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Extracellular vesicles (EVs) are heterogeneous membranous vesicles secreted by every cell type and offer significant potential in therapy and diagnostics. Differential ultracentrifugation is the gold standard for EV isolation, although other techniques including, polyethylene glycol (PEG) precipitation, immunoprecipitation, size exclusion chromatography, and immuno‐isolation approaches are common. Purified EVs can be characterized based on their physical characteristics, biochemical composition, or cell of origin. For size and concentration measurement, nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), and electron microscopy are commonly employed methods. Biochemical analyses of EVs are typically performed using flow cytometry, immunoblotting, or proteomic investigation. Based on tissue of origin, EVs have specific markers that can be used to isolate and purify specific cell‐associated EVs using an affinity selection approach. Despite existence of several methods for isolation and characterization, major limitations associated with each method hinder the progress of the field. Evolving concepts in EV biology possess great promise for better isolation and characterization leading to a better insight of biological function and have immense clinical implications. In this review, we discuss recent advancements in EV isolation and characterization approaches. [ABSTRACT FROM AUTHOR]

Context: Human and animal data suggest that low vitamin D (25-hydroxyvitamin D) status is associated with impaired fertility, endometriosis, and polycystic ovary syndrome. Vitamin D regulates antimullerian hormone (AMH), FSH, mRNA, and expression of genes in reproductive tissues, implicating a role in female reproduction. Aims: To study the vitamin D levels in infertile females and to know the correlation of vitamin D deficiency (VDD) with serum AMH in infertile females compare to fertile females. Settings And Design: This prospective study was conducted in department of Maternal and Reproductive Health in between April 2014 and April 2016. Materials and Methods: After matching inclusion and exclusion criteria out of total 70 infertile females, 45 were found to have VDD. Of these 35 patients were identified as cases; in whom, the AMH levels were assessed. As control 35 fertile normal females were taken, in which vitamin D and AMH were taken. In both groups, correlation of VDD with AMH was studied. Statistical Analysis Used: To analyze the correlation between vitamin D and AMH linear regression test and for comparison of both the groups, two sample t tests were used. Results: The VDD was present in 64.28% of infertile females. In vitamin D deficient cases, the mean for vitamin D was 6.18 ± 2.09 and AMH was 1.94 ± 1.30. In vitamin D deficient controls, the mean for vitamin D was 4.85 ± 3.02 and AMH was 3.47 ± 2.59. On comparison, the vitamin D levels were lower in fertile than infertile females, which was significant (P = 0.04), and AMH levels were lower in cases than control group (P = 0.003). Conclusion: The VDD was present in 64.28% of infertile females. No significant correlation was found in between VDD and AMH levels in both the groups. [ABSTRACT FROM AUTHOR]

R3124 --> 723.3 --> Gluconeogenesis is one of the key processes through which the kidney contributes to glucose homeostasis. Recently, we provided evidence to suggest that early insulin resistance may impair fast‐fed regulation of Phosphoenolpyruvate Carboxykinase (PEPCK), rate‐limiting enzyme for gluconeogenesis in humans. To confirm this and to elucidate a link between insulin resistance and fast‐fed regulation of PEPCK in the kidneys, we studied primary proximal tubule cells (hPT) and urinary extracellular vesicles (uEVs) from humans. We demonstrate that fasted hPTs had higher PEPCK and insulin receptor substrate‐2 (IRS2) mRNA levels than fed cells. The fast‐fed regulation was, however, attenuated in insulin receptor knockdown hPTs (IRKO). The insulin receptor knockdown was confirmed by a blunted insulin‐induced suppression in PEPCK and PGC1α mRNA and p‐IR and p‐AKT protein expression in IRKO cells. Exosomes secreted by the wild‐type or IRKO hPT showed similar regulation as observed in the respective hPT. Similarly, in human uE, the relative abundance of IRS‐2 mRNA (to IRS1) was higher in the fasted state relative to the fed condition. However, the fast‐fed difference was absent in subjects with early insulin resistance. These subjects had higher circulating glucagon levels relative to subjects with optimal insulin sensitivity. Furthermore, in hPT cells, glucagon significantly induced PEPCK mRNA, IRS2 mRNA, and gluconeogenesis. Insulin receptor deletion further increased their levels. Together the data suggest that reduced insulin sensitivity and high glucagon in early insulin resistance may impair renal gluconeogenesis via IRS2 regulation. Funding from ICMR‐CARE and SGPGI intramural to ST and CSIR to RS. [ABSTRACT FROM AUTHOR]

An increase in blood pressure (BP) due to angiotensin II (ANG II) infusion or other means is associated with adaptive pressure natriuresis due to reduced sodium reabsorption primarily in proximal tubule (PT) and thick ascending limb (TAL). We tested the hypothesis that male and female mice would show differential response to ANG II infusion with regard to the regulation of the protein abundance of sodium transporters in the PT and TAL and that these responses would be modulated by aging. Young (-3 mo) and old (-21 mo) male and female mice were infused with ANG II at 800 ng∙kg body wt-1∙min-1 by osmotic minipump for 7 days or received a sham operation. ANG II increased mean arterial pressure (MAP), measured by radiotelemetry, significantly more in male mice of both ages (increased ∼30-40 mmHg), compared with females (increased ∼15-25 mmHg). On day 1, MAP was also significantly increased in old mice, relative to young (P = 0.01). ANG II infusion was associated with a significant decline in plasma testosterone (to <30% of control male) in male mice and rise in young female mice (to 478% of control female). No sex differences were found in the upregulation of the sodium hydrogen exchanger abundance on Western blots observed with ANG II infusion or the downregulation of the sodium phosphate cotransporter; however, aging did impact on some of these changes. Male mice (especially young) also had significantly reduced levels of the TAL bumetanidesensitive Na-K-2C1 cotransporter (to 60% of male control), while young females showed an increase (to 126% of female control) with ANG II infusion. These sex differences do not support impaired pressure natriuresis in male mice, but might reflect a greater need and attempt to mount an appropriately BP-metered natriuretic response by additional downregulation of TAL sodium reabsorption. [ABSTRACT FROM AUTHOR]

Riazi S, Tiwari S, Sharma N, Rash A, Ecelbarger CM. Abundance of the Na-K-2C1 cotransporter NKCC2 is increased by high-fat feeding in Fischer 344 X Brown Norway (Fl) rats. Am J Physiol Renal Physiol 296: F762-F770, 2009. First published February 4, 2009; doi: 10.11 52/ajprenal.90484.2008.-Insulin resistance is associated with hypertension by mechanisms likely involving the kidney. To determine how the major apical sodium transporter of the thick ascending limb, the bumetanide-sensitive Na-K-2C1 cotransporter (NKCC2) is regulated by high-fat feeding, we treated young male, Fischer 344 X Brown Norway (F344BN) rats for 8 wk with diets containing either normal (NF, 4%) or high (HF, 36%) fat, by weight, primarily as lard. HF-fed rats had impaired glucose tolerance, increased urine excretion of 8-isoprostane (a marker of oxidative stress), increased protein levels for NKCC2 (50-125%) and the renal outer medullary potassium channel (106%), as well as increased natriuretic response to furosemide (20-40%). To test the role of oxidative stress in this response, in study 2, rats were fed the NF or HF diet plus plain drinking water, or water containing N[supG]-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor (100 mg/I), or tempol, a superoxide dismutase mimetic (I mmolll). The combination of tempol with HF nullified the increase in medullary NKCC2, while L-NAME with HF led to the highest expression of medullary NKCC2 (to 498% of NF mean). However, neither of these drugs dramatically affected the elevated natriuretic response to furosemide with HF. Finally, L-NAME led to a marked increase in blood pressure (measured by radiotelemetry), which was significantly enhanced with HF. Mean arterial blood pressure at 7 wk was as follows (mmHg): NF, 100 ± 2; NF plus L-NAME, 122 ± 3; and HF plus L-NAME, 131 ± 2. Overall, HF feeding increased the abundance of NKCC2. Inappropriately high sodium reabsorption in the thick ascending limb via NKCC2 may contribute to hypertension with insulin resistance. [ABSTRACT FROM AUTHOR]

Increased renal expression of the angiotensin II, type-1 receptor (AT1R) has been associated with increased blood pressure (BP) and progression of renal disease. We tested whether common medications used to treat hypertension and the metabolic syndrome alter renal AT1R; and whether urine AT1R can be used as a reasonable noninvasive marker of renal levels in the obese Zucker rat, a model for human metabolic syndrome. Immunoblotting revealed that renal and urinary levels of AT1R were significantly higher in obese versus lean rats and correlated (R = 0.62, p < 0.05). Chronic treatment with BP lowering, candesartan, an AT1R antagonist, increased renal levels of AT1R in both lean (282% of lean controls) and obese (178% of obese controls) rats, but decreased urine AT1R levels in obese rats (72% of obese controls). Similarly, chronic treatment with rosiglitazone (RGZ), a peroxisome proliferator activated receptor (subtype γ) agonist, significantly decreased urine (43% of obese controls) but not renal AT1R (105%) in obese rats. Blood pressure, measured by radiotelemetry, was significantly correlated in untreated and RGZ-treated rats to renal AT1R (R = 0.57, p = 0.0035). Finally, high- (4%) and medium- (0.4%) NaCl diets increased excretion of AT1R in obese rats to ∼400% of low- (0.04%) NaCl diet. This effect was markedly blunted in lean rats. Overall, we demonstrate increased renal AT1R levels in obese rats. Urine AT1R correlated with renal levels only in the untreated state. Relative salt-sensitivity of AT1R excretion in obese, relative to lean rats, may have implications for both BP and renal disease in the metabolic syndrome. [ABSTRACT FROM AUTHOR]

The obese Zucker rat reportedly has increased activity of the intrarenal renin-angiotensin- aldosterone system, which conceptually could contribute to elevated salt sensitivity and blood pressure (BP). Our aim was to determine whether there was increased angiotensin II type 1 receptor mediated upregulation of expression or activity of the bumetanide- sensitive Na-K-2C1 cotransporter, the thiazide-sensitive Na-Cl co- transporter (NCC), and/or the epithelial sodium channel (ENaC) in obese vs. lean Zucker rats. Male obese and lean Zucker rats (l0-wk old) were fed either 1) control chow (1% NaCI) or 2) chow with candesartan (CAN), an AT1R antagonist (25 mg/kgdiet) for 14 wk (n = 8/treatment body type). BP measured by radiotelemetry, was markedly reduced by CAN (∼-20-25 mmHg) in both lean and obese rats with no body-type differences. Obese rats had significantly greater net natriuretic response to single injections of hydrochlorothi- azide and benzamil, suggesting increased activity of NCC and ENaC, respectively; however, only the response to benzamil was reduced by CAN. CAN led to a significant reduction in whole kidney levels of NCC and Υ-ENaC (70-kD~ band) in both lean and obese rats. However, it significantly increased a-ENaC and Na-K-2C1 cotransporter levels, and these increases were greater in obese rats. These studies suggest that relatively increased ENaC, but not NCC activity, in obese rats is due to enhanced AT1R activity. CAN attenuated the reduction of several renal transporters in the obese rat kidney. Finally, differences in intrarenal AT1R activity do not seem directly responsible for BP differences between lean and obese rats. [ABSTRACT FROM AUTHOR]

Abstract: Peroxisome-proliferator-activated receptor (PPAR-γ) agonists improve insulin sensitivity, but are associated with edema. Increased distal tubule sodium and water reabsorption through the epithelial sodium channel (ENaC) and aquaporin-2 (AQP-2) have been suggested to play mechanistic roles. To determine the molecular regulation of these proteins, we treated male, Sprague–Dawley rats daily by gavage with either vehicle, rosiglitazone (RGZ, 50mg/kgbw), or PD168 (a test compound causing marked edema, 10mg/kgbw), for 1, 3, or 5 days (n =6/treatment/time). On day 1, urine sodium excretion was significantly reduced by RGZ with a strong trend for PD168 (p-values 0.047 and 0.053, respectively) indicating early sodium retention. Blood pressure was lowered by RGZ- or PD168 treatment by 12h. Immunoblotting of whole kidney homogenates (WKHs) and a membrane-enriched fraction (MF) revealed increased band densities for AQP-2 in WKH (29kDa and glycosylated bands) by both drugs at 1 day. However, at 5 days, the 29-kDa band was significantly decreased (∼30% of vehicle). α-ENaC was increased by RGZ at 3 days; however both agents decreased α-ENaC by 5 days. In contrast, β- and γ-ENaC (85kDa) were unchanged or decreased at all times by both agents. However, the 70-kDa band of γ-ENaC (active band) in MF was increased in density (120–600%) by both agents on days 3–5. Overall, both agents resulted in early alterations in banding patterns for AQP-2 and ENaC subunits, many of which are described as activating changes. However, later reduction in AQP-2 and α-ENaC may represent an attempt to re-establish sodium and water balance. [Copyright &y& Elsevier]

Renal tubule epithelial cells express the insulin receptor (IR); however, their value has not been firmly established. We generated mice with renal epithelial cell-specific knockout of the IR by Cre-recombinase-loxP recombination using a kidney-specific (Ksp) cadherin promoter. KO mice expressed significantly lower levels of IR mRNA and protein in kidney cortex (49-56% of the WT) and medulla (32-47%) homogenates. Immunofluorescence showed the greatest relative reduction in the thick ascending limb and collecting duct cell types. Body weight, kidney weight, and food and water intakes were not different from WT littermates. However, KO mice had significantly increased basal systolic blood pressure (BP, 15 mm Hg higher) as measured by radiotelemetry. In response to a volume load by gavage (20 ml/kg of body weight, 0.9% NaCl, 15% dextrose), KO mice had impaired natriuresis (37 ± 10 versus 99 ± 9 mmol of Na+ per 2 h in WT). Furthermore, volume load led to a sustained increase in BP in KO mice only. In contrast, insulin administration i.p. (0.5 units/kg of body weight) resulted in a significant fall in BP in WT, but not in KO mice. To test the role of reduced renal nitric oxide (NO) production in these responses, basal urinary nitrates plus nitrites excretion (UNOx) was measured and found to be 61% lower in KO vs. WT mice. Furthermore, acute insulin increased UNOx by 202% in the WT, relative to a significantly blunted rise (67%) in KO animals. These results illuminate a previously uncharacterized role for renal IR to reduce BP and facilitate sodium and water excretion, possibly via NO production. [ABSTRACT FROM AUTHOR]

lnsulin has been shown to have antinatriuretic actions in humans and animal models. Moreover, endogenous hyperinsulinemia and insulin infusion have been correlated to increased blood pressure in some models. In this review, we present the current state of understanding with regard to the regulation of the major renal sodium transporters by insulin in the kidney. Several groups, using primarily cell culture, have demonstrated that insulin can directly increase activity of the epithelial sodium channel, the sodium-phosphate cotransporter, the sodium-hydrogen exchanger type III, and Na-K-ATPase. We and others have demonstrated alterations in the expression at the protein level of many of these same proteins with insulin infusion or in hyperinsulinemic models. We also discuss how this regulation is perturbed in type I and type II diabetes mellitus. Finally, we discuss a potential role for regulation of insulin receptor signaling in the kidney in contributing to sodium balance and blood pressure. [ABSTRACT FROM AUTHOR]

Studies done in cell culture have demonstrated that insulin activates the epithelial sodium channel (ENaC) via a variety of mechanisms. However, to date, upregulation of ENaC in native renal tissue by in vivo administration of insulin has not been demonstrated. To address this, we injected 6-mo-old male C57BL/CBA mice (n = 14/group) intra- peritoneally with vehicle or 0.5 U/kg body wt insulin and examined short-term (1-2 h) sodium excretion and kidney ENaC subunits (α, α, and γ) and serum and glucocorticoid-induced kinase (SGK-1) regulation. Insulin resulted in a significant reduction in urine sodium (by ~80%) that was restored by intraperitoneal administration of the ENaC antagonist, benzamil (1.4 mg/kg body wt). Differential centrifugation followed by Western blotting of whole kidney revealed significantly increased band densities (by 26-103%) for insulin- relative to vehicle-treated mice for α- and γ-ENaC in the homogenate (H), and plasma membrane- enriched fraction (MF), with no difference in the vesicle-enriched fraction (VF). Similarly, β-ENaC was significantly increased in MF (by 45%) but no change in the H. It was, however, significantly decreased in the VF (by 28%) with insulin. In agreement, immunoperoxidase labeling demonstrated relatively stronger apical, relative to cytosolic, localization of α-, β-, and γ-ENaC with insulin, whereas, with vehicle, labeling was fairly evenly dispersed throughout collecting duct principal cells. Furthermore, Western blotting showed insulin increased SGK-1 (by 75%) and phosphorylated-SGK band densities (by 30%) but only in the MF. These studies demonstrate novel in vivo regulation of renal ENaC activity and subunit proteins and SGK-1 by insulin in the acute time frame in the mouse. [ABSTRACT FROM AUTHOR]

Atherosclerosis is an inflammatory process characterized by proliferation and dedifferentiation of vascular smooth muscle cells (VSMC). Caν1.2 calcium channels may have a role in atherosclerosis because they are essential for Ca2+-signal transduction in VSMC. The pore-forming Caν1.2α1 subunit of the channel is subject to alternative splicing. Here, we investigated whether the Caν1.2α1 splice variants are affected by atherosclerosis. VSMC were isolated by laser-capture microdissection from frozen sections of adjacent regions of arteries affected and not affected by atherosclerosis. In VSMC from nonatherosclerotic regions, RT-PCR analysis revealed an extended repertoire of Caν1.2α1 transcripts characterized by the presence of exons 21 and 41A. In VSMC affected by atherosclerosis, expression of the Caν1.2α1 transcript was reduced and the Cav1.2α1 splice variants were replaced with the unique exon-22 isoform lacking exon 41A. Molecular remodeling of the Caν1.2α1 subunits associated with atherosclerosis caused changes in electrophysiological properties of the channels, including the kinetics and voltage-dependence of inactivation, recovery from inactivation, and rundown of the Ca2+ current. Consistent with the pathophysiological state of VSMC in atherosclerosis, cell culture data pointed to a potentially important association of the exon-22 isoform of Caν1.2α1 with proliferation of VSMC. Our findings are consistent with a hypothesis that localized changes in cytokine expression generated by inflammation in atherosclerosis affect alternative splicing of the Caν1.2α1 gene in the human artery that causes molecular and electrophysiological remodeling of Caν1.2 calcium channels and possibly affects VSMC proliferation. [ABSTRACT FROM AUTHOR]

Previously, we demonstrated that rats undergoing vasopressin escape had increased mean arterial blood pressure (MAP), plasma and urine aldosterone, and increased renal protein abundance of the α-subunit of the epithelial sodium channel (ENaC), the thiazide-sensitive Na-Cl cotransporter (NCC), and the 70-kDa band of γ-ENaC (Song J, Hu X, Khan 0, Tian Y, Verbahis JG, and Ecelbarger CA. Am J Physiol Renal Physiol 287: F1076-F1083, 2004; Ecelbarger CA, Knepper MA, and Verbalis JG. J Am Soc Nephrol 12: 207-217, 2001). Here, we determine whether changes in these renal proteins and MAP require elevated aldosterone levels. We performed adrenalectomies (ADX) or sham surgeries on male Sprague-Dawley rats. Corticosterone and aldosterone were replaced to clamp these hormone levels. MAP was monitored by radiotelemetry. Rats were infused with 1-deamino-[8-D-arginine]-vasopressin (dDAVP) via osmotic minipumps (5 ng/h). At day 3 of dDAVP infusion, seven rats in each group were offered a liquid diet [water load (WL)] or continued on a solid diet (SD). Plasma aldosterone and corticosterone and urine aldosterone were increased by WL in sham rats. ADX-WL rats escaped, as assessed by early natriuresis followed by diuresis; however, urine volume and natriuresis were somewhat blunted. WL did not reduce the abundance or activity of 11-β-hydroxsteroid dehydrogenase type 2. Furthermore, the previously observed increase in renal aldosterone-sensitive proteins and escape-associated increased MAP persisted in clamped rats. The densitometry of immunoblots for NCC, α- and γ-70 kDa ENaC, respectively, were (% sham-SD): sham-WL, 159, 278, 233; ADX-SD, 69, 212, 171; ADX-WL, 116, 302, 161. However, clamping corticosteroids blunted the rise at least for NCC and γ-ENaC (70 kDa). Overall, the increase in aldosterone observed in vasopressin escape is not necessary for the increased expression of NCC, α- or γ-ENaC or increased MAP associated with "escape." [ABSTRACT FROM AUTHOR]

Voltage-gated Cav1.2 channels are composed of the pore-forming α1C and auxillary β and α2δ subunits. Voltage-dependent conformational rearrangements of the α1C subunit C-tail have been implicated in Ca2+ signal transduction. In contrast, the α1C N-tail demonstrates limited voltage-gated mobility. We have asked whether these properties are critical for the channel function. Here we report that transient anchoring of the α1C subunit C-tail in the plasma membrane inhibits Ca2+-dependent and slow voltage-dependent inactivation. Both α2δ and β subunits remain essential for the functional channel. In contrast, if α1C subunits are expressed with α2δ but in the absence of a β subunit, plasma membrane anchoring of the α1C N terminus or its deletion inhibit both voltage- and Ca2+-dependent inactivation of the current. The following findings all corroborate the importance of the α1C N-tail/β interaction: (i) co-expression of β restores inactivation properties, (ii) release of the α1C N terminus inhibits the β-deficient channel, and (iii) voltage-gated mobility of the α1C N-tail vis à vis the plasma membrane is increased in the β-deficient (silent) channel. Together, these data argue that both the α1C N- and C-tails have important but different roles in the voltage- and Ca2+-dependent inactivation, as well as β subunit modulation of the channel. The α1C N-tail may have a role in the channel trafficking and is a target of the β subunit modulation. The β subunit facilitates voltage gating by competing with the N-tail and constraining its voltage-dependent rearrangements. Thus, cross-talk between the α1C C and N termini, β subunit, and the cytoplasmic pore region confers the multifactorial regulation of Cav1.2 channels. [ABSTRACT FROM AUTHOR]

Cell-fusion mediated generation of multinucleated syncytia represent critical feature during viral infection and in development. Efficiency of syncytia formation is usually illustrated as fusion efficiency under given condition by quantifying total number of nuclei in syncytia normalized to total number of nuclei (both within syncytia and unfused cell nuclei) in unit field of view. However heterogeneity in multinucleated syncytia sizes poses challenge in quantification of cell-fusion multinucleation under diverse conditions. Taking in-vitro SARS-CoV-2 spike-protein variants mediated virus-cell fusion model and placenta trophoblast syncytialization as cell-cell fusion model; herein we emphasize wide application of simple unbiased detailed measure of virus-cell and cell-cell multinucleation using experiential cumulative distribution function (CDF) and fusion number events (FNE) approaches illustrating comprehensive metrics for syncytia interpretation. • Multinucleated syncytia formation is an important event during virus infection and in placenta development. • Combination of fusion number events (FNE) and cumulative distribution function (CDF) define syncytia sizes. • FNE and CDF represent unbiased approach for syncytia interpretation both in virus-cell and cell-fusion models. [ABSTRACT FROM AUTHOR]

Context: Aneuploidy screening is done in the early second trimester of pregnancy among all pregnant women as compulsory, with a special focus on those who had abnormal ultrasound parameters, higher dual marker risk, or other comorbidities. Recently, all individual quad markers of conventional trisomy screening have been suggested as useful in predicting adverse pregnancy outcomes (APO) such as preeclampsia, preterm labor, small for gestational age, and placental abruptions. However, similar studies on Indian pregnant women are limited. Hence, this study was intended to find the relation of quadruple markers with any other APO than aneuploidy. Materials and Methods: A retrospective study was conducted in a Tertiary Care multi-specialty hospital in North India. Data from 252 pregnant women's quadruple test was analyzed. The association of abnormal value of quadruple markers (human chorionic gonadotropin [HCG]/ alpha-fetoprotein/uE3/Inhibin A) with adverse outcomes was evaluated. Multiple logistic regression analysis and classification and regression tree were used to predict the significant risk factor in high-risk pregnancies. Results: In the study, a total (n = 252) of pregnant women, 190 were screened as high-risk pregnancies, whereas the remaining 62 were reported as low-risk using trisomy screening in the quadruple test. Baby birth weight was observed to be significantly associated with Inhibin-A, and HCG (P < 0.001), whereas Corrected (Corr)-multiple of median (MoM)-HCG (>1.415) and Inhibin-A Corr-MoM (>364.175) were the suitable predictor for the LBW. Both parameters were significantly higher in the high-risk group as compared to the low-risk group (each P < 0.05). Conclusion: Abnormal deviation of biochemical markers from aneuploidy screening assessment could help predict other perinatal adverse outcomes such as low birth weight babies. [ABSTRACT FROM AUTHOR]

Previous research has identified intravascular platelet thrombi in regions affected by myocardial ischemia-reperfusion (MI/R) injury and neighbouring areas. However, the occurrence of arterial thrombosis in the context of MI/R injury remains unexplored. This study utilizes intravital microscopy to investigate carotid artery thrombosis during MI/R injury in rats, establishing a connection with the presence of prothrombotic cellular fibronectin containing extra domain A (CFN-EDA) protein. Additionally, the study examines samples from patients with coronary artery disease (CAD) both before and after coronary artery bypass grafting (CABG). Levels of CFN-EDA significantly increase following MI with further elevation observed following reperfusion of the ischemic myocardium. Thrombotic events, such as thrombus formation and growth, show a significant increase, while the time to complete cessation of blood flow in the carotid artery significantly decreases following MI/R injury induced by ferric chloride. The acute infusion of purified CFN-EDA protein accelerates in-vivo thrombotic events in healthy rats and significantly enhances in-vitro adenosine diphosphate and collagen-induced platelet aggregation. Treatment with anti-CFN-EDA antibodies protected the rat against MI/R injury and significantly improved cardiac function as evidenced by increased end-systolic pressure-volume relationship slope and preload recruitable stroke work compared to control. Similarly, in a human study, plasma CFN-EDA levels were notably elevated in CAD patients undergoing CABG. Post-surgery, these levels continued to rise over time, alongside cardiac injury biomarkers such as cardiac troponin and B-type natriuretic peptide. The study highlights that increased CFN-EDA due to CAD or MI initiates a destructive positive feedback loop by amplifying arterial thrombus formation, potentially exacerbating MI/R injury. [Display omitted] • The study emphasizes a direct and robust connection between arterial thrombosis and MI/R injury, previously unidentified. • Increased CFN-EDA levels post-CAD or MI injury potentially accelerate arterial thrombosis events in reperfusion injury. • The significant increase in CFN-EDA following CABG, along with elevated cardiac-injury biomarkers, underscores its clinical significance. • Targeting CFN-EDA before treatment may offer promising strategy to alleviate the hastened thrombotic events linked to MI/R injury. [ABSTRACT FROM AUTHOR]

Recent studies have highlighted the therapeutic potential of small extracellular bodies derived from mesenchymal stem cells (MSC-sEVs) for various diseases, notably through their ability to alter T-cell differentiation and function. The current study aimed to explore immunomodulatory pathway alterations within T cells through mRNA sequencing of activated T cells cocultured with bone marrow-derived MSC-sEVs. mRNA profiling of activated human T cells cocultured with MSC-sEVs or vehicle control was performed using the QIAGEN Illumina sequencing platform. Pathway networks and biological functions of the differentially expressed genes were analyzed using Ingenuity pathway analysis (IPA)® software, KEGG pathway, GSEA and STRING database. A total of 364 differentially expressed genes were identified in sEV-treated T cells. Canonical pathway analysis highlighted the RhoA signaling pathway. Cellular development, movement, growth and proliferation, cell-to-cell interaction and inflammatory response-related gene expression were altered. KEGG enrichment pathway analysis underscored the apoptosis pathway. GSEA identified enrichment in downregulated genes associated with TNF alpha and interferon gamma response, and upregulated genes related to apoptosis and migration of lymphocytes and T-cell differentiation gene sets. Our findings provide valuable insights into the mechanisms by which MSC-sEVs implement immunomodulatory effects on activated T cells. These findings may contribute to the development of MSC-sEV-based therapies. [ABSTRACT FROM AUTHOR]

Diabetes is associated with increased activity of the renal renin angiotensin system (RAS). Previously, we demonstrated that streptozotocin (STZ)-induced diabetic rats have significantly increased abundances of renal distal sodium transporters and channels, e.g., the thiazide-sensitive Na-Cl cotransporter (NCC) and the epithelial sodium channel (ENaC). To determine the role of elevated RAS activity in these changes, male Sprague-Dawley rats (100-150 g) were made diabetic by intravenous injection of STZ (65 mg/kg·bw) or treated with vehicle (n = 12/group). Rats were fed standard 1% NaCl rodent chow and watered ad libitum. After 14 days, rats were further divided (n = 6/group) to receive candesartan in the drinking water (2 mg/kg/day) or plain water for 7 additional days. Immunoblotting revealed, in cortex, diabetes caused a modest increase in NCC, β- and γ-ENaC (85-kDa band) abundances (band density ∼ 150% of non-diabetic controls). Candesartan not only reversed these increases, but significantly reduced abundance of NCC and ENaC subunits to ∼50% of non-candesartan-treated rats in both diabetic and non-diabetic rats. In contrast, in the medulla, diabetes caused a much greater increase in ENaC subunits and α-1-Na-K-ATPase (band densities 200-600% of non-diabetic controls) and these increases were irreversible by candesartan. Moreover, candesartan increased β-and γ-ENaC in the non-diabetic rats, in the medulla tip. Thus the increased abundances of sodium transporters in the cortex, but not the medulla, with diabetes, are candesartan-sensitive, and therefore, likely due to enhanced ATl-mediated signaling. [ABSTRACT FROM AUTHOR]

Insulin affects sodium excretion and blood pressure. In order to examine the role of the insulin receptor (IR) in the kidney, we selectively knocked out the IR from the thick ascending limb (TAL) through collecting duct (CD) of the renal tubule by using a cre-recombinase/lox P cross-breeding strategy with Cre driven by Ksp-cadherin in mice. Immunoblotting revealed band density for the β-subunit of IR was reduced to 22% of the WT mean in the inner stripe of the outer medulla (OMH) of the KO mice. Baseline systolic blood pressures, measured by radiotelemetry, were significantly increased in the KO mice in the light period (mm Hg): 123 ± 3 (KO) versus 109 ± 5 (WT), but not different in the dark period. Moreover, OMH endothelial nitric oxide synthase protein abundance was significantly decreased in KO mice to 63% of the WT level. To examine pressure natriuresis, we administered, by gavage, 25 ml/kg/bw of 15% dextrose in 0.9% saline. Dextrose was included to raise endogenous insulin levels. Urine was collected every two hours after gavage. Blood pressure was monitored. The mean arterial blood pressure (MAP) was significantly increased, relative to baseline, in both KO and WT mice at 30 minutes. After 30 minutes, MAP came back to baseline in WT mice, however in KO mice it remained elevated significantly until 120 minutes. Furthermore, the urinary sodium excretion between 2 and 4 hours after gavage was significantly increased in WT, but not in KO mice. When dextrose was not included there were no differences between genotypes in blood pressure or sodium excretion. These data suggest a role for the IR in the renal TAL through CD in facilitating pressure natriuresis which may involve endothelial nitric oxide synthase. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cell (MSC) therapy is an innovative approach in diabetes due to its capacity to modulate tissue microenvironment and regeneration of glucose-responsive insulin-producing cells. In this study, we investigated the role of MSC-derived exosomes in pancreatic regeneration and insulin secretion in mice with streptozotocin-induced diabetes. Mesenchymal stem cells (MSCs) were isolated and characterized from umbilical cord blood (UCB). Exosomes were isolated and characterized from these MSCs. Diabetes was induced in male C57Bl/6 mice by streptozotocin (STZ; 40 mg/kg body weight, i.p.) for five consecutive days. The diabetic mice were administered (i.v.) with MSC (1 × 10 5 umbilical cord blood MSC cells/mice/day), their derived exosomes (the MSC-Exo group that received exosomes derived from 1 × 10 5 MSC cells/mice/day), or the same volume of PBS. Before administration, the potency of MSCs and their exosomes was evaluated in vitro by T cell activation experiments. After day 7 of the treatments, blood samples and pancreatic tissues were collected. Histochemistry was performed to check cellular architecture and β cell regeneration. In body weight, blood glucose level, and insulin level, cell proliferation assay was done to confirm regeneration of cells after MSC and MSC-Exo treatments. Hyperglycemia was also attenuated in these mice with a concomitant increase in insulin production and an improved histological structure compared to mice in the PBS-treated group. We found increased expression of genes associated with tissue regeneration pathways, including Reg2, Reg3, and Amy2b in the pancreatic tissue of mice treated with MSC or MSC-Exo relative to PBS-treated mice. MicroRNA profiling of MSC-derived exosomes showed the presence of miRs that may facilitate pancreatic regeneration by regulating the Extl3-Reg-cyclinD1 pathway. These results demonstrate a potential therapeutic role of umbilical cord blood MSC-derived exosomes in attenuating insulin deficiency by activating pancreatic islets' regenerative abilities. [ABSTRACT FROM AUTHOR]

Objectives: Objectives: SARS-CoV-2 virus dynamics in the human body and its correlation with disease severity and biomarkers have not been elucidated. This study aims to evaluate the viral load in the blood sample of COVID-19 patients and viral load association with disease severity and progression. Methods: Blood samples from COVID-19 positive inpatients were collected sequentially, at admission, and during hospitalization. Depending on the clinical presentation, the patients were classified into mild, moderate, and severe categories. The blood samples were subjected to SARS-CoV-2 Real-time polymerase chain reaction (RT-PCR); positive samples were then tested for viral load using digital drop RT-PCR. Patients were followed up until they were discharged. Results: Of 83 enrolled patients, 15 (18.1%) were mild, 45 (54%) were moderate, and 23 (27.7%) had severe symptoms. The blood PCR in 21 patients was positive (rate was 25.3); one was mild, four moderate, and 16 severe on the day of admission. The mean cycle threshold (CT) of RNAemia in these 21 patients was 24.7+3.1. The viral load was significantly higher in patients with severe symptoms than others. Serum ferritin, D-dimer, and plasma fibrinogen were found raised along with lymphopenia in all patients. Viremia was found persistently detectable (or even rising) in patients in the severe category and associated with a fatal outcome. Conclusion: A prolonged elevation of D-dimer, serum ferritin, and plasma fibrinogen together with lymphopenia lead to a greater risk of mortality and morbidity. Quantitative estimation of virus in blood has a clinical significance which may help in prognostication and management of patients of COVID-19. [ABSTRACT FROM AUTHOR]

• IAV blocks autophagy and induces apoptosis. • Vitamin D restores autophagic flux. • Vitamin D reduces IAV induced apoptosis. Vitamin D3/Calcitriol supplementation in humans is associated with reduced incidence and severity during influenza A virus (IAV) infection. Apoptosis in response to IAV infection is a major contributor to host cell death and tissue damage; however, its modulation by Vitamin D3 remains unclear. In this study, we demonstrate the efficacy of Vitamin D3 in preventing apoptosis induction by pandemic influenza A (H1N1)pdm09 virus in human alveolar cells (A549). Human alveolar epithelial cell line A549 was used to assess the cytotoxic effects of IAV infection. Immunoblotting and fluorescence microscopy were used to study apoptosis and autophagy. The results of the present study demonstrate that IAV induces apoptosis by subversion of host autophagy via down-regulating components of autophagic machinery involved in autophagosome-lysosome fusion and lysosomal activity. Vitamin D3 restores the autophagic flux inhibited by IAV by upregulating the expression of Syntaxin-17 (STX17) and V-type proton ATPase subunit (ATP6V0A2) thereby causing a concomitant decrease in cellular apoptosis via a Vitamin D3 receptor (VDR) dependent mechanism. The present study suggests that Vitamin D3 is a potentially useful agent for limiting IAV-induced cellular injury via its pro-autophagic action. [ABSTRACT FROM AUTHOR]

Role of autophagy in Japanese encephalitis viral (JEV) infection is not well known. In the present study, we reported the role of autophagy flux in microglia activation, neurobehavioral function and neuronal death using a mouse model of JEV. Markers for autophagy (LC3-II/I, SQSTM1/P62, phos-Akt, phos-AMPK), and neuronal death (cleaved caspase 12, H2Ax, polyubiquitin) were investigated by western blot at 1, 3 and 7 days post inoculation. Cathepsin D was measured in cerebral cotex of JEV infected mice spectrophotometrically. Microglia activation and pro-inflammatory cytokines (IL1β, TNF-α, IFNγ, IL6) were measured by immunohistochemistry, western blot and qPCR analysis. In order to determine the neuroinflammatory changes and autophagy mediated neuronal cell death, BV2-microglia and N2a-neuronal cells were used. Autophagy activation marker LC3-II/I and its substrate SQSTM1/P62 were significantly increased while cathepsin D activity was decreased on day 7 post inoculation in cerebral cortex. Microglia in cortex were activated and showed higher expression of proinflammatory mRNA of IL1β, TNF-α, IFNγ and IL6, with increased DNA damage (H2AX) and neuronal cell death pathways in hippocampus and neurobehavioral dysfunction. Similar observations on JEV infection mediated autophagy flux inhibition and neuronal cell death was found in N2a neuronal cell. Collectively, our study provides evidence on the role of autophagy regulation, microglial activation and neurodegeneration following JEV infection. [ABSTRACT FROM AUTHOR]

miRNAs provide fine tuning of gene expression via inhibition of translation. miR-451 has a modulatory role in cell cycling via downregulation of mechanistic target of rapamycin. We aimed to test whether chronic systemic inhibition of miR-451 would enhance renal fibrosis (associated with deranged autophagy). Adult TallyHo/Jng mice (obese insulin resistant) were randomized to two treatment groups to receive either miR-451 inhibition [via a locked nucleic acid construct] or a similar scrambled locked nucleic acid control for 8 wk. All mice were fed a high-fat diet (60% kcal from fat) ad libitum and humanely euthanized after 12 wk. Kidneys and blood were collected for analysis. Renal expression of miR-451 was sixfold lower in inhibitor-treated mice compared with control mice. miR-451 inhibition increased kidney weight and collagen and glycogen deposition. Blood chemistry revealed significantly higher Na+ and anion gap (relative metabolic acidosis) in inhibitor-treated mice. Western blot analysis and immunohistochemistry of the kidney revealed that the inhibitor increased markers of renal injury and fibrosis, e.g., kidney injury molecule 1, neutrophil gelatinase-associated lipocalin, transforming growth factor-β, 14-3-3 protein-, mechanistic target of rapamycin, AMP-activated protein kinase-α, calcium-binding protein 39, matrix metallopeptidase-9, and the autophagy receptor sequestosome 1. In contrast, the inhibitor reduced the epithelial cell integrity marker collagen type IV and the autophagy markers microtubuleassociated protein 1A/1B light chain 3B and beclin-1. Taken together, these results support a protective role for miR-451 in reducing renal fibrosis by enhancing autophagy in obese mice. [ABSTRACT FROM AUTHOR]

We determined the role of cellular fibronectin (CFN) containing the alternatively spliced extra domain A (FN-EDA) in causing insulin resistance (IR) through toll-like receptor 4 (TLR4). Circulating FN-EDA level was evaluated in mouse and rat IR models. Specific anti-FN-EDA antibody and TLR4 inhibitor were used to study its role in IR in mice. CFN protein was injected to evaluate TLR4 dependent effect of FN-EDA in IR. Furthermore, FN-EDA was estimated in blood plasma and correlated with demographic and clinical characteristics in healthy human participants (n = 38). High-fat diet feeding significantly increased circulating FN-EDA in both mouse (P = 0.03) and rat (P = 0.02) IR models. Antibody against FN-EDA protected mice from IR by increasing glucose disposal rate following glucose (P = 0.02) and insulin (P = 0.01) tolerance tests. CFN protein injection caused IR, however, TLR4 inhibitor protected the mice from CFN induced IR. Multivariate regression analysis predicted an independent positive correlation between circulating FN-EDA and fasting plasma glucose (P = 0.003) in healthy human participants. In conclusion, FN-EDA may cause IR through TLR4 by decreasing glucose disposal rate following glucose and insulin load. Targeting FN-EDA thus can be considered as a possible therapeutic strategy to delay prediabetes progression to diabetes. [ABSTRACT FROM AUTHOR]

Background/Aims: Metabolic syndrome and type 2 diabetes are associated with some degree of acidosis. Acidosis has also been shown to upregulate renal gluconeogenesis. Whether impaired insulin or insulin-like-growth factor 1 receptor (IGF1) signaling alter this relationship is not known. Our aim was to determine the effects of deletion of insulin and IGF1 receptors (Insr and Igf1r) from renal proximal tubule (PT) on the gluconeogenic response to acidosis. Methods: We developed a mouse model with PT-targeted dual knockout (KO) of the Insr/Igf1r by driving Cre-recombinase with the gamma-glutamyl transferase (gGT) promoter. Male and female mice were maintained as control or acidotic by treatment with NH4 Cl in the drinking water for 1-week. Results: Acidosis in both genotypes increased renal expression of phosphoenolpyruvate carboxykinase (PEPCK) and fructose-1-bisphosphatase (FBP1), but not glucose6-phosphatase catalytic subunit (G6PC), which showed significantly lower expression in the KO regardless of treatment. Several differences between KO and WT suggested a protective role for insulin/IGF1 receptor signaling in maintaining relative euglycemia in the face of acidosis. First, the increase in FBP1 with acid was greater in the KO (significant interactive term). Secondly, proximal-tubule-associated FOXO1 and AKT overall protein levels were suppressed by acid loading in the KO, but not in the WT. Robust intact insulin signaling would be needed to reduce gluconeogenesis in PT. Third, phosphorylated FOXO1 (pS256) levels were markedly reduced by acid loading in the KO PT, but not in the WT. This reduction would support greater gluconeogenesis. Fourth, the sodium-glucose cotransporter (SGLT1) was increased by acid loading in the KO kidney, but not the WT. While this would not necessarily affect gluconeogenesis, it could result in increased circulatory glucose via renal reabsorption. Reduced susceptibility to glucose-homeostatic dysregulation in the WT could potentially relate to the sharp (over 50%) reduction in renal levels of sirtuin-1 (SIRT1), which deacetylates and regulates transcription of a number of genes. This reduction was absent in the KO. Conclusion: Insulin resistance of the kidney may increase whole-body glucose instability a major risk factor for morbidity in diabetes. High dietary acid loads provide a dilemma for the kidney, as ammoniagenesis liberates α-ketoglutarate, which is a substrate for gluconeogenesis. We demonstrate an important role for insulin and/or IGF1 receptor signaling in the PT to facilitate this process and reduce excursions in blood glucose. Thus, medications and lifestyle changes that improve renal insulin sensitivity may also provide added benefit in type 2 diabetes especially when coupled with metabolic acidosis. [ABSTRACT FROM AUTHOR]

Impaired insulin-induced suppression of renal gluconeogenesis could be a risk for hyperglycemia. Diabetes is associated with elevated renal gluconeogenesis; however, its regulation in early insulin resistance is unclear in humans. A noninvasive marker of renal gluconeogenesis would be helpful. Here, we show that human urine exosomes (uE) contain three gluconeogenic enzymes: phosphoenolpyruvate carboxykinase (PEPCK), fructose 1,6-bisphosphatase, and glucose 6-phosphatase. Their protein levels were positively associated with whole body insulin sensitivity. PEPCK protein in uE exhibited a meal-induced suppression. However, subjects with lower insulin sensitivity had blunted meal-induced suppression. Also, uE from subjects with prediabetes and diabetic rats had higher PEPCK relative to nondiabetic controls. Moreover, uE-PEPCK was higher in drug-naïve subjects with diabetes relative to drug-treated subjects with diabetes. To determine whether increased renal gluconeogenesis is associated with hyperglycemia or PEPCK expression in uE, acidosis was induced in rats by 0.28 M NH4Cl with 0.5% sucrose in drinking water. Control rats were maintained on 0.5% sucrose. At the seventh day posttreatment, gluconeogenic enzyme activity in the kidneys, but not in the liver, was higher in acidotic rats. These rats had elevated PEPCK in their uE and a significant rise in blood glucose relative to controls. The induction of gluconeogenesis in human proximal tubule cells increased PEPCK expression in both human proximal tubules and human proximal tubule-secreted exosomes in the media. Overall, gluconeogenic enzymes are detectable in human uE. Elevated PEPCK and its blunted meal-induced suppression in human urine exosomes are associated with diabetes and early insulin resistance. [ABSTRACT FROM AUTHOR]

[Display omitted] Exosomes, biogenic nano-vesicles, are renowned for their ability to encapsulate diverse payloads, however the systematic development and validation of exosomal formulation with significant biological implications have been overlooked. Herein, we developed and validated Exo-DTX , a QbD-driven optimized RAW 264.7 cell derived exosomal anti-cancer formulation of docetaxel (DTX) and evaluate its anti-metastatic and apoptotic efficacy in TNBC 4T1 cells. RAW264.7-derived exosomes were having particle size (112.5 ± 21.48 nm) and zeta-potential (−10.268 ± 3.66 mV) with polydispersity (PDI:0.256 ± 0.03). The statistical optimization of exosomes (200 μg) with Exo: DTX ratio 4:1 confirmed encapsulation of 23.60 ± 1.54 ng DTX/ µg exosomes. Exo-DTX (∼189 nm, −11.03 mV) with 100 ng/ml DTX as payload exhibited ∼5 folds' improvement in IC 50 of DTX and distinct cytoskeletal deformation in TNBC 4T1 cells. It also has shown enormous Filamentous actin (F-actin) degradation and triggered apoptosis explained Exo-DTX's effective anti-migratory impact with just 2.6 ± 6.33 % wound closure and 4.56 ± 1.38 % invasion. The western blot confirmed that Exo-DTX downregulated migratory protein EGFR and β1-integrin but raised cleaved caspase 3/caspase 3 (CC3/C3) ratio and BAX/BCL-2 ratio by about 2.70 and 4.04 folds respectively. The naive RAW 264.7 exosomes also contributed positively towards the effect of Exo-DTX formulation by suppressing β1-integrin expression and increasing the CC3/C3 ratio in TNBC 4T1 cells as well. Additionally, significant improvement in PK parameters of Exo-DTX was observed in comparison to Taxotere, 6-folds and 3.04-folds improved t 1/2 and V d , proving the translational value of Exo-DTX formulation. Thus, the Exo-DTX so formulated proved beneficial in controlling the aggressiveness of TNBC wherein, naive exosomes also demonstrated beneficial synergistic anti-proliferative effect in 4T1. [ABSTRACT FROM AUTHOR]

Maternal nutrition has become a major public health concern over recent years and is a known predictor of adverse long-term metabolic derangement in offspring. Time-restricted feeding (TRF), wherein food consumption is restricted to the metabolically active phase of the day, is a dietary approach that improves metabolic parameters when consuming a high-fat diet (HFD). Here, we tested whether TRF could reduce maternal HFD associated inflammation and thereby mitigate defects in fetal organ developmental. Female rats were kept on following three dietary regimens; Ad libitum normal chow diet (NCD-AL), Ad libitum HFD (HFD-AL) and Time-restricted fed HFD (HFD-TRF) from 5 months prior to mating and continued throughout pregnancy. Rat dams were sacrificed at embryonic day 18.5 (ED18.5) and placental tissues from these rats were processed for the analysis of cellular apoptosis, inflammatory cytokines (TNFα and IL-6), oxidative stress, endoplasmic reticulum (ER) stress and autophagy. Furthermore, fetal hepatic triglyceride (TG) content and fetal lung maturation were assessed at ED18.5. Biochemical analysis revealed that HFD-TRF rat had significantly lower serum TG levels and body weight compared to HFD-AL rats. Additionally, TRF significantly blocked HFD-induced placental apoptosis and inflammation via minimizing cellular stress, and restoring autophagic flux. In addition, fetal hepatosteatosis and delayed fetal lung maturation induced by HFD was significantly ameliorated in HFD-TRF compared to HFD-AL. Collectively, our results suggest that reducing placental inflammation via TRF could prevent adverse fetal metabolic outcomes in pregnancies complicated by maternal obesity. Image 1 • Time restricted Feeding (TRF) limits HFD induced placental apoptosis and inflammation. • TRF reduced HFD induced oxidative stress and ER-stress. • TRF mitigates HFD induced fetal developmental defects. [ABSTRACT FROM AUTHOR]

Relative band intensities of C-I on (C) BN-PAGE and (D) in-gel assay were quantitated using ImageJ and normalized with total mitochondrial protein loaded (80 µg). (E) Equal loading of mitochondrial proteins is shown by immunoblotting results of an aliquot of the same samples used for BN-PAGE/in-gel assay, with mitochondrial marker protein VDAC. C-, complex; VDAC, Voltage-dependent anion channel; BN-PAGE, Blue native polyacrylamide gel electrophoresis. [Extracted from the article]

Exosomes, as a nanocarrier for the co-delivery of biologicals and small anticancer molecules is yet in its infancy. Herein, we investigated hUCBMSC derived exosomes as a biogenic nanocarrier for the co-delivery of tumor suppressor miR-125a and microtubule destabilizing Docetaxel (DTX) to target the proliferative and migratory aggressiveness of the murine TNBC 4T1 cells. In this study, hUCBMSCs from the human umbilical cord blood cells (hUCB) were successfully transfected with miR-125a. Thereafter, DTX was encapsulated into both non-transfected and transfected exosomes by optimized mild sonication-incubation technique. The anticancer efficiency of hUCBMSC Exo-DTX and miR-125a Exo-DTX was compared by MTT and morphometric assay. The prominent anti-metastatic behaviour of the latter was confirmed by in-vitro wound healing and transwell invasion assay. Further, the synergistic effect of miR-125a and DTX was confirmed by F-actin and nuclear degradation by confocal and FESEM assay. hUCBMSC exosomes exhibited DTX payload of 8.86 ± 1.97 ng DTX/ μg exosomes and miRNA retention capacity equivalent to 12.31 ± 5.73 %. The co-loaded formulation (miR-125a Exo-DTX) exhibited IC 50 at 192.8 ng/ml in 4T1 cells, which is almost 2.36 folds' lower than the free DTX IC 50 (472.8 ng/ml). Additionally, miR-125a Exo-DTX treatment caused wound broadening upto 6.14 ± 0.38 % while treatment with free DTX and miR-125a exosomes alone caused 18.71 ± 4.5 % and 77.36 ± 10.4 % of wound closure respectively in 36 h. miR-125a Exo-DTX treatment further exhibited significantly reduced invasiveness of 4T1 cells (by 3.5 ± 1.8 %) along with prominent cytoskeletal degradation and nuclear deformation as compared to the miR-125a exosomes treated group. The miR-125a expressing DTX loaded exosomal formulation clearly demonstrated the synergistic apoptotic and anti-migratory efficiency of the miR-125a Exo-DTX. The synergistic anticancer and anti-metastatic effect of miR-125a Exo-DTX was observed due to presence of both DTX and miR-125a as the cargo of hUCBMSC derived exosomes. [Display omitted] [ABSTRACT FROM AUTHOR]

Manganese nanoparticles (MnNPs) are increasingly being used in biomedical and industrial fields; however, their adverse effects on human health have not been fully investigated. In this study, we focused on some of the toxicological aspects of MnNPs. Here, we have attempted to investigate the role of MnNPs in endoplasmic reticulum (ER) stress during apoptosis using primary hippocampal neuron. We have determined dose dependent cytotoxicity of MnNPs for 24 h and assessed oxidative stress related parameters namely reactive oxygen species (ROS), glutathione (GSH) and Ca 2+ . We observed that SiNPs caused oxidative stress by increasing ROS production and intracellular calcium levels together with depletion of intracellular GSH levels. We also demonstrated the expression of key apoptosis and ER stress related proteins and found that, MnNPs, potentially induced expression of PERK, EIF2α, caspase 9, caspase 3, caspase 12, GADD153 and GRP78 which was indicative of ER stress. Overall our findings suggest that MnNPs induces ER stress through ROS generation which compromises the antioxidant defenses and thereby promoting apoptosis in primary hippocampal neuron. [ABSTRACT FROM AUTHOR]

The major site of fructose metabolism in the kidney is the proximal tubule (PT). To test whether insulin and/or IGF1 signaling in the PT is involved in renal structural/functional responses to dietary fructose, we bred mice with dual knockout (KO) of the insulin receptor (IR) and the IGF1 receptor (IGF1R) in PT by Cre-lox recombination, using a γ-glutamyl transferase promoter. KO mice had slightly (~10%) reduced body and kidney weights, as well as, a reduction in mean protein-to-DNA ratio in kidney cortex suggesting smaller cell size. Under control diet, IR and IGF1R protein band densities were 30-50% (P < 0.05) lower than WT, and the relative difference was greater in male animals. Male, but not female KO, also had significantly reduced band densities for Akt (protein kinase B), phosphorylated AktT308 and IRY1162/1163 A high-fructose diet (1-month) led to a significant increase in kidney weight in WT males (12%), but not in KO males or in either genotype of female mice. Kidney enlargement in the WT males was accompanied by a small, insignificant fall in protein-to-DNA ratio, supporting hyperplasia rather than hypertrophy. Fructose feeding of male WT mice led to significantly higher sodium bicarbonate exchanger (NBCe1), sodium hydrogen exchanger (NHE3), sodium phosphate co-transporter (NaPi-2), and transforming growth factor-β (TGF-β) abundances, as compared to male KO, suggesting elevated transport capacity and an early feature of fibrosis may have accompanied the renal enlargement. Overall, IR and/or IGF1R appear to have a role in PT cell size and enlargement in response to high-fructose diet. [ABSTRACT FROM AUTHOR]

Women consuming molecular iodine (l2) through seaweeds suffer the least from breast cancers. Zoledronate (Zol) is in clinical use for alleviation of bone pain in cancer patients. Triple negative breast cancers exhibit high mortality due to lack of neoadjuvant chemotherapy. l2 and Zol independently cause weak antiproliferative and apoptotic effect. So far, their combined effects have not been tested. We analyzed the effect of combination of l2 with Zol as a potent adjuvant therapeutic agent for triple negative breast cancer cells (MDA-MBA-231) and in the mice model of breast cancer. Cell viability, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, Western blotting, real-time PCR, flow cytometry, and other assays were performed for assessing cell death, calcium levels, and migration potential, respectively, in treated cells. The increased caspase 8, increased [Ca2+]c levels, and endoplasmic reticulum (ER) stress resulted in apoptosis. Real time and fluorescence-based analysis demonstrated that the combination treatment targets ER Ca2+ homeostasis chaperons leading to apoptosis. Combination therapy reduces metalloproteinases 2 and 9, inhibits invasion/migration of cells, and prevents growth of tumor in mice. l2 C Zol combination treatment induces synergistic increase in ER-mediated apoptosis, reduces invasion/migration potential of MDA-MB-231 cells, and exhibits antiproliferative property in vivo demonstrating its potential as combination therapy. [ABSTRACT FROM AUTHOR]

Non-invasive renal signatures can help in serial monitoring of diabetic patients. We tested whether urinary exosomal (UE) microRNA (miR) analysis could non-invasively predict renal pathology in diabetic rats during the course of diabetes. Diabetes mellitus (DM) was induced in male Wistar rats by a single intraperitoneal injection of streptozotocin (STZ, 50 mg/kg body weight). Non-diabetic control (CTRL) rats were injected with vehicle. Insulin (INS) treatment (5U/d, s.c.) was provided to 50% of the DM rats. Urine samples were collected at weeks 3, 6, and 9 following injections and UE prepared. An increase in miR-451-5p and miR-16, observed by pilot small RNA sequencing of UE RNA, was confirmed by quantitative real-time polymerase chain reaction (qPCR) and selected for further study. Subsets of rats were euthanized after 3, 6, and 9 weeks of diabetes for renal pathology analysis, including determination of the tubulointerstitial fibrotic index (TFI) and glomerulosclerotic index (GI) scores. qPCR showed a substantial rise in miR-451-5p in UE from DM rats during the course of diabetes, with a significant rise (median fold change >1000) between 3 and 6 weeks. Moreover, UE miR-451-5p at 6 weeks predicted urine albumin at 9 weeks (r = 0.76). A delayed but significant rise was also observed for miR-16. In contrast, mean urine albumin only increased 21% between 3 and 6 weeks (non-significant rise), and renal TFI and GI were unchanged till 9 weeks. Renal expression of miR-451-5p and miR-16 (at 10 weeks) did not correlate with urine levels, and moreover, was negatively associated with indices of renal pathology (r≥-0.70, p = 0.005 for TFI and r≥-0.6, p≤0.02 for GI). Overall, a relative elevation in renal miR-451-5p and miR-16 in diabetes appeared protective against diabetes-induced kidney fibrosis; while UE miR-451-5p may hold prognostic value as an early and sensitive non-invasive indicator of renal disease. [ABSTRACT FROM AUTHOR]

Aims: CKD-EPI (chronic kidney disease-epidemiological) serum creatinine equation is widely accepted for calculating estimated glomerular filtration rate (eGFR). The effect of transitioning from the older 2009 to the newer race-independent 2021 CKD-EPI equation on the estimated kidney disease burden (eKDB) was studied in an Asian-Indian population.Methods: The study included 1156 adults, the two equations were compared for agreement (Bland-Altman and Cohen's kappa) and concordance (Lin's correlation and test for proportions).Results: The 2021 CKD-EPI increased the eGFR (positive-bias), independent of age-group, gender or presence of type 2 diabetes mellitus (T2DM) and hypertension (HTN). Thus, the eKDB was significantly decreased by 2021 CKD-EPI equation. The agreement was highest for the age-group 31-40 years (95.8 % versus 87.5 % for > 50 years). Besides, the eGFR category was shifted from G3 to G1 in 8.2 % (95 % CI: 6.8-9.9) individuals by 2021 CKD-EPI. The effect of transition on eKDB was greater in individuals > 50 years (7.4 %) or with HTN (6.3 %).Conclusion: In comparison to the old equation, the 2021 CKD-EPI equation increased the eGFR, lowering the eKDB in this Asian-Indian cohort. The degree of lowering was affected by age-group, and presence of T2DM /HTN, but independent of gender. [ABSTRACT FROM AUTHOR]

The kidney is an important organ for arterial blood pressure (BP) maintenance. Reduced NO generation in the kidney is associated with hypertension in insulin resistance. NO is a critical regulator of vascular tone; however, whether insulin regulates NO production in the renal inner medullary collecting duct (IMCD), the segment with the greatest enzymatic activity for NO production in kidney, is not clear. Using an NO-sensitive 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) fluorescent dye, we found that insulin increased NO production in mouse IMCD cells (mIMCD) in a time- and dose-dependent manner. A concomitant dose-dependent increase in the NO metabolite (NOx) was also observed in the medium from insulin- stimulated cells. NO production peaked in mIMCD cells at a dose of 100 nM insulin with simultaneously increased Nox levels in the medium. At this dose, insulin significantly increased p-eNOSSer1177 levels in mIMCD cells. Pretreatment of cells with a PI 3-kinase inhibitor or insulin receptor silencing with RNA interference abolished these effects of insulin, whereas insulinlike growth factor-1 receptor (IGF-1R) silencing had no effect. We also showed that chronic insulin infusion to normal C57BL/6J mice resulted in increased endothelial NOS (eNOS) protein levels and NO production in the inner medulla. However, insulin-infused IRKO mice, with targeted deletion of insulin receptor from tubule epithelial cells of the kidney, had~50% reduced eNOS protein levels in their inner medulla along with a significant rise in BP relative to WT littermates. We have previously reported increased baseline BP and reduced urine NOx in IRKO mice. Thus, reduced insulin receptor signaling in IMCD could contribute to hypertension in the insulin-resistant state. [ABSTRACT FROM AUTHOR]

Background: Podocyte injury is an early feature of diabetic nephropathy (DN). Recently, urinary exosomal Wilm's tumor-1 protein (WT1), shed by renal epithelial cells, has been proposed as a novel biomarker for podocyte injury. However, its usefulness as biomarker for early diabetic nephropathy has not been verified yet. We investigated urinary exosomal WT1 in type-1 diabetic patients to confirm its role as a non-invasive biomarker for predicting early renal function decline. Methods: The expression of WT1 protein in urinary exosomes from spot urine samples of type-1 diabetes mellitus patients (n = 48) and healthy controls (n = 25) were analyzed. Patients were divided based on their urinary albumin excretion, ACR (mg/g creatinine) into non- proteinuria group (ACR<30 mg/g, n = 30) and proteinuria group (ACR>30 mg/g, n = 18). Regression analysis was used to assess the association between urinary exosomal levels of WT1 with parameters for renal function. Receiver Operating Characteristic (ROC) curve analysis was used to determine the diagnostic performance of exosomal WT-1. Results: WT1 protein was detected in 33 out of 48 diabetic patients and in only 1 healthy control. The levels of urinary exosomal WT1 protein is significantly higher (p = 0.001) in patients with proteinuria than in those without proteinuria. In addition, all the patients with proteinuria but only half of the patients without proteinuria were positive for exosomal WT1. We found that the level of exosomal WT1 were associated with a significant increase in urine protein-to-creatinine ratio, albumin-to-creatinine ratio, and serum creatinine as well as a decline in eGFR. Furthermore, patients exhibiting WT1-positive urinary exosomes had decreased renal function compared to WT1-negative patients. ROC analysis shows that WT-1 effectively predict GFR<60 ml. min-1/1.73 m2. Conclusion: The predominant presence of WT1 protein in urinary exosomes of diabetic patients and increase in its expression level with decline in renal function suggest that it could be useful as early non-invasive marker for diabetic nephropathy. [ABSTRACT FROM AUTHOR]

To elucidate the role of the insulin receptor (IR) in collecting duct (CD), we bred mice with IR selectively deleted from CD principal cells using an aquaporin-2 promoter to drive Cre-recombinase expression. Young, adult male knockout (KO) mice had altered plasma and electrolyte homeostasis under high- (HS) and low-sodium (LS) diets, relative to wild-type (WT) littermates. One week of LS feeding led to a significant reduction in urine potassium (K+) and sodium (Na+) excretion in KO, and a reduction in the ratio of Na+ to chloride (Cl-) in plasma, relative to WT. HS diet (1 wk) increased plasma K+ and reduced urine Na+ to Cl- ratio in the KO. Furthermore, KO mice had a significantly (P = 0.025) blunted natriuretic response to benzamil, an epithelial sodium channel (ENaC) antagonist. Western blotting of cortex homogenates revealed modestly, but significantly (~15%), lower band density for the β-subunit of ENaC in the KO vs. WT mice, with no differences for the α- or γ-subunits. Moreover, blood pressure (BP), measured by radiotelemetry, was significantly lower in KO vs. WT mice under basal conditions (mmHg): 112 ± 5 (WT), 104 ± 2 (KO), P = 0.023. Chronic insulin infusion reduced heart rate in the WT, but not in the KO, and modestly reduced BP in the WT only. Overall, these results support a fundamental role for insulin through its classic receptor in the modulation of electrolyte homeostasis and BP. [ABSTRACT FROM AUTHOR]

Maternal hypothyroidism affects postnatal lung structure. High prevalence of hypothyroxinemia (low T4, normal T3) in iodine-deficient pregnant women and associated risk for neuropsychological development along with high infant/neonatal mortality ascribed to respiratory distress prompted us to study the effects of maternal hypothyroxinemia on postnatal lung development. Female Sprague Dawley rats were given a low-iodine diet (LID) with 1% KClO4 in drinking water for 10 days, to minimize thyroid hormone differences. Half of these rats were continued on iodine-deficient diet; ID (LID with 0.005% KClO4) for 3 mo, whereas the rest were switched to an iodine-sufficient diet; IS [LID + potassium iodide (10μg iodine/20 g of diet + normal drinking water)]. Pups born to ID mothers were compared with age-matched pups from IS mothers at postnatal days 8 (P8) and 16 (P16) (n = 6-8/group). ID pups had normal circulating T3 but significantly low T4 levels (P < 0.05) and concomitantly approximately sixfold higher thyroid hormone receptor-β mRNA in alveolar epithelium. Lung histology revealed larger and irregularly shaped alveoli in ID pups relative to controls. Lung function was assessed at P16 using a double-chambered plethysmograph and observed reduced tidal volume, peak inspiratory and expiratory flow, and dynamic lung compliance in ID pups compared with IS pups. Significant lowering of surfactant protein (SP)-B and SP-C mRNA and protein found in ID pups at P16. ID pups had 16-fold lower matrix metalloproteinase-9 mRNA levels in their alveolar epithelium. In addition, mRNA levels of thyroid transcription factor-1 and SP-D were significantly higher (3-fold) compared with IS pups. At P16, significantly lower levels of SP-B and SP-C found in ID pups may be responsible for immature lung development and reduced lung compliance. Our data suggest that maternal hypothyroxinemia may result in the development of immature lungs that, through respiratory distress, could contribute to the observed high infant mortality in ID neonates. [ABSTRACT FROM AUTHOR]

Abstract: Estrogen receptor negative (ER−ve) and p53 mutant breast tumors are highly aggressive and have fewer treatment options. Previously, we showed that molecular Iodine (I2) induces apoptosis in hormone responsive MCF-7 breast cancer cells, and non-apoptotic cell death in ER−ve–p53 mutant MDA-MB231 cells (Shrivastava, 2006). Here we show that I2 (3μM) treatment enhanced the features of autophagy in MDA-MB231 cells. Since autophagy is a cell survival response to most anti-cancer therapies, we used both in vitro and in vivo systems to determine whether ER−ve mammary tumors could be sensitized to I2-induced apoptosis by inhibiting autophagy. Autophagy inhibition with chloroquine (CQ) and inhibitors for PI3K (3MA, LY294002) and H+/ATPase (baflomycin) resulted in enhanced cell death in I2 treated MDA-MB231 cells. Further, CQ (20μM) in combination with I2, showed apoptotic features such as increased sub-G1 fraction (∼5-fold), expression of cleaved caspase-9 and -3 compared to I2 treatment alone. Flowcytometry of I2 and CQ co-treated cells revealed increase in mitochondrial membrane permeability (p <0.01) and translocation of cathepsin D activity to cytosol relative to I2 treatment. For in vivo studies ICRC mice were transplanted subcutaneously with MMTV-induced mammary tumors. A significant reduction in tumor volumes, as measured by MRI, was found in I2 and CQ co-treated mice relative to I2 or vehicle treated mice. These data indicate that inhibition of autophagy renders ER−ve breast tumor cells more sensitive to I2 induced apoptosis. Thus, I2 together with autophagy inhibitor could have a potential tumorostatic role in ER−ve aggressive breast tumors that may be evaluated in future studies. [Copyright &y& Elsevier]

The effect of candesartan, an angiotensin-II type-1 receptor antagonist, on the metabolic profile and renal inflammation is unclear. We evaluated this relationship by feeding male lean (LZ) and obese (OZ) Zucker rats chow or chow with candesartan (23.5mg/kg·diet) for 14 weeks (n = 6-8/treatment/body type). Candesartan reduced serum triglycerides, plasma creatinine, urine albumin, and renal cortical collagen and glycogen deposition in the OZ. An ELISA-based cytokine array revealed that candesartan normalized elevated renal interleukin (IL) 1-β and monocyte chemoattractant protein-1 (MCP-1) levels in OZ. Nonetheless, candesartan impaired glucose tolerance, and did not lower blood insulin or glucose levels. Moreover, renal IL-1α, -2, -4, -6 and -10 tumor necrosis factor-α, interferon-γ, were significantly reduced in OZ relative to LZ, and increased by candesartan. Furthermore, candesartan increased growth-regulated oncogene, transforming growth factor-β1 and IL-18 in OZ kidneys to a level higher than LZ or untreated OZ. Candesartan did not affect renal cytokine levels in LZ. Overall, candesartan attenuated renal disease and improved renal function in OZ, despite mixed effects on metabolic factors and cytokines. Reduced plasma triglycerides and/or renal MCP-1 and IL-1β may have had a role in this protection. However, these effects were clearly independent of any improvement in glucose tolerance. [ABSTRACT FROM AUTHOR]

PPAR-γ agonists have been associated with heart failure (HF) in diabetic patients. These incidences have been reported mostly in patient populations who were at high risk for HF or had pre-existing impaired cardiovascular function. However, whether there are similar effects of these agents in subjects with no or reduced cardiovascular pathophysiology is not clear. In this study, the effects of chronic treatment with PD168, a potent peroxisome proliferator activated receptor (PPAR) subtype-γ agonist with weak activity at PPAR-α, and rosiglitazone (RGZ), a less potent PPAR-γ agonist with no PPAR-α activity, were evaluated on the cardiovascular-renal system in healthy male Sprague-Dawley (SD) rats by serial echocardiography and radiotelemetry. Rats were treated with vehicle (VEH), PD168, @ 10 or 50 mg/kg·bw/day (PD-10 or PD-50, resp.) or RGZ @ 180 mg/kg·bw/day for 28 days (n= 10/group). Relative to VEH, RGZ, and both doses of PD168 resulted in a significant fall in blood pressure. Furthermore, RGZ and PD168 increased plasma volume (% increase from baseline) 18%, 22%, and 48% for RGZ, PD-10, and PD-50, respectively. PD168 and RGZ significantly increased urinary aldosterone excretion and heart-to-body weight ratio relative to VEH. In addition, PD168 significantly decreased (10-16%) cardiac ejection fraction (EF) and increased left ventricular area (LVA) in systole (s) and diastole (d) in PD-10 and -50 rats. RGZ significantly increased LVAd; however, it did not affect EF relative to VEH. In conclusion, chronic PPAR-γ therapy may predispose the cardiorenal system to a potential sequela of structural and/or functional changes that may be deleterious with regard to morbidity and mortality. [ABSTRACT FROM AUTHOR]

Hyperinsulinemia is associated with hypertension. Dysregulation of renal distal tubule sodium reabsorption may play a role. We evaluated the regulation of the epithelial sodium channel (ENaC) and the thiazide-sensitive Na-Cl cotransporter (NCC) during chronic hyperinsulinemia in rats and correlated these changes to blood pressure as determined by radiotelemetry. Male Sprague-Dawley rats (∼270 g) underwent one of the following three treatments for 4 wk (n = 6/group): 1) control; 2) insulin-infused plus 20% dextrose in drinking water; or 3) glucose water-drinking (20% dextrose in water). Mean arterial pressures were increased by insulin and glucose (mmHg at 3 wk): 98 ± 1 (control), 107 ± 2 (insulin), and 109 ± 3 (glucose), P < 0.01. Insulin (but not glucose) increased natriuretic response to benzamil (ENaC inhibitor) and hydrochlorothiazide (NCC inhibitor) on average by 125 and 60%, respectively, relative to control rats, suggesting increased activity of these reabsorptive pathways. Neither insulin nor glucose affected the renal protein abundances of NCC or the ENaC subunits (α, β, and γ) in kidney cortex, outer medulla, or inner medulla in a major way, as determined by immunoblotting. However, insulin and to some extent glucose increased apical localization of these subunits in cortical collecting duct principal cells, as determined by immunoperoxidase labeling. In addition, insulin decreased cortical ‘with no lysine’ kinase (WNK4) abundance (by 16% relative to control), which may have increased NCC activity. Overall, insulin infusion increased blood pressure, and NCC and ENaC activity in rats. Increased apical targeting of ENaC and decreased WNK4 expression may be involved. [ABSTRACT FROM AUTHOR]

Aim: Type 2 diabetes (T2DM) and hypertension (HTN) are the main modifiable risk factors of chronic kidney disease (CKD), among the known traditional and non-traditional risk factors.Methods: We determined the prevalence and care-cascade of these modifiable CKD-risk factors and their association with socioeconomic status in adjoining Lucknow and Puducherry cities of India.Results: 439 participants reported no CKD were recruited. Serum analysis revealed an Estimated Glomerular Filtration Rate (eGFR) ≥ 90 ml/min/1.73 m2 in 60.36% of the population. Of them, 55.85% had HTN and/or T2DM as CKD-risk factors; however, less than half of this population was unaware of their CKD-risk status. Awareness and treatment were significantly higher in Puducherry and were associated with literacy, occupation, and residence place. Although the CKD-risk population was about two times higher in Puducherry than Lucknow, the populations with mild-low eGFR were comparable in the two regions. Moreover, in Lucknow, mild-low eGFR and low awareness were more prevalent among the younger participants (<30 years), relative to Puducherry.Conclusions: Despite a higher prevalence of CKD-risk factors in Puducherry, populations with mild-low eGFR were comparable to Lucknow. More heightened awareness and better care cascade for CKD-risk factors in Puducherry may prevent or delay eGFR reduction. [ABSTRACT FROM AUTHOR]

Mitochondria serve a vital role in cellular homeostasis as they regulate cell proliferation and death pathways, which are attributed to mitochondrial bioenergetics, free radicals and metabolism. Alterations in mitochondrial functions have been reported in various diseases, including cancer. Colorectal cancer (CRC) is one of the most common metastatic cancer types with high mortality rates. Although mitochondrial oxidative stress has been associated with CRC, its specific mechanism and contribution to metastatic progression remain poorly understood. Therefore, the aims of the present study were to investigate the role of mitochondria in CRC cells with low and high metastatic potential and to evaluate the contribution of mitochondrial respiratory chain (RC) complexes in oncogenic signaling pathways. The present results demonstrated that cell lines with low metastatic potential were resistant to mitochondrial complex I (C-I)-mediated oxidative stress, and had C-I inhibition with impaired mitochondrial functions. These adaptations enabled cells to cope with higher oxidative stress. Conversely, cells with high metastatic potential demonstrated functional C-I with improved mitochondrial function due to coordinated upregulation of mitochondrial biogenesis and metabolic reprogramming. Pharmacological inhibition of C-I in high metastatic cells resulted in increased sensitivity to cell death and decreased metastatic signaling. The present findings identified the differential regulation of mitochondrial functions in CRC cells, based on CRC metastatic potential. Specifically, it was suggested that a functional C-I is required for high metastatic features of cancer cells, and the role of C-I could be further examined as a potential target in the development of novel therapies for diagnosing high metastatic cancer types. [ABSTRACT FROM AUTHOR]

COVID-19 novel coronavirus (CoV) disease caused by severe acquired respiratory syndrome (SARS)-CoV-2 manifests severe lethal respiratory illness in humans and has recently developed into a worldwide pandemic. The lack of effective treatment strategy and vaccines against the SARS-CoV-2 poses a threat to human health. An extremely high infection rate and multi-organ secondary infection within a short period of time makes this virus more deadly and challenging for therapeutic interventions. Despite high sequence similarity and utilization of common host-cell receptor, human angiotensin-converting enzyme-2 (ACE2) for virus entry, SARS-CoV-2 is much more infectious than SARS-CoV. Structure-based sequence comparison of the N-terminal domain (NTD) of the spike protein of Middle East respiratory syndrome (MERS)-CoV, SARS-CoV, and SARS-CoV-2 illustrate three divergent loop regions in SARS-CoV-2, which is reminiscent of MERS-CoV sialoside binding pockets. Comparative binding analysis with host sialosides revealed conformational flexibility of SARS-CoV-2 divergent loop regions to accommodate diverse glycan-rich sialosides. These key differences with SARS-CoV and similarity with MERS-CoV suggest an evolutionary adaptation of SARS-CoV-2 spike glycoprotein reciprocal interaction with host surface sialosides to infect host cells with wide tissue tropism. [ABSTRACT FROM AUTHOR]

Insulin therapy is often needed to overcome insulin receptor resistance in type 2 diabetes; however, the impact of providing additional insulin to already hyperinsulinemic subjects is not clear. We infused male TALLYHO/Jng (TH) mice (insulin resistant) with insulin (50 U/kg·bw/d) or vehicle (control) by osmotic minipump for 14 days. One group of insulin-infused mice was switched to 4% NaCl diet (high-sodium diet, HSD) in the second week. Blood chemistry revealed a significantly higher anion gap and blood sodium concentrations with insulin infusion, i.e., relative metabolic acidosis. Systolic BP and heart rate were slightly (~5 mm Hg) higher in insulin-infused versus control mice. HSD resulted in a modest and transient rise in mean arterial blood pressure (BP), relative to control or insulin-infused, normal-NaCl-fed mice. In kidney, insulin infusion: (1) increased total and phosphorylated (serine-1177) endothelial nitric oxide synthase (eNOS) band densities; (2) reduced band density of the uncoupled form of eNOS; and (3) increased renal homogenate nitric oxide synthase (NOS) activity. Despite this, plasma and urine levels of nitrates plus nitrites (NOx) fell with insulin infusion, by day 14 (40–50%) suggesting worsening of resistance. Overall, insulin infusion ramps up the cellular means in kidney to increase vasodilatory and natriuretic NO, but in the long term may be associated with worsening of insulin receptor resistance. [ABSTRACT FROM AUTHOR]