Aging and comprehensive molecular profiling in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aging-related and heterogeneous hematopoietic malignancy. In this study, a total of 1,474 newly diagnosed AML patients with RNA sequencing data were enrolled, and targeted or whole exome sequencing data were obtained in 94% cases. The correlation of aging-related factors including age and clonal hematopoiesis (CH), gender, and genomic/transcriptomic profiles (gene fusions, genetic mutations, and gene expression networks or pathways) was systematically analyzed. Overall, AML patients aged 60 y and older showed an apparently dismal prognosis. Alongside age, the frequency of gene fusions defined in the World Health Organization classification decreased, while the positive rate of gene mutations, especially CH-related ones, increased. Additionally, the number of genetic mutations was higher in gene fusion-negative (GF-) patients than those with GF. Based on the status of CH- and myelodysplastic syndromes (MDS)-related mutations, three mutant subgroups were identified among the GF- AML cohort, namely, CH-AML, CH-MDS-AML, and other GF- AML. Notably, CH-MDS-AML demonstrated a predominance of elderly and male cases, cytopenia, and significantly adverse clinical outcomes. Besides, gene expression networks including HOXA/B, platelet factors, and inflammatory responses were most striking features associated with aging and poor prognosis in AML. Our work has thus unraveled the intricate regulatory circuitry of interactions among different age, gender, and molecular groups of AML.

Laparoscopic Sigmoid Vaginoplasty for the Treatment of Mayer-Rokitansky-Kuster-Hauser Syndrome in a Single Center: 20 years' Experience.

INTRODUCTION AND HYPOTHESIS: We investigate the feasibility, safety, and clinical therapeutic effect of laparoscopic sigmoid vaginoplasty in women with Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome. METHODS: We performed a retrospective case review cohort study of 56 patients with MRKHs undergoing laparoscopic sigmoid vaginoplasty in Wuhan Union Hospital between 2000 and 2020, and all patients were followed up. RESULTS: The median operating time was 165 min (120-420 min). The median hospital stay was 10 days (rang 7-15 days). A functional neovagina was created 11-15 cm in length and two fingers in breadth in all patients. No introitus stenosis was observed. No intra- or post-operative complications occurred. Two patients were lost to follow-up after 3 months of outpatient visits. Six patients had no intercourse and were required to wear a vaginal mold occasionally. None of the patients had complained of local irritation or dyspareunia. Patients who had post-surgery sexual intercourse were satisfied with their sexual life and the mean total Female Sexual Function Index (FSFI) score was 25.17 ± 0.63. The cosmetic results were excellent. CONCLUSIONS: The laparoscopic sigmoid vaginoplasty can achieve the goal of making a functional neovagina. The main advantage of this surgical technique is that it is minimally invasive and that there are fewer complications post-operation. It is an acceptable procedure for patients with MRKH syndrome.

Inherent hepatocytic heterogeneity determines expression and retention of edited F9 alleles post-AAV/CRISPR infusion.

Infusing CRISPR/donor-loaded adeno-associated viral vectors (AAV/CRISPR) could enable in vivo hepatic gene editing to remedy hemophilia B (HB) with inherited deficiency of clotting factor IX (FIX). Yet, current regimens focus on correcting HB with simple mutations in the coding region of the F9, overlooking those carrying complicated mutations involving the regulatory region. Moreover, a possible adverse effect of treatment-related inflammation remains unaddressed. Here we report that a single DNA cutting-mediated long-range replacement restored the FIX-encoding function of a mutant F9 (mF9) carrying both regulatory and coding defects in a severe mouse HB model, wherein incorporation of a synthetic Alb enhancer/promoter-mimic (P2) ensured FIX elevation to clinically meaningful levels. Through single-cell RNA sequencing (scRNA-seq) of liver tissues, we revealed that a subclinical hepatic inflammation post-AAV/CRISPR administration regulated the vulnerability of the edited mF9-harboring host cells to cytotoxic T lymphocytes (CTLs) and the P2 activity in a hepatocytic subset-dependent manner via modulating specific sets of liver-enriched transcription factors (LETFs). Collectively, our study establishes an AAV/CRISPR-mediated gene-editing protocol applicable to complicated monogenetic disorders, underscoring the potentiality of improving therapeutic benefits through managing inflammation.

Integrating a phenotypic screening with a structural simplification strategy to identify 4-phenoxy-quinoline derivatives to potently disrupt the mitotic localization of Aurora kinase B.

We combined a mechanism-informed phenotypic screening (MIPS) assay with a structural simplification strategy to guide the discovery of compounds that disrupt the localization of the mitotic regulator, Aurora kinase B (AURKB), rather than inhibiting its catalytic activity. An initial hit 4-(4-methylthiophen-2-yl)-N-(4-(quinolin-4-yloxy)phenyl)phthalazin-1-amine was identified after screening an in-house library of small molecules and phenocopied the loss of function mutations in AURKB without inhibiting its catalytic activity. We isolated this hit compound activity to its 4-phenoxy-quinoline moiety. The fragment was further optimized into a class of new chemical entities that potently disrupt the mitotic localization of AURKB at low nanomolar concentrations and consequently elicit severe growth inhibition in diverse human cancer cell lines. A lead compound, N-(3-methoxy-5-(6-methoxyquinolin-4-yl)oxy)phenyl)acetamide possessed desirable pharmacokinetic properties such as AUC0-∞: 227.15 [ng∙h/mL/(mg/kg)]; Cmax: 3378.52 ng/mL T1/2: 3.52 h; and F%: 42 % and produced the AURKB-inhibitory phenotypes in a mouse xenograft model. A lead compound is a powerful tool for interrogating the regulation of AURKB and has the potential to be further developed as a first-in-class oncology therapeutic.

A novel mutation in human EMD gene and mitochondrial dysfunction in emerin knockdown cardiomyocytes.

Emerin is an inner nuclear envelope protein encoded by the EMD gene, mutations in which cause Emery-Dreifuss muscular dystrophy type 1 (EDMD1). Cardiac involvement has become a major threat to patients with EDMD1; however, the cardiovascular phenotype spectrums of emerinopathy and the mechanisms by which emerin regulates cardiac pathophysiology remain unclear. Here, we identified a novel nonsense mutation (c.C57G, p.Y19X) in the EMD gene in a Han Chinese family through high-throughput sequencing. Two family members were found to have EDMD1 with muscle weakness and cardiac arrhythmia. Mechanistically, we first discovered that knockdown of emerin in HL-1 or H9C2 cardiomyocytes lead to impaired mitochondrial oxidative phosphorylation capacity with downregulation of electron transport chain complex I and IV and upregulation of complex III and V. Moreover, loss of emerin in HL-1 cells resulted in collapsed mitochondrial membrane potential, altered mitochondrial networks and downregulated multiple factors in RNA and protein level, such as PGC1α, DRP1, MFF, MFN2, which are involved in regulation of mitochondrial biogenesis, fission and fusion. Our findings suggest that targeting mitochondrial bioenergetics might be an effective strategy against cardiac disorders caused by EMD mutations.

Whole-genome sequencing identifies potential candidate genes for reproductive traits in pigs.

Reproductive performance is a complex quantitative trait, that is determined by multiple genes, regulatory pathways and environmental factors. A list of major genes with large effect have been detected, although multiple QTLs are identified. To identify candidate genes for pig prolificacy, whole genome variants from five high- and five low-prolificacy Yorkshire sows were collected using whole-genome resequencing. A total of 13,955,609 SNPs and 2,666,366 indels were detected across the genome. Common differential SNPs and indels were identified between the two groups of sows. Genes encoding components of the TGF-beta signaling pathway were enriched with the variations, including BMP5, BMP6, BMP7, ACVR1, INHBA, ZFYVE9, TGFBR2, DCN, ID4, BAMBI, and ACVR2A. Several differential variants within these genes related to reproductive traits were identified to be associated with litter size. A comparison of selective regions and published QTL data suggests that NEDD9, SLC39A11, SNCA, and UNC5D are candidate genes for reproduction traits.

Oestrogen receptors and hypoxia inducible factor 1 alpha expression in abdominal wall endometriosis.

RESEARCH QUESTION: What are the protein levels and localization of oestrogen receptors (including ERa, ERb and G protein-coupled oestrogen receptor [GPER]) and hypoxia-inducible factor-1alpha (HIF-1a) in normal control endometrium (COEM) and ectopic endometrium from abdominal wall endometriosis (AWE). DESIGN: AWE (n = 20) were obtained during surgery; COEM (n = 40) were collected by curettage. All tissues were obtained during the proliferative or secretory phase. Formalin-fixed paraffin-embedded tissues were used for immunohistochemical study for oestrogen receptors and HIF-1a proteins. RESULT(S): The expression of oestrogen receptors and HIF-1a in AWE differed from that in the corresponding menstrual cycle phase of COEM. Compared with COEM, ERa and HIF-1a were decreased whereas ERb and GPER were increased in AWE. The greatest difference was in GPER, with increased protein expression in both the cytoplasm and nucleus of endometrial epithelial and stromal cells, as well as a distinct change in localization from cytoplasmic expression to nuclear and cytoplasmic expression, compared with COEM. CONCLUSIONS: Our data suggest that the expression changes of oestrogen receptors and HIF-1a, especially GPER, are associated with AWE, which may provide new clues to understanding the cause of endometriosis.

Economic and clinical burdens and associated health disparities in HIV/AIDS management using big data: potentially inappropriate use and deprescribing of benzodiazepines.

This study aimed to examine factors, healthcare utilization, and medical costs associated with potentially inappropriate use of benzodiazepines in persons living with HIV (PLWH). We used big data from Medicare claims in 2017. Potentially inappropriate use of benzodiazepines was defined as having any benzodiazepine claims in individuals 65+ years or having benzodiazepine claims for more than four weeks in individuals 18-64 years. Logistic regressions, zero-inflated negative binomial regressions, and generalized linear models were used. This study included 1,211 PLWH and 235 (19.41%) had potentially inappropriate use of benzodiazepines. PLWH who were 65+ years (OR: 0.56; 95% CI: 0.33, 0.96), non-Hispanic blacks (OR: 0.29; 95% CI: 0.20, 0.41), or Hispanics (OR: 0.55; 95% CI: 0.35, 0.88) were less likely to use benzodiazepines inappropriately. PLWH who had potentially inappropriate use of benzodiazepines had more inpatient (IRR: 1.46; 95% CI: 1.10, 1.94), outpatient (IRR: 1.14; 95% CI 1.02, 1.28), and emergency room (IRR: 1.32; 95% CI: 1.03, 1.68) visits. Potentially inappropriate use of benzodiazepines was associated with higher total (ß: 0.16; 95% CI: 0.07, 0.25), Medicare (ß: 0.18; 95% CI: 0.07, 0.28), and out-of-pocket (ß: 0.21; 95% CI: 0.05, 0.36) costs. This study provides real-world evidence to support deprescribing benzodiazepines in PLWH.

The circRNA circ_0000291 acts as a sponge of microRNA 326 to regulate E26 transformation-specific sequence-1 expression and promote breast cancer progression.

Accumulating studies authenticate that circular RNAs (circRNAs) are involved in the progression of cancers. However, their role in breast cancer (BC) remains largely unknown. In this study, real-time polymerase chain reaction was employed to detect the circ_0000291 and miR-326 expressions in BC tissues and cells. The correlation between the expression level of circ_0000291 and clinicopathological parameters of BC patients was analyzed. Western blot was used to detect the expression of E26 transformation-specific sequence-1 (ETS1), E-cadherin, N-cadherin and Vimentin in BC cells. Cell proliferation was measured using the cell counting kit-8 assay and the bromodeoxyuridine assay. Cell migration and invasion were detected by Transwell assay. The interactions between circ_0000291 and miR-326, miR-326 and ETS1 were verified using bioinformatics prediction, the dual-luciferase reporter gene assay or/and RNA binding protein immunoprecipitation assay. We demonstrated that circ_0000291 was significantly upregulated in BC, and its high expression was positively correlated with T stage and local lymph node metastasis. Functional assays validated that circ_0000291 promoted BC cell proliferation, migration and invasion. The mechanism studies indicated that circ_0000291 could decoy miR-326 and in turn upregulate the expression of ETS1. In conclusion, circ_0000291 is the novel oncogenic circRNA and promotes BC progression via modulating the miR-326/ETS1 axis.

Quantitative analysis of pH value in soil using laser-induced breakdown spectroscopy coupled with a multivariate regression method.

The quantitative analyses of pH value in soil have been performed using laser-induced breakdown spectroscopy (LIBS) technology. The aim of this work was to obtain a reliable and accurate method for rapid detection of pH value in soil. Seventy-four samples were used as a calibration set, and 24 samples were used as a prediction set. To eliminate the matrix effect, the multivariate models of partial least-squares regression (PLSR) and least-squares support vector regression (LS-SVR) were used to construct the models. The intensities of nine emission lines of C, Ca, Na, O, H, Mg, Al, and Fe elements were used to fit the models. For the PLSR model, the correlation coefficient was 0.897 and 0.906 for the calibration and prediction set, respectively. Furthermore, the analysis accuracy was improved effectively by the LS-SVR method, and the correlation coefficients for calibration and prediction set were improved to 0.991 and 0.987. The prediction mean absolute error was pH 0.1 units, and the root mean square error of the prediction was only 0.079. The results indicated that the LIBS technique coupled with LS-SVR could be a reliable and accurate method for determining pH value in soil.

LncRNA MEG3 inhibits trophoblast invasion and trophoblast-mediated VSMC loss in uterine spiral artery remodeling.

Extravillous trophoblasts (EVTs) migrate into uterine decidua and induce vascular smooth muscle cell (VSMC) loss through mechanisms thought to involve migration and apoptosis, achieving complete spiral artery remodeling. Long noncoding RNA maternally expressed gene 3 (MEG3) can regulate diverse cellular processes, such as proliferation and migration, and has been discovered highly expressed in human placenta tissues. However, little is known about the role of MEG3 in modulating EVT functions and EVT-induced VSMC loss. In this study, we first examined the location of MEG3 in human first-trimester placenta by in situ hybridization. Then, exogenous upregulation of MEG3 in HTR-8/SVneo cells was performed to investigate the effects of MEG3 on EVT motility and EVT capacity to displace VSMCs. Meanwhile, the molecules mediating EVT-induced VSMC loss, such as tumor necrosis factor-α (TNF-α), Fas ligand (FasL), and tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) were detected at transcriptional and translational levels. Finally, VSMCs were cocultured with MEG3-upregulated HTR-8/SVneo to explore the role of MEG3 on EVT-mediated VSMC migration and apoptosis. Results showed that MEG3 was expressed in trophoblasts in placental villi and decidua, and MEG3 enhancement inhibited HTR-8/SVneo migration and invasion. Meanwhile, the displacement of VSMCs by HTR-8/SVneo and the expression of TNF-α, FasL and TRAIL in HTR-8/SVneo were reduced following MEG3 overexpression in HTR-8/SVneo. Furthermore, HTR-8/SVneo with MEG3 upregulation impaired VSMC migration and apoptosis. The PI3K/Akt pathway, which is possibly downstream, was inactivated in MEG3-upregulated HTR-8/SVneo. These findings suggest that MEG3 might be a negative regulator of spiral artery remodeling via suppressing EVT invasion and EVT-mediated VSMC loss.

Theoretical Study on the Reaction of Nitric Oxide with Propargyl Radical.

The reaction of nitric oxide (NO) with propargyl radical (C3H3) was investigated at the CCSD(T)/cc-pVTZ//B3LYP/6-311++G(df, pd) level of theory. The rate coefficients of the system were determined by using the RRKM-CVT method with Eckart tunneling correction over a temperature range of 200-800 K and a pressure range of 1.0 × 10-4 to 10.0 bar. Eight channels proceeding via the barrierless formation of excited intermediate ONCH2CCH or CH2CCHNO at the first step were explored. Three favorable channels (i.e., channels producing adduct of ONCH2CCH and CH2CCHNO and products of HCN and H2CCO) were confirmed. The rate coefficients of channels producing adduct of ONCH2CCH and CH2CCHNO are comparable and have weak negative temperature dependence and positive pressure dependence. Channel producing products of HCN and H2CCO is more important at low pressure and high temperature and less important after pressure greater than 1.0 × 10-2 bar (with a branching ratio less than 6% at 0.1 bar).

The Scientific Information Model of Chang'e-4 Visible and Near-IR Imaging Spectrometer (VNIS) and In-Flight Verification.

The Chang'e-4 (CE-4) lunar rover, equipped with a visible and near-IR imaging spectrometer (VNIS) based on acousto-optic tunable filter spectroscopy, was launched to the far side of the moon on December 8, 2018. The detection band of the VNIS ranges from 0.45 to 2.4 µm. Because of the weak reflection of infrared radiation from the lunar surface, a static electronic phase-locked acquisition method is adopted in the infrared channel for signal amplification. In this paper, full-link simulations and modeling are conducted on the infrared channel information flow of the instrument. The signal characteristics of the VNIS are analyzed in depth, and the signal to noise ratio (SNR) prediction and laboratory verification are presented. On 4 January 2019, the VNIS started working successfully and acquired high-resolution spectrum data of the far side of the moon for the first time. Through analysis we have found that the SNR ratio is in line with our predictions, and the data obtained by VNIS in orbit are consistent with the information model proposed in this paper.

Prognostic value of long non-coding RNA BLACAT1 in patients with papillary thyroid carcinoma.

BACKGROUND: Long noncoding RNAs (lncRNAs) have been reported to have potential diagnostic and prognostic values for numerous cancers. However, the plasma expression of lncRNA BLACAT1 and its clinical value in patients with papillary thyroid cancer (PTC) remain unknown. METHODS: The expression profile of BLACAT1 in 87 PTC patients (case group) and 36 patients with nodular goiter (control group) were investigated by qRT-PCR. The Kaplan-Meier method was used for RFS curves, and the univariate survival differences were analyzed by the log-rank test. RESULTS: BLACAT1 expression was downregulated in the plasma of case group compared with control group (P = 0.003). We also found that low plasma BLACAT1 expression was correlated with lymph node metastasis (LNM) (P < 0.001). Multivariate analysis showed that BLACAT1 was an independent risk factor for lymph node metastasis and gender (P < 0.05). The area under the ROC curve for BLACAT1 was 0.825 in LNM prediction (P < 0.001). CONCLUSION: The present study demonstrated that BLACAT1 could act as a possible suppressor gene in PTC and may serve as a potential biomarker for prognosis prediction of PTC.

Support effects on adsorption and catalytic activation of O2 in single atom iron catalysts with graphene-based substrates.

The adsorption and catalytic activation of O2 on single atom iron catalysts with graphene-based substrates were investigated systematically by density functional theory calculation. It is found that the support effects of graphene-based substrates have a significant influence on the stability of the single atom catalysts, the adsorption configuration, the electron transfer mechanism, the adsorption energy and the energy barrier. The differences in the stable adsorption configuration of O2 on single atom iron catalysts with different graphene-based substrates can be well understood by the symmetrical matching principle based on frontier molecular orbital analysis. There are two different mechanisms of electron transfer, in which the Fe atom acts as the electron donor in single vacancy graphene-based substrates while the Fe atom mainly acts as the bridge for electron transfer in double vacancy graphene-based substrates. The Fermi softness and work function are good descriptors of the adsorption energy and they can well reveal the relationship between electronic structure and adsorption energy. This single atom iron catalyst with single vacancy graphene modified by three nitrogen atoms is a promising non-noble metal single atom catalyst in the adsorption and catalytic oxidation of O2. Furthermore, the findings can lay the foundation for the further study of graphene-based support effects and provide a guideline for the development and design of new non-noble-metal single atom catalysts.

Biomechanical efficacy of AP, PA lag screws and posterior plating for fixation of posterior malleolar fractures: a three dimensional finite element study.

BACKGROUND: Clinically there are different fixation methods used for fixation of the posterior malleolar fractures (PMF), but the best treatment modality is still not clear. Few studies have concentrated on this issue, least of all using a biomechanical comparison. The purpose of this study was to carry out a computational comparative biomechanics of three different commonly used fixation constructs for the fixation of PMF by finite element analysis (FEA). METHODS: Computed tomography (CT) images were used to reconstruct three dimensional (3D) model of the tibia. Computer aided design (CAD) software was used to design 3D models of PMF. Finally, 3D models of PMF fixed with two antero-posterior (AP) lag screws, two postero-anterior (PA) lag screws and posterior plate were simulated through computational processing. Simulated loads of 500 N, 1000 N and 1500 N were applied to the PMF and proximal ends of the models were fixed in all degrees of freedom. Output results representing the model von Mises stress, relative fracture micro-motion and vertical displacement of the fracture fragment were analyzed. RESULTS: The mean vertical displacement value in the posterior plate group (0.52 mm) was lower than AP (0.68 mm) and PA (0.69 mm) lag groups. Statistically significant low amount of the relative micro-motion (P < 0.05) was observed in the posterior plate group. CONCLUSIONS: It was concluded that the posterior plate is biomechanically the most stable fixation method for fixation of PMF.

Optical design, laboratory test, and calibration of airborne long wave infrared imaging spectrometer.

We discuss and evaluate a long wave infrared imaging spectrometer in terms of its opto-mechanical design and analysis, alignment, testing, and calibration. The instrument is a practical airborne sensor achieving high spectral resolution and sensitive noise equivalent delta temperature. The instrument operates in the 8 to 12.5 µm spectral region with 28.85 nm spectral sampling, 1 mrad instantaneous field of view, and >40° cross track field. The instrument comprises three uniform sub-modules with identical design parameters and performances. The sub-module design is based on a refractive foreoptics feeding an all-reflective spectrometer. The optical form of the spectrometer is a double-pass reflective triplet with a flat grating, which has a fast f/2 and high optical throughput. Cryogenic optics of 100 K is implemented only for the spectrometer. Assembly and thermal deformation and focusing adjustment design are particularly considered for this low temperature. All the mirrors of the spectrometer are opto-mechanical-integrated designed and manufactured by single-point diamond turning technology. We consider the center sub-module as an example, and we present its laboratory testing results and calibration; the results indicate the instrument's potential value in airborne sensing.

Impact of Soot on NOx Adsorption over Cu-Modified Hydrotalcite-Derived Lean NOx Trap Catalyst.

The impact of soot on NOx adsorption was studied over a Cu-modified hydrotalcite-derived lean NOx trap catalyst in a NO + O2 atmosphere. Powder X-ray diffraction, scanning electron microscopy, Raman scattering spectroscopy, and X-ray photoelectron spectroscopy were used to characterize the surface properties of the pure catalyst and the soot/catalyst mixture. The adsorbed NOx species on the samples were evaluated by in situ diffuse reflectance Fourier transform spectroscopy. The soot coverage decreases the available adsorption sites on the surface of the catalyst, and a portion of active oxygen species are consumed by the soot oxidation during He pretreatment process. The NOx adsorption on two catalyst samples simultaneously undergoes two routes: the "nitrite route" and the "nitrate route". The "nitrite route" is more dominant than the "nitrate route". During NOx adsorption, the soot oxidation weakens the NO oxidation to NO2, and the released CO2 competes with NOx on the adsorption sites. Moreover, the temperature-programmed desorption tests indicate that the presence of soot reduces the NOx storage capacity of the catalyst and shifts the NO desorption peak to the lower temperature range by 50 °C.

Vaccination with toxofilin DNA in combination with an alum-monophosphoryl lipid A mixed adjuvant induces significant protective immunity against Toxoplasma gondii.

BACKGROUND: A widely prevalent disease, toxoplasmosis poses serious health threats to both humans and animals; therefore, development of an ideal DNA vaccine against Toxoplasma gondii is needed eagerly. The purpose of the present study is to assess the protective efficacy of a DNA vaccine encoding the T. gondii toxofilin gene (pEGFP-toxofilin). In addition, toxofilin DNA vaccine combined with the individual adjuvants, alum or monophosphoryl lipid A (MPLA), or a mixture of alum-MPLA adjuvant were screened for their ability to enhance antibody responses. METHODS: Using bioinformatics, we analyzed the gene and amino acid sequences of the toxofilin protein, recognizing and identifying several potential linear B and T helper (Th)-1 cell epitopes. BALB/c mice were immunized three times with either toxofilin DNA vaccine alone or in combination with the adjuvants such as alum, MPLA or an alum-MPLA mixture. The systemic immune response was evaluated by cytokine, the percentage of CD4 (+) and CD8 (+) T cells and specific antibody measurement. Two weeks after the last immunization, protective efficacy was evaluated by challenging intraperitoneally with 1 × 104 tachyzoites of T. gondii or intragastrically with 20 cysts of T. gondii PRU strain. RESULTS: All experimentally immunized mice developed strong humoral and cellular immune responses compared with the control groups. Moreover, by comparison with the non-adjuvant toxofilin DNA vaccine group, adding alum adjuvant to toxofilin DNA vaccine resulted in an increase in humoral response and a skewed Th2 response. However, the MPLA adjuvant with toxofilin DNA vaccine induced significantly enhanced humoral and Th1-biased immune responses. Importantly, the co-administration of alum-MPLA adjuvant in combination with the toxofilin DNA vaccine shifted the Th2 immune response to a Th1 response compared with the alum-toxofilin group, and elicited the strongest humoral and Th1 responses among all the groups. At the same time, a longer survival time and less cyst amounts against T. gondii infection were also observed in the alum-MPLA-toxofilin group in comparison with single or no adjuvant groups. CONCLUSIONS: Toxoplasma gondii toxofilin is a promising vaccine candidate that warrants further development. Co-administration of the alum-MPLA adjuvant mixture with DNA vaccine could effectively enhance immunogenicity and strongly skew the cellular immune response towards a Th1 phenotype.

Streptococcusmarmotae sp. nov., isolated from the respiratory tract of Marmota himalayana.

Five strains of a Gram-stain-positive, catalase-negative, α-haemolytic, coccus-shaped chain-forming organism were isolated separately from the lower respiratory tracts of five animals of Marmota himalayana in the endemic area of plague, the Qinghai-Tibet Plateau, China. Based on their morphological characteristics, biochemical features and molecular phylogenetic studies, the strains were placed as representing a new member of the genus Streptococcus. Comparative 16S rRNA gene sequence studies indicated that strain HTS5T shared 96.5, 96.2 and 96.0 % similarity with Streptococcus gallinaceus CCUG 42692T, Streptococcus parasanguinis ATCC 15912T and Streptococcus suis ATCC 43765T, respectively. Sequence analysis of its rpoB and sodA genes showed that strain HTS5T was most closely related to Streptococcus cuniculi CCUG 65085T with 9.2 and 10.9 % interspecies divergence, respectively. The whole genome phylogenetic tree based on 339 core genes of 65 Streptococcus genomes confirmed that HTS5T belongs to a distinct lineage that is well separated from recognized species of the genus Streptococcus. In silico DNA-DNA hybridization using 65 available genomes from GenBank showed that HTS5T displayed less than 70 % DNA-DNA relatedness with the other 65 species of the genus Streptococcus deposited in the GenBank database. The genome of strain HTS5T (2 322 791 bp) contained 2377 genes and had a G+C content of 41.6 mol%. Therefore, the five strains are considered to represent a novel species of the genus Streptococcus for which the name Streptococcusmarmotae sp. nov. is proposed. The type strain is HTS5T (=DSM 101995T=CGMCC 1.15534T).