SNHG15-mediated feedback loop interplays with HNRNPA1/SLC7A11/GPX4 pathway to promote gastric cancer progression.

Dysregulation of long noncoding RNA (lncRNA) expression plays a pivotal role in the initiation and progression of gastric cancer (GC). However, the regulation of lncRNA SNHG15 in GC has not been well studied. Mechanisms for ferroptosis by SNHG15 have not been revealed. Here, we aimed to explore SNHG15-mediated biological functions and underlying molecular mechanisms in GC. The novel SNHG15 was identified by analyzing RNA-sequencing (RNA-seq) data of GC tissues from our cohort and TCGA dataset, and further validated by qRT-PCR in GC cells and tissues. Gain- and loss-of-function assays were performed to examine the role of SNHG15 on GC both in vitro and in vivo. SNHG15 was highly expressed in GC. The enhanced SNHG15 was positively correlated with malignant stage and poor prognosis in GC patients. Gain- and loss-of-function studies showed that SNHG15 was required to affect GC cell growth, migration and invasion both in vitro and in vivo. Mechanistically, the oncogenic transcription factors E2F1 and MYC could bind to the SNHG15 promoter and enhance its expression. Meanwhile, SNHG15 increased E2F1 and MYC mRNA expression by sponging miR-24-3p. Notably, SNHG15 could also enhance the stability of SLC7A11 in the cytoplasm by competitively binding HNRNPA1. In addition, SNHG15 inhibited ferroptosis through an HNRNPA1-dependent regulation of SLC7A11/GPX4 axis. Our results support a novel model in which E2F1- and MYC-activated SNHG15 regulates ferroptosis via an HNRNPA1-dependent modulation of the SLC7A11/GPX4 axis, which serves as the critical effectors in GC progression, and provides a new therapeutic direction in the treatment of GC.

Degradation of TRIM32 is induced by RTA for Kaposi's sarcoma-associated herpesvirus lytic replication.

TRIM32 is often aberrantly expressed in many types of cancers. Kaposi's sarcoma-associated herpesvirus (KSHV) is linked with several human malignancies, including Kaposi's sarcoma and primary effusion lymphomas (PELs). Increasing evidence has demonstrated the crucial role of KSHV lytic replication in viral tumorigenesis. However, the role of TRIM32 in herpesvirus lytic replication remains unclear. Here, we reveal that the expression of TRIM32 is upregulated by KSHV in latency, and reactivation of KSHV lytic replication leads to the inhibition of TRIM32 in PEL cells. Strikingly, RTA, the master regulator of lytic replication, interacts with TRIM32 and dramatically promotes TRIM32 for degradation via the proteasome systems. Inhibition of TRIM32 induces cell apoptosis and in turn inhibits the proliferation and colony formation of KSHV-infected PEL cells and facilitates the reactivation of KSHV lytic replication and virion production. Thus, our data imply that the degradation of TRIM32 is vital for the lytic activation of KSHV and is a potential therapeutic target for KSHV-associated cancers. IMPORTANCE: TRIM32 is associated with many cancers and viral infections; however, the role of TRIM32 in viral oncogenesis remains largely unknown. In this study, we found that the expression of TRIM32 is elevated by Kaposi's sarcoma-associated herpesvirus (KSHV) in latency, and RTA (the master regulator of lytic replication) induces TRIM32 for proteasome degradation upon viral lytic reactivation. This finding provides a potential therapeutic target for KSHV-associated cancers.

Rationale for immune checkpoint inhibitors plus targeted therapy for advanced renal cell carcinoma.

Renal cell carcinoma (RCC) is a frequent urological malignancy characterized by a high rate of metastasis and lethality. The treatment strategy for advanced RCC has moved through multiple iterations over the past three decades. Initially, cytokine treatment was the only systemic treatment option for patients with RCC. With the development of medicine, antiangiogenic agents targeting vascular endothelial growth factor and mammalian target of rapamycin and immunotherapy, immune checkpoint inhibitors (ICIs) have emerged and received several achievements in the therapeutics of advanced RCC. However, ICIs have still not brought completely satisfactory results due to drug resistance and undesirable side effects. For the past years, the interests form researchers have been attracted by the combination of ICIs and targeted therapy for advanced RCC and the angiogenesis and immunogenic tumor microenvironmental variations in RCC. Therefore, we emphasize the potential principle and the clinical progress of ICIs combined with targeted treatment of advanced RCC, and summarize the future direction.

An intranasal combination vaccine induces systemic and mucosal immunity against COVID-19 and influenza.

Despite prolonged surveillance and interventions, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses continue to pose a severe global health burden. Thus, we developed a chimpanzee adenovirus-based combination vaccine, AdC68-HATRBD, with dual specificity against SARS-CoV-2 and influenza virus. When used as a standalone vaccine, intranasal immunization with AdC68-HATRBD induced comprehensive and potent immune responses consisting of immunoglobin (Ig) G, mucosal IgA, neutralizing antibodies, and memory T cells, which protected the mice from BA.5.2 and pandemic H1N1 infections. When used as a heterologous booster, AdC68-HATRBD markedly improved the protective immune response of the licensed SARS-CoV-2 or influenza vaccine. Therefore, whether administered intranasally as a standalone or booster vaccine, this combination vaccine is a valuable strategy to enhance the overall vaccine efficacy by inducing robust systemic and mucosal immune responses, thereby conferring dual lines of immunological defenses for these two viruses.

STAT3-mediated up-regulation of DAB2 via SRC-YAP1 signaling axis promotes Helicobacter pylori-driven gastric tumorigenesis.

BACKGROUND: Helicobacter pylori (H pylori) infection is the primary cause of gastric cancer (GC). The role of Disabled-2 (DAB2) in GC remains largely unclear. This study aimed to investigate the role of DAB2 in H pylori-mediated gastric tumorigenesis. METHODS: We screened various datasets of GC to analyze DAB2 expression and cell signaling pathways. DAB2 expression was assessed in human GC tissue microarrays. H pylori infection in vivo and in vitro models were further explored. Immunostaining, immunofluorescence, chromatin immunoprecipitation, co-immunoprecipitation, Western blot, quantitative polymerase chain reaction, and luciferase reporter assays were performed in the current study. RESULTS: The bioinformatic analysis verified that DAB2 was 1 of the 8 genes contributed to tumorigenesis and associated with poor prognosis in GC. The median overall survival and disease-free survival rates in DAB2high group were significantly less than those in DAB2low group. These findings demonstrated that H pylori transcriptionally activated DAB2 expression via signal transducer and activator of transcription 3 (STAT3)-dependent pathway. By bioinformatics analysis and knockdown or overexpression of DAB2, we found that DAB2 upregulated Yes-associated protein 1 (YAP1) transcriptional activity. Mechanistically, DAB2 served as a scaffold protein for integrin beta 3 (ITGB3) and SRC proto-oncogene non-receptor tyrosine kinase (SRC), facilitated the phosphorylation of SRC, promoted the small GTPase ras homolog family member A (RHOA) activation and phosphorylation of YAP1, and ultimately enhanced the YAP1 transcriptional activity. CONCLUSIONS: Altogether, these findings indicated that DAB2 is a key mediator in STAT3-regulated translation of YAP1 and plays crucial roles in H pylori-mediated GC development. DAB2 might serve as a novel therapeutic target for GC.

Vitamin D and Its Receptors in Cervical Cancer.

Several studies have investigated the relationship between vitamin D (VD) and its receptors (VDR) and the risk of cervical cancer. However, the underlying mechanisms that underpin these associations remain incompletely comprehended. In this review, we analyzed the impacts of VD and VDR on cervical cancer and related mechanisms, and discussed the effects of VD, calcium, and other vitamins on cervical cancer. Our literature research found that VD, VDR and their related signaling pathways played indispensable roles in the occurrence and progression of cervical cancer. Epidemiological studies have established associations between VD, VDR, and cervical cancer susceptibility. Current studies have shown that the inhibitory effect of VD and VDR on cervical cancer may be attributed to a variety of molecules and pathways, such as the EAG potassium channel, HCCR-1, estrogen and its receptor, p53, pRb, TNF-α, the PI3K/Akt pathway, and the Wnt/ß-catenin pathway. This review also briefly discussed the association between VDR gene polymorphisms and cervical cancer, albeit a comprehensive elucidation of this relationship remains an ongoing research endeavor. Additionally, the potential ramifications of VD, calcium, and other vitamins on cervical cancer has been elucidated, yet further exploration into the precise mechanistic underpinnings of these potential effects is warranted. Therefore, we suggest that further studies should focus on explorations into the intricate interplay among diverse molecular pathways and entities, elucidation of the mechanistic underpinnings of VDR polymorphic loci changes in the context of HPV infection and VD, inquiries into the mechanisms of VD in conjunction with calcium and other vitamins, as well as investigations of the efficacy of VD supplementation or VDR agonists as part of cervical cancer treatment strategies in the clinical trials.

Cross-reactive antibody response to Monkeypox virus surface proteins in a small proportion of individuals with and without Chinese smallpox vaccination history.

BACKGROUND: After the eradication of smallpox in China in 1979, vaccination with the vaccinia virus (VACV) Tiantan strain for the general population was stopped in 1980. As the monkeypox virus (MPXV) is rapidly spreading in the world, we would like to investigate whether the individuals with historic VACV Tiantan strain vaccination, even after more than 40 years, could still provide ELISA reactivity and neutralizing protection; and whether the unvaccinated individuals have no antibody reactivity against MPXV at all. RESULTS: We established serologic ELISA to measure the serum anti-MPXV titer by using immunodominant MPXV surface proteins, A35R, B6R, A29L, and M1R. A small proportion of individuals (born before 1980) with historic VACV Tiantan strain vaccination exhibited serum ELISA cross-reactivity against these MPXV surface proteins. Consistently, these donors also showed ELISA seropositivity and serum neutralization against VACV Tiantan strain. However, surprisingly, some unvaccinated young adults (born after 1980) also showed potent serum ELISA activity against MPXV proteins, possibly due to their past infection by some self-limiting Orthopoxvirus (OPXV). CONCLUSIONS: We report the serum ELISA cross-reactivity against MPXV surface protein in a small proportion of individuals both with and without VACV Tiantan strain vaccination history. Combined with our serum neutralization assay against VACV and the recent literature about mice vaccinated with VACV Tiantan strain, our study confirmed the anti-MPXV cross-reactivity and cross-neutralization of smallpox vaccine using VACV Tiantan strain. Therefore, it is necessary to restart the smallpox vaccination program in high risk populations.

Broad-spectrum vaccine via combined immunization routes triggers potent immunity to SARS-CoV-2 and its variants.

IMPORTANCE: The development of broad-spectrum SARS-CoV-2 vaccines will reduce the global economic and public health stress from the COVID-19 pandemic. The use of conserved T-cell epitopes in combination with spike antigen that induce humoral and cellular immune responses simultaneously may be a promising strategy to further enhance the broad spectrum of COVID-19 vaccine candidates. Moreover, this research suggests that the combined vaccination strategies have the ability to induce both effective systemic and mucosal immunity, which may represent promising strategies for maximizing the protective efficacy of respiratory virus vaccines.

Associations between an obesity-related dietary pattern and incidence of overall and site-specific cancers: a prospective cohort study.

BACKGROUND: A dietary pattern (DP) may impact on cancer incidence more strongly than individual foods, but this association remains uncertain. Here, we aimed to broadly explore the associations of an obesity-related DP with overall and 19 site-specific cancers. METHODS: This study included 114,289 cancer-free participants with at least two dietary assessments. A total of 210 food items were classified into 47 food groups, and the mean amount of each food group was used in reduced-rank regression to derive the obesity-related DP. Cox regressions were conducted to explore the associations of the obesity-related DP with overall and 19 site-specific cancers. The parallel mediation model was constructed to quantify the mediating roles of potential mediators. RESULTS: During a median follow-up period of 9.4 years, 10,145 (8.9%) incident cancer cases were documented. The derived-DP was characterized by a higher intake of beer and cider, processed meat, high sugar beverages, red meat, and artificial sweetener, and a lower intake of fresh vegetables, olive oil, tea, and high fiber breakfast cereals. Observational analysis showed that a higher obesity-related DP Z-score was linearly associated with an increased risk of overall cancer (adjusted hazard ratio (HR) = 1.02, 95% CI: 1.01, 1.04 per 1-SD increase, corrected P < 0.001). For site-specific cancer, positive linear associations for six cancer sites (oral, colorectal, liver, lung, endometrium, and thyroid) and nonlinear associations for six cancer sites (esophagus, malignant melanoma, prostate, kidney, bladder, and multiple myeloma) were observed. The paralleled mediation analysis suggested that the association between the obesity-related DP and overall cancer is mediated by the body mass index (BMI), the waist-to-hip ratio (WHR), C-reactive protein, high-density lipoproteins (HDLs), and triglycerides. CONCLUSIONS: The developed obesity-related DP is strongly associated with overall and multiple cancer sites. Our findings highlight the complicated and diverse associations between an obesity-related DP and cancers and provide clues for future research directions.

Lactate-mediated Fascin protrusions promote cell adhesion and migration in cervical cancer.

Background: Lactate is associated with the poor prognosis of many human malignancies. Cervical cancer, one of main causes of women mortality worldwide, is aggressive and absent of effective pharmacological treatment, and its underlying mechanisms of progression remain elusive. Methods: The regulation of ß-catenin to fascin protrusion formation upon acidic lactate (Lactic acid [LA]) stimulation was evaluated through in ß-catenin or fascin deficiency cell line models by immunofluorescence assays, and subcellular fractionation. The effect of ß-catenin and fascin relocation by LA and its antagonist were evaluated by immunohistochemistry assay in patient tissues and mouse tumor xenograft model. Trypsin digestion, Transwell assay, cell proliferation in vitro was performed to explore the role of LA in the cell growth, adhesion and migration. Results: Low concentration of LA significantly promotes cytoskeleton remodeling via `protrusion formation to increase cell adhesion and migration. Mechanistically, upon LA stimulation, ß-catenin diffuses from the cytoplasmic membrane into the nucleus, which in turn induces fascin nuclear-cytoplasm redistribution to the protrusion compartment. Moreover, the antagonist of LA sufficiently blocks the LA-mediated ß-catenin nuclear import, fascin nuclear export, and the growth and invasion of cervical cancer cells in vitro and in vivo using a murine xenograft model. Conclusions: This study uncovers ß-catenin-fascin axis as a key signal in response to extracellular lactate and indicates that antagonist of LA may serve as a potential clinical intervention for cancer development.

Aberrant expression of GOLM1 protects ALK+ anaplastic large cell lymphoma from apoptosis by enhancing BCL-XL stability.

Golgi membrane protein 1 (GOLM1) is aberrantly expressed in many types of solid tumors and contributes to cancer development; however, its role in hematopoietic and lymphoid neoplasms remains unknown. Here, we report that GOLM1 was significantly upregulated in anaplastic large cell lymphoma (ALCL), particularly in anaplastic lymphoma kinase-positive (ALK+) ALCL. Mechanistically, the expression of GOLM1 was induced by nucleophosmin-ALK in both ALK-transformed T cells and ALCL cell lines through AKT/mTOR pathway. Knockdown of GOLM1 expression led to a reduction in the growth and viability of ALCL cells with increased spontaneous apoptosis, whereas ectopic expression of GOLM1 protected ALCL cells from apoptosis induced by staurosporine treatment. Moreover, GOLM1 directly interacted with B-cell lymphoma-extra large protein (a crucial anti-apoptosis regulator) and significantly prolonged its stability. Introduction of GOLM1 promoted ALK+ ALCL cells colony formation in vitro and tumor growth in a murine xenograft model. Taken together, our findings demonstrate, to our knowledge, for the first time that GOLM1 plays a critical role in suppressing apoptosis and promoting the progression of ALK+ ALCL and provide evidence that GOLM1 is a potential biomarker and therapeutic target in ALK-induced hematological malignancies.

Parainfluenza virus 5 is a next-generation vaccine vector for human infectious pathogens.

Parainfluenza virus 5 (PIV5) is a negative-sense, single-stranded RNA virus that can infect humans and many species of animals. Infection in these reservoir hosts is generally asymptomatic and has few safety concerns. Emerging evidence has shown that PIV5 is a promising vector for developing vaccines against human infectious diseases caused by coronaviruses, influenza, respiratory syncytial virus, rabies, HIV, or bacteria. In this review, we summarize recent progress and highlight the advantages and strategies of PIV5 as a vaccine vector to improve future vaccine design and application for clinical trials.

Illuminating the Live-Cell Dynamics of Hepatitis B Virus Covalently Closed Circular DNA Using the CRISPR-Tag System.

The covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is the major obstacle to curing chronic hepatitis B (CHB). Current cccDNA detection methods are mostly based on biochemical extraction and bulk measurements. They nevertheless generated a general sketch of its biological features. However, an understanding of the spatiotemporal features of cccDNA is still lacking. To achieve this, we established a system combining CRISPR-Tag and recombinant HBV minicircle technology to visualize cccDNA at single-cell level in real time. Using this system, we found that the observed recombinant cccDNA (rcccDNA) correlated quantitatively with its active transcripts when a low to medium number of foci (<20) are present, but this correlation was lost in cells harboring high copy numbers (≥20) of rcccDNA. The disruption of HBx expression seems to displace cccDNA from the dCas9-accessible region, while HBx complementation restored the number of observable cccDNA foci. This indicated regulation of cccDNA accessibility by HBx. Second, observable HBV and duck HBV (DHBV) cccDNA molecules are substantially lost during cell division, and the remaining ones were distributed randomly to daughter cells. In contrast, Kaposi's sarcoma-associated herpesvirus (KSHV)-derived episomes can be retained in a LANA (latency-associated nuclear antigen)-dependent manner. Last, the dynamics of rcccDNA episomes in nuclei displayed confined diffusion at short time scales, with directional transport over longer time scales. In conclusion, this system enables the study of physiological kinetics of cccDNA at the single-cell level. The differential accessibility of rcccDNA to dCas9 under various physiological conditions may be exploited to elucidate the complex transcriptional and epigenetic regulation of the HBV minichromosome. IMPORTANCE Understanding the formation and maintenance of HBV cccDNA has always been a central issue in the study of HBV pathobiology. However, little progress has been made due to the lack of robust assay systems and its resistance to genetic modification. Here, a live-cell imaging system by grafting CRISPR-Tag into the recombinant cccDNA was established to visualize its molecular behavior in real time. We found that the accessibility of rcccDNA to dCas9-based imaging is related to HBx-regulated mechanisms. We also confirmed the substantial loss of observable rcccDNA in one-round cell division and random distribution of the remaining molecules. Molecular dynamics analysis revealed the confined movement of the rcccDNA episome, suggesting its juxtaposition to chromatin domains. Overall, this novel system offers a unique platform to investigate the intranuclear dynamics of cccDNA within live cells.

Application of Improved Robot-assisted Laparoscopic Telesurgery with 5G Technology in Urology.

The demand for telesurgery is rising rapidly, but robust evidence regarding the feasibility of its application in urology is still rare. From March to October 2021, a surgeon-controlled surgical robot in a tertiary hospital in Qingdao was used to remotely conduct robot-assisted laparoscopic radical nephrectomy (RN) in 29 patients located in eight primary hospitals. The median round-trip delay was 26 ms (interquartile range [IQR] 5) and the median distance between the primary hospital and the surgeon was 187 km (IQR 57). Both the master unit and the slave unit were guaranteed by network and mechanical engineers, and surgical assistants were well prepared on the patient side to prevent complications. The primary evaluation metric was the success rate, defined as the percentage of patients who underwent successful remote RN without conversion to other surgical procedures and no major intraoperative or postoperative complications. The results demonstrate that the combination of 5G technology and surgical robots is a novel potential telemedicine-based therapy choice for renal tumors. PATIENT SUMMARY: Our study shows that telesurgery using 5G technology is a safe and feasible treatment option for patients with kidney tumors. The total delay between the remote location and the operating rooms where surgery was being performed was just 200 ms. This approach could reduce health care costs and improve the quality of medical services accessed by patients.

Erratum: Long non-coding RNA SNHG5 affects the invasion and apoptosis of renal cell carcinoma by regulating the miR-363-3p-Twist1 interaction.

[This corrects the article on p. 697 in vol. 12, PMID: 32194916.].

Identification of hACE2-interacting sites in SARS-CoV-2 spike receptor binding domain for antiviral drugs screening.

The key structure of the interface between the spike protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and human angiotensin-converting enzyme 2 (hACE2) acts as an essential switch for cell entry by the virus and drugs targets. However, this is largely unknown. Here, we tested three peptides of spike receptor binding domain (RBD) and found that peptide 391-465 aa is the major hACE2-interacting sites in SARS-CoV-2 spike RBD. We then identified essential amino acid residues (403R, 449Y, 454R) of peptide 391-465 aa that were critical for the interaction between the RBD and hACE2. Additionally, a pseudotyped virus containing SARS-CoV-2 spike with individual mutation (R454G, Y449F, R403G, N439I, or N440I) was determined to have very low infectivity compared with the pseudotyped virus containing the wildtype (WT) spike from reference strain Wuhan 1, respectively. Furthermore, we showed the key amino acids had the potential to drug screening. For example, molecular docking (Docking) and infection assay showed that Cephalosporin derivatives can bind with the key amino acids to efficiently block infection of the pseudoviruses with wild type spike or new variants. Moreover, Cefixime inhibited live SARS-CoV-2 infection. These results also provide a novel model for drug screening and support further clinical evaluation and development of Cephalosporin derivatives as novel, safe, and cost-effective drugs for prevention/treatment of SARS-CoV-2.

Deciphering the myth of icariin and synthetic derivatives in improving erectile function from a molecular biology perspective: a narrative review.

Background and Objective: Although epimedium herb (EH) has been widely used in ancient Chinese medicine to enhance sexual activity, its pharmacological mechanism is not clear. Modern studies have shown that epimedium herb is rich in icariin (ICA, a flavonoid compound), and 91.2% of icariin is converted to icariside II (ICA II) by hydrolytic enzymes in intestinal bacteria after oral administration. YS-10 is a synthetic derivative of icariside II. The aim of this review was to summarize the contemporary evidence regarding the pharmacokinetics, therapeutic properties, and molecular biological mechanisms of ICA and some ICA derivatives for erectile dysfunction therapy. Methods: A detailed search was conducted in the PubMed database using keywords and phrases, such as "icariin" AND "erectile dysfunction", "icariside II" AND "erectile dysfunction". The publication time is limited to last 20 years. Articles had to be published in peer reviewed journals. Key Content and Findings: ICA and its some derivatives showed the specific inhibition on phosphodiesterase type 5 (PDE5) and the promotion of testosterone synthesis. In addition, by regulating various reliable evidence of signaling pathways such as PI3K/AKT, TGFß1/Smad2, p38/MAPK, Wnt and secretion of various cytokines, ICA and ICA derivatives can activate endogenous stem cells (ESCs) leading to endothelial cell and smooth muscle cell proliferation, nerve regeneration and fibrosis inhibition, repair pathological changes in penile tissue and improve erectile function. Conclusions: ICA and some of its derivatives could be a potential treatment for restoring spontaneous erections. In addition ICA and his derivatives may also be valuable as a regenerative medicine approach for other diseases, but more clinical and basic researches with high quality and large samples are recommended.

Correction: STAT6 degradation and ubiquitylated TRIML2 are essential for activation of human oncogenic herpesvirus.

[This corrects the article DOI: 10.1371/journal.ppat.1007416.].

Rapid establishment of murine gastrointestinal organoids using mechanical isolation method.

Gastrointestinal (GI) diseases, including pathological dysplasia, inflammation, neoplasia and injury, suffer millions of patients globally per year. Organoids, three-dimensional cell mass structures supported by biomaterials in dishes, are currently used as a research model for diseases of the small intestine. However, the traditional enzymatic-digestion method for establishing small-intestinal organoids (EnzyOs) is time consuming and often loses the bulk of crypts, a more efficient and reliable method needs to be developed. In this study, using mouse GI organoids as a model, we formulated a rapid mechanical isolation method that could efficiently isolate and culture villi-crypts into small intestinal organoids (MechOs). Primary duodenum organoids generated by MechOs displayed three types of morphology: spheroid, semi-budding and budding, while EnzyOs produced much less budding. Moreover, primary duodenum organoids from MechOs could be subcultured and presented similar gene expression profiles of small intestine specific markers as that from EnzyOs. Importantly, the MechOs method could also be used to generate other types of organoids derived from the stomach, jejunum-ileum, sigmoid-rectum and bile cysts. Taken together, the results show that MechOs could efficiently and economically generate digestive system organoids, providing a potential basis of epithelial organoids for the clinical treatment of gastroenterological diseases.