistant Enterococcus (VRE), ESBL- and carbapenemase-producing Enterobacteriaceae (E). We assessed T2R performance in detecting these resistant bacteria in whole blood (WB) and analyzed possible impact on time to appropriate Ab. Appropriate antibiotic (Ab) therapy of bloodstream infections (BSI) is often delayed by time to blood culture (BC) positivity, species (sp) identification and Ab sensitivity (sensi). The T2Resistance (T2R) Panel is a direct-from-blood (culture-independent) diagnostic that detects 13 genetic markers associated with methicillin-resistant We performed T2R using WB samples obtained from patients (pts) on the same day as BCs from July 2019-2020. Receipt of appropriate Ab was assessed at time of empiric, Gram stain-directed, MALDI-directed (sp identification) and sensi-directed therapy. T2R results were not available to care teams. Teams were notified of positive BCs. Stewardship optimized Abs based on sensi. S. aureus , Enterococcus , P. aeruginosa and others (n=10). 12 ESBL-E produced CTX-M 14/15. T2R sensitivity and specificity was 78% and 99%, respectively, compared to sequencing of resistance markers. Sensitivity was excellent for vanA/B, KPC (100% each), and CTX-M14/15 (92%); sensitivity was 58% for mecA/C. T2R detected resistance determinants in 3-7h. Median time to appropriate Ab was 16.3h, which was significantly longer for VRE (25.6h) and ESBL- or KPC-E (50.9h) BSIs than for T2R marker-negative bacteria . Pts with VRE or ESBL-/KPC-E BSI were less likely to received appropriate empiric Ab  than pts with T2R marker-negative BSI . Median times to achieve ≥80% appropriate Ab therapy of marker-negative, VRE and CTX-M/KPC-E BSIs were 15.5h (after Gram stain), 43.9h (after MALDI) and 63.5h (after sensi), respectively.BC from 103 pts grew 114 bacterial sp: E , Antibiotic TherapyThere was a significant delay in appropriate Ab therapy of BSIs, especially in pts infected with VRE and ESBL/KPC-E. T2R rapidly and accurately detected BSI caused by VRE and ESBL/KPC-E, and has the potential to significantly shorten time to appropriate Ab.Cornelius J. Clancy, MD, Merck (Grant/Research Support) Ryan K. Shields, PharmD, MS, Shionogi  Minh-Hong Nguyen, MD, Merck (Grant/Research Support)


NC_045512.2).We report a coding-complete genome sequence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain SARS-CoV-2/BGD/GC001, isolated from a Bangladeshi patient with respiratory symptoms. Phylogenetic analysis assigned this strain to lineage B.1.1.7, which presented a total of 36 mutations in the spike and other genomic regions compared to strain Wuhan Hu-1 (GenBank accession number  Coronaviridae and genus Betacoronavirus (Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) belongs to the family onavirus . Lineagehttp://www.sanger.ac.uk/science/tools/smalt-0) and SAMtools v1.9   from the symptomatic patient was processed for nucleic acid isolation utilizing a QIAamp viral RNA minikit . SARS-CoV-2 was confirmed using a TaqMan real-time PCR (RT-PCR) assay . A cDNA ols v1.9 . De novo v3.11.1 . RATT wa45512.2) . The Nex45512.2)  and MEGA45512.2)  software45512.2) . DefaultNC_045512.2)  . Strain 45512.2) . MoreovePRJNA702998, BioSample accession number SAMN17993365, and GenBank accession number MW624725.1. The Illumina raw reads have been deposited in the NCBI Sequence Read Archive under accession number SRR13744683.The genome sequence of SARS-CoV-2/BGD/GC001 was deposited in the NCBI database under the BioProject accession number


Chimeric antigen receptor (CAR-T) T-cell therapy is a novel immunotherapy for cancer treatment in which patients are treated with targeted, genetically-modified T-cells. Common side effects include cytokine release syndrome, neurotoxicity, hypogammaglobulinemia, and increased susceptibility to infections. Long-term infectious outcomes are poorly characterized.We retrospectively examined patients who received CAR-T therapy at BIDMC & MGH from July 2016 to March 2020 and evaluated bacterial, fungal, viral, and parasitic infections at 3 months intervals to 1 year following cell infusion. The incidence, timing, and outcomes of the infectious complications were evaluated.In total, there were 47 patients; averaging 61.4 years of age (±12 years). Primary indications for CAR-T therapy included diffuse large b-cell lymphoma (65%) and multiple myeloma (25%), chronic lymphocytic leukemia (2%) and mantle cell lymphoma (2%). Patients had received an average 4 ± 2.9 lines of chemotherapy prior to CAR-T infusion; 19 subjects (40%) had a history of prior autologous stem cell transplant. All patients received acyclovir for antiviral prophylaxis and most received either trimethoprim-sulfamethoxazole  or atovaquone  for pneumocystis prophylaxis. In the first year, 35/47 (74.5%) of subjects experienced at least one infection with an infection rate of 84.4/10,000 person days. Median time to first infection was 59 days (range 1-338 patient days). 31/47 (66.0%) subjects had at least one bacterial infection, with pulmonary  sources being the most common site of infection. 13/47 (27.7%) of patients had a viral infection  and 6/47 (12.8%) had a proven or probable fungal infection. Death attributed to infection was noted in 2 subjects (4.3%), both related to COVID-19. Baseline IgG levels were significantly lower in the group with infections (p=0.028), while white blood cell count and absolute neutrophil counts were comparable.Table 1. Baseline Demographic, Clinical Characteristics, and Outcomes of 47 Recipients of CAR-T Cell Therapy by Infection StatusBMI: body mass index; DLBCL: diffuse large B-cell lymphoma; CLL: chronic lymphocytic leukemia; Flu/Cy: Fludarabine/cyclophosphamide; IVIG: intravenous immunoglobulin; WBC: white blood cell count; ANC: absolute neutrophil count; ALC: absolute lymphocyte count.Table 2. Characteristics of the 113 Infections in the 35 Subjects Who Developed InfectionsInfectious complications, particularly of bacterial etiology, are common in the first year following CAR-T therapy. These data may inform future prophylactic strategies in this patient population.Matthew Frigault, MD, Arcellx (Consultant)BMS (Consultant)Iovance (Consultant)Kite (Consultant)Novartis (Consultant) Jay A. Fishman, MD, Nothing to disclose Jon Arnason, MD, BMS/Juno (Advisor or Review Panel member)Regeneron (Advisor or Review Panel member)