Myocardial ischemia-reperfusion injury released cellular fibronectin containing domain A (CFN-EDA): A destructive positive loop amplifying arterial thrombosis formation and exacerbating myocardial reperfusion injury.

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.

Macrophage derived Exosomal Docetaxel (Exo-DTX) for pro-metastasis suppression: QbD driven formulation development, validation, in-vitro and pharmacokinetic investigation.

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 IC50 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 ß1-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 ß1-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 t1/2 and Vd, 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.

Association of Wilms tumor-1 protein in urinary exosomes with kidney injury: a population-based cross-sectional study.

Objective: Loss of Wilms tumor-1 (WT1) protein, a podocytopathy marker, through urine exosome (uE), could be an early indication of kidney injury. We examined WT1 in uE (uE-WT1), along with other urine markers of glomerular and kidney tubule injury, in individuals without chronic kidney disease (CKD). Methodology: The cross-sectional study included individuals who reported having no evidence of chronic kidney disease (CKD). Albumin-to-creatinine ratio (ACR) and estimated glomerular filtration rate (eGFR) were used to assess kidney function. eGFR was calculated using the 2009 CKD-EPI (CKD-Epidemiological) equation. WT1 was analyzed in uE from humans and Wistar rats (before and after the 9th week of diabetes, n = 20). uE-WT1, urinary neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule-1 (KIM-1) were estimated using ELISA. The Kruskal-Wallis H test, Mann-Whitney U test, and stepwise multivariable linear regression were performed. Results: Urine NGAL and ACR increase with uE-WT1 quartiles (n = 146/quarter). Similarly, uE-WT1, KIM-1, and NGAL were positively associated with ACR. Furthermore, KIM-1, NGAL, and uE-WT1 correlated with ACR. uE-WT1 outperformed KMI-1 and NGAL to explain ACR variability (25% vs. 6% or 9%, respectively). Kidney injury in streptozotocin-induced diabetic rats was associated with a significant rise in uE-WT1. Moreover, the findings were confirmed by the histopathology of kidney tissues from rats. Conclusion: uE-WT1 was strongly associated with kidney function in rats. In individuals without CKD, uE-WT1 outperformed NGAL as a determinant of differences in ACR.

Mesenchymal Stromal Cell-Derived Small Extracellular Vesicles Modulate Apoptosis, TNF Alpha and Interferon Gamma Response Gene mRNA Expression in T Lymphocytes.

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.

Erratum: [Corrigendum] Differential regulation of mitochondrial complex I and oxidative stress based on metastatic potential of colorectal cancer cells.

[This corrects the article DOI: 10.3892/ol.2020.12176.].

Therapeutic potential of urine exosomes derived from rats with diabetic kidney disease.

Kidney disease is prevalent in diabetes. Urinary exosomes (uE) from animal models and patients with Diabetic nephropathy (DN) showed increased levels of miRs with reno-protective potential. We examined whether urinary loss of such miRs is associated with their reduced renal levels in DN patients. We also tested whether injecting uE can leverage kidney disease in rats. In this study (study-1) we performed microarray profiling of miRNA in uE and renal tissues in DN patients and subjects with diabetes without DN (controls). In study-2, diabetes was induced in Wistar rats by Streptozotocin (i.p. 50 mg/kg of body weight). Urinary exosomes were collected at 6th, 7th and 8th weeks, and injected back into the rats (100ug/biweekly, uE-treated n=7) via tail vein on weeks 9 and 10. Equal volume of vehicle was injected in controls (vehicle, n=7). uE from the human and rat showed the presence of exosome-specific proteins by immunoblotting. Microarray profiling revealed a set of 15 miRs having high levels in the uE, while lower in renal biopsies, from DN, compared to controls (n=5-9/group). Bioinformatic analysis also confirmed the Renoprotective potential of these miRs. Taqman qPCR confirmed the opposite regulation of miR-200c-3p and miR-24-3p in paired uE and renal biopsy samples from DN patients (n=15), relative to non-DN controls. A rise in 28 miRs levels, including miR-200c-3p, miR-24-3p, miR-30a-3p and miR-23a-3p were observed in the uE of DN rats, collected between 6th-8th weeks, relative to baseline (before diabetes induction). uE- treated DN rats had significantly reduced urine albumin-to-creatinine ratio, attenuated renal pathology, and lower miR-24-3p target fibrotic/inflammatory genes (TGF-beta, and Collagen IV), relative to vehicle treated DN rats. In uE treated rats, the renal expression of miR-24-3p, miR-30a-3p, let-7a-5p and miR-23a-3p was increased, relative to vehicle control. Patients with diabetic nephropathy had reduced renal levels, while higher uE abundance of miRs with reno-protective potential. Reverting the urinary loss of miRs by injecting uE attenuated renal pathology in diabetic rats.

TRAIL and EGFR Pathways Targeting microRNAs are Predominantly Regulated in Human Diabetic Nephropathy.

BACKGROUND: Unbiased microRNA profiling of renal tissue and urinary extracellular vesicles (uEVs) from diabetic nephropathy (DN) subjects may unravel novel targets with diagnostic and therapeutic potential. Here we used the miRNA profile of uEVs and renal biopsies from DN subjects available on the GEO database. METHODS: The miR expression profiles of kidney tissue (GSE51674) and urinary exosomes (GSE48318) from DN and control subjects were obtained by GEO2R tools from Gene Expression Omnibus (GEO) databases. Differentially expressed miRNAs in DN samples, relative to controls, were identified using a bioinformatic pipeline. Targets of miRs commonly regulated in both sample types were predicted by miRWalk, followed by functional gene enrichment analysis. Gene targets were identified by MiRTarBase, TargetScan and MiRDB. RESULTS: Eight miRs, including let-7c, miR-10a, miR-10b and miR-181c, were significantly regulated in kidney tissue and uEVs in DN subjects versus controls. The top 10 significant pathways targeted by these miRs included TRAIL, EGFR, Proteoglycan syndecan, VEGF and Integrin Pathway. Gene target analysis by miRwalk upon validation using ShinyGO 70 targets with significant miRNA-mRNA interaction. CONCLUSION: In silico analysis showed that miRs targeting TRAIL and EGFR signaling are predominately regulated in uEVs and renal tissue of DN subjects. After wet-lab validation, the identified miRstarget pairs may be explored for their diagnostic and/or therapeutic potential in diabetic nephropathy.

Human umbilical cord blood-mesenchymal stem cell derived exosomes as an efficient nanocarrier for Docetaxel and miR-125a: Formulation optimization and anti-metastatic behaviour.

AIM: 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. MAIN METHODS: 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. KEY FINDINGS: 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 IC50 at 192.8 ng/ml in 4T1 cells, which is almost 2.36 folds' lower than the free DTX IC50 (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. SIGNIFICANCE: 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.

Immunocyte Derived Exosomes: Insight into the Potential Chemo-immunotherapeutic Nanocarrier Targeting the Tumor Microenvironment.

"Cancer" is a dreadful immune-pathological condition that is characterized by anti-inflammatory and tumorigenic responses, elicited by the infiltrating immune cells in the vicinity of an uncontrollably proliferative tumor in the tumor microenvironment (TME). The TME offers a conducive microenvironment that supports cancer cell survival by modulating the host immune defense. Recent advancement in exosomal research has shown exosomes, originating from immune cells as well as the cancer cells, have immense potential for suppressing cancer progression and survival in the TME. Additionally, exosomes, irrespective of their diverse sources, have been reported to be efficient nanocarriers for cancer therapeutics with the ability for targeted delivery due to their biogenic nature, ease of cellular uptake, and scope for functionalization with biomolecules like peptides, aptamers, targeting ligands, etc. Immune cell-derived exosomes per se have been found efficacious against cancer owing to their immune-stimulant properties (in either naive or antigen primed form) even without loading any of cancer therapeutics or targeting ligand conjugation. Nevertheless, exosomes are being primarily explored as nanovesicular carriers for therapeutic molecules with different loading and targeting strategies, and the synergism between immunotherapeutic behavior of exosomes and the anticancer effect of the therapeutic molecules is yet to be explored. Hence, this review focuses specifically on the possible strategies to modulate the immunological nature of the source immune cells to obtain immune stimulant exosomes and bring these into the spotlight as chemo-immunotherapeutic nanovesicles, that can easily target and modulate the TME.

An In-Silico Approach to Identify Therapeutic Target and Markers Associated with Diabetic Nephropathy.

BACKGROUND: Renal disease in T2DM could arise independent of hyperglycemia, aka non diabetic kidney disease. Their prevalence ranges from 33%to72.5% among T2DM patients. Specific molecular signatures that distinguish Diabetic Nephropathy from NDKD (FSGS) in T2DM might provide new targets for CKD management. METHODS: Five original GEO microarray DN and FSGS datasets were evaluated (GSE111154, GSE96804, GSE125779, GSE129973 and GSE121233). Each of the three groups (DN, FSGS, and Controls) had equal renal transcriptome data (n=32) included in the analysis to eliminate bias. The DEGs were identified using TAC4.0. Pathway analysis was performed on the discovered genes that aligned to official gene symbols using Reactome, followed by functional gene enrichment analysis using Funrich,Enrichr. STRING and Network analyst investigated PPI, followed by Webgestalt's pathway enrichment. Finally, using the Targetscan7.0 and DIANA tools, filtered differential microRNAs downregulated in DN were evaluated for target identification. RESULT: Between the three groups, DN, FSGS, and Control, a total of 194 DEGs. with foldchange >2&<-2 and P-value0.01 were found in the renal transcriptome. In comparison to control, 45 genes were elevated particularly in DN, whereas 43 were upregulated specifically in FSGS. DN datasets were compared to FSGS in a separate analysis. FABP4, EBF1, ADIRF, and ART4 were shown to be among the substantially up-regulated genes unique to DN in both analyses. The transcriptional regulation of white adipocytes was discovered by a pathway analysis. CONCLUSION: The molecular markers revealed might be employed as specific targets in the aetiology of DN, as well as in T2DM patients' therapeutic care.

Development of RNA-Based Assay for Rapid Detection of SARS-CoV-2 in Clinical Samples.

INTRODUCTION: The ongoing spread of pandemic coronavirus disease-19 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of growing concern. Rapid diagnosis and management of SARS-CoV-2 are crucial for controlling the outbreak in the community. Here, we report the development of a first rapid-colorimetric assay capable of detecting SARS-CoV-2 in the human nasopharyngeal RNA sample in less than 30 min. METHOD: We utilized a nanomaterial-based optical sensing platform to detect RNA-dependent RNA polymerase gene of SARS-CoV-2, where the formation of oligo probe-target hybrid led to salt-induced aggregation and change in gold-colloid color from pink to blue visibility range. Accordingly, we found a change in colloid color from pink to blue in assay containing nasopharyngeal RNA sample from the subject with clinically diagnosed COVID-19. The colloid retained pink color when the test includes samples from COVID-19 negative subjects or human papillomavirus-infected women. RESULTS: The results were validated using nasopharyngeal RNA samples from positive COVID-19 subjects (n = 136). Using real-time polymerase chain reaction as gold standard, the assay was found to have 85.29% sensitivity and 94.12% specificity. The optimized method has detection limit as little as 0.5 ng of SARS-CoV-2 RNA. CONCLUSION: We found that the developed assay rapidly detects SARS-CoV-2 RNA in clinical samples in a cost-effective manner and would be useful in pandemic management by facilitating mass screening.

Lipid Metabolism and Mitochondria: Cross Talk in Cancer.

Metabolic reprogramming is considered a major event in cancer initiation, progression and metastasis. The metabolic signature of cancer cells includes alterations in glycolysis, mitochondrial respiration, fatty acid/lipid and amino acid metabolism. Being at a junction of various metabolic pathways, mitochondria play a key role in fueling cancer growth through regulating bioenergetics, metabolism and cell death. Increasing evidence suggests that alteration in lipid metabolism is a common feature of metastatic progression, including fatty acid synthesis as well as fatty acid oxidation. However, the interplay between lipid metabolism and mitochondria in carcinogenesis remains obscure. The present review focuses on key lipid metabolic pathways associated with mitochondrial regulation that drive cancer phenotype and metastasis. We also review potential targets of lipid metabolism and mitochondria to improve the therapeutic regime in cancer patients. This review aims to improve our current understanding of the intricate relation of lipids with mitochondria and provides insights into new therapeutic approaches.

Exosomes Secreted by Umbilical Cord Blood-Derived Mesenchymal Stem Cell Attenuate Diabetes in Mice.

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 × 105 umbilical cord blood MSC cells/mice/day), their derived exosomes (the MSC-Exo group that received exosomes derived from 1 × 105 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 ß 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.

Association of kidney functions with a cascade of care for diabetes and hypertension in two geographically distinct Indian cohorts.

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.

Modeling Type 1 Diabetes Using Pluripotent Stem Cell Technology.

Induced pluripotent stem cell (iPSC) technology is increasingly being used to create in vitro models of monogenic human disorders. This is possible because, by and large, the phenotypic consequences of such genetic variants are often confined to a specific and known cell type, and the genetic variants themselves can be clearly identified and controlled for using a standardized genetic background. In contrast, complex conditions such as autoimmune Type 1 diabetes (T1D) have a polygenic inheritance and are subject to diverse environmental influences. Moreover, the potential cell types thought to contribute to disease progression are many and varied. Furthermore, as HLA matching is critical for cell-cell interactions in disease pathogenesis, any model that seeks to test the involvement of particular cell types must take this restriction into account. As such, creation of an in vitro model of T1D will require a system that is cognizant of genetic background and enables the interaction of cells representing multiple lineages to be examined in the context of the relevant environmental disease triggers. In addition, as many of the lineages critical to the development of T1D cannot be easily generated from iPSCs, such models will likely require combinations of cell types derived from in vitro and in vivo sources. In this review we imagine what an ideal in vitro model of T1D might look like and discuss how the required elements could be feasibly assembled using existing technologies. We also examine recent advances towards this goal and discuss potential uses of this technology in contributing to our understanding of the mechanisms underlying this autoimmune condition.

Circulating Placental Alkaline Phosphatase Expressing Exosomes in Maternal Blood Showed Temporal Regulation of Placental Genes.

Background: Analysis of placental genes could unravel maternal-fetal complications. However, inaccessibility to placental tissue during early pregnancy has limited this effort. We tested if exosomes (Exo) released by human placenta in the maternal circulation harbor crucial placental genes. Methods: Placental alkaline phosphate positive exosomes (ExoPLAP) were enriched from maternal blood collected at the following gestational weeks; 6-8th (T1), 12-14th (T2), 20-24th (T3), and 28th-32nd (T4). Nanotracking analysis, electron microscopy, dynamic light scattering, and immunoblotting were used for characterization. We used microarray for transcriptome and quantitative PCR (qPCR) for gene analysis in ExoPLAP. Results: Physical characterization and presence of CD63 and CD9 proteins confirmed the successful ExoPLAP enrichment. Four of the selected 36 placental genes did not amplify in ExoPLAP, while 32 showed regulations (n = 3-8/time point). Most genes in ExoPLAP showed significantly lower expression at T2-T4, relative to T1 (p < 0.05), such as NOS3, TNFSF10, OR5H6, APOL3, and NEDD4L. In contrast, genes, such as ATF6, NEDD1, and IGF2, had significantly higher expression at T2-T4 relative to T1. Unbiased gene profiling by microarray also confirmed expression of above genes in ExoPLAP-transcriptome. In addition, repeated measure ANOVA showed a significant change in the ExoPLAP transcriptome from T2 to T4 (n = 5/time point). Conclusion: Placental alkaline phosphate positive exosomes transcriptome changed with gestational age advancement in healthy women. The transcriptome expressed crucial placental genes involved in early embryonic development, such as actin cytoskeleton organization, appropriate cell positioning, DNA replication, and B-cell regulation for protecting mammalian fetuses from rejection. Thus, ExoPLAP in maternal blood could be a promising source to study the placental genes regulation for non-invasive monitoring of placental health.

Critical role of three-dimensional tumorsphere size on experimental outcome.

Three-dimensional in vitro spheroids are a reliable model to study tumor biology and drug toxicity. However, inconsistencies exist in terms of seeding cell density that governs spheroid size and shape, influencing the experimental outcome. We investigated the effect of varying cell densities using glioblastoma cells on tumorsphere formation and their responsiveness to drug treatment. Our results demonstrated that in comparison with spheroids formed with lower cell density, spheroids formed with higher cell density were not only larger in size but also had a larger necrotic core surrounded by a higher number of quiescent cells and were irresponsive to drug treatment. Our study highlights the importance of predetermination of cell density to obtain desired/appropriate spheroid size to produce consistent and reliable data on drug toxicity studies in tumor cells.

Differential regulation of mitochondrial complex I and oxidative stress based on metastatic potential of colorectal cancer cells.

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.

Systemic inhibition of miR-451 increases fibrotic signaling and diminishes autophagic response to exacerbate renal damage in Tallyho/Jng mice.

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-ß, 14-3-3 protein-ζ, 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 microtubule-associated 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.

Acid Loading Unmasks Glucose Homeostatic Instability in Proximal-Tubule-Targeted Insulin/Insulin-Like-Growth-Factor-1 Receptor Dual Knockout Mice.

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 NH4Cl 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 glucose-6-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.