The apicoplast link to fever-survival and artemisinin-resistance in the malaria parasite.

The emergence and spread of Plasmodium falciparum parasites resistant to front-line antimalarial artemisinin-combination therapies (ACT) threatens to erase the considerable gains against the disease of the last decade. Here, we develop a large-scale phenotypic screening pipeline and use it to carry out a large-scale forward-genetic phenotype screen in P. falciparum to identify genes allowing parasites to survive febrile temperatures. Screening identifies more than 200 P. falciparum mutants with differential responses to increased temperature. These mutants are more likely to be sensitive to artemisinin derivatives as well as to heightened oxidative stress. Major processes critical for P. falciparum tolerance to febrile temperatures and artemisinin include highly essential, conserved pathways associated with protein-folding, heat shock and proteasome-mediated degradation, and unexpectedly, isoprenoid biosynthesis, which originated from the ancestral genome of the parasite's algal endosymbiont-derived plastid, the apicoplast. Apicoplast-targeted genes in general are upregulated in response to heat shock, as are other Plasmodium genes with orthologs in plant and algal genomes. Plasmodium falciparum parasites appear to exploit their innate febrile-response mechanisms to mediate resistance to artemisinin. Both responses depend on endosymbiont-derived genes in the parasite's genome, suggesting a link to the evolutionary origins of Plasmodium parasites in free-living ancestors.

Isotope discrimination by form IC RubisCO from Ralstonia eutropha and Rhodobacter sphaeroides, metabolically versatile members of 'Proteobacteria' from aquatic and soil habitats.

RubisCO, the CO2 fixing enzyme of the Calvin-Benson-Bassham (CBB) cycle, is responsible for the majority of carbon fixation on Earth. RubisCO fixes 12 CO2 faster than 13 CO2 resulting in 13 C-depleted biomass, enabling the use of δ13 C values to trace CBB activity in contemporary and ancient environments. Enzymatic fractionation is expressed as an ε value, and is routinely used in modelling, for example, the global carbon cycle and climate change, and for interpreting trophic interactions. Although values for spinach RubisCO (ε = ~29‰) have routinely been used in such efforts, there are five different forms of RubisCO utilized by diverse photolithoautotrophs and chemolithoautotrophs and ε values, now known for four forms (IA, B, D and II), vary substantially with ε = 11‰ to 27‰. Given the importance of ε values in δ13 C evaluation, we measured enzymatic fractionation of the fifth form, form IC RubisCO, which is found widely in aquatic and terrestrial environments. Values were determined for two model organisms, the 'Proteobacteria' Ralstonia eutropha (ε = 19.0‰) and Rhodobacter sphaeroides (ε = 22.4‰). It is apparent from these measurements that all RubisCO forms measured to date discriminate less than commonly assumed based on spinach, and that enzyme ε values must be considered when interpreting and modelling variability of δ13 C values in nature.

Developing Sexual and Reproductive Health Educational Resources for Young Women with Cystic Fibrosis: A Structured Approach to Stakeholder Engagement.

PURPOSE: Stakeholder engagement, specifically integration of patient and family perspectives about what matters, is increasingly recognized as a critical component of patient-centered healthcare delivery. This study describes a structured approach to and evaluation of stakeholder engagement in the development of novel sexual and reproductive health (SRH) educational resources for adolescent and young adult (AYA) women with cystic fibrosis (CF). METHODS: Key stakeholders participated in a systematic series of steps to iteratively develop and adapt patient educational resources. Process measures (stakeholder recruitment, composition, co-learning, and transparency) and proximal outcomes of stakeholder involvement (impact on the development process and satisfaction) were measured via recorded stakeholder recommendations and a stakeholder survey. RESULTS: Seventeen stakeholders participated in seven group and two patient-only conference calls. The majority of stakeholders understood their roles, had their expectations met or exceeded, and were satisfied with the frequency and quality of engagement in the project. All stakeholders in attendance provided multiple concrete recommen-dations during the development process. Stakeholders explored the motivations of AYA women with CF related to SRH and agreed that the ideal resource should be online. After reviewing the design of existing resources, stakeholders decided by consensus to partner with a pre-existing young women's health website and created 11 CF-specific SRH guides. CONCLUSIONS: This study illuminates a path for a formal process of stakeholder engagement and evaluation in educational resource development centered on the SRH care needs of AYA women with CF. Similar systematic, planned processes could be extended to other populations and aspects of healthcare.

Phenotypic Screens Identify Parasite Genetic Factors Associated with Malarial Fever Response in Plasmodium falciparum piggyBac Mutants.

Malaria remains one of the most devastating parasitic diseases worldwide, with 90% of the malaria deaths in Africa in 2013 attributable to Plasmodium falciparum. The clinical symptoms of malaria include cycles of fever, corresponding to parasite rupture from red blood cells every 48 h. Parasite pathways involved in the parasite's ability to survive the host fever response, and indeed, the functions of ~40% of P. falciparum genes as a whole, are still largely unknown. Here, we evaluated the potential of scalable forward-genetic screening methods to identify genes involved in the host fever response. We performed a phenotypic screen for genes linked to the parasite response to febrile temperatures by utilizing a selection of single-disruption P. falciparum mutants generated via random piggyBac transposon mutagenesis in a previous study. We identified several mutants presenting significant phenotypes in febrile response screens compared to the wild type, indicating possible roles for the disrupted genes in this process. We present these initial studies as proof that forward genetics can be used to gain insight into critical factors associated with parasite biology. IMPORTANCE Though the P. falciparum genome sequence has been available for many years, ~40% of its genes do not have informative annotations, as they show no detectable homology to those of studied organisms. More still have not been evaluated via genetic methods. Scalable forward-genetic approaches that allow interrogation of gene function without any pre-existing knowledge are needed to hasten understanding of parasite biology, which will expedite the identification of drug targets and the development of future interventions in the face of spreading resistance to existing frontline drugs. In this work, we describe a new approach to pursue forward-genetic phenotypic screens for P. falciparum to identify factors associated with virulence. Future large-scale phenotypic screens developed to probe other such interesting phenomena, when considered in parallel, will prove a powerful tool for functional annotation of the P. falciparum genome, where so much remains undiscovered.

Atypical mitogen-activated protein kinase phosphatase implicated in regulating transition from pre-S-Phase asexual intraerythrocytic development of Plasmodium falciparum.

Intraerythrocytic development of the human malaria parasite Plasmodium falciparum appears as a continuous flow through growth and proliferation. To develop a greater understanding of the critical regulatory events, we utilized piggyBac insertional mutagenesis to randomly disrupt genes. Screening a collection of piggyBac mutants for slow growth, we isolated the attenuated parasite C9, which carried a single insertion disrupting the open reading frame (ORF) of PF3D7_1305500. This gene encodes a protein structurally similar to a mitogen-activated protein kinase (MAPK) phosphatase, except for two notable characteristics that alter the signature motif of the dual-specificity phosphatase domain, suggesting that it may be a low-activity phosphatase or pseudophosphatase. C9 parasites demonstrated a significantly lower growth rate with delayed entry into the S/M phase of the cell cycle, which follows the stage of maximum PF3D7_1305500 expression in intact parasites. Genetic complementation with the full-length PF3D7_1305500 rescued the wild-type phenotype of C9, validating the importance of the putative protein phosphatase PF3D7_1305500 as a regulator of pre-S-phase cell cycle progression in P. falciparum.

Should progressive perineal dilation be considered first line therapy for vaginal agenesis?

PURPOSE: In women with vaginal agenesis progressive perineal dilation provides a minimally invasive method to create a functional vagina without the attendant risks or complications of traditional surgical options. We report our 12-year experience with this technique. MATERIALS AND METHODS: Patients with vaginal agenesis treated at our institution were analyzed retrospectively and followed prospectively using case report forms and semistructured interviews. Patients diagnosed with vaginal agenesis were counseled on vaginal reconstruction options. Those electing progressive perineal dilation were instructed on the proper use of vaginal dilators by one of us (MRL) and advised to dilate 2 or 3 times daily for 20 minutes. All patients received physician, nursing and social work education and counseling. Parameters reviewed included primary diagnosis, start and end of vaginal dilation, dilation frequency, dilator size, sexual activity and whether the patient experienced pain or bleeding with dilation or sexual activity. Functional success was defined as the ability to achieve sexual intercourse, vaginal acceptance of the largest dilator without discomfort or a vaginal length of 7 cm. Univariate and multivariate analysis was performed to identify factors associated with successful neovaginal creation. RESULTS: From 1996 to 2008 we enrolled 69 females with vaginal agenesis in a progressive perineal dilation program. The primary diagnosis was Mayer-Rokitansky-Küster-Hauser syndrome in 64 patients. Mean age at the start of vaginal dilation was 17.5 years (range 14 to 35) Mean followup was 19 months (range 0 to 100). Four patients (5.7%) were lost to followup. In 7 of the remaining 65 patients (12%) treatment failed due to noncompliance and 50 (88%) achieved functional success at a median of 18.7 months. Patients who dilated frequently (once daily or greater) achieved a functional neovagina at a mean +/- SD of 4.3 +/- 2.4 months. Functional success correlated positively with frequent (once daily or greater) dilation and the initiation of sexual activity. Complications were minor. Three patients reported infrequent pain and 2 reported a single episode of bleeding with dilation. A total of 18 sexually active patients reported satisfactory intercourse without dyspareunia. CONCLUSIONS: Progressive perineal dilation for neovaginal creation is a valuable, minimally invasive therapy to create a functional vagina with a high success rate and a much lower complication rate than that in published surgical series. Given these findings, progressive perineal dilation should be offered as first line therapy in adolescents with a congenitally absent vagina.

Japanese-style acupuncture for endometriosis-related pelvic pain in adolescents and young women: results of a randomized sham-controlled trial.

STUDY OBJECTIVE: To assess feasibility, and collect preliminary data for a subsequent randomized, sham-controlled trial to evaluate Japanese-style acupuncture for reducing chronic pelvic pain and improving health-related quality of life (HRQOL) in adolescents with endometriosis. DESIGN: Randomized, sham-controlled trial. SETTINGS: Tertiary-referral hospital. PARTICIPANTS: Eighteen young women (13-22y) with laparoscopically-diagnosed endometriosis-related chronic pelvic pain. INTERVENTIONS: A Japanese style of acupuncture and a sham acupuncture control. Sixteen treatments were administered over 8 weeks. MAIN OUTCOME MEASURES: Protocol feasibility, recruitment numbers, pain not associated with menses or intercourse, and multiple HRQOL instruments including Endometriosis Health Profile, Pediatric Quality of Life, Perceived Stress, and Activity Limitation. RESULTS: Fourteen participants (out of 18 randomized) completed the study per protocol. Participants in the active acupuncture group (n = 9) experienced an average 4.8 (SD = 2.4) point reduction on a 11 point scale (62%) in pain after 4 weeks, which differed significantly from the control group's (n = 5) average reduction of 1.4 (SD = 2.1) points (P = 0.004). Reduction in pain in the active group persisted through a 6-month assessment; however, after 4 weeks, differences between the active and control group decreased and were not statistically significant. All HRQOL measures indicated greater improvements in the active acupuncture group compared to the control; however, the majority of these trends were not statistically significant. No serious adverse events were reported. CONCLUSION: Preliminary estimates indicate that Japanese-style acupuncture may be an effective, safe, and well-tolerated adjunct therapy for endometriosis-related pelvic pain in adolescents. A more definitive trial evaluating Japanese-style acupuncture in this population is both feasible and warranted.

The genome of deep-sea vent chemolithoautotroph Thiomicrospira crunogena XCL-2.

Presented here is the complete genome sequence of Thiomicrospira crunogena XCL-2, representative of ubiquitous chemolithoautotrophic sulfur-oxidizing bacteria isolated from deep-sea hydrothermal vents. This gammaproteobacterium has a single chromosome (2,427,734 base pairs), and its genome illustrates many of the adaptations that have enabled it to thrive at vents globally. It has 14 methyl-accepting chemotaxis protein genes, including four that may assist in positioning it in the redoxcline. A relative abundance of coding sequences (CDSs) encoding regulatory proteins likely control the expression of genes encoding carboxysomes, multiple dissolved inorganic nitrogen and phosphate transporters, as well as a phosphonate operon, which provide this species with a variety of options for acquiring these substrates from the environment. Thiom. crunogena XCL-2 is unusual among obligate sulfur-oxidizing bacteria in relying on the Sox system for the oxidation of reduced sulfur compounds. The genome has characteristics consistent with an obligately chemolithoautotrophic lifestyle, including few transporters predicted to have organic allocrits, and Calvin-Benson-Bassham cycle CDSs scattered throughout the genome.

Adolescent endometriosis-related pelvic pain treated with acupuncture: two case reports.

BACKGROUND: Chronic pelvic pain in adolescents accounts for 10% of outpatient gynecology visits, and 70% of adolescent patients whose pelvic pain is unresponsive to initial therapy have endometriosis. To date, there has been no published research investigating the use of acupuncture for adolescents with chronic pelvic pain and/or endometriosis. METHODS: This paper presents two case reports describing the impact of a course of acupuncture on adolescent girls with endometriosis-related chronic pelvic pain of more than 1 year. RESULTS: Both patients, undergoing between 9 and 15 treatments over a 7- to 12-week period, experienced modest improvement in pain as measured by oral self-reports of pain on a scale from 1 to 10, as well as self or family-reported improvement in headaches, nausea and fatigue. No adverse effects were reported. CONCLUSIONS: These case reports provide preliminary evidence that acupuncture may be an acceptable and safe adjunct treatment therapy for some adolescents with endometriosis-related pelvic pain refractory to standard antiendometriosis therapies. These observations suggest that a prospective, randomized controlled trial of the safety and efficacy of acupuncture for this population may be warranted.