Efficacy and Safety of Ivarmacitinib in Patients With Moderate-to-Severe, Active, Ulcerative Colitis: A Phase II Study.

BACKGROUND & AIMS: Current therapies for ulcerative colitis (UC) fail to achieve satisfactory disease control. Selective inhibition of Janus kinase (JAK) type 1 may improve clinical outcomes in patients with UC while avoiding the side effects associated with pan-JAK inhibition. The safety and efficacy of the selective JAK1 inhibitor ivarmacitinib (formerly SHR0302) were evaluated in patients with moderate-to-severe, active UC. METHODS: AMBER2 was a double-blind, placebo-controlled, phase II trial conducted at 63 clinical centers in China, the United States, and Europe. Patients (N = 164) were randomized 1:1:1:1 to receive oral ivarmacitinib 8 mg once daily (QD), 4 mg twice daily (BID), or 4 mg QD, or placebo for 8 weeks, followed by an 8-week extension period. The primary endpoint was clinical response rate at week 8. Hochberg's procedure was used to control the study-wise type 1 error at alpha=0.1. RESULTS: A total of 146 (89.0%) patients completed 8 weeks of treatment. Week 8 clinical response rates were significantly higher in the 8 mg QD (46.3%; P = .066), 4 mg BID (46.3%; P = .059), and 4 mg QD (43.9%; P = .095) groups vs placebo (26.8%). Week 8 rates of clinical remission were 22.0% (P = .020), 24.4% (P = .013), and 24.4% (P = .011) in the 3 ivarmacitinib treatment groups, respectively, vs 4.9% for placebo. During the initial 8-week period, treatment-emergent adverse events occurred in 43.9% to 48.8% of ivarmacitinib-treated patients and in 39.0% of the placebo group, and were predominantly mild. There were no deaths, or major adverse cardiovascular or thromboembolic events. CONCLUSION: Ivarmacitinib demonstrated clinical efficacy and was well tolerated in patients with moderate-to-severe, active, UC. Ivarmacitinib represents a promising new treatment for moderate-to-severe UC. CLINICALTRIALS: gov number, NCT03675477.

Application of bilingual simulated patients in the medical history collection for international medical students in China.

BACKGROUND: In all international medical student (IMS) programs in China, language barriers between IMSs and Chinese patients greatly reduced the learning in clinical practice and brought great challenges to IMSs in their transition from preclinical to clinical practice. This study aimed to investigate the role of bilingual simulated patients (B-SPs) in IMSs learning of medical history collection in China. METHODS: 48 IMSs of grade 4 between October 2020 to Jan 2021 were enrolled in this study. During the training of medical history collection, students were randomly arranged into two groups trained with either B-SPs (B-SP group) or English-speaking SP (E-SP group). All SPs in Objective Structured Clinical Exam station (OSCE) were trained in the Affiliated Hospital of Wuhan University. Clinical skills in medical history collection were assessed by instructors during pre-clinical, post-clinical OSCE and clinical rotations. RESULTS: The scores of IMSs in each group were analyzed in terms of medical history collection including the ability to effectively consult for information and key communication skills related to patient care. Our results indicated that IMS in B-SP group obtained similar scores in preclinical training for history collection (67.3 ± 8.46 vs 67.69 ± 8.86, P < 0.05) compared to E-SP group, while obtaining significantly higher score improvements between pre- and post-OSCE (17.22 (95% CI 12.74 to 21.70) vs 10.84 (95% CI 3.53 to 18.15), P = 0.0007). CONCLUSION: B-SPs are more conducive to doctor-patient communication and actually improve IMSs learning in medical history collection in China.

Sclerotinia sclerotiorum utilizes host-derived copper for ROS detoxification and infection.

Necrotrophic plant pathogen induces host reactive oxygen species (ROS) production, which leads to necrosis in the host, allowing the pathogen to absorb nutrients from the dead tissues. Sclerotinia sclerotiorum is a typical necrotrophic pathogen that causes Sclerotinia stem rot in more than 400 species, resulting in serious economic losses. Here, we found that three S. sclerotiorum genes involved in copper ion import/transport, SsCTR1, SsCCS and SsATX1, were significantly up-regulated during infection of Brassica oleracea. Function analysis revealed that these genes involved in fungal ROS detoxification and virulence. On the host side, four genes putatively involved in copper ion homeostasis, BolCCS, BolCCH, BolMT2A and BolDRT112, were significantly down-regulated in susceptible B. oleracea, but stably expressed in resistant B. oleracea during infection. Their homologs were found to promote resistance to S. sclerotiorum and increase antioxidant activity in Arabidopsis thaliana. Furthermore, copper concentration analysis indicated that copper flow from healthy area into the necrotic area during infection. A model was proposed that S. sclerotiorum utilizes host copper to detoxify ROS in its cells, whereas the resistant hosts may restrict the supply of essential copper nutrients to S. sclerotiorum by maintaining copper ion homeostasis during infection.

CaMKII Mediates TGFß1-Induced Fibroblasts Activation and Its Cross Talk with Colon Cancer Cells.

INTRODUCTION: Cancer-associated fibroblasts (CAFs), as the activated fibroblasts in tumor stroma, are important modifiers of tumor progression. TGFß1 has been the mostly accepted factor to fuel normal fibroblasts transformation into CAFs. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is thought to play an important role in fibroblasts activation induced by TGFß1. The aim of this study is to investigate the potential role of CaMKII in TGFß1-induced fibroblasts activation and CAF-like differentiation. Cross talk between CaMKII-dependent fibroblasts and colon cancer in colon cancer progression also was addressed RESULTS: Immunostaining demonstrated that in colon cancer stroma, CaMKII overexpressed in stromal CAFs. In vitro, TGFß1 increased CAF markers expression in human colon fibroblasts CCD-18Co, but not in CaMKII depletion fibroblasts. CaMKII knockdown by CaMKII shRNA significantly inhibited TGFß1-induced fibroblasts activation and CAF-like differentiation. Smad3, AKT, and MAPK were targeted in TGFß1-CaMKII-mediated pathway. Human colon cancer cell line HCT-116 activated fibroblasts directly, whereas CaMKII depletion dragged CCD-18Co fibroblasts undergoing CAF-associated trans-differentiation. Furthermore, increased proliferation, migration, and invasion of colon cancer cells were stimulated when co-cultured with normal fibroblasts, but not with CaMKII depletion fibroblasts. CONCLUSIONS: These findings provide evidence that CaMKII is a critical mediator in TGFß1-induced fibroblasts activation and is involved in the cross talk with colon cancer cells. CaMKII is a potentially effective target for future treatment of colon cancer.

Gastrointestinal Symptoms Onset in COVID-19 Patients in Wuhan, China.

BACKGROUND: Early detection is critical in limiting the spread of 2019 novel coronavirus (COVID-19). Although previous data revealed characteristics of GI symptoms in COVID-19, for patients with only GI symptoms onset, their diagnostic process and potential transmission risk are still unclear. METHODS: We retrospectively reviewed 205 COVID-19 cases from January 16 to March 30, 2020, in Renmin Hospital of Wuhan University. All patients were confirmed by virus nuclei acid tests. The clinical features and laboratory and chest tomographic (CT) data were recorded and analyzed. RESULTS: A total of 171 patients with classic symptoms (group A) and 34 patients with only GI symptoms (group B) were included. In patients with classical COVID-19 symptoms, GI symptoms occurred more frequently in severe cases compared to non-severe cases (20/43 vs. 91/128, respectively, p < 0.05). In group B, 91.2% (31/34) patients were non-severe, while 73.5% (25/34) patients had obvious infiltrates in their first CT scans. Compared to group A, group B patients had a prolonged time to clinic services (5.0 days vs. 2.6 days, p < 0.01) and a longer time to a positive viral swab normalized to the time of admission (6.9 days vs. 3.3 days, respectively, p < 0.01). Two patients in group B had family clusters of SARS-CoV-2 infection. CONCLUSION: Patients with only GI symptoms of COVID-19 may take a longer time to present to healthcare services and receive a confirmed diagnosis. In areas where infection is rampant, physicians must remain vigilant of patients presenting with acute gastrointestinal symptoms and should do appropriate personal protective equipment.

Immunosuppression as a risk factor for COVID-19: a meta-analysis.

BACKGROUND: While immunosuppression poses a theoretical increase in the risk of COVID-19, the nature of this relationship is yet to be ascertained. AIMS: To determine whether immunosuppressed patients are at higher risk of COVID-19 to help inform the management of patients receiving immunosuppressant therapies during the pandemic. METHODS: We performed a random-effects meta-analysis of data from studies that reported on the prevalence of immunosuppression among patient cohorts with COVID-19. RESULTS: Sixty full-text publications were identified. In total, six individual studies were included in the final analysis, contributing a total of 10 049 patients with COVID-19 disease. The prevalence of immunosuppressed patients among the study cohorts with COVID-19 ranged from 0.126% to 1.357%. In the pooled cohort a total of 64/10 049 (0.637%) patients with COVID-19 disease was immunosuppressed. Observed to expected ratios were used to compare the prevalence of immunosuppression in cohorts with confirmed COVID-19 disease to the background prevalence of immunosuppression in the general community. The observed to expected ratio of immunosuppression among patients with COVID-19 illness, relative to the general community, was 0.12 (95% confidence interval: 0.05-0.27). CONCLUSIONS: Compared to the general population, immunosuppressed patients were not at significantly increased risk of COVID-19 infection. This finding provides support for current expert consensus statements, which have recommended the continuation of immunosuppressant therapy in the absence of COVID-19.

Glandular orientation and shape determined by computational pathology could identify aggressive tumor for early colon carcinoma: a triple-center study.

BACKGROUND: Identifying the early-stage colon adenocarcinoma (ECA) patients who have lower risk cancer vs. the higher risk cancer could improve disease prognosis. Our study aimed to explore whether the glandular morphological features determined by computational pathology could identify high risk cancer in ECA via H&E images digitally. METHODS: 532 ECA patients retrospectively from 2 independent data centers, as well as 113 from The Cancer Genome Atlas (TCGA), were enrolled in this study. Four tissue microarrays (TMAs) were constructed across ECA hematoxylin and eosin (H&E) stained slides. 797 quantitative glandular morphometric features were extracted and 5 most prognostic features were identified using minimum redundancy maximum relevance to construct an image classifier. The image classifier was evaluated on D2/D3 = 223, D4 = 46, D5 = 113. The expression of Ki67 and serum CEA levels were scored on D3, aiming to explore the correlations between image classifier and immunohistochemistry data and serum CEA levels. The roles of clinicopathological data and ECAHBC were evaluated by univariate and multivariate analyses for prognostic value. RESULTS: The image classifier could predict ECA recurrence (accuracy of 88.1%). ECA histomorphometric-based image classifier (ECAHBC) was an independent prognostic factor for poorer disease-specific survival [DSS, (HR = 9.65, 95% CI 2.15-43.12, P = 0.003)]. Significant correlations were observed between ECAHBC-positive patients and positivity of Ki67 labeling index (Ki67Li) and serum CEA. CONCLUSION: Glandular orientation and shape could predict the high risk cancer in ECA and contribute to precision oncology. Computational pathology is emerging as a viable and objective means of identifying predictive biomarkers for cancer patients.

Introgression and pyramiding of genetic loci from wild Brassica oleracea into B. napus for improving Sclerotinia resistance of rapeseed.

KEY MESSAGE: Resistant rapeseed lines pyramided with multiple resistant QTLs derived from Brassica oleracea were developed via a hexaploidy strategy. Rapeseed (Brassica napus L.) suffers heavily from Sclerotinia stem rot, but the breeding of Sclerotinia-resistant rapeseed cultivar has been unsuccessful. During the study, interspecific hexaploids were generated between rapeseed variety 'Zhongshuang 9' and a wild B. oleracea which was highly resistant to S. sclerotiorum, followed by backcrossing with Zhongshuang 9 and successive selfing. By molecular marker-assisted selection, three major resistant QTLs were transferred and pyramided from B. oleracea into two BC1F8 lines which exhibited ~ 35% higher resistance level than Zhongshuang 9 and produced good seed yield and seed quality. It is the first report on successful development of Sclerotinia-resistant rapeseed lines by introducing multiple resistant loci from wild B. oleracea. This study revealed the effectiveness of pyramiding multiple QTLs in improving Sclerotinia resistance in rapeseed and provided a novel breeding strategy on utilization of B. oleracea in rapeseed improvement.

Emergent Double Balloon Enteroscopy in Overt Suspected Small Bowel Bleeding: Diagnosis and Therapy.

BACKGROUND Double balloon enteroscopy (DBE) is a diagnosis and therapy method for suspected small bowel bleeding (SSBB). The data for emergent DBE is limited in overt SSBB cases. The aim of this study was to investigate the role of diagnosis and therapy of emergent DBE in patients with overt SSBB. MATERIAL AND METHODS The clinical and endoscopic data for patients with overt SSBB undergoing DBE in a single center from January 2010 to December 2017 were collected and analyzed. Emergent DBE was defined as DBE performed less than 3 days of last bleeding onset. RESULTS A total of 265 DBEs in 265 patients with overt SSBB were enrolled (mean age, 44.7±17.3 years; 66.8% males). The patients were divided into 3 groups according to the timing of DBE: less than 3 days (n=32), more than 3 days and less than 7 days (n=146), and more than 7 days (n=87) (first group was the emergent group, the latter 2 groups were the non-emergent groups). The diagnosis yield for the emergent group was significantly higher than the non-emergent groups (84.4% versus 65.1% or 59.8%, respectively, P<0.05), but was not different between the 2 non-emergent groups (P>0.05). The top 3 diagnoses were angioectasias (19.6%), diverticulum (16.2%), and tumor (12.1%). For therapy yield, there was a remarkable reducing trend in the emergent group (<3 days), and the 2 non-emergent groups (3 to 7 days group and >7 days group: 78.1%, 58.2% and 39.1%, respectively, P<0.05. The top 3 endoscopic treatments were hemostatic clips (21.9%), argon plasma coagulation (15.8%), and epinephrine injection (14.0%). CONCLUSIONS The emergent DBE had the highest yields for diagnosis and therapy. The study finding showed a pivotal role of emergent DBE in overt SSBB.

Simultaneous Transcriptome Analysis of Host and Pathogen Highlights the Interaction Between Brassica oleracea and Sclerotinia sclerotiorum.

White mold disease caused by Sclerotinia sclerotiorum is a devastating disease of Brassica crops. Here, we simultaneously assessed the transcriptome changes from lesions produced by S. sclerotiorum on disease-resistant (R) and -susceptible (S) B. oleracea pools bulked from a resistance-segregating F2 population. Virulence genes of S. sclerotiorum, including polygalacturonans, chitin synthase, secretory proteins, and oxalic acid biosynthesis, were significantly repressed in lesions of R B. oleracea at 12 h postinoculation (hpi) but exhibited similar expression patterns in R and S B. oleracea at 24 hpi. Resistant B. oleracea induced expression of receptors potentially to perceive Sclerotinia signals during 0 to 12 hpi and deployed complex strategies to suppress the pathogen establishment, including the quick accumulation of reactive oxygen species via activating Ca2+ signaling and suppressing pathogen oxalic acid generation in S. sclerotiorum. In addition, cell wall degradation was inhibited in the resistant B. oleracea potentially to prevent the expansion of Sclerotinia hyphae. The transcriptome changes in S. sclerotiorum and host revealed that resistant B. oleracea produces strong responses against S. sclerotiorum during early infection.

A genome-wide survey with different rapeseed ecotypes uncovers footprints of domestication and breeding.

Rapeseed (Brassica napus L.) is an important oilseed crop. Despite a short period of domestication and breeding, rapeseed has formed three diverse ecotype groups, namely spring, winter, and semi-winter. However, the genetic changes among the three ecotype groups have remained largely unknown. To detect selective signals, a set of 327 accessions from a worldwide collection were genotyped using a Brassica array, producing 33 186 high-quality single nucleotide polymorphisms (SNPs). Linkage disequilibrium (LD) was unevenly distributed across the genome. A total of 705 (78.2%) weak LD regions were found in the A subgenome, whereas 445 (72.6%) strong LD regions were in the C subgenome. By calculating the nucleotide diversity and population differentiation indices, a total of 198 selective sweeps were identified across ecotype groups, spanning 5.91% (37.9 Mb) of the genome. Within these genome regions, a few known functional genes or loci were found to be in association with environmental adaptability and yield-related traits. In particular, all 12 SNPs detected in significant association with flowering time among accessions were in the selection regions between ecotype groups. These findings provide new insights into the structure of the B. napus genome and uncover the footprints of domestication and breeding.

Transfer of sclerotinia resistance from wild relative of Brassica oleracea into Brassica napus using a hexaploidy step.

KEY MESSAGE: Sclerotinia resistance was transferred into rapeseed from a wild relative of Brassica oleracea (B. incana) using hexaploids derived from crosses between B. incana and rapeseed as a bridge. A high level of resistance against Sclerotinia sclerotiorum has been documented in wild Brassica oleracea, but not in cultivated rapeseed (Brassica napus). To transfer sclerotinia resistance from a wild relative into rapeseed, a strategy was proposed using hexaploids (AACCCC) derived from crosses between the wild B. oleracea-related B. incana genotype 'C01' and the Chinese rapeseed variety 'Zhongshuang 9' as a bridge. Progenies (BC1F1) generated by backcrossing the hexaploid to 'Zhongshuang 9' could be generated with a high crossability (average 18.3 seeds per pod). Seventy-three individuals in BC1F1 were firstly screened for resistance with five molecular markers linked to the major resistance QTL on chromosome C09 in 'C01', and 11 individuals harboring resistance loci were selected to develop vegetative clones. Of these, five exhibited significantly higher resistance than 'Zhongshuang 9' and the most resistant individual was chosen to develop the BC1F2 progeny. Finally, five individual genotypes with nearly twofold higher resistance than 'Zhongshuang 9' were found among 100 BC1F2 individuals by using marker-assisted selection and resistance evaluation. Hereof, one rapeseed-type individual with 38 chromosomes and good self-fertility (15.0 ± 3.56 seeds/pod) was identified. Our results indicate that the proposed strategy is effective for transferring sclerotinia resistance from a wild relative of B. oleracea into rapeseed.

Identification of genomic regions involved in resistance against Sclerotinia sclerotiorum from wild Brassica oleracea.

The lack of resistant source has greatly restrained resistance breeding of rapeseed (Brassica napus, AACC) against Sclerotinia sclerotiorum which causes severe yield losses in rapeseed production all over the world. Recently, several wild Brassica oleracea accessions (CC) with high level of resistance have been identified (Mei et al. in Euphytica 177:393-400, 2011), bringing a new hope to improve Sclerotinia resistance of rapeseed. To map quantitative trait loci (QTL) for Sclerotinia resistance from wild B. oleracea, an F2 population consisting of 149 genotypes, with several clones of each genotypes, was developed from one F1 individual derived from the cross between a resistant accession of wild B. oleracea (B. incana) and a susceptible accession of cultivated B. oleracea var. alboglabra. The F2 population was evaluated for Sclerotinia reaction in 2009 and 2010 under controlled condition. Significant differences among genotypes and high heritability for leaf and stem reaction indicated that genetic components accounted for a large portion of the phenotypic variance. A total of 12 QTL for leaf resistance and six QTL for stem resistance were identified in 2 years, each explaining 2.2-28.4 % of the phenotypic variation. The combined effect of alleles from wild B. oleracea reduced the relative susceptibility by 22.5 % in leaves and 15 % in stems on average over 2 years. A 12.8-cM genetic region on chromosome C09 of B. oleracea consisting of two major QTL intervals for both leaf and stem resistance was assigned into a 2.7-Mb genomic region on chromosome A09 of B. rapa, harboring about 30 putative resistance-related genes. Significant negative corrections were found between flowering time and relative susceptibility of leaf and stem. The association of flowering time with Sclerotinia resistance is discussed.

Efficacy of Ustekinumab Optimization by 2 Initial Intravenous Doses in Adult Patients With Severe Crohn's Disease.

BACKGROUND: Although increasing studies have reported that dose escalation can improve treatment response to ustekinumab in patients with Crohn's disease (CD), their strategies mainly focus on maintenance regimen. Evidence of ustekinumab dose escalation in induction regimen, particularly in severe CD, remains limited. This study evaluated the efficacy and safety of intravenous ustekinumab with 2 initial doses in patients with severely active CD. METHODS: A retrospective observational study of 99 adult patients with severe CD treated with ustekinumab from 3 IBD centers included 48 patients with standard and 51 with optimized induction treatment. Clinical outcomes, inflammatory biomarkers including fecal calprotectin (FC) normalization, and endoscopic outcomes were evaluated at weeks 16 and 48. Adverse events and treatment decisions after initial induction were also collected. RESULTS: Compared with the standard group, 2 initial intravenous injections of ustekinumab achieved higher clinical response (92.2%, 47 of 51, P = .656), clinical remission (88.2%, 45 of 51, P = .221), endoscopic response (75.8%, 25 of 33, P = .125), and FC normalization (70.6%, 36 of 51, P = .138) at week 16. The mucosal healing rate at week 16 (63.6%, P = .022) was statistically higher in the optimization group. At week 48, patients with optimized treatment achieved higher clinical response (80.4%, 41 of 51, P = .003), clinical remission (70.6%, 36 of 51, P = .007), FC normalization (66.7%, 34 of 51, P = .031), endoscopic response (72.7%, 24 of 33, P = .006), and mucosal healing (57.6%, 19 of 33, P = .004). At the last follow-up, 82.4% of optimally treated patients adhered to continued treatment with ustekinumab (P < .001). CONCLUSIONS: Optimization of ustekinumab by 2 initial intravenous inductions is more effective than standard therapy for adult patients with severe CD.

This study used an optimization strategy in severe adult Crohn's disease with 2 initial intravenous doses of ustekinumab. This new strategy proved to be effective and safe.

An education-based telehealth group improves the management and clinical outcomes of patients with inflammatory bowel disease in China (THEIM study).

BACKGROUND: Although poor medication adherence has a negative impact on disease prognosis in patients with inflammatory bowel disease (IBD), finding proven solutions remains a challenge. In this study, we developed a telehealth management model based on education and patient-centered medical care (PCEB) using the social media platform WeChat. OBJECTIVE: To investigate the effect of PCEB on adherence and clinical outcomes. METHODS: In this retrospective cohort, 543 IBD patients (274 in the PCEB group and 269 in the routine group) at the IBD center of Renmin Hospital (Wuhan University, Wuhan, China) were enrolled between January 2020 and September 2022. The routine group received routine follow-up and management, while for PCEB patients, a comprehensive IBD education program and PCEB were conducted. Medication adherence and clinical outcomes were also evaluated. RESULTS: There were no differences between the PCEB and routine groups in terms of patient demographics and clinical characteristics, including disease classification, duration, biological treatment, and educational background at baseline. Compared with routine treatment, PCEB greatly improved patient medication adherence, as assessed by compliance with oral medication, enteral nutrition, biological infusion, and scheduled endoscopic assessment. Clinical and endoscopic remission in patients with PCEB increased during short-term (month 4) and long-term (month 12) follow-ups, along with a decrease in relapse rates for CD (13.3% vs. 31.8%) and UC (19.8% vs. 37.2%). CONCLUSION: The telehealth model applied to the PCEB group improved medication adherence and clinical outcomes in patients with IBD. This is a new and powerful solution for the long-term management of this chronic and progressive disease.

Achieving Total Enteroscopy by Consecutive Bidirectional Double-balloon Enteroscopy Procedures.

OBJECTIVE: To investigate the feasibility and safety of achieving total enteroscopy by consecutive bidirectional double-balloon enteroscopy (DBE) procedures. METHODS: The demographic data, indication, initial insertion route, examination time for each insertion and the entire procedure, total enteroscopy rate, diagnostic yield and adverse events of patients who attempted to achieve total enteroscopy by consecutive bidirectional DBE procedures from January 2014 to December 2019 were retrospectively analyzed. RESULTS: A total of 189 patients were included, and the total enteroscopy rate was 87.3%. Initiating the DBE procedure via the retrograde approach as the initial insertion route achieved a higher total enterosocpy rate (90.9% vs. 78.9%, P=0.023), with shorter overall examination time (134.2±36.2 vs. 156.9±47.6 min, P=0.017) and shorter examination time for the opposite insertion route (23.8±19.9 vs. 53.1±27.6 min, P=0.000) compared with anteograde approach as the initial insertion route. The overall diagnostic yield was 37.6%. The diagnostic yield for successfully achieving total enteroscopy was higher, when compared to the yield for not successfully achieving total enteroscopy (39.4% vs. 25%, P=0.029). The overall rate of adverse events was 2.1% (4/189). There was no significant difference in adverse event rate between the overall examination time ≥2 h group and <2 h group (2.1% vs. 2.0%, P=0.593). CONCLUSION: Consecutive bidirectional DBE procedure is an effective and safe strategy for achieving total enteroscopy with a considerable success rate. This may be a promising option and alternative to traditional methods, and helpful to more promptly establish a definite diagnosis. The retrograde approach, as the initial insertion route, is preferred in clinical practice.

Host-induced gene silencing reveals the role of Sclerotinia sclerotiorum oxaloacetate acetylhydrolase gene in fungal oxalic acid accumulation and virulence.

Sclerotinia sclerotiorum, the causal agent of Sclerotinia stem rot, is a devastating necrotrophic pathogen which causes severe yield losses to oilseed production worldwide. Most of efforts at the genetic mitigation of the disease have not been successful. Present investigation was conducted to functionally characterize the effect of down-regulating Ssoah1 during host infection and explore the possibility of boosting host resistance by silencing this gene. We utilized host-induced gene silencing (HIGS) to silence Ssoah1 gene in the S. sclerotiorum fungus. A HIGS based vector was constructed and transformed into Arabidopsis thaliana. The pathogenicity assays in the transgenic A. thaliana lines revealed three T3 transformants with significantly higher resistance to S. sclerotiorum in comparison to untransformed controls. There was a concomitant reduction in expression of Ssoah1 and accumulation of oxalic acid in the necrotic regions of transgenic lines as compared to the non-transgenic controls. Specific Ssoah1-siRNA was highly expressed in HIGS Ssoah1 transgenic lines, as compared with WT and EV plants. The outcomes of oxalic acid estimation revealed that silencing of Ssoah1 results in decreased OA accumulation. The recovered mycelium plugs from HIGS Ssoah1 transgenic lines showed decreased Ssoah1 expression and pathogenesis. These results provide the possibility of using HIGS of Ssoah1 for engineering resistance against S. sclerotiorum.

SsCox17, a copper chaperone, is required for pathogenic process and oxidative stress tolerance of Sclerotinia sclerotiorum.

Stem rot, caused by Sclerotinia sclerotiorum has emerged as one of the major fungal pathogens of oilseed Brassica across the world. The pathogenic development is exquisitely dependent on reactive oxygen species (ROS) modulation. Cox17 is a crucial factor that shuttles copper ions from the cytosol to the mitochondria for the cytochrome c oxidase (CCO) assembly. Currently, no data is available regarding the impact of Cox17 in fungal pathogenesis. The present research was carried out to functionally characterize the role of Cox17 in S. sclerotiorum pathogenesis. SsCox17 transcripts showed high expression levels during inoculation on rapeseed. Intramitochondrial copper content and CCO activity were decreased in SsCox17 gene-silenced strains. The SsCox17 gene expression was up-regulated in the hyphae under oxidative stress and a deficiency response to oxidative stress was detected in SsCox17 gene-silenced strains. Compared to the S. sclerotiorum wild-type strain, there was a concomitant reduction in the virulence of SsCox17 gene-silenced strains. The SsCox17 overexpression strain was further found to increase copper content, CCO activity, tolerance to oxidative stress and virulence. We also observed a certain correlation of appressoria formation and SsCox17. These results provide evidence that SsCox17 is positively associated with fungal virulence and oxidative detoxification.

Sclerotinia sclerotiorum Thioredoxin1 (SsTrx1) is required for pathogenicity and oxidative stress tolerance.

Sclerotinia sclerotiorum infects host plant tissues by inducing necrosis to source nutrients needed for its establishment. Tissue necrosis results from an enhanced generation of reactive oxygen species (ROS) at the site of infection and apoptosis. Pathogens have evolved ROS scavenging mechanisms to withstand host-induced oxidative damage. However, the genes associated with ROS scavenging pathways are yet to be fully investigated in S. sclerotiorum. We selected the S. sclerotiorum Thioredoxin1 gene (SsTrx1) for our investigations as its expression is significantly induced during S. sclerotiorum infection. RNA interference-induced silencing of SsTrx1 in S. sclerotiorum affected the hyphal growth rate, mycelial morphology, and sclerotial development under in vitro conditions. These outcomes confirmed the involvement of SsTrx1 in promoting pathogenicity and oxidative stress tolerance of S. sclerotiorum. We next constructed an SsTrx1-based host-induced gene silencing (HIGS) vector and mobilized it into Arabidopsis thaliana (HIGS-A) and Nicotiana benthamiana (HIGS-N). The disease resistance analysis revealed significantly reduced pathogenicity and disease progression in the transformed genotypes as compared to the nontransformed and empty vector controls. The relative gene expression of SsTrx1 increased under oxidative stress. Taken together, our results show that normal expression of SsTrx1 is crucial for pathogenicity and oxidative stress tolerance of S. sclerotiorum.