Systematic Evaluation of the Metabolic to Mitogenic Potency Ratio for B10-Substituted Insulin Analogues

Background: Insulin analogues comprising acidic amino acid substitutions at position B10 have previously been shown to display increased mitogenic potencies compared to human insulin and the underlying molecular mechanisms have been subject to much scrutiny and debate. However, B10 is still an attractive position for amino acid substitutions given its important role in hexamer formation. The aim of this study was to investigate the relationships between the receptor binding properties as well as the metabolic and mitogenic potencies of a series of insulin analogues with different amino acid substitutions at position B10 and to identify a B10-substituted insulin analogue without an increased mitogenic to metabolic potency ratio. Methodology/Principal Findings: A panel of ten singly-substituted B10 insulin analogues with different amino acid side chain characteristics were prepared and insulin receptor (both isoforms) and IGF-I receptor binding affinities using purified receptors, insulin receptor dissociation rates using BHK cells over-expressing the human insulin receptor, metabolic potencies by lipogenesis in isolated rat adipocytes, and mitogenic potencies using two different cell types predominantly expressing either the insulin or the IGF-I receptor were systematically investigated. Only analogues B10D and B10E with significantly increased insulin and IGF-I receptor affinities as well as decreased insulin receptor dissociation rates displayed enhanced mitogenic potencies in both cell types employed. For the remaining analogues with less pronounced changes i

Energizing Star Formation: The Cosmic Ray Ionization Rate in NGC 253 Derived From ALCHEMI Measurements of H3_3O+^+ and SO

The cosmic ray ionization rate (CRIR) is a key parameter in understanding the physical and chemical processes in the interstellar medium. Cosmic rays are a significant source of energy in star formation regions, which impacts the physical and chemical processes which drive the formation of stars. Previous studies of the circum-molecular zone (CMZ) of the starburst galaxy NGC 253 have found evidence for a high CRIR value; $10^3-10^6$ times the average cosmic ray ionization rate within the Milky Way. This is a broad constraint and one goal of this study is to determine this value with much higher precision. We exploit ALMA observations towards the central molecular zone of NGC 253 to measure the CRIR. We first demonstrate that the abundance ratio of H$_3$O$^+$ and SO is strongly sensitive to the CRIR. We then combine chemical and radiative transfer models with nested sampling to infer the gas properties and CRIR of several star-forming regions in NGC 253 due to emission from their transitions. We find that each of the four regions modelled has a CRIR in the range $(1-80)\times10^{-14}$ s$^{-1}$ and that this result adequately fits the abundances of other species that are believed to be sensitive to cosmic rays including C$_2$H, HCO$^+$, HOC$^+$, and CO. From shock and PDR/XDR models, we further find that neither UV/X-ray driven nor shock dominated chemistry are a viable single alternative as none of these processes can adequately fit the abundances of all of these species.Comment: 24 pages, 15 figures, accepted for publication in Ap

Starburst Energy Feedback Seen through HCO+/HOC+Emission in NGC 253 from ALCHEMI

Molecular abundances are sensitive to the UV photon flux and cosmic-ray ionization rate. In starburst environments, the effects of high-energy photons and particles are expected to be stronger. We examine these astrochemical signatures through multiple transitions of HCO+ and its metastable isomer HOC+ in the center of the starburst galaxy NGC 253 using data from the Atacama Large Millimeter/submillimeter Array large program ALMA Comprehensive High-resolution Extragalactic Molecular inventory. The distribution of the HOC+(1-0) integrated intensity shows its association with "superbubbles,"cavities created either by supernovae or expanding H ii regions. The observed HCO+/HOC+ abundance ratios are ∼10-150, and the fractional abundance of HOC+ relative to H2 is ∼1.5 × 10-11-6 × 10-10, which implies that the HOC+ abundance in the center of NGC 253 is significantly higher than in quiescent spiral arm dark clouds in the Galaxy and the Galactic center clouds. Comparison with chemical models implies either an interstellar radiation field of G 0 ⪆ 103 if the maximum visual extinction is ⪆5, or a cosmic-ray ionization rate of ζ ⪆ 10-14 s-1 (3-4 orders of magnitude higher than that within clouds in the Galactic spiral arms) to reproduce the observed results. From the difference in formation routes of HOC+, we propose that a low-excitation line of HOC+ traces cosmic-ray dominated regions, while high-excitation lines trace photodissociation regions. Our results suggest that the interstellar medium in the center of NGC 253 is significantly affected by energy input from UV photons and cosmic rays, sources of energy feedback.N.H. acknowledges support from JSPS KAKENHI grant No. JP21K03634. K.S. has been supported by grants MOST 108-2112-M-001-015 and 109- 2112-M-001-020 from the Ministry of Science and Technology, Taiwan. Y.N. is supported by the NAOJ ALMA Scientific Research grant No. 2017-06B. V.M.R. and L.C. are funded by the Comunidad de Madrid through the Atracción de Talento Investigador (Doctores con experiencia) Grant (COOL: Cosmic Origins Of Life; 2019-T1/TIC-15379)

Mental Disorders in Megacities: Findings from the São Paulo Megacity Mental Health Survey, Brazil

Background: World population growth is projected to be concentrated in megacities, with increases in social inequality and urbanization-associated stress. São Paulo Metropolitan Area (SPMA) provides a forewarning of the burden of mental disorders in urban settings in developing world. The aim of this study is to estimate prevalence, severity, and treatment of recently active DSM-IV mental disorders. We examined socio-demographic correlates, aspects of urban living such as internal migration, exposure to violence, and neighborhood-level social deprivation with 12-month mental disorders. Methods and Results: A representative cross-sectional household sample of 5,037 adults was interviewed face-to-face using the WHO Composite International Diagnostic Interview (CIDI), to generate diagnoses of DSM-IV mental disorders within 12 months of interview, disorder severity, and treatment. Administrative data on neighborhood social deprivation were gathered. Multiple logistic regression was used to evaluate individual and contextual correlates of disorders, severity, and treatment. Around thirty percent of respondents reported a 12-month disorder, with an even distribution across severity levels. Anxiety disorders were the most common disorders (affecting 19.9%), followed by mood (11%), impulse-control (4.3%), and substance use (3.6%) disorders. Exposure to crime was associated with all four types of disorder. Migrants had low prevalence of all four types compared to stable residents. High urbanicity was associated with impulse-control disorders and high social deprivation with substance use disorders. Vulnerable subgroups were observed: women and migrant men living in most deprived areas. Only one-third of serious cases had received treatment in the previous year. Discussion: Adults living in São Paulo megacity had prevalence of mental disorders at greater levels than similar surveys conducted in other areas of the world. Integration of mental health promotion and care into the rapidly expanding Brazilian primary health system should be strengthened. This strategy might become a model for poorly resourced and highly populated developing countries

Engineering of Insulin Receptor Isoform-Selective Insulin Analogues

BACKGROUND: The insulin receptor (IR) exists in two isoforms, A and B, and the isoform expression pattern is tissue-specific. The C-terminus of the insulin B chain is important for receptor binding and has been shown to contact the IR just adjacent to the region where the A and B isoforms differ. The aim of this study was to investigate the importance of the C-terminus of the B chain in IR isoform binding in order to explore the possibility of engineering tissue-specific/liver-specific insulin analogues. METHODOLOGY/PRINCIPAL FINDINGS: Insulin analogue libraries were constructed by total amino acid scanning mutagenesis. The relative binding affinities for the A and B isoform of the IR were determined by competition assays using scintillation proximity assay technology. Structural information was obtained by X-ray crystallography. Introduction of B25A or B25N mutations resulted in analogues with a 2-fold preference for the B compared to the A isoform, whereas the opposite was observed with a B25Y substitution. An acidic amino acid residue at position B27 caused an additional 2-fold selective increase in affinity for the receptor B isoform for analogues bearing a B25N mutation. Furthermore, the combination of B25H with either B27D or B27E also resulted in B isoform-preferential analogues (2-fold preference) even though the corresponding single mutation analogues displayed no differences in relative isoform binding affinity. CONCLUSIONS/SIGNIFICANCE: We have discovered a new class of IR isoform-selective insulin analogues with 2-4-fold differences in relative binding affinities for either the A or the B isoform of the IR compared to human insulin. Our results demonstrate that a mutation at position B25 alone or in combination with a mutation at position B27 in the insulin molecule confers IR isoform selectivity. Isoform-preferential analogues may provide new opportunities for developing insulin analogues with improved clinical benefits

Mutation analysis in Bardet-Biedl syndrome by DNA pooling and massively parallel resequencing in 105 individuals

Bardet–Biedl syndrome (BBS) is a rare, primarily autosomal-recessive ciliopathy. The phenotype of this pleiotropic disease includes retinitis pigmentosa, postaxial polydactyly, truncal obesity, learning disabilities, hypogonadism and renal anomalies, among others. To date, mutations in 15 genes (BBS1–BBS14, SDCCAG8) have been described to cause BBS. The broad genetic locus heterogeneity renders mutation screening time-consuming and expensive. We applied a strategy of DNA pooling and subsequent massively parallel resequencing (MPR) to screen individuals affected with BBS from 105 families for mutations in 12 known BBS genes. DNA was pooled in 5 pools of 21 individuals each. All 132 coding exons of BBS1–BBS12 were amplified by conventional PCR. Subsequent MPR was performed on an Illumina Genome Analyzer II(™) platform. Following mutation identification, the mutation carrier was assigned by CEL I endonuclease heteroduplex screening and confirmed by Sanger sequencing. In 29 out of 105 individuals (28%), both mutated alleles were identified in 10 different BBS genes. A total of 35 different disease-causing mutations were confirmed, of which 18 mutations were novel. In 12 additional families, a total of 12 different single heterozygous changes of uncertain pathogenicity were found. Thus, DNA pooling combined with MPR offers a valuable strategy for mutation analysis of large patient cohorts, especially in genetically heterogeneous diseases such as BBS

Gastric opioid peptides : their biochemical forms, receptor distribution, release and actions

Gastric opioid peptides were characterized on the basis of their biochemical forms, sites of action, mode of release and effects on endocrine secretions of the stomach. Partial purification of opioid-like material from extracts of the corpus/antrum region of the rat stomach was carried out by Sephadex G-50 gel filtration chromatography followed by adsorption onto Amberlite XAD-2 resin. A single peak of opioid activity was determined by both radioreceptor assay (RRA) and bioassay. By high performance liquid chromatography (HPLC) this peak was resolved into several distinct components identified by their retention times and measurement by RRA and/or radioimmunoassay (RIA) as corresponding to methionine-enkephalin (met-enk), leucine-enkephalin (leu-enk), met-enk-arg-gly-leu, met-enk-arg-phe, as well as dynorphins 1-13, 1-17, and 1-8. Trypsin digestion of partially purified extracts resulted in an overall increase in opioid activity, suggesting the presence of larger, inactive forms which may function as precursors. The sites of opioid peptide action were inferred from autoradiographic demonstrations of the distribution of tritium-labelled opioid ligand binding. In the fundic region of the rat stomach mu- and delta-type opioid receptors were localized in the circular muscle, muscularis mucosae, suggesting that here the opioid peptides may be involved in the regulation of motor activity. In the corpus and antrum, these two opioid receptor types were found to be associated with the deepmuscular plexus in some areas, but predominantly in the submucosal plexus and mucosa where the opioid peptides may act to directly affect gastric endocrine and exocrine secretions. Mu-type ligands also bound to the circular muscle and myenteric plexus. Intestinal tissue demonstrated mu- and delta-opioid binding sites throughout the mucosa, extending to the tips of the villi, implicating their involvement in the regulation of intestinal fluid and electrolyte absorption. Using the isolated, perfused rat stomach preparation, endogenous leu-enk was found to be released into the gastric vasculature in response to potassium depolarization and to the nicotinic cholinergic agonist dimethyl-phenyl-piperazinium (DMPP). The muscarinic cholinergic agonist, methacholine, had no effect. Exogenously infused met-enk caused a prompt dose-dependent, naloxone sensitive inhibition of gastric inhibitory polypeptide (GlP)-stimulated somatostatin (SLI) secretion. A similar, but reduced effect was observed with dynorphin 1-13. Neither atropine nor hexamethonium affected the met-enk inhibition of GIP-stimulated SLI; therefore, it was proposed that met-enk was acting directly on the D-cells to inhibit somatostatin secretion, possibly by interacting with mucosal opioid receptors demonstrated by autoradiography. These results, demonstrating the presence of endogenous gastric opioid peptides of both the enkephalin and dynorphin families and the widespread distribution of opioid receptors in the gastrointestinal tract, coupled with the observed release of endogenous gastric leu-enk, and potent effects of opioid peptides on SLI secretion from the stomach, indicate that theopioid peptides are important physiological regulators of gastric and intestinal functions.Medicine, Faculty ofCellular and Physiological Sciences, Department ofGraduat

Free fatty acid-induced PP2A hyperactivity selectively impairs hepatic insulin action on glucose metabolism.

In type 2 Diabetes (T2D) free fatty acids (FFAs) in plasma are increased and hepatic insulin resistance is "selective", in the sense that the insulin-mediated decrease of glucose production is blunted while insulin's effect on stimulating lipogenesis is maintained. We investigated the molecular mechanisms underlying this pathogenic paradox. Primary rat hepatocytes were exposed to palmitate for twenty hours. To establish the physiological relevance of the in vitro findings, we also studied insulin-resistant Zucker Diabetic Fatty (ZDF) rats. While insulin-receptor phosphorylation was unaffected, activation of Akt and inactivation of the downstream targets Glycogen synthase kinase 3α (Gsk3α and Forkhead box O1 (FoxO1) was inhibited in palmitate-exposed cells. Accordingly, dose-response curves for insulin-mediated suppression of the FoxO1-induced gluconeogenic genes and for de novo glucose production were right shifted, and insulin-stimulated glucose oxidation and glycogen synthesis were impaired. In contrast, similar to findings in human T2D, the ability of insulin to induce triglyceride (TG) accumulation and transcription of the enzymes that catalyze de novo lipogenesis and TG assembly was unaffected. Insulin-induction of these genes could, however, be blocked by inhibition of the atypical PKCs (aPKCs). The activity of the Akt-inactivating Protein Phosphatase 2A (PP2A) was increased in the insulin-resistant cells. Furthermore, inhibition of PP2A by specific inhibitors increased insulin-stimulated activation of Akt and phosphorylation of FoxO1 and Gsk3α. Finally, PP2A mRNA levels were increased in liver, muscle and adipose tissue, while PP2A activity was increased in liver and muscle tissue in insulin-resistant ZDF rats. In conclusion, our findings indicate that FFAs may cause a selective impairment of insulin action upon hepatic glucose metabolism by increasing PP2A activity