Brush Border Research Articles

Myeloid Ptpn2 Deficiency in Mice Causes Functional Iron Deficiency

Background: Anemia is the most prevalent extraintestinal complication of inflammatory bowel disease (IBD) and is associated with the manifestation of Crohn’s disease. Anemia significantly reduces the quality of life, increases hospitalization visits, and increases medical care expenses of IBD patients. Iron deficiency is the leading cause of anemia in IBD; however, the physiological mechanisms involved are still poorly understood. Macrophages significantly contribute to iron homeostasis by mechanisms including iron recycling by phagocytosing senescent erythrocytes and damaged cells, and nurturing erythropoiesis in the bone marrow. In this study, we investigated the role of the IBD candidate gene, protein tyrosine phosphatase non-receptor type 2 ( PTPN2), in regulating macrophage iron homeostasis. Methods: Myeloid cell-specific knockout mice ( Ptpn2LysMCre) and control littermates ( Ptpn2fl/fl) were analyzed for serum iron content and liver and spleen non-heme iron. Duodenal epithelial cell (DEC) proteins were assayed by Western blotting. Localization of the brush border ferrous iron transporter, DMT1, and the coupled ferrous iron absorption driver, NHE3, in duodenal tissue was determined by immunohistochemistry (IHC). Gene expression profiles of macrophages were analyzed by Nanostring. Results: Ptpn2LysMCre mice had reduced serum iron ( p<0.0001; n=12) and reduced spleen non-heme iron (p=0.0017; n=6), whereas liver non-heme iron was unaltered (n=6), indicating iron deficiency in select tissues. DMT1 (n=8) and NHE3 (n=5) protein expression in DECs was unaltered and IHC confirmed the unaltered localization of DMT1 and NHE3 (n=6) in the duodenal epithelium of Ptpn2LysMCre mice, suggesting the reduction in serum iron was not due to an impairment in intestinal non-heme iron uptake. Nanostring analyses revealed that the gene expression of the iron transporter Dmt1, iron exporter Fpn, and iron importer Tfr1 were significantly downregulated (p<0.05; n=5) in macrophages from Ptpn2LysMCre mice, indicating an iron sequestering phenotype. Conclusions: Loss of myeloid Ptpn2 in mice causes functional iron deficiency. These findings indicate a major role of myeloid PTPN2 in regulating systemic iron homeostasis. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Acute kidney injury causes subclinical renal and endothelial injury in female rats which predisposes to adverse outcomes of pregnancy

Clinical data report a 3-5-fold increase in adverse pregnancy outcomes, including preeclampsia and small for gestational age neonates, among women with a history of acute kidney injury (AKI) despite clinical recovery of renal function. We developed a rat model of pregnancy post-AKI: Sprague-Dawley (SD) rats undergo a bilateral renal-ischemia reperfusion and fully recover renal function as measured by creatinine clearance 30 days post-AKI. Rats are then mated and during pregnancy recovered AKI rats exhibit decreased creatinine clearance, increased uterine artery resistance, reduced plasma volume expansion, and decreased fetal growth compared to pregnant rats that received sham surgery. Healthy pregnancy demands robust renal and vascular adaptations. Whether subclinical renal and vascular injury persists following AKI in females, and whether this is exacerbated in pregnancy to drive the adverse pregnancy outcomes remains unknown. We tested the hypothesis that AKI results in subclinical renal and vascular injury that lingers into pregnancy. Female SD rats were randomized to 45-minute warm bilateral renal ischemia reperfusion or sham (N=6-8/grp) and renal and vascular injury assessments were made 30 days post-AKI/Sham (pre-pregnancy) or during pregnancy on gestational day 20 (GD20). Pre-pregnancy urine volume measured via metabolic cage was greater in post-AKI rats compared to sham (10±1 vs 21±2 ml/daily urine, p<0.05). Urinary kidney injury marker-1 (KIM-1) measured via ELISA 30 days post-AKI/Sham was greater in pre-pregnant AKI rats compared to sham (9685±871 vs. 6399±198 pg/24 hrs urine, p<0.05). Vascular function, measured via wire myography on 3rd order mesenteric arteries 30 days post-AKI/Sham, revealed decreased endothelial-dependent relaxation to acetylcholine (ACh) (concentration-response, 1 nM-10 μM, 2-way ANOVA, RM, *p<0.05). GD20 urine volume (31±5 vs 19±2 ml/daily urine, p<0.05) and KIM-1 levels (9125±673 vs 6380±297 pg/daily urine, p<0.05) were elevated in post-AKI pregnant rats compared to sham pregnant rats. Mason Trichrome renal staining revealed greater glomerular fibrosis (0.93±0.08 vs 0.04±0.04, *p<0.05) and tubular fibrosis (1.7±0.19 vs 0.0±0.0, *p<0.05) in post-AKI pregnant rats vs. sham pregnant. Ultrastructural changes to the kidneys were evaluated via electron microscopy and further revealed extensive brush border loss, loss of mitochondrial structure and mitochondrial swelling in post-AKI pregnant rats compared to sham pregnant. Endothelial-dependent relaxation to ACh was also lower (2-way ANOVA, *p<0.05), suggesting enhanced endothelial dysfunction in post-AKI pregnant rats vs. sham pregnant. Taken together, these data suggest that with a history of AKI, subclinical renal and vascular injury is present despite “recovery” of renal function using clinical definitions and may predispose to adverse pregnancy outcomes in pregnant women with a history of AKI. 1R01HL169576 4R00HL146948 1RO1DK134695 AHA967662. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Regulation of renal acid-base balance by Epac isoforms in proximal tubule and collecting duct

Proper regulation of acid-base homeostasis has utmost significance for well-being and survival of the living organisms. Up to 50% of emergency room patients have notable disturbances in pH and associated morbific manifestations, which impedes effective treatment. Kidneys play a central role in regulation of acid-base balance by reabsorbing HCO3− via NHE-3 in the proximal tubule (PT) and secreting H+ via V-ATPase pump by the intercalated cells of collecting duct (CD). Exchange proteins directly activated by cAMP (Epac) are most prominently expressed in the proximal tubule and collecting ducts, the sites critical for acid-base transport in the renal tubule. However, the significance and physiological relevance of Epac isoforms (Epac1 and Epac2) for controlling HCO3− reabsorption and H+ secretion and by extension, the systemic pH regulation is not known. Here, we used transgenic mouse models lacking both or individual Epac1 and Epac2 isoforms to examine the systemic adaptation to acid-base insults and to inquire on the perturbed molecular signaling mechanisms and end-effectors in freshly isolated split-opened PTs and CDs using biochemical and fluorescent imaging tools. Deletion of Epac1&2 caused a dramatic decrease in NHE-3 protein levels in an additive manner. Moreover, we detected a marked redistribution of NHE-3 to the base of brush border in proximal tubule cells rendering its inactivation. Consistently, NHE-3 activity was more than 70% decreased upon Epac1&2 deletion, as was assessed by a rate of recovery after intracellular acidification in freshly isolated split-opened PTs. Furthermore, V-ATPase activity was similarly decreased in A-type of intercalated cells in freshly isolated CDs despite compensatory accumulation on the apical side, as monitored with confocal microscopy in cortical and medullary kidney sections of the knockout. In unstressed conditions, Epac1&2 deletion did not affect arterial pH and HCO3− levels, while urinary pH was significantly reduced. Dietary acid load (280 mM NH4Cl in drinking water for 3 days) unmasked severe hyperchloremic metabolic acidosis with pH 7.17±0.01 and HCO3− 14.5±0.6 mM and impaired urinary acidification. Deletion of Epac2 alone recapitulated the phenotype of double knockout, whereas Epac1−/− mice did not demonstrate notable deficiencies in response to dietary acid load. Overall, we concluded that Epac signaling plays a major role in urinary acidification with the dominant contribution of Epac2 isoform by regulating acid-base transport in both proximal tubule (via NHE-3) and the collecting duct (via V-ATPase). Epac1&2 and Epac2 deletion of impairs renal adaptation to the acid load resulting in severe metabolic acidosis. This research was supported by NIH-NIDDK DK117865, DK119170, AHA EIA35260097 (to O. Pochynyuk) and AHA-23POST1020372 (to K. Pyrshev). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Syntaxin5 is essential for survival by ensuring midgut epithelial homeostsis and regulating feeding in Locusta migratoria

Syntaxin5 (Syx5) belongs to SNAREs family, which play important roles in fusion of vesicles to target membranes. Most of what we know about functions of Syx5 originates from studies in fungal or vertebrate cells, how Syx5 operates during the development of insects is poorly understood. In this study, we investigated the role of LmSyx5 in the gut development of the hemimetabolous insect Locusta migratoria. LmSyx5 was expressed in many tissues, with higher levels in the gut. Knockdown of LmSyx5 by RNA interference (RNAi) considerably suppressed feeding in both nymphs and adults. The dsLmSyx5-injected locusts lost body weight and finally died at a mortality of 100%. Furthermore, hematoxylin-eosin staining indicated that the midgut is deformed in dsLmSyx5-treated nymphs and the brush border in midgut epithelial cells is severely damaged, suggesting that LmSyx5 is involved in morphogenesis of the midgut. TEM further showed that the endoplasmic reticulum of midgut cells have a bloated appearance. Taken together, these results suggest that LmSyx5 is essential for midgut epithelial homeostsis that affects growth and development of L. migratoria. Thus, Syx5 is a promising RNAi target for controlling L. migratoria, and even other pests.

Changes in growth, morphology, and levels of digestive enzymes and growth-related hormones in early ontogeny of black scraper, Thamnaconus modestus

IntroductionThe black scraper, Thamnaconus modestus, is a highly valued marine fish species, but its output has gradually decreased in recent years, which may be due to its low survival rate during early ontogenesis.MethodsTherefore, in this study, we assessed the changes in growth, morphology, digestive enzymes, and hormone levels in T. modestus from 0–60 days post-hatching (dph) and revealed growth turning points by morphological measurement and determination of digestive enzyme activities and hormone levels. We found that ontogenesis could be divided into the larval (0–20 dph) and juvenile (20–60 dph) stages. Acid and alkaline protease activity significantly increased and decreased, respectively, from 12–25 dph, likely due to the development of stomach and gastric glands. Acid phosphatase levels significantly increased at 0 and 4 dph, which may be related to the regulation of metabolism and immune protection. A sharp increase in alkaline phosphatase levels at 20 and 25 dph was observed and was likely due to the development of the brush border membrane of enterocytes. The amylase level was significantly higher at 25, 30, and 35 dph, possibly due to better digestion and absorption during the transition from consuming Artemia to compound feed. In newly hatched larvae, the level of thyroid hormones triiodothyronine (T3) and thyroxine (T4) gradually increased and peaked at 35 dph, highlighting the importance of these hormones during the development of T. modestus. Growth hormone (GH) levels first increased from 0–8 dph, with a plateau at 8–20 dph, and then increased at 25–30–35 dph. For insulin-like growth factor 1 (IGF-1), a significant increase with a subsequent plateau was observed between 8 and 20 dph, followed by a substantial decrease between 30 and 35 dph. These results suggest that the regulating functions of GH and IGF-1 are synchronised. Digestive enzyme activity and hormone levels of abnormal fry at 30 dph were lower than those of normal fish, highlighting the importance of specific hormones, especially T4 and IGF-1, in the development of T. modestus.

Pediatric early-phase anorexia nervosa without hypokalemia exhibiting acute tubular injury: a case report.

Anorexia nervosa can lead to kidney complications. Few studies reported kidney biopsy results in young adults, most of whom had chronic anorexia nervosa, and kidney biopsy findings in pediatric patients with early-phase anorexia nervosa are rarely reported. A 14-year-old girl who lost weight due to excessive exercise and reduced diet was admitted for kidney dysfunction. She was 147cm tall and weighed 32.9kg, with a body mass index of 15.2kg/m2. She was 39kg about a year earlier. Her heart rate and blood pressure were 30-40 beats/min and 108/68mmHg, respectively. She had kidney dysfunction (estimated glomerular filtration rate, 56.6mL/min/1.73 m2). Urine β2-microglobulin was slightly elevated (393μg/L), and percent tubular phosphate reabsorption was low (75.2%), suggesting tubular damage; however, hypokalemia was absent. Kidney dysfunction did not improve with fluid loading. Kidney biopsy revealed that all glomeruli were intact, with no vasculitis, interstitial inflammation or fibrosis on light microscopy. However, proximal tubular epithelial walls were flattened and the brush border was absent, suggesting acute tubular injury. Immunofluorescent staining was negative for immunoglobulins and complement proteins, and electron microscopy showed no significant electron-dense deposition. The patient's serum creatinine gradually declined, normalizing on the 17th day of admission. Unlike previous reports in young adults, kidney dysfunction was observed even in the absence of hypokalemia in the current pediatric patient with early-phase anorexia nervosa. Proximal tubular injury in early-phase anorexia nervosa may be caused by bradycardia without hypokalemia, leading to subsequent kidney dysfunction.

Integrated histopathological lesion (IHL) analysis - a histopathological tool for the evaluation of UV-B exposure on the gill, liver and intestine ofCatla catla.

The present study aimed to analyse the integrated histopathological lesions (IHLs) of the gill, liver and intestine of Catla catla exposed to the different doses of UV-B radiation. Gill exhibited the lesions like hypertrophy, hyperplasia, vacuolation, fusion of the gill filaments, rupture in the gill lamellae, epithelial cell lifting and necrosis. The UV-B-exposed liver of Catla showed the lesions like the degeneration of nucleus, the disarrangement of hepatocytes, sinusoidal vacuolation, epitheliod histiocyst, hepatocellular adenoma, exocrine adenoma, cyst formation and diffused epithelial necrosis (DEN). UV-B-exposed intestine showed the lesions like the distortion of columnar epithelial cells (CECs), distortion in lamina propria (LP), disruption in brush border (BB), vacuolation in LP, the presence of submucosal mass (SM), the degeneration of nucleus, the presence of tactoid bodies, the presence of aschoff nodules and metatypical cell carcinoma. These histopathological alterations can be considered as the main blocking alterations of the growth and absorption as well as the final production of fish which can cause a serious loss in total yield to fish farmers which can interrupt the profitable economical production of fish.

Infant milk formula, produced by membrane filtration, promotes mucus production in the upper small intestine of young pigs

Human breast milk promotes maturation of the infant gastrointestinal barrier, including the promotion of mucus production. In the quest to produce next generation infant milk formula (IMF), we have produced IMF by membrane filtration (MEM-IMF). With a higher quantity of native whey protein, MEM-IMF more closely mimics human breast milk than IMF produced using conventional heat treatment (HT-IMF). After a 4-week dietary intervention in young pigs, animals fed a MEM-IMF diet had a higher number of goblet cells, acidic mucus and mucin-2 in the jejunum compared to pigs fed HT-IMF (P < 0.05). In the duodenum, MEM-IMF fed pigs had increased trypsin activity in the gut lumen, increased mRNA transcript levels of claudin 1 in the mucosal scrapings and increased lactase activity in brush border membrane vesicles than those pigs fed HT-IMF (P < 0.05). In conclusion, MEM-IMF is superior to HT-IMF in the promotion of mucus production in the young gut.

Parental recovered acute kidney injury causes prenatal renal dysfunction and fetal growth restriction with sexually dimorphic implications for adult offspring.

Introduction: Acute kidney injury (AKI) is rapidly increasing in global incidence and a healthcare burden. Prior maternal AKI diagnosis correlates with later pregnancy complications. As pregnancy influences developmental programming, we hypothesized that recovered parental AKI results in poor pregnancy outcomes, impaired fetal growth, and adult offspring disease. Methods: Using a well-characterized model of rhabdomyolysis-induced acute kidney injury (RIAKI), a form of AKI commonly observed in young people, we confirmed functional renal recovery by assessing glomerular filtration rate (GFR) 2weeks following RIAKI. We bred sham and recovered RIAKI sires and dams in timed, matched matings for gestational day (GD) 16.5 and offspring (birth-12weeks, 6months) study. Results: Despite a normal GFR pre-pregnancy, recovered RIAKI dams at GD16.5 had impaired renal function, resulting in reduced fetoplacental ratios and offspring survival. Pregnant RIAKI dams also had albuminuria and less renal megalin in the proximal tubule brush border than shams, with renal subcapsular fibrosis and higher diastolic blood pressure. Growth-restricted offspring had a reduced GFR as older adults, with evidence of metabolic inefficiency in male offspring; this correlated with reduced renal AngII levels in female offspring from recovered RIAKI pairings. However, the blood pressures of 6-month-old offspring were unaffected by parental RIAKI. Conclusions: Our mouse model demonstrated a causal relationship among RIAKI, gestational risk, and developmental programming of the adult-onset offspring GFR and metabolic dysregulation despite parental recovery.

Evaluation of the efficacy of silymarin and dexmedetomidine on kidney and lung tissue in the treatment of sepsis in rats with cecal perforation.

Sepsis is a systemic inflammatory response syndrome that develops in the host against microorganisms. This response develops away from the primary infection site and results in end-organ damage. The present study aimed to investigate the protective and therapeutic effects on lung and kidney tissue of silymarin (S) and dexmedetomidine (DEX) applied 1 h before and after sepsis induced by the cecal ligation and puncture (CLP) method in rats. A total of 62 rats was randomly divided into eight groups: i) Control (n=6); ii) cecal perforation (CLP; n=8); iii) S + CLP (n=8; S + CLP; S administered 1 h before CPL); iv) CLP + S (n=8; S administered 1 h after CLP); v) DEX + CLP (n=8; D + CLP; DEX administered 1 h before CLP); vi) CLP + D (n=8; DEX administered 1 h after CLP); vii) SD + CLP (n=8; S and DEX administered 1 h before CLP) and viii) CLP + SD (n=8; S and DEX administered 1 h after CLP). After the cecum filled with stool, it was tied with 3/0 silk under the ileocecal valve and the anterior surface of the cecum was punctured twice with an 18-gauge needle. A total of 100 mg/kg silymarin and 100 µg/kg DEX were administered intraperitoneally to the treatment groups. Lung and kidney tissue samples were collected to evaluate biochemical and histopathological parameters. In the histopathological examination, all parameters indicating kidney injury; interstitial edema, peritubular capillary dilatation, vacuolization, ablation of tubular epithelium from the basement membrane, loss of brush border in the proximal tubule epithelium, cell swelling and nuclear defragmentation; were increased in the CLP compared with the control group. Silymarin administration increased kidney damage, including ablation of tubular epithelium from the basement membrane, compared with that in the CLP group. DEX significantly reduced kidney damage compared with the CLP and silymarin groups. The co-administration of DEX + silymarin decreased kidney damage, although it was not as effective as DEX-alone. To conclude, intraperitoneal DEX ameliorated injury in CLP rats. DEX + silymarin partially ameliorated injury but silymarin administration increased damage. As a result, silymarin has a negative effects with this dosage and DEX has a protective effect. In the present study, it was determined that using the two drugs together had a greater therapeutic effect than silymarin and no differences in the effects were not observed any when the application times of the agents were changed.

N-terminal proteolysis determines the differential activity of Bacillus thuringiensis Cry2A toxins towards Aedes aegypti

It has long been known that while both the Bacillus thuringiensis pesticidal proteins Cry2Aa and Cry2Ab have wide-ranging activities against lepidopteran insects only the former has activity against the mosquito Aedes aegypti. We have previously shown that this differential specificity is influenced by the N-terminal region of these proteins and here demonstrate that this is due to these sections affecting proteolytic activation. Enzymes from the midgut of A. aegypti cleave Cry2Aa at the C-terminal side of amino acid 49 resulting in a 58 kDa fragment whereas these enzymes do not cleave Cry2Ab at this position. The 58 kDa, but not the protoxin, form of Cry2Aa is capable of interacting with brush border membrane vesicles from A. aegypti.

The calcium-sensing receptor has only a parathyroid hormone-dependent role in the acute response of renal phosphate transporters to phosphate intake.

The kidney controls systemic inorganic phosphate (Pi) levels by adapting reabsorption to Pi intake. Renal Pi reabsorption is mostly mediated by sodium-phosphate cotransporters NaPi-IIa (SLC34A1) and NaPi-IIc (SLC34A3) that are tightly controlled by various hormones including parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23). PTH and FGF23 rise in response to Pi intake and decrease NaPi-IIa and NaPi-IIc brush border membrane abundance enhancing phosphaturia. Phosphaturia and transporter regulation occurs even in the absence of PTH and FGF23 signaling. The calcium-sensing receptor (CaSR) regulates PTH and FGF23 secretion, and may also directly affect renal Pi handling. Here, we combined pharmacological and genetic approaches to examine the role of the CaSR in the acute phosphaturic response to Pi loading. Animals pretreated with the calcimimetic cinacalcet were hyperphosphatemic, had blunted PTH levels upon Pi administration, a reduced Pi-induced phosphaturia, and no Pi-induced NaPi-IIa downregulation. The calcilytic NPS-2143 exaggerated the PTH response to Pi loading but did not abolish Pi-induced downregulation of NaPi-IIa. In mice with a dominant inactivating mutation in the Casr (CasrBCH002), baseline NaPi-IIa expression was higher, whereas downregulation of transporter expression was blunted in double CasrBCH002/PTH knockout (KO) transgenic animals. Thus, in response to an acute Pi load, acute modulation of the CaSR affects the endocrine and renal response, whereas chronic genetic inactivation, displays only subtle differences in the downregulation of NaPi-IIa and NaPi-IIc renal expression. We did not find evidence that the CaSR impacts on the acute renal response to oral Pi loading beyond its role in regulating PTH secretion.NEW & NOTEWORTHY Consumption of phosphate-rich diets causes an adaptive response of the body leading to the urinary excretion of phosphate. The underlying mechanisms are still poorly understood. Here, we examined the role of the calcium-sensing receptor (CaSR) that senses both calcium and phosphate. We confirmed that the receptor increases the secretion of parathyroid hormone involved in stimulating urinary phosphate excretion. However, we did not find any evidence for a role of the receptor beyond this function.

CGMP-dependent kinase 2, Na+/H+ exchanger NHE3, and PDZ-adaptor NHERF2 co-assemble in apical membrane microdomains.

Trafficking, membrane retention, and signal-specific regulation of the Na+/H+ exchanger 3 (NHE3) are modulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adapter proteins. This study explored the assembly of NHE3 and NHERF2 with the cGMP-dependent kinase II (cGKII) within detergent-resistant membrane microdomains (DRMs, "lipid rafts") during in vivo guanylate cycle C receptor (Gucy2c) activation in murine small intestine. Small intestinal brush border membranes (siBBMs) were isolated from wild type, NHE3-deficient, cGMP-kinase II-deficient, and NHERF2-deficient mice, after oral application of the heat-stable Escherichia coli toxin (STa) analog linaclotide. Lipid raft and non-raft fractions were separated by Optiprep density gradient centrifugation of Triton X-solubilized siBBMs. Confocal microscopy was performed to study NHE3 redistribution after linaclotide application in vivo. In the WT siBBM, NHE3, NHERF2, and cGKII were strongly raft associated. The raft association of NHE3, but not of cGKII, was NHERF2 dependent. After linaclotide application to WT mice, lipid raft association of NHE3 decreased, that of cGKII increased, while that of NHERF2 did not change. NHE3 expression in the BBM shifted from a microvillar to a terminal web region. The linaclotide-induced decrease in NHE3 raft association and in microvillar abundance was abolished in cGKII-deficient mice, and strongly reduced in NHERF2-deficient mice. NHE3, cGKII, and NHERF2 form a lipid raft-associated signal complex in the siBBM, which mediates the inhibition of salt and water absorption by Gucy2c activation. NHERF2 enhances the raft association of NHE3, which is essential for its close interaction with the exclusively raft-associated activated cGKII.

RNA interference-mediated silencing of coat protein II (COPII) genes affects the gut homeostasis and cuticle development in Locusta migratoria

The coat protein II (COPII) complex consists of five primary soluble proteins, namely the small GTP-binding protein Sar1, the inner coat Sec23/Sec24 heterodimers, and the outer coat Sec13/Sec31 heterotetramers. COPII is essential for cellular protein and lipid trafficking through cargo sorting and vesicle formation at the endoplasmic reticulum. However, the roles of COPII assembly genes remain unknown in insects. In present study, we identified five COPII assembly genes (LmSar1, LmSec23, LmSec24, LmSec13 and LmSec31) in Locusta migratoria. RT-qPCR results revealed that these genes showed different expression patterns in multiple tissues and developmental days of fifth-instar nymphs. Injection of double-stranded RNA against each LmCOPII gene induced a high RNAi efficiency, and considerably suppressed feeding, and increased mortality to 100 %. Results from the micro-sectioning and hematoxylin–eosin staining of midguts showed that the brush border was severely damaged and the number of columnar cells was significantly reduced in dsLmCOPII-injected nymphs, as compared with the control. The dilated endoplasmic reticulum phenotype of columnar cells was observed by transmission electron microscopy. RT-qPCR results further indicated that silencing any of the five genes responsible for COPII complex assembly repressed the expression of genes involved in insulin/mTOR-associated nutritional pathway. Therefore, COPII assembly genes could be promising RNAi targets for insect pest management by disrupting gut and cuticle development.

Characterization of the mode of action of eCry1Gb.1Ig, a fall armyworm (Spodoptera frugiperda) active protein, with a novel site of action

Insect pests cause immense agronomic losses worldwide. One of the most destructive of major crops is the Fall Armyworm (Spodoptera frugiperda, FAW). The ability to migrate long distances, a prodigious appetite, and a demonstrated ability to develop resistance to insecticides, make it a difficult target to control. Insecticidal proteins, for example those produced by the bacterium Bacillus thuringiensis, are among the safest and most effective insect control agents. Genetically modified (GM) crops expressing such proteins are a key part of a successful integrated pest management (IPM) program for FAW. However, due to the development of populations resistant to commercialized GM products, new GM traits are desperately needed. Herein, we describe a further characterization of the newly engineered trait protein eCry1Gb.1Ig. Similar to other well characterized Cry proteins, eCry1Gb.1Ig is shown to bind FAW midgut cells and induce cell-death. Binding competition assays using trait proteins from other FAW-active events show a lack of competition when binding FAW brush border membrane vesicles (BBMVs) and when utilizing non-pore-forming versions as competitors in in vivo bioassays. Similarly, insect cell lines expressing SfABCC2 and SfABCC3 (well characterized receptors of existing commercial Cry proteins) are insensitive to eCry1Gb.1Ig. These findings are consistent with results from our previous work showing that eCry1Gb.1Ig is effective in controlling insects with resistance to existing traits. This underscores the value of eCry1Gb.1Ig as a new GM trait protein with a unique site-of-action and its potential positive impact to global food production.

Therapeutic Potential of Aloe vera and Aloe vera-Conjugated Silver Nanoparticles on Mice Exposed to Hexavalent Chromium.

Hexavalent chromium (Cr (VI)) is a hazardous heavy metal that induces hepatotoxicity and nephrotoxicity. Thus, this study was planned to explore the ameliorating capacity of Aloe vera leaf gel extract (AV) and their conjugated silver nanoparticles (AVNP) against Cr (VI) induced hepatotoxicity and renal toxicity. The organ indices, level of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, malondialdehyde, total protein, and creatinine in blood serum were measured. The histopathological and micrometric analysis of the hepatic and renal tissue sections were studied. The hepatosomatic index was raised significantly (0.098 ± 0.13g) in Cr treated group. The blood serum level of AST (484 ± 10.7 U/L), ALT (163 ± 5.5 U/L), ALP (336.7 ± 9.5 U/L), MDA (642.3 ± 28.3 U/L), and creatinine (4.0 ± 0.1mg/dL) were increased significantly, whereas total protein level was declined (2.8 ± 0.3g/dL) significantly in Cr exposed group. In the histopathological study, necrosis, disturbed hepatic cords, impaired glomeruli, and Bowman's capsule were noted. Micrometric data from the liver and kidney revealed a significant surge in the size of hepatocytes and their nuclei (1188.2 ± 467.7µ2 and 456.5 ± 205.6µ2) and CSA of glomeruli and Bowman's capsule (9051.8 ± 249.8µ2 and 11,835.5 ± 336.7µ2) in Cr (VI) exposed group, whereas the brush border (10.2 ± 4.0µ) size declined significantly. The administration of AV and AVNP reduced the oxidative stress induced by Cr (VI).

Glucose stockpile in the intestinal apical brush border in C. elegans

Revealing the mechanisms of glucose transport is crucial for studying pathological diseases caused by glucose toxicities. Numerous studies have revealed molecular functions involved in glucose transport in the nematode Caenorhabditis elegans, a commonly used model organism. However, the behavior of glucose in the intestinal lumen-to-cell remains elusive. To address that, we evaluated the diffusion coefficient of glucose in the intestinal apical brush border of C. elegans by using fluorescent glucose and fluorescence recovery after photobleaching. Fluorescent glucose taken in the intestine of worms accumulates in the apical brush border, and its diffusion coefficient of ∼10−8 cm2/s is two orders of magnitude slower than that in bulk. This result indicates that the intestinal brush border is a viscous layer. ERM-1 point mutations at the phosphorylation site, which shorten the microvilli length, did not significantly affect the diffusion coefficient of fluorescent glucose in the brush border. Our findings imply that glucose enrichment is dominantly maintained by the viscous layer composed of the glycocalyx and molecular complexes on the apical surface.

Pyroptosis Tuning in Intestinal Cryptosporidiosis via the Natural Histone Deacetylase Inhibitor Romidepsin.

Cryptosporidium is an opportunistic protozoan, with many species of cross-human infectivity. It causes life-threatening diarrhoea in children and CD4-defective patients. Despite its limited efficacy, nitazoxanide remains the primary anti-cryptosporidial drug. Cryptosporidium infects the intestinal brush border (intracellular-extracytoplasmic) and down-regulates pyroptosis to prevent expulsion. Romidepsin is a natural histone deacetylase inhibitor that triggers pyroptosis. Romidepsin's effect on cryptosporidiosis was assessed in immunocompromised mice via gasdermin-D (GSDM-D) immunohistochemical expression, IFN-γ, IL-1β and IL-18 blood levels by ELISA, and via parasite scanning by modified Ziehl-Neelsen staining and scanning electron microscopy (SEM). Oocyst deformity and local cytokines were also assessed in ex vivo ileal explants. Following intraperitoneal injection of romidepsin, oocyst shedding significantly reduced at the 9th, 12th and 15th d.p.i. compared with infected-control and drug-control (nitazoxanide-treated) mice. H&E staining of intestinal sections from romidepsin-treated mice showed significantly low intestinal scoring with marked reduction in epithelial hyperplasia, villous blunting and cellular infiltrate. SEM revealed marked oocyst blebbing and paucity (in vivo and ex vivo) after romidepsin compared with nitazoxanide. Regarding pyroptosis, romidepsin triggered significantly higher intestinal GSDM-D expression in vivo, and higher serum/culture IFN-γ, IL-1β and IL-18 levels in romidepsin-treated mice than in the control groups. Collectively, in cryptosporidiosis, romidepsin succeeded in enhancing pyroptosis in the oocysts and infected epithelium, reducing infection and shifting the brush border towards normalisation.

Alistipes indistinctus-derived hippuric acid promotes intestinal urate excretion to alleviate hyperuricemia

Hyperuricemia induces inflammatory arthritis and accelerates the progression of renal and cardiovascular diseases. Gut microbiota has been linked to the development of hyperuricemia through unclear mechanisms. Here, we show that the abundance and centrality of Alistipes indistinctus are depleted in subjects with hyperuricemia. Integrative metagenomic and metabolomic analysis identified hippuric acid as the key microbial effector that mediates the uric-acid-lowering effect of A.indistinctus. Mechanistically, A.indistinctus-derived hippuric acid enhances the binding of peroxisome-proliferator-activated receptor γ (PPARγ) to the promoter of ATP-binding cassette subfamily G member 2 (ABCG2), which in turn boosts intestinal urate excretion. Tofacilitate this enhanced excretion, hippuric acid also promotes ABCG2 localization to the brush border membranes in a PDZ-domain-containing 1 (PDZK1)-dependent manner. These findings indicate that A.indistinctus and hippuric acid promote intestinal urate excretion and offer insights into microbiota-host crosstalk in the maintenance of uric acid homeostasis.

Modulation of Duodenal TfR Expression in IDA Induced Rat with Date Palm and Goat Milk Intake

Iron deficiency anemia (IDA) is a medical condition characterized by insufficient iron levels in the body, resulting in a reduced ability to produce hemoglobin (Hb), a critical component of red blood cells. IDA is often associated with chronic fatigue, impaired cognitive function, and diminished well-being. Transferrin (Tf) is one of the major proteins in iron homeostasis, responsible for transporting iron through the blood to various tissues, while its carrier protein, transferrin receptor (TfR), mediates the cellular uptake of transferrin-bound iron into the cell. This study aims to evaluate the expression of TfR mRNA and protein in the small intestine following intervention with date palm and goat milk in IDA-induced rats. Twenty-four male Wistar rats were induced with IDA for 2 weeks using a low-iron diet. Following IDA detection, rats were supplemented with date palm and goat milk, singly and in combination. After four weeks, the rats were sacrificed, and the expression of TfR mRNA and protein in the small intestine was assessed using qPCR and immunohistochemistry, respectively. Data were analyzed using SPSS 24.0, with a significance level set at p&lt;0.05. Results demonstrated that date palm and goat milk significantly improved Hb, serum iron, Tf saturation levels, and modulated the expression of TfR mRNA in the IDA-induced rats. Expression of TfR on the crypt region and brush border membrane of the small intestine was normalized following intervention. The findings indicate that supplementation of date palm and goat milk improved Hb and Tf saturation levels and significantly modulated duodenal TfR expression in IDA-induced rats.