Risk factors for herpes zoster infections: a systematic review and meta-analysis unveiling common trends and heterogeneity patterns.

PURPOSE: The burden of herpes zoster (HZ) is substantial and numerous chronic underlying conditions are known as predisposing risk factors for HZ onset. Thus, a comprehensive study is needed to synthesize existing evidence. This study aims to comprehensively identify these risk factors. METHODS: A systematic literature search was done using MEDLINE via PubMed, EMBASE and Web of Science for studies published from January 1, 2003 to January 1, 2023. A random-effects model was used to estimate pooled Odds Ratios (OR). Heterogeneity was assessed using the I2 statistic. For sensitivity analyses basic outlier removal, leave-one-out validation and Graphic Display of Heterogeneity (GOSH) plots with different algorithms were employed to further analyze heterogeneity patterns. Finally, a multiple meta-regression was conducted. RESULTS: Of 6392 considered records, 80 were included in the meta-analysis. 21 different conditions were identified as potential risk factors for HZ: asthma, autoimmune disorders, cancer, cardiovascular disorders, chronic heart failure (CHF), chronic obstructive pulmonary disorder (COPD), depression, diabetes, digestive disorders, endocrine and metabolic disorders, hematological disorders, HIV, inflammatory bowel disease (IBD), mental health conditions, musculoskeletal disorders, neurological disorders, psoriasis, renal disorders, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and transplantation. Transplantation was associated with the highest risk of HZ (OR = 4.51 (95% CI [1.9-10.7])). Other risk factors ranged from OR = 1.17-2.87, indicating an increased risk for all underlying conditions. Heterogeneity was substantial in all provided analyses. Sensitivity analyses showed comparable results regarding the pooled effects and heterogeneity. CONCLUSIONS: This study showed an increased risk of HZ infections for all identified factors.

The Relationship of Continuity of Care, Polypharmacy and Medication Appropriateness: A Systematic Review of Observational Studies.

INTRODUCTION: Worldwide, polypharmacy and medication appropriateness-related outcomes (MARO) are growing public health concerns associated with potentially inappropriate prescribing, adverse health effects, and avoidable costs to health systems. Continuity of care (COC) is a cornerstone of high-quality care that has been shown to improve patient-relevant outcomes. However, the relationship between COC and polypharmacy/MARO has not been systematically explored. OBJECTIVE: The aim of this systematic review was to investigate the operationalization of COC, polypharmacy, and MARO as well as the relationship between COC and polypharmacy/MARO. METHODS: We performed a systematic literature search in PubMed, Embase, and CINAHL. Quantitative observational studies investigating the associations between COC and polypharmacy and/or COC and MARO by applying multivariate regression analysis techniques were eligible. Qualitative or experimental studies were not included. Information on the definition and operationalization of COC, polypharmacy, and MARO and reported associations was extracted. COC measures were assigned to the relational, informational, or management dimension of COC and further classified as objective standard, objective non-standard, or subjective. Risk of bias was assessed by using the NIH Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies. RESULTS: Twenty-seven studies were included. Overall, substantial differences existed in terms of the COC dimensions and related COC measures. Relational COC was investigated in each study, while informational and management COC were only covered among three studies. The most frequent type of COC measure was objective non-standard (n = 16), followed by objective standard (n = 11) and subjective measures (n = 3). The majority of studies indicated that COC is strongly associated with both polypharmacy and MARO, such as potentially inappropriate medication (PIM), potentially inappropriate drug combination (PIDC), drug-drug interaction (DDI), adverse drug events (ADE), unnecessary drug use, duplicated medication, and overdose. More than half of the included studies (n = 15) had a low risk of bias, while five studies had an intermediate and seven studies a high risk of bias. CONCLUSIONS: Differences regarding the methodological quality of included studies as well as the heterogeneity in terms of the operationalization and measurement of COC, polypharmacy, and MARO need to be considered when interpreting the results. Yet, our findings suggest that optimizing COC may be helpful in reducing polypharmacy and MARO. Therefore, COC should be acknowledged as an important risk factor for polypharmacy and MARO, and the importance of COC should be considered when designing future interventions targeting these outcomes.

Health economic perspective on a community-based intervention for older people at risk of care dependency - results of a prospective quasi-experimental study.

This prospective, quasi-experimental study aims to compare healthcare resource utilization (HCRU) and costs of a multi-component care approach for older people in a community setting (intervention group (IG)) with usual care in a matched control group (CG) during a 21-month observation period. The reablement-oriented intervention included a geriatric assessment, a case and network management and digital supporting tools. Regression models were applied to determine intervention effects regarding hospitalization, total hospital length of stay (LOS), number of physician consultations, and healthcare costs using claims data. 872 subjects were included in the IG and 1,768 in the CG. The analyses showed that the intervention did not affect hospitalization (OR = 1.153; 95% CI: 0.971-1.369, p = .105). However, participating in the IG lead to a small but significant increase of physician contacts by a factor of 1.078 (Exp(ß) = 1.078; 95% CI: 1.011-1.149; p = .022). A non-significant mean difference in costs of €1,183 (95% CI: €-261.6 to €2,627.6, p = .108) per participant was identified. Further research is needed to generate robust evidence on the optimal design of care approaches for older people and the health economic implications of such interventions to improve care and resource allocation decision-making. Trial registration: The study was registered at the German Clinical Trials Register (DRKS00027866).

An evaluation of fusion partner proteins for paratransgenesis in Asaia bogorensis.

Mosquitoes transmit many pathogens responsible for human diseases, such as malaria which is caused by parasites in the genus Plasmodium. Current strategies to control vector-transmitted diseases are increasingly undermined by mosquito and pathogen resistance, so additional methods of control are required. Paratransgenesis is a method whereby symbiotic bacteria are genetically modified to affect the mosquito's phenotype by engineering them to deliver effector molecules into the midgut to kill parasites. One paratransgenesis candidate is Asaia bogorensis, a Gram-negative bacterium colonizing the midgut, ovaries, and salivary glands of Anopheles sp. mosquitoes. Previously, engineered Asaia strains using native signals to drive the release of the antimicrobial peptide, scorpine, fused to alkaline phosphatase were successful in significantly suppressing the number of oocysts formed after a blood meal containing P. berghei. However, these strains saw high fitness costs associated with the production of the recombinant protein. Here, we report evaluation of five different partner proteins fused to scorpine that were evaluated for effects on the growth and fitness of the transgenic bacteria. Three of the new partner proteins resulted in significant levels of protein released from the Asaia bacterium while also significantly reducing the prevalence of mosquitoes infected with P. berghei. Two partners performed as well as the previously tested Asaia strain that used alkaline phosphatase in the fitness analyses, but neither exceeded it. It may be that there is a maximum level of fitness and parasite inhibition that can be achieved with scorpine being driven constitutively, and that use of a Plasmodium specific effector molecule in place of scorpine would help to mitigate the stress on the symbionts.

Effectiveness of a multi-component community-based care approach for older people at risk of care dependency - results of a prospective quasi-experimental study.

BACKGROUND: Due to demographic changes, the elderly population in western countries is constantly growing. As the risk of functional decline and multimorbidity increases with age, health care systems need to face the challenge of high demand for health care services and related costs. Therefore, innovative health care approaches and geriatric screenings are needed to provide individualised care. This study aims to expand the state of research by investigating the effectiveness of a multi-component care approach for the elderly in a German community setting. METHODS: A prospective, quasi-experimental study was initiated by statutory health insurance (SHI) companies. The innovative care approach includes a geriatric assessment, a case and network management as well as digital supporting tools and was implemented at the Center for Geriatrics and Gerontology (Albertinen Haus, Hamburg-Eimsbuettel). Participants of the intervention were compared to matched controls recruited in comparable urban areas. The primary outcome measure was the progression in long-term care grade during the period of observation (21 months), which was analysed on the basis of SHI claims data. Secondary endpoints were morbidity, mortality and self-reported health-related quality of life (HRQoL) measured by SF-36. RESULTS: Overall, 2,670 patients (intervention group (IG) n=873; control group (CG) n=1,797) were analysed. Logistic regression analysis showed no statistically significant difference in progression of long-term care grade between IG and CG (Odds Ratio (OR)=1.054; 95% confidence interval (CI) 0.856-1.296; p-value=0.616). Differentiated analyses indicated an initial effect, which might be attributable to the geriatric assessment. However, an adapted regression model resulted in a reversed but even non-significant effect (OR=0.945; 95% CI 0.757-1.177; p-value=0.619). While secondary analyses of long-term care grade, mortality and HRQoL did not show intervention effects, a statistically significant relative change of 0.865 (95% CI 0.780, 0.960; p-value=0.006) in morbidity indicated a potential benefit for the IG. CONCLUSIONS: The analyses did not reveal a significant effect of the community-based intervention on the primary outcome and thus we are not able to recommend a transfer into SHI standard care. Tendencies in secondary analyses need to be proved in further research. TRIAL REGISTRATION: German Clinical Trials Register, retrospective registration on February 01, 2022 ( DRKS00027866 ).

Progress towards an inducible, replication-proficient transposon delivery vector for Chlamydia trachomatis.

Background Genetic systems have been developed for Chlamydia but the extremely low transformation frequency remains a significant bottleneck.  Our goal is to develop a self-replicating transposon delivery vector for C. trachomatis which can be expanded prior to transposase induction. Methods We made E. coli/ C. trachomatis shuttle vectors bearing the Himar1 C9  transposase under control of the tet promoter and a novel rearrangement of the Himar1 transposon with the ß-lactamase gene.  Activity of the transposase was monitored by immunoblot and by DNA sequencing. Results We constructed pSW2-mCh-C9, a C. trachomatis plasmid designed to act as a self-replicating vector carrying both the Himar1 C9  transposase under tet promoter control and its transposon.  However, we were unable to recover this plasmid in C. trachomatis following multiple attempts at transformation. Therefore, we assembled two new deletion plasmids pSW2-mCh-C9-ΔTpon carrying only the Himar1 C9  transposase (under tet promoter control) and a sister vector (same sequence backbone) pSW2-mCh-C9-ΔTpase carrying its cognate transposon.  We demonstrated that the biological components that make up both pSW2-mCh-C9-ΔTpon and pSW2-mCh-C9-ΔTpase are active in E. coli.  Both these plasmids could be independently recovered in C. trachomatis. We attempted to perform lateral gene transfer by transformation and mixed infection with C. trachomatis strains bearing pSW2-mCh-C9-ΔTpon and pSW2-RSGFP-Tpon (a green fluorescent version of pSW2-mCh-C9-ΔTpase).  Despite success in achieving mixed infections, it was not possible to recover progeny bearing both versions of these plasmids. Conclusions We have designed a self-replicating plasmid vector pSW2-mCh-C9 for C. trachomatis carrying the Himar1 C9  transposase under tet promoter control.  Whilst this can be transformed into E. coli it cannot be recovered in C. trachomatis.  Based on selected deletions and phenotypic analyses we conclude that low level expression from the tet inducible promoter is responsible for premature transposition and hence plasmid loss early on in the transformation process.

Novel Asaia bogorensis Signal Sequences for Plasmodium Inhibition in Anopheles stephensi.

Mosquitoes vector many pathogens that cause human disease, such as malaria that is caused by parasites in the genus Plasmodium. Current strategies to control vector-transmitted diseases are hindered by mosquito and pathogen resistance, so research has turned to altering the microbiota of the vectors. In this strategy, called paratransgenesis, symbiotic bacteria are genetically modified to affect the mosquito's phenotype by engineering them to deliver antiplasmodial effector molecules into the midgut to kill parasites. One paratransgenesis candidate is Asaia bogorensis, a Gram-negative, rod-shaped bacterium colonizing the midgut, ovaries, and salivary glands of Anopheles sp. mosquitoes. However, common secretion signals from E. coli and closely related species do not function in Asaia. Here, we report evaluation of 20 native Asaia N-terminal signal sequences predicted from bioinformatics for their ability to mediate increased levels of antiplasmodial effector molecules directed to the periplasm and ultimately outside the cell. We tested the hypothesis that by increasing the amount of antiplasmodials released from the cell we would also increase parasite killing power. We scanned the Asaia bogorensis SF2.1 genome to identify signal sequences from extra-cytoplasmic proteins and fused these to the reporter protein alkaline phosphatase. Six signals resulted in significant levels of protein released from the Asaia bacterium. Three signals were successfully used to drive the release of the antimicrobial peptide, scorpine. Further testing in mosquitoes demonstrated that these three Asaia strains were able to suppress the number of oocysts formed after a blood meal containing P. berghei to a significantly greater degree than wild-type Asaia, although prevalence was not decreased beyond levels obtained with a previously isolated siderophore receptor signal sequence. We interpret these results to indicate that there is a maximum level of suppression that can be achieved when the effectors are constitutively driven due to stress on the symbionts. This suggests that simply increasing the amount of antiplasmodial effector molecules in the midgut is insufficient to create superior paratransgenic bacterial strains and that symbiont fitness must be considered as well.

["Alternative study designs" for the evaluation of digital health applications - a real alternative?] / "Alternative Studiendesigns" zur Bewertung digitaler Gesundheitsanwendungen ­ eine echte Alternative?

INTRODUCTION: After the Digital Healthcare Act (Digitale-Versorgung-Gesetz, DVG) reformed digital health applications' (Digitale Gesundheitsanwendungen, DiGAs) access to German Statutory Health Insurance (SHI) reimbursement, the discussion concerning necessary evidence requirements has intensified. In the past, different "alternative study designs" have been proposed to replace randomized controlled trials (RCTs) in the DiGA efficacy and benefit assessments. The present paper examines the suitability of these alternative designs for informing SHI reimbursement decisions. METHODS: The four alternative study designs primarily discussed in the context of DiGA - "Continuous Evaluation of Evolving Behavioral Intervention Technologies" (CEEBIT), "Multiphase Optimization Strategy" (MOST), "Sequential Multiple Assignment Randomized Trial" (SMART) and "Micro-Randomized Trial" (MRT) - are characterized and compared on the basis of relevant primary and secondary sources. Subsequently, their suitability for effectiveness and benefit evaluation in the context of SHI reimbursement decisions is discussed. RESULTS: None of the study designs examined aims primarily at conclusively demonstrating efficacy and benefit. Three of the four designs (MOST, SMART, MRT) focus on the development and optimization of interventions. In order to reduce resource requirements, the approaches presented sometimes deviate considerably from the methodological approach in traditional RCTs. This is especially true for their applied statistical error tolerance and their underlying randomization logic. Three of the four concepts (MOST, SMART, MRT) therefore still require RCTs after the development phase in order to demonstrate the effectiveness and benefit of the optimized intervention. DISCUSSION: The methodological differences of the alternative study designs compared to classical RCTs are accompanied by serious potentials for bias and uncertainties with regard to the identified intervention effects. These may be acceptable in the context of intervention development, but do not appear to be appropriate for use in collective SHI reimbursement decisions. CONCLUSION: The alternative study designs presented cannot be regarded as a suitable RCT alternative for efficacy and benefit assessments. A pragmatic study design, which continues to meet high methodological standards, and better utilization of real-world data could, in the future, contribute to a compromise between the justified claims to sufficient certainty of results on the one hand and appropriate procedural effort on the other.

An inducible transposon mutagenesis approach for the intracellular human pathogen Chlamydia trachomatis.

Background: Chlamydia trachomatis is a prolific human pathogen that can cause serious long-term conditions if left untreated. Recent developments in Chlamydia genetics have opened the door to conducting targeted and random mutagenesis experiments to identify gene function. In the present study, an inducible transposon mutagenesis approach was developed for C. trachomatis using a self-replicating vector to deliver the transposon-transposase cassette - a significant step towards our ultimate aim of achieving saturation mutagenesis of the Chlamydia genome. Methods: The low transformation efficiency of C. trachomatis necessitated the design of a self-replicating vector carrying the transposon mutagenesis cassette (i.e. the Himar-1 transposon containing the beta lactamase gene as well as a hyperactive transposase gene under inducible control of the tet promoter system with the addition of a riboswitch). Chlamydia transformed with this vector (pSW2-RiboA-C9Q) were induced at 24 hours post-infection. Through dual control of transcription and translation, basal expression of transposase was tightly regulated to stabilise the plasmid prior to transposition. Results: Here we present the preliminary sequencing results of transposon mutant pools of both C. trachomatis biovars, using two plasmid-free representatives: urogenital strain   C. trachomatis SWFP- and the lymphogranuloma venereum isolate L2(25667R). DNA sequencing libraries were generated and analysed using Oxford Nanopore Technologies' MinION technology. This enabled 'proof of concept' for the methods as an initial low-throughput screen of mutant libraries; the next step is to employ high throughput sequencing to assess saturation mutagenesis. Conclusions: This significant advance provides an efficient method for assaying C. trachomatis gene function and will enable the identification of the essential gene set of C. trachomatis. In the long-term, the methods described herein will add to the growing knowledge of chlamydial infection biology leading to the discovery of novel drug or vaccine targets.

Prevalence and predictors of dropout from high-intensity interval training in sedentary individuals: A meta-analysis.

Recent evidence suggests that high-intensity interval training (HIIT) is an effective method to improve fitness and various health markers. However, the tolerability and acceptability of HIIT among sedentary individuals is currently controversially discussed. Therefore, our objective was to investigate the prevalence and predictors of dropout among sedentary individuals in HIIT-based exercise interventions. MEDLINE/PubMed, SPORTDiscus, and Web of Science were searched systematically for relevant articles until 06/2018. Studies included were required to (a) be written in English, (b) include sedentary healthy adults, (c) use some form of HIIT without any complementary intervention, (d) last ≥4 weeks, (e) report detailed description of the applied HIIT protocol, and (f) report data that allow calculation of a dropout rate. Fifty-five studies reporting results from 67 HIIT interventions with 1318 participants met the eligibility criteria. The trim and fill adjusted pooled dropout rate across all interventions was 17.6% (95% confidence interval 14.2-21.5%). Dropout rates were significantly lower in cycling-based interventions compared with studies using running/walking as exercise modality (P < 0.001). Longer session time (ß = 0.02, P < 0.05), higher time effort/week (ß = 0.005, P < 0.05), and overall time effort/intervention (ß = 0.0003, P < 0.05) predicted greater dropout. Exercise intensity was not related to dropout. Our data suggest that HIIT-based interventions are tolerable and acceptable for previously sedentary individuals, exhibiting generally lower dropout rates than commonly reported for traditional exercise programs. Given the association between HIIT volume and dropouts, future studies should further focus on identifying the minimally effective dose of practical HIIT for improving health status. Such efforts would be important to increase implementation and public health impact of HIIT.

Blood meal-induced inhibition of vector-borne disease by transgenic microbiota.

Vector-borne diseases are a substantial portion of the global disease burden; one of the deadliest of these is malaria. Vector control strategies have been hindered by mosquito and pathogen resistances, and population alteration approaches using transgenic mosquitos still have many hurdles to overcome before they can be implemented in the field. Here we report a paratransgenic control strategy in which the microbiota of Anopheles stephensi was engineered to produce an antiplasmodial effector causing the mosquito to become refractory to Plasmodium berghei. The midgut symbiont Asaia was used to conditionally express the antiplasmodial protein scorpine only when a blood meal was present. These blood meal inducible Asaia strains significantly inhibit pathogen infection, and display improved fitness compared to strains that constitutively express the antiplasmodial effector. This strategy may allow the antiplasmodial bacterial strains to survive and be transmitted through mosquito populations, creating an easily implemented and enduring vector control strategy.

Charles Darwin Synthetic Interview: A 19th Century Scientist Speaks in the 21st Century.

Charles Darwin is largely unknown and poorly understood as a historical figure. Similarly the fundamental principles of evolution are often misstated, misunderstood, or entirely rejected by large numbers of Americans. Simply trying to communicate more facts about Darwin, or facts supporting the principles of evaluation, is inadequate; neither students nor members of the public will care or retain the information. On the contrary, building facts into a one-on-one conversational narrative creates a memorable opportunity to learn. Here we create a digital media, self-guided question and answer 'synthetic interview' with Charles Darwin. Questions are derived from a survey of nearly 1,000 people. Answers spoken by an actor portraying Darwin are derived from Darwin's own writings. Questions on modern topics are answered by scientists, theologians, and lawyers. First produced as a museum exhibit and then later reproduced as an app (iOS/Android), the Darwin Synthetic Interview has been evaluated with more than 3,000 surveyed users, of which 69% indicated that they learned and more than 75% would recommend the experience. Students who interacted with the synthetic interview in a classroom setting found answers were unexpected and clarifying. Using a format of personal narrative, the Darwin Synthetic Interview creates a new way to engage students and the public in a process of self-directed discovery of a topic that is often considered difficult to teach.

Inhibition of Plasmodium berghei Development in Mosquitoes by Effector Proteins Secreted from Asaia sp. Bacteria Using a Novel Native Secretion Signal.

Novel interventions are needed to prevent the transmission of the Plasmodium parasites that cause malaria. One possible method is to supply mosquitoes with antiplasmodial effector proteins from bacteria by paratransgenesis. Mosquitoes have a diverse complement of midgut microbiota including the Gram-negative bacteria Asaia bogorensis. This study presents the first use of Asaia sp. bacteria for paratransgenesis against P. berghei. We identified putative secreted proteins from A. bogorensis by a genetic screen using alkaline phosphatase gene fusions. Two were secreted efficiently: a siderophore receptor protein and a YVTN beta-propeller repeat protein. The siderophore receptor gene was fused with antiplasmodial effector genes including the scorpine antimicrobial peptide and an anti-Pbs21 scFv-Shiva1 immunotoxin. Asaia SF2.1 secreting these fusion proteins were fed to mosquitoes and challenged with Plasmodium berghei-infected blood. With each of these effector constructs, significant inhibition of parasite development was observed. These results provide a novel and promising intervention against malaria transmission.

Current perspectives on unconventional shale gas extraction in the Appalachian Basin.

The Appalachian Basin is home to three major shales, the Upper Devonian, Marcellus, and Utica. Together, they contain significant quantities of tight oil, gas, and mixed hydrocarbons. The Marcellus alone is estimated to contain upwards of 500 trillion cubic feet of natural gas. The extraction of these deposits is facilitated by a combination of horizontal drilling and slick water stimulation (e.g., hydraulic fracturing) or "fracking." The process of fracking requires large volumes of water, proppant, and chemicals as well as a large well pad (3-7 acres) and an extensive network of gathering and transmission pipelines. Drilling can generate about 1,000 tons of drill cuttings depending on the depth of the formation and the length of the horizontal bore. The flowback and produced waters that return to the surface during production are high in total dissolved solids (TDS, 60,000-350,000 mg L(-1)) and contain halides (e.g., chloride, bromide, fluoride), strontium, barium, and often naturally occurring radioactive materials (NORMs) as well as organics. The condensate tanks used to store these fluids can off gas a plethora of volatile organic compounds. The waste water, with its high TDS may be recycled, treated, or disposed of through deep well injection. Where allowed, open impoundments used for recycling are a source of air borne contamination as they are often aerated. The gas may be "dry" (mostly methane) or "wet," the latter containing a mixture of light hydrocarbons and liquids that need to be separated from the methane. Although the wells can produce significant quantities of natural gas, from 2-7 bcf, their initial decline rates are significant (50-75%) and may cease to be economic within a few years. This review presents an overview of unconventional gas extraction highlighting the environmental impacts and challenges.

Draft Genome Sequence of Asaia sp. Strain SF2.1, an Important Member of the Microbiome of Anopheles Mosquitoes.

Asaia spp. are abundant members of the microbiota of Anopheles mosquitoes, the principle vectors of malaria. Here, we report the draft genome sequence of Asaia sp. strain SF2.1. This strain is under development as a platform to deliver antimalarial peptides and proteins to adult female Anopheles mosquitoes.

Functional characterization of the human mariner transposon Hsmar2.

DNA transposons are mobile elements with the ability to mobilize and transport genetic information between different chromosomal loci. Unfortunately, most transposons copies are currently inactivated, little is known about mariner elements in humans despite their role in the evolution of the human genome, even though the Hsmar2 transposon is associated to hotspots for homologous recombination involved in human genetic disorders as Charcot-Marie-Tooth, Prader-Willi/Angelman, and Williams syndromes. This manuscript describes the functional characterization of the human HSMAR2 transposase generated from fossil sequences and shows that the native HSMAR2 is active in human cells, but also in bacteria, with an efficiency similar to other mariner elements. We observe that the sub-cellular localization of HSMAR2 is dependent on the host cell type, and is cytotoxic when overexpressed in HeLa cells. Finally, we also demonstrate that the binding of HSMAR2 to its own ITRs is specific, and that the excision reaction leaves non-canonical footprints both in bacteria and eukaryotic cells.

Fighting malaria with engineered symbiotic bacteria from vector mosquitoes.

The most vulnerable stages of Plasmodium development occur in the lumen of the mosquito midgut, a compartment shared with symbiotic bacteria. Here, we describe a strategy that uses symbiotic bacteria to deliver antimalaria effector molecules to the midgut lumen, thus rendering host mosquitoes refractory to malaria infection. The Escherichia coli hemolysin A secretion system was used to promote the secretion of a variety of anti-Plasmodium effector proteins by Pantoea agglomerans, a common mosquito symbiotic bacterium. These engineered P. agglomerans strains inhibited development of the human malaria parasite Plasmodium falciparum and rodent malaria parasite Plasmodium berghei by up to 98%. Significantly, the proportion of mosquitoes carrying parasites (prevalence) decreased by up to 84% for two of the effector molecules, scorpine, a potent antiplasmodial peptide and (EPIP)(4), four copies of Plasmodium enolase-plasminogen interaction peptide that prevents plasminogen binding to the ookinete surface. We demonstrate the use of an engineered symbiotic bacterium to interfere with the development of P. falciparum in the mosquito. These findings provide the foundation for the use of genetically modified symbiotic bacteria as a powerful tool to combat malaria.

Ribosome display of combinatorial antibody libraries derived from mice immunized with heat-killed Xylella fastidiosa and the selection of MopB-specific single-chain antibodies.

Pierce's disease is a devastating lethal disease of Vitus vinifera grapevines caused by the bacterium Xylella fastidiosa. There is no cure for Pierce's disease, and control is achieved predominantly by suppressing transmission of the glassy-winged sharpshooter insect vector. We present a simple robust approach for the generation of panels of recombinant single-chain antibodies against the surface-exposed elements of X. fastidiosa that may have potential use in diagnosis and/or disease transmission blocking studies. In vitro combinatorial antibody ribosome display libraries were assembled from immunoglobulin transcripts rescued from the spleens of mice immunized with heat-killed X. fastidiosa. The libraries were used in a single round of selection against an outer membrane protein, MopB, resulting in the isolation of a panel of recombinant antibodies. The potential use of selected anti-MopB antibodies was demonstrated by the successful application of the 4XfMopB3 antibody in an enzyme-linked immunosorbent assay (ELISA), a Western blot assay, and an immunofluorescence assay (IFA). These immortalized in vitro recombinant single-chain antibody libraries generated against heat-killed X. fastidiosa are a resource for the Pierce's disease research community that may be readily accessed for the isolation of antibodies against a plethora of X. fastidiosa surface-exposed antigenic molecules.

Secretion of anti-Plasmodium effector proteins from a natural Pantoea agglomerans isolate by using PelB and HlyA secretion signals.

The insect-vectored disease malaria is a major world health problem. New control strategies are needed to supplement the current use of insecticides and medications. A genetic approach can be used to inhibit development of malaria parasites (Plasmodium spp.) in the mosquito host. We hypothesized that Pantoea agglomerans, a bacterial symbiont of Anopheles mosquitoes, could be engineered to express and secrete anti-Plasmodium effector proteins, a strategy termed paratransgenesis. To this end, plasmids that include the pelB or hlyA secretion signals from the genes of related species (pectate lyase from Erwinia carotovora and hemolysin A from Escherichia coli, respectively) were created and tested for their efficacy in secreting known anti-Plasmodium effector proteins (SM1, anti-Pbs21, and PLA2) in P. agglomerans and E. coli. P. agglomerans successfully secreted HlyA fusions of anti-Pbs21 and PLA2, and these strains are under evaluation for anti-Plasmodium activity in infected mosquitoes. Varied expression and/or secretion of the effector proteins was observed, suggesting that the individual characteristics of a particular effector may require empirical testing of several secretion signals. Importantly, those strains that secreted efficiently grew as well as wild-type strains under laboratory conditions and, thus, may be expected to be competitive with the native microbiota in the environment of the mosquito midgut.

Bacterial genetic methods to explore the biology of mariner transposons.

Mariners are small DNA mediated transposons of eukaryotes that fortuitously function in bacteria. Using bacterial genetics, it is possible to study a variety of properties of mariners, including transpositional ability, dominant-negative regulation, overexpresson inhibition, and the function of cis-acting sequences like the inverted terminal repeats. In conjunction with biochemical techniques, the structure of the transposase can be elucidated and the activity of the elements can be improved for genetic tool use. Finally, it is possible to uncover functional transposase genes directly from genomes given a suitable bacterial genetic screen.