Quantification and Genotyping of Torque Teno Virus at a Wastewater Treatment Plant in Japan

Torque teno virus (TTV) DNA was quantitatively detected in influent and effluent samples collected from a wastewater treatment plant in Japan, with the highest concentration being 4.8 × 10⁴ copies/liter. Genogroup-specific nested PCR demonstrated that TTV of genogroup 3 was the most abundant in wastewater among the five genogroups tested.     

Impact of Colic Pain as a Significant Factor for Predicting the Stone Free Rate of One-Session Shock Wave Lithotripsy for Treating Ureter Stones: A Bayesian Logistic Regression Model Analysis

Purpose

This study was conducted to evaluate colic pain as a prognostic pretreatment factor that can influence ureter stone clearance and to estimate the probability of stone-free status in shock wave lithotripsy (SWL) patients with a ureter stone.

Materials and Methods

We retrospectively reviewed the medical records of 1,418 patients who underwent their first SWL between 2005 and 2013. Among these patients, 551 had a ureter stone measuring 4–20 mm and were thus eligible for our analyses. The colic pain as the chief complaint was defined as either subjective flank pain during history taking and physical examination. Propensity-scores for established for colic pain was calculated for each patient using multivariate logistic regression based upon the following covariates: age, maximal stone length (MSL), and mean stone density (MSD). Each factor was evaluated as predictor for stone-free status by Bayesian and non-Bayesian logistic regression model.

Results

After propensity-score matching, 217 patients were extracted in each group from the total patient cohort. There were no statistical differences in variables used in propensity- score matching. One-session success and stone-free rate were also higher in the painful group (73.7% and 71.0%, respectively) than in the painless group (63.6% and 60.4%, respectively). In multivariate non-Bayesian and Bayesian logistic regression models, a painful stone, shorter MSL, and lower MSD were significant factors for one-session stone-free status in patients who underwent SWL.

Conclusions

Colic pain in patients with ureter calculi was one of the significant predicting factors including MSL and MSD for one-session stone-free status of SWL.     

Shrinkage Pre-Test Estimator of the Intercept Parameter for a Regression Model with Multivariate Student-t Errors

In the presence of an uncertain prior information about the value of the slope parameter, the estimation of the intercept parameter of a simple regression model with a multivariate Student-t error distribution is investigated. The unrestricted, restricted and shrinkage preliminary test maximum likelihood estimators are defined. The expressions for the bias and the mean square error of the three estimators are provided and the relative efficiences are analyzed. A maximin criterion is established, and graphs are constructed for an arbitrary number of degrees of freedom (D.F.) as well as sample sizes. A criterion to select optimal significance level is also discussed.     

Characterization of a Planctomycetal Organelle: a Novel Bacterial Microcompartment for the Aerobic Degradation of Plant Saccharides

Bacterial microcompartments (BMCs) are organelles that encapsulate functionally linked enzymes within a proteinaceous shell. The prototypical example is the carboxysome, which functions in carbon fixation in cyanobacteria and some chemoautotrophs. It is increasingly apparent that diverse heterotrophic bacteria contain BMCs that are involved in catabolic reactions, and many of the BMCs are predicted to have novel functions. However, most of these putative organelles have not been experimentally characterized. In this study, we sought to discover the function of a conserved BMC gene cluster encoded in the majority of the sequenced planctomycete genomes. This BMC is especially notable for its relatively simple genetic composition, its remote phylogenetic position relative to characterized BMCs, and its apparent exclusivity to the enigmatic Verrucomicrobia and Planctomycetes. Members of the phylum Planctomycetes are known for their morphological dissimilarity to the rest of the bacterial domain: internal membranes, reproduction by budding, and lack of peptidoglycan. As a result, they are ripe for many discoveries, but currently the tools for genetic studies are very limited. We expanded the genetic toolbox for the planctomycetes and generated directed gene knockouts of BMC-related genes in Planctomyces limnophilus. A metabolic activity screen revealed that BMC gene products are involved in the degradation of a number of plant and algal cell wall sugars. Among these sugars, we confirmed that BMCs are formed and required for growth on l-fucose and l-rhamnose. Our results shed light on the functional diversity of BMCs as well as their ecological role in the planctomycetes, which are commonly associated with algae.     

Estimating Treatment Effects for Recurrent Events in the Presence of Rescue Medications: An Application to the Immune Thrombocytopenia Study

In many clinical studies, patients may experience the same type of event of interest repeatedly over time. However, the assessment of treatment effects is often complicated by the rescue medication uses due to ethical reasons. For example, in the motivating trial in studying the Immune Thrombocytopenia (ITP), when the interest lies in evaluating the treatment benefit of investigational product (IP) on reducing patient’s repeated bleeding, rescue medication such as platelet transfusions may be allowed to raise platelet counts. Both the intention-to-treat analysis and treating the intermediate rescue medication as covariate tend to attenuate the treatment benefit, and the estimates can be biased if interpreted as causal. In this paper, we propose a general causal framework when intermediate rescue medications are informative. We adopt the inverse weighted estimation approach to estimate the treatment effect, where weights are constructed to reflect time-dependent medication use probabilities. The proposed estimators are shown to be asymptotically normal and are demonstrated to perform well in small-sample simulation studies. The application to the ITP studies reveals a stronger benefit of using IP in reducing bleeding.     

Indirect Estimates of Total Fertility Rate Using Child Woman/Ratio: A Comparison with the Bogue-Palmore Method

Indirect estimation methodologies of the total fertility rate (TFR) have a long history within demography and have provided important techniques applied demographers can use when data is sparse or lacking. However new methodologies for approximating the total fertility rate have not been proposed in nearly 30 years. This study presents a novel method for indirectly approximating the total fertility rate using an algebraic rearrangement of the general fertility rate (GFR) through the known relationship between GFR and TFR. It then compares the proposed method to the well-known Bogue-Palmore method. These methods are compared in 196 countries and include overall errors as well as characteristics of the countries that contribute to fertility behavior. Additionally, these methods were compared geographically to find any geographical patterns. We find this novel method is not only simpler than the Bogue-Palmore method, requiring fewer data inputs, but also has reduced algebraic and absolute errors when compared with the Bogue-Palmore method and specifically outperforms the Bogue-Palmore method in developing countries. We find that our novel method may be useful estimation procedure for demographers.     

Living Side by Side with a Virus: Characterization of Two Novel Plasmids from Thermococcus prieurii,a Host for the Spindle-Shaped Virus TPV1

Microbial cells often serve as an evolutionary battlefield for different types of mobile genetic elements, such as viruses and plasmids. Here, we describe the isolation and characterization of two new archaeal plasmids which share the host with the spindle-shaped Thermococcus prieurii virus 1 (TPV1). The two plasmids, pTP1 and pTP2, were isolated from the hyperthermophilic archaeon Thermococcus prieurii (phylum Euryarchaeota), a resident of a deep-sea hydrothermal vent located at the East Pacific Rise at 2,700-m depth (7°25′24 S, 107°47′66 W). pTP1 (3.1 kb) and pTP2 (2.0 kb) are among the smallest known plasmids of hyperthermophilic archaea, and both are predicted to replicate via the rolling-circle mechanism. The two plasmids and the virus TPV1 do not have a single gene in common and stably propagate in infected cells without any apparent antagonistic effect on each other. The compatibility of the three genetic elements and the high copy number of pTP1 and pTP2 plasmids (50 copies/cell) might be useful for developing new genetic tools for studying hyperthermophilic euryarchaea and their viruses.     

A Novel Imidazole Nucleoside Containing a Diaminodihydro-S-triazine as a Substituent: Inhibitory Activity Against the West Nile Virus NTPase/Helicase

The attempted synthesis of a ring-expanded guanosine (1) containing the imidazo[4,5-e][1,3]diazepine ring system by condensation of 1-(2′-deoxy-β-D-erythropentofuranosyl)-4-ethoxycarbonylimidazole-5-carbaldehyde (2) with guanidine resulted in the formation of an unexpected product, 1-(2′-deoxy-β-D-erythropentofuranosyl)-5-(2,4-diamino-3,6-dihydro-1,3,5-triazin-6-yl)imidazole-4-carboxamide (7). The structure as well as the pathway of formation of 7 was corroborated by isolation of the intermediate, followed by its conversion to the product. Nucleoside 7 showed promising in vitro anti-helicase activity against the West Nile virus NTPase/ helicase with an IC ₅₀ of 3-10 μg/mL.     

Nine-Year Study of the Occurrence of Culturable Viruses in Source Water for Two Drinking Water Treatment Plants and the Influent and Effluent of a Wastewater Treatment Plant in Milwaukee, Wisconsin (August 1994 through July 2003)

Reoviruses, enteroviruses, and adenoviruses were quantified by culture for various ambient waters in the Milwaukee area. From August 1994 through July 2003, the influent and effluent of a local wastewater treatment plant (WWTP) were tested monthly by a modified U.S. Environmental Protection Agency Information Collection Rule (ICR) organic flocculation cell culture procedure for the detection of culturable viruses. Modification of the ICR procedure included using Caco-2, RD, and HEp-2 cells in addition to BGM cells. Lake Michigan source water for two local drinking water treatment plants (DWTPs) was also tested monthly for culturable viruses by passing 200 liters of source water through a filter and culturing a concentrate representing 100 liters of source water. Reoviruses, enteroviruses, and adenoviruses were detected frequently (105 of 107 samples) and, at times, in high concentration in WWTP influent but were detected less frequently (32 of 107 samples) in plant effluent and at much lower concentrations. Eighteen of 204 samples (8.8%) of source waters for the two DWTPs were positive for virus and exclusively positive for reoviruses at relatively low titers. Both enteroviruses and reoviruses were detected in WWTP influent, most frequently during the second half of the year.     

Docking of PRAK/MK5 to the Atypical MAPKs ERK3 and ERK4 Defines a Novel MAPK Interaction Motif

ERK3 and ERK4 are atypical MAPKs in which the canonical TXY motif within the activation loop of the classical MAPKs is replaced by SEG. Both ERK3 and ERK4 bind, translocate, and activate the MAPK-activated protein kinase (MK) 5. The classical MAPKs ERK1/2 and p38 interact with downstream MKs (RSK1–3 and MK2–3, respectively) through conserved clusters of acidic amino acids, which constitute the common docking (CD) domain. In contrast to the classical MAPKs, the interaction between ERK3/4 and MK5 is strictly dependent on phosphorylation of the SEG motif of these kinases. Here we report that the conserved CD domain is dispensable for the interaction of ERK3 and ERK4 with MK5. Using peptide overlay assays, we have defined a novel MK5 interaction motif (FRIEDE) within both ERK4 and ERK3 that is essential for binding to the C-terminal region of MK5. This motif is located within the L16 extension lying C-terminal to the CD domain in ERK3 and ERK4 and a single isoleucine to lysine substitution in FRIEDE totally abrogates binding, activation, and translocation of MK5 by both ERK3 and ERK4. These findings are the first to demonstrate binding of a physiological substrate via this region of the L16 loop in a MAPK. Furthermore, the link between activation loop phosphorylation and accessibility of the FRIEDE interaction motif suggests a switch mechanism for these atypical MAPKs in which the phosphorylation status of the activation loop regulates the ability of both ERK3 and ERK4 to bind to a downstream effector.Mitogen-activated protein kinase (MAPK)² phosphorylation cascades play important roles in the regulation of diverse cellular functions such as cell proliferation, differentiation, migration, and apoptosis (1, 2). A characteristic and conserved feature of this family of signaling pathways is their organization into modules comprising a sequential three-tiered kinase cascade. This contains a MAPK kinase kinase, a MEK, and the MAPK itself. Four such MAPK signaling modules have been described in mammals: ERK1 and ERK2, the c-Jun N-terminal kinases 1–3, the p38 kinases (p38α/β/γ/δ), and ERK5 (3–7). The MAPK kinase kinases phosphorylate and activate the MEKs, which in turn activate the MAPKs by dual phosphorylation on both the threonine and the tyrosine residue of a highly conserved TXY motif in the kinase activation loop. MAPKs are Ser/Thr kinases, which phosphorylate a wide range of substrates with the minimal consensus sequence (S/T)P (2).ERK4 and its close relative ERK3 are regarded as atypical members of the MAPK family. In contrast to the classical MAPKs, ERK3 and ERK4 harbor an SEG motif in the activation loop and thus lack a second phosphoacceptor site. In addition, protein kinases all possess a conserved APE motif located just C-terminal to the phosphoacceptor sites within subdomain VIII, in which the conserved glutamate is important for maintaining the stability of the kinase domain. In both ERK3 and ERK4, this motif is substituted by SPR, and ERK3 and ERK4 are the only two protein kinases in the human genome with an arginine residue in this position (8). Although they display significant sequence homology (44% identity) with ERK1 and ERK2 within their kinase domains, both ERK3 and ERK4 have unique C-terminal extensions, which account for the large differences in size observed between ERK1/2 (∼360 amino acids) and ERK3/ERK4 (721/587 amino acids). Whereas classical MAPKs have been highly conserved throughout evolution, with examples found in both unicellular and multicellular organisms, ERK3 and ERK4 are only present in vertebrates. Finally, in contrast to many of the classical MAPKs, the regulation, substrate specificity, and physiological functions of ERK3 and ERK4 are poorly understood. Although ERK3 and ERK4 are very similar to each other, there are significant differences between them. For instance, whereas ERK4, like most classical MAPKs, is a stable protein, ERK3 is highly unstable and subject to rapid proteosomal degradation. Thus, ERK3 activity may be regulated at the level of cellular abundance, and taken together these features indicate that ERK3 and ERK4 may perform specialized functions and enjoy different modes of regulation when compared with classical MAPKs (9–11).Despite the striking differences between ERK3 and ERK4 and the classical MAPKs, they do share one property with the ERK1/2, p38, and ERK5, namely the ability to interact with a group of downstream Ser/Thr protein kinases, termed MAPK-activated protein kinases (MAPKAPKs or MKs) (12, 13). In the case of ERK3 and ERK4, both proteins interact with, translocate, and activate the MK5 protein kinase. Several studies have drawn attention to the role of specific docking interactions that contribute to both substrate selectivity and regulation in MAPK pathways (14–17). These interactions involve docking domains, which specifically recognize small peptide docking motifs (D motifs) located on functional MAPK partner proteins including downstream substrates, scaffold proteins, as well as positive and negative regulators. The docking domains, although located within the kinase domains, are distinct from the active site. Similarly the D motifs, which these docking domains recognize, are also distinct from the phosphoacceptor sites within protein substrates (18). There are several classes of D motifs. The motifs found in MAPKAP kinases including MK5 have the consensus sequence LX_1–2(K/R)_2–5 where X is any amino acid (12). The corresponding docking domains within the MAPKs have also been characterized (16, 19, 20). The common docking (CD) domain is a cluster of negatively charged amino acids located in the L16 extension directly C-terminal to the kinase domain in the MAPK primary structure. A second domain termed ED (Glu-Asp) also contributes to binding specificity. This latter site is located near the CD domain in the MAPK tertiary structure. Whereas the CD domain is considered commonly important for all docking interactions, the ED site is thought to be important for the determination of specificity (16). Other residues and regions distinct from the ED and CD domains have also been shown to be important for docking.(21–25).This work has so far been largely confined to analysis of the classical MAPKs, and much less is known about the nature of substrate or regulatory docking interactions for the atypical MAPKs. We and others (9, 11, 26) have recently reported that the region encompassing residues 326–340 within both ERK3 and ERK4 is required for their ability to interact with and activate MK5. Furthermore, a truncated mutant of MK5, which lacks the 50 C-terminal residues (MK5 1–423), was unable to bind to ERK4 despite the fact that it retains its D domain. Finally, in contrast to conventional MAPKs, the interaction between ERK3 and ERK4 and MK5 requires activation loop phosphorylation of ERK3 and ERK4 (27, 28). Taken together these observations suggest that the mechanism by which the atypical MAPKs recognize and bind to at least one important class of effector kinases may be distinct to that found in the classical MAPKs such as ERK1/2 and p38.Here we demonstrate that two separate C-terminal regions, encompassing residues 383–393 and 460–465, respectively, are necessary for MK5 to interact with both ERK3 and ERK4. These regions are distinct from the D motif previously identified within MK5, suggesting that binding to ERK3 and ERK4 may be mediated by a different mechanism to that seen in the classical MAPKs. In support of this, the conserved CD domains within ERK3 and ERK4 are shown to be completely dispensable for MK5 interaction. Using peptide overlay assays, we have defined a minimal MK5 interaction motif FRIEDE in ERK4. Furthermore, we demonstrate that a single point mutation (ERK3 I334K or ERK4 I330K) within this FRIEDE motif is sufficient to disrupt the binding of both ERK3 and ERK4 to MK5 and consequently their ability to both translocate and activate MK5. The FRIEDE motif is located within the L16 extension C-terminal to the CD domain in both ERK3 and ERK4. Interestingly, molecular modeling of the corresponding region in ERK2 suggests that it undergoes a significant conformational change as a result of activation loop phosphorylation, making this part of the L16 extension more accessible (29). We propose that the FRIEDE motif represents a novel MAPK interaction motif, the function of which is linked to activation loop phosphorylation and MAPK activation.     

A Model for Seed Transmission of a Plant Virus: Genetic and Structural Analyses of Pea Embryo Invasion by Pea Seed-Borne Mosaic Virus

Pea seed-borne mosaic virus (PSbMV), a seed-transmitted virus in pea and other legumes, invades pea embryos early in development. This process is controlled by maternal genes and, in a cultivar that shows no seed transmission, is prevented through the action of multiple host genes segregating as quantitative trait loci. These genes control the ability of PSbMV to spread into and/or multiply in the nonvascular testa tissues, thereby preventing the virus from crossing the boundary between the maternal and progeny tissues. Immunocytochemical and in situ hybridization studies suggested that the virus uses the embryonic suspensor as the route for the direct invasion of the embryo. The programmed degeneration of the suspensor during embryo development may provide a transient window for embryo invasion by the virus and could explain the inverse relationship between the age of the mother plant for virus infection and the extent of virus seed transmission.     

Auditory-Motor Mapping Training: Comparing the Effects of a Novel Speech Treatment to a Control Treatment for Minimally Verbal Children with Autism

This study compared Auditory-Motor Mapping Training (AMMT), an intonation-based treatment for facilitating spoken language in minimally verbal children with autism spectrum disorder (ASD), to a matched control treatment, Speech Repetition Therapy (SRT). 23 minimally verbal children with ASD (20 male, mean age 6;5) received at least 25 sessions of AMMT. Seven (all male) were matched on age and verbal ability to seven participants (five male) who received SRT. Outcome measures were Percent Syllables Approximated, Percent Consonants Correct (of 86), and Percent Vowels Correct (of 61) produced on two sets of 15 bisyllabic stimuli. All subjects were assessed on these measures several times at baseline and after 10, 15, 20, and 25 sessions. The post-25 session assessment timepoint, common to all participants, was compared to Best Baseline performance. Overall, after 25 sessions, AMMT participants increased by 19.4% Syllables Approximated, 13.8% Consonants Correct, and19.1% Vowels Correct, compared to Best Baseline. In the matched AMMT-SRT group, after 25 sessions, AMMT participants produced 29.0% more Syllables Approximated (SRT 3.6%);17.9% more Consonants Correct (SRT 0.5); and 17.6% more Vowels Correct (SRT 0.8%). Chi-square tests showed that significantly more AMMT than SRT participants in both the overall and matched groups improved significantly in number of Syllables Approximated per stimulus and number of Consonants Correct per stimulus. Pre-treatment ability to imitate phonemes, but not chronological age or baseline performance on outcome measures, was significantly correlated with amount of improvement after 25 sessions. Intonation-based therapy may offer a promising new interventional approach for teaching spoken language to minimally verbal children with ASD.     

Multivariate logistic regression for predicting total culturable virus presence at the intake of a potable-water treatment plant: novel application of the atypical coliform/total coliform ratio

Predicting the presence of enteric viruses in surface waters is a complex modeling problem. Multiple water quality parameters that indicate the presence of human fecal material, the load of fecal material, and the amount of time fecal material has been in the environment are needed. This paper presents the results of a multiyear study of raw-water quality at the inlet of a potable-water plant that related 17 physical, chemical, and biological indices to the presence of enteric viruses as indicated by cytopathic changes in cell cultures. It was found that several simple, multivariate logistic regression models that could reliably identify observations of the presence or absence of total culturable virus could be fitted. The best models developed combined a fecal age indicator (the atypical coliform [AC]/total coliform [TC] ratio), the detectable presence of a human-associated sterol (epicoprostanol) to indicate the fecal source, and one of several fecal load indicators (the levels of Giardia species cysts, coliform bacteria, and coprostanol). The best fit to the data was found when the AC/TC ratio, the presence of epicoprostanol, and the density of fecal coliform bacteria were input into a simple, multivariate logistic regression equation, resulting in 84.5% and 78.6% accuracies for the identification of the presence and absence of total culturable virus, respectively. The AC/TC ratio was the most influential input variable in all of the models generated, but producing the best prediction required additional input related to the fecal source and the fecal load. The potential for replacing microbial indicators of fecal load with levels of coprostanol was proposed and evaluated by multivariate logistic regression modeling for the presence and absence of virus.     

First Experimental Evidence for the Presence of a CRISPR Toxin in Sulfolobus

Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (cas) genes constitute the adaptive immune system in bacteria and archaea. Although the CRISPR-Cas systems have been hypothesized to encode potential toxins, no experimental data supporting the hypothesis are available in the literature. In this work, we provide the first experimental evidence for the presence of a toxin gene in the type I-A CRISPR system of hyperthermophilic archaeon Sulfolobus. csa5, under the control of its native promoter in a shuttle vector, could not be transformed into CRISPR-deficient mutant Sulfolobus solfataricus Sens1, demonstrating a strong toxicity in the cells. A single-amino-acid mutation destroying the intersubunit bridge of Csa5 attenuated the toxicity, indicative of the importance of Csa5 oligomerization for its toxicity. In line with the absence of Csa5 toxicity in S. solfataricus InF1 containing functional CRISPR systems, the expression of csa5 is repressed in InF1 cells. Induced from the arabinose promoter in Sens1 cells, Csa5 oligomers resistant to 1% SDS co-occur with chromosome degradation and cell death, reinforcing the connection between Csa5 oligomerization and its toxicity. Importantly, a rudivirus was shown to induce Csa5 expression and the formation of SDS-resistant Csa5 oligomers in Sulfolobus cells. This demonstrates that the derepression of csa5 and the subsequent Csa5 oligomerization take place in native virus-host systems. Thus, csa5 is likely to act as a suicide gene under certain circumstances to inhibit virus spreading.     

Application of a Hybrid Model for Predicting the Incidence of Tuberculosis in Hubei,China

Background

A prediction model for tuberculosis incidence is needed in China which may be used as a decision-supportive tool for planning health interventions and allocating health resources.

Methods

The autoregressive integrated moving average (ARIMA) model was first constructed with the data of tuberculosis report rate in Hubei Province from Jan 2004 to Dec 2011.The data from Jan 2012 to Jun 2012 were used to validate the model. Then the generalized regression neural network (GRNN)-ARIMA combination model was established based on the constructed ARIMA model. Finally, the fitting and prediction accuracy of the two models was evaluated.

Results

A total of 465,960 cases were reported between Jan 2004 and Dec 2011 in Hubei Province. The report rate of tuberculosis was highest in 2005 (119.932 per 100,000 population) and lowest in 2010 (84.724 per 100,000 population). The time series of tuberculosis report rate show a gradual secular decline and a striking seasonal variation. The ARIMA (2, 1, 0) × (0, 1, 1)₁₂ model was selected from several plausible ARIMA models. The residual mean square error of the GRNN-ARIMA model and ARIMA model were 0.4467 and 0.6521 in training part, and 0.0958 and 0.1133 in validation part, respectively. The mean absolute error and mean absolute percentage error of the hybrid model were also less than the ARIMA model.

Discussion and Conclusions

The gradual decline in tuberculosis report rate may be attributed to the effect of intensive measures on tuberculosis. The striking seasonal variation may have resulted from several factors. We suppose that a delay in the surveillance system may also have contributed to the variation. According to the fitting and prediction accuracy, the hybrid model outperforms the traditional ARIMA model, which may facilitate the allocation of health resources in China.