Rangaprasad Sarangarajan

Molecular and functional characterization of sodium--hydrogen exchanger in skin as well as cultured keratinocytes and melanocytes.

The sodium--hydrogen (Na(+)/H(+)) exchanger is one of the few transporter proteins involved in the regulation and maintenance of intracellular pH and cell volume in most eukaryotic cell types. The current study investigates the expression of isoforms of the Na(+)/H(+) exchanger (NHE) in human skin and in cultured keratinocytes, melanocytes, and melanoma cells by reverse transcription-polymerase chain reaction (RT--PCR), immunohistochemical analysis and functional studies. Neonatal foreskins were used to isolate RNA from epidermis and dermis, and to initiate cultures of keratinocytes and melanocytes. RT--PCR on RNA isolated from epidermis, dermis, keratinocytes, melanocytes and melanoma cells using PCR primers specific for NHE-1 yielded a 463 bp PCR product. RT--PCR performed using primers specific for NHE isoforms 2, 3, 4 and 5 did not yield any products. Western blotting analysis (of keratinocyte and melanocyte cell cultures) and indirect immunohistochemistry on neonatal foreskin, keratinocytes, melanocytes and melanoma cells using a NHE-1-specific polyclonal antibody demonstrated NHE-1 expression at the protein level. Physiological regulation of intracellular pH using a pH-sensitive dye, BCECF, detected an amiloride-sensitive NHE activity in human keratinocyte, melanocyte and melanoma cell cultures. These results indicate that cultures of human keratinocytes and melanocytes established from human skin and melanoma cells express the NHE-1 isoform of the sodium--hydrogen exchanger.

PIG3V, an immortalized human vitiligo melanocyte cell line, expresses dilated endoplasmic reticulum.

SummaryVitiligo is an enigmatic pigmentary disorder of the skin. Factors potentially involved in the progressive loss of melanocytes from the basal layer of the epidermis include genetically determined aberrancies of the vitiligo melanocyte. It follows that analysis of melanocytes cultured from vitiligo donors can contribute to a further understanding of the etiopathomechanism. A setback for vitiligo research has been the limited availability of vitiligo-derived melanocytes. To overcome this limitation, we have generated a vitiligo melanocyte cell line according to a protocol established previously for the immortalization of normal human melanocytes. Vitiligo melanocytes Ma9308P4 were transfected with HPV16 E6 and E7 genes using the retroviral construct LXSN16E6E7. Successful transformants were selected using geneticin and subsequently cloned to ensure genetic homogeneity. The resulting cell line PIG3V has undergone more than 100 cell population doublings ince its establishment as a confluent primary culture, whereas untransfected melanocytes derived from adult skin senesce after a maximum of 50 population doublings. Cells immortalized by this transfection procedure retain lineage-specific characteristics and proliferate significantly faster than parental cells. In this study, the phenotype of PIG3V resembled melanocytes rather than melanoma cells in culture. Tyrosinase was processed properly and melanosomes remained pigmented. Importantly, ultrastructural characterization of PIG3V cells revealed dilated endoplasmic reticulum profiles characteristic of vitiligo melanocytes. An explanation for this dilation may be found in the retention of proteins with molecular weight of 37.5, 47.5, and 56.5 kDa, as determined by gel electrophoresis of microsomal proteins isolated from radiolabeled cells.

NHE-1 is the sodium–hydrogen exchanger isoform present in erythroid cells

Erythrocyte sodium hydrogen exchanger (NHE) represents one of a limited number of sodium entry pathway in erythrocytes. At least five NHE isoforms have been identified, differing in tissue specificity, regulatory characteristics, and pharmacological sensitivities. Although physiological characteristics of erythrocyte NHE suggest that the widely expressed NHE-1 isoform may be present, evidence is not conclusive and does not exclude the existence of other isoforms. In this study, Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses were used to test for five NHE isoforms in erythroid cells. Blood from patients with sickle cell disease was depleted of white blood cells (WBC) by passage through leukocyte filters and cellulose column. RT-PCR performed on WBC depleted reticulocyte RNA using a NHE-1 primer set yielded product a of expected size, the sequence of which was identical to the published human NHE-1 sequence. Northern blot analysis of the reticulocyte RNA using a 1.6 kb probe revealed a message of approximately 5.0 kb in size. RT-PCR analysis of rat kidney RNA using primers specific for NHE isoforms -2, -3, -4 and rat brain RNA using primer specific for NHE-5 isoform yielded products of expected size, whereas WBC depleted RNA under identical conditions yielded no products. These results identify the erythroid isoform of the sodium hydrogen exchanger as NHE-1.

Effect of route of administration and dose on diabetes-induced protection against cisplatin nephrotoxicity.

Abstract The kidneys of diabetic rats exhibit resistance to the actions of a variety of nephrotoxic chemicals. Using the streptozotocin (STZ) diabetic rat as a model, the experiments in this study examined the effect of the diabetic state on bioavailability, renal accumulation and renal toxicity following intraperitoneal (ip) and intravenous (iv) injection of cisplatin at various doses. Comparison of the areas under the plasma concentration versus time curves up to 220 min after cisplatin injection (5 mg/kg body wt) demonstrated that bioavailability of cisplatin was significantly impaired in STZ diabetic versus nondiabetic rats after the ip route of administration, but not after the iv route. Regardless of the route of cisplatin injection, both renal cortex platinum level and renal toxicity as quantified by blood urea nitrogen (BUN) increase, were significantly lower in STZ diabetic versus nondiabetic rats. Thus, while decreased bioavailability probably plays a role in the protection seen after ip injection, renal mechanisms must also be involved since protection was also noted after iv injection despite equal bioavailability. To determine whether the protection can be overcome by increasing the dose of cisplatin, renal platinum and BUN levels in STZ diabetics and nondiabetics were compared after ip and iv cisplatin injections at 2.5, 5, 7.5, and 10 mg/kg body wt. The results demonstrate that the diabetes-induced renal protection can be partially reversed by increased cisplatin dose, but only by the iv route. Reversal was accompanied by increased renal platinum accumulation. These results suggest that STZ diabetes alters the tubular cells in a manner that confers protection that more closely resembles tolerance than absolute inherent resistance to the nephrotoxic actions of cisplatin.

Intrinsic Properties of Brown and White Adipocytes Have Differential Effects on Macrophage Inflammatory Responses

Obesity is marked by chronic, low-grade inflammation. Here, we examined whether intrinsic differences between white and brown adipocytes influence the inflammatory status of macrophages. White and brown adipocytes were characterized by transcriptional regulation of UCP-1, PGC1α, PGC1β, and CIDEA and their level of IL-6 secretion. The inflammatory profile of PMA-differentiated U937 and THP-1 macrophages, in resting state and after stimulation with LPS/IFN-gamma and IL-4, was assessed by measuring IL-6 secretion and transcriptional regulation of a panel of inflammatory genes after mono- or indirect coculture with white and brown adipocytes. White adipocyte monocultures show increased IL-6 secretion compared to brown adipocytes. White adipocytes cocultured with U937 and THP-1 macrophages induced a greater increase in IL-6 secretion compared to brown adipocytes cocultured with both macrophages. White adipocytes cocultured with macrophages increased inflammatory gene expression in both types. In contrast, macrophages cocultured with brown adipocytes induced downregulation or no alterations in inflammatory gene expression. The effects of adipocytes on macrophages appear to be independent of stimulation state. Brown adipocytes exhibit an intrinsic ability to dampen inflammatory profile of macrophages, while white adipocytes enhance it. These data suggest that brown adipocytes may be less prone to adipose tissue inflammation that is associated with obesity.

Non-obvious correlations to disease management unraveled by Bayesian artificial intelligence analyses of CMS data

OBJECTIVE Given the availability of extensive digitized healthcare data from medical records, claims and prescription information, it is now possible to use hypothesis-free, data-driven approaches to mine medical databases for novel insight. The goal of this analysis was to demonstrate the use of artificial intelligence based methods such as Bayesian networks to open up opportunities for creation of new knowledge in management of chronic conditions. MATERIALS AND METHODS Hospital level Medicare claims data containing discharge numbers for most common diagnoses were analyzed in a hypothesis-free manner using Bayesian networks learning methodology. RESULTS While many interactions identified between discharge rates of diagnoses using this data set are supported by current medical knowledge, a novel interaction linking asthma and renal failure was discovered. This interaction is non-obvious and had not been looked at by the research and clinical communities in epidemiological or clinical data. A plausible pharmacological explanation of this link is proposed together with a verification of the risk significance by conventional statistical analysis. CONCLUSION Potential clinical and molecular pathways defining the relationship between commonly used asthma medications and renal disease are discussed. The study underscores the need for further epidemiological research to validate this novel hypothesis. Validation will lead to advancement in clinical treatment of asthma & bronchitis, thereby, improving patient outcomes and leading to long term cost savings. In summary, this study demonstrates that application of advanced artificial intelligence methods in healthcare has the potential to enhance the quality of care by discovering non-obvious, clinically relevant relationships and enabling timely care intervention.

Monoacylglycerol Analysis Using MS/MSALL Quadruple Time of Flight Mass Spectrometry

Monoacylglycerols (MAGs) are structural and bioactive metabolites critical for biological function. Development of facile tools for measuring MAG are essential to understand its role in different diseases and various pathways. A data-independent acquisition method, MS/MSALL, using electrospray ionization (ESI) coupled quadrupole time of flight mass spectrometry (MS), was utilized for the structural identification and quantitative analysis of individual MAG molecular species. Compared with other acylglycerols, diacylglycerols (DAG) and triacylglycerols (TAG), MAG characteristically presented as a dominant protonated ion, [M + H]+, and under low collision energy as fatty acid-like fragments due to the neutral loss of the glycerol head group. At low concentrations (<10 pmol/µL), where lipid-lipid interactions are rare, there was a strong linear correlation between ion abundance and MAG concentration. Moreover, using the MS/MSALL method the major MAG species from human plasma and mouse brown and white adipose tissues were quantified in less than 6 min. Collectively, these results demonstrate that MS/MSALL analysis of MAG is an enabling strategy for the direct identification and quantitative analysis of low level MAG species from biological samples with high throughput and sensitivity.

Identification of Filamin-A and -B as potential biomarkers for prostate cancer

Aim: A novel strategy for prostate cancer (PrCa) biomarker discovery is described. Materials & methods: In vitro perturbation biology, proteomics and Bayesian causal analysis identified biomarkers that were validated in in vitro models and clinical specimens. Results: Filamin-B (FLNB) and Keratin-19 were identified as biomarkers. Filamin-A (FLNA) was found to be causally linked to FLNB. Characterization of the biomarkers in a panel of cells revealed differential mRNA expression and regulation. Moreover, FLNA and FLNB were detected in the conditioned media of cells. Last, in patients without PrCa, FLNA and FLNB blood levels were positively correlated, while in patients with adenocarcinoma the relationship is dysregulated. Conclusion: These data support the strategy and the potential use of the biomarkers for PrCa.

Clinical metabolomics: a pivotal tool for companion diagnostic development and precision medicine

Translation of companion diagnostic (CDx) development into a personalized clinical application remains a challenging task. It requires integration of multidimensional molecular and clinical data into patient-centric models. Family history, clinical history, and physical examination are mandatory for the interpretation of laboratory results. The inclusion of metabolomics data into the molecular architecture of the patient analyses allows for a more robust biological narrative. To empower broad spectrum utility in population health, adherence to protocol standardization and data accuracy will enable more predictive outcomes. A transformative evolution in healthcare has been galvanized, engaging patient and population biology for theranostic drug development. Theranostics employs diagnostic testing to select a targeted therapy for individuals. This approach serves as the catalyst for implementing mainstream precision medicine. Precision medicine focuses on identifying effective quantitative approaches aligning single-agent or combinational regimens based on genetics; demographics; socioeconomic status; ethnicity; gender; and environmental, metabolic, and lifestyle factors of the patient. For implementation, two essential guiding principles should be followed for CDx development. First, identification of valid biomarker panels which define positive or negative treatment outcomes, as well as adverse event risk factors for an individual or a population. Second, a reproducible method is required for detecting the biomarker panel in the population. This scheme aligns the intervention and the selectionmethod for CDx development in precisionmedicine. The FDA defines CDx as a tool or imaging device that provides information essential for the safe and effective use of a corresponding treatment [1]. This approach demands patient stratification, i.e. identifying populations which are more likely to benefit from the therapy. In the past, drug development began with preclinical studies and progressed toward NDA submission. However, CDx development also includes individual biomarker (or panel development) and advances toward regulatory submission and CDx launch, which is often in addition to the therapeutic launch. The impact of CDx implementation as a method for the development of new drugs is profound. Preferably, CDx incorporation should be early in the drug development process, although it can also occur at later stages of clinical development. In the era of precision medicine, a novel panel of diagnostic markers can change the economics of drug development as well as the economics of the market size significantly, depending on whether the biomarker panel is labeled as a CDx or a complementary diagnostic [2]. These processes are all dependent on the path of the specific drug in CDx development, and the regulatory and compliance decisions enacted upon in pivotal clinical studies. Additional processes are guided by population selection based on diagnostic cutoff points and determination of the evasive zones’ positive and negative predictive assessment. The complexity of CDx development, particularly in parallel to a new drug development course, requires coordination across several cross-functional units and potentially also across organizations. Incorporating CDx into drug development infuses a more precise and accurate approach to matching patient populations to effective therapeutics. Moreover, in reimbursement environments that are leaning toward outcome-based models, CDx may emerge as the cardinal tool for clinical focus and revenue forecasting [3].

Reduced expression of collagen VI alpha 3 (COL6A3) confers resistance to inflammation-induced MCP1 expression in adipocytes

Collagen VI alpha 3 (COL6A3) is associated with insulin resistance and adipose tissue inflammation. In this study, the role of COL6A3 in human adipocyte function was characterized.