Quantification of Antiviral Drug Tenofovir (TFV) by Surface-Enhanced Raman Spectroscopy (SERS) Using Cumulative Distribution Functions (CDFs).

Surface-enhanced Raman spectroscopy (SERS) is an ultrasensitive spectroscopic technique that generates signal-enhanced fingerprint vibrational spectra of small molecules. However, without rigorous control of SERS substrate active sites, geometry, surface area, or surface functionality, SERS is notoriously irreproducible, complicating the consistent quantitative analysis of small molecules. While evaporatively prepared samples yield significant SERS enhancement resulting in lower detection limits, the distribution of these enhancements along the SERS surface is inherently stochastic. Acquiring spatially resolved SERS spectra of these dried surfaces, we have shown that this enhancement is governed by a power law as a function of analyte concentration. Consequently, by definition, there is no true mean of SERS enhancement, requiring an alternative approach to achieve reproducible quantitative results. In this study, we introduce a new method of analysis of SERS data using a cumulative distribution function (CDF). The antiviral drug tenofovir (TFV) in an aqueous matrix was quantified down to a clinically relevant concentration of 25 ng/mL using hydroxylamine-reduced silver colloids evaporated to dryness. The data presented in this study provide a rationale for the benefits of combining a novel statistical approach using CDFs with simple and inexpensive experimental techniques to increase the precision, accuracy, and analytical sensitivity of aqueous TFV quantification by SERS. TFV calibration curves generated using CDF analysis showed higher analytical sensitivity (in the form of a normalized calibration curve average slope increase of 0.25) compared to traditional SERS intensity calculations. A second aliquot of nanoparticles and analyte dried on the SERS surface followed by CDF analysis showed further analytical sensitivity with a normalized calibration curve slope increase of 0.23 and decreased variation among replicates represented by an average standard deviation decrease of 0.02 with a second aliquot. The quantitative analysis of SERS data using CDFs presented here shows promise to be a reproducible method for quantitative analysis of SERS data, a significant step toward implementing SERS as an analytical method in clinical and industrial settings.

National and Sub-National Estimates of Household Coverage of Iodized Salt and Urinary Iodine Status among Women of Reproductive Age in India: Insights from the India Iodine Survey, 2018-19.

BACKGROUND: Iodine deficiency is a significant public health problem for many populations worldwide, including India, particularly during the "first 1000 days" of life. Though Universal Salt iodization (USI) is mandatory in India, prior to 2018-19, there was no state-wide survey with estimates of iodine concentrations in salt using iodometric titration. Taking cognizance of this fact, Nutrition International commissioned the first-of-its-kind national-level survey in India, titled the India Iodine Survey 2018-19. OBJECTIVES: The study was conducted across the country to provide national and subnational estimates of iodine concentrations in household salt using iodometric titration and iodine nutrition status among women of reproductive age (15-49 y). METHODS: The survey adopted a multi-stage randomcluster probability proportional to size sampling design, covering 21,406 households in all the states and union territories (UTs) of India. RESULTS: At the national level, the household coverage of edible salt with adequate iodine (content ≥15 parts/million) was 76.3%. At the sub-national level, the coverage varied, with 10 states and 3 UTs achieving USI and 11 states and 2 UTs falling below the national average, with the highest among all the states and UTs, being Jammu and Kashmir and the lowest being Tamil Nadu. At the national level, the median urinary iodine concentration for pregnant women was 173.4 µg/L, for lactating women was 172.8 µg/L, and for non-pregnant, non-lactating women, it was 178.0 µg/L, which is within the adequate iodine nutrition range according to the WHO guidelines. CONCLUSIONS: The survey results can be widely used by various stakeholders, including government, academia, and industry, to understand the iodine nutrition status of the population, enable the scale-up of sustained efforts toward consolidating gains and achieving USI, leading to the reduction and elimination of Iodine Deficiency Disorders.

Quantitative proteomic analytic approaches to identify metabolic changes in the medial prefrontal cortex of rats exposed to space radiation.

NASA's planned mission to Mars will result in astronauts being exposed to ∼350 mSv/yr of Galactic Cosmic Radiation (GCR). A growing body of data from ground-based experiments indicates that exposure to space radiation doses (approximating those that astronauts will be exposed to on a mission to Mars) impairs a variety of cognitive processes, including cognitive flexibility tasks. Some studies report that 33% of individuals may experience severe cognitive impairment. Translating the results from ground-based rodent studies into tangible risk estimates for astronauts is an enormous challenge, but it would be germane for NASA to use the vast body of data from the rodent studies to start developing appropriate countermeasures, in the expectation that some level of space radiation (SR) -induced cognitive impairment could occur in astronauts. While some targeted studies have reported radiation-induced changes in the neurotransmission properties and/or increased neuroinflammation within space radiation exposed brains, there remains little information that can be used to start the development of a mechanism-based countermeasure strategy. In this study, we have employed a robust label-free mass spectrometry (MS) -based untargeted quantitative proteomic profiling approach to characterize the composition of the medial prefrontal cortex (mPFC) proteome in rats that have been exposed to 15 cGy of 600 MeV/n28Si ions. A variety of analytical techniques were used to mine the generated expression data, which in such studies is typically hampered by low and variable sample size. We have identified several pathways and proteins whose expression alters as a result of space radiation exposure, including decreased mitochondrial function, and a further subset of proteins differs in rats that have a high level of cognitive performance after SR exposure in comparison with those that have low performance levels. While this study has provided further insight into how SR impacts upon neurophysiology, and what adaptive responses can be invoked to prevent the emergence of SR-induced cognitive impairment, the main objective of this paper is to outline strategies that can be used by others to analyze sub-optimal data sets and to identify new information.

Space Radiation-Induced Alterations in the Hippocampal Ubiquitin-Proteome System.

Exposure of rodents to <20 cGy Space Radiation (SR) impairs performance in several hippocampus-dependent cognitive tasks, including spatial memory. However, there is considerable inter-individual susceptibility to develop SR-induced spatial memory impairment. In this study, a robust label-free mass spectrometry (MS)-based unbiased proteomic profiling approach was used to characterize the composition of the hippocampal proteome in adult male Wistar rats exposed to 15 cGy of 1 GeV/n 48Ti and their sham counterparts. Unique protein signatures were identified in the hippocampal proteome of: (1) sham rats, (2) Ti-exposed rats, (3) Ti-exposed rats that had sham-like spatial memory performance, and (4) Ti-exposed rats that impaired spatial memory performance. Approximately 14% (159) of the proteins detected in hippocampal proteome of sham rats were not detected in the Ti-exposed rats. We explored the possibility that the loss of the Sham-only proteins may arise as a result of SR-induced changes in protein homeostasis. SR-exposure was associated with a switch towards increased pro-ubiquitination proteins from that seen in Sham. These data suggest that the role of the ubiquitin-proteome system as a determinant of SR-induced neurocognitive deficits needs to be more thoroughly investigated.

Parallel Glyco-SPOT Synthesis of Glycopeptide Libraries.

Glycan recognition is typically studied using free glycans, but glycopeptide presentations represent more physiological conditions for glycoproteins. To facilitate studies of glycopeptide recognition, we developed Glyco-SPOT synthesis, which enables the parallel production of diverse glycopeptide libraries at microgram scales. The method uses a closed system for prolonged reactions required for coupling Fmoc-protected glycoamino acids, including O-, N-, and S-linked glycosides, and release conditions to prevent side reactions. To optimize reaction conditions and sample reaction progress, we devised a biopsy testing method. We demonstrate the efficient utilization of such microscale glycopeptide libraries to determine the specificity of glycan-recognizing antibodies (e.g., CTD110.6) using microarrays, enzyme specificity on-array and in-solution (e.g., ST6GalNAc1, GCNT1, and T-synthase), and binding kinetics using fluorescence polarization. We demonstrated that the glycosylation on these peptides can be expanded using glycosyltransferases both in-solution and on-array. This technology will promote the discovery of biological functions of peptide modifications by glycans.

Identification of Tn antigen O-GalNAc-expressing glycoproteins in human carcinomas using novel anti-Tn recombinant antibodies.

The Tn antigen is a neoantigen abnormally expressed in many human carcinomas and expression correlates with metastasis and poor survival. To explore its biomarker potential, new antibodies are needed that specifically recognize this antigen in tumors. Here we generated two recombinant antibodies to the Tn antigen, Remab6 as a chimeric human IgG1 antibody and ReBaGs6 as a murine IgM antibody and characterized their specificities using multiple biochemical and biological approaches. Both Remab6 and ReBaGs6 recognize clustered Tn structures, but most importantly do not recognize glycoforms of human IgA1 that contain potential cross-reactive Tn antigen structures. In flow cytometry and immunofluorescence analyses, Remab6 recognizes human cancer cell lines expressing the Tn antigen, but not their Tn-negative counterparts. In immunohistochemistry (IHC), Remab6 stains many human cancers in tissue array format but rarely stains normal tissues and then mostly intracellularly. We used these antibodies to identify several unique Tn-containing glycoproteins in Tn-positive Colo205 cells, indicating their utility for glycoproteomics in future biomarker studies. Thus, recombinant Remab6 and ReBaGs6 are useful for biochemical characterization of cancer cells and IHC of tumors and represent promising tools for Tn biomarker discovery independently of recognition of IgA1.

Influenza binds phosphorylated glycans from human lung.

Influenza A viruses can bind sialic acid-terminating glycan receptors, and species specificity is often correlated with sialic acid linkage with avian strains recognizing α2,3-linked sialylated glycans and mammalian strains preferring α2,6-linked sialylated glycans. These paradigms derive primarily from studies involving erythrocyte agglutination, binding to synthetic receptor analogs or binding to undefined surface markers on cells or tissues. Here, we present the first examination of the N-glycome of the human lung for identifying natural receptors for a range of avian and mammalian influenza viruses. We found that the human lung contains many α2,3- and α2,6-linked sialylated glycan determinants bound by virus, but all viruses also bound to phosphorylated, nonsialylated glycans.

Thyroid transcription factor 1 enhances cellular statin sensitivity via perturbing cholesterol metabolism.

We have discovered an unexpected connection between a critical lung development and cancer gene termed thyroid transcription factor 1 (TTF-1 also known as NKX2-1) and cholesterol metabolism. Our published work implicates that TTF-1 positively regulates miR-33a which is known to repress ATP-binding cassette transporter 1 (ABCA1) and thus its cholesterol efflux activity. We set out to demonstrate that a higher TTF-1 expression would presumably inhibit cholesterol efflux and consequently raise intracellular cholesterol level. Surprisingly, raising TTF-1 expression actually lowers intracellular cholesterol level, which, we believe, is attributed to a direct transactivation of ABCA1 by TTF-1. Subsequently, we show that lung cancer cells primed with a TTF-1-driven decrease of cholesterol were more vulnerable to simvastatin, a frequently prescribed cholesterol biosynthesis inhibitor. In view of the fact that pathologists routinely interrogate human lung cancers for TTF-1 immunopositivity to guide diagnosis and the prevalent use of statins, TTF-1 should be further investigated as a putative biomarker of lung cancer vulnerability to statins.

A Lipidomics Approach to Identifying Key Lipid Species Involved in VEGF-Inhibitor Mediated Attenuation of Bleomycin-Induced Pulmonary Fibrosis.

PURPOSE: Poor molecular characterization of idiopathic pulmonary fibrosis (IPF) has led to insufficient understanding of the pathogenesis of the disease, resulting in lack of effective therapies and poor prognosis. Particularly, the role of lipid imbalance due to impaired lipid metabolism in the pathogenesis of IPF has been poorly studied. EXPERIMENTAL DESIGN: The authors have used shotgun lipidomics in a bleomycin (BLM) mouse model of pulmonary fibrosis with vascular endothelial growth factor (VEGF)-inhibitor CBO-P11 as a therapeutic measure, to identify a comprehensive set of lipids that contribute to the pathogenesis of pulmonary fibrosis. RESULTS: The authors report that attenuation of BLM-induced fibrotic response with CBO-P11 cotreatment is accompanied by a decrease in total lipid content and specific downregulation of lipids, which are upregulated in response to BLM treatment. CONCLUSION AND CLINICAL RELEVANCE: Dysregulated lipids identified in this study hold the potential of being future biomarkers for IPF.

Quantitative Proteomic Analysis of the Hippocampus of Rats with GCR-Induced Spatial Memory Impairment.

NASA is planning future missions to Mars, which will result in astronauts being exposed to ∼13 cGy/year of galactic cosmic radiation (GCR). Previous ground-based experiments have demonstrated that low (15 cGy) doses of 1 GeV/n 56Fe ions impair hippocampus-dependent spatial memory in rats. However, some irradiated rats maintain a spatial memory performance comparable to that seen in the sham-irradiated rats, suggesting that some of these animals are able to ameliorate the deleterious effects of the GCR, while others are not. This rat model provides a unique opportunity to increase our understanding of how GCR affects neurophysiology, what adaptive responses can be invoked to prevent the emergence of GCR-induced spatial memory impairment, as well as the pathways that are altered when spatial memory impairment occurs. A label-free, unbiased proteomic profiling approach involving quantitative protein/peptide profiling followed by Cytoscape analysis has established the composition of the hippocampal proteome in male Wistar rats after exposure to 15 cGy of 1 GeV/n 56Fe, and identified proteins whose expression is altered with respect to: 1. radiation exposure and 2. impaired spatial memory performance. We identified 30 proteins that were classified as "GCR exposure marker" (GEM) proteins (expressed solely or at higher levels in the irradiated rats but not related to spatial memory performance), most notably CD98, Cadps and GMFB. Conversely, there were 252 proteins that were detected only in the sham-irradiated samples, i.e., they were not detected in either of the irradiated cohorts; of these 10% have well-documented roles in neurotransmission. The second aspect of our data mining was to identify proteins whose expression was associated with either impaired or functional spatial memory. While there are multiple changes in the hippocampal proteome in the irradiated rats that have impaired spatial memory performance, with 203 proteins being detected (or upregulated) only in these rats, it would appear that spatial memory impairment may also arise from an inability of these rats to express "good spatial memory" (GSM) proteins, many of which play an important role in neuronal homeostasis and function, axonogenesis, presynaptic membrane organization and G-protein coupled receptor (GCPR) signaling. It may be possible to use this knowledge to develop two alternative countermeasure strategies, one that preserves critical pathways prophylactically and one that invokes restorative pathways after GCR exposure.

Ticks elicit variable fibrinogenolytic activities upon feeding on hosts with different immune backgrounds.

Ticks secrete several anti-hemostatic factors in their saliva to suppress the host innate and acquired immune defenses against infestations. Using Ixodes scapularis ticks and age-matched mice purchased from two independent commercial vendors with two different immune backgrounds as a model, we show that ticks fed on immunodeficient animals demonstrate decreased fibrinogenolytic activity in comparison to ticks fed on immunocompetent animals. Reduced levels of D-dimer (fibrin degradation product) were evident in ticks fed on immunodeficient animals in comparison to ticks fed on immunocompetent animals. Increased engorgement weights were noted for ticks fed on immunodeficient animals in comparison to ticks fed on immunocompetent animals. Furthermore, the LC-MS/MS and quantitative real-time-PCR analysis followed by inhibitor and antibody-blocking assays revealed that the arthropod HSP70-like molecule contributes to differential fibrinogenolysis during tick feeding. Collectively, these results not only indicate that ticks elicit variable fibrinogenolysis upon feeding on hosts with different immune backgrounds but also provide insights for the novel role of arthropod HSP70-like molecule in fibrinogenolysis during blood feeding.

Changes in the Hippocampal Proteome Associated with Spatial Memory Impairment after Exposure to Low (20 cGy) Doses of 1 GeV/n 56Fe Radiation.

Exposure to low (∼20 cGy) doses of high-energy charged (HZE) particles, such as 1 GeV/n 56Fe, results in impaired hippocampal-dependent learning and memory (e.g., novel object recognition and spatial memory) in rodents. While these findings raise the possibility that astronauts on deep-space missions may develop cognitive deficits, not all rats develop HZE-induced cognitive impairments, even after exposure to high (200 cGy) HZE doses. The reasons for this differential sensitivity in some animals that develop HZE-induced cognitive failure remain speculative. We employed a robust quantitative mass spectrometry-based workflow, which links early-stage discovery to next-stage quantitative verification, to identify differentially active proteins/pathways in rats that developed spatial memory impairment at three months after exposure to 20 cGy of 1 GeV/n 56Fe (20/impaired), and in those rats that managed to maintain normal cognitive performance (20/functional). Quantitative data were obtained on 665-828 hippocampal proteins in the various cohorts of rats studied, of which 580 were expressed in all groups. A total of 107 proteins were upregulated in the irradiated rats irrespective of their spatial memory performance status, which included proteins involved in oxidative damage response, calcium transport and signaling. Thirty percent (37/107) of these "radiation biomarkers" formed a functional interactome of the proteasome and the COP9 signalosome. These data suggest that there is persistent oxidative stress, ongoing autophagy and altered synaptic plasticity in the irradiated hippocampus, irrespective of the spatial memory performance status, suggesting that the ultimate phenotype may be determined by how well the hippocampal neurons compensate to the ongoing oxidative stress and associated side effects. There were 67 proteins with expression that correlated with impaired spatial memory performance. Several of the "impaired biomarkers" have been implicated in poor spatial memory performance, neurodegeneration, neuronal loss or neuronal susceptibility to apoptosis, or neuronal synaptic or structural plasticity. Therefore, in addition to the baseline oxidative stress and altered adenosine metabolism observed in all irradiated rats, the 20/impaired rats expressed proteins that led to poor spatial memory performance, enhanced neuronal loss and apoptosis, changes in synaptic plasticity and dendritic remodeling. A total of 46 proteins, which were differentially upregulated in the sham-irradiated and 20/functional rat cohorts, can thus be considered as markers of good spatial memory, while another 95 proteins are associated with the maintenance of good spatial memory in the 20/functional rats. The loss or downregulation of these "good spatial memory" proteins would most likely exacerbate the situation in the 20/impaired rats, having a major impact on their neurocognitive status, given that many of those proteins play an important role in neuronal homeostasis and function. Our large-scale comprehensive proteomic analysis has provided some insight into the processes that are altered after exposure, and the collective data suggests that there are multiple problems with the functionality of the neurons and astrocytes in the irradiated hippocampi, which appear to be further exacerbated in the rats that have impaired spatial memory performance or partially compensated for in the rats with good spatial memory.

Human placenta hydrogel reduces scarring in a rat model of cardiac ischemia and enhances cardiomyocyte and stem cell cultures.

INTRODUCTION: Xenogeneic extracellular matrix (ECM) hydrogels have shown promise in remediating cardiac ischemia damage in animal models, yet analogous human ECM hydrogels have not been well development. An original human placenta-derived hydrogel (hpECM) preparation was thus generated for assessment in cardiomyocyte cell culture and therapeutic cardiac injection applications. METHODS AND RESULTS: Hybrid orbitrap-quadrupole mass spectrometry and ELISAs showed hpECM to be rich in collagens, basement membrane proteins, and regenerative growth factors (e.g. VEGF-B, HGF). Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes synchronized and electrically coupled on hpECM faster than on conventional cell culture environments, as validated by intracellular calcium measurements. In vivo, injections using biotin-labeled hpECM confirmed its spatially discrete localization to the myocardium proximal to the injection site. hpECM was injected into rat myocardium following an acute myocardium infarction induced by left anterior descending artery ligation. Compared to sham treated animals, which exhibited aberrant electrical activity and larger myocardial scars, hpECM injected rat hearts showed a significant reduction in scar volume along with normal electrical activity of the surviving tissue, as determined by optical mapping. CONCLUSION: Placental matrix and growth factors can be extracted as a hydrogel that effectively supports cardiomyocytes in vitro, and in vivo reduces scar formation while maintaining electrophysiological activity when injected into ischemic myocardium. STATEMENT OF SIGNIFICANCE: This is the first report of an original extracellular matrix hydrogel preparation isolated from human placentas (hpECM). hpECM is rich in collagens, laminin, fibronectin, glycoproteins, and growth factors, including known pro-regenerative, pro-angiogenic, anti-scarring, anti-inflammatory, and stem cell-recruiting factors. hpECM supports the culture of cardiomyocytes, stem cells and blood vessels assembly from endothelial cells. In a rat model of myocardial infarction, hpECM injections were safely deliverable to the ischemic myocardium. hpECM injections repaired the myocardium, resulting in a significant reduction in infarct size, more viable myocardium, and a normal electrophysiological contraction profile. hpECM thus has potential in therapeutic cardiovascular applications, in cellular therapies (as a delivery vehicle), and is a promising biomaterial for advancing basic cell-based research and regenerative medicine applications.

Demineralized bone matrix fibers formable as general and custom 3D printed mold-based implants for promoting bone regeneration.

INTRODUCTION: Bone repair frequently requires time-consuming implant construction, particularly when using un-formed implants with poor handling properties. We therefore developed osteoinductive, micro-fibrous surface patterned demineralized bone matrix (DBM) fibers for engineering both defect-matched and general three-dimensional implants. METHODS AND RESULTS: Implant molds were filled with demineralized human cortical bone fibers there were compressed and lyophilized, forming mechanically strong shaped DBM scaffolds. Enzyme linked immunosorbent assays and mass spectrometry confirmed that DBM fibers contained abundant osteogenic growth factors (bone morphogenetic proteins, insulin-like growth factor-I) and extracellular matrix proteins. Mercury porosimetry and mechanical testing showed interconnected pores within the mechanically stable, custom DBM fiber scaffolds. Mesenchymal stem cells readily attached to the DBM and showed increasing metabolic activity over time. DBM fibers further increased alkaline phosphatase activity in C2C12 cells. In vivo, DBM implants elicited osteoinductive potential in a mouse muscle pouch, and also promoted spine fusion in a rat arthrodesis model. SIGNIFICANCE: DBM fibers can be engineered into custom-shaped, osteoinductive and osteoconductive implants with potential for repairing osseous defects with precise fitment, potentially reducing operating time. By providing pre-formed and custom implants, this regenerative allograft may improve patient outcomes following surgical bone repair, while further advancing personalized orthopedic and craniomaxillofacial medicine using three-dimensional-printed tissue molds.

A proteomics approach to identifying key protein targets involved in VEGF inhibitor mediated attenuation of bleomycin-induced pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with a life expectancy of less than 5 years post diagnosis for most patients. Poor molecular characterization of IPF has led to insufficient understanding of the pathogenesis of the disease, resulting in lack of effective therapies. In this study, we have integrated a label-free LC-MS based approach with systems biology to identify signaling pathways and regulatory nodes within protein interaction networks that govern phenotypic changes that may lead to IPF. Ingenuity Pathway Analysis of proteins modulated in response to bleomycin treatment identified PI3K/Akt and Wnt signaling as the most significant profibrotic pathways. Similar analysis of proteins modulated in response to vascular endothelial growth factor (VEGF) inhibitor (CBO-P11) treatment identified natural killer cell signaling and PTEN signaling as the most significant antifibrotic pathways. Mechanistic/mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase (ERK) were identified to be key mediators of pro- and antifibrotic response, where bleomycin (BLM) treatment resulted in increased expression and VEGF inhibitor treatment attenuated expression of mTOR and ERK. Using a BLM mouse model of pulmonary fibrosis and VEGF inhibitor CBO-P11 as a therapeutic measure, we identified a comprehensive set of signaling pathways and proteins that contribute to the pathogenesis of pulmonary fibrosis that can be targeted for therapy against this fatal disease.

MicroRNA abundance is altered in synaptoneurosomes during prion disease.

Discrepancy in synaptic structural plasticity is one of the earliest manifestations of the neurodegenerative state. In prion diseases, a reduction in synapses and dendritic spine densities is observed during preclinical disease in neurons of the cortex and hippocampus. The underlying molecular mechanisms of these alterations have not been identified but microRNAs (miRNAs), many of which are enriched at the synapse, likely regulate local protein synthesis in rapid response to stressors such as replicating prions. MiRNAs are therefore candidate regulators of these early neurodegenerative changes and may provide clues as to the molecular pathways involved. We therefore determined changes in mature miRNA abundance within synaptoneurosomes isolated from prion-infected, as compared to mock-infected animals, at asymptomatic and symptomatic stages of disease. During preclinical disease, miRNAs that are enriched in neurons including miR-124a-3p, miR-136-5p and miR-376a-3p were elevated. At later stages of disease we found increases in miRNAs that have previously been identified as deregulated in brain tissues of prion infected mice, as well as in Alzheimer's disease (AD) models. These include miR-146a-5p, miR-142-3p, miR-143-3p, miR-145a-5p, miR-451a, miR-let-7b, miR-320 and miR-150-5p. A number of miRNAs also decreased in abundance during clinical disease. These included almost all members of the related miR-200 family (miR-200a-3p, miR-200b-3p, miR-200c-3p, miR-141-3p, and miR-429-3p) and the 182 cluster (miR-182-5p and miR-183-5p).

Scaling-up public sector childhood diarrhea management program: Lessons from Indian states of Gujarat, Uttar Pradesh and Bihar.

BACKGROUND: Diarrhea remains a leading cause of death among children under five in India. Public health sector is an important source for diarrhea treatment with oral rehydration salts (ORS) and zinc. In 2010, Micronutrient Initiative started a project to improve service delivery for childhood diarrhea management through public health sector in Gujarat, Uttar Pradesh (UP) and Bihar. This paper aims to highlight feasible strategies, experiences and lessons learned from scaling-up zinc and ORS for childhood diarrhea management in the public sector in three Indian states. METHODS: The project was implemented in six districts of Gujarat, 12 districts of UP and 15 districts of Bihar, which includes 10.5 million children. Program strategies included capacity building of health care providers, expanding service delivery through community health workers (CHWs), providing supportive supervision to CHWs, ensuring supplies and conducting monitoring and evaluation. The lessons described in this paper are based on program data, government documents and studies that were used to generate evidence and inform program scale-up. RESULTS: 140 000 health personnel, including CHWs, were trained in childhood diarrhea management. During three years, CHWs had sustained knowledge and have treated and reported more than three million children aged 2-59 months having diarrhea, of which 84% were treated with both zinc and ORS. The successful strategies were scaled-up. CONCLUSION: It is feasible and viable to introduce and scale-up zinc and ORS for childhood diarrhea treatment through public sector. Community-based service delivery, timely and adequate supplies, trained staff and pro-active engagement with government were essential for program success.

Dysregulated expression of cell surface glycoprotein CDCP1 in prostate cancer.

CUB-domain-containing protein 1 (CDCP1) is a trans-membrane protein regulator of cell adhesion with a potent pro-migratory function in tumors. Given that proteolytic cleavage of the ectodomain correlates with outside-in oncogenic signaling, we characterized glycosylation in the context of cellular processing and expression of CDCP1 in prostate cancer. We detected 135 kDa full-length and proteolytic processed 70 kDa species in a panel of PCa cell models. The relative expression of full-length CDCP1 correlated with the metastatic potential of syngeneic cell models and an increase in surface membrane expression of CDCP1 was observed in tumor compared to adjacent normal prostate tissues. We demonstrated that glycosylation of CDCP1 is a prerequisite for protein stability and plasma membrane localization, and that the expression level and extent of N-glycosylation of CDCP1 correlated with metastatic status. Interestingly, complex N-linked glycans with sialic acid chains were restricted to the N-terminal half of the ectodomain and absent in the truncated species. Characterization of the extracellular expression of CDCP1 identified novel circulating forms and revealed that extracellular vesicles provide additional processing pathways. Employing immunoaffinity mass spectrometry, we detected elevated levels of circulating CDCP1 in patient urine with high-risk disease. Our results establish that differential glycosylation, cell surface presentation and extracellular expression of CDCP1 are hallmarks of PCa progression.

Liver Perilipin 5 Expression Worsens Hepatosteatosis But Not Insulin Resistance in High Fat-Fed Mice.

Perilipin 5 (PLIN5) is a lipid droplet (LD) protein highly expressed in oxidative tissues, including the fasted liver. However, its expression also increases in nonalcoholic fatty liver. To determine whether PLIN5 regulates metabolic phenotypes of hepatosteatosis under nutritional excess, liver targeted overexpression of PLIN5 was achieved using adenoviral vector (Ad-PLIN5) in male C57BL/6J mice fed high-fat diet. Mice treated with adenovirus expressing green fluorescent protein (GFP) (Ad-GFP) served as control. Ad-PLIN5 livers increased LD in the liver section, and liquid chromatography with tandem mass spectrometry revealed increases in lipid classes associated with LD, including triacylglycerol, cholesterol ester, and phospholipid classes, compared with Ad-GFP liver. Lipids commonly associated with hepatic lipotoxicity, diacylglycerol, and ceramides, were also increased in Ad-PLIN5 liver. The expression of genes in lipid metabolism regulated by peroxisome proliferator-activated receptor-α was reduced suggestive of slower mobilization of stored lipids in Ad-PLIN5 mice. However, the increase of hepatosteatosis by PLIN5 overexpression did not worsen glucose homeostasis. Rather, serum insulin levels were decreased, indicating better insulin sensitivity in Ad-PLIN5 mice. Moreover, genes associated with liver injury were unaltered in Ad-PLIN5 steatotic liver compared with Ad-GFP control. Phosphorylation of protein kinase B was increased in Ad-PLIN5-transduced AML12 hepatocyte despite of the promotion of fatty acid incorporation to triacylglycerol as well. Collectively, our data indicates that the increase in liver PLIN5 during hepatosteatosis drives further lipid accumulation but does not adversely affect hepatic health or insulin sensitivity.