Molecular phylogeny of Rhacophoridae (Anura): A framework of taxonomic reassignment of species within the genera Aquixalus, Chiromantis, Rhacophorus, and Philautus

Phylogenetic relationships among representative species of the family Rhacophoridae were investigated based on 2904bp of sequences from both mitochondrial (12S rRNA, 16S rRNA, the complete t-RNA for valine), and nuclear (tyrosinase, rhodopsin) genes. Maximum parsimony, maximum likelihood, and Bayesian analyses were employed to reconstruct the phylogenetic trees. This analysis, combined with previous phylogenetic studies, serves as a framework for future work in rhacophorid systematics. The monophyly of Rhacophorus is strongly confirmed except for the species R.hainanus, which is the sister taxon to A.odontotarsus. The non-monophyly of the newly designated genus Aquixalus by Delorme et al. [Delorme, M., Dubois, A., Grosjean, S., Ohler, A., 2005. Une nouvelle classification générique et subgénérique de la tribu des Philautini (Amphibia, Anura, Ranidae, Rhacophorinae). Bull. Mens. Soc. Linn. Lyon 74, 165-171] is further confirmed. Aquixalus (Aquixalus) forms a well-supported monophyletic group within Kurixalus, whereas, Aquixalus (Gracixalus) is more closely related to species of Rhacophorus, Polypedates, and Chiromantis. Philautus as currently understood, does not form a monophyletic group. Philautus (Kirtixalus) is the sister group to the clade comprising Kurixalus and Aquixalus (Aquixalus), and more remotely related to Philautus (Philautus). Chiromantisromeri does not cluster with species of Chiromantis, and forms a basal clade to all rhacophorids save Buergeria. We propose some taxonomic changes that reflect these findings, but further revision should await more detailed studies, which include combined morphological and molecular analyses, with greater species sampling.     

αCT1 peptide sensitizes glioma cells to temozolomide in a glioblastoma organoid platform

Glioblastoma (GBM) is the most common form of brain cancer. Even with aggressive treatment, tumor recurrence is almost universal and patient prognosis is poor because many GBM cell subpopulations, especially the mesenchymal and glioma stem cell populations, are resistant to temozolomide (TMZ), the most commonly used chemotherapeutic in GBM. For this reason, there is an urgent need for the development of new therapies that can more effectively treat GBM. Several recent studies have indicated that high expression of connexin 43 (Cx43) in GBM is associated with poor patient outcomes. It has been hypothesized that inhibition of the Cx43 hemichannels could prevent TMZ efflux and sensitize otherwise resistance cells to the treatment. In this study, we use a three-dimensional organoid model of GBM to demonstrate that combinatorial treatment with TMZ and αCT1, a Cx43 mimetic peptide, significantly improves treatment efficacy in certain populations of GBM. Confocal imaging was used to visualize changes in Cx43 expression in response to combinatorial treatment. These results indicate that Cx43 inhibition should be pursued further as an improved treatment for GBM.     

Characterization of the Denitrification-Associated Phosphorus Uptake Properties of “Candidatus Accumulibacter phosphatis” Clades in Sludge Subjected to Enhanced Biological Phosphorus Removal

To characterize the denitrifying phosphorus (P) uptake properties of “Candidatus Accumulibacter phosphatis,” a sequencing batch reactor (SBR) was operated with acetate. The SBR operation was gradually acclimated from anaerobic-oxic (AO) to anaerobic-anoxic-oxic (A2O) conditions by stepwise increases of nitrate concentration and the anoxic time. The communities of “Ca. Accumulibacter” and associated bacteria at the initial (AO) and final (A2O) stages were compared using 16S rRNA and polyphosphate kinase genes and using fluorescence in situ hybridization (FISH). The acclimation process led to a clear shift in the relative abundances of recognized “Ca. Accumulibacter” subpopulations from clades IIA > IA > IIF to clades IIC > IA > IIF, as well as to increases in the abundance of other associated bacteria (Dechloromonas [from 1.2% to 19.2%] and “Candidatus Competibacter phosphatis” [from 16.4% to 20.0%]), while the overall “Ca. Accumulibacter” abundance decreased (from 55.1% to 29.2%). A series of batch experiments combined with FISH/microautoradiography (MAR) analyses was performed to characterize the denitrifying P uptake properties of the “Ca. Accumulibacter” clades. In FISH/MAR experiments using slightly diluted sludge (∼0.5 g/liter), all “Ca. Accumulibacter” clades successfully took up phosphorus in the presence of nitrate. However, the “Ca. Accumulibacter” clades showed no P uptake in the presence of nitrate when the sludge was highly diluted (∼0.005 g/liter); under these conditions, reduction of nitrate to nitrite did not occur, whereas P uptake by “Ca. Accumulibacter” clades occurred when nitrite was added. These results suggest that the “Ca. Accumulibacter” cells lack nitrate reduction capabilities and that P uptake by “Ca. Accumulibacter” is dependent upon nitrite generated by associated nitrate-reducing bacteria such as Dechloromonas and “Ca. Competibacter.”     

ProSAAS processing in mouse brain and pituitary

ProSAAS is a newly discovered protein with a neuroendocrine distribution generally similar to that of prohormone convertase 1 (PC1), a peptide-processing endopeptidase. Several proSAAS-derived peptides were previously identified in the brain and pituitary of the Cpe(fat)/Cpe(fat) mouse based on the accumulation of C-terminally extended peptides due to the absence of enzymatically active carboxypeptidase E, a peptide-processing exopeptidase. In the present study, antisera against different regions of proSAAS were used to develop radioimmunoassays and examine the processing profile of proSAAS in wild type and Cpe(fat)/Cpe(fat) mouse tissues following gel filtration and reverse phase high performance liquid chromatography. In wild type mouse brain and pituitary, the majority of proSAAS is processed into smaller peptides. These proSAAS-derived peptides elute from the reverse-phase column in the same positions as synthetic peptides that correspond to little SAAS, PEN, and big LEN. Mass spectrometry revealed the presence of peptides with the expected molecular masses of little SAAS and big LEN in the fractions containing immunoreactive peptides. The processing of proSAAS is slightly impaired in Cpe(fat)/Cpe(fat) mice, relative to wild-type mice, leading to the accumulation of partially processed peptides. One of these peptides, the C-terminally extended form of PEN, is known to inhibit PC1 activity and this could account for the reduction in enzymatically active PC1 seen in Cpe(fat)/Cpe(fat) mice. The observation that little SAAS and big LEN are the major forms of these peptides produced in mouse brain and pituitary raises the possibility that these peptides function as neurotransmitters or hormones.     

Examination of the rate of peptide biosynthesis in neuroendocrine cell lines using a stable isotopic label and mass spectrometry

The biosynthesis of neuroendocrine peptides is typically examined by following the rate of appearance of a radioactive amino acid into mature forms of peptides. In the present study, we labeled cell lines with L-leucine containing 10 deuterium residues (d(10)-Leu) and used mass spectrometry to measure the biosynthetic rate of gamma-lipotropin in the AtT-20 cell line and insulin in the INS-1 cell line. After 3 h of labeling, both peptides show detectable levels of the d-labeled form in the cells and media. The relative levels of the d-labeled forms are greater in the media than in the cells, consistent with previous studies that found that newly synthesized peptides are secreted at a higher rate than older peptides under basal conditions. When AtT-20 cells were stimulated with KCl or forskolin, the ratio of d- to H-labeled gamma-lipotropin in the medium decreased, suggesting that the older peptide was in a compartment that could be released upon the appropriate stimulation. Overexpression of proSAAS in AtT-20 cells reduced the ratio of d- to H-labeled gamma-lipotropin, consistent with the proposed role of proSAAS as an endogenous inhibitor of prohormone convertase-1. Labeling with d10-Leu was also used to test whether altering the pH of the secretory pathway with chloroquine affected the rate of peptide biosynthesis. In AtT-20 cells, 30 microm chloroquine for 3 or 6 h significantly reduced the rate of formation of gamma-lipotropin in both cells and media. Similarly, INS-1 cells treated with 10, 30, or 60 microm chloroquine for 6 h showed a significant decrease in the rate of formation of insulin in both cells and media. These results are consistent with the acidic pH optima for peptide processing enzymes. Stable isotopic labeling with d10-Leu provides a sensitive method to examine the rate of peptide formation in neuroendocrine cell lines.     

The population of Odonata (dragonflies) in small tropical rivers with reference to asynchronous growth patterns

The odonate larval communities in three small rivers in Penang Island were studied. More species of dragonflies were found in the Botanical Garden and Titi Teras rivers (13 and 11 respectively) of relatively similar environmental parameters. Fewer (nine) dragonfly species were collected from the Youth Park River which has a lower dissolved oxygen (DO) and a higher biological oxygen demand (BOD), conductivity and turbidity. A mixture of sand, gravel and pebble substrate of Botanical Garden River with dense growth of submerged Hydrilla, grasses and Cladias (Araceae) provided suitable habitats for the dragonflies. The sandy substrate and relatively fast flowing water of Titi Teras River was highly preferred by gomphids. In the Youth Park River, the small community of dragonfly larvae was dominated by tolerant Pseudagrion rubriceps, P. microcephalum, Orthetrum chrysis and Crocothemis servilia. Based on the larval instar distribution of Ictinogomphus decoratus and O. chrysis, very asynchronous populations of these dragonflies occurred in each river. Young larvae were continuously introduced into the populations resulting in undulating growth rate curves. The growth rates of these two species were higher in the Titi Teras River when compared to those in other rivers. Density-dependent mortality, asynchronous cannibalism and fish predation could play important roles in regulating the larval dragonfly population in these rivers.     

Pierce's Disease of Grapevines in Taiwan: Isolation,Cultivation and Pathogenicity of Xylella fastidiosa

Characteristic symptoms of Pierce's disease (PD) in grapevines (Vitis vinifera L.) were observed in 2002 in the major grape production fields of central Taiwan. Disease severity in vineyards varied, and all investigated grape cultivars were affected. Diseased tissues were collected from fields for subsequent isolation and characterization of the causal agent of the disease (Xylella fastidiosa). Koch's postulates were fulfilled by artificially inoculating two purified PD bacteria to grape cultivars Kyoho, Honey Red and Golden Muscat. The inoculated plants developed typical leaf‐scorching symptoms, and similar disease severity developed in the three cultivars from which the bacterium was readily re‐isolated, proving that the leaf scorch of grapevines in Taiwan is caused by the fastidious X. fastidiosa. This confirmed PD of grapevines is also the first report from the Asian Continent. Phylogenetic analyses were performed by comparing the 16S rRNA gene and 16S‐23S rRNA internal transcribed spacer region (16S‐23S ITS) of 12 PD strains from Taiwan with the sequences of 13 X. fastidiosa strains from different hosts and different geographical areas. Results showed that the PD strains of Taiwan were closely related to the American X. fastidiosa grape strains but not to the pear strains of Taiwan, suggesting that the X. fastidiosa grape and pear strains of Taiwan may have evolved independently from each other.     

肠道菌群与呼吸道感染患者免疫球蛋白的关系及其肠内营养的疗效

目的分析肠道菌群紊乱对呼吸道感染患者免疫球蛋白水平的影响,探讨肠内营养对此类患者的疗效。方法选取2016年2月至2018年12月在我院接受治疗的116例呼吸道感染患者作为观察组,进一步将其分为非菌群失调组(50例)和菌群失调组(66例),选取同期60例健康体检者作为对照组,比较各组对象肠道菌群失调情况和血清免疫球蛋白水平,同时对菌群失调患者在常规治疗基础上联合应用肠内营养治疗,观察其治疗效果。结果 (1)观察组患者肠道乳杆菌、双歧杆菌数量及双歧杆菌/大肠埃希菌比值(B/E)显著低于对照组,而肠球菌、大肠埃希菌数量显著高于对照组(均P0.05);观察组患者血清IgM、IgA、IgG水平均显著低于对照组(均P0.05);菌群失调组患者血清IgM、IgA、IgG水平均显著低于非菌群失调组(均P0.05)。(2)观察组患者肠道乳杆菌、双歧杆菌数量及B/E值与血清IgM、IgA、IgG水平呈显著正相关(均P0.05),而大肠埃希菌和肠球菌数量与之呈显著负相关(均P0.05)。(3)菌群失调组患者血清ALB、PA水平随治疗时间的延长而逐渐升高,CRP、PCT水平逐渐降低,IgM、IgA、IgG水平逐渐升高,乳杆菌、双歧杆菌数量和B/E值逐渐增加,大肠埃希菌和肠球菌数量逐渐减少(均P0.05)。结论呼吸道感染患者存在明显的肠道菌群失调情况,其与患者免疫球蛋白水平的降低有关,这可能是引发呼吸道感染的主要危险因素。肠内营养支持疗法能有效改善患者肠道菌群失调,降低血清炎性因子水平,增强机体免疫力,提高患者营养状况及治疗效果。     

Chitosan-modified poly(acrylonitrile-co-acrylic acid) nanofibrous membranes for the immobilization of concanavalin A

Lectin affinity membranes have been receiving much attention for the separation and detection of various glycoconjugates. In this work, we present a simple and efficient method for the preparation of lectin affinity nanofibrous membranes. Chitosan-modified poly(acrylonitrile-co-acrylic acid) (PANCAA) nanofibrous membranes were first prepared by a coupling reaction between the primary amino groups of chitosan and the carboxyl groups of PANCAA electrospun membranes. Surface characterizations by attenuated total reflectance Fourier transform infrared spectroscopy (FT-IR/ATR), X-ray photoelectron spectroscopy (XPS) and field-emission scanning electron microscopy (FESEM) confirm the chemical and morphological changes of the studied nanofibrous membranes. Fluorescence-labeled concanavalin A (FL-Con A) was then immobilized on these membranes via noncovalent binding. Analyses by fluorescence spectrophotometer (FS) and confocal laser scanning microscopy (CLSM) reveal that the immobilization of Con A onto the modified nanofibrous membranes has been successfully achieved on the basis of the electrostatic interaction and the specific recognition between Con A and chitosan. The results show that the amount of adsorbed FL-Con A increases dramatically with the increasing coupling degree of chitosan (CDC) on the nanofibrous membrane. Moreover, Con A immobilized on the chitosan-modified nanofibrous membranes (CMNMs) can remain relatively stable at pH 5.3. Therefore, it is believed that this work may provide a new kind of material for affinity application.