Intermittent fasting: consider the risks of disordered eating for your patient.

This is a commentary on "Intermittent fasting: is there a role in the treatment of diabetes? A review of the literature and guide for primary care physicians" by Albosta et al. While this article adequately summarized the biochemical clinical advantages and limitations, we feel it failed to mention a few drawbacks, primarily the risk for disordered eating and eating disorders. Here we delve into the emerging data on intermittent fasting or time-restricted feeding in patient populations and urge clinicians to consider these risks prior to encouragement of intermittent fasting.

Inducible Knockout of 14-3-3ß Attenuates Proliferation and Spheroid Formation in a Human Glioblastoma Cell Line U87MG.

Glioblastomas (GBs) are the most common and malignant brain tumors in adults. A protein encoded by the gene YWHAB, 14-3-3ß, is commonly found to be upregulated throughout the initiation and progression of GB. The 14-3-3ß has oncogenic roles in several different types of cancer cells through interactions with proteins such as Bad, FBI1, Raf-1, Cdc25b, and others. Previous RNA interference studies have shown that 14-3-3ß promotes proliferation, cell cycle progression, and migration and invasion of GB cells. However, despite the many oncogenic functions of 14-3-3ß, a CRISPR/Cas9 knockout model of 14-3-3ß has not been investigated. This study confirmed previous findings and showed that siRNA inhibition of 14-3-3ß results in reduced cellular proliferation in a human glioblastoma cell line, U87MG. We also used a YWHAB Tet-On CRISPR/Cas9 U87MG cell line that, upon doxycycline induction, leads to robust Cas9 expression and subsequent knockout of 14-3-3ß. Using this model, we show that loss of 14-3-3ß significantly reduces cellular proliferation and spheroid formation of U87MG cells.

Chronic depletion of vertebrate lipids in Aedes aegypti cells dysregulates lipid metabolism and inhibits innate immunity without altering dengue infectivity.

Aedes aegypti is the primary vector of dengue virus (DENV) and other arboviruses. Previous literature suggests that vertebrate and invertebrate lipids and the nutritional status of mosquitoes modify virus infection. Here, we developed a vertebrate lipid-depleted Ae. aegypti cell line to investigate if chronic depletion of vertebrate lipids normally present in a blood meal and insect cell culture medium would impact cell growth and virus infection. Chronic depletion of vertebrate lipids reduced cell size and proliferation, although cells retained equivalent total intracellular lipids per cell by reducing lipolysis and modifying gene expression related to sugar and lipid metabolism. Downregulation of innate immunity genes was also observed. We hypothesized that chronic depletion of vertebrate lipids would impact virus infection; however, the same amount of DENV was produced per cell. This study reveals how Ae. aegypti cells adapt in the absence of vertebrate lipids, and how DENV can replicate equally well in cells that contain predominately vertebrate or invertebrate lipids.

Identification of Essential Genes Using Sequential CRISPR and siRNA Screens.

Genome-wide CRISPR and siRNA screening methodologies are powerful tools that are aptly suited to the discovery of essential genes. In this chapter, we outline our methods to conduct sequential CRISPR and siRNA screens to quickly and efficiently identify essential genes within a collection of cell lines. The utilization of both screening methodologies provides a pipeline that minimizes costs and time while enabling the robust detection of candidate genes.

Intermittent fasting: is there a role in the treatment of diabetes? A review of the literature and guide for primary care physicians.

BACKGROUND: Type 2 Diabetes is a metabolic disorder characterized by hyperglycemia that causes numerous complications with significant long-term morbidity and mortality. The disorder is primarily due to insulin resistance particularly in liver, skeletal muscle, and adipose tissue. In this review, we detail the hormonal mechanisms leading to the development of diabetes and discuss whether intermittent fasting should be considered as an alternative, non-medicinal treatment option for patients with this disorder. METHODS: We searched PubMed, Ovid MEDLINE, and Google Scholar databases for review articles, clinical trials, and case series related to type 2 diabetes, insulin resistance, and intermittent fasting. Articles were carefully reviewed and included based on relevance to our topic. We excluded abstracts and any non-English articles. RESULTS: The majority of the available research demonstrates that intermittent fasting is effective at reducing body weight, decreasing fasting glucose, decreasing fasting insulin, reducing insulin resistance, decreasing levels of leptin, and increasing levels of adiponectin. Some studies found that patients were able to reverse their need for insulin therapy during therapeutic intermittent fasting protocols with supervision by their physician. CONCLUSION: Current evidence suggests that intermittent fasting is an effective non-medicinal treatment option for type 2 diabetes. More research is needed to delineate the effects of intermittent fasting from weight loss. Physicians should consider educating themselves regarding the benefits of intermittent fasting. Diabetic patients should consult their physician prior to beginning an intermittent fasting regimen in order to allow for appropriate oversight and titration of the patients medication regimen during periods of fasting.

KANSL2 and MBNL3 are regulators of pancreatic ductal adenocarcinoma invasion.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer. One major reason for this is that PDAC quickly metastasizes to other organs, thereby making its treatment difficult. The molecular machinery driving PDAC metastasis is still poorly understood. In this study, we applied an unbiased approach using CRISPR screening to identify genes that strongly regulate invasion (based on an in vitro assessment of their metastatic potential) in PANC-1, a PDAC cell line. Through CRISPR screening, we identified MBNL3 and KANSL2 as strong regulators of invasion in PANC-1 cells. We further validated MBNL3 and KANSL2 as regulators of PANC-1 cell invasion by using the doxycycline-inducible shRNA system. We also showed that MBNL3 and KANSL2 do not affect cell proliferation. Through our efforts, we have established a process to identify genes that regulate cell invasion and can be further investigated as potential targets for therapeutic intervention.

Pediatric patients with acute lymphoblastic leukemia generate abundant and functional neoantigen-specific CD8+ T cell responses.

Cancer arises from the accumulation of genetic alterations, which can lead to the production of mutant proteins not expressed by normal cells. These mutant proteins can be processed and presented on the cell surface by major histocompatibility complex molecules as neoepitopes, allowing CD8+ T cells to mount responses against them. For solid tumors, only an average 2% of neoepitopes predicted by algorithms have detectable endogenous antitumor T cell responses. This suggests that low mutation burden tumors, which include many pediatric tumors, are poorly immunogenic. Here, we report that pediatric patients with acute lymphoblastic leukemia (ALL) have tumor-associated neoepitope-specific CD8+ T cells, responding to 86% of tested neoantigens and recognizing 68% of the tested neoepitopes. These responses include a public neoantigen from the ETV6-RUNX1 fusion that is targeted in seven of nine tested patients. We characterized phenotypic and transcriptional profiles of CD8+ tumor-infiltrating lymphocytes (TILs) at the single-cell level and found a heterogeneous population that included highly functional effectors. Moreover, we observed immunodominance hierarchies among the CD8+ TILs restricted to one or two putative neoepitopes. Our results indicate that robust antitumor immune responses are induced in pediatric ALL despite their low mutation burdens and emphasize the importance of immunodominance in shaping cellular immune responses. Furthermore, these data suggest that pediatric cancers may be amenable to immunotherapies aimed at enhancing immune recognition of tumor-specific neoantigens.

Genome-wide CRISPR screen reveals PSMA6 to be an essential gene in pancreatic cancer cells.

BACKGROUND: Despite its relatively low incidence, pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer deaths because of the aggressive growth/metastasis of the tumor, the lack of early symptoms, and the poor treatment options. Basic research to identify potential therapeutic targets for PDAC is greatly needed. METHODS: We used a negative-selection genome-wide CRISPR screen to identify essential genes in the PANC-1 human pancreatic carcinoma cell line. We validated the top hits with follow-up siRNA screens, using the HPNE, HPAF-II, AsPC-1, and Mia PaCa-2 cell lines. RESULTS: The PSMA6 gene was an identified candidate hit after the CRISPR screen, siRNA validation screen, and siRNA deconvolution screen. Spheroid formation assays and flow cytometry analysis showed that PSMA6 is critical for survival in many pancreatic ductal carcinoma cell models. Lastly, as PSMA6 protein is a proteosomal subunit of the 20S core complex, we showed that bortezomib, a proteasome inhibitor, was especially toxic in PANC-1 cells. CONCLUSIONS: Further study of PSMA6 and the proteasome subunit that it encodes, along with other hits identified in our CRISPR screens, may provide valuable insights into potential therapeutic targets for PDAC.

HNRNPH1 is required for rhabdomyosarcoma cell growth and survival.

Rhabdomyosarcoma (RMS) is an aggressive and difficult to treat cancer characterized by a muscle-like phenotype. Although the average 5-y survival rate is 65% for newly diagnosed RMS, the treatment options for metastatic disease are limited in efficacy, with the 5-y survival rate plummeting to 30%. Heterogenous nuclear ribonucleoprotein H1 (HNRNPH1) is an RNA-binding protein that is highly expressed in many cancers, including RMS. To determine the role HNRNPH1 plays in RMS tumorigenesis, we investigated its expression and effect on growth in three cellular models of RMS: RD, RH30, and RH41 cells. Upon knockdown of HNRNPH1, growth of all cell lines was reduced, most likely through a combination of apoptosis and cell cycle arrest. We then recapitulated this finding by performing in vivo xenograft studies, in which knockdown of HNRNPH1 resulted in a reduction of tumor formation and growth. We used RNA sequencing to identify changes in gene expression after HNRNPH1 knockdown and found altered splicing of some oncogenes. Our data contribute to understanding the role of HNRNPH1 in RMS development.

Transcription factor ZNF148 is a negative regulator of human muscle differentiation.

Muscle differentiation is a complex process in which muscle progenitor cells undergo determination and eventually cellular fusion. This process is heavily regulated by such master transcription factors as MYOD and members of the MEF2 family. Here, we show that the transcription factor ZNF148 plays a direct role in human muscle cell differentiation. Downregulation of ZNF148 drives the formation of a muscle phenotype with rapid expression of myosin heavy chain, even in proliferative conditions. This phenotype was most likely mediated by the robust and swift upregulation of MYOD and MEF2C.

MAP4K4 regulates integrin-FERM binding to control endothelial cell motility.

Cell migration is a stepwise process that coordinates multiple molecular machineries. Using in vitro angiogenesis screens with short interfering RNA and chemical inhibitors, we define here a MAP4K4-moesin-talin-ß1-integrin molecular pathway that promotes efficient plasma membrane retraction during endothelial cell migration. Loss of MAP4K4 decreased membrane dynamics, slowed endothelial cell migration, and impaired angiogenesis in vitro and in vivo. In migrating endothelial cells, MAP4K4 phosphorylates moesin in retracting membranes at sites of focal adhesion disassembly. Epistasis analyses indicated that moesin functions downstream of MAP4K4 to inactivate integrin by competing with talin for binding to ß1-integrin intracellular domain. Consequently, loss of moesin (encoded by the MSN gene) or MAP4K4 reduced adhesion disassembly rate in endothelial cells. Additionally, α5ß1-integrin blockade reversed the membrane retraction defects associated with loss of Map4k4 in vitro and in vivo. Our study uncovers a novel aspect of endothelial cell migration. Finally, loss of MAP4K4 function suppressed pathological angiogenesis in disease models, identifying MAP4K4 as a potential therapeutic target.

Protein-tyrosine phosphatase 1B substrates and metabolic regulation.

Metabolic homeostasis requires integration of complex signaling networks which, when deregulated, contribute to metabolic syndrome and related disorders. Protein-tyrosine phosphatase 1B (PTP1B) has emerged as a key regulator of signaling networks that are implicated in metabolic diseases such as obesity and type 2 diabetes. In this review, we examine mechanisms that regulate PTP1B-substrate interaction, enzymatic activity and experimental approaches to identify PTP1B substrates. We then highlight findings that implicate PTP1B in metabolic regulation. In particular, insulin and leptin signaling are discussed as well as recently identified PTP1B substrates that are involved in endoplasmic reticulum stress response, cell-cell communication, energy balance and vesicle trafficking. In summary, PTP1B exhibits exquisite substrate specificity and is an outstanding pharmaceutical target for obesity and type 2 diabetes.

Proteomic analysis of cAMP-mediated signaling during differentiation of 3 T3-L1 preadipocytes.

Initiation of adipocyte differentiation is promoted by the synergistic action of insulin/insulin-like growth factor, glucocorticoids, and agents activating cAMP-dependent signaling. The action of cAMP is mediated via PKA and Epac, where at least part of the PKA function relates to strong repression of Rho kinase activity, whereas Epac counteracts the reduction in insulin/insulin-like growth factor signaling associated with complete repression of Rho kinase activity. However, detailed knowledge of the Epac-dependent branch and the interplay with PKA is still limited. In the present study, we present a comprehensive evaluation of Epac-mediated processes and their interplay with PKA during the initiation of 3 T3-L1 preadipocyte differentiation using a combination of proteomics, molecular approaches, and bioinformatics. Proteomic analyses revealed 7 proteins specifically regulated in response to Epac activation, 4 in response to PKA activation, and 11 in response to the combined activation of Epac and PKA during the initial phase of differentiation. Network analyses indicated that the identified proteins are involved in pathways of importance for glucose metabolism, inositol metabolism, and calcium-dependent signaling thereby adding a novel facet to our understanding of cAMP-mediated potentiation of adipocyte differentiation.

Regulation of the SNARE-interacting protein Munc18c tyrosine phosphorylation in adipocytes by protein-tyrosine phosphatase 1B.

BACKGROUND: Protein-tyrosine phosphatase 1B (PTP1B) is a physiological regulator of insulin signaling and adiposity and is a drug target for the treatment of obesity and diabetes. The molecular mechanisms underlying PTP1B metabolic actions require additional investigation. RESULTS: Herein, we identify Munc18c as a novel PTP1B substrate in adipocytes and in vivo. We demonstrate nutritional regulation of Munc18c in adipose tissue revealing decreased expression upon high fat feeding. In addition, PTP1B deficiency leads to elevated Munc18c tyrosine phosphorylation and dissociation from syntaxin4. At the molecular level, we identify Munc18c Tyr218/219 and Tyr521 as key residues that mediate Munc18c interaction with PTP1B. Further, we uncover an essential role of Munc18c total tyrosine phosphorylation in general, and Tyr218/219 and Tyr521 in particular, in regulating its interactions and glucose uptake in adipocytes. CONCLUSION: In conclusion, our findings identify PTP1B as the first known tyrosine phosphatase for Munc18c and a regulator of its phosphorylation and function in adipocytes.

Protein tyrosine phosphatase 1B regulates pyruvate kinase M2 tyrosine phosphorylation.

Protein-tyrosine phosphatase 1B (PTP1B) is a physiological regulator of glucose homeostasis and adiposity and is a drug target for the treatment of obesity and diabetes. Here we identify pyruvate kinase M2 (PKM2) as a novel PTP1B substrate in adipocytes. PTP1B deficiency leads to increased PKM2 total tyrosine and Tyr(105) phosphorylation in cultured adipocytes and in vivo. Substrate trapping and mutagenesis studies identify PKM2 Tyr-105 and Tyr-148 as key sites that mediate PTP1B-PKM2 interaction. In addition, in vitro analyses illustrate a direct effect of Tyr-105 phosphorylation on PKM2 activity in adipocytes. Importantly, PTP1B pharmacological inhibition increased PKM2 Tyr-105 phosphorylation and decreased PKM2 activity. Moreover, PKM2 Tyr-105 phosphorylation is regulated nutritionally, decreasing in adipose tissue depots after high-fat feeding. Further, decreased PKM2 Tyr-105 phosphorylation correlates with the development of glucose intolerance and insulin resistance in rodents, non-human primates, and humans. Together, these findings identify PKM2 as a novel substrate of PTP1B and provide new insights into the regulation of adipose PKM2 activity.

Hepatic Src homology phosphatase 2 regulates energy balance in mice.

The Src homology 2 domain-containing protein-tyrosine phosphatase Src homology phosphatase 2 (Shp2) is a negative regulator of hepatic insulin action in mice fed regular chow. To investigate the role of hepatic Shp2 in lipid metabolism and energy balance, we determined the metabolic effects of its deletion in mice challenged with a high-fat diet (HFD). We analyzed body mass, lipid metabolism, insulin sensitivity, and glucose tolerance in liver-specific Shp2-deficient mice (referred to herein as LSHKO) and control mice fed HFD. Hepatic Shp2 protein expression is regulated by nutritional status, increasing in mice fed HFD and decreasing during fasting. LSHKO mice gained less weight and exhibited increased energy expenditure compared with control mice. In addition, hepatic Shp2 deficiency led to decreased liver steatosis, enhanced insulin-induced suppression of hepatic glucose production, and impeded the development of insulin resistance after high-fat feeding. At the molecular level, LSHKO exhibited decreased hepatic endoplasmic reticulum stress and inflammation compared with control mice. In addition, tyrosine and serine phosphorylation of total and mitochondrial signal transducer and activator of transcription 3 were enhanced in LSHKO compared with control mice. In line with this observation and the increased energy expenditure of LSHKO, oxygen consumption rate was higher in liver mitochondria of LSHKO compared with controls. Collectively, these studies identify hepatic Shp2 as a novel regulator of systemic energy balance under conditions of high-fat feeding.

Differential regulation of endoplasmic reticulum stress by protein tyrosine phosphatase 1B and T cell protein tyrosine phosphatase.

Protein-tyrosine phosphatase 1B (PTP1B) and T cell protein-tyrosine phosphatase (TCPTP) are closely related intracellular phosphatases implicated in the control of glucose homeostasis. PTP1B and TCPTP can function coordinately to regulate protein tyrosine kinase signaling, and PTP1B has been implicated previously in the regulation of endoplasmic reticulum (ER) stress. In this study, we assessed the roles of PTP1B and TCPTP in regulating ER stress in the endocrine pancreas. PTP1B and TCPTP expression was determined in pancreases from chow and high fat fed mice and the impact of PTP1B and TCPTP over- or underexpression on palmitate- or tunicamycin-induced ER stress signaling assessed in MIN6 insulinoma ß cells. PTP1B expression was increased, and TCPTP expression decreased in pancreases of mice fed a high fat diet, as well as in MIN6 cells treated with palmitate. PTP1B overexpression or TCPTP knockdown in MIN6 cells mitigated palmitate- or tunicamycin-induced PERK/eIF2α ER stress signaling, whereas PTP1B deficiency enhanced ER stress. Moreover, PTP1B deficiency increased ER stress-induced cell death, whereas TCPTP deficiency protected MIN6 cells from ER stress-induced death. ER stress coincided with the inhibition of Src family kinases (SFKs), which was exacerbated by PTP1B overexpression and largely prevented by TCPTP knockdown. Pharmacological inhibition of SFKs ameliorated the protective effect of TCPTP deficiency on ER stress-induced cell death. These results demonstrate that PTP1B and TCPTP play nonredundant roles in modulating ER stress in pancreatic ß cells and suggest that changes in PTP1B and TCPTP expression may serve as an adaptive response for the mitigation of chronic ER stress.

Coenzyme Q10 in human blood: native levels and determinants of oxidation during processing and storage.

Coenzyme Q10 (Q10) is present in the circulation mainly in its reduced form (ubiquinol-10; UL10), but oxidizes quickly ex vivo to ubiquinone-10 (UN10). Therefore, native UL10:UN10 ratios, used as markers of redox status and disease risk, are difficult to measure. We established an RP-(U)HPLC method with coulometric detection to measure natively circulating UL10 and UN10 concentrations by adding a ubiquinol/ubiquinone mixture as an internal standard immediately after plasma preparation. This allowed adjustment for unavoidable artificial UL10 oxidation as well as for total losses (or gains) of analytes during sample storage, processing, and analysis because the internal standards exactly paralleled the chemical behavior of Q10. This technique applied to blood (n = 13) revealed Q10 levels of 680-3300 nM with a mean UL10:UN10 ratio of 95:5, which was inversely associated with total Q10 (r=-0.69; p=0.004). The oxidation of UL10 to UN10 was equimolar, increased by O(2), and decreased by lower temperatures or various degassing methods. Although UL10 was stable in blood or when pure in organic solvents at 22 degrees C, its oxidation was catalyzed dose dependently by alpha-tocopherol and butylated hydroxytoluene, particularly when present in combination. Key structural features for the catalytic pro-oxidant properties of phenolic antioxidants included two substituents vicinal to the phenolic hydroxyl group.