Colletotrichum gloeosporioides, a guava canker and dieback pathogen. The reference gene selection and validation in C. gloeosporioides were evaluated for germinated conidia and mycelium (in vitro) and in infected guava (Psidium guajava) (interaction with host plant). The CgCAL gene was determined as a highly stable reference gene, followed by the CgTUB2 in C. gloeosporioides for germinating conidia and mycelium. However, the CgTUB2 gene was determined to be a highly stable reference gene, followed by the CgCAL for expression analysis during its interaction with the plant. Expression profiling revealed stable and constant relative expression patterns of selected reference genes for both PR genes by determining their relative transcript level. This study is the first to describe reference gene selection and validation to quantify target gene expression in C. gloeosporioides.Quantitative real-time PCR is used to quantify gene expression, even to detect low-level transcripts. It detects and quantifies the inoculum level of fungal pathogens in infected hosts. However, reliable expression profiling data require accurate transcript normalization against a stable reference gene. Hence, using stably expressed reference genes under variable conditions is paramount in gene expression analysis. In the current study, reference genes were selected and validated in  The genus Colletotrichum includes a broad array of economically important fungal pathogens that infect various host plant species . Fungal Colletotrichum higginasianum, the actin (ACT) gene was used to quantify and normalize fungal growth and transporter (MFS) gene expression  showed better stability , Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA (18S rRNA), calmodulin (CAL), and β-tubulin (TUB2), are considered the most appropriate candidate reference genes for fungal plant pathogens because of their involvement in essential cellular functions  medium to get germinate conidia and mycelium. For this purpose, single spore and hyphae tip methods were employed. The culture plates were incubated at 27°C under a dark regime for 48-72 hrs, and growth was observed. Microscopical features were examined under stereomicroscopy.Germinating conidia and mycelial stage of fungal pathogen C. gloeosporioides with guava pants, fungal inoculation of plants was performed. A Guava cultivar named “Large Sorahie” (Pyriform) was used in this experiment. Plants were raised in the research area of the Department of Plant Pathology, University of Agriculture Faisalabad, under the greenhouse-controlled condition. Guava plants at the seedling stage (six months old) were inoculated by spraying the plants with a spore suspension (150 ml), which was adjusted to a concentration of 1×106 spores ml−1  was used for RNA extraction. The quantification of extracted RNA was determined using a UV-visible NANODROP 8000 Spectrophotometer (Thermo Scientific USA); however, integrity was analyzed by gel electrophoresis using 1.5% agarose. The isolated RNA from each sample was treated with a RapidOut DNA Removal kit (Thermo Scientific USA) to remove genomic DNA (gDNA).Total RNA was isolated from germinated conidia and mycelium of Treated RNA samples were reverse transcribed with RevertAid M-MuLV RT using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific USA) in a final volume, as per the manufacturer’s recommendations, 20-fold diluted and quantified by UV visible NANODROP 8000 Spectrophotometer (Thermo Scientific USA).C. gloeosporioides, including calmodulin (CgCAL), Glyceraldehyde-3-phosphate dehydrogenase (CgGAPDH), 18S rRNA, β-tubulin (CgTUB2), and actin (CgACT). The primers employed for qRT-PCR analysis were designed using PrimerQuest. To verify their specificity for C. gloeosporioides, we used general PCR to test amplification in guava. Only primers with no amplification for guava were retained for qRT−PCR analysis , NormFinder (https://moma.dk/normfnder-sofware), and BestKeeper (https://www.gene-quantifcation.de/bestkeeper.html), were applied to assess the candidate reference genes’ expression stability for transcript normalization. The best-ranked reference genes in C. gloeosporioides were assessed; the Cq (quantification cycle) conversion value to relative quantitates, except for the BestKeeper algorithm, was made. The overall comprehensive ranking was generated through the RefFinder tool (https://heartcure.com.au). The selected reference genes were validated for normalizing the expression data of qRT−PCR in C. gloeosporioides using the diacylglycerol acyltransferase (DGAT1) gene and 3-dehydroshikimate dehydratase (qutC) gene in qRT−PCR analysis. These pathogenicity-related genes were documented by C. gloeosporioides species complex.Three mathematical algorithms, geNorm  cannot be exactly matched. However, it could be similar in these samples. That is why the expression analysis directly in the pathogen and the pathogen in the infected plant could not be compared  for transcript normalization in C. gloeosporioides. For the statistical evaluation of the candidate reference genes, three statistical algorithms were employed to rank the CgCAL, CgTUB2, and CgGAPDH genes based on expression stability. For expression stability analysis, we selected infected leaves of the guava plant after the third and fourth weeks postinoculation. The pathogen’s inoculum level in infected leaves of these periods was higher than that of infected leaves two weeks postinoculation (WPI) based on symptoms that appeared.Target DNA fragments were amplified from the cDNA of compared . The exp samples Figure 1CgGAPDH, CgCAL, and CgTUB2) for normalizing qRT−PCR expression analysis of C. gloeosporioides at four different levels . Three Excel-based statistical software programs were used to select a highly stable reference gene for normalization in expression profiling. The generated Cq values were converted into relative quantities for the analysis. The geNorm analysis revealed that the candidate reference genes CgTUB2 and CgCAL in combination were projected to have the least stability values, “M,” in germinating conidia and mycelium (0.040) and infected leaves (0.025). They ranked as the best reference genes with the highest stability for accurate data normalization in the qRT-PCR analysis of C. gloeosporioides  ± SD value of 3.43 ± 0.61 was identified as the most stable reference gene for transcript normalization. However, CgGAPDH (5.52 ± 0.67) and CgCAL (4.83 ± 0.71) were ranked as the second and third reference genes, respectively, with stable expression for normalizing qRT−PCR analysis. In the case of infected leaves of guava plants, the ranking was observed; CgCAL ranked as the top best reference gene, with a CV (% CP) ± SD score of 3.86 ± 0.66, followed by CgTUB2, with a value of 4.09 ± 0.78. However, in the BestKeeper ranking, CgGAPDH was proven to be a reference gene with unstable expression by scoring an 8.82 ± 1.21 value because a gene with SD >1 is considered an unstable expression . The comprehensive ranking order, CgCAL>CgTUB2>CgGAPDH, for stable reference genes for transcript normalization of qRT−PCR in C. gloeosporioides, was found to be matched with the ranking order given by the NormFinder method  and qutC, were selected. The expression profiling of both genes was performed using qRT-PCR analysis by adopting the earlier profiles. The expression of these genes was determined relative to selected reference genes (CgCAL and CgTUB2) for normalization. DGAT1 gene expression was higher in germinating conidia, but its conspicuous downregulation was observed in mycelium. However, qutC gene expression was higher in the infected plant sample (4th-week postinoculation), followed by mycelium, which supports the role of this gene in infection  can be found in the article/supplementary material.Conceptualization, IH and SI. Data curation, IH and SI. Formal analysis, IH and SI. Funding acquisition, EA_A. Writing – original draft, IH and SI. Writing – review & editing, IH, SI, EA_A, AH, and GA-Q. All authors contributed to the article and approved the submitted version.


Patients with connective tissue disease have a poor prognosis after receiving cardiac surgery. This study described the clinical scenarios and investigated factors correlated with acute kidney injury (AKI) after on-pump cardiac surgery in patients with systemic lupus erythematosus (SLE) or vasculitis.Patients with SLE or vasculitis who underwent on-pump cardiac surgery from March 2002 to March 2022 were enrolled, while patients with preoperative renal dysfunction were excluded. AKI was defined according to the Kidney Disease: Improving Global Outcomes (KDIGO) criteria. Uni- and multivariable analyses were performed to identify potential factors associated with postoperative AKI.P < 0.001) and extubation time  than those in the non-AKI group. Multivariable logistic regression revealed that BMI over 24 kg/m2  and comorbid SLE  were independently correlated with postoperative AKI.Among 123 patients enrolled, 39 (31.7%) developed AKI within seven days after receiving on-pump cardiac surgery. Four patients died in the hospital, resulting in an overall in-hospital mortality of 3.3%, and all deaths occurred in the AKI group. Patients in the AKI group also had longer ICU stays (median difference 3.0 day, 95% CI: 1.0–4.0, Factors potentially correlated with AKI following on-pump cardiac surgery in patients with connective tissue disease were explored. Clinicians should pay more attention to preoperative evaluation and intraoperative management in patients with risk factors. AKI Cardiac involvement, such as ascending aortic involvement, cardiac valvular involvement, and coronary artery involvement, is fairly frequent in patients with connective tissue disease (CTD) –8. TheseThis single-center observational study aimed to describe the prevalence and prognosis of cardiac surgery–associated AKI and to investigate the potential predictors of cardiac surgery–associated AKI in patients with systemic lupus erythematosus (SLE) or vasculitis undergoing on-pump cardiac surgery.2.2.1.2]; (2) need for renal replacement therapy prior to cardiac surgery; and (3) intraoperative death.This study was retrospectively conducted at a tertiary teaching hospital in Beijing, China. Ethics approval was obtained from the Peking Union Medical College Hospital Institutional Review Board . No informed consent was required because the study was retrospective and the data were anonymized. The hospital medical record system was searched, and adult patients diagnosed with vasculitis (mainly including Takayasu arteritis and Behçet's disease) or SLE who underwent cardiac surgery from March 2002 to March 2022 were consecutively enrolled. All patients were diagnosed by an experienced rheumatologist. The following exclusion criteria were applied: (1) diagnosis of chronic kidney dysfunction at the time of surgery , and course of CTD. Furthermore, preoperative laboratory test results  were also extracted. The intraoperative data we collected included the type of surgery, time of surgery, time of cardiopulmonary bypass, time of aortic cross-clamping, quantities of blood component infusion, and vasoactive drug dose. The vasoactive-inotropic score was calculated at operating theater discharge based on a weighted sum of all administered inotropes and vasoconstrictors, reflecting pharmacological support of the cardiovascular system . PrognosAKI was defined according to the Kidney Disease: Improving Global Outcomes (KDIGO) criteria, as follows: an increase in serum creatinine concentration ≥0.3 mg/dl (26.5 µmol/L) within 48 h, or an increase in serum creatinine concentration ≥1.5 times of baseline that is known or presumed to have occurred within the prior 7 days, or urine output <0.5 ml/kg/h for 6 h , 12. The2.3.t-test, whereas nonnormally distributed continuous variables using the Mann‒Whitney U-test. Categorical variables were compared using the chi-square (χ2) test by default or Fisher's exact test if the expected count in each cell of the table was less than 5. Continuous variables were transformed into categorical variables based on standard cutoff values used in clinical practice or according to the clinicians' experience , N-terminal pro-B-type natriuretic peptide (NT-proBNP), ESR, and CRP). Median differences and corresponding 95% confidence intervals were calculated using Hodges-Lehmann method on the basis of Mann–Whitney U-test.The demographic and clinical characteristics of the patients were described as follows. The normality of the continuous variables were assessed using the Shapiro–Wilk test. Continuous variables were expressed as the mean ± standard deviation or medians (25th–75th percentiles). Categorical variables are expressed as case numbers and percentages. Normally distributed continuous variables were analyzed using an independent P < 0.1) in the univariable analysis were incorporated into the ultimate multivariable model. A logistic regression model with the enter method was then built to screen the correlated factors, in which the occurrence of postoperative AKI was analyzed as the binary outcome.Clinical factors potentially correlating with postoperative AKI occurrence were all first analyzed using univariable analysis. Variables with potential associations  were also incorporated into the analysis; (2) the multivariable analysis was further conducted among those with both NT-proBNP and cTnI available prior to surgery.Another 2 sensitive analyses were additional performed to examine the robustness of the results: (1) Besides patients with eGFR of 60 ml/min/1.73 mP-value less than 0.05 was regarded as statistically significant. SPSS  was used for the statistical analysis of the data.A two-sided 3.3.1.P = 0.954).A total of 152 adult patients with SLE or vasculitis received on-pump cardiac surgery from March 2002 to March 2022 in our hospital. Twenty-seven of them had preoperative chronic kidney dysfunction or were supported by renal replacement therapy, and two patient died intraoperatively, such that a total of 29 patients were excluded. Ultimately, there were 123 patients enrolled in our study. Ninety-two (74.8%) patients had accompanying vasculitis, including 55 (44.7%) patients with Takayasu arteritis, 31 (25.2%) patients with Behçet's disease and 6 (4.8%) patients with other types of vasculitis. The other 31 (25.2%) patients had accompanying SLE. The AKI incidence in males and females was 32.0% and 31.5%, respectively (P = 0.158). Obesity (BMI ≥ 24 kg/m2) was more prevalent in the AKI group than in the non-AKI group , and comorbid SLE as opposed to vasculitis was significantly more common in the AKI group than in the non-AKI group .Based on the KDIGO definition, AKI occurred in 39 (31.7%) patients within seven days postoperatively, and these patients were defined as the AKI group. The other 84 (68.3%) patients did not develop AKI and were defined as the non-AKI group. The demographic data and clinical characteristics of the patients are presented in P = 0.545).To evaluate the proportion of patients with active/non-active inflammation state, we checked patients' preoperative consultation performed by department of rheumatology. Patients without preoperative consultation were evaluated retrospectively by rheumatologist. CTD activity was checked by SLEDAI (SLE disease activity index), BDCAF (Behçet's disease current activity form), and Kerr index for patients with SLE, Behçet's disease, and Takayasu arteritis, respectively. 116 patients had preoperative disease activity result. 39.5% (32/81) patients in the AKI group and 45.7% (16/35) patients in the non-AKI group were in active state, and the difference was not statistically significant  patients were under medication treatment . 32 (26.0%) patients did not receive medical anti-inflammation treatment, and 4 patients' treatment information were not available.3.2.P = 0.891] or eGFR level  between the AKI group and the non-AKI group. In comparison with the non-AKI group (N = 64), the NT-proBNP level was significantly higher in the AKI group (N = 27) . The univariable analyses of intraoperative data did not show any significant differences between the two groups . The results are listed in detail in At baseline, no significant difference was revealed regarding serum creatinine level [69.2 (18.1) µmol/L vs. 68.8 (15.2) µmol/L, 101.1 21. ml/min/13.3.2  and comorbid SLE  were independently correlated with postoperative AKI occurrence.A multivariable logistic regression test was performed to identify potential correlated factors for AKI based on the comparison between the AKI and non-AKI groups. The analysis results  showed t2 and greater indicated that, in the context of AKI incidence of 34.1% (45/132), risk factors associated with postoperative AKI kept unaltered . Another sensitivity analysis restricted in those with both baseline NT-proBNP and cTnI available (N = 88) also showed the same results.The sensitivity analysis performed in patients with eGFR of 45 ml/min/1.73 m3.4.P = 0.003). Patients in the AKI group had a longer length of stay in intensive care unit  and time to extubation  than those in the non-AKI group. Regarding postoperative complications, more cerebrovascular accidents occurred in the AKI group than in the non-AKI group. The results are shown in Four patients died in the hospital, resulting in an overall in-hospital mortality of 3.3%. All deaths occurred in the AKI group, and the mortality difference between the two groups was significant  to combined surgery (31.8%) or valvuloplasty/valve replacement procedures (30.4%) to coronary artery bypass graft (26.3%). The effective improvement of renal perfusion may be secondary to swift and rapid alleviation of myocardial ischemia following coronary revascularization. Furthermore, there exists no surprise that renal impairment after aortic involving procedures was the most obtrusive, which was also highlighted by a previous study conducted within a large-cohort of patients . In oursSeveral limitations should be considered when interpreting the present results. First, this was a single-center retrospective study; therefore, it has limitations in terms of the applicability of its results to other clinical settings. Second, we only included patients with SLE or vasculitis in our study. Patients with other types of CTD might have completely different clinical features, and their risk factors require further investigation. Third, patients with preoperative renal dysfunction were excluded from our study; thus, the results could only partially reflect the overall scenario. Last but not least, the rarity of CTD led to a very small sample size, which gave the study limited statistical power to detect potential correlated factors. This also resulted in a compromised ability to appropriately control the confounding effects using multivariable regression. Confounding effects cannot be fully excluded in this study; hence, the results should be interpreted with caution. Multicenter studies are required to further validate these findings.2 or comorbid SLE were especially vulnerable to postoperative AKI after receiving on-pump cardiac surgery. Clinicians should pay more attention to preoperative evaluation and intraoperative management in patients with risk factors for postoperative AKI.In conclusion, CTD patients undergoing on-pump cardiac surgery were susceptible to postoperative AKI, which was associated with increased mortality and a worsened prognosis. We found that patients with BMI over 24 kg/m


The standard treatment for chronic anal fissures that have failed non-operative management is lateral internal sphincterotomy. Surgery can cause de novo incontinence. Fissurectomy has been proposed as a sphincter/saving procedure, especially in the presence of a deep posterior pouch with or without a crypt infection. This study investigated whether fissurectomy offers a benefit in terms of de novo post-operative incontinence.Patients surgically managed with fissurectomy or lateral internal sphincterotomy for chronic anal fissures from 2013 to 2019 have been included. Healing rate, changes in continence and patient satisfaction were investigated at long-term follow-up.p = 0.338). The mean Vaizey score in these patients was 10.37 [standard deviation (sd) 6.3] after lateral internal sphincterotomy (LIS) and 5.4 (sd 2.3) after fissurectomy Healing rate was 97.8% in the lateral internal sphincterotomy group and 75.8% in the fissurectomy group (p = 0.001). In the lateral internal sphincterotomy group, patients with de novo post-op incontinence showed a statistically significant lower satisfaction rate  while no differences were present in the fissurectomy group .One hundred twenty patients  were analysed: 29 patients underwent fissurectomy and 91 lateral internal sphincterotomy. Mean follow-up was 55 months [confidence interval (CI) 5–116 months]. Both techniques showed some rate of de novo post-operative incontinence (> +3 Vaizey score points): 8.9% lateral internal sphincterotomy, 17.8% fissurectomy (Lateral internal sphincterotomy is confirmed as the preferred technique in term of healing rate. Fissurectomy did not offer a lower rate of de novo post-operative incontinence, but resulted in lower Vaizey scores in patients in whom this occurred. Satisfaction was lower in patients suffering a de novo post-operative incontinence after lateral internal sphincterotomy. Anal fissure is the second most frequent cause for a proctologic consultation, after haemorrhoidal disease. The disease is more prevalent in young people, with both sexes equally affected, and is associated with an impairment of social and working life.The aetiology is still unclear, various anatomical considerations have been postulated, such as reduced vascular supply in the posterior midline region of the anal canal and the fixity of the anoderm to the muscle in this zone, resulting in both more frequent lacerations and difficulty of healing in this area.An infective aetiology has also been proposed: persistent bacterial overgrowth caused by the colonization of the anal crypts, leading to difficult healing of the fissure . This thIt is classified as acute if appeared recently (less than 6 weeks) and chronic if it has been present for a longer time and has developed a distinctive appearance, such as a proximal papilla, a perianal skin tag with or without a deep crypt and fibrotic borders . Acute aThere are also subgroups of patients in which the anal pressure is normal or low, and who may not benefit from sphincter tone reduction. These fissures can be associated with obstructive defaecation syndrome and dysfunctional toilet habits, and/or with regular anal intercourse; where clinically unclear, anorectal physiology/manometry could/should be considered pre-operatively.LIS has success rates exceeding 90%, but a major risk for post-operative faecal incontinence of up to 9.2% , 4. GeneModern proctology has increasingly focused on the preservation of anatomy, to avoid the post-operative sequelae of surgical incision of the ano-rectal structures. This occurs for anal fistulas and hemorrhoids, among others . To offeIn our department, fissurectomy as alternative to LIS has been used since 2015.No definitive data are present in literature about the benefits of this technique and its advantages over traditional LIS in CAF.The aim of this study was to compare the results of fissurectomy and LIS in terms of cure rate, continence preservation and patient’ satisfaction.This retrospective study was conducted at the department of Emergency Surgery of the University hospital ‘P. Giaccone’ of Palermo. No ethical approval was required. Informed consent was obtained from all individual participants included in the study.Patients surgically managed for CAF from 2013 to 2019, identified by the diagnostic code on admission of International Classification of Diseases (ICD)-9: 565.0, have been included.Data were collected from medical records and electronic archives. Follow-up was performed with outpatient visits and telephone interviews.Demographic data including age, number and type of previous perineal surgery, type and duration of symptoms were extracted from medical records and electronic archives. Pre-operative bowel function including frequency of defecation, urgency and anal continence was taken from patient records and confirmed in the follow-up interviews.Patients were divided into two groups according to the surgical technique used: fissurectomy or LIS.Homogeneity in terms of mean age, sex and previous perineal surgery between the groups was verified.In our practice, fissurectomy comprises the removal of fissure borders, hypertrophic papilla, sentinel pile, the coagulation of the base of the fissure and, if present, the lay open of a submucosal fistula (crypt) without sphincter section. The LIS technique is a closed one, performed with scissors up to a half of the internal sphincter. All patients underwent local anaesthesia and mild sedation as previously described . All theBoth techniques have been used according to surgeon preference, with fissurectomy usually but not exclusively the preferred treatment in patients with normal/low anal resting tone. At follow-up, healing rate, continence and patient’ satisfaction were analysed.Healing rate was defined as the resolution of symptoms without the need for further surgical interventions.Anal continence was evaluated using the Vaizey score, which ranges from 0  to 24 (severe incontinence) , assuminThe relationships among de novo post-operative incontinence and gender, previous ano-rectal surgery, age over 60 years and healing were analysed.Patient satisfaction was determined using a simple numerical scale from 0 (not satisfied) to 10 (completely satisfied).t-test or Wilcoxon’s rank sum test. The Mann–Whitney U test was used to evaluate patient satisfaction. A p-value of < 0.05 was considered statistically significant. Statistical analysis was conducted using SPSS software  and MedCalc Statistical Software .Descriptive data are presented as parametric and non-parametric. Comparisons between pre-operative and post-operative functional scores was performed using the paired One hundred twenty patients  who underwent surgery between 2013 and 2019 were analysed. Twenty-eight more patients were unavailable at follow-up and were excluded.Twenty-eight patients [average age 52.3 (sd 14.4) years] underwent fissurectomy and 89 patients LIS [average age 50.27 (sd 13.98) years].There were no statistically significant differences in mean age, number of females or prior perineal surgery between patients who underwent LIS or fissurectomy Table .Table 1PFifteen patients (16.5%) in the LIS group and five patients (17.3%) in the fissurectomy group had some degree of pre-operative incontinence.Mean follow-up was 55 months (CI 5–116 months).p = 0.001). Only one patient who had previously undergone LIS and was not cured subsequently underwent fissurectomy, without showing changes in continence.Healing rate was 97.8% in the LIS group and 75.8% in the fissurectomy group ; ].In both treatments, patients with pre-operative incontinence did not show a worsening of function. On the contrary, 7.14% of the patients in the fissurectomy group and 8.9% in the LIS group improved after surgery.The rate of patients in whom continence improved, remained unchanged or worsened is reported in Table p = 0.338). The mean Vaizey score in these patients was 10.37 (sd 6.3) after LIS and 5.4 (sd 2.3) after fissurectomy.De novo post-operative incontinence (> +3 Vaizey points) occurred in 8.9% of patients who underwent LIS and 17.8% of those who underwent fissurectomy (p = 0.148) nor in the fissurectomy group (p = 0.576).There was no relationship between age over 60 years and post-operative incontinence in the LIS group (p = 0.328) nor in the fissurectomy group (p = 1.001).There was no relationship between gender and post-operative incontinence in the LIS group (p = 0.154), nor in the LIS group (p = 0.631).There was no relationship between previous ano-rectal operation and post-operative incontinence in the fissurectomy group (p = 0.827) nor in the fissurectomy group (p = 1.000).There was no relation between healing and post-operative incontinence in the LIS group .Mean post-operative patient satisfaction was 9.2 (sd 1.57) in the LIS group and 8.87 (sd 1.69) in the fissurectomy group. The satisfaction rate decreased in both groups in patients with de novo post-operative incontinence . All patients with pre-operative incontinence reported improvement in mild incontinence. Excluding patients with overt incontinence, they found mild post-operative incontinence in 10% of patients .Aigner reported a historical series of 162 patients in whom, despite a higher recurrence rate in fissurectomy compared with LIS (8% versus 2%), fissurectomy showed a benefit in terms of any incontinence (2% versus 5%) .As expected, also in the present study, the cure rate was higher in patients who underwent LIS (97.8% versus 75.8%).Zeitun et al. presented a study on 50 patients who underwent fissurectomy with a cure rate of 93.6%, healing time of up to 10.3 ± 4.96 weeks and no statistically significant changes in Vaizey score .Schornagel et al. also reported a recurrence rate of 11.6% after fissurectomy but no de novo post-operative incontinence in patients with an intact sphincter who were pre-operatively continent. The only patient with a de novo post-operative Vaizey score of 14 points had previously undergone LIS [Levin et al. demonstrated that internal sphincterotomy may contribute or precipitate anal incontinence in the long term .In contrast, Mousavi et al., comparing LIS with fissurectomy in 62 patients, reported 6.2% faecal incontinence after fissurectomy and none after LIS, and 96.9% versus 100% healing rate for fissurectomy and LIS, respectively .Similar data are reported by Saeed et al., who found transient post-operative incontinence for flatus in 6.97% and faecal incontinence in 4.65% of patients after fissurectomy, compared with none in the LIS group .Due to these conflicting data, despite the fact that surgery may be appropriate without a trial of pharmacological treatment after failure of conservative therapy, as indicated by the ‘Practice parameters for the management of anal fissure’ , incontiAnalysing the factors related to the onset of de novo incontinence, no correlation was found with gender, age over 60 years, previous anorectal surgery or healing in either group.Therefore, it is very difficult to adequately select patients, and a full explanation of possible changes in continence must be provided, including that continence already seems affected in CAF and sometimes improves after surgical treatment.The results obtained in this study partially support the idea, because LIS is still considered the standard method in the treatment of CAF, but they leave a place for fissurectomy in clinical practice as it seems to offer a lower degree of post-operative incontinence and better satisfaction rates.The present study offers data on an ongoing question in modern proctology.This was a retrospective analysis, and the data need to be confirmed by prospective randomized studies.The healing rate was statistically higher in the LIS group. Post-operative incontinence was higher in the fissurectomy group but with lower Vaizey scores than in the LIS group; however, no statistical differences were found.Therefore, we cannot state that there is a clear advantage of fissurectomy over LIS in the treatment of CAF.


Salmonella (NTS) is a major cause of bacterial gastroenteritis. Although many countries have implemented whole genome sequencing (WGS) of NTS, there is limited knowledge on NTS diversity on food and its contribution to human disease. In this study, the aim was to characterise the NTS genomes from retail foods in a particular region of the UK and assess the contribution to human NTS infections. Raw food samples were collected at retail in a repeated cross-sectional design in Norfolk, UK, including chicken (n=311), leafy green (n=311), pork (n=311), prawn (n=279) and salmon (n=157) samples. Up to eight presumptive NTS isolates per positive sample underwent WGS and were compared to publicly available NTS genomes from UK human cases. NTS was isolated from chicken (9.6 %), prawn (2.9 %) and pork (1.3 %) samples and included 14 serovars, of which Salmonella Infantis and Salmonella Enteritidis were the most common. The S. Enteritidis isolates were only isolated from imported chicken. No antimicrobial resistance determinants were found in prawn isolates, whilst 5.1 % of chicken and 0.64 % of pork samples contained multi-drug resistant NTS. The maximum number of pairwise core non-recombinant single nucleotide polymorphisms (SNPs) amongst isolates from the same sample was used to measure diversity and most samples had a median of two SNPs (range: 0–251). NTS isolates that were within five SNPs to clinical UK isolates belonged to specific serovars: S. Enteritidis and S. Infantis (chicken), and S. I 4,[5],12:i- (pork and chicken). Most NTS isolates that were closely related to human-derived isolates were obtained from imported chicken, but further epidemiological data are required to assess definitively the probable source of the human cases. Continued WGS surveillance of Salmonella on retail food involving multiple isolates from each sample is necessary to capture the diversity of Salmonella and determine the relative importance of different sources of human disease.Non-typhoidal  This study describes NTS isolates collected from retail food from the UK using whole genome sequencing (WGS) and compares the isolates to those collected from humans in the UK to determine the degree to which these foods may contribute to human NTS infections. We isolated NTS from chicken, pork and prawns, but only the isolates from chicken and pork contained antimicrobial resistance determinants. NTS isolates collected from the same food sample varied in diversity, which has implications for outbreak analyses which typically analyse one NTS isolate per food sample. Fourteen serovars of NTS were isolated, but only three consisted of isolates that were closely related to human-derived UK isolates: Salmonella Enteritidis and Salmonella Infantis (chicken), and Salmonella I 4,[5],12:i- (pork and chicken). Most NTS isolates that were closely related to human-derived isolates were obtained from imported chicken, but further epidemiological data are required to assess definitively the probable source of the human cases. Continued WGS surveillance of Salmonella on retail food involving multiple isolates from each food sample is necessary to determine the diversity of Salmonella present and the relative importance of different sources of human disease.Non-typhoidal Salmonella (NTS) includes all serovars of Salmonella enterica subspecies enterica not associated with typhoid or paratyphoid fever. The majority of illnesses caused by NTS manifest as gastroenteritis, but it can also cause systemic infections. Salmonellosis is a worldwide problem ,12:i:- that was found in both pork and chicken samples (Fourteen  samples .S. Newport (n=2 samples) and S. Enteritidis (n=9 samples) were only isolated from imported samples, whilst S. I 1,4,[5],12:i:- (n=1 sample), S. Kedougou (n=1 sample), S. Mbandaka (n=1 sample) and S. Ohio (n=2 samples) serovars were only isolated from domestic samples . The S. Infantis (n=14 samples) serovar was isolated from both domestic and imported chicken samples. Sample storage and sample origin were strongly linked in chicken in this study, with 85 % of imported chicken samples available as frozen compared to 1 % of domestic samples available as frozen. The exception was one chilled imported sample from which S. Enteritidis was isolated. The only serovar isolated from chicken samples obtained from butcher shops was S. Infantis.For chicken samples, Salmonella isolates contained at least one AMR determinant, 70 (35.2 %) contained no AMR determinants and 78 (39.2 %) were classified as MDR  d as MDR . MDR NTSserovars .Of the 42 samples that tested positive for NTS, 29 contained at least one AMR NTS isolate and 18 contained at least one MDR NTS isolate. No samples contained a mixture of AMR and non-AMR NTS isolates, but a single chicken sample contained a mixture of both MDR and non-MDR AMR isolates.For NTS-containing chicken samples, 80 % (24/30) contained at least one AMR determinant-containing NTS isolate and 53 % (16/30) contained at least one MDR NTS isolate . For the four NTS-containing pork samples, all contained at least one AMR determinant-containing NTS isolate and half (2/4) contained at least one MDR NTS isolate. None of the NTS isolates from prawns contained any AMR determinants.S. Infantis was the most commonly isolated serovar in this study and was only isolated from chicken . The AMR and metal-tolerance genes of the pESI plasmid are usually located in two regions ,12:b:- was isolated using the MKTTn-BSA (n=1 isolate), MSRV-BSA (n=1 isolate) and MSRV-XLD (n=1 isolate) methods.Of the 42 food samples from which NTS was cultured, 40 contained isolates belonging to a single serovar; one prawn sample contained isolates that belonged to two serovars (PR-0007) and one prawn sample contained isolates that belonged to three serovars (PR-0151). For PR-0007, S. Bovismorbificans serovar was not isolated using mKTTN enrichment. Comparisons between isolates belonging to each sample and method of detection found that the BSA culture method detected NTS from 42 positive samples, but only 40 cultured for NTS using the XLD culture method, 39 by the mKTTN enrichment method and 37 by the MSRV method.Comparisons between the isolates belonging to each serovar and the methods used to detect them found that all 14 serovars were detected by the BSA and XLD culture methods, and MSRV enrichment, but the S. Weltevreden isolates that differed by up to 251 SNPs  was measured by calculating the maximum number of pairwise core non-recombinant SNPs amongst these isolates. Most isolates from the same serovar–sample combination were similar with a median of two pairwise SNP differences (interquartile range: 0–6 SNPs). In total, 26 % (11/42) of NTS-positive samples contained isolates that differed by five or more SNPs, with one prawn sample containing 251 SNPs . There wP=8.7×10−3), but the culture method (P=0.87) was not associated with larger numbers of SNPs.To determine if any of the methods were better at detecting NTS diversity on food samples, the maximum number of pairwise core non-recombinant SNPs between isolates collected from the same serovar–sample combination using the same method were calculated . MSRV identified a larger number of SNPs than MKTTn (The isolates collected from food in this study were 0–462 (median=5.5) core non-recombinant SNPs from the closest UK human-derived isolate available . For two of these 21 samples the closest UK human isolate differed by a plasmid replicon, and for one of these samples the closest UK human isolate differed by an AMR determinant. However, the closest human UK isolate for the remaining 18 samples contained identical plasmid replicons and AMR determinants to the closest food isolate.P<0.00001) . Of the 225 chilled chicken samples, 2.7 % (95 % CI: 0.56–4.8 %) contained NTS isolates that were within five SNPs to clinical isolates compared to 15 % (95 % CI: 7.5–23 %) of the 86 frozen chicken samples investigated, and these percentages were significantly different (P=5.3×10−5).Of the 88 imported chicken samples investigated, 17 % : 9.2–25 %) contained NTS isolates that were within five SNPs to clinical isolates compared to 2.3 % (95 % CI: 0.31–4.4 %) of the 214 domestic samples investigated, and these percentages were significantly different  of S. I 4,[5],12:i- and 93 % (11/14) of S. Infantis samples sharing fewer than five SNPs with the closest human isolate. If the SNP cut-off was increased from five to ten SNPs between food- and UK human-derived isolates, some S. Weltevreden from prawn samples would also have been classified as being closely related to a UK human-derived isolate.The pairwise SNP distances between food samples and the closest UK human isolate were serovar dependent, with 78 % (7/9) of Salmonella are sporadic in nature and cannot be attributed to any specific source ,12:i-, S. Enteritidis and S. Infantis were the serovars collected from retail food samples that included isolates that were within five SNPs of clinical cases. S. Infantis was the most common serovar isolated in this study, found in domestic and imported chicken samples. S. Infantis has increased in prevalence in countries around the world over the last two decades ,12:i- serovar was the only serovar isolated from multiple food commodities in this study; all isolates were ST 34 and were collected from two domestic pork samples and one domestic chicken sample from three different chain supermarkets. S. I 4,[5],12:i- carries genes conferring resistance to copper, which is believed to enable its persistence in some settings as copper is used as a growth promoter in pig rearing ,12:i- has been previously isolated from poultry sources in the UK but not as commonly as from porcine sources [The  rearing . S. I 4, sources .S. Weltevreden isolates collected from food were within five SNPs of human UK sources, suggesting they are not a potential source of outbreaks [S. Weltevreden from some prawn sources were within ten SNPs of UK human isolates, suggesting they are related to isolates from potential outbreak sources. S. Weltevreden is usually associated with aquatic environments and animals that inhabit these environments, especially in South East Asia [S. Weltevreden-positive samples collected in this study were black tiger prawns (Penaeus monodon) from Vietnam (n=4 samples), Indonesia (n=1 sample) or of unknown (n=1 sample) origin. S. Weltevreden isolates have previously been shown to be less virulent than S. Typhimurium isolates, and few contain AMR genes [S. Weltevreden are required to better understand this emerging pathogen.No utbreaks . Howeverast Asia . All S. MR genes , but conMR genes . Global S. Enteritidis cases in the UK has decreased since the 1980s with the introduction of a vaccine against this serovar for hens [S. Enteritidis from domestic chicken samples in this study. However, S. Enteritidis is still a major cause of salmonellosis in the UK [S. Enteritidis from imported breaded chicken was associated with over 100 human cases in the UK [S. Enteritidis in this study were also all of Polish origin or originated from multiple countries including Poland, but belonged to ST 4747 and two novel STs, suggesting that imported chicken is exposing consumers to S. Enteritidis with STs other than ST 11.The number of for hens , which ln the UK ; recentln the UK  and a suSalmonella serovars were isolated from the food commodities examined in this study. Although no isolates were within five SNPs of publicly available Salmonella genomes from clinical cases, it is possible that isolates belonging to these serovars could acquire genetic factors that would allow them to colonise animal hosts more effectively, and therefore increase in prevalence, as was the case with the S. Infantis serovar and acquisition of the pESI plasmid [Ten other  plasmid . Continu−7 substitutions per site per year [Identifying where NTS contaminates foods such as chicken, pork and prawns is challenging, as it can derive from colonisation of the animal itself , or fromper year , which eper year , pigs foper year  and aquaper year . If thesSalmonella contamination on these food products was low, as previously reported [Salmonella on these foods, which may hinder outbreak investigations and source attribution. It is important to note that given the dynamic and shifting nature of food sources, farming and production practices and consumer behaviour, the types of foods associated with foodborne disease may change over time and by geographical location. Therefore, the prevention of future salmonellosis cases and outbreaks relies on continued surveillance of NTS found on retail food, with the high resolution offered by WGS allowing greater insight into its relationship to human clinical cases.In this study we investigated the genetic diversity of NTS from retail food sold in Norfolk, UK, and assessed the risk of infection by NTS based on how distantly related they were to clinical cases from the UK, the number of AMR determinants they contained and the diversity of isolates found on samples. Overall, the prevalence of reported , but theClick here for additional data file.


We conducted a systematic review and meta-analysis to summarize the predictive and prognostic ability of the log odds of positive lymph nodes (LODDS) staging system and compare it with pathological N (pN) classification and the ratio-based lymph node system (rN) for the overall survival (OS) of gastric cancer (GC).Through a systematic review till March 7, 2022, we identified population-based studies that reported the prognostic effects of LODDS in patients with GC. We compare the predictive effectiveness of the LODDS staging system with that of the rN and pN classification systems for the OS of GC.P-values were < 0.001) with the same rN and pN classifications. Meanwhile, for patients with different pN or rN classifications with the same LODDS classification, prognosis was highly similar.Twelve studies comprising 20,312 patients were included in this systematic review and meta-analysis. The results showed that LODDS1, LODDS2, LODDS3, and LODDS4 in GC patients were correlated with poor OS compared with LODDS0 ; LODDS2 vs. LODDS0: HR = 2.47, 95% CI ; LODDS3 vs. LODDS0: HR = 3.15, 95% CI ; LODDS4 vs. LODDS0: HR = 4.55, 95% CI ). Additionally, significant differences in survival were observed among patients with different LODDS classifications (all The findings show that LODDS is correlated with the prognosis of GC patients and is superior to the pN and rN classifications for prognostic assessment.The online version contains supplementary material available at 10.1186/s12885-023-10805-6. Gastric cancer (GC), a common highly recurrent malignant tumor, is the fifth most prevalent tumor, and is the third most frequent primary cause of tumor-related death worldwide . MultiplWe conducted a systematic review and meta-analysis to summarize the predictive and prognostic ability of the LODDS staging system and compare it with the rN and pN classification systems to address these limitations.https://www.crd.york.ac.uk/prospero/). The PROSPERO registration number is CRD42021274996.We searched PubMed, Medline, Embase, Web of Science and the Cochrane Library for relevant studies from inception to March 7, 2022, which formed the basis for evidence used to conduct the meta-analysis. The following keywords were used: “log odds of positive lymph nodes”, “LODDS”, and “gastric cancer”. We used the following strategy: (((((((((gastric neoplasms[MeSH Terms]) OR (stomach neoplasms)) OR (stomach carcinoma)) OR (gastric tumor)) OR (gastric carcinoma)) OR (stomach cancer)) OR (stomach tumor)) OR (gastric neoplasms)) OR (GC)) AND ((log odds of positive lymph nodes) OR (LODDS)). We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement for reporting systematic reviews  and regiWe included studies that enrolled ≥ 100 patients with GC (diagnosed with the gold standard test) who were classified by the LODDS, rN, and pN classification systems of the AJCC and followed for at least five years. We included studies that reported at least one of the outcomes of interest or studies wherein the outcome could be calculated according to data extracted from the published data. We excluded conference abstracts, reviews, case reports, ongoing trials, open-label trials, comments, letters, meeting records, and studies that enrolled patients with a total sample size of less than 100 patients, as studies with very small samples are more prone to bias and contribute little information to pooled analyses. We excluded articles that shared a study population with another article and those that did not provide crucial information needed for detailed stratification.Using a standardized form, two reviewers independently screened the titles and abstracts identified by the search, and the full text was obtained to check eligibility. When a disagreement arose and they were unable to reach a consensus, an adjudicator was consulted to resolve discrepancies.Using a standardized form, two reviewers worked independently to perform duplicate data abstraction in each eligible study. When a disagreement arose and they were unable to reach a consensus, an adjudicator helped to resolve disagreements. We collected information regarding study characteristics , patient characteristics , and all patient-important outcomes ).Prognostic values (OS) were used to compare the different LODDS groups. We compared the predictive and prognostic abilities of the LODDS staging system with those of the rN and pN classification systems.Using the Newcastle–Ottawa Scale (NOS) , two invThe data were analyzed using Stata statistical software version 16.0  and SPSS version 22 (IBM Corporation).P values for two-sided testing at α = 0.05. To assess the existence of heterogeneity among studies, we tested OS for heterogeneity across cohorts using Cochran’s Q statistic and used I2 to measure the extent of heterogeneity [I2 statistic, heterogeneity was defined as low (25%–50%), moderate (50%–75%) or high (> 75%) [P ≤ 0.1 was considered to indicate significant heterogeneity. In addition, subgroup analyses were performed based on the differences in baseline characteristics and risk factors in the data retrieved. Then, we also conducted a sensitivity analysis in which each study was removed in turn to evaluate the undue influence of the study on the overall summary estimates including Duval and Tweedie’s trim-and-fill method [P values for the pooled results were two-sided, and the significance level was 0.05.To statistically assess the prognostic effects of LODDS, we extracted the hazard ratio (HR) and 95% confidence interval (CI) of 5-year OS from the included studies. If not directly provided in the original literature, the estimated HR and 95% CI were used to assess prognostic effects based on the method described by Tierney et al. , and an ogeneity . For the (> 75%) . For thel method , and Gall method . Publical method . The groBased on the search strategy mentioned above, the original search yielded 209 records. Finally, we included 12 unique studies , 34–38 pTables We analyzed OS in different LODDS categories according to the data from the included articles. The results of the pooled analysis are summarized in Table I2 statistic = 63.5%, P heterogeneity = 0.003). The pooled results indicated that LODDS2 GC patients had a worse OS ) than LODDS0 GC patients. There was significant heterogeneity . Compared with LODDS0 GC patients, LODDS3 GC patients had a worse OS ), and the heterogeneity was significant . The results of the pooled analysis using the REM showed that LODDS4 GC patients were also associated with poorer OS ) than LODDS0 GC patients, and between-study heterogeneity was significant . Overall, as the LODDS grade increases, the prognosis of patients with GC becomes increasingly worse.When pooling the HR for OS, LODDS1, LODDS2, LODDS3, and LODDS4 in GC patients were correlated with poor OS compared with LODDS0 .To analyze the potential sources of between-study heterogeneity, we performed subgroup analysis according to differences in the variables, including the publication year, country, patient number and whether patients received neoadjuvant therapy. The results are summarized in Table I2 statistic = 81.3%, P heterogeneity < 0.001 to I2 statistic = 2.2%, P heterogeneity = 0.381; Fig. To explore the potential sources of heterogeneity in the LODDS4 vs. LODDS0 group, we also used Galbraith plots and Duval and Tweedie’s trim-and-fill method to further explore the source of heterogeneity in OS, and the results showed that the training set of the study by Jian-Hui C et al.  might hap = 0.608), LODDS2 vs. LODDS0 (p = 0.799), LODDS3 vs. LODDS0 (p = 0.943), and LODDS4 vs. LODDS0 (p = 0.216) for OS. The results with P values for Egger’s test are listed in Table Publication bias was assessed by funnel plots and Egger’s test. Formal evaluation using Egger’s test failed to identify significant publication bias in the analyses of LODDS1 vs. LODDS0 , I2 statistic = 86.2%, P heterogeneity < 0.001; LODDS3 vs. LODDS0: HR = 3.15, 95% CI , I2 statistic = 92.1%, P heterogeneity < 0.001; LODDS4 vs. LODDS0: HR = 4.55, 95% CI , I2 statistic = 96.6%, P heterogeneity < 0.001). As the LODDS grade increases, the prognosis of patients with GC becomes correspondingly worse. However, we discovered high levels of heterogeneity in all groups. To analyze the potential sources of between-study heterogeneity, we performed subgroup analysis according to differences in the variables, including the publication year, country, patient number and whether patients received neoadjuvant therapy. After subgroup analysis, it was found that the heterogeneity was mainly from whether patients received neoadjuvant therapy. Additionally, we found that patients without neoadjuvant therapy had poorer OS than patients with neoadjuvant therapy in the same group. Therefore, the results suggest that whether patients receive neoadjuvant therapy is one of the most important factors affecting the prognostic effectiveness of LODDS in patients with GC. Although we performed a subgroup analysis according to whether patients received neoadjuvant therapy, significant heterogeneity was still found in the pooled analysis of OS in the LODDS4 vs. LODDS0 group. To explore the potential sources of heterogeneity, we also used Galbraith plots and Duval and Tweedie’s trim-and-fill method to further explore the source of heterogeneity in OS, and the results showed that the training set of the study by Jian-Hui C et al. [This is the first meta-analysis that focused on the crucial roles of LODDS in predicting the prognosis of patients with GC. As a result, our study is more informative than any previous study. Our meta-analysis of twelve articles including 20,312 GC patients indicated that LODDS1, LODDS2, LODDS3, and LODDS4 patients have poor OS compared with LODDS0 patients, which shows that LODDS has prognostic value in patients with GC. When pooling the HR for OS, LODDS1, LODDS2, LODDS3, and LODDS4 in GC patients was correlated with poor OS compared with LODDS0 , C et al.  might haAdditionally, we assessed differences in survival among patients in different LODDS classifications for each of the pN or rN classifications. We summarized the results from four studies , 14, 38 However, several limitations of the current meta-analysis should be emphasized. First, as several studies did not report HRs, HRs in this meta-analysis were estimated based on the method described by Tierney et al. . Second,Our systematic review demonstrated that LODDS is correlated with the prognosis of GC patients and more accurately predicts the survival of GC patients than previous methods. As the LODDS grade increases, the prognosis of patients with GC becomes correspondingly worse. Additionally, we found that patients without neoadjuvant therapy had poorer OS than patients with neoadjuvant therapy in the same LODDS group. Therefore, the results suggest that whether patients receive neoadjuvant therapy is one of the strongest factors affecting the prognostic effectiveness of LODDS. Moreover, the LODDS classification is superior to the pN and rN classifications for prognostic assessment. Incorporating LODDS into the staging system of GC will enable clinicians to predict the prognosis of patients more accurately. Further high-quality, large-scale, international, well-designed multicenter prospective studies are needed to obtain the optimal cutoff point of LODDS and to find a simple and repeatable way to calculate LODDS values to facilitate the utilization of LODDS in the clinic.Additional file 1: Figure S1. Assessment of publication bias using Funnel plot analysis. a-b Funnel plot analysis of studies on OS ((a) LODDS1 vs. LODDS0, (b) LODDS2 vs. LODDS0, (c) LODDS1 vs. LODDS0, (d) LODDS2 vs. LODDS0); e–h Funnel plot analysis of studies which included the patients without neoadjuvant therapy on OS ((e) LODDS1 vs. LODDS0, (f) LODDS2 vs. LODDS0), (g) LODDS1 vs. LODDS0, (h) LODDS2 vs. LODDS0). Publication bias was not found in the meta-analyses of OS. All of the funnel plots of the included articles showed a symmetrical distribution. Thus, no significant publication bias was found in the meta-analyses of OS. Abbreviations: HR, hazard ratio; OS, overall survival; LODDS, log odds of positive lymph nodes.


Currently, the role of oncostatin M (OSM) in clear cell renal cell carcinoma (ccRCC) has not been investigated. This study will explore the impact of OSM on ccRCC expression, prognosis, and cell function.  In this study, we used The Cancer Genome Atlas (TCGA) database to evaluate OSM expression characteristics, pathogenic factor distribution, and prognostic aspects in ccRCC. We also combined this analysis with qRT-PCR to verify OSM mRNA expression levels at the tissue level. Then, the effects of OSM on the proliferation, invasion, and migration abilities of ccRCC cells were explored through CCK8, Transwell, Western blotting, and immunofluorescence experiments. Finally, the oncogenic mechanisms associated with OSM in ccRCC were explored through signaling pathway enrichment and single-cell analysis.  The results demonstrated that OSM was significantly more expressed in ccRCC than in normal tissues. According to the survival analysis, OSM in ccRCC was considerably worse in the group with high expression than in the group with low expression. Also, the univariate and multivariate Cox analyses of clinical characteristics show that OSM in ccRCC may be able to predict a poor prognosis on its own as a biomarker. In vitro cellular experiments demonstrated that high OSM expression had no discernible impact on ccRCC cell proliferation compared to the control group, but it did promote tumor cell invasion and migration. Signaling pathways and single-cell analysis revealed that OSM might promote ccRCC invasion and migration through M2 macrophages.  In conclusion, OSM may serve as an independent poor prognostic biomarker in ccRCC and promote tumor cell invasion and migration. This discovery is expected to provide a new therapeutic target for patients with recurrent and metastatic ccRCC. Renal cell carcinoma (RCC) is a prevalent malignant neoplasm affecting the urinary system, posing a global health burden. The International Agency for Research on Cancer has reported an alarming annual incidence of over 430,000 newly diagnosed cases of RCC worldwide . Clear cOncostatin M (OSM) is a secreted cytokine and growth regulator that belongs to the interleukin 6 cytokine family, one of the most important cytokine families in tumorigenesis and metastasis , 11. ThiIn cultured renal fibroblasts, OSM has been demonstrated to induce upregulation of inflammatory cytokines. OSM potentially exerts a direct influence on renal tubular epithelial cells and renal fibroblasts, leading to the production of crystal-binding molecules and inflammatory cytokines. These factors may contribute to the formation of renal crystal deposits . Howeverhttps://www.cancer.gov/) database, such as follow-up time, survival status, age, gender, total pathological stage, T stage, N stage, M stage, and pathological grading . We also carried out a survival analysis using the “survival” R package. Utilizing the “rms” program, OSM expression data and clinicopathological factors were combined to create the nomogram survival prediction scoring system.In this study, we obtained the ccRCC mRNA expression dataset  and clinicopathological parameters dataset from The Cancer Genome Atlas  provided the human ccRCC cell lines Caki-1 and 786-O in this study. The tumor cell lines were grown in high-sugar Dulbecco's Modified Eagle's Medium , which was then boosted with 10% foetal bovine serum and 1% penicillin-streptomycin solution . The cells were kept in a controlled setting with 5% CO3 cells. Cells underwent 24, 48, 72, and 96 h incubation.The OSM and control groups were set up in Caki-1 and 786-O cell lines, respectively. Cells in the midlogarithmic growth phase and in good growth condition were harvested, and single-cell suspensions were made by applying cell culture medium, and each well was then seeded with 5 × 10μl of freshly prepared CCK8 solution to each well and continuing incubation for 4 hours. The optical density value was then determined using an enzyme-linked immunoassay reader at a wavelength of 450 nm. Three independent experiments were conducted for each experiment.A CCK8 experiment was carried out to evaluate the cancer cells' capacity for proliferation. Cell growth was monitored using a CCK8 kit  by adding 50 μl of growth medium containing 10% FBS , while the upper chamber received 100 μl of cell suspension and 200 μl of serum-free medium. The plate was subsequently incubated for 48 hours at a controlled temperature of 37°C with 5% CO2. After the incubation period, the samples were fixed with 95% alcohol for 5 min and then stained for 1 hour with a 4 g/l crystal violet solution. Subsequently, five random fields at 100x magnification were selected under a microscope. Finally, the cell counts were quantified by ImageJ software.The Transwell assay was used to identify cancer cells' capacity for invasion and migration. The Matrigel matrix gel , frozen in the -20°C refrigerator, was left to liquefy overnight at 4°C. Liquefied Matrigel matrix gel was diluted as a working solution at a ratio of 1 : 5. The upper chamber surface of the Transwell membrane received this solution, and it was then incubated for 30 min at 37°C. In the Transwell, the lower chamber received 600 Total protein was extracted from well-cultured tumor cells by adding RIPA cell protein lysate . For protein quantification, the BCA protein quantification kit  was used in a 96-well plate with three replicate wells for each sample. Using 10% SDS-PAGE and PVDF membranes from Millipore (USA), cell lysates were separated from cell proteins for 90 min. The membranes were subsequently blocked for an hour in PBS containing 5% skim milk. The above solutions were incubated with primary antibodies OSM , E-cadherin , N-cadherin , vimentin , and GAPDH  at 4°C overnight. The membrane is washed the following day and incubated for two hours at room temperature with a horseradish peroxidase-coupled secondary antibody . Three independent experiments were conducted for each experiment. Finally, the band density was quantified by ImageJ software.The tumor cells were cultured in a 6-well plate and fixed using a 4% paraformaldehyde solution for 30 min. After fixation, the cells were treated with a membrane-breaking working solution obtained from Ribiology (China) for 10 min at room temperature. The cell well plates were then supplemented with primary antibodies directed against OSM, E-cadherin, and vimentin and incubated overnight at 4°C in a humid environment. The next day, an Alexa Fluor 555-labeled goat anti-mouse IgG antibody  was applied for detection. The cells restained with DAPI staining solution and incubated at room temperature for 10 min; then, the phosphate-buffered saline (PBS) was removed. A Nikon (Japan) fluorescence microscope was used to take immunofluorescence pictures.h (h = 83/178) was assigned to the upregulated gene set. In contrast, the downregulated gene collection was classified as belonging to phenotype l (l = 95/178), and the “h-versus-l” comparison was chosen to find enriched OSM-related pathways. Significant enrichment was considered for a normalized enrichment score > 1, normal p value < 0.05, and false discovery rate < 0.25.GSEA software (version 4.2.1) was used to do an enrichment analysis to look into the role of OSM carcinogenesis-related pathways in ccRCC. Then, phenotype http://tisch.comp-genomics.org/) website collects human tumor scRNA-seq datasets from the Gene Expression Omnibus (GEO) and ArrayExpress. The TISCH2 platform was used for uniform processing and analysis of these datasets, facilitating comprehensive exploration of the tumor single-cell transcriptome and corresponding patient information. The robust platform TISCH2 facilitates the investigation of the tumor microenvironment (TME) in many cancer types by enabling thorough cell type annotations at the individual cell level [The Tumor Immune Single-cell Hub 2 . The Wilcoxon rank sum test was used to look at variables with nonnormal distributions, while Student's p value < 0.05; p value < 0.05; p value < 0.05; Using the TCGA database, an unpaired differential analysis was used to examine the expression of OSM in ccRCC tumor tissues compared with normal tissues. The results showed that OSM expression was significantly higher in tumor tissues than in normal tissues  and low (n = 265) expression groups. The results demonstrated that the high OSM expression group had significantly worse overall survival and disease-specific survival than the low expression group  revealed that OSM has protein interactions with M2 macrophage surface markers. Therefore, we suggest that OSM forms immunosuppression through M2 macrophages, thereby promoting ccRCC invasion and migration.Then, we selected three datasets from different primary renal clear cell tumors . These datasets are referred to as the GSE111360, GSE159115, and GSE171306 datasets of ccRCC, and we performed single-cell analyses on them using the TISCH website. The results revealed that OSM expression was mainly located in the monocyte-macrophage subpopulation of ccRCC . MacrophCurrently, ccRCC is a severe threat to human health, and approximately hundreds of thousands of ccRCC patients die each year worldwide . With adThe molecular therapy for ccRCC has transitioned from gene-targeted therapy to immunotherapy . TargeteUsing the TCGA dataset and clinical samples, we found that OSM was overexpressed in ccRCC and could be an independent biomarker of a bad prognosis. Cell function experiments demonstrated that high OSM expression in ccRCC could promote tumor cell invasion and migration. Therefore, targeting OSM may hold promise as a potential therapeutic strategy for addressing ccRCC metastasis and recurrence. Studies have demonstrated that OSM overexpression in hepatocellular carcinoma can promote tumor cell invasion and angiogenesis . In breaWe also revealed by pathway enrichment and single-cell analysis that OSM may promote tumor metastasis through M2 macrophages, which are essential for tumor growth, metastasis, immune regulation, tumor angiogenesis, TME remodelling, and response to cancer therapy , 42, 43.However, there are some things that could be improved in our study. For example, our experimental studies were only at the in vitro cellular level and were not further validated in vivo. Because OSM promotes ccRCC cell invasion and migration, we have only analyzed the possibility in terms of single-cell data and lack further specific regulatory mechanisms. This will be investigated in more depth in our subsequent research projects.In conclusion, OSM can facilitate tumor cell invasion and migration and may act as a standalone biomarker for predicting poor prognosis in ccRCC. For patients with recurrent and metastatic ccRCC, this discovery may offer a novel treatment target.


Gallus gallus) facilitate the transfer of RNA cargo from an avian species to humans and mice, and their dietary depletion elicits phenotypes. sEVs were purified from raw egg yolk by ultracentrifugation and authenticated by transmission electron microscopy, nano-tracking device, and immunoblots. The miRNA profile was assessed by RNA-sequencing. Bioavailability of these miRNAs in humans was assessed by egg feeding study in adults, and by culturing human peripheral blood mononuclear cells (PBMCs) with fluorophore-labeled egg sEVs ex vivo. To further assess bioavailability, fluorophore-labeled miRNAs, encapsulated in egg sEVs, were administered to C57BL/6 J mice by oral gavage. Phenotypes of sEV RNA cargo depletion were assessed by feeding egg sEV and RNA-defined diets to mice and using spatial learning and memory in the Barnes and water mazes as experimental readouts. Egg yolk contained 6.30 × 1010 ± 6.06 × 109 sEVs/mL, which harbored eighty-three distinct miRNAs. Human PBMCs internalized sEVs and their RNA cargo. Egg sEVs, loaded with fluorophore-labeled RNA and administered orally to mice, accumulated primarily in brain, intestine and lungs. Spatial learning and memory (SLM) was compromised in mice fed on egg sEV- and RNA-depleted diet compared to controls. Egg consumption elicited an increase of miRNAs in human plasma. We conclude that egg sEVs and their RNA cargo probably are bioavailable. The human study is registered as a clinical trial and accessible at https://www.isrctn.com/ISRCTN77867213.Small extracellular vesicles (sEVs) and their RNA cargo in milk are bioavailable in humans, pigs, and mice, and their dietary depletion and supplementation elicits phenotypes. Little is known about the content and biological activity of sEVs in foods of animal origin other than milk. Here we tested the hypothesis that sEVs in chicken eggs ( Extracellular vesicles (EVs) are detectable in most, if not all, body fluids including saliva, plasma, urine, and milk , 2. Five11 sEVs/mL that harbor more than 200 distinct miRNAs  express miRNAs with nucleotide sequences that can be distinguished from endogenous miRNAs in humans and mice, thereby serving as endogenous tracers of dietary miRNAs . Egg yolk and albumen were separated manually. Samples (50 ug) were mixed with 5 uL of proteinase K (Thermo Fisher) and incubated at 37°C with light shaking for 60 min  were used as secondary antibodies. Transmission electron microscopy (Hitachi H7500) and nanoparticle tracking analysis  were conducted as previously described.Egg yolks were mixed with ten volumes of phosphate-buffered saline (PBS). sEVs were isolated by sequential ultracentrifugation as previously described . Some sa®) as previously described  were separated using density centrifugation . mRNAs were analyzed by RNA-sequencing analysis  in the DNA Sequencing Core at the University of Nebraska Medical Center  and selected miRNAs were analyzed by quantitative reverse transcriptase PCR (qRT-PCR)  30). Al. Al30). 12) were added to PBMC cultures (106 cells in 3 mL media) from four subjects (ages 26–35 years) and cultured at 37°C for 9 h. Controls were cultured with unlabeled sEVs. Extracellular sEVs were removed by washing PBMCs in PBS . The final PBMC pellet was resuspended in 3 mL PBS and fluorescence was measured using an EVOS FL Cell Imaging System .PBMCs were cultured in RPMI-1640 media supplemented with exosome-depleted fetal bovine serum. Exo-Glow labeled sEVs (2.5 × 1012/g body weight). Controls received unlabeled sEVs and free Exo-Glow. Note that the vendor has replaced the Exo-Glow Red kit with a new kit that labels polyA tails and therefore cannot be used to label miRNAs. Tissues were harvested 12 and 24 h after gavage, rinsed and perfused with PBS, and fluorescence was assessed in a LI-COR Odyssey® CLx. In another strategy, sEVs were loaded with two miRNAs found in egg yolk (12 sEVs /per g body weight) were administered by oral gavage to C57/BL6 mice age 6 weeks. Controls received unloaded sEVs. Tissues were harvested 12 h after gavage, rinsed and perfused with PBS, and fluorescence was assessed in a LI-COR Odyssey® CLx. All animal experiments were approved by the Institutional Animal Care Program at the University of Nebraska-Lincoln (protocol 1229).We used the following strategies for assessing the bioavailability distribution of egg sEV RNA in mice: In the first strategy, the entire pool of single-stranded RNA in egg sEVs was labeled with Exo-Glow following the manufacturer’s recommendations . Exo-Glow labeled sEVs were administered to C57/BL6 age 6 weeks  by oral gavage . RNA integrity was confirmed in the DNA Sequencing Core at the University of Nebraska Medical Center by using a Fragment Analyzer Automated CE System . Libraries were prepared by using the TrueSeq RNA Library Prep Kit v2  and sequenced using an Illumina HiSeq 2,500 platform and a 150 base-pair paired-end protocol (RNA-seq). The sequencing reads were annotated to the mouse reference genome  using RSEM .n = 8). In the Barnes maze, breeders were studied at age 12 weeks and F1 offspring was studied at age 16 weeks as previously described (Barnes and Morris water mazes were used to assess SLM. Tests were performed in C57BL/6 breeders fed ERS or ERD diets (ERS x ERS and ERD x ERD), and in F1 pups born to ERS and ERD parents and fed parental diets (escribed . Mice weescribed .p < 0.05 for multiple comparisons using Benjamini’s method). Differences were considered statistically significant at value of p < 0.05.Data variances were homogenous as per Bartlett’s test. Effect of eggs consumption on mRNA abundance in PBMCs was analyzed by the paired, two-sided t-test. miRNA concentrations in plasma were analyzed by the Wilcoxon signed-rank test. GraphPad Prism 6 (GraphPad Software Inc.) was used for statistical analyses. Differentially expressed transcripts were identified by DESeq2  using th10 ± 6.06 × 109 sEVs per mL yolk with a diameter of 110.0 ± 20.5 nm (mean ± SD)  . Consistan ± SD) . Iodixan103 sEVs . The dia103 sEVs , which i103 sEVs . Immunob103 sEVs .Two eggs yielded 0.141 and 0.319 ng RNA per μL yolk. Eighty-three miRNAs were identified in both yolks by RNA-sequencing analysis. Out of these eighty-three miRNAs, fifty-six were common in both yolk samples . Two eggn = 5). One hundred-seventeen miRNAs were identified by RNA-sequencing analysis. Twenty-six miRNAs were upregulated after egg consumption; the others were downregulated or remained unchanged . Their low abundance in plasma was attributed to their low abundance in egg yolk, e.g., miR-146c-5p .in vivo. We isolated PBMCs from human subjects before and after consumption of four eggs and analyzed the levels of three miRNAs by qRT-PCR. The levels of miR-142-5p, miR-181b-5p, and miR-30a-5p were more than 2-fold higher 9 hours after egg consumption compared to before consumption  in diet such as edible plant juices and milk has been the center of attention for the past few years. Studies suggest that these vesicles and their cargo (including miRNA) can be absorbed through intestine and travel This study provides compelling evidence that chicken egg yolk, but not albumen, contains detectable amounts of sEVs with features expected of exosomes, including morphology, size, marker proteins, and miRNA cargo. Yolk content is the sole source of nutrition for the avian embryo , 46 and ex vivo, and egg miRNAs accumulate in murine tissues, predominantly in the gastrointestinal mucosa, brain, and lungs. In contrast, we cannot state with a high degree of confidence that avian miRNAs entered human circulation. While postprandial changes in plasma miRNA levels were readily apparent in humans after egg consumption, the changes were limited to miRNAs for which the nucleotide sequence is conserved in humans and chickens. We attributed the absence of a postprandial increase in chicken-specific miRNAs in human plasma to their low content in yolk. For example, gga-miR-146c-5p was one of the least abundant miRNAs in yolk and not detected in human plasma. We acknowledge the limitation that we did not collect a 9-h sample from subjects who did not consume eggs, but used the baseline sample as control. Approximately a third of plasma miRNAs exhibit a diurnal variation .As for phenotypes of sEV and miRNA depletion, we focused on SLM because loss of SLM was among the strongest phenotypes of (milk) sEV and RNA depletion observed in previous studies in mice . In the We conclude that studies of milk sEVs are a more fruitful line of investigation than studies of chicken sEVs, primarily because of the unique role of milk in infant nutrition and the use of milk sEVs in the delivery of therapeutics , 49. Bechttps://www.ncbi.nlm.nih.gov/, PRJNA422156.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: The studies involving human participants were reviewed and approved by Institutional Review Board in the University of Nebraska-Lincoln (protocols no. 14585 and 16566). The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Institutional Animal Care Program at the University of Nebraska-Lincoln (protocol 1229).JZ conceived the project. DF, KH, and SB collected data. JM, DF, JS, KH, and JC analyzed data. JM and DF wrote the draft manuscript. JZ wrote the final manuscript. All authors contributed to the article and approved the submitted version.This study was supported by the National Institutes of Health (NIH 1P20GM104320), the National Institute of Food and Agriculture (NIFA 2016–67001-25301), the Egg Nutrition Center, United States Department of Agriculture (Hatch NEB-36-087 and W4002).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Hemorrhagic transformation (HT) after intravenous thrombolysis (IVT) in acute ischemic stroke seriously affects the prognosis of patients. This study aimed to investigate the risk factors of different types of HT and their correlation with prognosis after IVT.Based on the CASE II registry, we included patients with acute ischemic stroke who received IVT within 4.5 h of onset. HT was further divided into hemorrhagic infarction (HI) and parenchymal hemorrhage (PH). Poor outcome was defined as a modified Rankin Scale (mRS) score of 3–6 at 3 months. Multivariate logistic regression analysis was used to determine the independent influencing factors of HT subtypes and clinical outcome.p < 0.001), atrial fibrillation , baseline diastolic pressure , baseline NIHSS score  and onset to treatment time  independently predicted PH after IVT. In the patients with HT, PH  and remote hemorrhage  were independently related to poor outcome.Among 13108 included patients, 541 (4.1%) developed HI and 440 (3.4%) developed PH. In multivariate analysis, age (OR 1.038, 95% CI 1.028 to 1.049, Different types of HT after IVT had different risk factors and clinical significance. The occurrence of PH and remote hemorrhage independently increased the risk of poor outcome.The online version contains supplementary material available at 10.1186/s40001-023-01503-x. At present, intravenous thrombolysis (IVT) with alteplase is the standard treatment for acute ischemic stroke . DespiteThe purpose of this study is to explore the possible risk factors predicting different subtypes of HT, especially for PH type that may cause distinct deterioration in neurological status and functional outcome, in a large multicenter stroke database.The Computer-Based Online Database of Acute Stroke Patients for Stroke Management Quality Evaluation (CASE II) is a prospective multicenter registration study that aims to establish an online database of acute stroke patients for stroke management quality evaluation in China (NCT04487340). Based on CASE II registration, we included patients with acute ischemic stroke who received IVT within 4.5 h of stroke symptom onset from December 2016 to July 2021. We excluded patients who (1) were treated with endovascular therapy after IVT; (2) had no follow-up images. (3) experienced HT other than the types of HI and PH, such as remote hemorrhage, subarachnoid hemorrhage, or intraventricular hemorrhage; (4) lost to follow-up at 90 days.The following patient characteristics were recorded from the registry database, such as demographics, vascular risk factors, prior antithrombotic (antiplatelet and anticoagulant) drugs usage, onset to treatment time (OTT), baseline National Institutes of Health Stroke Scale (NIHSS) score, site and type of HT.The head CT/MR imaging taken 24 h after IVT was reviewed and evaluated by radiologist and neurologists, with a consensus reached in case of discrepancies. Referring to the ECASS-II classification standard , HT was The primary outcome of this analysis was modified Rankin Scale (mRS) score at 90 days (good outcome defined as a score of 0–2 and poor outcome defined as a score of 3–6). Secondary outcomes were mortality within 7 days, and stroke recurrence at discharge and 90 days.t test or Mann–Whitney U test was used for the continuous variables, and Chi-square test or Fisher's exact test was used to compare the dichotomous variables between groups, while as appropriate. One-way ANOVA or Kruskal–Wallis test was used between multiple groups. Variables with a p value of < 0.1 in univariate analyses were included in the binary logistic regression model. All statistical analyses were carried out with SPSS 24.0 statistical software, and the difference was statistically significant with p < 0.05.Study participants with HT were dichotomized HI and PH. Patients without HT at the same time were used as the control group. Continuous variables were expressed as mean with standard deviation (SD), while median (interquartile range [IQR]), or No. (%) for ordinal variables. Independent samples The flowchart of enrollment is shown in Fig. p < 0.05) among patients with HI and PH, and the control group. The 7-day mortality, stroke recurrence rate at discharge, and 90 day mRS score were higher in patients with PH. The baseline characteristics and functional outcomes stratified by presence of HT are shown in Additional file The baseline characteristics and functional outcomes stratified by HT type are shown in Table p < 0.1) in univariate analysis were estimated using binary logistic regression analysis, as shown in Tables The predictors with marginally significant difference (p < 0.001). In addition, combination of remote hemorrhage also independently increased the risk of poor outcome  , which might affect the primary outcome of this study. Second, due to the non-mandatory nature of certain data submission to the CASE II registry, crucial factors such as baseline Alberta Stroke Program Early CT Score, admission blood glucose levels and international normalized ratio were not assessed. Moreover, potential risk factors linked to HT, such as biomarkers, were not incorporated into this analysis. Third, the CASE II registry encompasses hospitals of varying levels, where the influence of different operational tiers on HT might surpass that of traditional risk factors. Hence, this study concentrates on patients only undergoing IVT. However, excluding patients who received endovascular therapy clearly limits the potential clinical application.Our study shows that different types of HT after IVT had different risk factors and clinical significance. The occurrence of PH and remote hemorrhage independently increased the risk of poor outcome in those developed HT after IVT.Additional file1: Table S1. Comparison of baseline characteristics and outcomes of patients with or without HT.