Alteration of cholesterol content and oxygen level in intestinal organoids after infection with Staphylococcus aureus.

The pathogenicity elicited by Staphylococcus (S.) aureus, one of the best-studied bacteria, in the intestine is not well understood. Recently, we demonstrated that S. aureus infection induces alterations in membrane composition that are associated with concomitant impairment of intestinal function. Here, we used two organoid models, induced pluripotent stem cell (iPSC)-derived intestinal organoids and colonic intestinal stem cell-derived intestinal organoids (colonoids), to examine how sterol metabolism and oxygen levels change in response to S. aureus infection. HPLC quantification showed differences in lipid homeostasis between infected and uninfected cells, characterized by a remarkable decrease in total cellular cholesterol. As the altered sterol metabolism is often due to oxidative stress response, we next examined intracellular and extracellular oxygen levels. Three different approaches to oxygen measurement were applied: (1) cell-penetrating nanoparticles to quantify intracellular oxygen content, (2) sensor plates to quantify extracellular oxygen content in the medium, and (3) a sensor foil system for oxygen distribution in organoid cultures. The data revealed significant intracellular and extracellular oxygen drop after infection in both intestinal organoid models as well as in Caco-2 cells, which even 48 h after elimination of extracellular bacteria, did not return to preinfection oxygen levels. In summary, we show alterations in sterol metabolism and intra- and extracellular hypoxia as a result of S. aureus infection. These results will help understand the cellular stress responses during sustained bacterial infections in the intestinal epithelium.

TRAPγ-CDG shows asymmetric glycosylation and an effect on processing of proteins required in higher organisms.

Newly synthesised glycoproteins enter the rough endoplasmic reticulum through a translocation pore. The translocon associated protein (TRAP) complex is located close to the pore. In a patient with a homozygous start codon variant in TRAPγ (SSR3), absence of TRAPγ causes disruption of the TRAP complex, impairs protein translocation into the endoplasmic reticulum and affects transport, for example, into the brush-border membrane. Furthermore, we observed an unbalanced non-occupancy of N-glycosylation sites. The major clinical features are intrauterine growth retardation, facial dysmorphism, congenital diarrhoea, failure to thrive, pulmonary disease and severe psychomotor disability.

Digestive enzyme expression in the large intestine of children with short bowel syndrome in a late stage of adaptation.

BACKGROUND AND AIMS: Intestinal adaptation in short bowel syndrome (SBS) includes morphologic processes and functional mechanisms. This study investigated whether digestive enzyme expression in the duodenum and colon is upregulated in SBS patients. METHOD: Sucrase-isomaltase (SI), lactase-phlorizin hydrolase (LPH), and neutral Aminopeptidase N (ApN) were analyzed in duodenal and colonic biopsies from nine SBS patients in a late stage of adaptation as well as healthy and disease controls by immunoelectron microscopy (IEM), Western blots, and enzyme activities. Furthermore, proliferation rates and intestinal microbiota were analyzed in the mucosal specimen. RESULTS: We found significantly increased amounts of SI, LPH, and ApN in colonocytes in most SBS patients with large variation and strongest effect for SI and ApN. Digestive enzyme expression was only partially elevated in duodenal enterocytes due to a low proliferation level measured by Ki-67 staining. Microbiome analysis revealed high amounts of Lactobacillus resp. low amounts of Proteobacteria in SBS patients with preservation of colon and ileocecal valve. Colonic expression was associated with a better clinical course in single cases. CONCLUSION: In SBS patients disaccharidases and peptidases can be upregulated in the colon. Stimulation of this colonic intestinalization process by drugs, nutrients, and pre- or probiotics might offer better therapeutic approaches.

Mechanism of drug extrusion by brain endothelial cells via lysosomal drug trapping and disposal by neutrophils.

The blood-brain barrier protects the brain against a variety of potentially toxic compounds. Barrier function results from tight junctions between brain capillary endothelial cells and high expression of active efflux transporters, including P-glycoprotein (Pgp), at the apical membrane of these cells. In addition to actively transporting drugs out of the cell, Pgp mediates lysosomal sequestration of chemotherapeutic drugs in cancer cells, thus contributing to drug resistance. Here, we describe that lysosomal sequestration of Pgp substrates, including doxorubicin, also occurs in human and porcine brain endothelial cells that form the blood-brain barrier. This is followed by shedding of drug-sequestering vesicular structures, which stay attached to the apical side of the plasma membrane and form aggregates ("barrier bodies") that ultimately undergo phagocytosis by neutrophils, thus constituting an as-yet-undescribed mechanism of drug disposal. These findings introduce a mechanism that might contribute to brain protection against potentially toxic xenobiotics, including therapeutically important chemotherapeutic drugs.

Mesenchymal to epithelial transition driven by canine distemper virus infection of canine histiocytic sarcoma cells contributes to a reduced cell motility in vitro.

Sarcomas especially of histiocytic origin often possess a poor prognosis and response to conventional therapies. Interestingly, tumours undergoing mesenchymal to epithelial transition (MET) are often associated with a favourable clinical outcome. This process is characterized by an increased expression of epithelial markers leading to a decreased invasion and metastatic rate. Based on the failure of conventional therapies, viral oncolysis might represent a promising alternative with canine distemper virus (CDV) as a possible candidate. This study hypothesizes that a CDV infection of canine histiocytic sarcoma cells (DH82 cells) triggers the MET process leading to a decreased cellular motility. Immunofluorescence and immunoblotting were used to investigate the expression of epithelial and mesenchymal markers followed by scratch assay and an invasion assay as functional confirmation. Furthermore, microarray data were analysed for genes associated with the MET process, invasion and angiogenesis. CDV-infected cells exhibited an increased expression of epithelial markers such as E-cadherin and cytokeratin 8 compared to controls, indicating a MET process. This was accompanied by a reduced cell motility and invasiveness. Summarized, these results suggest that CDV infection of DH82 cells triggers the MET process by an increased expression of epithelial markers resulting in a decreased cell motility in vitro.

A mutation map for human glycoside hydrolase genes.

Glycoside hydrolases (GHs) are found in all domains of life, and at least 87 distinct genes encoding proteins related to GHs are found in the human genome. GHs serve diverse functions from digestion of dietary polysaccharides to breakdown of intracellular oligosaccharides, glycoproteins, proteoglycans and glycolipids. Congenital disorders of GHs (CDGHs) represent more than 30 rare diseases caused by mutations in one of the GH genes. We previously used whole-exome sequencing of a homogenous Danish population of almost 2000 individuals to probe the incidence of deleterious mutations in the human glycosyltransferases (GTs) and developed a mutation map of human GT genes (GlyMAP-I). While deleterious disease-causing mutations in the GT genes were very rare, and in many cases lethal, we predicted deleterious mutations in GH genes to be less rare and less severe given the higher incidence of CDGHs reported worldwide. To probe the incidence of GH mutations, we constructed a mutation map of human GH-related genes (GlyMAP-II) using the Danish WES data, and correlating this with reported disease-causing mutations confirmed the higher prevalence of disease-causing mutations in several GH genes compared to GT genes. We identified 76 novel nonsynonymous single-nucleotide variations (nsSNVs) in 32 GH genes that have not been associated with a CDGH phenotype, and we experimentally validated two novel potentially damaging nsSNVs in the congenital sucrase-isomaltase deficiency gene, SI. Our study provides a global view of human GH genes and disease-causing mutations and serves as a discovery tool for novel damaging nsSNVs in CDGHs.

Dextran Sodium Sulfate-Induced Impairment of Protein Trafficking and Alterations in Membrane Composition in Intestinal Caco-2 Cell Line.

A key morphological feature of inflammatory bowel disease (IBD) is the loss of the barrier function of intestinal epithelial cells. The present study investigates endoplasmic reticulum (ER) stress in addition to alterations in protein and membrane trafficking in a dextran sulfate sodium (DSS)-induced IBD-like phenotype of intestinal Caco-2 cells in culture. DSS treatment significantly reduced the transepithelial electric resistance (TEER) and increased the epithelial permeability of Caco-2 cells, without affecting their viability. This was associated with an alteration in the expression levels of inflammatory factors in addition to an increase in the expression of the ER stress protein markers, namely immunoglobulin-binding protein (BiP), C/EBP homologous protein (CHOP), activation transcription factor 4 (ATF4), and X-box binding protein (XBP1). The DSS-induced ER-stress resulted in impaired intracellular trafficking and polarized sorting of sucrase-isomaltase (SI) and dipeptidyl peptidase-4 (DPPIV), which are normally sorted to the apical membrane via association with lipid rafts. The observed impaired sorting was caused by reduced cholesterol levels and subsequent distortion of the lipid rafts. The data presented confirm perturbation of ER homeostasis in DSS-treated Caco-2 cells, accompanied by impairment of membrane and protein trafficking resulting in altered membrane integrity, cellular polarity, and hence disrupted barrier function.

Different Trafficking Phenotypes of Niemann-Pick C1 Gene Mutations Correlate with Various Alterations in Lipid Storage, Membrane Composition and Miglustat Amenability.

Niemann-Pick Type C (NPC) is an autosomal recessive lysosomal storage disease leading to progressive neurodegeneration. Mutations in the NPC1 gene, which accounts for 95% of the cases, lead to a defect in intra-lysosomal trafficking of cholesterol and an accumulation of storage material including cholesterol and sphingolipids in the endo-lysosomal system. Symptoms are progressive neurological and visceral deterioration, with variable onset and severity of the disease. This study investigates the influence of two different NPC1 mutations on the biochemical phenotype in fibroblasts isolated from NPC patients in comparison to healthy, wild type (WT) cells. Skin derived fibroblasts were cultured from one patient compound-heterozygous for D874V/D948Y mutations, which presented wild-type like intracellular trafficking of NPC1, and a second patient compound- heterozygous for I1061T/P887L mutations, which exhibited a more severe biochemical phenotype as revealed in the delayed trafficking of NPC1. Biochemical analysis using HPLC and TLC indicated that lipid accumulations were mutation-dependent and correlated with the trafficking pattern of NPC1: higher levels of cholesterol and glycolipids were associated with the mutations that exhibited delayed intracellular trafficking, as compared to their WT-like trafficked or wild type (WT) counterparts. Furthermore, variations in membrane structure was confirmed in these cell lines based on alteration in lipid rafts composition as revealed by the shift in flotillin-2 (FLOT2) distribution, a typical lipid rafts marker, which again showed marked alterations only in the NPC1 mutant showing major trafficking delay. Finally, treatment with N-butyldeoxynojirimycin (NB-DNJ, Miglustat) led to a reduction of stored lipids in cells from both patients to various extents, however, no normalisation in lipid raft structure was achieved. The data presented in this study help in understanding the varying biochemical phenotypes observed in patients harbouring different mutations, which explain why the effectiveness of NB-DNJ treatment is patient specific.

Dietary starch breakdown product sensing mobilizes and apically activates α-glucosidases in small intestinal enterocytes.

Dietary starch is finally converted to glucose for absorption by the small intestine mucosal α-glucosidases (sucrase-isomaltase [SI] and maltase-glucoamylase), and control of this process has health implications. Here, the molecular mechanisms were analyzed associated with starch-triggered maturation and transport of SI. Biosynthetic pulse-chase in Caco-2 cells revealed that the high MW SI species (265 kDa) induced by maltose (an α-amylase starch digestion product) had a higher rate of early trafficking and maturation compared with a glucose-induced SI (245 kDa). The maltose-induced SI was found to have higher affinity to lipid rafts, which are associated with enhanced targeting to the apical membrane and higher activity. Accordingly, in situ maltose-hydrolyzing action was enhanced in the maltose-treated cells. Thus, starch digestion products at the luminal surface of small intestinal enterocytes are sensed and accelerate the intracellular processing of SI to enhance starch digestion capacity in the intestinal lumen.-Chegeni, M., Amiri, M., Nichols, B. L., Naim, H. Y., Hamaker, B. R. Dietary starch breakdown product sensing mobilizes and apically activates α-glucosidases in small intestinal enterocytes.

The Functions and Therapeutic Potential of Heat Shock Proteins in Inflammatory Bowel Disease-An Update.

Inflammatory bowel disease (IBD) is a multifactorial human intestinal disease that arises from numerous, yet incompletely defined, factors. Two main forms, Crohn's disease (CD) and ulcerative colitis (UC), lead to a chronic pathological form. Heat shock proteins (HSPs) are stress-responsive molecules involved in various pathophysiological processes. Several lines of evidence link the expression of HSPs to the development and prognosis of IBD. HSP90, HSP70 and HSP60 have been reported to contribute to IBD in different aspects. Moreover, induction and/or targeted inhibition of specific HSPs have been suggested to ameliorate the disease consequences. In the present review, we shed the light on the role of HSPs in IBD and their targeting to prevent further disease progression.

Functional variants in the sucrase-isomaltase gene associate with increased risk of irritable bowel syndrome.

OBJECTIVE: IBS is a common gut disorder of uncertain pathogenesis. Among other factors, genetics and certain foods are proposed to contribute. Congenital sucrase-isomaltase deficiency (CSID) is a rare genetic form of disaccharide malabsorption characterised by diarrhoea, abdominal pain and bloating, which are features common to IBS. We tested sucrase-isomaltase (SI) gene variants for their potential relevance in IBS. DESIGN: We sequenced SI exons in seven familial cases, and screened four CSID mutations (p.Val557Gly, p.Gly1073Asp, p.Arg1124Ter and p.Phe1745Cys) and a common SI coding polymorphism (p.Val15Phe) in a multicentre cohort of 1887 cases and controls. We studied the effect of the 15Val to 15Phe substitution on SI function in vitro. We analysed p.Val15Phe genotype in relation to IBS status, stool frequency and faecal microbiota composition in 250 individuals from the general population. RESULTS: CSID mutations were more common in patients than asymptomatic controls (p=0.074; OR=1.84) and Exome Aggregation Consortium reference sequenced individuals (p=0.020; OR=1.57). 15Phe was detected in 6/7 sequenced familial cases, and increased IBS risk in case-control and population-based cohorts, with best evidence for diarrhoea phenotypes (combined p=0.00012; OR=1.36). In the population-based sample, 15Phe allele dosage correlated with stool frequency (p=0.026) and Parabacteroides faecal microbiota abundance (p=0.0024). The SI protein with 15Phe exhibited 35% reduced enzymatic activity in vitro compared with 15Val (p<0.05). CONCLUSIONS: SI gene variants coding for disaccharidases with defective or reduced enzymatic activity predispose to IBS. This may help the identification of individuals at risk, and contribute to personalising treatment options in a subset of patients.

Structure-function analysis of human sucrase-isomaltase identifies key residues required for catalytic activity.

Sucrase-isomaltase (SI) is an intestinal membrane-associated α-glucosidase that breaks down di- and oligosaccharides to absorbable monosaccharides. SI has two homologous functional subunits (sucrase and isomaltase) that both belong to the glycoside hydrolase family 31 (GH31) and differ in substrate specificity. All GH31 enzymes share a consensus sequence harboring an aspartic acid residue as a catalytic nucleophile. Moreover, crystallographic structural analysis of isomaltase predicts that another aspartic acid residue functions as a proton donor in hydrolysis. Here, we mutagenized the predicted proton donor residues and the nucleophilic catalyst residues in each SI subunit. We expressed these SI variants in COS-1 cells and analyzed their structural, transport, and functional characteristics. All of the mutants revealed expression levels and maturation rates comparable with those of the wild-type species and the corresponding nonmutated subunits were functionally active. Thereby we determined rate and substrate specificity for each single subunit without influence from the other subunit. This approach provides a model for functional analysis of the single subunits within a multidomain protein, achieved without the necessity to express the individual subunits separately. Of note, we also found that glucose product inhibition regulates the activities of both SI subunits. We experimentally confirmed the catalytic function of the predicted proton donor residues, and sequence analysis suggested that these residues are located in a consensus region in many GH31 family members. In summary, these findings reveal the kinetic features specific for each human SI subunit and demonstrate that the activities of these subunits are regulated via product inhibition.

Posttranslational Processing and Function of Mucosal Maltases.

The final step of carbohydrate digestion in the intestine is performed by 2 major α-glucosidases of the intestinal mucosa, sucrase-isomaltase (SI) and maltase-glucoamylase. Both of these enzymes are type II membrane glycoproteins, which share a significant level of homology in gene and protein structures and yet have differences in the posttranslational processing, substrate specificity and functional capacity. Insufficient activity of these disaccharidases particularly SI as a result of genetic mutations or secondary intestinal pathologies is associated with carbohydrate maldigestion and gastrointestinal intolerances. This review will discuss the maturation profiles of SI and maltase-glucoamylase relative to their functional capacities and deficiencies.

Starch Tolerance and the Short Bowel.

Short bowel syndrome with intestinal failure is a rare disease with a massive impairment in quality of life, requiring a multidisciplinary team approach to medical, surgical, and nutritional therapy. Current pharmacological and surgical therapeutic options are limited; an important cornerstone is enteral and parenteral nutrition. The changed physiology of carbohydrate digestion plays a major role in the adaptation process and can be a target for specific enteral nutrition interventions. An important prognostic factor is the preservation of at least portions of the colon in continuity with small bowel. This strategy has to include an evaluation of the anatomical situation and small bowel absorptive capacity, adaptation processes, and luminal microbiota including its fermentative properties. Starch is probably the most important complex carbohydrate in short bowel syndrome nutrition, because it is absorbed or fermented almost completely. Benefits of supplementation with complex carbohydrates include improved adaptive processes, positive trophic effects on the mucosa and its hormonal response, longer transit time, and possibly a faster time to wean from parenteral nutrition, but supplementation advice needs to weigh carefully the risks and benefits, especially considering bacterial overgrowth, osmotic load, and D-lactate acidosis.

The HSP90 Family: Structure, Regulation, Function, and Implications in Health and Disease.

The mammalian HSP90 family of proteins is a cluster of highly conserved molecules that are involved in myriad cellular processes. Their distribution in various cellular compartments underlines their essential roles in cellular homeostasis. HSP90 and its co-chaperones orchestrate crucial physiological processes such as cell survival, cell cycle control, hormone signaling, and apoptosis. Conversely, HSP90, and its secreted forms, contribute to the development and progress of serious pathologies, including cancer and neurodegenerative diseases. Therefore, targeting HSP90 is an attractive strategy for the treatment of neoplasms and other diseases. This manuscript will review the general structure, regulation and function of HSP90 family and their potential role in pathophysiology.

Differential Glycosylation and Modulation of Camel and Human HSP Isoforms in Response to Thermal and Hypoxic Stresses.

Increased expression of heat shock proteins (HSPs) following heat stress or other stress conditions is a common physiological response in almost all living organisms. Modification of cytosolic proteins including HSPs by O-GlcNAc has been shown to enhance their capabilities for counteracting lethal levels of cellular stress. Since HSPs are key players in stress resistance and protein homeostasis, we aimed to analyze their forms at the cellular and molecular level using camel and human HSPs as models for efficient and moderate thermotolerant mammals, respectively. In this study, we cloned the cDNA encoding two inducible HSP members, HSPA6 and CRYAB from both camel (Camelus dromedarius) and human in a Myc-tagged mammalian expression vector. Expression of these chaperones in COS-1 cells revealed protein bands of approximately 25-kDa for both camel and human CRYAB and 70-kDa for camel HSPA6 and its human homologue. While localization and trafficking of the camel and human HSPs revealed similar cytosolic localization, we could demonstrate altered glycan structure between camel and human HSPA6. Interestingly, the glycoform of camel HSPA6 was rapidly formed and stabilized under normal and stress culture conditions whereas human HSPA6 reacted differently under similar thermal and hypoxic stress conditions. Our data suggest that efficient glycosylation of camel HSPA6 is among the mechanisms that provide camelids with a superior capability for alleviating stressful environmental circumstances.

Impact of Virtual Patients as Optional Learning Material in Veterinary Biochemistry Education.

Biochemistry and physiology teachers from veterinary faculties in Hannover, Budapest, and Lublin prepared innovative, computer-based, integrative clinical case scenarios as optional learning materials for teaching and learning in basic sciences. These learning materials were designed to enhance attention and increase interest and intrinsic motivation for learning, thus strengthening autonomous, active, and self-directed learning. We investigated learning progress and success by administering a pre-test before exposure to the virtual patients (vetVIP) cases, offered vetVIP cases alongside regular biochemistry courses, and then administered a complementary post-test. We analyzed improvement in cohort performance and level of confidence in rating questions. Results of the performance in biochemistry examinations in 2014, 2015, and 2016 were correlated with the use of and performance in vetVIP cases throughout biochemistry courses in Hannover. Surveys of students reflected that interactive cases helped them understand the relevance of basic sciences in veterinary education. Differences between identical pre- and post-tests revealed knowledge improvement (correct answers: +28% in Hannover, +9% in Lublin) and enhanced confidence in decision making ("I don't know" answers: -20% in Hannover, -7.5% in Lublin). High case usage and voluntary participation (use of vetVIP cases in Hannover and Lublin >70%, Budapest <1%; response rates in pre-test 72% and post-test 48%) indicated a good increase in motivation for the subject of biochemistry. Despite increased motivation, there was only a weak correlation between performance in final exams and performance in the vetVIP cases. Case-based e-learning could be extended and generated cases should be shared across veterinary faculties.