Reduced surface pH and upregulated AE2 anion exchange in SLC26A3-deleted polarized intestinal epithelial cells.

Loss of function mutations in the SLC26A3 gene cause chloride-losing diarrhea in mice and humans. Although systemic adaptive changes have been documented in these patients and in the corresponding knockout mice, how colonic enterocytes adapt to loss of this highly expressed and highly regulated luminal membrane anion exchanger remains unclear. To address this question, SLC26A3 was deleted in the self-differentiating Caco2BBe colonic cell line by the CRISPR/Cas9 technique. We selected a clone with loss of SLC26A3 protein expression and morphological features indistinguishable from those of the native cell line. Neither growth curves nor development of transepithelial electrical resistance (TEER) differed between wild-type (WT) and SLC26A3 knockout (KO) cells. Real-time qPCR and Western analysis in SLC26A3-KO cells revealed an increase in AE2 expression without significant change in NHE3 expression or localization. Steady-state pHi and apical and basolateral Cl-/HCO3- exchange activities were assessed fluorometrically in a dual perfusion chamber with independent perfusion of luminal and serosal baths. Apical Cl-/HCO3- exchange rates were strongly reduced in SLC26A3-KO cells, accompanied by a surface pH more acidic than that of WT cells. Steady-state pHi was not significantly different from that of WT cells, but basolateral Cl-/HCO3- exchange rates were higher in SLC26A3-KO than in WT cells. The data show that CRISPR/Cas9-mediated SLC26A3 deletion strongly reduced apical Cl-/HCO3- exchange rate and apical surface pH, but sustained a normal steady-state pHi due to increased expression and function of basolateral AE2. The low apical surface pH resulted in functional inhibition of NHE-mediated fluid absorption despite normal expression of NHE3 polypeptide.NEW & NOTEWORTHY SLC26A3 gene mutations cause chloride-losing diarrhea. To understand how colonic enterocytes adapt, SLC26A3 was deleted in Caco2BBe cells using CRISPR/Cas9. In comparison to the wild-type cells, SLC26A3 knockout cells showed similar growth and transepithelial resistance but substantially reduced apical Cl-/HCO3- exchange rates, and an acidic surface pH. Steady-state intracellular pH was comparable between the WT and KO cells due to increased basolateral AE2 expression and function.

The electroneutral Na+-HCO3- cotransporter NBCn1 (SLC4A7) modulates colonic enterocyte pHi, proliferation, and migration.

NBCn1 (SLC4A7) is one of the two major Na+-HCO3- cotransporters in the human colonic epithelium, expressed predominantly in the highly proliferating colonocytes at the cryptal base. Increased NBCn1 expression levels are reported in tumors, including colorectal cancer. The study explores its importance for maintenance of the intracellular pH (pHi), as well as the proliferative, adhesive, and migratory behavior of the self-differentiating Caco2BBe colonic tumor cell line. In the self-differentiating Caco2BBe cells, NBCn1 mRNA was highly expressed from the proliferative stage until full differentiation. The downregulation of NBCn1 expression by RNA interference affected proliferation and differentiation and decreased intracellular pH (pHi) of the cells in correlation with the degree of knockdown. In addition, a disturbed cell adhesion and reduced migratory speed were associated with NBCn1 knockdown. Murine colonic Nbcn1-/- enteroids also displayed reduced proliferative activity. In the migrating Caco2BBe cells, NBCn1 was found at the leading edge and in colocalization with the focal adhesion markers vinculin and paxillin, which suggests that NBCn1 is involved in the establishment of cell-matrix adhesion. Our data highlight the physiological significance of NBCn1 in modulating epithelial pH homeostasis and cell-matrix interactions in the proliferative region of the colonic epithelium and unravel the molecular mechanism behind pathological overexpression of this transporter in human colorectal cancers.NEW & NOTEWORTHY The transporter NBCn1 plays a central role in maintaining homeostasis within Caco2BBe colonic epithelial cells through its regulation of intracellular pH, matrix adhesion, migration, and proliferation. These observations yield valuable insights into the molecular mechanism of the aberrant upregulation of this transporter in human colorectal cancers.

ECL sensor for selective determination of citrate ions as a prostate cancer biomarker using polymer of intrinsic microporosity-1 nanoparticles/nitrogen-doped carbon quantum dots.

Urine citrate analysis is relevant in the screening and monitoring of patients with prostate cancer and calcium nephrolithiasis. A sensitive, fast, easy, and low-maintenance electrochemiluminescence (ECL) method with conductivity detection for the analysis of citrate in urine is developed and validated by employing polymer of intrinsic microporosity-1 nanoparticles/nitrogen-doped carbon quantum dots (nano-PIM-1/N-CQDs). Using optimum conditions, the sensor was applied in ECL experiments in the presence of different concentrations of citrate ions. The ECL signals were quenched gradually by the increasing citrate concentration. The linear range of the relationship between the logarithm of the citrate concentration and ΔECL (ECL of blank - ECL of sample) was obtained between 1.0 × 10-7 M and 5.0 × 10-4 M. The limit of detection (LOD) was calculated to be 2.2 × 10-8 M (S/N = 3). The sensor was successfully applied in real samples such as human serum and patient urine.

Human Colonoid-Myofibroblast Coculture for Study of Apical Na+/H+ Exchangers of the Lower Cryptal Neck Region.

Cation and anion transport in the colonocyte apical membrane is highly spatially organized along the cryptal axis. Because of lack of experimental accessibility, information about the functionality of ion transporters in the colonocyte apical membrane in the lower part of the crypt is scarce. The aim of this study was to establish an in vitro model of the colonic lower crypt compartment, which expresses the transit amplifying/progenitor (TA/PE) cells, with accessibility of the apical membrane for functional study of lower crypt-expressed Na+/H+ exchangers (NHEs). Colonic crypts and myofibroblasts were isolated from human transverse colonic biopsies, expanded as three-dimensional (3D) colonoids and myofibroblast monolayers, and characterized. Filter-grown colonic myofibroblast-colonic epithelial cell (CM-CE) cocultures (myofibroblasts on the bottom of the transwell and colonocytes on the filter) were established. The expression pattern for ion transport/junctional/stem cell markers of the CM-CE monolayers was compared with that of nondifferentiated (EM) and differentiated (DM) colonoid monolayers. Fluorometric pHi measurements were performed to characterize apical NHEs. CM-CE cocultures displayed a rapid increase in transepithelial electrical resistance (TEER), paralleled by downregulation of claudin-2. They maintained proliferative activity and an expression pattern resembling TA/PE cells. The CM-CE monolayers displayed high apical Na+/H+ exchange activity, mediated to >80% by NHE2. Human colonoid-myofibroblast cocultures allow the study of ion transporters that are expressed in the apical membrane of the nondifferentiated colonocytes of the cryptal neck region. The NHE2 isoform is the predominant apical Na+/H+ exchanger in this epithelial compartment.

The Anion Channel TMEM16a/Ano1 Modulates CFTR Activity, but Does Not Function as an Apical Anion Channel in Colonic Epithelium from Cystic Fibrosis Patients and Healthy Individuals.

Studies in human colonic cell lines and murine intestine suggest the presence of a Ca2+-activated anion channel, presumably TMEM16a. Is there a potential for fluid secretion in patients with severe cystic fibrosis transmembrane conductance regulator (CFTR) mutations by activating this alternative pathway? Two-dimensional nondifferentiated colonoid-myofibroblast cocultures resembling transit amplifying/progenitor (TA/PE) cells, as well as differentiated monolayer (DM) cultures resembling near-surface cells, were established from both healthy controls (HLs) and patients with severe functional defects in the CFTR gene (PwCF). F508del mutant and CFTR knockout (null) mice ileal and colonic mucosa was also studied. HL TA/PE monolayers displayed a robust short-circuit current response (ΔIeq) to UTP (100 µM), forskolin (Fsk, 10 µM) and carbachol (CCH, 100 µM), while ΔIeq was much smaller in differentiated monolayers. The selective TMEM16a inhibitor Ani9 (up to 30 µM) did not alter the response to luminal UTP, significantly decreased Fsk-induced ΔIeq, and significantly increased CCH-induced ΔIeq in HL TA/PE colonoid monolayers. The PwCF TA/PE and the PwCF differentiated monolayers displayed negligible agonist-induced ΔIeq, without a significant effect of Ani9. When TMEM16a was localized in intracellular structures, a staining in the apical membrane was not detected. TMEM16a is highly expressed in human colonoid monolayers resembling transit amplifying cells of the colonic cryptal neck zone, from both HL and PwCF. While it may play a role in modulating agonist-induced CFTR-mediated anion currents, it is not localized in the apical membrane, and it has no function as an apical anion channel in cystic fibrosis (CF) and healthy human colonic epithelium.

Characteristics, risk factors, and outcomes associated with readmission in COVID-19 patients: A systematic review and meta-analysis.

BACKGROUND: We aimed to determine the characteristics, risk factors, and outcomes associated with readmission in COVID-19 patients. METHODS: PubMed, Embase, Web of Science, and Scopus databases were searched to retrieve articles on readmitted COVID-19 patients, available up to September 25, 2021. All studies comparing characteristics of readmitted and non-readmitted COVID-19 patients were included. We also included articles reporting the reasons for readmission in COVID-19 patients. Data were pooled and meta-analyzed using random or fixed-effect models, as appropriate. Subgroup analyses were conducted based on the place and duration of readmission. RESULTS: Our meta-analysis included 4823 readmitted and 63,413 non-readmitted COVID-19 patients. The re-hospitalization rate was calculated at 9.3% with 95% Confidence Interval (CI) [5.5%-15.4%], mostly associated with respiratory or cardiac complications (48% and 14%, respectively). Comorbidities including cerebrovascular disease (Odds Ratio (OR) = 1.812; 95% CI [1.547-2.121]), cardiovascular (2.173 [1.545-3.057]), hypertension (1.608 [1.319-1.960]), ischemic heart disease (1.998 [1.495-2.670]), heart failure (2.556 [1.980-3.300]), diabetes (1.588 [1.443-1.747]), cancer (1.817 [1.526-2.162]), kidney disease (2.083 [1.498-2.897]), chronic pulmonary disease (1.601 [1.438-1.783]), as well as older age (1.525 [1.175-1.978]), male sex (1.155 [1.041-1.282]), and white race (1.263 [1.044-1.528]) were significantly associated with higher readmission rates (P < 0.05 for all instances). The mortality rate was significantly lower in readmitted patients (OR = 0.530 [0.329-0.855], P = 0.009). CONCLUSIONS: Male sex, white race, comorbidities, and older age were associated with a higher risk of readmission among previously admitted COVID-19 patients. These factors can help clinicians and policy-makers predict, and conceivably reduce the risk of readmission in COVID-19 patients.

Metabolomics approach to study in vivo toxicity of graphene oxide nanosheets.

Although graphene oxide (GO) nanosheets are widely used in different fields, the mechanism of their toxicity remains relatively unknown. NMR-based metabolomics was used to study in vivo time and dose-dependent toxicity of GO nanosheets in mice. Sixty serum samples from mice in four different time intervals including 24 and 72 h and 7 and 21 days after injection of 0-, 1-, and 10-mg/kg b.w. were analyzed based on 1 HNMR spectra of each sample and multivariate methods. In comparison with the control group, 12 changed metabolites were identified in GO nanosheet-treated mice groups. These metabolites are involved in steroid hormone biosynthesis and steroid biosynthesis pathways. It was seen that the time factor is more important than the dose factor and the groups were separated in a time direction, completely. We found that GO nanosheets has toxicity and can affect steroidal hormones. However, this study shows that after 21 days, the treated groups regardless of their GO nanosheet dose are very close to the control group. This means that in one step exposure to GO nanosheets, their toxicity diminished after 21 days.

Functional characterization of the sodium/hydrogen exchanger 8 and its role in proliferation of colonic epithelial cells.

Intestinal NaCl, HCO3-, and fluid absorption are strongly dependent on apical Na+/H+ exchange. The intestine expresses three presumably apical sodium-hydrogen exchanger (NHE) isoforms: NHE2, NHE3, and NHE8. We addressed the role of NHE8 [solute carrier 9A8 (SLC9A8)] and its interplay with NHE2 (SLC9A2) in luminal proton extrusion during acute and chronic enterocyte acidosis and studied the differential effects of NHE8 and NHE2 on enterocyte proliferation. In contrast to NHE3, which was upregulated in differentiated versus undifferentiated colonoids, the expression of NHE2 and NHE8 remained constant during differentiation of colonoids and Caco2Bbe cells. Heterogeneously expressed Flag-tagged rat (r)Nhe8 and human (h)NHE8 translocated to the apical membrane of Caco2Bbe cells. rNhe8 and hNHE8, when expressed in NHE-deficient PS120 fibroblasts showed higher sensitivity to HOE642 compared to NHE2. Lentiviral shRNA knockdown of endogenous NHE2 in Caco2Bbe cells (C2Bbe/shNHE2) resulted in a decreased steady-state intracellular pH (pHi), an increased NHE8 mRNA expression, and augmented NHE8-mediated apical NHE activity. Lentiviral shRNA knockdown of endogenous NHE8 in Caco2Bbe cells (C2Bbe/shNHE8) resulted in a decreased steady-state pHi as well, accompanied by decreased NHE2 mRNA expression and activity, which together contributed to reduced apical NHE activity in the NHE8-knockdown cells. Chronic acidosis increased NHE8 but not NHE2 mRNA expression. Alterations in NHE2 and NHE8 expression/activity affected proliferation, with C2Bbe/shNHE2 cells having lower and C2Bbe/shNHE8 having higher proliferative capacity, accompanied by amplified ERK1/2 signaling pathway and increased EGFR expression in the latter cell line. Thus, both Na+/H+ exchangers have distinct functions during cellular homeostasis by triggering different signaling pathways to regulate cellular proliferation and pHi control.

Expression, Localization and Functional Activity of the Major Na⁺/H⁺ Exchange Isoforms Expressed in the Intestinal Cell Line Caco-2BBe.

BACKGROUND/AIMS: Enterocytes express a number of NHE isoforms with presumed localization in the apical (NHE2, 3 and 8) or basolateral (NHE1) membrane. Functional activity and localization of enterocyte NHE isoforms were assessed using fully differentiated Caco-2BBe cells, whose genetic expression profile closely resembles mature enterocytes. METHODS: The activity of the different NHEs was analyzed by fluorometric pHi-metry in a perfusion chamber with separate apical and basolateral perfusion, using specific inhibitors and shRNA knockdown of NHE2. The expression of the NHEs and of other relevant acid extrusion transporters was quantified by qPCR. RESULTS: Quantitative comparison of the mRNA expression levels of the different NHE isoforms in 14 day-differentiated Caco-2BBe cells showed the following order: NHE2>NHE8>NHE3>NHE1. Acid-activated NHE exchange rates in the basolateral membrane were >6-fold higher than in the apical membrane. 79 ± 3 % of the acid-activated basolateral Na⁺/H⁺ exchange rate displayed a NHE1-typical inhibitor profile, and no NHE2/3/8 typical activity could be observed. Analysis of the apical Na⁺/H⁺ exchange rates revealed that approximately 51 ± 3 % of the total apical activity displayed a NHE2/8-typical inhibitor profile and 31 ± 6 % a NHE3-typical inhibitor profile. Because no selective NHE2 inhibitor is available, a stable NHE2 knockdown cell line (C2NHE2KD) was generated. C2NHE2KD displayed a reduced NHE2-typical apical Na⁺/H⁺ exchange rate and maintained a lower steady-state pHi, despite high expression levels of other acid extruders, in particular NBCn1 (Slc4a7). CONCLUSION: Differentiated Caco-2BBe cells display particularly high mRNA expression levels of NHE2, which can be functionally identified in the apical membrane. Although at low intracellular pH, NHE2 transport rate was far lower than that of NHE1. NHE2 activity was nevertheless essential for the maintenance of the steady-state pHi of these cells.

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.

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.

Apigenin inhibits growth of the Plasmodium berghei and disrupts some metabolic pathways in mice.

Due to the challenges in the control, prevention, and eradication of parasitic diseases like malaria, there is an urgent need to discover new therapeutic agents. Plant-derived medicines may open new ways in the field of antiplasmodial therapy. This study is aimed to investigate the toxicity and in vivo antiplasmodial activity of apigenin, a dietary flavonoid. Apigenin cytotoxicity was investigated on Huh7 cell line, brine shrimp (Artemia salina) larva, and human red blood cells. In vivo toxicity of apigenin was assessed by metabolomics approaches. Apigenin exhibited significant suppression of parasitemia in a dose-dependent manner; it suppressed Plasmodium berghei growth by 69.74%, 50.3%, and 49.23% at concentrations of 70, 35, and 15 mg/kg/day, respectively. The IC50 value for apigenin after 24 hr exposure to Huh7 cells was 225 µg/ml. Apigenin did not show noticeable toxicity on A. salina and also on the membrane integrity of red blood cells. After 24 hr exposure of mice to apigenin, alterations were seen in the metabolism of glucocorticoids and mineralocorticoids, bile acid metabolism (alternative pathway), sulfur metabolism, bile acid metabolism, metabolism of estrogens and androgens, cholesterol catabolism, and biosynthesis of cholesterol. These findings indicate that apigenin has potential in vivo antiplasmodial activity against P. berghei infected mice with high selectivity against malaria, but it can disrupt some metabolic pathways in mice.

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.

Molecular pathogenicity of novel sucrase-isomaltase mutations found in congenital sucrase-isomaltase deficiency patients.

BACKGROUND & AIMS: Congenital sucrase-isomaltase deficiency (CSID) is a genetic disorder associated with mutations in the sucrase-isomaltase (SI) gene. The diagnosis of congenital diarrheal disorders like CSID is difficult due to unspecific symptoms and usually requires invasive biopsy sampling of the intestine. Sequencing of the SI gene and molecular analysis of the resulting potentially pathogenic SI protein variants may facilitate a diagnosis in the future. This study aimed to categorize SI mutations based on their functional consequences. METHODS: cDNAs encoding 13 SI mutants were expressed in COS-1 cells. The molecular pathogenicity of the resulting SI mutants was defined by analyzing their biosynthesis, cellular localization, structure and enzymatic functions. RESULTS: Three biosynthetic phenotypes for the novel SI mutations were identified. The first biosynthetic phenotype was defined by mutants that are intracellularly transported in a fashion similar to wild type SI and with normal, but varying, levels of enzymatic activity. The second biosynthetic phenotype was defined by mutants with delayed maturation and trafficking kinetics and reduced activity. The third group of mutants is entirely transport incompetent and functionally inactive. CONCLUSIONS: The current study unraveled CSID as a multifaceted malabsorption disorder that comprises three major classes of functional and trafficking mutants of SI and established a gradient of mild to severe functional deficits in the enzymatic functions of the enzyme. GENERAL SIGNIFICANCE: This novel concept and the existence of mild consequences in a number of SI mutants strongly propose that CSID is an underdiagnosed and a more common intestinal disease than currently known.

Structural determinants for transport of lactase phlorizin-hydrolase in the early secretory pathway as a multi-domain membrane glycoprotein.

BACKGROUND: Lactase phlorizin-hydrolase (LPH) is a membrane anchored type I glycoprotein of the intestinal epithelium that is composed of four homologous structural domains. The role of each distinct domain in the intramolecular organization and function of LPH is not completely understood. METHODS: Here, we analyzed the early events of LPH biosynthesis and trafficking by directed restructuring of the domain compositions. RESULTS: Removal of domain I (LPH∆1) results in a malfolded ER-localized protein. By contrast, LPH without domain II (LPH∆2) is normally transported along the secretory pathway, but does not dimerize nor is enzymatically active. Interestingly a polypeptide stretch in domain II between L735-R868 exerts an intriguing role in modulating the trafficking behavior of LPH and its biological function. In fact, association of this stretch with transport-competent LPH chimeras results in their ER-arrest or aberrant trafficking. This stretch harbors a unique N-glycosylation site that is responsible for LPH retention in the ER via association with calnexin and facilitates proper folding of domains I and III before ER exit of LPH. Notably, a similar N-glycosylation site is also found in domain IV with comparable effects on the trafficking of LPH-derived molecules. CONCLUSIONS: Our study provides novel insights into the intramolecular interactions and the sequence of events involved in the folding, dimerization and transport of LPH. GENERAL SIGNIFICANCE: Elucidation of the structural-functional relevance of the domains in pro-LPH is crucial in unravelling and understanding the molecular basis of carbohydrate malabsorption disorders that are associated with lactase deficiency or lactase malfunction.

Long term differential consequences of miglustat therapy on intestinal disaccharidases.

Miglustat is an oral medication for treatment of lysosomal storage diseases such as Gaucher disease type I and Niemann Pick disease type C. In many cases application of Miglustat is associated with symptoms similar to those observed in intestinal carbohydrate malabsorption. Previously, we have demonstrated that intestinal disaccharidases are inhibited immediately by Miglustat in the intestinal lumen. Nevertheless, the multiple functions of Miglustat hypothesize long term effects of Miglustat on intracellular mechanisms, including glycosylation, maturation and trafficking of the intestinal disaccharidases. Our data show that a major long term effect of Miglustat is its interference with N-glycosylation of the proteins in the ER leading to a delay in the trafficking of sucrase-isomaltase. Also association with lipid rafts and plausibly apical targeting of this protein is partly affected in the presence of Miglustat. More drastic is the effect of Miglustat on lactase-phlorizin hydrolase which is partially blocked intracellularly. The de novo synthesized SI and LPH in the presence of Miglustat show reduced functional efficiencies according to altered posttranslational processing of these proteins. However, at physiological concentrations of Miglustat (≤50 µM) a major part of the activity of these disaccharidases is found to be still preserved, which puts the charge of the observed carbohydrate maldigestion mostly on the direct inhibition of disaccharidases in the intestinal lumen by Miglustat as the immediate side effect.

Role of autoantibodies targeting interferon type 1 in COVID-19 severity: A systematic review and meta-analysis.

Introduction: Impairment of the type I interferon (IFN-I) signaling pathway is associated with increased severity of COVID-19 disease. Here we have undertaken a systematic review and meta = analysis on the association between the severity of COVID-19 and IFN-1 autoantibodies (AAbs; aIFN-1, aIFN-α, aIFN-ω, and aIFN-ß). Methods: Four databases, including Medline [PubMed], Embase, Web of Science, and Scopus, were systematically searched to find papers on the role of aIFN-1 and its subtype AAbs in the severity of COVID-19 infection. Data on the prevalence of aIFN-1, aIFN-α, aIFN-ω, and aIFN-ß were pooled using random- or fixed-effects models. Subgroup analysis was performed based on disease severity. Odds ratios (OR) for COVID-19 disease outcome, including length of hospital stay, ICU admission and death, were calculated in relation to positive or negative plasma IFN-1 AAbs. Results: A total of 33 studies with 13023 patients were included. The overall prevalence of circulating aIFN-1, aIFN-α, and aIFN-ω AAbs was 17.8 % [13.8, 22.8], 7.2 % [4.7, 10.9], and 4.4 % [2.1, 8.6], respectively, and the overall prevalence of neutralizing aIFN-1, aIFN-α, aIFN-ω, and aIFN-ß AAbs was 7.1 % [4.9, 10.1], 7.5 % [5.9, 9.5], 8.0 % [5.7, 11.1] and 1.2 % [0.4, 3.5], respectively. Circulating aIFN-α (OR = 4.537 [2.247, 9.158]), neutralizing aIFN-α (O = 17.482 [8.899, 34.342]), and neutralizing aIFN-ω (OR = 12.529 [7.397, 21.222]) were significantly more frequent in critical/severe patients than in moderate/mild patients (p < 0.001 for all). Anti-IFN-1 was more common in male subjects (OR = 2.248 [1.366, 3.699], p = 0.001) and two COVID-19 outcomes including ICU admission (OR = 2.485 [1.409, 4.385], p = 0.002) and death (OR = 2.593 [1.199, 5.604], p = 0.015) occurred more frequently in patients with positive anti-IFN-1.Conclusion: aIFN-1 and its subtypes AAbs are associated with severe and critical COVID-19 disease and may be a predictive marker for a poor prognosis, particularly in men.

Anti-Plasmodial Effects of Different Ecotypes of Glycyrrhiza glabra Traditionally Used for Malaria in Iran.

Glycyrrhiza glabra L., Fabaceae, or licorice has shown potential therapeutic effects on fever, gastric ulcers, hepatic disorders, and malaria. This study aimed to assess the antimalarial activity of different fractions of root extract from twelve ecotypes from Iran. In this regard, mice were then randomly divided into 8 groups of 5 mice. Four hours after mice were infected by Plasmodium berghei, they received methanolic plant extract by intraperitoneal injection. The treatment was continued for 4 consecutive days (every 24 h), then on the fifth and seventh days, blood samples were taken from the tails of the mice and the parasitic percentages were calculated by microscopy technique. In comparison to control, every analyzed ecotype has a remarkable parasite inhibitory effect, whereas the source of the root also has a drastic difference in its antimalarial effects. The highest percentage of inhibition on days 5 and 7 was subjected to the extract of Semirom ecotype with suppression of 86.37 and 83%, respectively. On the other hand, 13.21 and 9.19% parasite growth inhibition was shown in the extracts of Shahrbabak and Haji Abad, respectively. The significant difference between these 12 ecotypes was shown with Mann-Whitney U pairwise comparison to variable parasitemia day 5 and parasitemia day 7 (p < 0.001). Supplementary Information: The online version contains supplementary material available at 10.1007/s43450-022-00353-8.

Cadherin-related protein 24 induces morphological changes and partial cell polarization by facilitating direct cell-cell interactions.

Cadherin-related protein 24 (CDHR24) is a potential tumor suppressor located apically as well as laterally in polarized cells. Here, the role of CDHR24 in contributing to cell morphology and polarity is examined. CDHR24 was predominantly localized at the nonattached part of nonpolarizing cells while another apically sorted protein, aminopeptidase N, was equally distributed over the plasma membrane. Furthermore, CDHR24 expression induced cell aggregation capacity, indicating direct cell-cell interaction. The transepithelial resistance, however, was elevated in polarized MDCK cells, but not in nonpolarizing CHO cells. Our data propose a model in which CDHR24 is directly involved in cell and tissue morphogenesis.