Disrupted oogenesis in the frog Xenopus tropicalis after exposure to environmental progestin concentrations

Levonorgestrel is a synthetic progesterone commonly used in pharmaceuticals (e.g., in contraceptives). It is found in sewage treatment plant effluents at concentrations up to 30 ng/L and was recently shown to pose a threat to egg laying in fish. Information on the susceptibility of adult amphibians to progestin toxicity is lacking. The present study aimed to 1) characterize progestogenic effects on the full cycle of oogenesis (egg development) in frogs and 2) determine female amphibians' susceptibility to reproductive impacts from progestogenic compounds in the environment. Sexually mature female Xenopus tropicalis were exposed to levonorgestrel via the surrounding water for 7 days (0, 51, or 307 ng/L) or 28 days (0, 1.3, 18, 160, or 1240 ng/L). Their ovaries were analyzed histologically with respect to frequencies of immature (in early meiotic prophase I), previtellogenic, vitellogenic, mature, and atretic oocytes. The 28-day exposure caused reduced proportions of oocytes at immature, vitellogenic, and mature stages, and increased proportions of previtellogenic oocytes compared with the control. The lowest tested concentration, 1.3 ng/L, increased the proportions of previtellogenic oocytes and reduced the proportions of vitellogenic oocytes, indicating inhibited vitellogenesis. The present study shows that progestin concentrations found in the aquatic environment impaired oogenesis in adult frogs. Our results indicate that progestogenic effects on oocyte development include interrupted germ cell progression into meiosis and inhibited vitellogenesis. Considering the crucial role of oogenesis in female fertility, our results indicate that progestogenic pollutants may pose a threat to reproduction in wild amphibian populations.     

Environmental impacts of hybrid and electric vehicles—a review

Purpose

A literature review is undertaken to understand how well existing studies of the environmental impacts of hybrid and electric vehicles (EV) address the full life cycle of these technologies. Results of studies are synthesized to compare the global warming potential (GWP) of different EV and internal combustion engine vehicle (ICEV) options. Other impacts are compared; however, data availability limits the extent to which this could be accomplished.

Method

We define what should be included in a complete, state-of-the-art environmental assessment of hybrid and electric vehicles considering components and life cycle stages, emission categories, impact categories, and resource use and compare the content of 51 environmental assessments of hybrid and electric vehicles to our definition. Impact assessment results associated with full life cycle inventories (LCI) are compared for GWP as well as emissions of other pollutants. GWP results by life cycle stage and key parameters are extracted and used to perform a meta-analysis quantifying the impacts of vehicle options.

Results

Few studies provide a full LCI for EVs together with assessment of multiple impacts. Research has focused on well to wheel studies comparing fossil fuel and electricity use as the use phase has been seen to dominate the life cycle of vehicles. Only very recently have studies begun to better address production impacts. Apart from batteries, very few studies provide transparent LCIs of other key EV drivetrain components. Estimates of EV energy use in the literature span a wide range, 0.10?C0.24?kWh/km. Similarly, battery and vehicle lifetime plays an important role in results, yet lifetime assumptions range between 150,000?C300,000?km. CO₂ and GWP are the most frequently reported results. Compiled results suggest the GWP of EVs powered by coal electricity falls between small and large conventional vehicles while EVs powered by natural gas or low-carbon energy sources perform better than the most efficient ICEVs. EV results in regions dependant on coal electricity demonstrated a trend toward increased SO _x emissions compared to fuel use by ICEVs.

Conclusions

Moving forward research should focus on providing consensus around a transparent inventory for production of electric vehicles, appropriate electricity grid mix assumptions, the implications of EV adoption on the existing grid, and means of comparing vehicle on the basis of common driving and charging patterns. Although EVs appear to demonstrate decreases in GWP compared to conventional ICEVs, high efficiency ICEVs and grid-independent hybrid electric vehicles perform better than EVs using coal-fired electricity.     

Risks of hormonally active pharmaceuticals to amphibians: a growing concern regarding progestagens

Most amphibians breed in water, including the terrestrial species, and may therefore be exposed to water-borne pharmaceuticals during critical phases of the reproductive cycle, i.e. sex differentiation and gamete maturation. The objectives of this paper were to (i) review available literature regarding adverse effects of hormonally active pharmaceuticals on amphibians, with special reference to environmentally relevant exposure levels and (ii) expand the knowledge on toxicity of progestagens in amphibians by determining effects of norethindrone (NET) and progesterone (P) exposure to 0, 1, 10 or 100 ng l⁻¹ (nominal) on oogenesis in the test species Xenopus tropicalis. Very little information was found on toxicity of environmentally relevant concentrations of pharmaceuticals on amphibians. Research has shown that environmental concentrations (1.8 ng l⁻¹) of the pharmaceutical oestrogen ethinylestradiol (EE₂) cause developmental reproductive toxicity involving impaired spermatogenesis in frogs. Recently, it was found that the progestagen levonorgestrel (LNG) inhibited oogenesis in frogs by interrupting the formation of vitellogenic oocytes at an environmentally relevant concentration (1.3 ng l⁻¹). Results from the present study revealed that 1 ng NET l⁻¹ and 10 ng P l⁻¹ caused reduced proportions of vitellogenic oocytes and increased proportions of previtellogenic oocytes compared with the controls, thereby indicating inhibited vitellogenesis. Hence, the available literature shows that the oestrogen EE₂ and the progestagens LNG, NET and P impair reproductive functions in amphibians at environmentally relevant exposure concentrations. The progestagens are of particular concern given their prevalence, the range of compounds and that several of them (LNG, NET and P) share the same target (oogenesis) at environmental exposure concentrations, indicating a risk for adverse effects on fertility in exposed wild amphibians.     

Evaluation of an Internet-Based Monitoring System for Influenza-Like Illness in Sweden

To complement traditional influenza surveillance with data on disease occurrence not only among care-seeking individuals, the Swedish Institute for Communicable Disease Control (SMI) has tested an Internet-based monitoring system (IMS) with self-recruited volunteers submitting weekly on-line reports about their health in the preceding week, upon weekly reminders. We evaluated IMS acceptability and to which extent participants represented the Swedish population. We also studied the agreement of data on influenza-like illness (ILI) occurrence from IMS with data from a previously evaluated population-based system (PBS) with an actively recruited random sample of the population who spontaneously report disease onsets in real-time via telephone/Internet, and with traditional general practitioner based sentinel and virological influenza surveillance, in the 2011–2012 and 2012–2013 influenza seasons. We assessed acceptability by calculating the participation proportion in an invited IMS-sample and the weekly reporting proportion of enrolled self-recruited IMS participants. We compared distributions of socio-demographic indicators of self-recruited IMS participants to the general Swedish population using chi-square tests. Finally, we assessed the agreement of weekly incidence proportions (%) of ILI in IMS and PBS with cross-correlation analyses. Among 2,511 invited persons, 166 (6.6%) agreed to participate in the IMS. In each season, 2,552 and 2,486 self-recruited persons participated in the IMS respectively. The weekly reporting proportion among self-recruited participants decreased from 87% to 23% (2011–2012) and 82% to 45% (2012–2013). Women, highly educated, and middle-aged persons were overrepresented among self-recruited IMS participants (p<0.01). IMS (invited and self-recruited) and PBS weekly incidence proportions correlated strongest when no lags were applied (r = 0.71 and r = 0.69, p<0.05). This evaluation revealed socio-demographic misrepresentation and limited compliance among the self-recruited IMS participants. Yet, IMS offered a reasonable representation of the temporal ILI pattern in the community overall during the 2011–2012 and 2012–2013 influenza seasons and could be a simple tool for collecting community-based ILI data.     

Highest Vaccine Uptake after School-Based Delivery - A County-Level Evaluation of the Implementation Strategies for HPV Catch-Up Vaccination in Sweden

Background

The Swedish school-based vaccination programme offers HPV vaccine to girls born ≥1999 in 5-6th grade. In 2012, all counties introduced free-of-charge catch-up vaccination campaigns targeting girls born 1993–1998. Varying vaccine uptake in the catch-up group by December 2012 suggested that some implementation strategies were more successful than others. In order to inform future vaccination campaigns, we assessed the impact of different implementation strategies on the county-level catch-up vaccine uptake.

Methods

We conducted an ecological study including all Swedish counties (n = 21), asking regional health offices about the information channels they used and where vaccination of the catch-up target group took place in their counties. The uptake of ≥1 dose by 30 September 2014 was estimated using data from the voluntary national vaccination register. We investigated associations between counties’ catch-up vaccine uptake, information channels and vaccination settings by calculating incidence rate ratios (IRR) and 95% confidence intervals (CI), using negative binomial regression models.

Results

County level catch-up vaccine uptake varied between 49–84%. All counties offered vaccination through primary health care settings. Apart from this eight (34%) also offered the vaccine in some of their schools, four (19%) in all their schools, and two (10%) in other health care centres. The information channels most frequently used were: information at the national on-line health care consulting web-page (100%), letter/invitations (90%), and advertisement (81%). Counties offering vaccination to girls in all schools and counties offering vaccination in some of their schools, reached higher vaccine uptake compared to counties not offering vaccination in any of their schools (all schools adjusted IRR: 1.3, 95% CI: 1.1–1.5, some schools adjusted IRR: 1.2, 95% CI: 1.1–1.3).

Conclusion

Counties offering HPV vaccination to catch-up groups in schools reached the highest vaccine uptake. No information channel explained differences in county-level vaccine uptake. Our findings suggest that catch-up vaccination outside the national vaccination program can reach a high uptake at the population level if it is implemented primarily with an organized delivery (e.g. in schools).     

Autoproteolysis of YscU of Yersinia pseudotuberculosis Is Important for Regulation of Expression and Secretion of Yop Proteins

YscU of Yersinia can be autoproteolysed to generate a 10-kDa C-terminal polypeptide designated YscU_CC. Autoproteolysis occurs at the conserved N↓PTH motif of YscU. The specific in-cis-generated point mutants N263A and P264A were found to be defective in proteolysis. Both mutants expressed and secreted Yop proteins (Yops) in calcium-containing medium (+Ca²⁺ conditions) and calcium-depleted medium (−Ca²⁺ conditions). The level of Yop and LcrV secretion by the N263A mutant was about 20% that of the wild-type strain, but there was no significant difference in the ratio of the different secreted Yops, including LcrV. The N263A mutant secreted LcrQ regardless of the calcium concentration in the medium, corroborating the observation that Yops were expressed and secreted in Ca²⁺-containing medium by the mutant. YscF, the type III secretion system (T3SS) needle protein, was secreted at elevated levels by the mutant compared to the wild type when bacteria were grown under +Ca²⁺ conditions. YscF secretion was induced in the mutant, as well as in the wild type, when the bacteria were incubated under −Ca²⁺ conditions, although the mutant secreted smaller amounts of YscF. The N263A mutant was cytotoxic for HeLa cells, demonstrating that the T3SS-mediated delivery of effectors was functional. We suggest that YscU blocks Yop release and that autoproteolysis is required to relieve this block.The type III secretion system (T3SS) occurs in many gram-negative pathogenic or symbiotic bacteria (6, 16, 19). The T3SS is evolutionarily related to the bacterial flagellum (19, 24), but while the flagellar apparatus is dedicated to bacterial motion, the T3SS specifically allows bacterial targeting of effector proteins across eukaryotic cell membranes into the lumen of the target cell (19). The main function of the effectors is to reprogram the cell to the benefit of the bacterium (28). The two organelles are superficially similar in form and can be divided into two physical substructures; a basal body is connected to a multimeric filamentous protein structure protruding from the bacterial surface. The basal body is embedded in the cell wall and spans from the cytosol to the surface of the bacterium with a cytosolic extension called the C-ring. The proximal center of the basal body is likely involved in the actual export of nonfolded substrates, which are thought to pass through the cell wall through this hollow structure (6, 16, 41). Early and elegant work by Macnab''s group showed that morphogenesis of the flagella is ordered such that first the cell-proximal hook structure is polymerized and then the flagellar filament is assembled on top of the hook structure (43). Thus, there is ordered switching from secretion of hook proteins to flagellin, which was called substrate specificity switching by Macnab et al. (15, 27). Mutants expressing extraordinarily long hooks have been isolated and connected to regulation and determination of hook buildup and subsequent substrate specificity switching (18, 29, 43). A central factor in this process is the integral 42-kDa cytoplasmic membrane protein FlhB, which has four putative transmembrane helices in its N-terminal domain, which is designated FlhB_TM. The hydrophilic C-terminal domain (FlhB_C) is predicted to protrude into the cytosol. In addition, FlhB_C can be further divided into two subdomains, FlhB_CN (amino acids 211 to 269) and FlhB_CC (amino acis 270 to 383), that are connected via a proposed flexible hinge region (27). The hinge region contains a highly conserved NPTH motif, which is found in all T3SSs. Interestingly, FlhB_C is specifically cleaved within this NPTH sequence (N269↓P270) (27). Site-specific mutagenesis of the NPTH site has a significant effect on the substrate switching, and the ability of flhB(N269A) and flhB(P270A) mutants to cleave FlhB is impaired, indicating that autoproteolysis is important (13, 15). Interestingly, the proteolysis is most likely the outcome of an autochemical process rather than an effect of external proteolytic enzymes (13). The FlhB homolog in the Yersinia pseudotuberculosis plasmid-encoded T3SS is the YscU protein, which has been shown to be essential for proper function of the T3SS since a yscU-null mutant is unable to secrete Yop proteins (Yops) into the culture supernatant (1, 21). YscU has been coupled to needle and Yop secretion regulation, as second-site suppressor mutations introduced into YscU_CC restore the yscP-null mutant phenotype. A yscP mutant is unable to exhibit substrate specificity switching and carries excess amounts of the needle protein YscF on the bacterial surface compared to the wild type. (11) Furthermore, YscP has been implicated in regulation of the T3SS needle length as a molecular ruler, where the size and helical content of YscP determine the length of the needle (20, 42). Together, these findings suggest that YscP and YscU interact and that this interaction is important for regulation of needle length, as well as for Yop secretion. As in FlhB, four predicted transmembrane helices followed by a cytoplasmic tail can be identified in YscU (1). In addition, the cytoplasmic part (YscU_C) can be divided into the YscU_CN and YscU_CC subdomains (Fig. ​(Fig.1A).1A). Variants of YscU with a single substitution in the conserved NPTH sequence (N263A) have been found to be unable to generate YscU_CC, suggesting that YscU of Yersinia also is autoproteolysed (21, 33, 38). The T3SS of Y. pseudotuberculosis secretes about 11 proteins, which collectively are called Yops (Yersinia outer proteins). These Yops have different functions during infection. Some are directly involved as effector proteins, attacking host cells to prevent phagocytosis and inflammation, while others have regulatory functions. Although the pathogen is extracellularly located, the Yop effectors are found solely in the cytosol of the target cell, and secretion of Yops occurs only at the zone of contact between the pathogen and the eukaryotic target cell (7, 36). Close contact between the pathogen and the eukaryotic cell also results in elevated expression and secretion of Yops (12, 30). Hence, cell contact induces the substrate switching; therefore, here we studied the connection between YscU autoproteolysis and expression, as well as secretion and translocation of Yops. Previous studies of YscU function were conducted mainly with in trans constructs instead of introduced YscU mutations in cis. Such studies reported loss of T3SS regulation (21). To avoid potential in trans problems, we introduced all mutations in cis with the aim of elucidating the function of YscU in type III secretion (T3S). Our results suggest that YscU autoproteolysis is not an absolute requirement either for Yop/LcrV secretion or for Yop translocation but is important for accurate regulation of Yop expression and secretion.Open in a separate windowFIG. 1.Autoproteolysis of YscU. (A) Schematic diagram of YscU in the bacterial inner membrane. The diagram shows the NPTH motif and the different parts of YscU after autoproteolysis and is the result of a prediction of transmembrane helices in proteins performed at the site http://www.cbs.dtu.dk/services/TMHMM. IM, inner membrane. (B) E. coli expressing C-terminally His-tagged YscU_C was induced with IPTG, which was followed by sonication and solubilization and denaturation of the protein in binding buffer (8 M urea and 10 mM imidazole). The lysate (lane L) was flushed over the Ni column, and the flowthrough (lane FT) was collected. The column was washed five times with binding buffer, and the wash fractions (lanes W1 to W5) were collected. Elution buffer (8 M urea and 300 mM imidazole) was flushed over the column to release proteins bound to the column, resulting in the eluate (lane E). The eluate was diluted 1:30 in 10 mM Tris (pH 7.4) to obtain a urea concentration of 0.2 M and incubated at 21°C overnight. The resulting overnight eluate fraction (lane E/ON) was TCA precipitated and taken up in binding buffer. Samples were analyzed by 15% Tris-Tricine SDS-PAGE. The cleavage of YscU_C-His₆ to YscU_CC-His₆ and YscU_CN was verified by N-terminal sequencing. All fractions were volume corrected. Lane ST contained a protein standard.     

YscP and YscU regulate substrate specificity of the Yersinia type III secretion system

Pathogenic Yersinia species use a type III secretion system to inhibit phagocytosis by eukaryotic cells. At 37 degrees C, the secretion system is assembled, forming a needle-like structure on the bacterial cell surface. Upon eukaryotic cell contact, six effector proteins, called Yops, are translocated into the eukaryotic cell cytosol. Here, we show that a yscP mutant exports an increased amount of the needle component YscF to the bacterial cell surface but is unable to efficiently secrete effector Yops. Mutations in the cytoplasmic domain of the inner membrane protein YscU suppress the yscP phenotype by reducing the level of YscF secretion and increasing the level of Yop secretion. These results suggest that YscP and YscU coordinately regulate the substrate specificity of the Yersinia type III secretion system. Furthermore, we show that YscP and YscU act upstream of the cell contact sensor YopN as well as the inner gatekeeper LcrG in the pathway of substrate export regulation. These results further strengthen the strong evolutionary link between flagellar biosynthesis and type III synthesis.     

A Randomized Clinical Trial of the Immunogenicity of 7-Valent Pneumococcal Conjugate Vaccine Compared to 23-Valent Polysaccharide Vaccine in Frail,Hospitalized Elderly

Background

Elderly people do not mount strong immune responses to vaccines. We compared 23-valent capsular polysaccharide (23vPPV) alone versus 7-valent conjugate (PCV7) vaccine followed by 23vPPV 6 months later in hospitalized elderly.

Methods

Participants were randomized to receive 23vPPV or PCV7-23vPPV. Antibodies against serotypes 3, 4, 6A, 6B, 9V, 14, 18C, 19A, 19F, 23F were measured by enzyme-linked immunosorbent (ELISA) and opsonophagocytic (OPA) assays at baseline, 6 months and 12 months.

Results

Of 312 recruited, between 40% and 72% of subjects had undetectable OPA titres at baseline. After one dose, PCV7 recipients had significantly higher responses to serotypes 9V (both assays) and 23F (OPA only), and 23vPPV recipients had significantly higher responses to serotype 3 (ELISA), 19F and 19A (OPA only). In subjects with undetectable OPA titres at baseline, a proportionately greater rise in OPA titre (P<0.01) was seen for all serotypes after both vaccines. The GMT ratio of OPA was significantly higher at 12 months in the PCV7-23vPPV group for serotypes 6A, 9V, 18C and 23F. OPA titre levels for these serotypes increased moderately after 6 months, whereas immunity waned in the 23vPPV only arm.

Conclusion

We did not show overwhelming benefit of one vaccine over the other. Low baseline immunity does not preclude a robust immune response, reiterating the importance of vaccinating the frail elderly. A schedule of PCV7-23vPPV prevents waning of antibody, suggesting that both vaccines could be useful in the elderly. Follow up studies are needed to determine persistence of immunity.

Trial Registration

The Australian Clinical Trials Registry ACTRN12607000387426     

DotU and VgrG, core components of type VI secretion systems, are essential for Francisella LVS pathogenicity

The Gram-negative bacterium Francisella tularensis causes tularemia, a disease which requires bacterial escape from phagosomes of infected macrophages. Once in the cytosol, the bacterium rapidly multiplies, inhibits activation of the inflammasome and ultimately causes death of the host cell. Of importance for these processes is a 33-kb gene cluster, the Francisella pathogenicity island (FPI), which is believed to encode a type VI secretion system (T6SS). In this study, we analyzed the role of the FPI-encoded proteins VgrG and DotU, which are conserved components of type VI secretion (T6S) clusters. We demonstrate that in F. tularensis LVS, VgrG was shown to form multimers, consistent with its suggested role as a trimeric membrane puncturing device in T6SSs, while the inner membrane protein DotU was shown to stabilize PdpB/IcmF, another T6SS core component. Upon infection of J774 cells, both ΔvgrG and ΔdotU mutants did not escape from phagosomes, and subsequently, did not multiply or cause cytopathogenicity. They also showed impaired activation of the inflammasome and marked attenuation in the mouse model. Moreover, all of the DotU-dependent functions investigated here required the presence of three residues that are essentially conserved among all DotU homologues. Thus, in agreement with a core function in T6S clusters, VgrG and DotU play key roles for modulation of the intracellular host response as well as for the virulence of F. tularensis.     

A single-step competitive binding assay for mapping of single DNA molecules

Optical mapping of genomic DNA is of relevance for a plethora of applications such as scaffolding for sequencing and detection of structural variations as well as identification of pathogens like bacteria and viruses. For future clinical applications it is desirable to have a fast and robust mapping method based on as few steps as possible. We here demonstrate a single-step method to obtain a DNA barcode that is directly visualized using nanofluidic devices and fluorescence microscopy. Using a mixture of YOYO-1, a bright DNA dye, and netropsin, a natural antibiotic with very high AT specificity, we obtain a DNA map with a fluorescence intensity profile along the DNA that reflects the underlying sequence. The netropsin binds to AT-tetrads and blocks these binding sites from YOYO-1 binding which results in lower fluorescence intensity from AT-rich regions of the DNA. We thus obtain a DNA barcode that is dark in AT-rich regions and bright in GC-rich regions with kilobasepair resolution. We demonstrate the versatility of the method by obtaining a barcode on DNA from the phage T4 that captures its circular permutation and agrees well with its known sequence.