Severe hypoglycemia and the risk of end stage renal disease in type 2 diabetes.

We investigated the association between the incidence of severe hypoglycemia and the risk of end-stage renal disease (ESRD) in patients with type 2 diabetes. Baseline and follow-up data for 988,333 participants with type 2 diabetes were retrieved from the National Health Insurance System database. The number of severe hypoglycemia episodes experienced from 2007 to 2009 was determined. The primary outcome was the development of ESRD after the baseline evaluation. Participants were followed from the baseline until death or December 31, 2016, during this period 14,545 participants (1.5%) developed ESRD. In the crude model, compared with those who experienced no severe hypoglycemia, the hazard ratios (95% confidential intervals) for developing ESRD were 4.96 (4.57-5.39), 6.84 (5.62-8.32), and 9.51 (7.14-12.66) in participants who experienced one, two, and three or more episodes of severe hypoglycemia, respectively. Further adjustment for various confounding factors attenuated the association between severe hypoglycemia and ESRD; the significance of the association between severe hypoglycemia and ESRD was maintained. Having three or more severe hypoglycemia episodes was associated with a nearly two-fold increased risk of developing ESRD. Prior episodes of severe hypoglycemia were associated with an increased risk of ESRD among Korean adults with type 2 diabetes.

Analysis of phototoxin taste closely correlates nucleophilicity to type 1 phototoxicity.

Pigments often inflict tissue-damaging and proaging toxicity on light illumination by generating free radicals and reactive oxygen species (ROS). However, the molecular mechanism by which organisms sense phototoxic pigments is unknown. Here, we discover that Transient Receptor Potential Ankyrin 1-A isoform [TRPA1(A)], previously shown to serve as a receptor for free radicals and ROS induced by photochemical reactions, enables Drosophila melanogaster to aphotically sense phototoxic pigments for feeding deterrence. Thus, TRPA1(A) detects both cause (phototoxins) and effect (free radicals and ROS) of photochemical reactions. A group of pigment molecules not only activates TRPA1(A) in darkness but also generates free radicals on light illumination. Such aphotic detection of phototoxins harboring the type 1 (radical-generating) photochemical potential requires the nucleophile-sensing ability of TRPA1. In addition, agTRPA1(A) from malaria-transmitting mosquitoes Anopheles gambiae heterologously produces larger current responses to phototoxins than Drosophila TRPA1(A), similar to their disparate nucleophile responsiveness. Along with TRPA1(A)-stimulating capabilities, type 1 phototoxins exhibit relatively strong photo-absorbance and low energy gaps between the highest occupied molecular orbital and the lowest unoccupied molecular orbital. However, TRPA1(A) activation is more highly concordant to type 1 phototoxicity than are those photochemical parameters. Collectively, nucleophile sensitivity of TRPA1(A) allows flies to taste potential phototoxins for feeding deterrence, preventing postingestive photo-injury. Conversely, pigments need to bear high nucleophilicity (electron-donating propensity) to act as type 1 phototoxins, which is consistent with the fact that transferring photoexcited electrons from phototoxins to other molecules causes free radicals. Thus, identification of a sensory mechanism in Drosophila reveals a property fundamental to type 1 phototoxins.

14-3-3γ Haploinsufficient Mice Display Hyperactive and Stress-sensitive Behaviors.

14-3-3γ plays diverse roles in different aspects of cellular processes. Especially in the brain where 14-3-3γ is enriched, it has been reported to be involved in neurological and psychiatric diseases (e.g. Williams-Beuren syndrome and Creutzfeldt-Jakob disease). However, behavioral abnormalities related to 14-3-3γ deficiency are largely unknown. Here, by using 14-3-3γ deficient mice, we found that homozygous knockout mice were prenatally lethal, and heterozygous mice showed developmental delay relative to wild-type littermate mice. In addition, in behavioral analyses, we found that 14-3-3γ heterozygote mice display hyperactive and depressive-like behavior along with more sensitive responses to acute stress than littermate control mice. These results suggest that 14-3-3γ levels may be involved in the developmental manifestation of related neuropsychiatric diseases. In addition, 14-3-3γ heterozygote mice may be a potential model to study the molecular pathophysiology of neuropsychiatric symptoms.

Ionotropic Receptor 76b Is Required for Gustatory Aversion to Excessive Na+ in Drosophila.

Avoiding ingestion of excessively salty food is essential for cation homeostasis that underlies various physiological processes in organisms. The molecular and cellular basis of the aversive salt taste, however, remains elusive. Through a behavioral reverse genetic screening, we discover that feeding suppression by Na+-rich food requires Ionotropic Receptor 76b (Ir76b) in Drosophila labellar gustatory receptor neurons (GRNs). Concentrated sodium solutions with various anions caused feeding suppression dependent on Ir76b. Feeding aversion to caffeine and high concentrations of divalent cations and sorbitol was unimpaired in Ir76b-deficient animals, indicating sensory specificity of Ir76b-dependent Na+ detection and the irrelevance of hyperosmolarity-driven mechanosensation to Ir76b-mediated feeding aversion. Ir76b-dependent Na+-sensing GRNs in both L- and s-bristles are required for repulsion as opposed to the previous report where the L-bristle GRNs direct only low-Na+ attraction. Our work extends the physiological implications of Ir76b from low-Na+ attraction to high-Na+ aversion, prompting further investigation of the physiological mechanisms that modulate two competing components of Na+-evoked gustation coded in heterogeneous Ir76b-positive GRNs.

Conditional deletion of Ndufs4 in dopaminergic neurons promotes Parkinson's disease-like non-motor symptoms without loss of dopamine neurons.

Reduction of mitochondrial complex I activity is one of the major hypotheses for dopaminergic neuron death in Parkinson's disease. However, reduction of complex I activity in all cells or selectively in dopaminergic neurons via conditional deletion of the Ndufs4 gene, a subunit of the mitochondrial complex I, does not cause dopaminergic neuron death or motor impairment. Here, we investigated the effect of reduced complex I activity on non-motor symptoms associated with Parkinson's disease using conditional knockout (cKO) mice in which Ndufs4 was selectively deleted in dopaminergic neurons (Ndufs4 cKO). This conditional deletion of Ndufs4, which reduces complex I activity in dopamine neurons, did not cause a significant loss of dopaminergic neurons in substantia nigra pars compacta (SNpc), and there was no loss of dopaminergic neurites in striatum or amygdala. However, Ndufs4 cKO mice had a reduced amount of dopamine in the brain compared to control mice. Furthermore, even though motor behavior were not affected, Ndufs4 cKO mice showed non-motor symptoms experienced by many Parkinson's disease patients including impaired cognitive function and increased anxiety-like behavior. These data suggest that mitochondrial complex I dysfunction in dopaminergic neurons promotes non-motor symptoms of Parkinson's disease and reduces dopamine content in the absence of dopamine neuron loss.

The effect of lead exposure on fatty acid composition in mouse brain analyzed using pseudo-catalytic derivatization.

We performed toxicological study of mice exposed to lead by quantifying fatty acids in brain of the mice. This study suggests that the introduced analytical method had an extremely high tolerance against impurities such as water and extractives; thus, it led to the enhanced resolution in visualizing the spectrum of fatty acid profiles in animal brain. Furthermore, one of the biggest technical advantages achieved in this study was the quantitation of fatty acid methyl ester profiles of mouse brain using a trace amount of sample (e.g., 100 µL mixture). Methanol was screened as the most effective extraction solvent for mouse brain. The behavioral test of the mice before and after lead exposure was conducted to see the effect of lead exposure on fatty acid composition of the mice' brain. The lead exposure led to changes in disease-related behavior of the mice. Also, the lead exposure induced significant alterations of fatty acid profile (C16:0, C 18:0, and C 18:1) in brain of the mice, implicated in pathology of psychiatric diseases. The alteration of fatty acid profile of brain of the mice suggests that the derivatizing technique can be applicable to most research fields associated with the environmental neurotoxins with better resolution in a short time, as compared to the current protocols for lipid analysis.

A case of gabapentin-induced rhabdomyolysis requiring renal replacement therapy.

Gabapentin is commonly used for controlling convulsions, restless pain syndrome, and pain in diabetic neuropathy. Common side effects include dizziness, somnolence, ataxia, peripheral edema, and confusion; gabapentin-induced rhabdomyolysis is rarely reported. To date, the reported cases of gabapentin-induced rhabdomyolysis have been associated with patients with multiple underlying diseases and assuming multiple medicines for various reasons. In this report, we describe a case of gabapentin-induced rhabdomyolysis in a 32-year-old woman with no medical history. We also review related literature and discuss the possible mechanism and the association with other factors. This case shows that gabapentin can induce rhabdomyolysis in healthy patients and that clinicians must consider the possible association between gabapentin and rhabdomyolysis.

Estimating total lipid content of Camelina sativa via pyrolysis assisted in-situ transesterification with dimethyl carbonate.

Direct derivatization of C. sativa seed into FAMEs without lipid extraction was conducted for the quantification of lipid analysis via in-situ thermal methylation with dimethyl carbonate as an acyl acceptor on silica (SiO2). The introduced method had an extraordinarily high tolerance against impurities such as pyrolytic products and moisture. To ensure the technical completeness of in-situ methylation, thermal cracking of FAMEs transformed from C. sativa seed was also explored. Thermal cracking of unsaturated FAMEs such as C18:1, C18:2, C18:3 and C20:1 occurred at temperatures higher than 365°C due to their thermal instability. Thus, experimental findings in this study suggests not only that qualitative analysis of fatty acid profile in C. sativa seed via in-situ methylation using SiO2 could be achieve, but also that the total lipid content (42.65wt.%) in C. sativa seed could be accurately estimated.

In-situ pyrogenic production of biodiesel from swine fat.

In-situ production of fatty acid methyl esters from swine fat via thermally induced pseudo-catalytic transesterification on silica was investigated in this study. Instead of methanol, dimethyl carbonate (DMC) was used as acyl acceptor to achieve environmental benefits and economic viability. Thermo-gravimetric analysis of swine fat reveals that swine fat contains 19.57wt.% of water and impurities. Moreover, the fatty acid profiles obtained under various conditions (extracted swine oil+methanol+NaOH, extracted swine oil+DMC+pseudo-catalytic, and swine fat+DMC+pseudo-catalytic) were compared. These profiles were identical, showing that the introduced in-situ transesterification is technically feasible. This also suggests that in-situ pseudo-catalytic transesterification has a high tolerance against impurities. This study also shows that FAME yield via in-situ pseudo-catalytic transesterification of swine fat reached up to 97.2% at 380°C. Therefore, in-situ pseudo-catalytic transesterification can be applicable to biodiesel production of other oil-bearing biomass feedstocks.

Nucleophile sensitivity of Drosophila TRPA1 underlies light-induced feeding deterrence.

Solar irradiation including ultraviolet (UV) light causes tissue damage by generating reactive free radicals that can be electrophilic or nucleophilic due to unpaired electrons. Little is known about how free radicals induced by natural sunlight are rapidly detected and avoided by animals. We discover that Drosophila Transient Receptor Potential Ankyrin 1 (TRPA1), previously known only as an electrophile receptor, sensitively detects photochemically active sunlight through nucleophile sensitivity. Rapid light-dependent feeding deterrence in Drosophila was mediated only by the TRPA1(A) isoform, despite the TRPA1(A) and TRPA1(B) isoforms having similar electrophile sensitivities. Such isoform dependence re-emerges in the detection of structurally varied nucleophilic compounds and nucleophilicity-accompanying hydrogen peroxide (H2O2). Furthermore, these isoform-dependent mechanisms require a common set of TRPA1(A)-specific residues dispensable for electrophile detection. Collectively, TRPA1(A) rapidly responds to natural sunlight intensities through its nucleophile sensitivity as a receptor of photochemically generated radicals, leading to an acute light-induced behavioral shift in Drosophila.

Enhanced thermal destruction of toxic microalgal biomass by using CO2.

This work confirmed that dominant microalgal strain in the eutrophic site (the Han River in Korea) was Microcystis aeruginosa (M. aeruginosa) secreting toxins. Collected and dried microalgal biomass had an offensive odor due to microalgal lipid, of which the content reached up to 2±0.2wt.% of microalgal biomass (dry basis). This study has validated that the offensive odor is attributed to the C3-6 range of volatile fatty acids (VFAs), which was experimentally identified by the non-catalytic transformation of triglycerides (TGs) and free fatty acids (FFAs) in microalgal biomass into fatty acid methyl esters (FAMEs). In particular, this study mechanistically investigated the influence of CO2 in the thermal destruction (i.e., pyrolysis) of hazardous microalgal biomass in order to achieve dual purposes (i.e., thermal disposal of hazardous microalgal biomass and energy recovery). The influence of CO2 in pyrolysis of microalgal biomass was identified as 1) the enhanced thermal cracking behaviors of volatile organic compounds (VOCs) from the thermal degradation of microalgal biomass and 2) the direct gas phase reaction between CO2 and VOCs. These identified influences of CO2 in pyrolysis of microalgal biomass significantly enhanced the generation of CO: the enhanced generation of CO in the presence of CO2 was 590% at 660°C, 1260% at 690°C, and 3200% at 720°C. In addition, two identified influences of CO2 (i.e., enhanced thermal cracking and direct gas phase reaction) occurred simultaneously and independently. The identified gas phase reaction in the presence of CO2 was only initiated at temperatures higher than 500°C, which was different from the Boudouard reaction. Lastly, the experimental work justified that exploiting CO2 as a reaction medium and/or chemical feedstock will provide new technical approaches for controlling syngas ratio and in-situ air pollutant control without using catalysts.

Second-look endoscopy and factors associated with delayed bleeding after endoscopic submucosal dissection.

Endoscopic submucosal dissection (ESD) is a widely used procedure as curative treatment for superficial gastric neoplasms, including early gastric cancer without lymph node metastasis. However, ESD requires advanced endoscopic skill and there is a major concern regarding complications from bleeding. So far, extensive efforts have been made to develop strategies to reduce post-ESD bleeding. Use of proton pump inhibitors and coagulating exposed vessels on the ulcer floor after ESD are strategies known to reduce the risk of delayed bleeding. Second-look endoscopy (SLE) is also carried out to reduce delayed bleeding following ESD in many institutions. However, the incidence of bleeding still remains around 5%, and further measures are needed to reduce delayed bleeding after gastric ESD. Recently, three randomized studies indicated that routine SLE was unnecessary. Although routine SLE may not be recommended for all patients after gastric ESD, SLE might be an important tool for the prevention of the delayed bleeding in selected high-risk patients. Thus, the identification of the risk factors, such as large size of resected specimen and treatment with multiple antiplatelet medications, may help to further guide clinicians in deciding whether to perform SLE. Studies carried out on larger cohorts are necessary to clarify the efficacy of SLE after ESD in the prevention of post-ESD bleeding in potentially high-risk patients.

TrpA1 Regulates Defecation of Food-Borne Pathogens under the Control of the Duox Pathway.

Pathogen expulsion from the gut is an important defense strategy against infection, but little is known about how interaction between the intestinal microbiome and host immunity modulates defecation. In Drosophila melanogaster, dual oxidase (Duox) kills pathogenic microbes by generating the microbicidal reactive oxygen species (ROS), hypochlorous acid (HOCl) in response to bacterially excreted uracil. The physiological function of enzymatically generated HOCl in the gut is, however, unknown aside from its anti-microbial activity. Drosophila TRPA1 is an evolutionarily conserved receptor for reactive chemicals like HOCl, but a role for this molecule in mediating responses to gut microbial content has not been described. Here we identify a molecular mechanism through which bacteria-produced uracil facilitates pathogen-clearing defecation. Ingestion of uracil increases defecation frequency, requiring the Duox pathway and TrpA1. The TrpA1(A) transcript spliced with exon10b (TrpA1(A)10b) that is present in a subset of midgut enteroendocrine cells (EECs) is critical for uracil-dependent defecation. TRPA1(A)10b heterologously expressed in Xenopus oocytes is an excellent HOCl receptor characterized with elevated sensitivity and fast activation kinetics of macroscopic HOCl-evoked currents compared to those of the alternative TRPA1(A)10a isoform. Consistent with TrpA1's role in defecation, uracil-excreting Erwinia carotovora showed higher persistence in TrpA1-deficient guts. Taken together, our results propose that the uracil/Duox pathway promotes bacteria expulsion from the gut through the HOCl-sensitive receptor, TRPA1(A)10b, thereby minimizing the chances that bacteria adapt to survive host defense systems.

The Mosquito Repellent Citronellal Directly Potentiates Drosophila TRPA1, Facilitating Feeding Suppression.

Citronellal, a well-known plant-derived mosquito repellent, was previously reported to repel Drosophila melanogaster via olfactory pathways involving but not directly activating Transient Receptor Potential Ankyrin 1 (TRPA1). Here, we show that citronellal is a direct agonist for Drosophila and human TRPA1s (dTRPA1 and hTRPA1) as well as Anopheles gambiae TRPA1 (agTRPA1). Citronellal-induced activity is isoform-dependent for Drosophila and Anopheles gambiae TRPA1s. The recently identified dTRPA1(A) and ag-TRPA1(A) isoforms showed citronellal-provoked currents with EC50s of 1.0 B1 0.2 and 0.1 B1 0.03 mM, respectively, in Xenopus oocytes, while the sensitivities of TRPA1(B)s were much inferior to those of TRPA1(A)s. Citronellal dramatically enhanced the feeding-inhibitory effect of the TRPA1 agonist N-methylmaleimide (NMM) in Drosophila at an NMM concentration that barely repels flies. Thus, citronellal can promote feeding deterrence of fruit flies through direct action on gustatory dTRPA1, revealing the first isoform-specific function for TRPA1(A).

Analysis of the lipid profiles in a section of bovine brain via non-catalytic rapid methylation.

The main focus of this study is to mechanistically introduce a new qualitative and quantitative technique for mapping the lipid profile of a sectional brain via non-catalytic transesterification reaction (i.e., pseudo catalytic reaction in the presence of porous materials). One of the biggest technical advances achieved in this study is the qualitative and quantitative analysis of lipid from bovine brain in trace quantities in the magnitude of µg via the non-catalytic pathway. Moreover, newly introduced derivatization in this work showed high tolerance against impurities (i.e., water and extractives).

SNAREs support atlastin-mediated homotypic ER fusion in Saccharomyces cerevisiae.

Dynamin-like GTPases of the atlastin family are thought to mediate homotypic endoplasmic reticulum (ER) membrane fusion; however, the underlying mechanism remains largely unclear. Here, we developed a simple and quantitative in vitro assay using isolated yeast microsomes for measuring yeast atlastin Sey1p-dependent ER fusion. Using this assay, we found that the ER SNAREs Sec22p and Sec20p were required for Sey1p-mediated ER fusion. Consistently, ER fusion was significantly reduced by inhibition of Sec18p and Sec17p, which regulate SNARE-mediated membrane fusion. The involvement of SNAREs in Sey1p-dependent ER fusion was further supported by the physical interaction of Sey1p with Sec22p and Ufe1p, another ER SNARE. Furthermore, our estimation of the concentration of Sey1p on isolated microsomes, together with the lack of fusion between Sey1p proteoliposomes even with a 25-fold excess of the physiological concentration of Sey1p, suggests that Sey1p requires additional factors to support ER fusion in vivo. Collectively, our data strongly suggest that SNARE-mediated membrane fusion is involved in atlastin-initiated homotypic ER fusion.

Genetic reduction of mitochondrial complex I function does not lead to loss of dopamine neurons in vivo.

Inhibition of mitochondrial complex I activity is hypothesized to be one of the major mechanisms responsible for dopaminergic neuron death in Parkinson's disease. However, loss of complex I activity by systemic deletion of the Ndufs4 gene, one of the subunits comprising complex I, does not cause dopaminergic neuron death in culture. Here, we generated mice with conditional Ndufs4 knockout in dopaminergic neurons (Ndufs4 conditional knockout mice [cKO]) to examine the effect of complex I inhibition on dopaminergic neuron function and survival during aging and on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment in vivo. Ndufs4 cKO mice did not show enhanced dopaminergic neuron loss in the substantia nigra pars compacta or dopamine-dependent motor deficits over the 24-month life span. These mice were just as susceptible to MPTP as control mice. However, compared with control mice, Ndufs4 cKO mice exhibited an age-dependent reduction of dopamine in the striatum and increased α-synuclein phosphorylation in dopaminergic neurons of the substantia nigra pars compacta. We also used an inducible Ndufs4 knockout mouse strain (Ndufs4 inducible knockout) in which Ndufs4 is conditionally deleted in all cells in adult to examine the effect of adult onset, complex I inhibition on MPTP sensitivity of dopaminergic neurons. The Ndufs4 inducible knockout mice exhibited similar sensitivity to MPTP as control littermates. These data suggest that mitochondrial complex I inhibition in dopaminergic neurons does contribute to dopamine loss and the development of α-synuclein pathology. However, it is not sufficient to cause cell-autonomous dopaminergic neuron death during the normal life span of mice. Furthermore, mitochondrial complex I inhibition does not underlie MPTP toxicity in vivo in either cell autonomous or nonautonomous manner. These results provide strong evidence that inhibition of mitochondrial complex I activity is not sufficient to cause dopaminergic neuron death during aging nor does it contribute to dopamine neuron toxicity in the MPTP model of Parkinson's disease. These findings suggest the existence of alternative mechanisms of dopaminergic neuron death independent of mitochondrial complex I inhibition.