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To assess prognostic influences of RAS mutational status and primary tumor site on cases with colorectal liver metastasis (CRLM) who underwent hepatectomy.Clinicopathological data of 762 patients with CRLM who underwent hepatectomy between January 2000 and November 2018 were retrospectively analyzed. The left-sided tumors (LST) included tumors located in the splenic flexure, descending colon, sigmoid colon, and rectum; while right-sided tumors (RST) included those located in the cecum, ascending colon, and transverse colon. RAS mutational status was determined using Sanger sequencing or next-generation sequencing, including KRAS (Codons 12, 13, and 61) and NRAS (Codons 12, 13, and 61), which were defined as wild-type (RASwt) and mutant-type (RASmut), respectively. Survival curves were plotted using the Kaplan-Meier plotter and compared by the log rank test. The clinicopathological data were analyzed using univariate and multivariate analyses.The 5-year overall survival (OS) in the LST group was longer than that in the RST group (OS: 47.1% vs. 31.0%, p = 0.000, respectively), and the OS in the RASwt group was longer compared with that in the RASmut group (OS: 53.6% vs. 24.0%, p = 0.000). Besides, overall survival of the patients after hepatectomy was alternative, which was stratified by primary tumor site, with the 1-, 3-, and 5-year survival rates of 93.1%, 62.1%, and 47.1% for patients with LST, and 91.1%, 42.8%, and 31.0% for patients with RST, respectively. OS and disease-free survival (DFS) were significantly different stratified by RAS mutational status, with the 1-, 3-, and 5-year rates of 96.9%, 67.9%, and 53.6% for patients with RASwt tumors, and 85.7%, 41.5%, and 24.0% for patients with RASmut tumors, respectively. The 1-, 3-, and 5-year DFS rates were 51.9%, 30.0%, and 26.7% for patients with RASwt tumors, and 35.8%, 18.2%, and 14.9% for patients with RASmut tumors, respectively. The results of multivariate analysis showed that RAS mutational status and primary tumor site were both independent influencing factors of OS.RAS mutational status and primary tumor site affect OS independently in CRLM patients undergoing hepatectomy. The worse prognosis of RST cannot be simply attributed to the imbalance of RAS mutational status in different primary tumor sites.

Dermatologists should be aware that the clinical manifestations of syphilis are very complex and changeable. Unilaterally distributed skin lesions and painless lip ulcers may also be the clinical manifestations of secondary syphilis.

Peroxisomes play a central role in lipid metabolism. We previously demonstrated that Pex11a deficiency impairs peroxisome abundance and fatty acid β‐oxidation and results in hepatic triglyceride accumulation. The role of Pex11a in dyslipidaemia and obesity is investigated here with Pex11a knockout mice (Pex11a−/−). Metabolic phenotypes including tissue weight, glucose tolerance, insulin sensitivity, cholesterol levels, fatty acid profile, oxygen consumption, physical activity were assessed in wild‐type (WT) and Pex11a−/− fed with a high‐fat diet. Molecular changes and peroxisome abundance in adipose tissue were evaluated through qRT‐PCR, Western blotting, and Immunofluorescence. Pex11a−/− showed increased fat mass, decreased skeletal muscle, higher cholesterol levels, and more severely impaired glucose and insulin tolerance. Pex11a−/− consumed less oxygen, indicating a decrease in fatty acid oxidation, which is consistent with the accumulation of very long‐ and long‐chain fatty acids. Adipose palmitic acid (C16:0) levels were elevated in Pex11a−/−, which may be because of dramatically increased fatty acid synthase mRNA and protein levels. Furthermore, Pex11a deficiency increased ventricle size and macrophage infiltration, which are related to the reduced physical activity. These data demonstrate that Pex11a deficiency impairs physical activity and energy expenditure, decreases fatty acid β‐oxidation, increases de novo lipogenesis and results in dyslipidaemia and obesity.

The flavonoid luteolin is a common flavone released from peanut (Arachis hypogaea L.) residues. Luteolin accumulates in consecutively monocultured soil and affects the growth and nodulation of continuously cropped peanut. A microcosm experiment was performed to determine the effects of luteolin released from peanut residue on peanut nodule formation and on the rhizosphere soil microbial community. Luteolin reached a maximum concentration of 0.42 μg g–¹ soil in litterbag experiments, and the half-life of luteolin at 2.5 to 40 μg g–¹ soil varied from 1.13 to 2.91 d. Results from pot experiments indicate that soil with luteolin treatment inhibited peanut seedling nodulation and growth, soil dehydrogenase activity, microbial biomass carbon content, biofilm formation, and chemotactic response of Rhizobium sp. and changed the microbial community structures and abundances at concentration of luteolin as low as 0.42 μg g–¹ in soil. This might explain why peanut performs poorly with continuous consecutive monocropping.

Aim The programmed death 1/programmed death 1 ligand (PD-1/PD-L1) pathway can decrease the immune clearance effects of antigen-presenting cells and T lymphocytes to promote immune evasion of cervical cancer cells. However, the effects of this pathway on cervical intraepithelial neoplasia (CIN) progression and squamous cell carcinoma (SCC) metastasis are not clear. We herein investigated whether human papillomavirus infection could affect PD-1 and PD-L1 expression in CIN, and whether their expression is associated with CIN progression and SCC metastasis. Methods We collected paraffin-embedded samples from two cohorts of patients: (i) CIN samples from cohort I (40 women who tested positive or negative for high-risk human papillomavirus [HR-HPV] with grades 0, I, and II–III CIN); and (ii) paired primary and metastatic tumor samples from cohort II (20 SCC patients with or without metastasis). Immunohistochemistry was used to detect expressions of PD-L1 in tumor cells and PD-1 in tumor-associated macrophages and tumor-infiltrating lymphocytes. We also measured P16INK4a expression and interferon-γ levels in the cervical tissues. Results The most common HPV type seen in both cohorts of patients was HPV16, followed by HPV18. Increase in PD-L1 and PD-1 expression was positively correlated with HPV-positivity, increase in CIN grade, and tumor metastasis. Furthermore, upregulation of the PD-1/PD-L1 pathway was associated with decreased expression of the pro-inflammatory cytokine, interferon-γ and increased expression of P16INK4a. Conclusion Expression of PD-L1 and PD-1 could be used as clinical prognostic biomarkers for evaluating CIN and cervical cancer because of its positive correlation with CIN progression and tumor metastasis.

SummaryBackground Striae distensae (SD) are a common dermatologic problem that plagues many people. Although there are many therapeutic modalities have been used to treat SD, effective method has been disappointing for striae Alba. Aims To evaluate the clinical and histopathologic efficacy and safety of the 2940-nm erbium yttrium aluminum garnet (Er:YAG) ablative fractional laser (AFL) with recombinant bovine basic fibroblast growth factor (rb-bFGF) and light-emitting diode-red light (LED-RL) for the treatment of striae alba. Patients and methods Thirty volunteers with striae distensae alba were enrolled. The subjects completed treatments with the 2940-nm Er:YAG AFL 6 times at 4-week intervals. Following this treatment, the subjects were required to spray rb-BFGF for 1 week at home. They then received LED-RL once every 7 days for three sessions between the two laser treatments. Two independent investigators evaluated clinical improvement at pretreatment and 1, 3, 6, and 12 months post-treatment, patients also provided self-assessments of clinical improvement. Two biopsies were obtained from two subjects, both of the same sites of striae alba, one before the first treatment and one 6 months after the last session. Results All 30 subjects demonstrated clinical improvement after treatment. Skin biopsies after treatment showed an increase in epidermal thickness, dermal thickness, and collagen and elastin density when compared to that at the baseline. Conclusions The combination of the 2940-nm Er:YAG laser with rb-bFGF and LED-RL for the treatment of striae alba was a safe and effective approach for improving the appearance of striae alba.

New Findings What is the central question of this study? Exercise training increases brain-derived neurotrophic factor (BDNF) in the hippocampus, which depends on a myokine, fibronectin type III domain-containing protein 5 (FNDC5). Whether exercise training after myocardial infarction induces parallel increases in FNDC5 and BDNF expression in skeletal muscle and the heart has not yet been studied. What is the main finding and its importance? Exercise training after myocardial infarction increases BDNF protein in skeletal muscle and the non-infarct area of the LV without changes in FNDC5 protein, suggesting that BDNF is not regulated by FNDC5 in skeletal muscle and heart. An increase in cardiac BDNF may contribute to the improvement of cardiac function by exercise training. Exercise training after myocardial infarction (MI) attenuates progressive left ventricular (LV) remodelling and dysfunction, but the peripheral stimuli induced by exercise that trigger these beneficial effects are still unclear. We investigated as possible mediators fibronectin type III domain-containing protein 5 (FNDC5) and brain-derived neurotrophic factor (BDNF) in the skeletal muscle and heart. Male Wistar rats underwent either sham surgery or ligation of the left descending coronary artery, and surviving MI rats were allocated to either a sedentary (Sed-MI) or an exercise group (ExT-MI). Exercise training was done for 4 weeks on a motor-driven treadmill. At the end, LV function was evaluated, and FNDC5 and BDNF mRNA and protein were assessed in soleus muscle, quadriceps and non-, peri- and infarct areas of the LV. At 5 weeks post MI, FNDC5 mRNA was decreased in soleus muscle and all areas of the LV, but FNDC5 protein was increased in the soleus muscle and the infarct area. Mature BDNF (mBDNF) protein was decreased in the infarct area without a change in mRNA. Exercise training attenuated the decrease in ejection fraction and the increase in LV end-diastolic pressure post MI. Exercise training had no effect on FNDC5 mRNA and protein, but increased mBDNF protein in soleus muscle, quadriceps and the non-infarct area of the LV. The mBDNF protein in the non-infarct area correlated positively with ejection fraction and inversely with LV end-diastolic pressure. In conclusion, mBDNF is induced by exercise training in skeletal muscle and the non-infarct area of the LV, which may contribute to improvement of muscle dysfunction and cardiac function post MI.

With the ability to resolve structures of macromolecules at atomic resolution, X-ray crystallography has been the most powerful tool in modern structural biology. At the same time, recent technical improvements have triggered a resolution revolution in the single particle cryo-EM method. While the two methods are different in many respects, from sample preparation to structure determination, they both have the power to solve macromolecular structures at atomic resolution. It is important to understand the unique advantages and caveats of the two methods in solving structures and to appreciate the complementary nature of the two methods in structural biology. In this review we provide some examples, and discuss how X-ray crystallography and cryo-EM can be combined in deciphering structures of macromolecules for our full understanding of their biological mechanisms.

We hypothesized that lack of the high-energy phosphates during liver storage may potentially cause persistent injury to the vascular endothelium. Biopsies were obtained from livers obtained from beating heart human donors, stored either in the standard storage solution, that is, University of Wisconsin solution (UWS) or Celsior, and examined for various markers related to progressive endothelial injury. The expression of P2Y1 receptor, the major signal transduction machinery for adenosine triphosphate/adenosine diphosphate, decreased in hepatic vascular endothelial cells over time. Despite unaltered endothelial nitric oxide synthase (eNOS) levels, serine1177-phosphorylated eNOS, the active form of eNOS, progressively decreased with time. The production of nitric oxide enzyme decreased with time when liver tissues were examined in vitro. This also coincided with decreased interaction of eNOS with actin nucleating proteins like myristoylated alanine-rich C kinase substrate and Rac1, which plays a role in modulating the cytoskeleton and helps position eNOS in a favorable cytosolic position for active enzymatic activity. Conversely, the interaction of eNOS with caveolin1 was significantly increased 6 H after ex vivo storage. Finally, we demonstrated by targeted contrast-enhanced ultrasound that membrane-bound vascular cell adhesion molecule-1 in the hepatic vascular endothelial cell increased after 6 H of ex vivo storage. Overall, the results of this study provide evidence of a progressive hepatic vascular endothelial injury during the ex vivo storage. This may be a causative factor for ischemic cholangiopathy and delayed graft function post liver transplantation. © 2015 IUBMB Life, 68(1):51-57, 2015.

BACKGROUND Continuous cropping practices cause a severe decline in peanut yield. The aim of this study was to investigate the remediation effect of Serratia marcescens on continuously cropped peanut soil. A pot experiment was conducted under natural conditions to determine peanut agronomic indices, soil microorganism characteristics, soil enzyme activities and antagonism ability to typical pathogens at different growth stages. Four treatments were applied to red soil as follows: an active fermentation liquor of S. marcescens (RZ-21), an equivalent sterilized fermentation liquor (M), an equivalent fermentation medium (P) and distilled water (CK). RESULTS S. marcescens significantly inhibited the two typical plant pathogens Fusarium oxysporum A1 and Ralstonia solanacearum B1 and reduced their populations in rhizosphere soil. The RZ-21 treatment significantly increased peanut yield, vine dry weight, root nodules and taproot length by 62.3, 33, 72 and 61.4% respectively, followed by the M treatment. The P treatment also increased root nodules and root length slightly. RZ-21 also enhanced the activities of soil urease, sucrase and hydrogen peroxidase at various stages. In addition, RZ-21 and M treatments increased the average population of soil bacteria and decreased the average population of fungi in the three critical peanut growth stages, except for M in the case of the fungal population at flowering, thus balancing the structure of the soil microorganism community. CONCLUSION This is the first report of S. marcescens being applied to continuously cropped peanut soil. The results suggest that S. marcescens RZ-21 has the potential to improve the soil environment and agricultural products and thus allow the development of sustainable management practices. © 2015 Society of Chemical Industry

Key points Chronic subcutaneous infusion of Ang II causes a progressive increase in blood pressure (BP) associated with significant increases in angiotensin type 1 receptor (AT1R) and mineralocorticoid receptor (MR) expression in hypothalamic nuclei. Intra-paraventricular nucleus (PVN) infusion of AAV- AT1aR-siRNA or of AAV-MR-siRNA markedly knockdown AT1a-R or MR expression in the PVN but not in the subfornical organ; or supraoptic nucleus, and prevent most of the increase in BP. These findings indicate that increased MR and AT1R activation in the PVN play a critical role in Ang II-induced hypertension in rats on regular salt intake. Abstract Circulating Ang II activates an aldosterone-mineralocorticoid receptor (MR) – angiotensin II (Ang II) – angiotensin type 1 receptor (AT1R) pathway in the hypothalamus. To obtain insights into the actual neuronal projections involved, adeno-associated virus carrying small interfering RNA against either AT1aR (AAV-AT1aR-siRNA) or MR (AAV-MR-siRNA) were infused into the paraventricular nucleus (PVN) in Wistar rats. Intra-PVN infusion of AAV-AT1aR-siRNA or AAV-MR-siRNA decreased AT1R or MR expression in the PVN but not in the subfornical organ (SFO) or supraoptic nucleus (SON). Subcutaneous infusion of Ang II at 500 ng kg−1 min−1 for 2 weeks increased mean arterial pressure by 60–70 mmHg, and increased AT1R and MR expression in the SFO, SON and PVN. Intra-PVN AT1aR-siRNA prevented the Ang II-induced increase in AT1R but not MR expression in the PVN, and MR-siRNA prevented MR but not AT1R expression in the PVN. The increases in AT1R and MR expression in both the SFO and the SON were not changed by the two AAV-siRNAs. Specific knockdown of AT1R or MR in the PVN by AAV-siRNA each prevented most of the Ang II-induced hypertension. Prevention of the subcutaneous Ang II-induced increase in MR but not the increase in AT1R by knockdown of MR and vice versa suggests an independent regulation of MR and AT1R expression in the PVN. Both AT1R and MR activation in the PVN play a critical role in Ang II-induced hypertension in rats.

Key points Central mineralocorticoid receptor (MR) and angiotensin II type 1 receptor (AT1R) activation play a critical role in sympathetic hyperactivity and progressive left ventricle (LV) remodelling and dysfunction after a myocardial infarction (MI). Intra-paraventricular nucleus (PVN) infusion of adeno-associated virus (AAV) carrying small interfering RNA (siRNA) against MR (AAV-MR-siRNA) markedly decreases both MR and AT1R expression in the PVN post MI, whereas AAV-AT1aR-siRNA only decreases AT1R expression. Both AAVs largely prevent sympathetic hyperactivity and inhibit part of LV remodelling and dysfunction post MI. These findings indicate that enhanced MR–AT1R signalling in the PVN is critical for sympathetic hyperactivity post MI, and contributes to part of LV dysfunction post MI. Abstract Intracerebroventricular infusion of a mineralocorticoid receptor (MR) or angiotensin II type 1 receptor (AT1R) blocker in rats attenuates sympathetic hyperactivity and progressive left ventricular (LV) dysfunction post myocardial infarction (MI). The present study examined whether knockdown of MRs or AT1Rs specifically in the paraventricular nucleus (PVN) contributes to these effects, and compared cardiac effects with those of systemic treatment with the β1-adrenergic receptor blocker metoprolol. The PVN of rats was infused with adeno-associated virus carrying small interfering RNA against either MR (AAV-MR-siRNA) or AT1R (AAV-AT1R-siRNA), or as control scrambled siRNA. At 4 weeks post MI, AT1R but not MR expression was increased in the PVN, excitatory renal sympathetic nerve activity and pressor responses to air stress were enhanced, and arterial baroreflex function was impaired; LV end-diastolic pressure (LVEDP) was increased and LV peak systolic pressure (LVPSP), ejection fraction (EF) and dP/dtmax decreased. AAV-MR-siRNA and AAV-AT1R-siRNA both normalized AT1R expression in the PVN, similarly ameliorated sympathetic and pressor responses to air stress, largely prevented baroreflex desensitization, and improved LVEDP, EF and dP/dtmax as well as cardiac interstitial (but not perivascular) fibrosis. In a second set of rats, metoprolol at 70 or 250 mg kg−1 day−1 in the drinking water for 4 weeks post MI did not improve LV function except for a decrease in LVEDP at the lower dose. These results suggest that in rats MR-dependent upregulation of AT1Rs in the PVN contributes to sympathetic hyperactivity, and LV dysfunction and remodelling post MI. In rats, normalizing MR–AT1R signalling in the PVN is a more effective strategy to improve LV dysfunction post MI than systemic β1 blockade.

The enormous biological diversity of endophytes, coupled with their potential to enhance the production of bioactive metabolites in plants, has driven research efforts focusing on endophytes. However, limited information is available on the impacts of bacterial endophytes on plant secondary metabolism and signaling pathways involved. This work showed that an endophytic Acinetobacter sp. ALEB16, capable of activating accumulation of plant volatile oils, also induced abscisic acid (ABA) and salicylic acid (SA) production in Atractylodes lancea. Pre-treatment of plantlets with biosynthetic inhibitors of ABA or SA blocked the bacterium-induced volatile production. ABA inhibitors suppressed not only the bacterium-induced volatile accumulation but also the induced ABA and SA generation; nevertheless, SA inhibitors did not significantly inhibit the induced ABA biosynthesis, implying that SA acted downstream of ABA production. These results were confirmed by observations that exogenous ABA and SA reversed the inhibition of bacterium-induced volatile accumulation by inhibitors. Transcriptional activities of genes in sesquiterpenoid biosynthesis also increased significantly with bacterium, ABA and SA treatments. Mevalonate pathway proved to be the main source of isopentenyldiphosphate for bacterium-induced sesquiterpenoids, as assessed in experiments using specific terpene biosynthesis inhibitors. These results suggest that Acinetobacter sp. acts as an endophytic elicitor to stimulate volatile biosynthesis of A. lancea via an ABA/SA-dependent pathway, thereby yielding additional insight into the interconnection between ABA and SA in biosynthesis-related signaling pathways.

Many studies have examined pair-wise interactions between plants and endophytes, while overlooking the interplays among multiple endosymbionts and their combined impacts on hosts. In this study, Atractylodes lancea plantlets were inoculated with endophytic fungus Acremonium strictum AL16, or endophytic bacterium Acinetobacter sp., or both, to investigate the impacts of the three-way symbiosis on the host. Our results showed that defense-related responses of the co-inoculated plantlets were delayed and weakened relative to plantlets with single inoculants, but no detrimental effects on phyto-physiology (growth, photosynthesis) were observed after combined inoculations. Quantitative PCR analysis verified a decrease in AL16 colonization density within plants after co-inoculation with the endobacteria. An in vitro assay was then performed to elucidate the suppressed plant defense responses and reduced fungal colonization by dual inoculation. The data showed that the presence of Acinetobacter sp. reduced AL16 colony diameter and spore germination rate without negatively affecting fungal morphology. Additionally, direct hyphal attachment of the bacterium to AL16 in vitro was visualized by scanning electronic microscopy. Therefore, we propose that a balanced and compatible symbiosis might require constraints conferred by the antagonistic endophyte Acinetobacter sp. on the fungus AL16 in the tripartite endophytic bacterium-fungus-plant system.

Impaired local cellular immunity contributes to the pathogenesis of persistent high-risk human papillomavirus (HR-HPV) infection and related cervical intraepithelial neoplasia (CIN), but the underlying molecular mechanisms remain unclear. Recently, the programmed death 1/programmed death 1 ligand (PD-1/PD-L1; CD279/CD274) pathway was demonstrated to play a critical role in attenuating T-cell responses and promoting T-cell tolerance during chronic viral infections. In this study, we examined the expression of PD-1 and PD-L1 on cervical T cells and dendritic cells (DCs), respectively, from 40 women who were HR-HPV-negative (−) or HR-HPV-positive (+) with CIN grades 0, I and II–III. We also measured interferon-γ, interleukin-12 (IL-12) and IL-10 in cervical exudates. The most common HPV type was HPV 16, followed by HPV 18, 33, 51 and 58. PD-1 and PD-L1 expression on cervical T cells and DCs, respectively, was associated with HR-HPV positivity and increased in parallel with increasing CIN grade. The opposite pattern was observed for CD80 and CD86 expression on DCs, which decreased in HR-HPV(+) patients in parallel with increasing CIN grade. Similarly, reduced levels of the T helper type 1 cytokines interferon-γ and IL-12 and increased levels of the T helper type 2 cytokine IL-10 in cervical exudates correlated with HR-HPV positivity and CIN grade. Our results suggest that up-regulation of the inhibitory PD-1/PD-L1 pathway may negatively regulate cervical cell-mediated immunity to HPV and contribute to the progression of HR-HPV-related CIN. These results may aid in the development of PD-1/PD-L1 pathway-based strategies for immunotherapy of HR-HPV-related CIN.

The protein tyrosine phosphatase N22 (PTPN22) gene C1858T polymorphism has been reported to be associated with susceptibility to type 1 diabetes (T1D) in relatively small sample sizes. This study aimed at investigating the pooled association by carrying out a meta-analysis on the published studies. The Medline, EBSCO, and BIOSIS databases were searched to identify eligible studies published in English before June 2012. The association was assessed by odds ratio (OR) with 95% confidence intervals (CI). The presence of heterogeneity and publication bias was explored by using meta-regression analysis and Begg's test, respectively. A total of 28 studies were involved in this meta-analysis. Across all populations, significant associations were found between the PTPN22 C1858T polymorphism and susceptibility to T1D under genotypic (TT vs. CC [OR = 3.656, 95% CI: 3.139-4.257], CT vs. CC [OR = 1.968, 95% CI: 1.683-2.300]), recessive (OR = 3.147, 95% CI: 2.704-3.663), and dominant models (OR = 1.957, 95% CI: 1.817-2.108). In ethnicity- and sex-stratified analyses, similar associations were found among Caucasians and within Caucasian male and female strata. The meta-analysis results suggest that the PTPN22 C1858T polymorphism was associated with susceptibility to T1D among the Caucasian population, and males who carried the -1858T allele were more susceptible to T1D than females.

Four new limonoids, azadiraindins A-D (1-4, resp.), together with seven known analogs, were isolated from the MeOH extract of Azadirachta indica. The structures of 1-4 were elucidated by NMR and MS spectroscopic analyses, and the relative configuration of 1 was determined by single-crystal X-ray crystallography. The compounds isolated in comparatively large amount were evaluated for their antifeedant activities against Plutella xylostella; the antifeedant rate of 10 was 90.6% and the corrected mortality of 8 was 79.2%.

1. The serotoninergic system is known to be involved in the control of multiple behavioural and physiological functions. The serotonin (5-hydroxtryptamine; 5-HT) transporter (SERT), which controls the synaptic 5-HT concentration through re-uptake of this neurotransmitter into presynaptic terminals, has been a primary therapeutic target for various psychiatric and peripheral disorders. The aim of the present study was to identify the regulatory mechanism(s) of the human SERT (hSERT) in heterologously expressed oocytes. 2. The hSERT cRNA was transcribed in vitro and injected into Xenopus oocytes. The 5-HT-induced transporter currents were measured by voltage clamp. The effects of extracellular sodium or chloride were studied by replacement perfusion with tetramethylammonium-chloride (96 mmol/L) or sodium acetate (96 mmol/L). In addition, to alter the internal calcium concentration, CaCl2 (50 micromol/L) and inositol triphosphate (IP3; 50 micromol/L), with or without EGTA (2.5 mmol/L), were injected into oocytes. The specificity of 5-HT-sensitive currents was determined by the use of the SERT antagonist desipramine and niflumic acid to block background chloride currents. 3. The hSERT-expressing oocytes displayed voltage-dependent, 5-HT-induced currents that increased at negative potentials. Replacing extracellular sodium or chloride significantly decreased the hSERT currents by 89 and 45%, respectively (P < 0.05, n = 7 each). Injection of IP3 or CaCl2 increased the hSERT currents by approximately 65% (P < 0.05; n = 10 each) and the effect of IP3 was abolished by preinjection of EGTA. 4. These results demonstrate that hSERT activity is not only voltage dependent, but is also affected by intracellular calcium and extracellular sodium and chloride.

Cathaya argyrophylla is an endangered conifer restricted to subtropical mountains of China. To study phylogeographical pattern and demographic history of C. argyrophylla, species-wide genetic variation was investigated using sequences of maternally inherited mtDNA and biparentally inherited nuclear DNA. Of 15 populations sampled from all four distinct regions, only three mitotypes were detected at two loci, without single region having a mixed composition (G(ST) = 1). Average nucleotide diversity (theta(ws) = 0.0024; pi(s) = 0.0029) across eight nuclear loci is significantly lower than those found for other conifers (theta(ws) = 0.003 approximately 0.015; pi(s) = 0.002 approximately 0.012) based on estimates of multiple loci. Because of its highest diversity among the eight nuclear loci and evolving neutrally, one locus (2009) was further used for phylogeographical studies and eight haplotypes resulting from 12 polymorphic sites were obtained from 98 individuals. All the four distinct regions had at least four haplotypes, with the Dalou region (DL) having the highest diversity and the Bamian region (BM) the lowest, paralleling the result of the eight nuclear loci. An AMOVA revealed significant proportion of diversity attributable to differences among regions (13.4%) and among populations within regions (8.9%). F(ST) analysis also indicated significantly high differentiation among populations (F(ST) = 0.22) and between regions (F(ST) = 0.12-0.38). Non-overlapping distribution of mitotypes and high genetic differentiation among the distinct geographical groups suggest the existence of at least four separate glacial refugia. Based on network and mismatch distribution analyses, we do not find evidence of long distance dispersal and population expansion in C. argyrophylla. Ex situ conservation and artificial crossing are recommended for the management of this endangered species.

1. Decreasing heart rate during development is known to be the result of parasympathetic nervous system maturation that depresses the pacemaker current (If) by acetylcholine (ACh). However, a direct effect of ACh on If has been ruled out and the involvement of other secondary messengers, such as cAMP, was verified in previous studies. Therefore, we hypothesized that reduced basal cAMP production in sinoatrial (SA) nodal cells may contribute to the slowing of heart rate after birth. 2. The electrocardiogram and heart rate variability (HRV) were documented and measured in vivo and in vitro (in isolated perfused Langendorff preparations) for rabbits aged 2, 4, 6, 8 and 12 weeks. Sinoatrial node action potential (AP) recording and perforated patch-clamp analyses were used to investigate the spontaneous depolarization rate and pacemaker If currents. Concentrations of cAMP in SA nodal tissues were determined by radioimmunoassay. Relative expression of adenylate cyclases (ADCY1, 5) and phosphodiesterases (PDE1A, 4A and 8A) were quantified by real-time reverse transcription-polymerase chain reaction. 3. Significantly reduced heart rate, but unchanged HRV, was observed in perfused hearts in the older age groups, accompanied with a slowed phase 4 spontaneous depolarization rate (90.5 +/- 4.7 vs 49.6 +/- 2.6 mV/s for 2 week vs 4 week hearts, respectively; n = 5; P < 0.05), a negative shift of the If threshold potential (-45.5 +/- 3.0 vs -51.1 +/- 6.0 mV for 2 week vs 4 week hearts, respectively; n = 9; P < 0.05) and decreasing basal levels of SA nodal cAMP (0.31 +/- 0.05 vs 0.025 +/- 0.002 micromol/L for 2 week vs 4 week hearts, respectively; n = 6; P < 0.05). Gene expression levels of PDE1A, 4A and 8A were increased in the 12 week group compared with the 2 week group 1.5-, 2- and 1.8-fold, respectively (P < 0.05), with little change in ADCY1 and 5. 4. These data suggest that, in addition to autonomic innervation, slowing of heart rate during postnatal maturation can be attributed to a negative shift of the If activation caused by diminished baseline cAMP content in SA nodal cells.

IL-15 induces proliferation, inhibits apoptosis and increases IL-4 production in murine mast cells. There is evidence that these activities are mediated via the uncharacterised receptor, IL-15R-X, rather than the classical three-chain IL-15 receptor. Effects of IL-15 on important aspects of mast cell biology, such as migration and degranulation, are unknown. We report that IL-15 induces migration of murine and human mast cells in a dose-dependent and biphasic manner, with peaks of migration occurring at approximately 10(-15) and approximately 10(-9) M. The potency of the response was similar to that induced by other well-established mast cell chemoattractants. Competition assays performed with murine and human mast cells indicate that both peaks of migration are due to chemotaxis. Pre-treatment of cells with pertussis toxin (PTX), a guanine nucleotide-binding regulatory protein (G-protein) inhibitor, resulted in complete inhibition of murine mast cell migration at approximately 10(-15) M IL-15, and human mast cell migration at approximately 10(-15) and approximately 10(-9) M. This demonstrates that murine and human mast cells express a PTX-sensitive receptor, activated in response to IL-15. Additionally, IL-15 did not induce degranulation in murine mast cells. Locally-produced IL-15 may contribute to mast cell recruitment during inflammatory responses, thereby acting as a linking cytokine between innate and adaptive arms of the immune system.

The tight skin (Tsk) mouse develops many pathological changes seen in human scleroderma, such as increased collagen content and mast cell density. Although associations between mast cell expansion and skin fibrosis have been reported, the mechanisms underlying mast cell accumulation remain unclear. In this study, we have measured the density of skin mast cells in Tsk mice and their normal littermates (pa/pa) of 4-36 weeks of age, and in the skin heterografted between Tsk and pa/pa mice. Cytokines related to mast cell differentiation, proliferation and migration were examined by using RNase protection assays. Skin mast cell density in Tsk mice was significantly increased from 12 weeks of age, compared to that in pa/pa mice. The expression of transforming growth factor-beta1 (TGF-beta1), and to a lesser extent, stem cell factor (SCF) and interleukin-15 (IL-15) mRNA was higher in Tsk mice, compared to that in control mice. Mast cell density was unchanged in Tsk skin grafted onto pa/pa hosts, but dramatically increased in pa/pa skin grafted onto Tsk hosts. This latter mast cell hyperplasia was associated with the increases in mRNA levels of TGF-beta1, SCF and IL-15, whereas little change in cytokine levels was seen in heterografted Tsk skin. These results suggest that locally produced cytokines in Tsk skin influence mast cell accumulation in this animal model of human scleroderma.

Most of our knowledge of the function of the angiotensin type 2 receptor (AT(2)R) has been obtained from transgenic mouse models. The aim of the present study was to investigate the role of the AT(2)R in normotensive Sprague-Dawley (SD) rats by using antisense gene transfer technology to 'knockdown' this specific receptor subtype. A retroviral vector containing full-length AT(2)R antisense cDNA (AT(2)R-AS) was constructed and the effectiveness of the transduction of AT(2)R-AS was studied in vitro. In subsequent in vivo studies, 5-day-old normotensive SD rats received a single intracardiac bolus (25 microl) of AT(2)R-AS viral particles. When animals reached adulthood, direct blood pressure (BP), and both pressor and dipsogenic responses to angiotensin II were investigated. Long-lasting expression of the AT(2)R-AS transcript and a reduction in mRNA and binding of the AT(2)R was observed in vitro. Expression of AT(2)R-AS transcript was maintained for 90 days in heart, kidney, lung and brain, indicating a high degree of transgene transduction in vivo. As adults, systolic BP and the pressor responses to angiotensin were significantly elevated in AT(2)R-AS-treated rats. However, AT(2)R-AS-treated rats displayed significantly reduced dipsogenic responses to both angiotensin and water deprivation. Collectively, these data demonstrate that a single neonatal injection of the retroviral vector containing antisense to the AT(2) receptors in rats results in similar cardiovascular and dipsogenic responses as reported in AT(2)R knockout mice. The actions of the AT(2) receptors appear to be antagonistic to the cardiovascular actions of the AT(1) receptors, whereas AT(1) and AT(2) receptors appear to act synergistically in the regulation of water intake.

SUMMARY Morphological observations and molecular analyses of the north-western Pacific species of the red algal genus Grateloupia (Halymeniaceae) indicate the presence of an entity, which is somewhat similar in gross morphology to G. asiatica Kawaguchi et Wang but is distinguished from the latter species by some morphological features. These include: (i) a somewhat fleshy texture; (ii) wider and much thicker (4.5‐10 mm wide and up to 1300 µ m thick) axes, of which an inner cortex consists of more (6‐9) cells; (iii) generally longer (up to 17 cm), marginal and surface proliferations that are clearly constricted (terete) at bases; and (iv) much elongated, oblong auxiliary cells. Phylogenetic analysis using the ribulose-1,5-bisphosphate carboxylase/ oxygenase ( rbc L) gene of G. asiatica and the alga in question shows them to be distantly related and strongly supports the differentiation of these two entities at the species level. Judging from the literature, this entity is actually Grateloupia subpectinata Holmes, which has been placed into synonymy under G. asiatica [as G. filicina (Lamouroux) C. Agardh] or G. prolongata J. Agardh in previous reports, and therefore the Holmes name is reinstated.

Yonagunia Kawaguchi et Masuda, gen. nov. (Halymeniaceae, Rhodophyta) is proposed to accommodate a new species, Yonagunia tenuifolia Kawaguchi et Masuda and the species currently known as Prionitis formosana (Okamura) Kawaguchi et Nguyen. Based on auxiliary cell ampullar features, Yonagunia is included in the group of genera with the simplest type of ampulla (the Grateloupia type) that comprises Dermocorynus, Grateloupia, Kintokiocolax, Phyllymenia, and Zymurgia. However, Yonagunia differs from these genera in the behavior of cells in the ampullar filaments immediately after diploidization, most cells of the primary and secondary filaments simultaneously dividing to form grape-like clusters of small globular cells that subsequently elongate and produce involucral filaments to laxly surround the maturing carposporophyte. Yonagunia is resolved by our rbcL gene sequence analyses as one of five monophyletic clades within the Halymeniaceae (an Aeodes/Pachymenia, a Polyopes, a Carpopertis/Cryptonemia/Halymenia, a Yonagunia, and a Grateloupia clade) that is positioned as sister to the Grateloupia clade. Carpogonial branch apparatuses are identified as a potential taxonomic significance on the same level as auxiliary cell ampullae.

An imbalance in helper T-cell type 1 (Th1) and type 2 (Th2) cytokines is suggested to play an important role in the pathogenesis of acute graft-versus-host disease (aGVHD). The aim of this study was to investigate the cytokine bias acquired by T cells after transplantation and its possible influence on relapse of original malignancy. Cytokine levels by peripheral CD4 and CD8 T cells were tested at various pre- and post-transplant time points with fluorescein isothiocyanate-based intracellular cytokine assay after short-term in vitro mitogenic stimulation (phorbol myristate acetate + ionomycin). In both CD4+ and CD8+ cells, interferon (IFN)-γ-producing cell populations increased, indicating a shift to a Th1 cytokine profile with aGVHD. IFN-γ-producing T cells was significantly lower in patients who experienced relapse of original disease compared to those who showed no signs of relapse and compared to normal controls. Our studies demonstrate that aGVHD correlates with a Th1 bias and that Th1 response may potentiate an effective immune surveillance. Am. J. Hematol. 75:78–83, 2004. © 2004 Wiley-Liss, Inc.

Morphological observations and molecular analyses of the red alga Grateloupia filicina (Halymeniaceae) from two geographically distant regions, eastern Asia (Japan and northern China) in the northwestern Pacific and Italy in the Mediterranean, reveal the presence of two distinct entities. Morphologically, the eastern Asian entity differs substantially from the Italian entity in the following ways: 1) thin and soft thalli with wider axes, 2) denser medullary filaments, 3) scattered reproductive structures over the entire thallus, and 4) a mature auxiliary cell that is oval and slightly larger than other ampullary cells. Phylogenetic analysis based on the ribulose-1,5-bisphosphate carboxylase/oxygenase gene (rbcL) sequences revealed that the eastern Asian and Italian entities are phylogenetically far apart, strongly supporting the differentiation of these two entities at the species level. The eastern Asian entity is therefore described as a new species, Grateloupia asiatica. This species can be distinguished from most known species of Grateloupia that have widely flattened thalli by its compressed to narrowly flattened axes with numerous pinnate proliferations and from a few species with similar thalli by a particular combination of features, including a gelatinous texture, mostly simple and narrower axes, a thinner cortex, and the absence of catenate proliferations.

BackgroundCervical cancer is the second most common cancer among women worldwide. However, information about miRNAs in cervical cancer remains limited. ObjectiveTo examine miR-146a and miR-218 expr...

Supramolecular photochemistry is concerned with systems where non-covalent interactions become significant or dominate in determining the chemistry of guest/host systems. The supramolecular photochemistry of phenyl phenylacetates, photocycloaddition of diaryl compounds and the photooxidation of alkenes included in zeolites and Nafion membranes is the subject of this report.

Blockade of central and peripheral MR in rats post MI attenuates sympathetic hyperactivity and ameliorates LV remodeling. The mechanisms involved are not yet clear. We studied mRNA expression of MR...