Anxiety is common in youth on the autism spectrum and cognitive behavioural therapy (CBT) has been adapted to address associated symptoms. The aim of the current systematic review and meta-analysis was to examine the efficacy of CBT for reducing anxiety in autistic youth.Searches of PubMed and Scopus databases were undertaken from January 1990 until December 2020. Studies were included if they consisted of randomised controlled trials (RCTs) using CBT to reduce anxiety in autistic youth. Separate random effects meta-analyses assessed anxiety ratings according to informant , both at end-of-trial and at follow-up.g = 0.88, 95% CI 0.55, 1.12, k = 11), while those for both parent  and child-reported anxiety  were smaller, but significant. These benefits were not however maintained at follow-up. Moderator analyses showed that CBT was more efficacious for younger children (for clinician and parent ratings) and when delivered as individual therapy (for clinician ratings). Using the Cochrane Risk of Bias 2 tool, we found concerns about reporting bias across most trials.A total of 19 RCTs met our inclusion criteria . Random effects meta-analyses revealed a large effect size for clinician rated symptoms . Follow-up analyses failed to reveal sustained benefits, though few studies have included this data. It will be important for future trials to address robustness of treatment gains overtime and to further explore inconsistency in efficacy by informant. We also recommend pre-registration of methods by trialists to address concerns with reporting bias.The online version contains supplementary material available at 10.1186/s40359-021-00658-8. Autism Spectrum Disorder (ASD) is a lifelong, neurodevelopmental condition characterised by difficulties in social interaction, social communication and restricted and/or repetitive patterns of behaviour . As manyCognitive behavioural therapy (CBT) has been advanced as an effective, evidence-based intervention for anxiety –13. ThisThe past decade has seen significant interest in the adaption of CBT for autistic children and adolescents. Intuitively, several factors might suggest that CBT may not be suited to this context. For example, treatment protocols often require consideration of multiple possibilities such as different causes or outcomes in situations. Restricted behavioural and cognitive profiles associated with ASD might therefore be expected to interfere with treatment engagement . CBT alsAn emerging body of evidence supports the use of adapted CBT protocols for autistic youth –31, inclg = − 0.66) on the reduction of anxiety in autistic youth aged under 18 years. The researchers also highlighted important moderators of treatment gains. Specifically, parent involvement and longer treatment protocols were found to advance more benefit. Although Perihan et al. is the most current evidence synthesis, its conclusions are limited by the inclusion of 7 out of 24 samples (30%) being from non-randomised studies. Whilst it is useful to summarise all available evidence, in meta-analysis, this requires consideration of study design features and adjustment of estimates of effect sizes ). The studies were heterogeneous , with an I2 value of 75.86. However, one study [g = 4.34) was an outlier showing no overlap of 95% CI with any other studies—this same study by Chalfant et al. was also excluded by previous meta-analyses [g effect size to 0.40 [95% CI 0.24–0.56], showing that CBT remains efficacious at reducing anxiety compared with controls  = 22.03, p = 0.018], with an I2 value of 22.84.Parent reported outcomes for anxiety in autistic youth showed CBT as an efficacious treatment in comparison to control conditions. Using a random effects model, the overall pooled effect size was medium . g = 0.36, [95% CI 0.19–0.54]).Observation of funnel plots and the Trim and fill analysis suggested the possibility of one potentially missing trial reducing the effect size (g = 0.50 [95% CI − 0.10 to 1.12]). Heterogeneity was high .Nine trials (10 samples) contained follow-up data, but only 5 samples presented follow-up data for the controls , 53–55. Ten studies (11 samples) compared CBT with a control group using clinician-rated measures. Three of the studies used two clinician-rated measures , 27, 52 g = 0.88, 95% CI 0.55–1.21. The studies were heterogeneous, (Q(10) = 26.74, p < 0.001), with an I2 value of 62.61 . The studies were heterogeneous, (Q(12) = 78.77, p < 0.001), with an I2 value of 84.76. One study , with an I2 = 0.00 and the effect size although it remained significant (g = 0.25 [95% CI 0.06–0.43]) . Heterogeneity was low (Q(2) = 0.67, p = 0.71; I2 = 0%).Seven trials provided follow-up data, but only three provided follow-up data for controls , 53, 54.Four continuous variables  were examined as moderators using meta-regression analyses (see Table g = 0.58 [0.24–0.91]); however, self-ratings were non-significant and comparable to those obtained in the larger set (g = 0.26 [− 0.01 to 0.53]). Clinician ratings were significantly greater than child ratings  and just failed to reach significance when compared to parental ratings ; while child and parental ratings did not significantly differ .As effect sizes for clinicians, parents and child ratings differed substantially in the analyses outlined above, we performed a subgroup analysis comparing effect sizes in 6 trials where all three types of ratings were administered in the same groups of children , 49, 50.g = 0.53 [0.07–0.99], k = 2) versus g = 1.16 [0.74–1.58], k = 7) identified a significantly larger effects for individual therapy . Parent ratings for group versus individual formats  vs g = 0.50 [0.33–0.67]; k = 9) did not differ significantly ; nor did child ratings for group versus individual  versus  differ significantly .We also used subgroup analysis to compare group versus individual format therapies for each rating informant. Clinician rating for group and individual formats (p = 0.31), parents  or children themselves .Earlier trials, using different diagnostic criteria tended to also include a number of children who were given a diagnosis of Pervasive Developmental Disorder-Not Otherwise Specified (PDD-NOS): Wood et al.  (85%); SWe intended to analyse the categorical impact of blind versus non-blind outcome assessment. Of course, both parents and children/young people were aware of assignment and so non-blind. By contrast, all clinician ratings appeared to be blind at outcome and thus we could not analyse as a moderator within any rating type.All trials were evaluated for risk of bias using the Cochrane Risk of Bias Tool (RoB2)  by two aThis systematic review and meta-analysis aimed to assess the evidence base on the efficacy of CBT in reducing anxiety in children and young people on the autism spectrum. We included data derived only from RCTs and considered treatment efficacy from the perspective of multiple informants including parent, clinician and child/ self-report. Importantly, we also summarised what is known about the durability of treatment gains. This has been missing in previous evidence syntheses in this area , 36, 38.Nineteen studies were included with data from 833 participants. The most common modality of intervention delivery across trials was group (n = 9) or individual therapy (n = 8), with two trials using a combination of these approaches. Some degree of consistency was apparent in the duration of intervention across trials, with the modal number being 16 sessions. All studies reported the use of CBT protocols that had been adapted for use with autistic youth. Most included parent and or clinician ratings of anxiety, with 13 also including self-rating by the child/young person. Interestingly, a wide range of assessments were used to profile symptoms of anxiety, but not all have support for their use in ASD trials , 62 or ag = 0.88) versus a moderate effect for parent rated symptoms (g = 0.58), although the latter reduced following the removal of one extreme outlier (g = 0.42). For child/self-rated symptoms, we found a small to moderate effect (g = 0.47), which again reduced after removal of one extreme outlier (g = 0.25). A direct analysis in 6 trials using all three informant ratings confirmed a larger effect size for clinicians (g =1.71 [0.66–1.69]) than for both parents (g = 0.58 [0.24–0.91]) and the self-ratings of children, with the latter being non-significant (g = 0.26 [− 0.01 to 0.53]) in this sub-analysis.The current meta-analyses confirm the efficacy of CBT in reducing anxiety in autistic youth, and thus concurs with previous reviews , 36, 38,Turning to factors that might moderate the efficacy of CBT, we could divide them broadly into those relating to the participants, and those relating to the therapy itself. Turning to participant variables, meta-regression suggested that the benefits of CBT may be more pronounced in younger children. This may suggest better developmental tailoring of intervention protocols, with room to enhance therapeutic techniques for older children. Gender was not a significant moderator, although females are generally under-represented in autism research, impacting the power of an analysis to detect a true effect. We also found no effect for the proportion of those identified as having Asperger syndrome or PDD-NOS. Turning to effects related to the therapy itself, the duration of CBT was not found to be a contributing factor, but this likely reflects most trials including a similar number of sessions (i.e. 16). Our subgroup comparison of group versus individual CBT revealed only one significant advantage for individual therapy for clinician ratings; all other informant rating revealed no differences. The advantage for individual therapy for clinician ratings however needs to be interpreted cautiously as it involved just 2 group versus 7 individual trials.An important difference between the current meta-analysis and those of both Ung et al.  and PeriThe most notable feature of course is the large effect size reported for clinicians and the fact that the children/young people themselves report little or no reduction of anxiety following CBT. The clinician and self-ratings reported here also differ in one other critical manner and that is that all clinician ratings were made blind. Arguably, blinded clinician ratings reporting greater symptom improvement might reflect the ability of skilled professionals to unpick the overlap of anxiety symptoms from features of autism per se, though this distinction is a source of debate . Our finThe lack of consensus on the ‘gold standard’ measure of anxiety for autistic youth  adds nuaWhere outcome reporting beyond post-treatment effects was included in trials, we restricted our analyses to studies that did this consistently (i.e. control participants included). Although we identified 23 sets of follow-up data from various informant groups, only 8 followed-up the control group and these all came from 5 trials. This resulted in synthesis of parent and child rated anxiety only and yielded no support for sustained treatment effects at follow-up (range 1–9 months). Whilst data from studies in the general population have supported the long-term effectiveness of CBT for anxiety in youth , we mighSome caveats need to be acknowledged when interpreting the current findings. The first relates to the assessment of risk of bias, which had not been examined in the three previous meta-analyses , 36, 38.This study provides the largest meta-analysis (19 RCTs and 833 participants) to date on the efficacy of CBT for anxiety in children and adolescents on the autism spectrum. Our primary findings concur with previous reviews showing that CBT reduces anxiety in the immediate posttreatment period. Indeed, ratings by all three informant types were consistent in documenting a significant reduction of anxiety; and showed little or no evidence of publication bias. Nevertheless, the substantial variability in effect sizes across clinicians, parents and children would advise against future meta-analyses pooling across informants. It is notable that risk of bias was low except in the domain of reporting bias, where risk was generally higher. The latter seems to reflect the lack of preregistration of trials.Recommendations for future trials include a focus on selecting autism-specific measures of anxiety or at least making necessary adaptions to scales; prospective registration of trials to overcome potential reporting biases; and the inclusion of follow-up analyses to assess whether treatment gains are sustained. We also note the absence of reporting on adverse events and the relative absence of health economic evaluation across trials, both of which are crucial to determine the wider value of the intervention beyond the reduction of symptomatology.Additional file 1. PRISMA Checklist.Additional file 2. Additional study information on included studies.


A2M) and transforming growth factor beta 3 (TGFB3), was correlated with BMI, pre-operative prostate volume, and age at surgery.Benign prostatic obstruction (BPO) due to benign prostatic hyperplasia (BPH) is a leading cause of morbidity in men over the age of 40. This study examined whether there was an association between body mass index (BMI) and pre-operative prostate volume and whether expression of two genes, alpha-2-macroglobulin . In linear regression analysis, BMI was positively associated with prostate volume , while hyperlipidemia was negatively associated with prostate volume . A trend for a positive association was also observed for diabetes . In the race/ethnicity stratified analysis, age at surgery showed a trend for significantly positive association with prostate volume in European Americans , but not in Hispanic Americans. Expression of the A2M gene in the stroma was negatively correlated with age at surgery (P = 0.006). A2M expression in the gland was positively correlated with prostate volume among older men  and overweight men . TGFB3 expression in the gland was positively correlated with BMI (P = 0.007) among older men.A total of 278 patients were identified, including 62.9% European Americans (n = 175) and 27.7% Hispanic Americans (n = 77). BMI was significantly correlated with prostate volume (Spearman’s TGFB3 and A2M was correlated with BMI, prostate volume, and age at surgery.This study demonstrated the positive correlation between BMI and prostate volume. Expression of Supplementary information accompanies this paper at 10.1186/s12894-020-00753-9. Benign prostatic obstruction (BPO) due to benign prostatic hypertrophy (BPH), enlargement of the prostate gland, is a leading cause of morbidity in men over the age of 40. The prevalence of BPO/BPH increases with advancing age reaching 100% in the ninth decade of life [The current guidelines for the management of BPH recommend to treat initially with medical therapy until the disease process progresses to a point at which surgery is a necessity . UnfortuAlthough current BPH and lower urinary tract symptoms (LUTS) studies rarely report racial/ethnic prevalence, it has been reported that men from minority groups, such as Hispanic-Americans (HAs) and African Americans, are at higher risk of developing BPH –9. AdditIt has been hypothesized that pro-inflammatory states due to obesity may be triggers for developing BPH and there may be reactive changes characterized by alterations in the stromal and epithelial microenvironment, reflected in changes in expression of genes involved in inflammatory response and cell growth and development , 20. HowIGF2), alpha-2-macroglobulin (A2M), and transforming growth factor beta 3 (TGFB3), was correlated with BMI, pre-operative prostate volume, and age at surgery. We also assessed if age and self-identified race/ethnicity modified these relationships.Two main goals of this study were 1) to examine whether there was an association between higher BMI or obesity and prostate volume including HAs who are previously underrepresented in BPH research and 2) to investigate whether expression of three genes, Insulin like growth factor 2  surgery for treatment of BPO/BPH between October 2012 and June 2018 were retrospectively reviewed. Patients who had histologically confirmed BPH were included. Patients with (a) pathology diagnoses other than BPH, (b) a history of prostate cancer, or (c) a prior surgical intervention for BPO/BPH were not included. Chart review was conducted on every eligible patient in the identified timeframe. Clinical  and demographic  characteristics as well as BMI, smoking status, and comorbidities  were extracted. The pre-operative prostate volume was estimated using the ellipsoid formula (length x width x height x (π/6) based on either transrectal ultrasound or cross-sectional imaging (MRI or CT). The study protocol was approved by the University of Arizona Institutional Review Board.2 punches from each identified area  were taken.To prepare the surgical specimens for gene expression analysis, hematoxylin and eosin (H&E) slides associated with formalin-fixed paraffin-embedded (FFPE) tissue samples underwent a review by board certified genitourinary pathologists to identify different architectural and cellular components  on the slides. Then, from the FFPE tissue sample, 1.5 mmIGF2, A2M, and TGFB3, were selected from Luo et al. [A2M and TGFB3, were successfully amplified, but due to technical issues IGF2 was not successfully amplified. The samples from 30 patients yielded quantifiable RNA expression data using a real time PCR approach. ΔCt method and a housekeeping gene, 18SrRNA, were used for normalization. ΔCt was log2 transformed for statistical analysis.The Roche high Pure FFPET kit was used to extract RNA, and RNA was quantified using Nanodrop. Three genes, o et al. . Two genChi-square test, independent sample T-test, and Mann–Whitney U-test were performed to characterize eligible patients for this study and patients included for the gene expression study. Linear regression analysis was performed to test the association between BMI and pre-operative prostate volume. Pre-operative prostate volume was log-transformed to normalize. Backward selection approach was used to determine variables to include in the regression mode. Age and race/ethnicity were added to the model to assess if they altered the association between BMI and prostate size. Spearman’s correlation was used to examine whether gene expression was correlated with BMI, prostate volume, and age at surgery. Separate analysis was performed for younger men (age < 70) and older men (age ≥ 70) as well as European Americans (EAs) and HAs to assess if associations were different in these groups.rho = 0.123, P = 0.045, Fig. rho = −0.169, P = 0.005). The correlation was stronger in EAs than in HAs. Median pre-operative prostate volume was 75 mL . Characteristics of patients with smaller prostate volume (≤ 75 mL) and larger prostate (> 75 mL) were compared (Table P = 0.03). Urinary retention was more common in men with larger prostate .A total of 278 patients who met inclusion criteria were identified. The mean age of patients was 70.6 (SD 8.5). The largest group of patients was EAs (62.9%), and HAs were the second largest group of patients (27.7%). BMI was significantly correlated with pre-operative prostate volume . Hyperlipidemia was negatively associated with prostate volume , and a trend for a positive association was observed for diabetes . Additionally, including age at surgery and race/ethnicity in the model did not change the association between BMI and prostate volume. In the race/ethnicity stratified analysis, the association between BMI and prostate volume was similar in both groups, but association was no longer significant in HAs, possibly due to small sample size , but not in HAs. The analysis stratified based on age revealed similar association in younger and older men.The linear regression analysis was performed to assess if BMI was associated with pre-operative prostate volume Table . BMI, hyA2M gene in the stroma was significantly negatively correlated with age at surgery (P = 0.006). In the stratified analysis, the correlation was significant in HAs, men with normal BMI (BMI < 25), and obese men (BMI > 30)  and overweight men . Among older men, TGFB3 expression in the gland was significantly positively correlated with BMI  and showed a trend for significant correlation with prostate volume (P = 0.08).We then assessed the correlation of gene expression with age at surgery, BMI, and pre-operative prostate volume Table . The chaTGFB3 and A2M and its associations with BMI, preoperative prostate volume, and age at surgery. We also investigated if age and race/ethnicity modified the patterns of correlations.In this study, we demonstrated the positive correlation between BMI and pre-operative prostate volume. We also demonstrated gene expression of Although multiple studies have examined the relationships between obesity, particularly abdominal obesity, and BPH risk , 12, thePrevious studies also demonstrated that Hispanic ethnicity was associated with BPH risk with increased rates of reporting LUTS , 8. ObesContrary to our hypothesis, hyperlipidemia was negatively associated with pre-operative prostate volume, and we did not observe significant association for diabetes or hypertension. There have been multiple studies interested in demonstrating the link between metabolic syndrome and BPH, but findings have been inconsistent. Some studies have reported how diabetes , 14 and A2M and TGFB3, were correlated with age at surgery, BMI, or preoperative prostate volume. These two genes along with IGF2 were selected as genes of interest in BPH because they were over-expressed in BPH compared to normal prostate tissue in a previous study by Luo et al. [A2M was also over-expressed in BPH compared to normal prostate tissue in another study [TGFB3 and IGF2 are both growth factors, and TGFB3 encodes a ligand of the transforming growth factor-beta (TGF-beta) superfamily. A2M encodes a protease inhibitor and cytokine transporter, and the protein encoded by A2M can inhibit inflammatory cytokines and interrupt inflammatory cascades. A more recent molecular profiling study using RNA sequencing further demonstrated that A2M and TGFB3 were over-expressed in BPH [BMP5 and CXCL13 as the two most significantly over-expressed genes in BPH. BMP5 (bone morphogenetic protein 5) also encodes a ligand of the TGF-beta superfamily. Ligands of this protein family bind TGF-beta receptors and activate SMAD family transcription factors. Dysregulation of TGFB3 and BMP5 may affect expression of downstream genes that are involved in BPH progression. CXCL13 (C-X-C motif chemokine ligand 13) is an antimicrobial peptide and CXC chemokine, a small cytokine. Both growth factors and immune response are likely to be involved in epithelial and stromal growth in the prostate promoting development and progression of BPH [To explore the biological basis of obesity-driven BPH, we examined the correlations between obesity and expression of genes that are potentially related to obesity. We demonstrated how expression of two genes, o et al.  and theso et al. –33. A2M er study . TGFB3 ad in BPH . Howevern of BPH , 36–38, n of BPH . This cuIGF2. All the patients included were had advanced BPH that required a surgical treatment and they were receiving medical management with alpha blockade and/or 5-alpha reductase inhibitors. Medications may have altered the correction between BMI and prostate volumes or between metabolic diseases and prostate volumes. Inclusion of patients with less severe BPH or BPO who have not undergone surgical or therapeutic treatment and a multi-institutional study with a larger sample size and a more diverse patient population is warranted to further investigate the impact of obesity or obesity related diseases on BPH and BPO characteristics. Finally, there is a paucity of data regarding molecular risk factors in patients with BPH. Further studies are needed to identify genes linked to BPH, so precision medicine can be used to predict disease progression, treatment response, and clinical outcomes.The limitations of this study include its retrospective nature and small sample size at a single institution. Only 278 patients met our inclusion criteria, but we provide surgical care for a diverse patient population due to our location in the Southwestern United States. To our knowledge, this is the first study to explore the relationship between obesity and BPH in HA men in the United States. Secondly, the prostate volume was measured using ultrasound, CT, or MR. Different measurement methods may create systematic bias causing false positive or negative associations. Moreover, we explored the correlation between expression of only three genes and BPH characteristics in a small subset of patients, and we failed to amplify one of the genes, TGFB3 and A2M with BMI, preoperative prostate volume and age at surgery. Further studies are needed to confirm these findings.We demonstrated the positive association between BMI and pre-operative prostate volume. We also demonstrated the association between gene expression of Additional file 1. Suppmentary Tables.


Patients with gastric cancer are aging in Japan. It is not clear which patients and which surgical procedures have survival benefits after gastrectomy. A multivariate analysis was performed.The medical records of 166 patients aged ≥ 80 years who underwent gastrectomy without macroscopic residual tumors were retrospectively reviewed. Univariate and multivariate analyses using Cox proportional hazard models were performed to detect prognostic factors for overall survival.In univariate analyses, age , performance status (3 vs. 0), American Society of Anesthesiologists physical status (ASA-PS) , Onodera’s prognostic nutritional index (< 40 vs. ≥ 45), the physiological score of the Physiological and Operative Severity Score for the enUmeration of Mortality and morbidity (POSSUM) , surgical approach (laparoscopic vs. open), extent of gastrectomy , extent of lymphadenectomy (D1 vs. ≥ D2), pathological stage (II–IV vs. I), and residual tumor (R1 vs. R0) were significantly correlated with worse overall survival. Multivariate analysis revealed that ASA-PS , extent of gastrectomy  , extent of lymphadenectomy , and pathological stage were independent risk factors for mortality.ASA-PS was a useful predictor for postoperative mortality. Gastrectomy including cardia is best avoided. The population of Japan is aging year by year. According to official Japanese statistics, 9.3% of Japan's population was estimated to be ≥ 80 years in 2021 , extent of lymphadenectomy (D1 vs. ≥ D2), pathological stage (II–IV vs. I), and residual tumor (R1 vs. R0) were significantly correlated with worse OS , worse general, physical, nutritional, or physiological condition , open surgery, total or proximal gastrectomy, D1 lymphadenectomy, advanced cancer stage (pStage II–IV), and R1 resection were prognostic factors for worse OS. A multivariate analysis revealed that ASA-PS 3,4, total or proximal gastrectomy, D0 lymphadenectomy, and pStage II–IV were independent risk factors for worse OS.This result may mean that, among several parameters that may predict postoperative mortality, ASA-PS, while simple, is the keenest classification. A disadvantage of ASA-PS is that it can vary among evaluators even for the same patient. To address this, specific examples and explanations were added in 2014 as follows . The defThe extent of gastrectomy was an independent risk factor for mortality. Patients who underwent gastrectomy including the cardia  had shorter survival than those who underwent distal gastrectomy or subtotal resection of the remnant stomach. The former patients were more likely to die of pneumonia and senility  than were the latter patients , although no significant differences were found. Preserving the cardia may contribute to preventing regurgitation and malnutrition after gastrectomy. A previous report with patients aged ≥ 85 years also mentioned that the prognosis of patients undergoing total gastrectomy was worse than that of patients after distal gastrectomy .As well as the extent of gastrectomy, the extent of lymphadenectomy is also recommended to be limited in patients aged ≥ 80 years . A recenThe pros and cons of adjuvant chemotherapy for gastric cancer in the elderly are also controversial. In the ACTS-GC trial , which sThe present study had several potential limitations. First, this study was limited by its retrospective nature. Second, some patients were not followed up for a sufficient period of time. Third, this study was conducted with a relatively small number of patients from two institutions. We would like to collect and reexamine more patients from more institutions in the future.We retrospectively analyzed the prognostic factors for gastric cancer patients aged ≥ 80 years after gastrectomy. The multivariate analyses showed that ASA-PS 3,4, gastrectomy including cardia, D0 lymphadenectomy, and pathological stage II-IV had worse prognoses. Gastrectomy for elderly patients with severe systemic disease should be carefully performed, and the gastric cardia should be preserved if possible.


Heart failure (HF) is an epidemic disease affecting a large population worldwide. Traditional Chinese medicine (TCM) is playing an increasingly important role in the clinical treatment of HF. According to the TCM theory, HF could be classified into Yang deficiency and Qi-yin deficiency; however, there are few objective and biological lines of evidence for differentiation of TCM HF syndromes to date. In this study, data-independent acquisition (DIA) mass spectrometry was applied to comparatively analyze the protein expression in serum samples obtained from 12 Yang deficiency patients, 12 Qi-yin deficiency patients, and 12 healthy volunteers. Compared to the healthy controls, a total of 121 differentially expressed proteins (DEPs) (77 upregulated and 44 downregulated proteins) were identified in Yang deficiency samples, while 59 DEPs (49 upregulated and 10 downregulated proteins) were detected in Qi-yin deficiency samples. Enrichment analyses of these DEPs based on the GO and KEGG databases revealed functional clusters associated with the immune system, signal transduction, and infectious disease. Several previously reported HF biomarker proteins were found to be the hub proteins in a protein-protein interaction network analysis. Three novel hub DEPs were identified as potential biomarkers for differentiation between different TCM syndromes of HF. The results provide biological insight into the differences of different TCM HF syndromes and an opportunity for specific biomarker identification for different TCM HF syndromes. Heart failure (HF) is a progressive and complex clinical syndrome characterized by symptoms such as breathlessness, ankle swelling, and fatigue and is associated with high morbidity and high mortality. HF is the end stage of various cardiac diseases, such as valvular heart disease, coronary artery disease, and hypertension, which primarily have myocardial damage in common as pathogenesis. In developed countries, the prevalence of HF is approximately 1%-2% in the adult population, while it is ≥10% among people above 70 years of age [In modern Western medicine practice, HF patients are mainly treated with angiotensin-converting enzyme inhibitors, cardiotonic drugs, and diuretics . In ChinIn TCM practice, the diagnosis and treatment of diseases are based on syndrome differentiation (defined as Bian Zheng) and mainly depend on observation, listening, questioning, and pulse analysis . AccordiSince a decade, systems biology technology, in particular proteomics, has been increasingly adopted in TCM research –13. The This study was performed in accordance with the Declaration of Helsinki. The use of human serum samples was reviewed and approved by the research medical ethics committee of Hunan Academy of Traditional Chinese Medicine Affiliated Hospital (registration number: ChiCTR2000033155), and signed informed consent was obtained from all participants prior to enrollment.P > 0.05).A total of 24 treatment-naïve HF patients (12 Yang deficiency syndrome and 12 Qi-yin deficiency syndrome) and 12 healthy controls were enrolled from the Department of Cardiology of Hunan Academy of Traditional Chinese Medicine Affiliated Hospital (China) between June 2020 and December 2020. Clinical data of the enrolled participants are shown in expert consensus on TCM treatment of chronic heart failure, Yang deficiency syndrome is mainly defined by shortness of breath or wheezing, fatigue, and palpitation. Secondary symptoms include being afraid of cold and (or) preference for warmth, cold sensation of abdomen, waist, or limbs, cold sweat, complexion, dark purple lips, dark purple tongue (or with ecchymosis or purplish veins under the tongue), fat and large tongue with possible tooth marks, and a thin, deep, and weak pulse. Meanwhile Qi-yin deficiency syndrome is defined by palpitation, shortness of breath, tiredness, fatigue, nighttime perspiration, dry mouth, dark red zygoma, red tongue with little coating, and a thready and rapid pulse or irregular intermittent pulse. The following exclusion criteria were applied: (a) Patients who did not sign the informed consent or participated in other studies in the past month; (b) diagnosed with other severe primary disease(s) such as severe liver, kidney, hematopoietic system diseases, and/or malignant tumors; (c) patients with acute coronary syndrome, pulmonary embolism, or acute cerebrovascular disease; (d) patients with a history of organ transplantation or psychosis with poor control; (e) drug addicts without detoxification; and (f) age <18 or >85 years.All the participants were diagnosed by chief physicians according to both western medicine  and TCM After thorough clinical evaluation, patients were excluded according to the criteria stated above. For proteomics analysis, blood samples of 12 Yang deficiency patients, 12 Qi-yin deficiency patients, and 12 healthy controls were obtained. The peripheral blood samples (4 mL) were collected from HF patients and controls, following fasting overnight, without anticoagulation. Within 1 hr after collection, the samples were centrifuged at 3500 rpm for 10 min at 4°C. All the plasma samples were stored at −80°C for further analysis.μL SDS-free L3 lysate solution was added to 100 μL of serum sample. The following was conducted to reduce and alkylate the proteins in order to disrupt the disulfide bonds: addition of 10 mM dithiothreitol (DTT) to the samples and incubation for 30 min at 37°C; then, the samples were alkylated with 55 mM iodoacetamide and incubated in the dark for 30 min at room temperature; and lastly, after the treatments, the proteins were enriched using a Gemini C18 column for liquid phase separation of the samples. Protein concentration was quantified using Bradford Protein Assay Kit  and the prepared proteins were stored at −80°C for subsequent analysis.A total of 36 serum samples were obtained and screened. To reduce individual differences and biased parameter estimation, the 12 serum samples per group  were divided into four subgroups. This resulted in three samples in each subgroup, which were pooled. Thus, there were four biological replicates for each clinical phenotype and 12 in total for analysis. Then 900 μg/sample), followed by dilution of 20 μg mixture with 2 mL of buffer A , which was injected into an LC-20AB liquid phase system. Subsequently, a Gemini C18 column  was used for liquid phase separation. Gradient elution was applied to the samples at a flow rate of 4 μL/min in the following gradients: 2% buffer B  for 7 min, 2% to 7% buffer B for 3 min, 7% to 25% buffer B for 27 min, 25% to 60% buffer B for 2 min, 60% to 80% buffer B for 1 min, with buffer B held for 3 min, and 80% to 2% buffer B for 2 min, and 5% buffer B was equilibrated for 5 min. The elution peaks were observed at a wavelength of 214 nm, while one fraction was obtained every 3.15 min. The samples were mixed with the elution peaks of the chromatogram to collect 10 fractions in total and then freeze-dried.The same amount of peptides was taken from each sample and mixed equally . The peptides were separated at a flow rate of 500 nL/min with the following effective gradient: 0–5 min, 5% buffer B ; 5–90 min, 5% to 25% buffer B; 90–100 min, 25% to 35% buffer B; 100–108 min, 35% to 80% buffer B; 108–113 min, 80% buffer B; and 113–120 min, 5% buffer B. The end of the nanoliter liquid phase separator was directly linked to the MS.The dried peptide samples were redissolved in buffer A  and centrifuged for 10 min at 20,000 g, and the supernatant was injected into an UltiMate 3000 UHPLC  for separation. The samples were first enriched and desalted in a trap column and then eluted onto a self-packed C18 column  for data-dependent acquisition (DDA) and DIA analysis. The main settings of the MS and HCD-MS/MS were as follows for DDA analysis: MS: scan range 350–1,500 (m/z), mass resolution 120,000, and maximum injection time (MIT) 100 ms; HCD-MS/MS: collision energy 28 eV, mass resolution 30,000, MIT 100 ms, and 30 s of dynamic exclusion. For DIA analysis, the parameters were set as follows: (1) MS: scan range 400–1,250 (m/z), mass resolution 120,000, and MIT 50 ms; (2) HCD-MS/MS: isolation window 1.7 m/z, mass resolution 30,000, automatic MIT, and 30 s of dynamic exclusion.H. sapiens proteome database using MaxQuant software . The final spectral library was built with peptide/protein entries that satisfied the false-discovery rate (FDR) of ≤1%. Search parameters in the MaxQuant software were selected as follows: enzyme = Trypsin/P; minimal peptide length = 7; PSM-level FDR and protein FDR = 0.01; fixed modifications: carbamidomethyl (C); and variable modifications: oxidation (M) and acetyl (protein N-term). DIA data were analyzed using Spectronaut, which uses iRT peptides to calibrate the retention time. FDR was estimated using the mProphet scoring algorithm, which accurately reflects the matching degree of ion pairs. Then, based on the target-decoy model applicable to SWATH-MS, the false-positive control was completed with FDR ≤1%. DEPs between the three groups were screened using MSstats and fold change ≥2.0 and P < 0.05 were the criteria for determining significant differences.Raw mass spectral DDA data were searched against the UniProtKB/Swiss-Prot https://www.ebi.ac.uk/GOA/). The molecular function, biological processes, and cellular components of the identified proteins were analyzed.Gene Ontology (GO) annotation was performed using the UniProt-GOA Database  database. The KEGG Automatic Annotation Server (KAAS) was first used to annotate the DEPs, followed by mapping the results with the KEGG mapper.The protein-protein interaction (PPI) network was analyzed using the STRING database, from which interactions with a high confidence score of ≥0.7 were selected.Statistical analyses were conducted with GraphPad Prism and SPSS software.P value <0.05 was considered as statistically significant.Pearson's correlation coefficient was calculated between samples to demonstrate correlation of protein quantification and represented as a heat map. The clinical data were analyzed by one-way ANOVA and expressed as mean ± standard deviation (SD). The degree of GO terms enrichment was analyzed with Fisher's exact test. A P > 0.05). However, hemoglobin, blood platelets, and natrium were significantly lower, while creatinine and BNP were significantly higher in the Yang deficiency and Qi-yin deficiency groups compared to the healthy controls. Liver functions and C-Reactive Protein (CRP) did not show any significant differences between the groups.The clinical data of HF patients and the control group are shown in The serum samples of Yang deficiency, Qi-yin deficiency, and healthy controls were analyzed by DDA-based MS. A total of 20322 peptides and 2738 proteins were obtained after applying an FDR of ≤1%.The mass distribution of the identified proteins  revealedP < 0.05 were identified as DEPs. As shown in the volcano plots, 121 DEPs, 77 upregulated  enriched in immune system (hsa04657), signal transduction , cardiovascular diseases (hsa05410 and hsa05414), and infectious disease (hsa04114)  is a member of the IGFBP family which is present in the heart. Recent studies demonstrated the diagnostic and prognostic potential of IGFBP2 in the field of HF nonredundant with BNP –41. IGFBBesides these HF related biomarker proteins, several disease related proteins also showed a different level of upregulation between Yang deficiency and Qi-yin deficiency patients, such as serum amyloid A-2  and Myosin-15 . These results indicated that Yang deficiency patients had a more severe condition than Qi-yin deficiency patients, which is consistent with the clinical diagnosis syndromes .The identification of proteins with a difference in level of induction between the two types of TCM HF syndromes could facilitate the clinical diagnosis and treatment. Adiponectin is the major insulin sensitizing adipokine, mainly secreted by hepatocytes and adipocytes. Previous studies demonstrated that adiponectin exhibits anti-inflammatory, antihyperglycemic, and antiatherogenic properties . In addiIn addition, we found that MBL2 was significantly upregulated in Qi-yin deficiency patients (upregulated by 2.26-fold) but significantly downregulated in Yang deficiency patients . MBL is a pattern recognition molecule of the innate immune system. Due to the important role of innate immune in producing an inflammatory response during microbial infection and tissue regeneration, MBL could be used as a potential biomarker for many human diseases . FurtherThe present study has several limitations. First, the sample size was limited with a total of 12 Yang deficiency patients, 12 Qi-yin deficiency patients, and 12 healthy controls. Although the potential biomarkers found in this study have been reported in other studies, more rigorous researches to study their specific functions and to clinically validate their sensitivity and specificity need to be conducted in another large cohort. Second, BNP, the most widely used biomarker in clinical diagnosis, was not detected in our proteomic analysis. This could be due to two reasons; one is that BNP has a very short half-life, which made it undetectable , and anoThe DEPs, discovered by DIA-based MS and bioinformatics analyses, have shown biological differences between the Yang deficiency and Qi-yin deficiency TCM syndromes of HF. According to the PPI network and different expression levels, ADIPOQ, MBL2, and IGFBP4 were identified as potential biomarkers for syndrome differentiation. These results present a biological insight for diagnosing and differentiating between different TCM syndromes of HF on a scientific basis.


Human Immuno-Deficiency Virus (HIV) remains one of the world’s significant public health challenges. Viral suppression is the key indicator for treatment success in People living with HIV (PLHIV). We determined the level of viral suppression, and its associated factors among PLHIV attending Federal Medical Centre Katsina (FMC Katsina), Nigeria.This retrospective descriptive cross-sectional study was conducted on 913 HIV positive adults enrolled in care between January 2009 and December 2019. Information on socio-demographics, clinical, immunological, Viral load (VL), and other relevant parameters were extracted from the patients’ care records. The primary outcome was the proportion of patients that achieved viral suppression. We also analyzed variables that were associated with VL suppression.Of 913, records of 831 (91.0%) registered patients were analyzed. During the period, 751 (90.4%) achieved viral suppression, 427 (51.4%) had CD4 counts  ≥ 500 and 477 (57.4%) were on HAART for ≥ 5 years. Majority, 793 (95.4%) were on first-line HAART regimen (Tenofovir-Lamivudine-Dolutegravir or Abacavir-Lamivudine-Dolutegravir), and 809 (97.4%) in the non-advanced stage (WHO stages 1 and 2). The median (interquartile range) of viral load was 20 (20–40) vs 19,989  cp/ml in virally suppressed, and unsuppressed  respectively. Factors associated with viral suppression included being unemployed , educated , having a baseline CD4 count ≥ 500 cells/µl , and being on first line HAART regimen .Our study demonstrated a good viral suppression among PLHIV on HAART. Variables associated with viral suppression included unemployment, formal education, high baseline CD4 count, and first line HAART regimen. Human Immuno-Deficiency Virus (HIV) is a leading cause of infections worldwide despite the success of highly active antiretroviral therapy (HAART) . It remaThe 90-90-90 target by UNAIDS envisages that by 2020, 90% of PLHIV will know their HIV status, 90% of people who know their HIV-positive status will be accessing treatment and 90% of people on treatment will have suppressed viral loads . HoweverViral load (VL) measurement is the gold standard for monitoring treatment success. PLHIV on HAART with unsuppressed viral load have a higher risk of disease progression, transmission, and mortality . AvailabThe objective of this study was to determine the level of viral suppression and its associated factors among HIV positive adults attending the HIV clinic at FMC Katsina, Nigeria.This retrospective cross-sectional descriptive study was conducted from January 2009 to December 2019 at FMC, Katsina. The hospital is a 1000 bed capacity, tertiary health facility. It serves as a referral center for both private and other public health facilities in the state, neighboring states, and the Niger Republic. The HIV clinic otherwise referred to as Action Aid clinic was established in 2006 and provides services for the diagnosis, treatment, and prevention of HIV, inclusive of free counseling and testing. It can accommodate about two hundred patients per clinic day and further subdivided into units. The HIV clinic units included the adult ART clinic, Paediatrics ART unit, the Prevention of Mother-to-Child Transmission (PMTCT) unit, HIV/TB co-infection unit, Pharmacy unit and the laboratory.The source of data was from the database of the HIV clinic obtained from all PLHIV enrolled in ART care from January 2009 to December 2019 at FMC Katsina. A total of 1034 clients were registered within this period. The database provided information on socio-demographics, clinical, immunological, virological and other relevant parameters of patients. A total number of 913 adult clients were enrolled within the study period and they were all considered as the study subjects. We excluded children and adolescents less than 18 years on HAART. We used VL testing data for samples corresponding to HIV positive patients who had been on HAART for at least 6 months. Where there were more than one VL results, the most recent  was used for the data. We abstracted data on VL testing results (for plasma) measured in terms of viral RNA copies/ml of blood. Before 2017, virological monitoring was carried out only in those with clinical suspicion of failure or poor adherence. Following policy change in the country and update of National guideline in 2018, all enrolled patients among the PLHIV had a minimum of yearly viral load monitoring. However, where the viral load was above the thresholds of 1000 copies/ml, intensified adherence counseling were carried out and viral load assays repeated within three to 6 months.The primary outcome was virological suppression, defined as having ≤ 1000 copies of viral RNA/ml of blood for plasma, as provided by the National guideline for treatment and control of HIV/AIDS 2018. The secondary outcome of this study was factors associated with viral suppression.Base line CD4 count: This was the CD4 count at the commencement of HAART.2), normal or healthy weight (18.5 to 24.9 kg/m2), overweight (25 to 29.9 kg/m2) and obese (≥ 30 kg/m2).Body mass index (BMI): The body mass index was determined by dividing the weight in kilogram with height in metre squared and classified using Centres for Disease Control (CDC) into underweight . The VL measurements were carried-out in 2018 and 2019.One of the authors extracted the data into Microsoft Excel spreadsheet and this was verified independently by two other co-authors to ensure accuracy and consistency of the data.This was done using Epi-info version 7 and IBM SPSS statistics for Windows, version 25 . The proportion of subjects with virological suppression was expressed as percent. We compared the socio-demograhics , BMI categories and distance from the health facility using chi-square and Fischer’s exact test where apporiate between the subjects that were virally suppressed and those without viral suppression. Besides, clinical and laboratory parameters  were also compared between those that were virologically suppressed and unsuppressed subjects using Chi-square and Fischer’s exact test as appropriate. The viral loads were summarized as median with interquartile range (IQR) and compared using Man-Whitney U test between the two groups. Based on the literature, some variables  along with other variables  with p-value less than 0.25 on the bivariate analysis were entered into binary logistic regression model to determine factors that were associated with virological suppression. The p-value for level of statistical significance was set at < 0.05.During the study period, a total of 1034 subjects including children were enrolled into care at the centre. Out of the 1034 registered subjects, 913 (88.3%) were adults. Of the 913 adults, we excluded 82 subjects . Thus, we included 831 (91.0%) in the final data analysis as highlighted in Fig. The subjects age ranged from 18 to 77 years. The mean age (standard deviation) of the subjects was 34.5 ± 9.3 years. Most of the subjects were below the age of 50 years . There were more females, 578 (69.6%) than males. A total of 709 (85.3%) were unemployed while 78.6% were married as shown in Table The median viral load was 20 (20–74) cp/ml in the study subjects. The median viral load in those that achieved viral suppression range from 20 to 925 cp/ml, while in those without viral suppression, VL range from 1170 to 2,468,443.0). In 2018, out of 87 subjects, 77 (88.5%) had viral suppression. Similarly, in 2019, out of 774 subjects, 704 (91.0%) had achieved viral suppression. The duration on HAART, TB status, WHO staging of the disease, and pregnancy status were not related to viral suppression. However, baseline CD4 count and HAART regimens were associated with viral suppression  due to missing information. Although we compared with global set target of 90-90-90, the samples were not randomly drawn. Hence, our findings may not be generalizable. Furthermore, being a retrospective study, we could not adjust for some potential confounders, which may have affected our findings.Our study demonstrates a low level of viral non-suppression among PLHIV on HAART and the target of 90% viral suppression in the PLHIV on HAART by the USAID is achievable in resource constraint settings. Also, having a high baseline CD4 count, being employed, uneducated and being on first-line HAART are predictive of viral load suppression.


The aim of this study was to evaluate the effects of a modified Xiaohua Funing decoction (Xfd) on acute liver failure (ALF) and determine whether the protective mechanisms are related to alterations in the gut microbiota. μg/kg). Male BALB/c mice were randomly divided into the following 4 groups: the control group , model group , silymarin pretreatment group , and modified Xfd pretreatment group . The Sil and Xfd groups received the respective intervention orally for 14 days and 2 h before D-Gal/LPS treatment. The liver injury markers included alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and liver histology. 16S rRNA gene sequencing was performed to assess the effects on the caecum content.  An animal model of ALF was induced by intraperitoneal injection of D-galactosamine  and lipopolysaccharide . Interestingly, a negative correlation was observed between the abundances of the gut microbiota constituents, specifically Clostridia_UCG-014, and ALT and AST levels.  D-Gal/LPS treatment caused severe ALF, illustrating that the ALF model was successfully established. The administration of Sil and Xfd greatly reduced the serum ALT and AST levels and improved the pathological signs of liver injury. However, no significant difference was found between the two groups. In contrast to the Mod group, the Sil and Xfd groups showed a shift toward the Con group in terms of the gut microbiota structure. The abundances of Firmicutes and Bacteroidetes and the Bacteroidetes/Firmicutes ratio in the Mod group significantly differed from those in the Con group. The Sil and Xfd groups showed restoration of the disordered microbiota. Significantly increased relative abundances of Lachnospiraceae_NK4A136_group and Candidatus_Saccharimonas and a markedly decreased Muribaculaceae abundance were found in the Sil and Xfd mice compared with those in the Mod mice ( In summary, our results indicate that Xfd may protect the liver and modify the gut microbiota in ALF mice. Acute liver failure (ALF) caused by extensive hepatocyte necrosis is a fatal clinical syndrome and has been well characterized. However, the underlying mechanisms are not fully understood . StudiesBambusae Caulis in Taenias, Bupleuri Radix, Pinelliae Rhizoma Praeparatum cum Zingibere et Alumine, Aurantii Fructus, Setariae Fructus Germinatus, Paeoniae Radix Alba, Schisandrae Fructus Chinensis, Salviae Miltiorrhizae Radix et Rhizoma, Glehniae Radix, Citri Reticulatae Pericarpium, and Mume Fructus. For over five decades, Xfd has been known to have a significant impact on liver diseases. Our previous clinical studies suggested that Xfd improved liver function and other symptoms, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein, total bilirubin, and alkaline phosphatase levels; lateral thorax pain; stomach pain; loss of appetite; abdominal distension; loose stools; and jaundice. Xfd not only is effective but also has no other adverse effects, such as induction of kidney failure and diarrhea [In China, Chinese herbal medicine (CHM) therapy for ALF has been widely used. Several clinical trials have shown that CHM can provide ALF patients with important alternative and complementary therapy benefits, such as improvement in systemic and liver recovery, regulation of inflammatory mediators, and induction of antiapoptotic genes –9. Modifdiarrhea , 11. Sevdiarrhea .The classical liver injury model induced by D-galactosamine /lipopolysaccharide (LPS) has been used to evaluate abnormal liver function, regulation of metabolic disorders, and variations in the gut microbiota , 14. ThiBambusae Caulis in Taenias, Bupleuri Radix, Pinelliae Rhizoma Praeparatum cum Zingibere et Alumine, Aurantii Fructus, Setariae Fructus Germinatus, Paeoniae Radix Alba, Schisandrae Fructus Chinensis, Salviae Miltiorrhizae Radix et Rhizoma, Glehniae Radix, Citri Reticulatae Pericarpium, and Mume Fructus, at a ratio of 15 : 10 : 10 : 15 : 30 : 20 : 10 : 12 : 15 : 12 : 10. Xfd has been authenticated and standardized based on marker compounds according to the Chinese Pharmacopoeia 2020 [Escherichia coli, 055:B5) and D-Gal were obtained from Sigma-Aldrich Chemical Co. . ALT and AST assay kits were provided by Nanjing Jiancheng Bioengineering Institute . A paraformaldehyde solution, acetonitrile, and formic acid were acquired from Macklin Biochemical Co., Inc. .Xfd contains 11 medicinal components, namely, eia 2020 . A dose c mice  were obtained from the Shandong Laboratory Animal Center . The animals were acclimated in a specific pathogen-free animal laboratory at 22 ± 2°C with 50–70% relative humidity under a 12/12 h light/dark cycle. The mice were fed standard laboratory chow and given water ad libitum.Thirty-two male BALB/n = 8). The control group (Con) and model group (Mod) received the same volume of saline intragastrically once daily, the pretreatment silymarin group (Sil) received 200 mg/kg silymarin intragastrically once daily, and the pretreatment Xfd group (Xfd) received 650 mg/kg Xfd intragastrically once daily. The pretreatment course was 14 days. The bodyweight of the mice was measured every week.The mice were randomly divided into 4 groups (μg/kg) via intraperitoneal injection to induce ALF; D-Gal and LPS were freshly dissolved  in saline. The mice in the control group were treated with the same volume of saline and with the same procedures. The experimental design and mouse handling procedures were in accordance with the guidelines for the care and use of laboratory animals.Two hours after the last intragastric treatment, the mice in the Mod, Sil, and Xfd groups were treated with D-Gal (0.5 g/kg)/LPS .μm sections, and stained with hematoxylin-eosin (HE). The sections were analyzed under a Zeiss Axioskop 2 Plus upright light microscope equipped with a camera .The liver tissue was weighed, washed with saline, and immediately placed into a tube containing 4% paraformaldehyde solution. The liver tissues were washed with phosphate-buffered saline (PBS), embedded in paraffin wax, sliced into 5 R packages (v3.3.1) [The cecal contents were collected in sterile tubes and stored at −80°C until gut microbiota analysis. Total bacterial DNA was extracted using a TIANamp Stool DNA Kit  following the manufacturer's instructions. The 16S rRNA gene in the DNA samples was amplified using the conventional barcoded universal bacterial primers F338/R806 targeting the V3–V4 region, which were selected to analyze the taxonomic composition of the gut microbiota. The concentrations of the PCR products were determined using an Illumina HiSeq platform  for paired-end reads with 2 × 300 bp (468 bp) sequencing. The trimmed sequences were uploaded to the Quantitative Insights Into Microbial Ecology  and (v3.3.1) , 18. Theα diversity was represented according to the Shannon, Simpson, ACE, and Chao indexes. The taxonomy-based analyses were visualized based on GraPhlAn. The data were visualized via principal component analysis (PCA) [The number of shared and unique operational taxonomic units (OTUs) and taxonomic classifications were determined by BLAST searching the sequences against the Greengenes database at the 97% nucleotide similarity level . The α dis (PCA) .P < 0.05 was considered statistically significant. The results were graphically visualized using GraphPad Prism , and correlation heatmaps were generated using R software (v3.3.1).All data are expressed as the mean ± SD and were subjected to one-way analysis of variance (ANOVA), Fisher's least significant difference (LSD) test, or Spearman's correlation test using SPSS 23.0 . The PCA score plots based on phylogenetic statistical analysis methods indicated clustering of the gut microbiota within groups. The Wilcoxon rank-sum test was used to compare the group data of two samples. In a preliminary experiment, it was observed that the mice began to die 10 h after D-Gal (0.5 g/kg)/LPS injection; thus, to ensure that no mice died during the experiment, we chose to perform euthanasia at 8 h.There was a gradual increase in body weight in all groups. In week 2, the body weights of the mice were not significantly increased in any of the four groups .The liver weight of the mice did not significantly differ among the four groups . The livThe livers of the Con mice had clear liver lobules, and no degeneration or necrosis of liver cells was observed. In contrast, D-Gal/LPS treatment caused severe liver failure. The livers of the Mod mice had a large amount of hepatocyte degeneration, mainly cellular edema. Nuclear lysis, necrosis, inflammatory cell infiltration, massive bleeding, venous congestion and expansion, and no cell proliferation were observed. Compared with the Mod group, the Sil and Xfd groups showed reduced hepatocyte necrosis, bleeding, and inflammation, and the ALF changes were significantly improved .To characterize the composition of the gut microbiota, 1,542,312 cleaned sequences were generated from 32 samples. In total, 567, 608, 501, and 540 OTUs were found in the Con, Mod, Sil, and Xfd groups, respectively. In total, 747 operational OTUs were obtained. Moreover, there were 364 common OTUs across the four groups, and 16, 47, 19, and 41 specific OTUs were found in the Con, Mod, Sil, and Xfd groups, respectively .α diversity metrics. There was an obvious increase in the ACE- and Chao-based diversity (P < 0.05) following Sil or Xfd treatment (P=0.055) and Simpson (P=0.217) indexes.The Shannon, Simpson, ACE, and Chao indexes were analyzed as reatment . There wThe PCA of both unweighted and weighted UniFrac distances showed that the first principal component (PC1) accounted for 71.68% and the second principal component (PC2) accounted for 16% of the variation. PCA also showed a reversal of the changes that occurred in the gut microbiota in response to the Sil and Xfd treatments, especially an obvious shift along PC1 Figures .P < 0.01). The proportion of Firmicutes showed an increasing trend in the Sil and Xfd groups  compared with that in the Mod groups . The abundance of Bacteroidetes in the Mod group (34.62 ± 21.23%) was significantly higher than that in the Con group (19.95 ± 13.92%) (P < 0.05). The proportion of Bacteroidetes showed a decreasing trend in the Sil and Xfd groups  compared with that in the Mod group . The gut microbiota of the Mod group mice was characterized by an increased Bacteroidetes/Firmicutes ratio (0.90 ± 0.71), and the ratio showed significant differences, with lower proportions in the Sil and Xfd group mice  . There was no statistically significant difference in the abundances of Firmicutes and Bacteroidetes or the Bacteroidetes/Firmicutes ratio between the Sil and Xfd groups (P < 0.05). There were no significant differences in the Actinobacteriota and Desulfobacterota abundances among the four groups . Otherwise, compared with the Sil treatment, the Xfd treatment did not induce remarkable increases in abundance (P < 0.05) . Xfd treatment did not induce a significantly reduced relative abundance compared to that in the Mod group (P < 0.01)   .ALF is a devastating clinical syndrome with high mortality and a substantial disease burden . AlthougMassive liver cell necrosis and extensive inflammation are the major changes in ALF , 24. D-Gc mice are widely used in animal immunology experiments. Since the weight change in the mice after 2 weeks was not very obvious, a significant difference was not observed. Moreover, Xfd had no effect on the weight of the liver. ALT and AST are major biochemical indicators of liver function. The ALT and AST levels in the Mod group were significantly increased compared with those in the Con group. Additionally, these elevations were rescued after Sil and Xfd pretreatment, indicating protective effects on ALF. The major histopathological changes in the livers of the mice after D-Gal/LPS exposure were the development of necrotic areas and severe hepatocyte necrosis. The liver appearance and texture associated with ALF were improved. Moreover, the Sil and Xfd treatments could also protect the liver tissue microstructure. The improvement in ALT and AST levels may be the most important reason.BALB/Salviae Miltiorrhizae Radix et Rhizoma. Ma [Schisandrae Fructus Chinensis prevented elevations in serum biochemical parameters, including AST and ALT levels. The mechanisms of Schisandrae Fructus Chinensis against drug-induced liver damage involve the regulation of inflammatory factors and oxidative stress [Schisandrae Fructus Chinensis can accelerate the repair and regeneration of liver cells. In this experiment, this phenomenon was not observed due to the short time span from establishing the model to killing the mice. Saikosaponins, which are major bioactive compounds in Bupleuri Radix, possess anti-inflammatory and antiviral activities, regulate cell apoptosis, and have hepatoprotective effects, as described in a comprehensive review [Citri Reticulatae Pericarpium was shown to inhibit adipogenesis in 3T3-L1 preadipocytes. A marked reduction in PPAR-γ, C/EBP-α, and SREBP-1 levels played a positive role in protection against ALF [Mume Fructus might play a therapeutic role in the improvement in ALT and AST levels by enhancing superoxide dismutase (SOD) activity and lowering malondialdehyde (MDA) content [These data clearly reveal that D-Gal/LPS-induced ALF was attenuated by Xfd treatment. We did not perform a high-performance liquid chromatography analysis of Xfd. This is a shortcoming of this study. We will perform this analysis in future research to obtain a clearer understanding of the main compounds present in Xfd. Tanshinone IIA is the primary active component extracted from zoma. Ma  reportedzoma. Ma  demonstre stress . Studiese review . Furthere review . Citri Rinst ALF . Mume Fr content . Accordi content .Accumulating evidence indicates that multiple pathogenic factors that accelerate ALF are associated with gut microbiome imbalance –39. AlteG found that walnut green husk polysaccharide consumption decreased the relative abundance of norank_f_Muribaculaceae at the genus level during a liver injury in Kunming male mice [α and TLR2 were negatively correlated with the abundance of Candidatus_Saccharimonas. In their research, Qingluo Tongbi decoction was shown to play a therapeutic role by decreasing the inflammatory responses regulated by the gut microbiota [At the genus level, the microbiota composition greatly varied among the four groups. Currently, knowledge regarding the relationships between the members of Muribaculaceae and ALF is limited. Our data revealed that the decreased Muribaculaceae abundance might be a protective effect of Sil treatment. However, the effect of Xfd was not very obvious. Consistent with our results, Wang ale mice . Polyeneale mice . After pale mice . Surana ale mice . Patientale mice . Huang pcrobiota . In our crobiota , 51. We In conclusion, our findings demonstrate that Sil and Xfd treatment could notably alleviate D-Gal/LPS-induced ALF in mice, which might be correlated with decreased ALT and AST levels and reduced liver pathological damage. These protective effects may be mediated by alteration of the gut microbiota in ALF. Our findings provide new insight into the pretreatment of ALF and suggest that Xfd has probiotic potential against ALF and that its hepatoprotective effect on ALF patients needs to be proven in future clinical trials.


However, resolving the output signals of individual cells within the tissue sample, i.e., single cell segmentation, remains challenging. To address this problem, we developed MATISSE (iMaging mAss cyTometry mIcroscopy Single cell SegmEntation), a method that combines high-resolution fluorescence microscopy with the multiplex capability of IMC into a single workflow to achieve improved segmentation over the current state-of-the-art.Visualizing and quantifying cellular heterogeneity is of central importance to study tissue complexity, development, and physiology and has a vital role in understanding pathologies. Mass spectrometry-based methods including imaging mass cytometry (IMC) have in recent years emerged as powerful approaches for assessing cellular heterogeneity in tissues. IMC is an innovative multiplex imaging method that combines imaging using up to 40 metal conjugated antibodies and provides distributions of protein markers in tissues with a resolution of 1 μmMATISSE results in improved quality and quantity of segmented cells when compared to IMC-only segmentation in sections of heterogeneous tissues. Additionally, MATISSE enables more complete and accurate identification of epithelial cells, fibroblasts, and infiltrating immune cells in densely packed cellular areas in tissue sections. MATISSE has been designed based on commonly used open-access tools and regular fluorescence microscopy, allowing easy implementation by labs using multiplex IMC into their analysis methods.MATISSE allows segmentation of densely packed cellular areas and provides a qualitative and quantitative improvement when compared to IMC-based segmentation. We expect that implementing MATISSE into tissue section analysis pipelines will yield improved cell segmentation and enable more accurate analysis of the tissue microenvironment in epithelial tissue pathologies, such as autoimmunity and cancer.The online version contains supplementary material available at 10.1186/s12915-021-01043-y. Multiplex imaging technologies have revolutionized our ability to study cellular heterogeneity in tissues. These methods allow visualization of spatial organization of the tissue and quantification of different cell types. Moreover, these methods can yield precise views of cell-to-cell differences, quantify differences in signaling status, map signaling network topologies, and lead to important mechanistic insights. Visualizing and quantifying cellular heterogeneity in tissue samples is of increasing importance in many areas of biology, most notably in cancer research and oncology, where we now appreciate that the heterogeneous tumor microenvironment has profound implications for study, diagnosis, prognosis, and treatment of cancer .2 tissue area at a time. Cytometry time-of-flight (CyTOF) mass spectrometry is then used to analyze metal isotope distribution as a readout for protein markers. Unlike fluorescence-based microscopy imaging methods where only 4 or 5 markers can be labeled and visualized simultaneously, mass spectrometry-based imaging methods can provide simultaneous labeling and readout of approximately 40 markers. However, one of the main challenges for mass spectrometry-based imaging methods, such as IMC, is the difficulty of single cell segmentation, i.e., distinguishing signals coming from individual cells. Currently available pipelines, such as an ilastik [The most commonly used multiplex imaging technologies fall into two main categories: microscopy based multiplex immune-histochemistry (IHC) methods , 3 and m ilastik  and Cell ilastik  extensio ilastik , use memWhile IMC resolution is limited by a fixed 1 μM pixel size, fluorescent microscopy allows acquisition at variable resolutions well below 1 μM pixel size. Therefore, to provide a solution for the single cell segmentation problem in IMC, we have developed MATISSE  . We usedHere, we prepared the tissue sections according to the procedure described in the “Methods” section  tissue blocks of colonic biopsies from patients with inflammatory bowel disease (IBD) were collected and included. Informed consent was obtained from all patients. Ten tissue sections were included from 3 patients with IBD, of 3 separate timepoints of biopsy per patient. The study was approved by the medical ethical board of the UMC Utrecht . Furthermore, TMA sections of rest-material, including intestine, liver, and non-small cell lung cancer, were included according to the no-objection-agreement, approved by the UMC Utrecht biobank committee (protocol #18-222).For a comprehensive list of antibodies, compounds, and kits, see Tissue sections of 4 μM thickness were cut and placed on a glass slide .2O for 5 min. Samples were mounted in 90% glycerol and covered with a coverslip for microscopy.Slides were baked for an hour at 60 °C. Samples were deparaffinized in xylene twice for 10 minutes, followed by rehydration in a graded series of ethanol , washed in Milli Q water 3 min, and finally PBST (TBS containing 0.1% Tween) for 10 min. Antigen retrieval was performed for 30 min at 96 °C in Tris-EDTA  pH 9, followed by a cool-down period of 10 min at room temperature. Samples were incubated in TBST for 10 min. Tissue sections were encircled using a PAP pen. Blocking was performed with TBST containing 3% BSA and FC-block 1:100 for 1 h at room temperature. Metal-conjugated antibodies were diluted according to dilutions stated in Slides were imaged on a Zeiss CellObserver using a × 20 dry objective . A Colibri 7 was used as light source, in combination with a Zeiss 90 HE filter set. The system was equipped with a Hamamatsu Orca Flash4.0 V2+ camera C11440-22CU). Images were acquired in a tiled Z-stack format with 10% overlap between tiles and 9 Z-slices using ZEN software (2.3). ZEN was again used to export imaging data to individual 16-bit tiff tiles. Z-stacks were converted to single in-focus images using the Extended Depth of Field plugin in Fiji at highest quality settings 40-22CU. . Tile im2O. Samples were stained with toluidine blue for 5 min at RT, washed for 3 min with ddH2O, and dried. Mass cytometry imaging was performed on a Hyperion (Fluidigm) laser ablation module, coupled to a Helios (Fluidigm) mass cytometer. Tuning was performed according to manufacturer instructions. Laser ablation frequency was set to 200 Hz. Data files were converted to 32-bit tiff files using the imctools library (https://github.com/BodenmillerGroup/imctools).After microscopy, samples were unmounted by dipping and washing in ddHHistone H3 imaging results typically show nuclear localization with a different staining pattern compared to DNA intercalator , since they bind to different targets, and Histone H3 is an antibody staining. Therefore, we used both intercalators for registration (see below).https://imagej.net/Landmark_Correspondences). The method for transformation used was moving least squares and transformation class similarity.Fluorescent microscopy images of DAPI were registered to mass cytometry images of the DNA-intercalator signal, while retaining the resolution of the fluorescent images. Registration was performed using key points generated using the MOPS algorithm . Images The plot profile function in Fiji  was usedBefore training on imaging mass cytometry data, signal intensity was manually scaled and converted to a 16-bit range. Annotations were generated by experienced users using Ilastik [Training data was generated for 5 classes, namely non-epithelial cellular membranes, non-epithelial nuclei, epithelial cytoplasm/membrane, epithelial nuclei, and background. Training and probability map generation was performed in Ilastik using only IMC data as input. In Ilastik all features with sigma between 0.3 and 1.6 were selected. Probability maps were saved to individual 32-bit tiff files for each class.Training data was generated on a random subset of 100 tile regions of fluorescent imaging data. Annotations were made by 4 experienced users for classes nuclei, edges of nuclei, and background, using Fiji  based onSegmentation was performed using Cellprofiler  (v3.1.9)For IMC-based segmentation, only the probability maps generated using the IMC-data were used. For MATISSE segmentation, identification of nuclei was based on the probability maps generated using the fluorescent images of DAPI, at high resolution. The identified nuclei were next downscaled to the resolution of the IMC data and expanded to the full extent of the cells using the membrane probability generated using only the IMC data.Trained experts were asked to manually annotate individual nuclei in a subset of 30 images of nuclear staining (100 × 100 μm). These images were a composite of both fluorescent and IMC data, of DAPI and DNA-intercalator respectively, after co-registration, at high resolution. In total 2642 nuclei were annotated. The annotations were converted to a binary mask and downscaled to the resolution of IMC data.For calculation of a recall score, first the intersection over union (IOU) was calculated. The IOU is determined for all manually annotated nuclei that overlap with any nuclear outline generated by either segmentation pipeline separately. IOU is calculated as the surface area of the intersection area between nuclear outlines, divided by the surface area of the union of both nuclear outlines. For each manual event only the interaction with highest IOU was taken for recall calculation, in case of multiple identified overlaps . Recall The proportion of split events is calculated as the fraction of ground truth nuclear annotations that overlap with multiple nuclear events from either segmentation pipeline, over the total number of events that have any overlap. Overlap being defined as at least 20% of surface area of the ground truth object.A probability score was calculated for intersection of cell boundaries, derived from segmentation maps from automated pipelines, with manually annotated nuclei . This waFor calculation of fragmentation per phenocluster, we calculated the proportion of events generated by either segmentation pipeline that was identified as part of a fragmentation event, from the total number of events that overlap for at least 20% with any ground truth annotation. Fragmentation events being at least two events overlapping with a single ground truth annotation for at least 20% of the ground truth surface area per interaction.Single cell data was generated in R (v4.0)  by extraSegmentation maps were converted to polygons in R using packages sf  and starSingle cell clusters were generated with the Rphenograph package , based oPolygons representing cell outlines were plotted and given a random color fill corresponding to their assigned phenocluster number. Clusters with a large difference in number of cells across segmentation methods were selected by taking the top 6 with the highest or lowest ratio. Clusters assumed to be equally represented in both segmentation methods were selected based on a ratio between 0.9 and 1.1. Random regions were generated by selecting a region of 100 by 100 μm from 4 randomly selected ROIs.Additional file 1. MATISSE_MANUAL: an interactive PDF outlining all steps of computational methods, to allow smooth implementation of MATISSE methods. All code required is publicly accessible in Github: https://github.com/VercoulenLab/MATISSE-Pipeline.Additional file 2: Supplementary Table 1. Antibody panel resourcesAdditional file 3: Figure S1. a) Representative examples of IMC images, b) nuclear staining profiles DAPI versus Ir193, and c) predicted cell outlines of different tissues. Figure S2. a) Representative example of overlap between manual annotations and predictions, b) Recall scores calculated for different tissues, and c) Ir193 signal intensity across all analyzed images. Figure S3. Comparison of IMC and MATISSE performance per phenocluster. a) Cell numbers identified per phenocluster across all analyzed images. b) representative examples of cell outlines, density and phenoclusters, c) representative examples of cells colored by specific phenoclusters, d) fragmentation events per phenocluster.Additional file 4: Supplementary Table 2. Phenocluster names.


Alterations of microcirculation are associated with organ hypoperfusion and high mortality in septic shock. This study is aimed at investigating the effects of unfractionated heparin (UFH) on intestinal microcirculatory perfusion and systemic circulation in a septic shock model. Escherichia coli, (c) basic therapy group: septic shock animals treated with antibiotics and 10 ml/kg/h saline, and (d) heparin group: septic shock animals treated with basic therapy plus UFH. Hemodynamic variables were measured within 24 h after E. coli administration. The intestinal microcirculation was simultaneously investigated with a sidestream dark-field imaging technique. Additionally, the function of vital organs was evaluated at 12 h postadministration (T12).  Twenty-four beagle dogs were randomly allocated into four groups: (a) sham group: healthy controls, (b) shock group: septic shock induced by E. coli induced a progressive septic shock in which the mean arterial pressure (MAP) decreased and lactate levels sharply increased, accompanied by deteriorated microvessel perfusion. While basic therapy partially improved the microvascular flow index and the perfused microvessel density in the jejunal villi, UFH significantly restored major microcirculation variables at T12. Physiological variables, including MAP, urine output, and lactate levels, were improved by UFH, whereas some hemodynamic indices were not affected by UFH. With respect to organ function, UFH increased the platelet count and decreased the creatinine level.  UFH improves microcirculatory perfusion of the small intestine independently of the changes in systemic hemodynamic variables in a canine model of septic shock, thereby improving coagulation and renal function. Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection and is associated with high mortality . Fluid rTissue perfusion insufficiency, including systemic or regional alteration, plays a crucial role in organ dysfunction. Research has proven that organ dysfunction may persist, despite apparent restoration of systemic macrohemodynamics . TherefoUnfractionated heparin (UFH) is an acidic glycosaminoglycan mainly generated by mast cells and basophils. It acts as an anticoagulant, preventing thrombosis by increasing the affinity between antithrombin III and proteases, thereby accelerating the inactivation of proteases. Except for anticoagulant and anti-inflammatory properties, UFH can efficiently restore the morphology and function of vascular endothelial cells, prevent glycocalyx shedding, and improve organ perfusion . Our preEscherichia coli-induced deterioration of jejunal mucosa microcirculation was accompanied by hemodynamic disorder and organ dysfunction in a canine septic shock model. While UFH slightly improved some physiological variables, such as the mean arterial pressure (MAP), urine output (UO), and the lactate clearance rate, it (i) significantly improved jejunal microcirculatory dysfunction and (ii) accompanies the normalization of the coagulation and renal function in a canine septic shock model.Here, we report that Guide for the Care and Use of Laboratory Animals  and were approved by the Ethics Committee for Animal Research of China Medical University (protocol number L2011001).Twenty-four adult beagle dogs , weighing 13.31 ± 1.65 kg, were purchased from the Laboratory Animal Center of Dalian Medical University . All of the animals had free access to standard dog food and water throughout the experiments. They were kept on a strict day/night light cycle at a room temperature of 22 ± 1°C and a relative humidity of 30%-60% for a minimum of 10 days before surgery. All of the experiments were performed following the Escherichia coli  bacteria strain was provided by the Department of Laboratory Medicine of the First Hospital of China Medical University [2, and colony counts were determined. The McBurney turbidity (Mc-fold) of the bacterial suspension was measured using spectrophotometry. According to the preliminary experiment, a bacterial amount of 3.5 × 108 colony-forming units (cfu)/kg was administrated to each animal. The bacterial suspension was mixed with 20 ml sterile saline before injection.The iversity . This stμg/kg/h) until the animals were euthanized. Intravascular catheters were placed in the femoral arteries and external jugular veins. The animals were orally intubated with a 7.5 ID tracheal tube for mechanical ventilation , in order to maintain SpO2 > 95%, pO2 > 80 mmHg, and pCO2 = 35 ± 5 mmHg. A heating pad was used to maintain the core body temperature at 36.5 ± 1.0°C. Cystostomy was performed with a urethral catheter for adults (14F) fixed to the abdominal wall through a paramedian incision. Additionally, a gastric tube was orally placed for gastrointestinal decompression. Jejunostomy was performed over a length of 15-20 cm from the Treitz ligament, and the orificium fistulae were fixed at the lateral abdomen for intestinal microcirculation observation.The animals underwent surgery under sterile conditions. Anesthesia was induced by intramuscular injections of pentobarbital (25 mg/kg). Sedation and analgesia were maintained through the continuous intravenous infusion of pentobarbital (2-3 mg/kg/h) combined with fentanyl (0.3-0.4 E. coli suspension (3.5 × 108 cfu/kg) was injected intravenously into the animals at 3 ml/kg/h. When the MAP decreased to 80% of the baseline, the timepoint (approximately 1 h postinjection) was designated as the establishment of the septic shock model [The 24 canines were randomly allocated into four groups. (a) Sham group: intravascular catheters were placed and microcirculation of intestinal villi was monitored. (b) Shock group: ck model . (c) Basck model  (No.1 BiE. coli injection), T1, T2, T3, T4, T6, T8, T12, T18, and T24. The internal surface of the intestine was washed with saline at 37°C to maintain the same temperature conditions for microcirculation observation. The vascular video analysis was conducted manually and blindly by two independent investigators (Z. Heng and A. Xin). We used the following parameters to assess intestinal microcirculation, as suggested by Schiffer et al. [2), perfused vascular density , proportion of perfused vessels , and microvascular flow index (MFI). The TVD was calculated as the total length of vessels divided by the total area of the image. The PVD was calculated as the length of perfused vessels divided by the total area of the image. The PPV was calculated as follows: PPV = /total number of vessels. The MFI was evaluated as described by Schiffer et al. [The microcirculation of the jejunal villi was visualized with a sidestream dark-field (SDF) illumination imaging system . The probe of the SDF was inserted to the same depth (5-7 cm) from the fistula of the abdominal wall. We collected five high-quality steady images of at least 20 s in different regions of the jejunum using a hand-held SDF device according to recommended techniques for video acquisition . All ther et al. : total vr et al. . The imaE. coli administration was designated as T0. E. coli suspension (3.5 × 108 cfu/kg) was continuously injected from T0 to T2 via the external jugular vein. Blood samples were taken, and hemodynamic measurements were performed at T0, T1, T2, T3, T4, T6, T8, T12, T18, and T24. Hemodynamic variables were monitored by a pulse indicator continuous cardiac output (PICCO) system  through the catheter in the right femoral artery. MAP, heart rate (HR), cardiac index (CI), systemic vascular resistance index (SVRI), and stroke volume variation (SVV) were measured at designated timepoints. The PICCO system was calibrated by injecting 5 ml saline at 0°C three times via the external jugular vein. Samples for blood gas analysis were collected in heparinized 1 ml syringes. The variables representing organ function, including the platelet count, oxygenation index, hepatic alanine transaminase (ALT), and creatinine (Cr) levels, were measured at T12.The timepoint of U tests with adjustment for multiple comparisons. Two-tailed P values < 0.05 were considered statistically significant. All statistical analyses were conducted using SPSS 22.0 software  and GraphPad Prism 7.0 software .Normally distributed data are expressed as the mean ± standard error of the mean. Dynamic data were analyzed over time and between groups by two-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test. For a single timepoint, the four groups were compared by one-way ANOVA, followed by Dunnett's test or the LSD multiple comparisons test. Nonnormally distributed data are expressed as medians (interquartile range) and were analyzed with the Kruskal-Wallis test, followed by post hoc Mann-Whitney E. coli infusion. When the MAP decreased to 80% of the baseline (approximately at T1), this was defined as the establishment of the septic shock model. From this moment, therapy was administrated into the animals (P > 0.05).Twenty-four age- and gender-matched beagle dogs were randomly allocated into four groups. Each group included three female and three male animals. The MAP sharply decreased after  animals . Two of E. coli infusion aggravated major microcirculatory variables, including the PPV, PVD, and MFI. The PPV sharply and continuously declined until the end of the study in the shock group . PPV decreased to zero at T4 and then slowly increased in the basic therapy group. Compared with the basic therapy group, UFH normalized the PPV from T3 . The MFI did not significantly increase in the basic therapy group. However, the MFI significantly increased from T4 after a transient decline in the heparin group . UO tended to increase from T4 in the basic therapy group. Furthermore, UFH dramatically improved UO from T4 to T24 compared to the basic therapy group . While the lactate level consistently rose in the shock animals, it tended to decrease from T12 in the basic therapy group and from T6 in the heparin group , and the lactate clearance rate increased in the basic therapy group  across 24 h in the four groups. ScvO2 in the shock group showed a sharp decrease relative to the sham animals. Additionally, UFH improved ScvO2  strategy effectively improved the microcirculation of the jejunum, resulting in renal and coagulation functional recovery, in an Here, we demonstrated that UFH ameliorated intestinal microcirculation by enhancing the PVD and microvessel flow following septic shock. First, UFH is widely used as an anticoagulant to prevent deep vein thrombosis in the clinic. Further, it has been demonstrated that UFH improves disseminated intravascular coagulation-associated microvascular thrombosis in sepsis . BesidesApart from increasing intestinal microvascular perfusion, UFH tended to improve renal function, as demonstrated by an increase in UO and a decrease in blood Cr concentration. Although we could not detect renal microvascular flow via SDF, this implies that UFH may exert beneficial effects on improving renal function in septic shock. The optimal dosage, duration, and safety of UFH treatment in sepsis remain controversial. In the present study, the dosage of UFH was determined on the basis of our preliminary experiments and by referring to the study by Schiffer et al. in an ovine endotoxin shock model . Our finWhile UFH significantly improved sepsis-induced microcirculatory dysfunction, some hemodynamic variables were slightly affected by either basic therapy or UFH. A possible explanation for this observation is that young beagle dogs suffer from arrhythmia and tachycardia. HR increased upon septic shock and even more after fluid resuscitation. PICCO variables, such as CI, may be affected by tachycardia. It is noteworthy that the observed changes in macrocirculation were not parallel with changes in microcirculation in the septic shock model. Our results are consistent with previous experimental and clinical observations, which suggest that sepsis-related microcirculatory alteration is independent of systemic variables , 28. NajWe used fluid resuscitation and antibiotics as the standard treatment strategy. The PVD and MFI were significantly increased by basic therapy, compared with the shock group, suggesting the effectiveness of fluid resuscitation and antibiotics. However, it was also found that the increased fluid volume did not improve the hemodynamic parameters. In contrast, tissue edema and increased effusion in the lung were observed upon autopsy of animals. Consistent with previous studies, we confirmed that excessive fluid resuscitation and an inadequate response to fluid resuscitation led to tissue edema and increased mortality . The addThe present study has several limitations. First, an analysis bias of the MFI cannot be ruled out as we have added undetected vessel segments and deleted vague vessels during SDF video analysis. However, this method has proven feasible and valuable in animal studies of cardiopulmonary resuscitation and sepsis. Second, alterations in microcirculation of other organs should be simultaneously monitored. Nonetheless, SDF can be applied to the microvascular system of superficial tissue, such as intestinal mucosa or sublingual mucosa. It is not suitable for other solid organs. Third, we did not investigate the mechanisms underlying the beneficial effects of UFH on microcirculation, which need to be elucidated in future studies.The present study revealed that UFH could improve microcirculatory dysfunction independently of hemodynamic variables in a large animal model of septic shock. Our findings firstly reported that the intestinal microcirculation was improved significantly by the administration of UFH in a large animal model of septic shock, which imitates clinical septic shock in humans. Further, the result indicates that UFH improves coagulation and renal function. Taken together, we demonstrate that UFH could be a promising therapeutic candidate for the clinical treatment of Gram-negative sepsis.