Structure-guided product determination of the bacterial type II diterpene synthase Tpn2.

A grand challenge in terpene synthase (TS) enzymology is the ability to predict function from protein sequence. Given the limited number of characterized bacterial TSs and significant sequence diversities between them and their eukaryotic counterparts, this is currently impossible. To contribute towards understanding the sequence-structure-function relationships of type II bacterial TSs, we determined the structure of the terpentedienyl diphosphate synthase Tpn2 from Kitasatospora sp. CB02891 by X-ray crystallography and made structure-guided mutants to probe its mechanism. Substitution of a glycine into a basic residue changed the product preference from the clerodane skeleton to a syn-labdane skeleton, resulting in the first syn-labdane identified from a bacterial TS. Understanding how a single residue can dictate the cyclization pattern in Tpn2, along with detailed bioinformatics analysis of bacterial type II TSs, sets the stage for the investigation of the functional scope of bacterial type II TSs and the discovery of novel bacterial terpenoids.

Doppler Echocardiographic Features of Pulmonary Vein Stenosis in Ex-Preterm Children.

BACKGROUND: Echocardiography is used to screen for the presence of pulmonary vein stenosis (PVS) in ex-preterm infants and children. However, there are no standard accepted criteria for screening or diagnosis of PVS by echocardiography. The aim of this study was to identify Doppler waveform features and Doppler systolic and diastolic velocity cutoff values associated with a diagnosis of PVS by cardiac catheterization. METHODS: In this retrospective observational study, the echocardiograms of ex-preterm children <3 years old who underwent cardiac catheterization at a single institution were reviewed. PVS on cardiac catheterization was defined by a mean pressure gradient of >3 mm Hg in the pulmonary vein, with angiographic evidence of stenosis. Pulmonary vein Doppler waveforms, from echocardiograms obtained before catheterization, in children with and without PVS were compared. Nonstenosed veins in patients with PVS were excluded. The systolic and diastolic velocities of blood flow, phasic flow, and return of the Doppler waveform to baseline were analyzed. RESULTS: Forty-seven children were analyzed in the study, 18 children with 25 stenosed pulmonary veins and 29 children with 78 nonstenosed pulmonary veins. Stenosed pulmonary veins had higher peak systolic and diastolic velocities and higher peak and mean pressure gradients as measured by spectral Doppler. Peak systolic and diastolic velocities had areas under the receiver operating characteristic curve of 0.89 (95% CI, 0.79-0.99) and 0.93 (95% CI, 0.85-0.99) for PVS, respectively, and a threshold velocity of 0.7 m/sec had sensitivity of 80% and 84% and specificity of 94%. There was no correlation between Doppler-derived pulmonary vein mean gradient and measured pulmonary vein mean gradient during cardiac catheterization in stenosed pulmonary veins. Presence of phasic flow in the pulmonary vein and return of the Doppler waveform to baseline were associated with a nonstenosed pulmonary vein (sensitivity of 94% and 60% and specificity of 52% and 60%, respectively). CONCLUSIONS: Systolic and diastolic Doppler velocities and features of the waveform can discriminate stenosed pulmonary veins confirmed by cardiac catheterization in ex-preterm children. These results suggest the use of lower systolic and diastolic Doppler velocity cutoff values than currently published to screen for PVS in ex-preterm children. These cutoff values require validation in prospective studies.

ATP-citrate lyase reduction mediates palmitate-induced apoptosis in pancreatic beta cells.

Elevated extracellular lipids, such as the free fatty acid palmitate, can induce pancreatic beta cell endoplasmic reticulum (ER) stress and apoptosis, thereby contributing to the initiation and progression of type 2 diabetes. ATP-citrate lyase (ACLY), a key enzyme in cellular lipid production, was identified as a palmitate target in a proteomic screen. We investigated the effects of palmitate on ACLY activity and phosphorylation and its role in beta cell ER stress and apoptosis. We demonstrated that treatment of MIN6 cells, mouse islets and human islets with palmitate reduced ACLY protein levels. These in vitro results were validated by our finding that islets from high fat-fed mice had a significant decrease in ACLY, similar to that previously observed in type 2 diabetic human islets. Palmitate decreased intracellular acetyl-CoA levels to a similar degree as the ACLY inhibitor, SB-204990, suggesting a reduction in ACLY activity. ACLY inhibitors alone were sufficient to induce CCAAT/enhancer-binding protein homologues protein (CHOP)-dependent ER stress and caspase-3-dependent apoptosis. Similarly, even modest shRNA-mediated knockdown of ACLY caused a significant increase in beta cell apoptosis and ER stress. The effects of chemical ACLY inhibition and palmitate were non-additive and therefore potentially mediated by a common mechanism. Indeed, overexpression of ACLY prevented palmitate-induced beta cell death. These observations provide new evidence that ACLY expression and activity can be suppressed by exogenous lipids and demonstrate a critical role for ACLY in pancreatic beta cell survival. These findings add to the emerging body of evidence linking beta cell metabolism with programmed cell death.

Carboxypeptidase E mediates palmitate-induced beta-cell ER stress and apoptosis.

Obesity is a principal risk factor for type 2 diabetes, and elevated fatty acids reduce beta-cell function and survival. An unbiased proteomic screen was used to identify targets of palmitate in beta-cell death. The most significantly altered protein in both human islets and MIN6 beta-cells treated with palmitate was carboxypeptidase E (CPE). Palmitate reduced CPE protein levels within 2 h, preceding endoplasmic reticulum (ER) stress and cell death, by a mechanism involving CPE translocation to Golgi and lysosomal degradation. Palmitate metabolism and Ca(2+) flux were also required for CPE proteolysis and beta-cell death. Chronic palmitate exposure increased the ratio of proinsulin to insulin. CPE null islets had increased apoptosis in vivo and in vitro. Reducing CPE by approximately 30% using shRNA also increased ER stress and apoptosis. Conversely, overexpression of CPE partially rescued beta-cells from palmitate-induced ER stress and apoptosis. Thus, carboxypeptidase E degradation contributes to palmitate-induced beta-cell ER stress and apoptosis. CPE is a major link between hyperlipidemia and beta-cell death pathways in diabetes.

Muscle Tissue Damage and Recovery After EV71 Infection Correspond to Dynamic Macrophage Phenotypes.

Enterovirus 71 (EV71) is a positive single-stranded RNA virus from the enterovirus genus of the Picornaviridae family. Most young children infected with EV71 develop mild symptoms of hand, foot and mouth disease, but some develop severe symptoms with neurological involvement. Limb paralysis from EV71 infection is presumed to arise mainly from dysfunction of motor neurons in the spinal cord. However, EV71 also targets and damages skeletal muscle, which may also contribute to the debilitating symptoms. In this study, we have delineated the impacts of EV71 infection on skeletal muscle using a mouse model. Mouse pups infected with EV71 developed limb paralysis, starting at day 3 post-infection and peaking at day 5-7 post-infection. At later times, mice recovered gradually but not completely. Notably, severe disease was associated with high levels of myositis accompanied by muscle calcification and persistent motor end plate abnormalities. Interestingly, macrophages exhibited a dynamic change in phenotype, with inflammatory macrophages (CD45+CD11b+Ly6Chi) appearing in the early stage of infection and anti-inflammatory/restorative macrophages (CD45+CD11b+Ly6Clow/-) appearing in the late stage. The presence of inflammatory macrophages was associated with severe inflammation, while the restorative macrophages were associated with recovery. Altogether, we have demonstrated that EV71 infection causes myositis, muscle calcification and structural defects in motor end plates. Subsequent muscle regeneration is associated with a dynamic change in macrophage phenotype.

TLR7 Is Critical for Anti-Viral Humoral Immunity to EV71 Infection in the Spinal Cord.

Enterovirus 71 (EV71) is a positive single-stranded RNA (ssRNA) virus from the enterovirus genus of Picornaviridae family and causes diseases ranged from the mild disease of hand, foot and mouth disease (HFMD) to the severe disease of neurological involvement in young children. TLR7 is an intracellular pattern recognition receptor (PRR) recognizing viral ssRNA. In this study, we investigated the role of TLR7 in EV71 infection in mouse pups (10-12 days old) and found that wild-type (WT) and TLR7 knock-out (TLR7KO) mice infected with EV71 showed similar limb paralysis at the onset and peak of the disease, comparable loss of motor neurons, and similar levels of antiviral molecules in the spinal cord. These results suggest that TLR7 is not the absolute PRR for EV71 in the spinal cord. Interestingly, TLR7KO mice infected with EV71 exhibited significantly delayed recovery from limb paralysis compared with WT mice. TLR7KO mice infected with EV71 showed significantly decreased levels of IgM and IgG2, important antibodies for antiviral humoral immunity. Furthermore, TLR7KO mice infected with EV71 showed a decrease of germinal center B cells in the spleen compared with WT mice. Altogether, our study suggests that TLR7 plays a critical role in anti-viral humoral immunity rather than in being a PRR in the spinal cord during EV71 infection in young mice.

CCR6 Deficiency Impairs IgA Production and Dysregulates Antimicrobial Peptide Production, Altering the Intestinal Flora.

Intestinal immunity exists as a complex relationship among immune cells, epithelial cells, and microbiota. CCR6 and its ligand-CCL20 are highly expressed in intestinal mucosal tissues, such as Peyer's patches (PPs) and isolated lymphoid follicles (ILFs). In this study, we investigated the role of the CCR6-CCL20 axis in intestinal immunity under homeostatic conditions. CCR6 deficiency intrinsically affects germinal center reactions in PPs, leading to impairments in IgA class switching, IgA affinity, and IgA memory B cell production and positioning in PPs, suggesting an important role for CCR6 in T-cell-dependent IgA generation. CCR6 deficiency impairs the maturation of ILFs. In these follicles, group 3 innate lymphoid cells are important components and a major source of IL-22, which stimulates intestinal epithelial cells (IECs) to produce antimicrobial peptides (AMPs). We found that CCR6 deficiency reduces IL-22 production, likely due to diminished numbers of group 3 innate lymphoid cells within small-sized ILFs. The reduced IL-22 levels subsequently decrease the production of AMPs, suggesting a critical role for CCR6 in innate intestinal immunity. Finally, we found that CCR6 deficiency impairs the production of IgA and AMPs, leading to increased levels of Alcaligenes in PPs, and segmented filamentous bacteria in IECs. Thus, the CCR6-CCL20 axis plays a crucial role in maintaining intestinal symbiosis by limiting the overgrowth of mucosa-associated commensal bacteria.

A flexible and miniaturized hair dye based photodetector via chemiluminescence pathway.

A flexible and miniaturized metal semiconductor metal (MSM) biomolecular photodetector was developed as the core photocurrent system through chemiluminescence for hydrogen peroxide sensing. The flexible photocurrent sensing system was manufactured on a 30-µm-thick crystalline silicon chip by chemical etching process, which produced a flexible silicon chip. A surface texturization design on the flexible device enhanced the light-trapping effect and minimized reflectivity losses from the incident light. The model protein streptavidin bound to horseradish peroxidase (HRP) was successfully immobilized onto the sensor surface through high-affinity conjugation with biotin. The luminescence reaction occurred with luminol, hydrogen peroxide and HRP enzyme, and the emission of light from the catalytic reaction was detected by underlying flexible photodetector. The chemiluminescence in the miniaturized photocurrent sensing system was successfully used to determine the hydrogen peroxide concentration in real-time analyses. The hydrogen peroxide detection limit of the flexible MSM photodetector was 2.47mM. The performance of the flexible MSM photodetector maintained high stability under bending at various bending radii. Moreover, for concave bending, a significant improvement in detection signal intensity (14.5% enhancement compared with a flat configuration) was observed because of the increased photocurrent, which was attributed to enhancement of light trapping. Additionally, this detector was used to detect hydrogen peroxide concentrations in commercial hair dye products, which is a significant issue in the healthcare field. The development of this novel, flexible and miniaturized MSM biomolecular photodetector with excellent mechanical flexibility and high sensitivity demonstrates the applicability of this approach to future wearable sensor development efforts.

Effect of high-fat diet on hepatic proteomics of hamsters.

A high-fat diet contributes to the etiology of metabolic diseases. As the liver plays a crucial role in metabolism, an insight into the hepatic proteomics will help to illustrate the physiological effect of a high-fat diet. Fourteen nine-week old male Syrian hamsters were maintained on either control (C) or high-fat (HF) diets (0.2% cholesterol +22% fat) for 8 weeks. Hamsters were chosen because they show close similarity to human lipid metabolism. At the end of study, blood and livers were collected for analysis. Liver proteins were fractionated by electrophoresis, digested by trypsin, and then separated by label-free nano-LC/MS/MS. The TurboSequest algorithm was used to identify the peptide sequences against the hamster database in Universal Proteins Resource Knowledgebase (UniProt). The results indicate that 1191 hepatic proteins were identified and 135 of them were expressed differentially in the high-fat group (p < 0.05). Some of these 135 proteins that involve in metabolic diseases were further validated by Western blotting. The animals maintained on the high-fat diet had significantly (p < 0.05) higher serum triglyceride, cholesterol, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and uric acid. Animals consuming a high-fat diet also had significantly (p < 0.05) more accumulation of triglyceride and cholesterol in livers. Xanthine dehydrogenase (XDH), which plays an important role in uric acid synthesis, was up-regulated by the high-fat diet (p < 0.05). The α-subunit of hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (HADHA), which catalyzes the second and third reactions of ß-oxidation, was down-regulated by the high-fat diet (p < 0.05). Aconitate hydratase 2 (ACO2), which catalyzes the conversion of citrate to isocitrate in TCA cycle, was down-regulated in animals of the high-fat group (p < 0.05). Inflammatory markers annexin A3 (ANXA3) and annexin A5 (ANXA5) were up-regulated by the high-fat diet (p < 0.05). Moreover, enzymes involved in the urea cycle were suppressed by high-fat diet, including carbamoyl phosphate synthase 1 (CPS1), ornithine transcarbamoylase (OTC), argininosuccinate synthase (ASS), argininosuccinate lyase (ASL), and arginase 1 (ARG 1). Post-translational modifications (PTM) of ANXA3, ANXA5, and XDH were also analyzed. A set of differentially expressed proteins were identified as molecular markers for elucidating the pathological mechanism of high-fat diet.

Methyl protodioscin increases ABCA1 expression and cholesterol efflux while inhibiting gene expressions for synthesis of cholesterol and triglycerides by suppressing SREBP transcription and microRNA 33a/b levels.

Sterol regulatory element-binding proteins (SREBPs) regulate homeostasis of LDL, HDL and triglycerides. This study was aimed to determine if inhibition of SREBPs by methyl protodioscin (MPD) regulates downstream gene and protein expressions of lipid metabolisms. In THP-1 macrophages, MPD increases levels of ABCA1 mRNA and protein in dose- and time-dependent manners, and apoA-1-mediated cholesterol efflux. The underlying mechanisms for the effects is that MPD inhibits the transcription of SREBP1c and SREBP2, and decreases levels of microRNA 33a/b hosted in the introns of SREBPs, which leads to reciprocally increase ABCA1 levels. In HepG2 cells, MPD shows the same effects as these observed in THP-1 macrophages. MPD also decreases the gene expressions of HMGCR, FAS and ACC for cholesterol and fatty acid synthesis. MPD further promotes LDL receptor through reducing the PCSK9 level. Collectively, the study demonstrates that MPD potentially increase HDL cholesterol while reducing LDL cholesterol and triglycerides.

2,4,5-TMBA, a natural inhibitor of cyclooxygenase-2, suppresses adipogenesis and promotes lipolysis in 3T3-L1 adipocytes.

Obesity is a global health problem. Because of the high costs and side effects of obesity-treatment drugs, the potential of natural products as alternatives for treating obesity is under exploration. 2,4,5-Trimethoxybenzaldehyde (2,4,5-TMBA) present in plant roots, seeds, and leaves was reported to be a significant inhibitor of cyclooxygenase-2 (COX-2) activity at the concentration of 100 µg/mL. Because COX-2 is associated with differentiation of preadipocytes, the murine 3T3-L1 cells were cultured with 100 µg/mL of 2,4,5-TMBA during differentiation and after the cells were fully differentiated to study the effect of 2,4,5-TMBA on adipogenesis and lipolysis. Oil Red O staining and triglyceride assay revealed that 2,4,5-TMBA inhibited the formation of lipid droplets during differentiation; moreover, 2,4,5-TMBA down-regulated the protein levels of adipogenic signaling molecules and transcription factors MAP kinase kinase (MEK), extracellular signal-regulated kinase (ERK), CCAAT/enhancer binding protein (C/EBP)α, ß, and δ, peroxisome proliferator-activated receptor (PPAR)γ, adipocyte determination and differentiation-dependent factor 1 (ADD1), and the rate-limiting enzyme for lipid synthesis acetyl-CoA carboxylase (ACC). In fully differentiated adipocytes, treatment with 2,4,5-TMBA for 72 h significantly decreased lipid accumulation by increasing the hydrolysis of triglyceride through suppression of perilipin A (lipid droplet coating protein) and up-regulation of hormone-sensitive lipase (HSL). The results of this in vitro study will pioneer future in vivo studies on antiobesity effects of 2,4,5-TMBA and selective COX-2 inhibitors.

Exposure of rice seedlings to heat shock protects against subsequent Cd-induced decrease in glutamine synthetase activity and increase in specific protease activity in leaves.

In the present study, we investigated the effect of heat shock (HS) on the subsequent Cd-induced decrease in the activity of glutamine synthetase (GS) and increase in the specific activity of protease in rice leaves. HS exposure of rice seedlings for 3h in the dark was effective in reducing subsequent Cd-induced decrease in the activity of glutamine synthetase and increase in the specific activity of protease. The effect of HS can be mimicked by pretreatment of rice seedlings with exogenous H(2)O(2) or reduced glutathione (GSH) under non-HS conditions. We also found that HS protected against subsequent Cd-induced decrease in the activity of GS and increase in the specific activity of protease can be counteracted by imidazole, a NADPH oxidase inhibitor. Pretreatment with buthione sulfoximine (a GSH synthesis inhibitor) under HS conditions enhanced subsequent Cd effects on the activity of GS and the specific activity of protease. Moreover, the effect of BSO can be reversed by the addition of GSH. The mechanisms of the protective effect of HS effect against subsequent Cd effects are discussed.

Effects of palmitate on ER and cytosolic Ca2+ homeostasis in beta-cells.

There are strong links between obesity, elevated free fatty acids, and type 2 diabetes. Specifically, the saturated fatty acid palmitate has pleiotropic effects on beta-cell function and survival. In the present study, we sought to determine the mechanism by which palmitate affects intracellular Ca2+, and in particular the role of the endoplasmic reticulum (ER). In human beta-cells and MIN6 cells, palmitate rapidly increased cytosolic Ca2+ through a combination of Ca2+ store release and extracellular Ca2+ influx. Palmitate caused a reversible lowering of ER Ca2+, measured directly with the fluorescent protein-based ER Ca2+ sensor D1ER. Using another genetically encoded indicator, we observed long-lasting oscillations of cytosolic Ca2+ in palmitate-treated cells. In keeping with this observed ER Ca2+ depletion, palmitate induced rapid phosphorylation of the ER Ca2+ sensor protein kinase R-like ER kinase (PERK) and subsequently ER stress and beta-cell death. We detected little palmitate-induced insulin secretion, suggesting that these Ca2+ signals are poorly coupled to exocytosis. In summary, we have characterized Ca2+-dependent mechanisms involved in altered beta-cell function and survival induced by the free fatty acid palmitate. We present the first direct evidence that free fatty acids reduce ER Ca2+ and shed light on pathways involved in lipotoxicity and the pathogenesis of type 2 diabetes.

Platelet aggregation induced by serotype polysaccharides from Streptococcus mutans.

Platelet aggregation plays an important role in the pathogenesis of infective endocarditis induced by viridans streptococci or staphylococci. Aggregation induced in vitro involves direct binding of bacteria to platelets through multiple surface components. Using platelet aggregometry, we demonstrated in this study that two Streptococcus mutans laboratory strains, GS-5 and Xc, and two clinical isolates could aggregate platelets in an irreversible manner in rabbit platelet-rich plasma preparations. The aggregation was partially inhibited by prostaglandin I(2) (PGI(2)) in a dose-dependent manner. Whole bacteria and heated bacterial cell wall extracts were able to induce aggregation. Cell wall polysaccharides extracted from the wild-type Xc strain, containing serotype-specific polysaccharides which are composed of rhamnose-glucose polymers (RGPs), could induce platelet aggregation in the presence of plasma. Aggregation induced by the serotype-specific RGP-deficient mutant Xc24R was reduced by 50% compared to the wild-type strain Xc. In addition, cell wall polysaccharides extracted from Xc24R failed to induce platelet aggregation. The Xc strain, but not the Xc24R mutant, could induce platelet aggregation when preincubated with plasma. Both Xc and Xc24R failed to induce platelets to aggregate in plasma depleted of immunoglobulin G (IgG), but aggregation was restored by replenishment of anti-serotype c IgG. Analysis by flow cytometry showed that S. mutans RGPs could bind directly to rabbit and human platelets. Furthermore, cell wall polysaccharides extracted from the Xc, but not the Xc24R, strain could induce pseudopod formation of both rabbit and human platelets in the absence of plasma. Distinct from the aggregation of rabbit platelets, bacterium-triggered aggregation of human platelets required a prolonged lag phase and could be blocked completely by PGI(2). RGPs also trigger aggregation of human platelets in a donor-dependent manner, either as a transient and reversible or a complete and irreversible response. These results indicated that serotype-specific RGPs, a soluble product of S. mutans, could directly bind to and activate platelets from both rabbit and human. In the presence of plasma containing IgG specific to RGPs, RGPs could trigger aggregation of both human and rabbit platelets, but the degree of aggregation in human platelets depends on the donors.