Molecular detection of Cryptosporidium: an emerging parasite in different water sources of 2010 flood-affected district Nowshera, Pakistan.

Cryptosporidium is a water-borne zoonotic parasite worldwide, usually found in lakes and rivers contaminated with sewage and animal wastes, causing outbreaks of cryptosporidiosis. In this study, 300 water samples were collected from four designated places of flood-affected district Nowshera consist of different water sources to find out the prevalence of Cryptosporidium via polymerase chain reaction (PCR). The overall prevalence of Cryptosporidium was 30.33% (91/300) with more prevalent 44% in drain water and low 5% in bore/tube well water. The prevalence in open well and tap water was recorded 33% and 20%, respectively. The highest prevalence was recorded in summer (June-September). The result of this study ensures enormous contamination of drinking water that requires appropriate treatment, cleaning and filtration to provide safe drinking water. Preventing water-borne disease and proper treatment of water supplies is essential to public health.

Comparative assessment of impact analysis methods applied to large commercial aircraft crash on reinforced concrete containment.

The precise evaluation of the potential damage caused by large commercial aircraft crash into civil structures, especially nuclear power plants (NPPs), has become essential design consideration. In this study, impact of Boeing 767 against rigid wall and outer containment building (reinforced concrete) of an NPP are simulated in ANSYS/LS-DYNA by using both force time history and missile target interaction methods with impact velocities ranging from 100 m/s to 150 m/s. The results show that impact loads, displacements, stresses for concrete and steel reinforcement, and damaged elements are higher in case of force time history method than missile target interaction method, making the former relatively conservative. It is observed that no perforation or scabbing takes place in case of 100 m/s impact speed, thus preventing any potential leakage. With full mass of Boeing 767 and impact velocity slightly above 100 m/s, the outer containment building can prevent local failure modes. At impact velocity higher than 120 m/s, scabbing and perforations are dominant. This concludes that in design and assessment of NPP structures against aircraft loadings, sufficient thickness or consideration of steel plates are essential to account for local failure modes and overall structural integrity. Furthermore, validation and application of detail 3D finite element and material models to full-scale impact analysis have been carried out to expand the existing database. In rigid wall impact analysis, the impact forces and impulses from FE analysis and Riera's method correspond well, which satisfies the recommendations of relevant standards and further ensure the accuracy of results in full-scale impact analysis. The methodology presented in this paper is extremely effective in simulating structural evaluation of full-scale aircraft impact on important facilities such as NPPs.

Integrated analysis of mRNA and miRNA expression profiles reveals muscle growth differences between fast- and slow-growing king ratsnakes (Elaphe carinata).

In some countries, snakes are important protein sources in human diets, and their economic value depends predominantly on their muscle production, including in the king ratsnake (Elaphe carinata). Muscle growth in the king ratsnake clearly differs among individuals. To date, few potential molecular mechanisms underlying these differences in muscle growth and development have been reported. Here, we integrated mRNA and miRNA expression profiles to screen for genes, pathways, and predicted miRNA-mRNA networks associated with muscle growth and development in fast-growing and slow-growing King ratsnakes. Six hundred eight differentially expressed genes (DEGs) were identified, 48 of which were associated with muscle growth. The 37 genes upregulated in fast-growing individuals (FGIs) may be related to the promotion of muscle growth, whereas the 11 upregulated genes in slow-growing individuals (SGIs) may be related to the inhibition of muscle growth. Seven DEGs were enriched in the PI3K-AKT-MTOR signaling pathway, which appears to promote muscle growth in FGIs. Eleven DEGs were enriched in the ubiquitin-proteasome pathway, which appears to inhibit muscle growth in SGIs. It may interpret why muscle growth differences. Furthermore, 698 miRNA were identified, including 125 novel miRNAs. 63 differentially expressed miRNA (DEMs) were screened, and 950 negative miRNA-mRNA interactions with the 63 DEMs and 608 DEGs were predicted. The miRNA-targeted genes were enriched in pathways related to muscle growth, protein synthesis, and protein degradation. Therefore, in addition to the identified DEGs, miRNAs may play important roles in the differential regulation of muscle growth in FGIs and SGIs of the king ratsnake.

Molecular characterization, expression analysis of myostatin gene and its negative regulation by miR-29b-3p in Chinese concave-eared frogs (Odorrana tormota).

The molecular characteristics, expression patterns and functions of the amphibian myostatin (MSTN) gene are unknown. Here, we isolated a full-length Odorrana tormota MSTN cDNA sequence of 1701 bp (Ot-MSTN), containing a putative N-terminal signal peptide, a TGF-ß propeptide domain and an active peptide. Ot-MSTN was expressed in 9 selected tissues examined, and the highest level of expression was in thigh muscle, followed by brain and female gonadal tissue. The expression of Ot-MSTN in multiple O. tormota tissues supported that the activities of MSTN may be not limited to skeletal muscle. Ot-MSTN expression was decreased from stage 31 to stage 40, while the growth rate was increased. The expression of Ot-MSTN in adult male frogs increased with age, indicating that adult male frogs may inhibit the continued hypertrophy of thigh muscle fibers and decrease the growth rate of thigh muscle to ensure muscles do not grow too large. Luciferase reporter assays showed that miR-29b-3p directly targeted the 3'-UTR of Ot-MSTN. miR-29b-3p expression in the thigh muscle of 2 yrs. females who grew faster was significantly lower than that of the slow-growing 2 yrs. male individuals, which showed an opposite trend with Ot-MSTN expression. In addition，miR-29b-3p expression reversed trends of Ot-MSTN expression at different developmental stages in thigh muscle. Therefore, these data indicate that miR-29-3p may negatively regulate the expression of MSTN and regulate thigh muscle growth and development in O. tormota.

Modulation of human Th17 cell responses through complement receptor 3 (CD11 b/CD18) ligation on monocyte-derived dendritic cells.

OBJECTIVE: Apoptotic cell receptors contribute to the induction of tolerance by modulating dendritic cell function following the uptake of apoptotic cells or microparticles. Dendritic cells that have bound or ingested apoptotic cells produce only low amounts of pro-inflammatory cytokines and fail to prime effector T cell responses. Specifically, ligation of the apoptotic cell receptor CR3 (CD11 b/CD18) on human monocyte-derived dendritic cells (moDC) down-modates proinflammatory cytokine secretion, but the consequences for human Th17 cell homeostasis and effector responses remain unknown. Here, we aimed to establish whether CD11b-ligated moDC modulate Th17 cell effector reponses to assess their potential for future use in moDC-based suppressive immunotherapy. METHODS: We generated a bead-based surrogate system to target CD11b on monocyte-derived human dendritic cells and examined the effects of CD11b ligation on Th17-skewing cytokine secretion, priming, expansion and functional plasticity in DC/T cell co-culture systems at the poly- and monoclonal level. RESULTS: We show that Th17 cell expansion within the human memory CD4+ T cell compartment was efficiently constricted by targeting the CD11b receptor on moDC. This tolerogenic capacity was primarily dependent on cytokine skewing. Furthermore, ligation of CD11b on healthy homozygous carriers of the rs11143679 (ITGAM) variant - a strong genetic susceptibility marker for human systemic lupus erythematosus - also down-modulated the secretion of Th17-skewing cytokines. CONCLUSION: Overall, our findings underline the potential of targeted CD11b ligation on human dendritic cells for the engineering of suppressive immunotherapy for Th17-related autoimmune disorders.

Molecular detection of Toxoplasma gondii in water sources of district Nowshehra, Khyber Pakhtunkhwa, Pakistan.

Toxoplasmosis is spread through contamination of water sources and results in morbidity globally. In the current study 300 water samples were processed by polymerase chain reaction (PCR) for detection of Toxoplasma gondii. The overall prevalence in different water sources was 6.6% (17/300). Among different water sources the highest prevalence was recorded in drain water at 7% (7/100), followed by tube well water at 7.5% (3/40) and open well water at 5% (5/100) ,and the lowest was recorded in tap water at 3.33% (2/60). The highest prevalence was recorded in summer. Evidence indicates that cleaning and filtration need to be adopted to avoid the health hazards of waterborne zoonotic parasites.

Active systemic lupus erythematosus is associated with decreased blood conventional dendritic cells.

OBJECTIVE: The aim of the study is to determine the frequency and functionality of blood conventional dendritic cells (cDCs) in relation to disease activity in systemic lupus erythematosus. METHODS: Blood cDCs were enumerated for 34 SLE patients, defined as "active" (SLEDAI ≥ 4) or "inactive" (SLEDAI < 4), 26 RA subjects and 8 healthy subjects by FACS. cDC activation was measured by IL-12p40/70 staining following resiquimod stimulation. RESULTS: The frequency of blood cDCs was significantly lower in active compared to inactive patients, however, with comparable cDC functionality. CONCLUSION: cDC frequency in active SLE is decreased with no perturbation in cDC function, possibly due to enhanced turnover and/or tissue-specific migration.

HIV-1 infection-induced apoptotic microparticles inhibit human DCs via CD44.

Acute HIV-1 infection results in dysregulated immunity, which contributes to poor control of viral infection. DCs are key regulators of both adaptive and innate immune responses needed for controlling HIV-1, and we surmised that factors elicited during acute HIV-1 infection might impede DC function. We derived immature DCs from healthy donor peripheral blood monocytes and treated them with plasma from uninfected control donors and donors with acute HIV-1 infections. We found that the plasma from patients with HIV specifically inhibited DC function. This suppression was mediated by elevated apoptotic microparticles derived from dying cells during acute HIV-1 infection. Apoptotic microparticles bound to and inhibited DCs through the hyaluronate receptor CD44. These data suggest that targeting this CD44-mediated inhibition by apoptotic microparticles could be a novel strategy to potentiate DC activation of HIV-specific immunity.

Lysine trimethylation of retinoic acid receptor-alpha: a novel means to regulate receptor function.

Retinoic acid receptors (RARs) belong to the nuclear receptor superfamily. The mechanism of ligand-dependent activation of RARs is well known. The effect of protein phosphorylation on the activity of RARs has also been demonstrated. However, it is unclear whether other types of modifications exist and if so whether they can affect the activity of RARs. In a mass spectrometric analysis of mouse RARalpha expressed in insect cells, we identified a trimethylation site on Lys(347) in the ligand binding domain. The modification site was verified in mammalian cells, and site-directed mutagenesis studies revealed the functionality of Lys(347) methylation in vivo. Constitutive negative mutants, mimicking hypomethylated RARalpha, were prepared by replacing methylated Lys(347) with either alanine or glutamine. A constitutive positive mutant partially mimicking the hypermethylated RARalpha was generated by replacing the methylated lysine residue with phenylalanine, a bulky hydrophobic amino acid, to introduce a site-specific hydrophobicity similar to that contributed by lysine methylation. Studies of these mutants revealed that trimethylation of Lys(347) of RARalpha facilitated its interactions with cofactors p300/CREB-binding protein-associated factor and receptor-interacting protein 140 as well as its heterodimeric partner retinoid X receptor, suggesting that site-specific hydrophobicity at Lys(347) enhanced molecular interaction of RARalpha with its modulators. This study uncovers the first example of lysine trimethylation on a mammalian non-histone protein that has an important biological consequence. Our finding also provides the evidence for lysine methylation for the family of nuclear receptors for the first time.

Paired values of serum fructosamine and blood glucose for the screening of gestational diabetes mellitus: A retrospective study of 165 Saudi pregnant women.

This study reports the utilization of serum fructosamine and blood glucose for the screening of gestational diabetes mellitus (GDM). Blood samples from 165 pregnant women were analyzed for fasting blood glucose (FBG), random blood glucose (RBG) and serum fructosamine. The actual fructosamine levels were corrected for serum protein (c-Fruct) for more precise presentation. Two cut-off values of FBG (>5.3 mmol/L and >7.0 mmol/L) and RBG (>7.8 mmol/L and >11.0 mmol/L) were used to classify hyperglycemic subjects for subsequent evaluation. The average values±standard deviations for FBG, RBG and cFruct were 5.865±1.95, 7.767±3.21 and 2.387±0.47 mmol/L, respectively. FBG levels were significantly correlated with RBG (Pearson correlation=0.597, P<0.001). Significant correlations were also observed between cFruct and FBG (Pearson correlation=0.673, P<0.001) or RBG (Pearson correlation=0.641, P<0.001). Out of 165 subjects, 24 (14.5%) cases were classified as hyperglycemic on the basis of FBG>7.0 mmol/L or RBG>11.0 mmol/L; use of lower cut-off values resulted higher frequencies of hyperglycemia. Whereas, a combined criteria of FBG>5.3 mmol/L and cFruct >2.5 mmol/L predicted 35 patients as the most probable hyperglycemic as compared to 32 patients identified using the criteria of RBG >7.8 mmol/L and cFruct >2.5 mmol/L. These criteria were associated with 4.8% and 3.6% false-positivity at the expense of 3.6% and 3.0% false-negative outcomes, respectively. The levels of FBG, RBG and cFruct were significantly higher in hyperglycemic groups (irrespective of grouping criteria) as compared to the respective normal groups. In conclusion, these findings clearly indicate that the paired values of cFruct with FBG or RBG could help in filtering high-risk individuals for OGTT and therefore avoiding a unnecessary OGTT.

Ligand-independent orphan receptor TR2 activation by phosphorylation at the DNA-binding domain.

In a previous report we demonstrated protein kinase C (PKC)-mediated phosphorylation of the ligand-binding domain (LBD) of orphan nuclear receptor TR2. In this report, we provide the evidence of PKC-mediated phosphorylation of the DNA-binding domain (DBD) of TR2. Two PKC target sites were predicted within the DBD, at Ser-170 and Ser-185, but only Ser-185 was confirmed by MS. Phosphorylation of DBD facilitated DNA binding of the TR2 receptor and its recruiting of coactivator p300/CBP-associated factor (P/CAF). Ser-185 was required for DNA binding, whereas both Ser-170 and Ser-185 were necessary for receptor interaction with P/CAF. The P/CAF-interacting domain of TR2 was located in its DBD. A double mutant (Ser-170 and Ser-185) of TR2 significantly lowered the activation of its target gene RARbeta2. This study provides the first evidence for ligand-independent activation of TR2 orphan receptor through PTM at the DBD, which enhanced its DNA-binding ability and interaction with coactivator P/CAF.

Protein kinase C-mediated phosphorylation of orphan nuclear receptor TR2: effects on receptor stability and activity.

In vivo metabolic labeling showed that orphan nuclear receptor TR2 could be phosphorylated. Systematic studies were conducted using specific kinases/phosphatase inhibitors to determine the enzymes responsible for TR2 phosphorylation and the effects of TR2 phosphorylation on its protein stability and activation of its target gene. The data showed that protein kinase C (PKC)-mediated phosphorylation enhanced the activating ability of TR2 on target gene RARbeta as well as its stability through protection from proteosome-mediated degradation. Several PKC-mediated potential serine/threonine phosphorylation sites on TR2 protein were predicted from the computer analysis using NetPhos software (http://us.expasy.org) and were commensurate by in vitro phosphorylation of purified TR2 protein using PKC enzyme. Two phosphorylation sites at Ser-461 and Ser-568 were identified by LC-ESI-MS/MS. Point mutations at Ser-568 or Ser-461 were prepared and evaluated for their biological activity. Ser-568, but not Ser-461, mutation significantly reduced PKC-mediated TR2 protein stability and its transcriptional activity.

Regulation of co-repressive activity of and HDAC recruitment to RIP140 by site-specific phosphorylation.

Receptor interacting protein 140 (RIP140) is a versatile transcriptional co-repressor that contains several autonomous repressive domains (RDs). The N-terminal RD acts by recruiting histone deacetylases (HDACs). In a comprehensive proteomic analysis of RIP140 by MS, 11 phosphorylation sites of RIP140 are identified; among them five sites are located in the N-terminal RD including Ser104, Thr202, Thr207, Ser358, and Ser380. The role of phosphorylation of RIP140 in regulating its biological activity and the underlying mechanism are examined using a site-directed mutagenesis approach. Mutations mimicking constitutive phosphorylation or dephosphorylation are introduced. The N-terminal RD phosphorylation, mediated by the mitogen-activated protein kinase (MAPK), enhances its repressive activity through increased recruitment of HDAC. Mutations mimicking constitutive dephosphorylation at Thr202 or Thr207 significantly impair its repressive activity and HDAC recruitment, whereas mutation at Ser358 only slightly affects its HDAC recruitment and the repressive activity. Consistently, mutations mimicking constitutive phosphorylation at either Thr202 or Thr207 convert RIP140 into a more potent repressor, which is less responsive to a disturbance in the MAPK system. Furthermore, constitutive phosphorylation at both Thr202 and Thr207 residues renders RIP140 fully repressive and strongly interacting with HDAC. The activity of this mutant is resistant to the MAPK inhibitor, indicating an essential role for Thr202 and Thr207 in MAPK-mediated modulation of RIP140 function. The study provides insights into the modulation of RIP140 biological activity through a specific cellular signaling pathway that augments phosphorylation at specific residues of RIP140 molecule and alters its cofactor recruitment.

Mapping of phosphorylation sites of nuclear corepressor receptor interacting protein 140 by liquid chromatography-tandem mass spectroscopy.

Receptor interacting protein (RIP140) is a versatile coregulator for many nuclear receptors and transcription factors. Analysis by liqid chromatography tandem mass spectroscopy led to the identification of 11 phosphopeptides from tryptic digests of His6-RIP140 purified from Sf21 insect cells. No phosphopeptides were detected on RIP140 expressed in E. coli in a parallel experiment, suggesting that RIP140 phosphorylation occurred specifically only in eukaryotic cells. The tandem mass spectra of the precursor ions of the phosphopeptides were analyzed to map the exact phosphorylation sites on RIP140. All the phosphopeptides displayed intact phosphate containing y- or b-ion signals along with their beta-eliminated product ions, due to neutral loss of phosphoric acid. Phosphorylation occurred specifically on nine serine and a single threonine residues, including Ser-104, Thr-207, Ser-358, Ser-380, Ser-488, Ser-519, Ser-531, Ser-543, Ser-672, and Ser-1003. No tyrosine phosphorylation was found. These data suggested that the central region of RIP140, one major repressive domain, was extensively modified by phosphorylation. These phosphorylation sites can be the targets in future studies addressing post-translational modification of RIP140 with regards to its biological activities.