l-cysteine reversibly inhibits glucose-induced biphasic insulin secretion and ATP production by inactivating PKM2

Increase in the concentration of plasma l-cysteine is closely associated with defective insulin secretion from pancreatic β-cells, which results in type 2 diabetes (T2D). In this study, we investigated the effects of prolonged l-cysteine treatment on glucose-stimulated insulin secretion (GSIS) from mouse insulinoma 6 (MIN6) cells and from mouse pancreatic islets, and found that the treatment reversibly inhibited glucose-induced ATP production and resulting GSIS without affecting proinsulin and insulin synthesis. Comprehensive metabolic analyses using capillary electrophoresis time-of-flight mass spectrometry showed that prolonged l-cysteine treatment decreased the levels of pyruvate and its downstream metabolites. In addition, methyl pyruvate, a membrane-permeable form of pyruvate, rescued l-cysteine–induced inhibition of GSIS. Based on these results, we found that both in vitro and in MIN6 cells, l-cysteine specifically inhibited the activity of pyruvate kinase muscle isoform 2 (PKM2), an isoform of pyruvate kinases that catalyze the conversion of phosphoenolpyruvate to pyruvate. l-cysteine also induced PKM2 subunit dissociation (tetramers to dimers/monomers) in cells, which resulted in impaired glucose-induced ATP production for GSIS. DASA-10 (NCGC00181061, a substituted N,N′-diarylsulfonamide), a specific activator for PKM2, restored the tetramer formation and the activity of PKM2, glucose-induced ATP production, and biphasic insulin secretion in l-cysteine–treated cells. Collectively, our results demonstrate that impaired insulin secretion due to exposure to l-cysteine resulted from its direct binding and inactivation of PKM2 and suggest that PKM2 is a potential therapeutic target for T2D.A metabolite, l-cysteine, is found in blood plasma, and its concentration is closely associated with an increase in fat mass and the body-mass index. These values are used as an index of obesity (1, 2), which is a major risk factor for type 2 diabetes (T2D) (3). The relationship between l-cysteine and diabetes has attracted attention because there is increasing evidence for a positive correlation between increases in plasma l-cysteine concentrations and the development and progression of diabetes. For example, increased plasma l-cysteine concentrations were associated with T2D in African American women (4), renal insufficiency [reduced glomerular filtration rate (GFR)] in T2D patients (5), obstructive sleep apnea [a risk factor for diabetes (6, 7)], and insulin resistance among Europeans (8).Reduced insulin secretion from pancreatic β-cells is the major cause of T2D (9, 10). Many investigators have studied the molecular mechanisms of glucose-stimulated insulin secretion (GSIS), which have been elucidated in detail. Elevated extracellular glucose concentration results in the enhancement of ATP production, an increased ATP/ADP ratio, the closure of ATP-sensitive K channels (K_ATP channels), and depolarization (11). The resulting activation of voltage-dependent Ca²⁺ channels (VDCCs) induces an influx of calcium ions and elevated intracellular Ca²⁺ concentrations, which triggers insulin secretion (11). Perifusion experiments have shown that insulin secretion could be categorized into two phases. The first phase involves a sharp increase in insulin secretion within ∼5 min, followed by a second phase, during which moderate insulin secretion lasts for hours (9, 12). A loss of the GSIS first phase is closely associated with the future development of T2D (9, 13, 14).Many recent studies have reported that l-cysteine is involved in GSIS. In mouse pancreatic islets and mouse insulinoma 6 (MIN6) cells, l-cysteine treatment decreased both intracellular ATP levels and insulin secretion (15). Ammon et al. also reported that the total amount and GSIS second phase were specifically inhibited by l-cysteine for rat pancreatic islets (16). Several groups showed that an increase in the H₂S moiety, which is generated from l-cysteine in cells (17), was one possible cause for l-cysteine–induced impairment of GSIS by inhibiting K_ATP channels and VDCCs (18–20). However, opposite results were reported in that l-cysteine increased the amount of total and first-phase insulin secretion by rat pancreatic islets (16, 21). Thus, the effects of l-cysteine on GSIS remain controversial. It should be noted that most of these studies on the effects of l-cysteine on GSIS were performed using experimental conditions in which insulin-secreting cells were only transiently exposed (∼1 h) to a high glucose solution that contained l-cysteine. However, in obese or T2D patients, insulin-secreting cells can be exposed to plasma that contains l-cysteine for prolonged periods of time; thus, continuous exposure to an l-cysteine–containing solution is necessary to investigate the precise effects of l-cysteine on GSIS in insulin-secreting cells.In this study, we found that l-cysteine treatment of statically incubated or perifused MIN6 cells and mouse pancreatic islets resulted in reversibly inhibiting GSIS. A comprehensive analysis of charged metabolites in l-cysteine–treated MIN6 cells by capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS) showed significant accumulations of l-cysteine and, concomitantly, decreased levels of pyruvate and its downstream metabolites in the tricarboxylic acid (TCA) cycle in these cells. Biochemical experiments for pyruvate kinase activity in vitro and in MIN6 cells showed that l-cysteine specifically inhibited the activity of pyruvate kinase muscle isoform 2 (PKM2), an isoform of major pyruvate kinases in pancreatic islets (22) that catalyzes the glycolytic conversion of phosphoenolpyruvate (PEP) to pyruvate. PKM2 inactivation resulted from l-cysteine–induced subunit dissociation (tetramers to dimers/monomers) and inhibited glucose-induced ATP production for GSIS in MIN6 cells. Impaired GSIS due to l-cysteine was restored by treatment with methyl pyruvate, a membrane-permeable form of pyruvate, or DASA-10 (NCGC00181061, a substituted N,N′-diarylsulfonamide), a specific activator of PKM2 (23). Thus, we concluded that reduced PKM2 activity due to l-cysteine inhibited ATP production and subsequently inhibited GSIS by insulin-secreting cells.     

Repeatability of Blood Gas Parameters,Pco2 Gap,and Pco2 Gap to Arterial-to-Venous Oxygen Content Difference in Critically Ill Adult Patients

The objective of this study was to examine the repeatability of blood gas (BG) parameters and their derived variables such as the central venous-to-arterial carbon dioxide tension difference (▵Pco₂) and the ratio of ▵Pco₂ over the central arteriovenous oxygen content difference (▵Pco₂/C(a-cv)O₂) and to determine the smallest detectable changes in individual patients.A total of 192 patients with arterial and central venous catheters were included prospectively. Two subsequent arterial and central venous blood samples were collected immediately one after the other and analyzed using the same point-of-care BG analyzer. The samples were analyzed for arterial and venous BG parameters, ▵Pco₂, and ▵Pco₂/C(a-cv)O₂ ratio. Repeatability was expressed as the smallest detectable difference (SDD) and the least significant change (LSC). A change in value of these parameters exceeding the SDD or the LSC should be regarded as real.The SDDs for arterial carbon dioxide tension, arterial oxygen saturation, central venous oxygen saturation (ScvO₂)_, and ▵Pco₂ were small: ±2.06 mm Hg, ±1.23%, 2.92%, and ±1.98 mm Hg, respectively, whereas the SDDs for arterial oxygen tension (Pao₂) and ▵Pco₂/C(a-cv)O₂ were high: ±9.09 mm Hg and ±0.57 mm Hg/mL, respectively. The LSCs (%) for these variables were 5.06, 1.27, 4.44, 32.4, 9.51, and 38.5, respectively.The repeatability of all these variables was good except for Pao₂ and ▵Pco₂/C(a-cv)O₂ ratio for which we observed an important inherent variability. Expressed as SDD, a ScvO₂ change value of at least ±3% should be considered as true. The clinician must be aware that an apparent change in these variables in an individual patient might represent only an inherent variation.     

Tissue Response to,and Degradation Rate of,Photocrosslinked Trimethylene Carbonate-Based Elastomers Following Intramuscular Implantation

Cylindrical elastomers were prepared through the UV-initiated crosslinking of terminally acrylated, 8,000 Da star-poly(trimethylene carbonate-co-ε-caprolactone) and star-poly(trimethylene carbonate-co-d,l-lactide). These elastomers were implanted intramuscularly into the hind legs of male Wistar rats to determine the influence of the comonomer on the weight loss, tissue response, and change in mechanical properties of the elastomer. The elastomers exhibited only a mild inflammatory response that subsided after the first week; the response was greater for the stiffer d,l-lactide-containing elastomers. The elastomers exhibited weight loss and sol content changes consistent with a bulk degradation mechanism. The d,l-lactide-containing elastomers displayed a nearly zero-order change in Young’s modulus and stress at break over the 30 week degradation time, while the ε-caprolactone-containing elastomers exhibited little change in modulus or stress at break.     

Structural and functional analysis of the yeast N-acetyltransferase Mpr1 involved in oxidative stress tolerance via proline metabolism

Mpr1 (sigma1278b gene for proline-analog resistance 1), which was originally isolated as N-acetyltransferase detoxifying the proline analog l-azetidine-2-carboxylate, protects yeast cells from various oxidative stresses. Mpr1 mediates the l-proline and l-arginine metabolism by acetylating l-Δ¹-pyrroline-5-carboxylate, leading to the l-arginine–dependent production of nitric oxide, which confers oxidative stress tolerance. Mpr1 belongs to the Gcn5-related N-acetyltransferase (GNAT) superfamily, but exhibits poor sequence homology with the GNAT enzymes and unique substrate specificity. Here, we present the X-ray crystal structure of Mpr1 and its complex with the substrate cis-4-hydroxy-l-proline at 1.9 and 2.3 Å resolution, respectively. Mpr1 is folded into α/β-structure with eight-stranded mixed β-sheets and six α-helices. The substrate binds to Asn135 and the backbone amide of Asn172 and Leu173, and the predicted acetyl-CoA–binding site is located near the backbone amide of Phe138 and the side chain of Asn178. Alanine substitution of Asn178, which can interact with the sulfur of acetyl-CoA, caused a large reduction in the apparent k_cat value. The replacement of Asn135 led to a remarkable increase in the apparent K_m value. These results indicate that Asn178 and Asn135 play an important role in catalysis and substrate recognition, respectively. Such a catalytic mechanism has not been reported in the GNAT proteins. Importantly, the amino acid substitutions in these residues increased the l-Δ¹-pyrroline-5-carboxylate level in yeast cells exposed to heat stress, indicating that these residues are also crucial for its physiological functions. These studies provide some benefits of Mpr1 applications, such as the breeding of industrial yeasts and the development of antifungal drugs.     

Genomic reconstruction of Shewanella oneidensis MR-1 metabolism reveals a previously uncharacterized machinery for lactate utilization

The ability to use lactate as a sole source of carbon and energy is one of the key metabolic signatures of Shewanellae, a diverse group of dissimilatory metal-reducing bacteria commonly found in aquatic and sedimentary environments. Nonetheless, homology searches failed to recognize orthologs of previously described bacterial d- or l-lactate oxidizing enzymes (Escherichia coli genes dld and lldD) in any of the 13 analyzed genomes of Shewanella spp. By using comparative genomic techniques, we identified a conserved chromosomal gene cluster in Shewanella oneidensis MR-1 (locus tag: SO_1522–SO_1518) containing lactate permease and candidate genes for both d- and l-lactate dehydrogenase enzymes. The predicted d-LDH gene (dld-II, SO_1521) is a distant homolog of FAD-dependent lactate dehydrogenase from yeast, whereas the predicted l-LDH is encoded by 3 genes with previously unknown functions (lldEGF, SO_1520–SO_1518). Through a combination of genetic and biochemical techniques, we experimentally confirmed the predicted physiological role of these novel genes in S. oneidensis MR-1 and carried out successful functional validation studies in Escherichia coli and Bacillus subtilis. We conclusively showed that dld-II and lldEFG encode fully functional d-and l-LDH enzymes, which catalyze the oxidation of the respective lactate stereoisomers to pyruvate. Notably, the S. oneidensis MR-1 LldEFG enzyme is a previously uncharacterized example of a multisubunit lactate oxidase. Comparative analysis of >400 bacterial species revealed the presence of LldEFG and Dld-II in a broad range of diverse species accentuating the potential importance of these previously unknown proteins in microbial metabolism.     

Lactate promotes plasticity gene expression by potentiating NMDA signaling in neurons

l-lactate is a product of aerobic glycolysis that can be used by neurons as an energy substrate. Here we report that in neurons l-lactate stimulates the expression of synaptic plasticity-related genes such as Arc, c-Fos, and Zif268 through a mechanism involving NMDA receptor activity and its downstream signaling cascade Erk1/2. l-lactate potentiates NMDA receptor-mediated currents and the ensuing increase in intracellular calcium. In parallel to this, l-lactate increases intracellular levels of NADH, thereby modulating the redox state of neurons. NADH mimics all of the effects of l-lactate on NMDA signaling, pointing to NADH increase as a primary mediator of l-lactate effects. The induction of plasticity genes is observed both in mouse primary neurons in culture and in vivo in the mouse sensory-motor cortex. These results provide insights for the understanding of the molecular mechanisms underlying the critical role of astrocyte-derived l-lactate in long-term memory and long-term potentiation in vivo. This set of data reveals a previously unidentified action of l-lactate as a signaling molecule for neuronal plasticity.The transfer of l-lactate from astrocytes to neurons was recently shown to be necessary for the establishment of long-term memory (LTM) in an inhibitory avoidance (IA) paradigm and for the maintenance of in vivo long-term potentiation (LTP) in the rodent hippocampus (1). This key role of l-lactate in neuronal plasticity mechanisms was demonstrated in experiments in which specific pharmacological and gene expression down-regulation interventions were implemented to prevent the production of l-lactate from glycogen—which is exclusively localized in astrocytes—and its release from these cells in the hippocampus during behavioral training (1). Such interventions completely prevented the establishment of LTM and their effect was fully reversed by the intrahippocampal administration of l-lactate during the training session. The fact that glucose at equicaloric concentrations only marginally mimicked the rescuing effect of l-lactate was taken as an unexpected indication that the primary mechanism of action of l-lactate on plasticity mechanisms was independent of its ability to act as an energy substrate. A role of l-lactate in memory processes was also recently shown in other behavioral paradigms (2, 3). We therefore set out to investigate the molecular mechanisms at the basis of the function of l-lactate on neuronal plasticity.Molecular mechanisms underlying both LTM and long-term plasticity include the induction of expression of a group of immediate early genes (IEGs) such as early growth response 1 (Zif268 or Egr1), CCAAT/enhancer binding protein (C/EBP), and proto-oncogene c-Fos (c-Fos) as well as activity-regulated cytoskeletal-associated protein (Arc or Arg3.1) as a direct effector protein at the synapse, which all participate to different physiological processes associated with neuronal plasticity (4–6). Although stimulation of expression of these IEGs is not restricted to plasticity processes, they are considered as key plasticity-related genes in sustaining such phenomena. In addition, late response genes such as brain-derived neurotrophic factor (BDNF) have also been demonstrated to be major intermediates of plasticity-related processes (7). A role of NMDA receptors (NMDARs) in such plasticity mechanisms is well-established (5, 8).In this article we describe a cascade of molecular events demonstrating that l-lactate stimulates plasticity-related gene expression in neurons through modulation of NMDAR activity associated with changes in redox cellular state. The induction of plasticity gene expression by l-lactate was observed in primary cultures of neurons as well as in vivo in the sensory-motor cortex of mice.     

The Combination of d-Dimer and Peritoneal Irritation Signs as a Potential Indicator to Exclude the Diagnosis of Intestinal Necrosis

Intestinal necrosis is a life-threatening disease, and its prompt and accurate diagnosis is very important. This study aimed to evaluate the value of d-dimer as a marker for early diagnosis of bowel necrosis.From 2009 to 2013, patients undergoing operation due to acute intestinal obstruction were retrospectively analyzed. Clinicopathologic characteristics were compared among no ischemia group, reversible ischemia group, and bowel necrosis group.There were totally 274 patients being included for analyses. Patients with bowel necrosis had a significant highest level of d-dimer compared with other 2 groups (P = 0.007) when FEU unit was applied. The optimal cutoff value of d-dimer levels as an indicator in diagnosing bowel necrosis was projected to be 1.965 mg/L, which yielded a sensitivity of 84.0%, a specificity of 45.6%, a positive predictive value of 60.7%, and a negative predictive value of 74.0%. And the sensitivity of 84.0% and specificity of 70.0% were detected, when 1.65 mg/L of d-dimer was set as the cutoff value to distinguish the reversible ischemia and bowel necrosis. The corresponding results in patients with no or slight peritoneal irritation signs were 85.2%, 44.7%, 35.4% and 89.5% respectively. The sensitivity and negative predictive value were 96.0% and 91.7%, respectively, when d-dimer and peritoneal irritation signs were combined to perform the parallel analysis.The combination of d-dimer and peritoneal irritation signs could generate a reliable negative predictive value, which is helpful to exclude the diagnosis of intestinal necrosis. However, it should also be proved in well-designed large-scale prospective study.     

Distribution of the activity of the angiotensin-converting enzyme in the rat aorta and changes in the activity with aging and by the action of l-NAME

The activity of the angiotensin-converting enzyme (ACE) of the inner surface (the endothelium surface) of rat aorta sections has been studied depending on their distance from the aortic arch, age of rats, and the duration of treatment of rats with the NO synthase inhibitor, N_ω-nitro-l-arginine (l-NAME). The activity of ACE of aorta sections was determined by measuring the hydrolysis of hippuryl-l-histidyl-l-leucine and was expressed as picomoles of Hip–His–Leu hydrolyzed per minute per square millimeter of the endothelium surface. It was found that the ACE activity considerably varies along the aorta of young rats. This variability decreases with increasing age of rats and by the action of l-NAME. The average ACE activity in the aorta increases with the age of rats and with increasing time of l-NAME treatment. Enalapril normalizes the distribution of the ACE activity along the aorta and decreases the average ACE activity. The changes in the distribution of the ACE activity along the aorta and in the average ACE activity in the aorta with increasing age of the rat and by the action of l-NAME may play a role in the development of atherosclerosis of vessels on aging and the inhibition of formation of nitric oxide.     

Structural basis for the evolution of vancomycin resistance D,D-peptidases

Vancomycin resistance in Gram-positive bacteria is due to production of cell-wall precursors ending in d-Ala-d-Lac or d-Ala-d-Ser, to which vancomycin exhibits low binding affinities, and to the elimination of the high-affinity precursors ending in d-Ala-d-Ala. Depletion of the susceptible high-affinity precursors is catalyzed by the zinc-dependent d,d-peptidases VanX and VanY acting on dipeptide (d-Ala-d-Ala) or pentapeptide (UDP-MurNac-l-Ala-d-Glu-l-Lys-d-Ala-d-Ala), respectively. Some of the vancomycin resistance operons encode VanXY d,d-carboxypeptidase, which hydrolyzes both di- and pentapeptide. The molecular basis for the diverse specificity of Van d,d-peptidases remains unknown. We present the crystal structures of VanXY_C and VanXY_G in apo and transition state analog-bound forms and of VanXY_C in complex with the d-Ala-d-Ala substrate and d-Ala product. Structural and biochemical analysis identified the molecular determinants of VanXY dual specificity. VanXY residues 110–115 form a mobile cap over the catalytic site, whose flexibility is involved in the switch between di- and pentapeptide hydrolysis. Structure-based alignment of the Van d,d-peptidases showed that VanY enzymes lack this element, which promotes binding of the penta- rather than that of the dipeptide. The structures also highlight the molecular basis for selection of d-Ala–ending precursors over the modified resistance targets. These results illustrate the remarkable adaptability of the d,d-peptidase fold in response to antibiotic pressure via evolution of specific structural elements that confer hydrolytic activity against vancomycin-susceptible peptidoglycan precursors.The emergence of high-level resistance to vancomycin, a last resort antibiotic against Gram-positive bacteria, in Enterococcus spp. and its spread to methicillin-resistant Staphylococcus aureus is a serious threat to public health (1). Vancomycin acts by binding to the d-alanyl-d-alanine moiety of the uncross-linked N-acetyl-muramyl-l-Ala-d-γ-Glu-l-Lys-d-Ala-d-Ala (pentapeptide[d-Ala]) peptidoglycan precursor blocking the extracellular steps in peptidoglycan synthesis. Resistance is mediated by nine types of operons that replace the d-Ala-d-Ala terminus of peptidoglycan precursors with d-Ala-d-lactate (VanA, -B, -D, and -M types) or d-Ala-d-serine (VanC, -E, -G, -L, and -N types), to which vancomycin exhibits lower binding affinities (2).A critical step in vancomycin resistance involves depletion of d-Ala–terminating precursors to prevent interaction of vancomycin with its target. This step is facilitated by the d,d-dipeptidase VanX and the d,d-pentapeptidase VanY, which hydrolyze, respectively, d-Ala-d-Ala and the C-terminal d-Ala residue from pentapeptide[d-Ala] (3–6). In the d-Ala-d-Ser form of resistance, a single VanXY enzyme evolved to mediate both d,d-dipeptidase and d,d-pentapeptidase activities (7, 8). Thus, Van d,d-peptidases demonstrate variation in peptidoglycan substrate selectivity that correlates with the specific resistance mechanism. As part of the d-Ala-d-Lac type resistance mechanism, the VanX enzyme shows 10⁵-fold higher catalytic efficiency against d-Ala-d-Ala compared with d-Ala-d-Lac substrates, thus facilitating accumulation of the depsipeptide. However, VanX retains significant activity against d-Ala-d-Ser dipeptide (9). Appropriate to its role in resistance, the VanY enzyme shows carboxypeptidase activity against pentapeptide[d-Ala] but lacks activity against d,d-dipeptide substrates (5, 10). The VanXY_C enzyme is selective against resistant dipeptide and pentapeptide peptidoglycan substrates ending in d-Ser (8). Finally, the VanXY_G enzyme, first assigned as a dual substrate active “XY” enzyme by sequence similarity (11), was later shown to lack d,d-pentapeptidase activity typical for bona fide VanXY enzymes (12). The molecular determinants responsible for such diversity of substrate specificity among Van d,d-peptidases are unknown, limiting the understanding of their evolution and hampering the development of inhibitors that could be used in combination with glycopeptides.With the exception of VanY_D, which is a penicillin-binding protein (13), all VanX, VanY, and VanXY d,d-peptidases are zinc-dependent enzymes classified into the metallopeptidase clan MD, family M15, according to the MEROPS database (14). Within clan MD, multiple families of enzymes, including M15 representatives, are involved in bacterial cell wall metabolism (15). Three members of this family have been structurally characterized: zinc d-Ala-d-Ala carboxypeptidase from Streptomyces albus (16) (subfamily M15A), bacteriophage l-Ala-d-Glu peptidase PLY500 (17) (subfamily M15C), and VanX (subfamily M15D) from Enterococcus faecium (18). These enzymes display a common core tertiary fold built on a central antiparallel β-sheet arrayed with multiple α-helices on either face of the β-sheet. The fold contains two consensus motifs that form the active site, His-X(6)-Asp and Glu-X(2)-His, with a zinc ion coordinated by the histidine and aspartate residues. In addition, the structure of VanX revealed a small and constricted active site that explains its specificity toward the d-Ala-d-Ala substrate (18). The VanY and VanXY enzymes belonging to subfamily M15B remain structurally uncharacterized.Given the evolution toward dual substrate specificity of VanXY enzymes, their importance in resistance, and their sequence and functional diversity, we undertook their detailed structural and functional characterization. We determined the crystal structures of VanXY_C and VanXY_G and performed extensive mutagenesis analysis. Our data led to the identification and characterization of the molecular features responsible for their substrate specificities and demonstrated an exceptional diversification and plasticity within their common metallopeptidase fold in response to drug selective pressure.     

N‐terminal pro‐B‐type natriuretic peptide and D‐dimer combined with left atrial diameter to predict the risk of ischemic stroke in nonvalvular atrial fibrillation

ObjectivesWe aimed to explore the potential role of N‐terminal pro‐B‐type natriuretic peptide (NT‐proBNP), d‐dimer, and the echocardiographic parameter left atrial diameter (LAD) in identifying and predicting the occurrence of ischemic stroke (IS) in patients with nonvalvular atrial fibrillation (NVAF).MethodsWe conducted a retrospective study of 445 patients with NVAF in the First Affiliated Hospital of Nanchang University. They were divided into the NVAF (309 cases) and NVAF with stroke (136 cases) groups according to whether acute ischemic stroke (AIS) occurred at admission. Multivariate logistic regression was used to analyze the odds ratio (OR) of NT‐proBNP, d‐dimer, and LAD for IS. The predictive value of NT‐proBNP, d‐dimer, and LAD in identifying the occurrence of IS in NVAF was determined by plotting the receiver operating characteristic (ROC) curves.ResultsNT‐proBNP, d‐dimer, and LAD levels were significantly higher in the NVAF with stroke group than in the NVAF group (p < .05). NT‐ProBNP, d‐dimer, and LAD were independently associated with IS in NVAF patients (odds ratio [OR] = 1.12, 95% confidence interval [CI]: 1.08–1.16; OR = 1.87, 95% CI: 1.37–2.55; OR = 1.21, 95% CI: 1.13–1.28, p < .01). The optimal cutoff points for NT‐ProBNP, d‐dimer, and LAD levels to distinguish the NVAF group from the NVAF with stroke group were 715.0 pg/ml, 0.515 ng/ml, and 38.5 mm, respectively, with the area under the curve (AUC) being [0.801 (95% CI: 0.76–0.84); 0.770 (95% CI: 0.72–0.85); 0.752 (95% CI: 0.71–0.80), p < .01]. The combined score of NT‐proBNP, d‐dimer, and LAD improved the predictive efficacy of the single index, with an AUC of 0.846 (95% CI: 0.81–0.88, p < .01), sensitivity of 77.2%, and specificity of 76.4%.ConclusionNT‐proBNP, d‐dimer, and the echocardiographic parameter LAD have outstanding value in predicting the risk of IS in patients with NVAF.     

Arginine racemization by coupled catabolic and anabolic dehydrogenases

d-Amino acids exist in living organisms as specialized components of many different machineries. Biosynthesis of d-amino acids from racemization of predominant l-enantiomers is catalyzed by a single enzyme. Here, we report the finding of a novel 2-component amino acid racemase for d-to-l inversion in d-arginine metabolism of Pseudomonas aeruginosa. From DNA microarray analysis, the putative dauBAR operon (for d-arginine utilization) of unknown functions was found to be highly induced by d-arginine. The importance of the dau operon in d-arginine metabolism was demonstrated by the findings that strains with a lesion at dauA or dauB failed to use d-arginine as sole carbon source. Two lines of evidence suggest that DauA and DauB are required for d-to-l racemization of arginine. First, growth complementation of an l-arginine auxotroph by d-arginine was abolished by a lesion at dauA or dauB. Second, d-arginine induced l-arginine-specific genes in the parental strain PAO1 but not in its dauA or dauB mutants. This hypothesis was further supported by activity measurements of the purified enzymes: DauA catalyzes oxidative deamination of d-arginine into 2-ketoarginine and ammonia, and DauB is able to use 2-ketoarginine and ammonia as substrates and convert them into l-arginine in the presence of NADPH or NADH. Thus, we propose that DauA and DauB are coupled catabolic and anabolic dehydrogenases to perform d-to-l racemization of arginine, which serves as prerequisite of d-arginine utilization through l-arginine catabolic pathways.     

Rescue of Notch signaling in cells incapable of GDP-l-fucose synthesis by gap junction transfer of GDP-l-fucose in Drosophila

Notch (N) is a transmembrane receptor that mediates cell–cell interactions to determine many cell-fate decisions. N contains EGF-like repeats, many of which have an O-fucose glycan modification that regulates N-ligand binding. This modification requires GDP-l-fucose as a donor of fucose. The GDP-l-fucose biosynthetic pathways are well understood, including the de novo pathway, which depends on GDP-mannose 4,6 dehydratase (Gmd) and GDP-4-keto-6-deoxy-d-mannose 3,5-epimerase/4-reductase (Gmer). However, the potential for intercellularly supplied GDP-l-fucose and the molecular basis of such transportation have not been explored in depth. To address these points, we studied the genetic effects of mutating Gmd and Gmer on fucose modifications in Drosophila. We found that these mutants functioned cell-nonautonomously, and that GDP-l-fucose was supplied intercellularly through gap junctions composed of Innexin-2. GDP-l-fucose was not supplied through body fluids from different isolated organs, indicating that the intercellular distribution of GDP-l-fucose is restricted within a given organ. Moreover, the gap junction-mediated supply of GDP-l-fucose was sufficient to support the fucosylation of N-glycans and the O-fucosylation of the N EGF-like repeats. Our results indicate that intercellular delivery is a metabolic pathway for nucleotide sugars in live animals under certain circumstances.     

Changes in hemostatic factors after kidney transplantation: A retrospective cohort study

Chronic kidney disease affects hemostasis in complex ways, producing both thrombotic and hemorrhagic diatheses. These changes may impact patient morbidity and mortality pre-transplantation, as well as allograft survival after kidney transplantation (KT). This study was conducted to analyze changes in hemostatic factors in the early post-KT period.We retrospectively analyzed 676 recipients of kidney allografts from December 2009 to December 2014. Patients receiving plasmapheresis pre- or post-KT, experiencing early allograft failure, or receiving anticoagulants or antiplatelet agents pre- or post-KT were excluded.Of the 367 included patients, acute (≤1 month) rejection occurred in 4.1% and delayed graft function occurred in 3.3%. Postoperative bleeding complications occurred in 7.9% of patients and thrombotic complications in 3.3%. Pre-transplantation, recipients had below normal hemoglobin, above normal d-dimer and homocysteine levels, and elevated rates of antiphospholipid antibodies. Hemoglobin increased to almost normal by postoperative day (POD) 28 (P < .001). d-dimer increased on POD7, 14, and 28, although the values were not significantly different from pre-KT. The pattern of d-dimer changes suggested that they were a nonspecific consequence of major surgery. Homocysteine decreased to normal by POD7 (P < .001). The percentage of patients with ≥1 prothrombotic factor was 82.0% pre-KT and only 14.2% on POD28 (P < .001).The most of patients exhibited prothrombotic tendencies, including increased d-dimer and homocysteine, and increased prevalence of antiphospholipid antibodies before transplantation. They also had pre-transplantation anemia, suggesting a concomitant bleeding diathesis. However, most of these abnormal hemostatic factors improved or resolved after KT.     

Left atrial volume provides independent and incremental information compared with exercise tolerance parameters in patients with heart failure and left ventricular systolic dysfunction

Objective

Left atrial volume (LAV) is a powerful predictor of outcome in patients with chronic heart failure (CHF) independently of symptomatic status, age and left ventricular (LV) function. It is unknown whether LAV provides independent and incremental information compared with exercise tolerance parameters.

Methods

273 patients with CHF (mean (SD) 62 (9) years; 13% female) prospectively underwent echocardiography and exercise testing with maximal oxygen consumption (Vo₂). The primary end point was composite and included cardiac death, hospitalisation for worsening heart failure or cardiac transplantation.

Results

At Cox proportional hazard analysis, LAV normalised for body surface area (LAV/BSA) was strongly associated with mortality (hazard ratio (HR) = 1.027 (95% CI 1.018 to 1.04), p<0.001). The predictive value of LAV/BSA was independent of Vo₂ and LV ejection fraction (EF) (HR = 1.014 (1.002 to 1.025), p = 0.02; HR = 0.95 (0.91 to 0.99), p = 0.02; HR = 0.89 (0.82 to 0.98), p = 0.02 for LAV/BSA, EF and Vo₂, respectively). Receiver operator characteristic (ROC) curve analysis identified the best cut‐off values for prediction of the end point. LAV/BSA >63 ml, EF <30% and Vo₂ <16 ml/kg/min were considered to be risk factors. Patients with three risk factors had an HR of 38 (95% CI 11 to 129) compared with patients with no risk factors.

Conclusion

LAV provides powerful prognostic information incrementally and independently of Vo₂. LAV, EF and Vo₂ can be used to build a risk prediction model, which can be used clinically.     

Pico2 Monitoring of Transferred Jejunum Perfusion Using an Air Tonometry Technique After Hypopharyngeal Cancer Surgery

This study aimed to investigate the usefulness of intraluminal Pco₂ (Pico₂) monitoring by air tonometry for the assessment of the vascular condition of the transferred jejunum after surgery for hypopharyngeal cancer.Pico₂ in the transplanted jejunum of 24 patients was monitored using air tonometry after radical surgery for hypopharyngeal cancer from 2003 to 2010.All but 1 patient, who removed the catheter before monitoring began, were monitored safely. Pico₂ in the transferred jejunum correlated with arterial Pco₂ (Paco₂) that was measured concurrently, and dissociation of Pico₂ from Paco₂ was observed in cases with vascular complication. In those cases without postoperative vascular complication, the Pico₂ value gradually increased for 3 hours but then decreased by 12 hours after surgery. Three patients experienced major vascular complication. All 3 patients had continuous elevation of Pico₂ >100 mm Hg, although vascular flow in 1 patient recovered by removal of a venous thrombosis and reanastomosis of the vein 7.5 hours after surgery. Four other patients who experienced elevation of Pico₂ had their skin suture released for decompression of their neck wound, resulting in a decrease in Pico₂ after treatment.The current results demonstrated that continuous monitoring of Pico₂ by air tonometry accurately reflects the vascular condition of the transferred jejunum, and this method is one of the best options for postoperative monitoring of jejunum blood perfusion.     

Induced fit substrate binding to an archeal glutamate transporter homologue

Excitatory amino acid transporters (EAATs) are a class of glutamate transporters that terminate glutamatergic synaptic transmission in the mammalian CNS. Glt_Ph, an archeal EAAT homolog from Pyrococcus horikoshii, is currently the only member with a known 3D structure. Here, we studied the kinetics of substrate binding of a single tryptophan mutant (L130W) Glt_Ph in detergent micelles. At low millimolar [Na⁺], the addition of l-aspartate resulted in complex time courses of W130 fluorescence changes over tens of seconds. With increasing [Na⁺], the kinetics were dominated by a fast component [k_obs,fast; K_D (Na⁺) = 22 ± 3 mM, n_Hill = 1.7 ± 0.3] with values of k_obs,fast rising in a saturable manner to ≈500 s⁻¹ (at 6 °C) with increasing [l-aspartate]. The binding kinetics of l-aspartate differed from the binding kinetics of two alternative substrates: l-cysteine sulfinic acid and d-aspartate. l-cysteine sulfinic acid bound with higher affinity than l-aspartate but involved lower saturating rates, whereas the saturating rates after d-aspartate binding were higher. Thus, after the association of two Na⁺ to the empty transporter, Glt_Ph binds amino acids by induced fit. Cross-linking and proteolysis experiments suggest that the induced fit results from the closure of helical hairpin 2. This conformational change is faster for Glt_Ph than for most mammalian homologues, whereas the amino acid association rates are similar. Our data reveal the importance of induced fit for substrate selection in EAATs and illustrate how high-affinity binding and the efficient transport of glutamate can be accomplished simultaneously by this class of transporters.     

Baseline neutrophil-lymphocyte ratio is associated with outcomes in patients with castration-resistant prostate cancer treated with Docetaxel in South China

The aim of this study is to investigate the association between baseline neutrophil-to-lymphocyte ratio (NLR) and progression-free survival (PFS), overall survival (OS) and radiological response in castration-resistant prostate cancer patients treated with docetaxel.Forty-one prostate cancer patients who were treated with docetaxel were selected. Univariable and multivariable Cox regression models were used to predict the association of baseline NLR as a dichotomous variable with PFS and OS after chemotherapy initiation.In Kaplan–Meier analysis, the median PFS (9.8 vs 7.5 months, P = .039, Fig. ​Fig.1)1) and OS (17.6 vs 14.2 months, P = .021, Fig. ​Fig.2)2) was higher in patients who did not have an elevated NLR than in those with an elevated NLR. In univariate analysis, the pretreatment NLR was significantly associated with PFS (P = .049) and OS (P = .023). In multivariable analysis, patients with a NLR of >3 were at significantly higher risk of tumor progress (hazard ratio 2.458; 95% confidence interval 1.186–5.093; P = .016) and death (hazard ratio 3.435; 95% CI 1.522–7.750; P = .003)than patients with a NLR of ⩽3.Open in a separate windowFigure 1Kaplan–Meier curves for progression-free survival of prostate cancer patients categorized by the neutrophil-to-lymphocyte ratio.Open in a separate windowFigure 2Kaplan–Meier curves for overall survival of prostate cancer patients categorized by the neutrophil-to-lymphocyte ratio.NLR may be an independent predictor of PFS and OS in castration-resistant prostate cancer patients treated with docetaxel. The findings require validation in further prospective, big sample-sized studies.     

Targeting β-arrestin2 in the treatment of l-DOPA–induced dyskinesia in Parkinson’s disease

Parkinson’s disease (PD) is characterized by severe locomotor deficits and is commonly treated with the dopamine (DA) precursor l-3,4-dihydroxyphenylalanine (l-DOPA), but its prolonged use causes dyskinesias referred to as l-DOPA–induced dyskinesias (LIDs). Recent studies in animal models of PD have suggested that dyskinesias are associated with the overactivation of G protein-mediated signaling through DA receptors. β-Arrestins desensitize G protein signaling at DA receptors (D1R and D2R) in addition to activating their own G protein-independent signaling events, which have been shown to mediate locomotion. Therefore, targeting β-arrestins in PD l-DOPA therapy might prove to be a desirable approach. Here we show in a bilateral DA-depletion mouse model of Parkinson’s symptoms that genetic deletion of β-arrestin2 significantly limits the beneficial locomotor effects while markedly enhancing the dyskinesia-like effects of acute or chronic l-DOPA treatment. Viral rescue or overexpression of β-arrestin2 in knockout or control mice either reverses or protects against LIDs and its key biochemical markers. In other more conventional animal models of DA neuron loss and PD, such as 6-hydroxydopamine–treated mice or rats and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine–treated nonhuman primates, β-arrestin2 overexpression significantly reduced dyskinesias while maintaining the therapeutic effect of l-DOPA. Considerable efforts are being spent in the pharmaceutical industry to identify therapeutic approaches to block LIDs in patients with PD. Our results point to a potential therapeutic approach, whereby development of either a genetic or pharmacological intervention to enhance β-arrestin2- or limit G protein-dependent D1/D2R signaling could represent a more mechanistically informed strategy.Dopamine (DA) is a major catecholamine neurotransmitter that is released by midbrain DA neurons. DA activates G protein-coupled receptors (GPCRs), which belong to the dopamine D1 (D1R and D5R) or D2 (D2R, D3R, and D4R) class of DA receptors that are known to signal via G protein-dependent mechanisms (1, 2). However, recent studies have shown that in addition to G protein-mediated signaling, many GPCRs, including DA receptors, signal through beta-arrestin 1 and 2 (βarr1 and βarr2)-dependent mechanisms (3, 4). The two isoforms of β-arrestin, β-arrestin1 (βarr1) and β-arrestin2 (βarr2) are widely coexpressed in the brain (5, 6). β-Arrestins were originally appreciated for their ability to desensitize (i.e., turn off) GPCR signaling in a GPCR kinase (GRK)-dependent manner (7, 8). Binding of β-arrestin to the GPCR sterically hinders G protein binding and in most instances initiates receptor endocytosis via interactions with adaptor protein complex-2 (AP-2) and clathrin (9–11). It is now appreciated that β-arrestins regulate physiology and behaviors independently of G protein signaling through their ability to scaffold multiple intracellular signaling molecules such as kinases and phosphatases (3, 4, 12, 13). Studies from our laboratory have shown that through both dopamine D1 and D2 receptors, βarr2-mediated signaling plays a major role in DA-dependent locomotion (14–16).Parkinson’s disease (PD) is a neurodegenerative disorder caused by the progressive loss of DA neurons projecting to the striatum that is characterized by severe locomotor deficits and is commonly treated with the DA precursor l-3,4-dihydroxyphenylalanine (l-DOPA) or D2 receptor agonists. Although l-DOPA treatment ameliorates the locomotor deficits, prolonged l-DOPA use causes dyskinesias, termed as l-DOPA–induced dyskinesias (LIDs) in humans or abnormal involuntary movements (AIMs) in animal models. Despite these drawbacks, l-DOPA is still the mainstay of PD treatment for its unsurpassed antiparkinsonian efficacy. Studies in animal models of PD suggest that dyskinesias are associated with enhanced G protein-mediated signaling at dopamine receptors (17–23) potentially leading to changes in gene expression and uncontrolled neuronal excitability (21, 24–28). Therefore, PD therapy strategies that moderate G protein signaling and neuronal excitability while maintaining normal movement may be an ideal way to eliminate DA receptor-associated dyskinesias. To moderate this uncontrolled signaling or neuronal excitability, several approaches have been explored such as reducing D1R surface expression (29, 30), dampening overactive intracellular signaling (20, 23, 31, 32), and inhibiting A2A (33, 34), mGluR5 (35–37) or NMDA receptors (24, 25, 38–40). Although these targets have clinical potential, several drugs to these targets have either failed clinical trials or have the potential to affect other key CNS physiological processes. As a novel approach, targeting βarr2 function in the DA system might be desirable because through its desensitization of G protein signaling, it can reduce dyskinesias and simultaneously through its signaling ability facilitate locomotion, without potentially affecting other neurotransmitter systems.In the current study we provide evidence supporting the hypothesis that up-regulating βarr2 expression ameliorates LIDs but enhances the therapeutic effects of l-DOPA. We use four different animal models of PD and LIDs to support this notion: (i) a nonconventional mouse model of acute PD symptoms: the bilateral DA-deficient dopamine transporter (DAT)-KO (DDD) mouse (41, 42), (ii) a unilateral 6-hydroxydopamine (6-OHDA)-lesioned mouse model, (iii) a unilateral 6-OHDA–lesioned rat model, and (iv) a bilateral 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned nonhuman primate (NHP) model of PD. Using these various animal models we show that deletion of βarr2 enhances LIDs and reduces forward locomotion but overexpression of βarr2 in the striatum reduces LIDs and enhances the therapeutic effects of l-DOPA.