SLC38A10 Regulate Glutamate Homeostasis and Modulate the AKT/TSC2/mTOR Pathway in Mouse Primary Cortex Cells.

Glutamate acts as a critical regulator of neurotransmitter balance, recycling, synaptic function and homeostasis in the brain and glutamate transporters control glutamate levels in the brain. SLC38A10 is a member of the SLC38 family and regulates protein synthesis and cellular stress responses. Here, we uncover the role of SLC38A10 as a transceptor involved in glutamate-sensing signaling pathways that control both the glutamate homeostasis and mTOR-signaling. The culture of primary cortex cells from SLC38A10 knockout mice had increased intracellular glutamate. In addition, under nutrient starvation, KO cells had an impaired response in amino acid-dependent mTORC1 signaling. Combined studies from transcriptomics, protein arrays and metabolomics established that SLC38A10 is involved in mTOR signaling and that SLC38A10 deficient primary cortex cells have increased protein synthesis. Metabolomic data showed decreased cholesterol levels, changed fatty acid synthesis, and altered levels of fumaric acid, citrate, 2-oxoglutarate and succinate in the TCA cycle. These data suggests that SLC38A10 may act as a modulator of glutamate homeostasis, and mTOR-sensing and loss of this transceptor result in lower cholesterol, which could have implications in neurodegenerative diseases.

Generation of a human iPSC line (UUIGPi015-A) from a patient with Dravet syndrome and a 2.9 Mb deletion spanning SCN1A on chromosome 2.

Dravet syndrome is an early onset devastating epilepsy syndrome usually caused by heterozygous mutations in SCN1A. We generated a human iPSC line (UUIGPi015-A) from dermal fibroblasts of a patient with Dravet syndrome carrying a deletion on chromosome 2 encompassing SCN1A and 9 flanking genes. Characterization of the iPSC line confirmed expression of pluripotency markers, tri-lineage differentiation capacity and absence of exogenous reprogramming factors. The iPSC line retained the deletion and was genomically stable. The iPSC line UUIGPi015-A provides a useful resource for studies on the pathophysiology of Dravet syndrome and seizures caused by haploinsufficiency of SCN1A and flanking gene products.

Molecular genetic analysis of neural stem cells after space flight and simulated microgravity on earth.

Understanding how stem cells adapt to space flight conditions is fundamental for human space missions and extraterrestrial settlement. We analyzed gene expression in boundary cap neural crest stem cells (BCs), which are attractive for regenerative medicine by their ability to promote proliferation and survival of cocultured and co-implanted cells. BCs were launched to space (space exposed cells) (SEC), onboard sounding rocket MASER 14 as free-floating neurospheres or in a bioprinted scaffold. For comparison, BCs were placed in a random positioning machine (RPM) to simulate microgravity on earth (RPM cells) or were cultured under control conditions in the laboratory. Using next-generation RNA sequencing and data post-processing, we discovered that SEC upregulated genes related to proliferation and survival, whereas RPM cells upregulated genes associated with differentiation and inflammation. Thus, (i) space flight provides unique conditions with distinctly different effects on the properties of BC compared to earth controls, and (ii) the space flight exposure induces postflight properties that reinforce the utility of BC for regenerative medicine and tissue engineering.

SLC38A10 Transporter Plays a Role in Cell Survival Under Oxidative Stress and Glutamate Toxicity.

Solute carrier (SLC) transporters regulate amino acids, glucose, ions, and metabolites that flow across cell membranes. In the brain, SLCs are the key regulators of neurotransmission, in particular, the glutamate/GABA-glutamine (GGG) cycle. Genetic mutations in SLCs are associated with various neurodevelopmental and neurodegenerative diseases. In this study, we have investigated the role of SLC38A10 under acute oxidative and glutamate stress in mouse primary cortical cells from SLC38A10 knockout (KO) mice. The ER/golgi localized transporter, SLC38A10, transports glutamate, glutamine, and alanine in brain cells, and the aim of this study was to determine the possible effects of removal of SLC38A10 in primary cortical cells under glutamate and oxidative challenges. Primary cortical neuronal cultures of wild-type (WT) cell and SLC38A10 KO mice were subjected to different concentrations of glutamate and hydrogen peroxide. There was no morphological change observed between KO and WT cortical neurons in culture. Interestingly, KO cells showed significantly lower cell viability and higher cell death compared to WT cells under both glutamate and hydrogen peroxide exposure. Further, we evaluated the possible role of p53 in neuronal cell apoptosis in KO cells. We found decreased intracellular p53 protein levels under glutamate and hydrogen peroxide treatment in KO cortical cells. In contrast, caspase 3/7 activity remains unaltered under all conditions. These results demonstrate an indirect relationship between the expression of SLC38A10 and p53 and a role in the cell defense mechanism against neurotoxicity.

Best Practices to Verify Ongoing Placement of NG or OG Tube After Initial X-ray Confirmation.

BACKGROUND: Many patients in intensive care units (ICU) require nasogastric (NG) or orogastric (OG) tubes. These patients often require a combination of sedatives that can alter level of consciousness and impair cough or gag reflexes. Such factors can lead to NG/OG tube displacement. Using a misplaced tube can lead to aspiration, lung injury, infection, and even death. AIMS: To standardize ongoing verification of NG tube placement practices in our 34-bed Medical-Surgical ICU. METHODS: The Johns Hopkins Nursing Model was utilized to guide this project. A literature review and critical appraisal were performed to establish NG/OG tube best practices. Best practices were implemented and assessed (via a survey and charting audits). RESULTS: Fifteen publications were identified and appraised as Level 4 and 5 sources. Best evidence supported that at the time of radiographic confirmation of the tube site, it should be marked with inedible ink or adhesive tape where it exits the nares; tube location should be checked at 4-hour intervals; and placement/patency should be checked in patients who complain of pain, vomiting, or coughing. Following the practice change, N = 40 nurses indicated improvement in verification of NG/OG tube knowledge, "OK to use" order was verified for 89% of patients, and 63% of tubes were marked with tape at the exit site. LINKING ACTION TO EVIDENCE: Adherence to current, evidence-based strategies for NG/OG tube verification promotes patient safety. Monitoring practice changes is critical to determine whether a best practice is sustained. Electronic health records must be current to guide and support evidence-based nursing practice.

Refunctionalization of Decellularized Organ Scaffold of Pancreas by Recellularization: Whole Organ Regeneration into Functional Pancreas.

BACKGROUND: Tissue engineering centers on creating a niche similar to the natural one, with a purpose of developing an organ construct. A natural scaffold can replace none while creating a scaffold unique to each tissue in composition, architecture and cues that regulate the character of cells. METHODS: Whole pancreas from mouse was decellularized using detergent and enzymes, followed by recellularizing with MSC from human placenta. This construct was transplanted in streptozotocin induced diabetic mice. Histopathology of both decellularized and recellularized transplanted pancreas and qPCR analysis were performed to assess its recovery. RESULTS: Decellularization removes the cells leaving behind extracellular matrix rich natural scaffold. After reseeding with mesenchymal stem cells, these cells differentiate into pancreas specific cells. Upon transplantation in streptozotocin induced diabetic mice, this organ was capable of restoring its histomorphology and functioning. Restoration of endocrine (islets), the exocrine region (acinar) and vascular network was seen in transplanted pancreas. The process of functional recovery of endocrine system took about 20 days when the mice start showing blood glucose reduction, though none achieved gluconormalization. CONCLUSION: Natural decellularized scaffolds of soft organs can be refunctionalized using recipient's mesenchymal stem cells to restore structure and function; and counter immune problems arising during transplantation.

Glucose Availability Alters Gene and Protein Expression of Several Newly Classified and Putative Solute Carriers in Mice Cortex Cell Culture and D. melanogaster.

Many newly identified solute carriers (SLCs) and putative transporters have the possibility to be intricately involved in glucose metabolism. Here we show that many transporters of this type display a high degree of regulation at both mRNA and protein level following no or low glucose availability in mouse cortex cultures. We show that this is also the case in Drosophila melanogaster subjected to starvation or diets with different sugar content. Interestingly, re-introduction of glucose to media, or refeeding flies, normalized the gene expression of a number of the targets, indicating a fast and highly dynamic control. Our findings demonstrate high conservation of these transporters and how dependent both cell cultures and organisms are on gene and protein regulation during metabolic fluctuations. Several transporter genes were regulated simultaneously maybe to initiate alternative metabolic pathways as a response to low glucose levels, both in the cell cultures and in D. melanogaster. Our results display that newly identified SLCs of Major Facilitator Superfamily type, as well as the putative transporters included in our study, are regulated by glucose availability and could be involved in several cellular aspects dependent of glucose and/or its metabolites. Recently, a correlation between dysregulation of glucose in the central nervous system and numerous diseases such as obesity, type 2 diabetes mellitus as well as neurological disease such as Alzheimer's and Parkinson's diseases indicate a complex regulation and fine tuning of glucose levels in the brain. The fact that almost one third of transporters and transporter-related proteins remain orphans with unknown or contradictive substrate profile, location and function, pinpoint the need for further research about them to fully understand their mechanistic role and their impact on cellular metabolism.

SLC38A10 (SNAT10) is Located in ER and Golgi Compartments and Has a Role in Regulating Nascent Protein Synthesis.

The solute carrier (SLC) family-38 of transporters has eleven members known to transport amino acids, with glutamine being a common substrate for ten of them, with SLC38A9 being the exception. In this study, we examine the subcellular localization of SNAT10 in several independent immortalized cell lines and stem cell-derived neurons. Co-localization studies confirmed the SNAT10 was specifically localized to secretory organelles. SNAT10 is expressed in both excitatory and inhibitory neurons in the mouse brain, predominantly in the endoplasmic reticulum, and in the Golgi apparatus. Knock-down experiments of SNAT10, using Slc38a10-specific siRNA in PC12 cells reduced nascent protein synthesis by more than 40%, suggesting that SNAT10 might play a role in signaling pathways that regulate protein synthesis, and may act as a transceptor in a similar fashion to what has been shown previously for SLC38A2 (SNAT2) and SNAT9(SLC38A9).

Nutritional Stress Induced by Amino Acid Starvation Results in Changes for Slc38 Transporters in Immortalized Hypothalamic Neuronal Cells and Primary Cortex Cells.

Amino acid sensing and signaling is vital for cells, and both gene expression and protein levels of amino acid transporters are regulated in response to amino acid availability. Here, the aim was to study the regulation of all members of the SLC38 amino acid transporter family, Slc38a1-11, in mouse brain cells following amino acid starvation. We reanalyzed microarray data for the immortalized hypothalamic cell line N25/2 subjected to complete amino acid starvation for 1, 2, 3, 5, or 16 h, focusing specifically on the SLC38 family. All 11 Slc38 genes were expressed in the cell line, and Slc38a1, Slc38a2, and Slc38a7 were significantly upregulated at 5 h and most strongly at 16 h. Here, protein level changes were measured for SLC38A7 and the orphan family member SLC38A11 which has not been studied under different amino acid starvation condition at protein level. At 5 h, no significant alteration on protein level for either SLC38A7 or SLC38A11 could be detected. In addition, primary embryonic cortex cells were deprived of nine amino acids, the most common amino acids transported by the SLC38 family members, for 3 h, 7 h or 12 h, and the gene expression was measured using qPCR. Slc38a1, Slc38a2, Slc38a5, Slc38a6, Slc38a9, and Slc38a10 were upregulated, while Slc38a3 and Slc38a7 were downregulated. Slc38a8 was upregulated at 5 h and downregulated at 12 h. In conclusion, several members from the SLC38 family are regulated depending on amino acid levels and are likely to be involved in amino acid sensing and signaling in brain.

Racial disparity in outcomes of a clinical trial population with metastatic renal cell carcinoma.

OBJECTIVES: Population studies have revealed that black Americans with renal cell carcinoma (RCC) have a shorter survival than do white Americans. Differences in socioeconomic status and treatment are frequently cited as the reasons for this disparity. The effect of these social obstacles may be reduced by studying a patient population with advanced RCC enrolled in clinical trials, because patients in these trials are likely to be similar in terms of their access to care, compliance, and performance status. METHODS: A retrospective review of all patients with metastatic RCC enrolled in clinical trials at Wayne State University from 1992 to 2002 was conducted. Log-rank survival analysis by age, sex, race, smoking history, nephrectomy history, prior therapy, type of protocol therapy (immunotherapy versus other), performance status (0 versus 1 to 2), and number of metastatic sites was conducted. Univariate and multivariate comparisons by race were performed for overall survival and time to progression. RESULTS: A total of 122 patients (median age 57 years) were enrolled; 21 (17%) were black and 101 (83%) were white. Overall survival was significantly shorter for the black Americans (P = 0.0027). The median survival for black Americans and white Americans was 6.9 and 11.5 months, respectively. On multivariate analysis, black race and performance status of 0 versus 1 and 2 were significant predictors of shorter survival. The presence of liver metastases and/or the absence of prior nephrectomy also influenced the length of overall survival through an interaction effect. CONCLUSIONS: Within a clinical trial patient population with RCC, race was a significant predictor of overall survival.