Abstract

Pyrroloquinoline quinone (PQQ) is a water-soluble, anionic, quinonoid substance that has been established as an essential nutrient in animals. Owing to the inherent properties of PQQ as an antioxidant and redox modulator in various systems, PQQ is expected to be used in pharmacological applications in the near future. Although many recent studies have investigated its neuroprotective effects, the effect of PQQ on traumatic brain injury (TBI) has not been examined. In this study we employed Morris water maze (MWM) training, the results of which showed that PQQ led to improved behavioral performance in post-TBI animals. Considering that many experiments have suggested that β-1,4-galactosyltransferase I (β-1,4-GalT-I) and -V play significant roles in inflammation and the nervous system, in the present study we used Western blot analysis to study the effect of PQQ on the expression of β-1,4-GalT-I and -V. We found apparent expression upregulation of β-1,4-GalT-I and -V after PQQ was systemically administered. Lectin-fluorescent staining with RCA-I also revealed that PQQ contributed to expression upregulation of the galactosidase β-1 (Gal β-1), 4-galactosyltransferase N-acylsphingosine (4-GlcNAc) group in microglia and neurons of the cortex and hippocampal CA2 region. In summary, our experiment established that PQQ may play an important role in recovery post-TBI.

Background

Treatment of diabetes mellitus with Traditional Chinese Medicine has a long history. The aim of this study is to establish the safety and efficacy of traditional Chinese medicine combined with glibenclamide to treat type 2 diabetes mellitus.

Methods

In a controlled, double blind, multicentre non-inferiority trial, 800 patients with unsatisfactory glycemic control (fasting glucose 7–13 mmol/L and HbA1c 7–11%) were randomly assigned to receive Xiaoke Pill, a compound of Chinese herbs combined with glibenclamide, or Glibenclamide in two study groups – drug naive group, and patients previously treated with metformin monotherapy (metformin group). Outcome measures at 48 weeks were the incidence and rate of hypoglycemia, mean difference in HbA1c, and proportion of patients with HbA1c<6.5%.

Findings

In drug naïve group, the total hypoglycemia rate and the mild hypoglycemic episode in the Xiaoke Pill arm were 38% (p = 0.024) and 41% (p = 0.002) less compared to Glibenclamide arm; in Metformin group, the average annual rate of hypoglycemia was 62% lower in Xiaoke Pill arm (p = 0.003). Respective mean changes in HbA1c from baseline were −0.70% and −0.66% for Xiaoke Pill and Glibenclamide, with a between-group difference (95% CI) of −0.04% (−0.20, 0.12) in the drug naïve group, and those in metformin group were −0.45% and −0.59%, 0.14% (−0.12, 0.39) respectively. The respective proportions of patients with a HbA1c level <6.5% were 26.6% and 23.4% in the drug naïve group and 20.1% and 18.9% in the metformin group.

Interpretation

In patients with type 2 diabetes and inadequate glycaemic control, treatment with Xiaoke Pill led to significant reduction in risk of hypoglycemia and similar improvements in glycemic control after 48 weeks compared to Glibenclamide.

Trial Registration

Chinese Clinical Trial Register number, ChiCTR-TRC-08000074

Adult stem cells are well known for their self-renewal and regenerative capacity. The mechanisms protecting these cells from inflammatory damage have not been well elucidated. This study investigated the immunoprotective properties of corneal epithelial stem cells from inflammation by producing glial cell-derived neurotrophic factor (GDNF). Primary human limbal epithelial cells (HLECs) cultured from limbal explants were treated with interleukin (IL)-17A, tumor necrosis factor (TNF)-α, or hyperosmotic media, with or without GDNF or nuclear factor kappa B (NF-κB) inhibitor (NF-κB-I) for 4–48 hours. Inflammatory mediators and Th17-inducing cytokines were determined by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and immunobead assays. NF-κB activation was detected by p65 phosphorylation, immunostaining and Western blotting. GDNF and its receptor, GDNF family receptor α-1, were exclusively immunolocalized in the basal layer of limbal epithelium, whereas IL-17 receptor was negative in these cells. Exogenous IL-17A stimulated the expression and production of inflammatory cytokines (TNF-α, IL-6, and IL-1β) and chemokine IL-8 by HLECs. Th17-inducing cytokines, transforming growth factor (TGF)-β1, IL-6, IL-23, and IL-1β, were significantly increased at mRNA and protein levels by HLECs exposed to TNF-α or hyperosmotic media. IL-17 activated NF-κB by p65 phosphorylation at serine 536 and nuclear translocation. GDNF or NF-κB-I blocked IL-17-induced NF-κB p65 activation and production of inflammatory mediators. Furthermore, GDNF suppressed the production of Th17-inducing cytokines through inhibiting NF-κB activation. These findings demonstrate that limbal progenitor cell-produced neurotrophic factor GDNF suppresses IL-17-mediated inflammation via NF-κB signaling pathway. This may represent a unique immunoprotective property of limbal stem cells against inflammatory challenges on the ocular surface.

Plant heat stress transcription factors (Hsfs) are the critical components involved in mediating responses to various environmental stressors. However, the detailed roles of many plant Hsfs are far from fully understood. In this study, an Hsf (SlHsfA3) was isolated from the cultivated tomato (Solanum lycopersicum, Sl) and functionally characterized at the genetic and developmental levels. The nucleus-localized SlHsfA3 was basally and ubiquitously expressed in different plant organs. The expression of SlHsfA3 was induced dramatically by heat stress, moderately by high salinity, and slightly by drought, but was not induced by abscisic acid (ABA). The ectopic overexpression of SlHsfA3 conferred increased thermotolerance and late flowering phenotype to transgenic Arabidopsis plants. Moreover, SlHsfA3 played a negative role in controlling seed germination under salt stress. RNA-sequencing data demonstrated that a number of heat shock proteins (Hsps) and stress-associated genes were induced in Arabidopsis plants overexpressing SlHsfA3. A gel shift experiment and transient expression assays in Nicotiana benthamiana leaves demonstrated that SlHsfA3 directly activates the expression of SlHsp26.1-P and SlHsp21.5-ER. Taken together, our results suggest that SlHsfA3 behaves as a typical Hsf to contribute to plant thermotolerance. The late flowering and seed germination phenotypes and the RNA-seq data derived from SlHsfA3 overexpression lines lend more credence to the hypothesis that plant Hsfs participate in diverse physiological and biochemical processes related to adverse conditions.

Neonates with intrauterine growth retardation (IUGR) are susceptible to decreases in cellular immunity. In recent years, a growing body of evidence indicates that Hsp70 may serve as a danger signal to the innate immune system and promote receptor-mediated apoptosis. Using neonatal pigs with IUGR, we investigated immune function of pigs and expression of heat shock protein 70 (Hsp70), nuclear factor-kappa B (NF-κB), and forkhead box O 3a (FoxO3a) in the intestinal tract. Samples from the blood, duodenum, jejunum, and ileum of normal body weight (NBW) piglets and IUGR piglets were collected at day 7 after birth. Furthermore, to test whether Hsp70 is associated with regulation of NF-κB and FoxO3a, Hsp70 was silenced using small RNA interference (siRNA) in IEC-6 cells. Body and intestinal weights were lower in IUGR piglets than in NBW piglets (p < 0.05). Proliferation of peripheral blood lymphocytes was decreased (p < 0.05) in IUGR piglets. Cytokine concentrations (IFN-γ, IL-4, IL-10, IL-1, and IL-8) were lower in serum of IUGR piglets. The levels of IFN-γ and IL-10 were decreased (p < 0.05) in the ileum of IUGR piglets, but IL-4 was increased (p < 0.05). The expressions of Hsp70 and FoxO3a were increased, and NF-κB activity was downregulated in IUGR piglets (p < 0.05). Furthermore, siRNA-mediated Hsp70 downregulation increased NF-κB activity, inhibited expression of FoxO3a, and decreased cell apoptosis. In contrast, overexpression of Hsp70 inhibited NF-κB activation. In conclusion, IUGR impairs immune functions in neonatal pigs. An inefficient immunity in IUGR piglets is associated with overexpression of Hsp70, which impairs NF-κB signaling and upregulates FoxO3a expression.

Background

Previous investigation has demonstrated that CD4+ T cells play a crucial role in effective immunity against Helicobacter pylori (H.pylori) infection. It has been well proved that Lpp20 is one of major protective antigens that induce immune responses after H.pylori invades host. Therefore it is valuable to identify CD4+ T cell epitopes on Lpp20, which is uncharacterized.

Methods

Putative epitopes of H-2d restricted CD4+ T cell on Lpp20 of H.pylori were predicted by the SYFPEITHI algorithm and then eight hypothetical epitope peptides were synthesized. After BALB/c mice were primed with recombinant Lpp20, splenic CD4+ T cells were isolated and stimulated with synthesized peptides to measure T cell proliferation and MHC restriction. Cytokine profile was determined by ELISA and real-time PCR. Two identified epitopes were used to immunize mice to investigate CD4+ T cell response by flow cytometry.

Results

Two of eight peptides were able to stimulate CD4+ T cell proliferation and were mapped to residues 83-97aa and 58-72aa on Lpp20 respectively. These two peptides additively stimulated Th1 cells to secrete IFN-γ. The percentage of CD4+ T cell from mice immunized with two identified epitopes respectively was higher than the control group.

Conclusion

The identification and characterization of two CD4+ T cell epitopes of Lpp20 helps understand the protective immunity of Lpp20 in H.pylori infection and design effective epitope vaccines against H.pylori.

Background

Allergic diseases affect large population. Pollen, a ubiquitous allergen, is the trigger of seasonal rhinitis, conjunctivitis and asthma, as well as an exacerbating factor of atopic dermatitis. However, the underlying mechanism by which pollen induces thymic stromal lymphopoietin (TSLP) triggered allergic inflammation via epithelial innate immunity is largely unknown.

Objective

To explore whether short ragweed (SRW) pollen induces TSLP/OX40L/OX40 signaling via Toll-like receptor (TLR) 4-dependent pathways in allergic disease.

Methods

Three models were used for this study, a well-characterized murine model of allergic conjunctivitis induced by SRW pollen, a topical challenge model on mouse ocular surface, and a culture model of primary human corneal epithelium exposed to aqueous extract of defatted SRW pollen (SRWe).

Results

The topical challenges with SRW pollen generated a typical allergic conjunctivitis in BALB/c mice. Clinical signs, stimulated TSLP/OX40L/OX40 signaling and Th2 cytokines in ocular mucosa and draining cervical lymph nodes were significantly reduced or eliminated in TLR4 deficient (Tlr4-d) or MyD88 knockout (MyD88−/−) mice, compared with their wild type littermates. SRWe stimulated TSLP production by ocular epithelia in wild type, but not Tlr4-d or MyD88−/− mice. SRWe stimulated TSLP was blocked by TLR4 antibody and NF-kB inhibitor in mouse and human corneal epithelia.

Conclusion

We for the first time uncovered a novel phenomenon that SRW pollen, acting as a functional TLR4 agonist, initiates TLR4-dependent TSLP/OX40L/OX40 signaling that triggers Th2-dominant allergic inflammation. These findings shed light on the understanding of mucosal epithelial innate immunity, and create new therapeutic targets to cure allergic diseases.

Tissue engineering holds great promise for corneal transplantation to treat blinding diseases. This study was to explore the use of natural corneal stroma as an optimal substrate to construct a native like corneal equivalent. Human corneal epithelium was cultivated from donor limbal explants on corneal stromal discs prepared by FDA approved Horizon Epikeratome system. The morphology, phenotype, regenerative capacity and transplantation potential were evaluated by hematoxylin eosin and immunofluorescent staining, a wound healing model, and the xeno-transplantation of the corneal constructs to nude mice. An optically transparent and stratified epithelium was rapidly generated on donor corneal stromal substrate and displayed native-like morphology and structure. The cells were polygonal in the basal layer and became flattened in superficial layers. The epithelium displayed a phenotype similar to human corneal epithelium in vivo. The differentiation markers, keratin 3, involucrin and connexin 43, were expressed in full or superficial layers. Interestingly, certain basal cells were immunopositive to antibodies against limbal stem/progenitor cell markers ABCG2 and p63, which are usually negative in corneal epithelium in vivo. It suggests that this bioengineered corneal epithelium shared some characteristics of human limbal epithelium in vivo. This engineered epithelium was able to regenerate in 4 days following from a 4mm-diameter wound created by a filter paper soaked with 1 N NaOH. This corneal construct survived well after xeno-transplantation to the back of a nude mouse. The transplanted epithelium remained multilayer and became thicker with a phenotype similar to human corneal epithelium. Our findings demonstrate that natural corneal stroma is an optimal substrate for tissue bioengineering, and a native-like corneal construct has been created with epithelium containing limbal stem cells. This construct may have great potential for clinical use in corneal reconstruction.

Background

Liraglutide is a glucagon-like peptide-1 analogue that stimulates insulin secretion and improves β-cell function. However, it is not clear whether liraglutide achieves its glucose lowering effect only by its known effects or whether other as yet unknown mechanisms are involved. The aim of this study was to examine the effects of liraglutide on Fibroblast growth factor-21 (FGF-21) activity in High-fat diet (HFD) fed ApoE−/− mice with adiponectin (Acrp30) knockdown.

Method

HFD-fed ApoE−/− mice were treated with adenovirus vectors expressing shAcrp30 to produce insulin resistance. Hyperinsulinemic-euglycemic clamp studies were performed to evaluate insulin sensitivity of the mouse model. QRT-PCR and Western blot were used to measure the mRNA and protein expression of the target genes.

Results

The combination of HFD, ApoE deficiency, and hypoadiponectinemia resulted in an additive effect on insulin resistance. FGF-21 mRNA expressions in both liver and adipose tissues were significantly increased while FGF-21 receptor 1 (FGFR-1) and β-Klotho mRNA levels in adipose tissue, as well as FGFR-1-3 and β-Klotho mRNA levels in liver were significantly decreased in this model. Liraglutide treatment markedly improved insulin resistance and increased FGF-21 expression in liver and FGFR-3 in adipose tissue, restored β-Klotho mRNA expression in adipose tissue as well as FGFR-1-3, β-Klotho levels and phosphorylation of FGFR1 up to the levels observed in control mice in liver. Liraglutide treatment also further increased FGF-21 proteins in liver and plasma. In addition, as shown by hyperinsulinemic-euglycemic clamp, liraglutide treatment also markedly improved glucose metabolism and insulin sensitivity in these animals.

Conclusion

These findings demonstrate an additive effect of HFD, ApoE deficiency, and adiponectin knockdown on insulin resistance and unveil that the regulation of glucose metabolism and insulin sensitivity by liraglutide may be partly mediated via increased FGF-21 and its receptors action.

Interleukin (IL) 33 has been recently identified as a ligand to the ST2 receptor that mediates Th2-dominant allergic inflammation. The purpose of this study was to explore the role of toll-like receptor (TLR)-mediated innate immunity in IL-33 induction by mucosal epithelium. Human corneal tissues and cultured primary human corneal epithelial cells (HCECs) were treated with a variety of viral or bacterial components without or with different inhibitors to evaluate the IL-33 regulation and signaling pathways. The level of mRNA expression was determined by reverse transcription and real time PCR, and protein was measured by ELISA, immunostaining and Western blotting. IL-33 mRNA and protein were largely induced by various microbial components, mainly by polyI:C and flagellin, the ligands to TLR3 and TLR5, respectively in human corneal epithelium ex vivo and in vitro cultures. Pro-IL-33 protein was normally restricted inside cells, and could be secreted outside when activated by ATP. The PolyI:C induced IL-33 production was blocked by TLR3 antibody or TRIF Inhibitory peptide, while flagellin stimulated IL-33 was blocked by TLR5 antibody or MyD88 Inhibitory peptide. Interestingly, IκB-α inhibitor (BAY11-7082) or NF-κB inhibitor (quinazoline) blocked NF-κB p65 protein nuclear translocation, and suppressed IL-33 production induced by PolyI:C and flagellin. These findings demonstrate that IL-33, an epithelium-derived pro-allergic cytokine, is induced by microbial ligands through TLR-mediated innate signaling pathways, suggesting a possible role of mucosal epithelium in Th2-dominant allergic inflammation.

Heat shock protein 27 (HSP27, or HSPB1) exerts cytoprotection against many cellular insults, including cerebral ischemia. We previously identified apoptosis signal-regulating kinase 1 (ASK1) as a critical downstream target of HSP27 conferring the neuroprotective effects of HSP27 against neuronal ischemia. However, the function of HSP27 is highly influenced by post-translational modification, with differential cellular effects based on phosphorylation at specific serine residues. The role of phosphorylation in neuronal ischemic neuroprotection is currently unknown. We have created transgenic mice and viral vectors containing HSP27 mutated at three critical serine residues (Ser15, Ser78 and Ser82) to either alanine (HSP27-A, non-phosphorylatable) or aspartate (HSP27-D, phospho-mimetic) residues. Under both in vitro and in vivo neuronal ischemic settings, overexpression of wild-type HSP27 (HSP27) and HSP27-D, but not HSP27-A, was neuroprotective and inhibited downstream ASK1 signaling pathways. Consistently, overexpressed HSP27 was phosphorylated by endogenous mechanisms when neurons were under ischemic stress, and single point mutations identified Ser15 and Ser82 as critical for neuroprotection. Using a panel of inhibitors and gene knockdown approaches, we identified the upstream kinase protein kinase D (PKD) as the primary kinase targeting HSP27 directly for phosphorylation. PKD and HSP27 co-immunoprecipitated, and inhibition or knockdown of PKD abrogated the neuroprotective effects of HSP27 as well as the interaction with and inhibition of ASK1 signaling. Taken together, these data demonstrate that HSP27 requires PKD-mediated phosphorylation for its suppression of ASK1 cell death signaling and neuroprotection against ischemic injury.

Plasmodium spp. are pathogenic to their vertebrate hosts and also apparently, impose a fitness cost on their insect vectors. We show here, however, that Plasmodium-infected mosquitoes survive starvation significantly better than uninfected mosquitoes. This survival advantage during starvation is associated with higher energy resource storage that infected mosquitoes accumulate during period of Plasmodium oocyst development. Microarray analysis revealed that the metabolism of sated mosquitoes is altered in the presence of rapidly growing oocysts, including the down-regulation of several enzymes involved in carbohydrate catabolism. In addition, enhanced expression of several insulin-like peptides was observed in Plasmodium-infected mosquitoes. Blocking insulin-like signaling pathway resulted in impaired Plasmodium development. We conclude that Plasmodium infection alters metabolic pathways in mosquitoes, epitomized by enhanced insulin-like signaling – thereby conferring a survival advantage to the insects during periods of starvation. Manipulation of this pathway might provide new strategies to influence the ability of mosquitoes to survive and transmit the protozoa that cause malaria.

We previously reported that XccR, a LuxR-type regulator of Xanthomonas campestris pv. campestris (Xcc), activates the downstream proline iminopeptidase virulence gene (pip) in response to certain host plant factor(s). In this report, we further show that the expression of the xccR gene was repressed in the culture medium by an NtrC-type response regulator, which we named XerR (XccR expression-related, repressor), and that this repression was relieved when the bacteria were grown in planta. Such a regulatory mechanism is reinforced by the observations that XerR directly bound to the xccR promoter in vitro, and that mutations at the phosphorylation-related residues of XerR resulted in the loss of its repressor function. Furthermore, the expression level of xccR increased even in XerR-overexpressing Xcc cells when they were vacuum infiltrated into cabbage plants. We also preliminarily characterized the host factor(s) involved in the above mentioned interactions between Xcc and the host plant, showing that a plant material(s) with molecular weight(s) less than 1 kDa abolished the binding of XerR to the xccR promoter, while the same material enhanced the binding of XccR to the luxXc box in the pip promoter. Taken together, our results implicate XerR in a new layer of the regulatory mechanism controlling the expression of the virulence-related xccR/pip locus and provide clues to the identification of plant signal molecules that interact with XerR and XccR to enhance the virulence of Xcc.

Previously 11 Elaphropeza species were known from Taiwan. The following two species of the genus Elaphropeza are described: Elaphropeza flaviscutum sp. n. and Elaphropeza trimacula sp. n. One species, Elaphropeza plumata Yang, Merz & Grootaert, is newly recorded from Taiwan. A key to 14 known species of Elaphropeza from Taiwan is presented.

Objective

The loss of estrogen in mRen2.Lewis rats leads to an exacerbation of diastolic dysfunction. Since specific neuronal nitric oxide synthase inhibition reverses renal damage in the same model, we assessed the effects of inhibiting neuronal nitric oxide on diastolic function, left ventricular remodeling, and the components of the cardiac nitric oxide system in ovariectomized and sham-operated mRen2.Lewis rats treated with L-VNIO (0.5 mg/kg/day for 28 days) or vehicle (saline).

Methods

Female mRen2.Lewis rats underwent either bilateral oophorectomy (OVX; n=15) or sham-operation (Sham; n=19) at 4 weeks of age. Beginning at 11 weeks of age, the rats were randomized to receive either L-VNIO or vehicle.

Results

The surgical loss of ovarian hormones, particularly estrogen, led to exacerbated hypertension, impaired myocardial relaxation, diminished diastolic compliance, increased perivascular fibrosis, and increased relative wall thickness. The cardiac tetrahydrobiopterin (BH4)-to-dihydrobiopterin (BH2) levels were lower among OVX rats compared to sham-operated rats and this altered cardiac biopterin profile was associated with enhanced myocardial superoxide production and decreased nitric oxide release. LVNIO decreased myocardial reactive oxygen species production, increased nitrite concentrations, attenuated cardiac remodeling and improved diastolic function.

Conclusions

Impaired relaxation, diastolic stiffness and cardiac remodeling were found among OVX mRen2.Lewis rats. A possible mechanism for this unfavorable cardiac phenotype may have resulted from a deficiency in available BH4 and subsequent increase in nNOS-derived superoxide and reduction in NO metabolites within the heart. Selective nNOS inhibition with L-VNIO attenuated cardiac superoxide production and limited remodeling, leading to improved diastolic function in OVX mRen2.Lewis rats.

Background

Studies have shown that type-specific persistence of high-risk human papillomavirus (HPV) infection contributed significantly to cervical carcinogenesis.

Methods

In this population-based study (on 24041 women), we report on the prevalent genotypes of HPVs and the prevalent genotypes of HPV persistent infection in the northeast of China.

Results

Our results showed that in HPV infected women (45.6% in total), (95% CI, 44.97%–46.23%), 17.35% (95%CI, 16.87%–17.83%) suffered persistent infection. The most common high-risk HPV types in persistent positivity were HPV-16 (18.21%; 95%CI, 17.04%–19.38%), HPV-58 (13.2%; 95%CI, 12.17%–14.23%), HPV-18 (8.66%; 95%CI, 7.81%–9.51%), HPV-52 (7.06%; 95% CI, 6.28%–7.84%) and HPV-33 (6.78%; 95% CI, 6.02%–7.54%). The prevalence of persistent infections with HPV-16,–58, −18, −52 and 33 in cervicitis were lower compared to those in CIN (all P < 0.05). HPV-58, −33 and multiple HPV persistent positivity were significantly associated with older age (all P < 0.05). HPV-18 persistent positivity was significantly associated with adenocarcinoma and lymphatic metastasis (all P < 0.05). HPV-18 persistent positivity was associated with cervical cancer prognosis (P <0.0001). Multivariate analyses showed that HPV-18 persistent positivity, (RR = 1.704, 95%CI = 1.095–2.654, p = 0.028) and lymphatic metastasis (RR = 2.304, 95%CI = 1.354–3.254, P = 0.015) were independent predictors for 3-year survival in cervical cancer.

Conclusions

we provided extensive results of HPV genotype prevalence and distribution in the northeast of China. HPV genotyping is worthwhile to perform because of its independent prognostic value in cervical cancer

Three species of Michotamia are recorded from China. Of these Michotamia aurata (Fabricius, 1794) was previously reported from Hainan and Taiwan. Michotamia assamensis Joseph & Parui, 1995 is recorded from China and Laos for the first time, and Michotamia yunnanensis sp. n., is described and figured. A key to the known species from China is provided. A new name, Michotamia subnigra, is given to Michotamia nigra Scarbrough & Hill, 2000, which is preoccupied by Michotamia nigra (Meijere, 1911).

Cotton fiber qualities including length, strength and fineness are known to be controlled by genes affecting cell elongation and secondary cell wall (SCW) biosynthesis, but the molecular mechanisms that govern development of fiber traits are largely unknown. Here, we evaluated an interspecific backcrossed population from G. barbadense cv. Hai7124 and G. hirsutum acc. TM-1 for fiber characteristics in four-year environments under field conditions, and detected 12 quantitative trait loci (QTL) and QTL-by-environment interactions by multi-QTL joint analysis. Further analysis of fiber growth and gene expression between TM-1 and Hai7124 showed greater differences at 10 and 25 days post-anthesis (DPA). In this two period important for fiber performances, we integrated genome-wide expression profiling with linkage analysis using the same genetic materials and identified in total 916 expression QTL (eQTL) significantly (P<0.05) affecting the expression of 394 differential genes. Many positional cis-/trans-acting eQTL and eQTL hotspots were detected across the genome. By comparative mapping of eQTL and fiber QTL, a dataset of candidate genes affecting fiber qualities was generated. Real-time quantitative RT-PCR (qRT-PCR) analysis confirmed the major differential genes regulating fiber cell elongation or SCW synthesis. These data collectively support molecular mechanism for G. hirsutum and G. barbadense through differential gene regulation causing difference of fiber qualities. The down-regulated expression of abscisic acid (ABA) and ethylene signaling pathway genes and high-level and long-term expression of positive regulators including auxin and cell wall enzyme genes for fiber cell elongation at the fiber developmental transition stage may account for superior fiber qualities.

The P2Y12 receptor, a Gi protein-coupled receptor, plays a central role in platelet activation. In this study, we did a mutational analysis of residues possibly involved in the ligand interactions with the human P2Y12 receptor. Mutant receptors were stably expressed in CHO-K1 cells with an HA-tag at the N-terminus. Expression of wild-type and mutant receptors was confirmed by detecting the HA-tag on the cell membrane. Residues in transmembrane helical domains (TMs) 3, 5, 6, and 7, which are homologous to residues important for P2Y1 receptor activation and ligand recognition, were replaced by site-directed mutagenesis. ADP-induced inhibition of forskolin-stimulated cAMP levels in the presence or absence of antagonist AR-C69931MX were investigated for each of the mutant receptors. F104S and S288P significantly increased agonist-induced receptor function without affecting the antagonism by AR-C69931MX. Arg256 in TM6 and Arg 265 in extracellular loop 3 (EL3) are more important for antagonist recognition than effect on agonist-mediated receptor function. Compared to wild-type P2Y12 receptor, mutations in Arg 256 or/and Arg 265 significantly increased the sensitivity to antagonist AR-C69931MX. Our study shows that the cytosolic side of TM3 and the exofacial side of TM5 are critical for P2Y12 receptor function, which is different from P2Y1. Arg 256 in TM6 and Arg265 in EL3 appear to play a role in antagonist recognition rather than effects on agonist-induced receptor function.

The peroxiredoxin (PRX) family of antioxidant enzymes helps maintain the intracellular reducing milieu and suppresses apoptosis in non-neuronal cells. However, whether PRX can inhibit neuronal apoptosis through specific signaling mechanisms remains poorly understood. Induction of PRX2, the most abundant neuronal PRX, occurs in Parkinson’s disease (PD) patient brains, but its functional impact is unclear. In the present study, we used the dopaminergic (DA) toxin 6-hydroxydopamine (6-OHDA) to model PD and explore the protective effect and mechanisms of PRX on DA neurons. Of the 2-cysteine PRXs that were tested in MN9D DA neurons, endogenous PRX2 was most beneficial to cell survival. Lentivirus-mediated PRX2 over-expression conferred marked in vitro and in vivo neuroprotection against 6-OHDA toxicity in DA neurons, and preserved motor functions involving the dopamine system in mouse. In addition to its role as an antioxidant enzyme, PRX2 exhibited anti-apoptotic effects in DA neurons via suppression of ASK1-dependent activation of the JNK/c-Jun and p38 pro-death pathways, which are also activated in DA neurons of post-mortem PD brains. PRX2 inhibited 6-OHDA-induced ASK1 activation by modulating the redox status of the endogenous ASK1 inhibitor thioredoxin (Trx). PRX2 over-expression maintained Trx in a reduced state by inhibiting the cysteine thiol-disulfide exchange, thereby preventing its dissociation from ASK1. This study describes a previously undefined mechanism by which redox-sensitive molecules signal via apoptotic pathways in response to PD-relevant toxic stress in DA neurons. Our results also suggest that PRX2 and ASK1 may be potential targets for neuroprotective intervention in PD.

Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted to humans by bite of Ixodes scapularis ticks. The mechanisms by which the bacterium is transmitted from vector to host are poorly understood. In this study, we show that the F(ab)2 fragments of BBE31, a B.burgdorferi outer-surface lipoprotein, interfere with the migration of the spirochete from tick gut into the hemolymph during tick feeding. The decreased hemolymph infection results in lower salivary glands infection, and consequently attenuates mouse infection by tick-transmitted B. burgdorferi. Using a yeast surface display approach, a tick gut protein named TRE31 was identified to interact with BBE31. Silencing tre31 also decreased the B. burgdorferi burden in the tick hemolymph. Delineating the specific spirochete and arthropod ligands required for B. burgdorferi movement in the tick may lead to new strategies to interrupt the life cycle of the Lyme disease agent.

Author Summary

Lyme disease, the most common tick-borne illness in North America, is caused by Borrelia burgdorferi. Currently, spirochete and tick molecules that facilitate Borrelia migration within the vector, a key step for mammalian infection by tick-transmitted spirochetes, have not yet been identified. In this study, we show that F(ab)2 fragments of BBE31, a B.burgdorferi outer-surface lipoprotein, interfere with the spirochete migration from the tick gut into the hemolymph. Our results indicated that decreased hemolymph infection by blocking BBE31 resulted in lower salivary glands infection, which eventually attenuated murine infection by tick-transmitted B.burgdorferi. We also found that a tick gut protein TRE31 enables Borrelia movement by interacting with BBE31. This finding provides novel insights into the transmission of spirochete within the vector and provides potential vaccine targets to block the microbial life cycle within the vector.

Despite the immense significance retrotransposons have had for genome evolution much about their biology is unknown, including the processes of forming their ribonucleoprotein (RNP) particles and transporting them about the cell. Suppression of retrotransposon expression, together with the presence of retrotransposon sequence within numerous mRNAs, makes tracking endogenous L1 RNP particles in cells problematic. We overcame these difficulties by assaying in living and fixed cells tagged-RNPs generated from constructs expressing retrotransposition-competent L1s. In this way, we demonstrate for the first time the subcellular colocalization of L1 RNA and proteins ORF1p and ORF2p, and show their targeting together to cytoplasmic foci. Foci are often associated with markers of cytoplasmic stress granules. Furthermore, mutation analyses reveal that ORF1p can direct L1 RNP distribution within the cell. We also assayed RNA localization of the non-autonomous retrotransposons Alu and SVA. Despite a requirement for the L1 integration machinery, each manifests unique features of subcellular RNA distribution. In nuclei Alu RNA forms small round foci partially associated with marker proteins for coiled bodies, suborganelles involved in the processing of non-coding RNAs. SVA RNA patterning is distinctive, being cytoplasmic but without prominent foci and concentrated in large nuclear aggregates that often ring nucleoli. Such variability predicts significant differences in the life cycles of these elements.

This is the first report of the ability of azithromycin to inhibit the production of inflammatory mediators by human corneal epithelial cells. The authors demonstrated that the fungal component zymosan induces proinflammatory responses through TLR2 and NF-κB signaling pathways, whereas azithromycin suppresses its stimulation by blocking NF-κB activation in human corneal epithelial cells, suggesting the potential efficacy of this antibiotic for treating ocular surface inflammatory disorders.

Purpose.

In addition to its antibiotic effects, azithromycin has been noted to have anti-inflammatory activity, particularly in the context of microbial infections. This study was conducted to explore the suppressive effects of azithromycin on the production of proinflammatory mediators by human corneal epithelial cells (HCECs) stimulated by a fungal component, zymosan.

Methods.

Primary HCECs were cultured from donor corneal limbal explants and grown to subconfluence. The cells were treated with toll-like receptor (TLR) 2 agonist zymosan (1–50 μg/mL) for 4 to 48 hours, with or without preincubation with azithromycin (1–50 μg/mL), TLR2 antibody, or NF-κB activation inhibitor quinazoline (NF-κB-I). The cells were subjected to total RNA extraction, reverse transcription (RT), and real-time PCR using gene expression assays. Cells treated for 48 hours were used for immunofluorescence staining and Western blot analysis, and their medium supernatants were collected for protein quantitation by immunobead assays.

Results.

The mRNA expression and protein production of proinflammatory cytokines (TNF-α and IL-1β), chemokines (IL-8 and RANTES), and matrix metalloproteinases (MMP-1, -3, and -9) by HCECs were stimulated by zymosan in a concentration-dependent manner, with peak levels noted at 4 hours. These stimulated levels of proinflammatory mediators by zymosan were significantly inhibited by TLR2 antibody, NF-κB-I, or azithromycin, which blocked zymosan-induced NF-κB activation as determined by p65 protein nuclear translocation.

Conclusions.

These findings demonstrated that the fungal component zymosan induces proinflammatory responses through TLR2 and NF-κB signaling pathways, whereas azithromycin suppresses its stimulation by blocking NF-κB activation in HCECs, suggesting the potential efficacy of this antibiotic for treating ocular surface inflammatory disorders.

The CDKN2A/ARF locus encompasses overlapping tumor suppressor genes p16(INK4A) and p14(ARF), which are frequently co-deleted in human malignant mesothelioma (MM). The importance of p16(INK4A) loss in human cancer is well established, but the relative significance of p14(ARF) loss has been debated. The tumor predisposition of mice singly deficient for either Ink4a or Arf, due to targeting of exons 1α or 1β, respectively, supports the idea that both play significant and nonredundant roles in suppressing spontaneous tumors. To further test this notion, we exposed Ink4a(+/−) and Arf(+/−) mice to asbestos, the major cause of MM. Asbestos-treated Ink4a(+/−) and Arf(+/−) mice showed increased incidence and shorter latency of MM relative to wild-type littermates. MMs from Ink4a(+/−) mice exhibited biallelic inactivation of Ink4a, loss of Arf or p53 expression and frequent loss of p15(Ink4b). In contrast, MMs from Arf(+/−) mice exhibited loss of Arf expression, but did not require loss of Ink4a or Ink4b. Mice doubly deficient for Ink4a and Arf, due to deletion of Cdkn2a/Arf exon 2, showed accelerated asbestos-induced MM formation relative to mice deficient for Ink4a or Arf alone, and MMs exhibited biallelic loss of both tumor suppressor genes. The tumor suppressor function of Arf in MM was p53-independent, since MMs with loss of Arf retained functional p53. Collectively, these in vivo data indicate that both CDKN2A/ARF gene products suppress asbestos carcinogenicity. Furthermore, while inactivation of Arf appears to be crucial for MM pathogenesis, the inactivation of both p16(Ink4a) and p19(Arf) cooperate to accelerate asbestos-induced tumorigenesis.

Since their arrival in the Tibetan Plateau during the Neolithic Age, Tibetans have been well-adapted to extreme environmental conditions and possess genetic variation that reflect their living environment and migratory history. To investigate the origin of Tibetans and the genetic basis of adaptation in a rigorous environment, we genotyped 30 Tibetan individuals with more than one million SNP markers. Our findings suggested that Tibetans, together with the Yi people, were descendants of Tibeto-Burmans who diverged from ancient settlers of East Asia. The valleys of the Hengduan Mountain range may be a major migration route. We also identified a set of positively-selected genes that belong to functional classes of the embryonic, female gonad, and blood vessel developments, as well as response to hypoxia. Most of these genes were highly correlated with population-specific and beneficial phenotypes, such as high infant survival rate and the absence of chronic mountain sickness.