Improved reference genome of Aedes aegypti informs arbovirus vector control.

Female Aedes aegypti mosquitoes infect more than 400 million people each year with dangerous viral pathogens including dengue, yellow fever, Zika and chikungunya. Progress in understanding the biology of mosquitoes and developing the tools to fight them has been slowed by the lack of a high-quality genome assembly. Here we combine diverse technologies to produce the markedly improved, fully re-annotated AaegL5 genome assembly, and demonstrate how it accelerates mosquito science. We anchored physical and cytogenetic maps, doubled the number of known chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites, provided further insight into the size and composition of the sex-determining M locus, and revealed copy-number variation among glutathione S-transferase genes that are important for insecticide resistance. Using high-resolution quantitative trait locus and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly disease vector.

Psychosocial Functioning of Parents of Youth Receiving Intensive Interdisciplinary Pain Treatment.

OBJECTIVE: Parents of youth with chronic pain report psychosocial difficulties, yet treatment often focuses on improving their child's functioning and pain. This study evaluated changes in parents' social and emotional functioning and explored predictors of change, as they completed a parent-focused intervention while their child was enrolled in an intensive interdisciplinary pain treatment (IIPT) program. METHODS: Parents (n = 69) completed questionnaires at baseline and weekly (average duration of 4 weeks) during their child's participation in IIPT. Parents engaged in 3 groups per week providing education, therapeutic art, and psychotherapy (3 hr/week total). RESULTS: At baseline, 38% of parents reported scores in the clinically elevated range for at least 1 psychosocial variable. Linear mixed modeling for the full sample indicated reduced parent anxiety (t = -2.72, p <.01) and depression (t = -3.59, p <.001), but not increased emotional support (t = 1.86, p >. 05) or reduced social isolation (t = -1.20, p >.05). For parents with at least moderately elevated psychosocial concerns, statistically significant improvements were observed for all 4 outcomes (all p's<.01). Psychological flexibility, cognitive reappraisal, and emotional suppression were found to be related to changes in parent outcomes (anxiety, depression, isolation, and support). CONCLUSIONS: Findings support the benefit of parent-focused interventions in addition to child-focused interventions. Many parents of youth participating in IIPT had elevated scores for at least 1 psychosocial concern at baseline. Brief, parent-focused intervention including psychoeducation, therapeutic art, and psychotherapy targeting mindfulness, acceptance, and values had a significant impact on these parents, particularly those with greater struggles at baseline.

The effects of joint hypermobility on pain and functional biomechanics in adolescents with juvenile fibromyalgia: secondary baseline analysis from a pilot randomized controlled trial.

BACKGROUND: Joint hypermobility is a common clinical finding amongst hereditary connective tissue disorders that is observed in pediatric rheumatological settings, and often associated with chronic pain. Joint hypermobility may also contribute to deficits in physical functioning and physical activity, but previous findings have been inconsistent. It is possible that physical activity impairment in joint hypermobility may be due to chronic aberrant movement patterns subsequent to increased joint laxity. METHOD: As part of a larger randomized pilot trial of juvenile onset fibromyalgia (JFM), a secondary analysis was conducted to explore whether adolescents with JFM and joint hypermobility differed from non-joint hypermobility peers in terms of pain, daily functioning, and biomechanics (i.e., kinetics and kinematics) during a moderately vigorous functional task. RESULTS: From the larger sample of adolescents with JFM (N = 36), 13 adolescents (36.1%) met criteria for joint hypermobility and 23 did not have joint hypermobility. Those with joint hypermobility exhibited poorer overall functioning (Md = 20, Q1,Q3 [5.8, 7.6] vs. Md = 29, Q1,Q3 [5.1, 7.6]) but there were no differences in pain (Md = 6.9, Q1,Q3 [22, 33], vs. Md = 6.45, Q1,Q3 [15, 29.5]). Inspection of time-series plots suggests those with joint hypermobility exhibited decreased hip flexion and frontal plane hip moment (e.g., resistance to dynamic valgus) during the landing phase (early stance) and greater hip and knee transverse plane moments during the propulsion phase (late stance) of the drop vertical jump task (DVJ). No other differences in lower extremity biomechanics were observed between study groups. CONCLUSIONS: In this exploratory study, there were small but notable differences in biomechanics between patients with JFM who also had joint hypermobility versus those without joint hypermobility during a landing and jumping task (e.g., DVJ). These differences may indicate decreased joint stiffness during landing, associated with increased joint laxity and decreased joint stability, which may put them at greater risk for injury. Further study with a larger sample size is warranted to examine whether these biomechanical differences in patients with JFM and joint hypermobility affect their response to typical physical therapy or exercise recommendations.

Current enzyme-mediated insecticide resistance status of Aedes aegypti populations from a dengue-endemic city in southern Mexico.

OBJECTIVE: To identify the enzyme-mediated insecticide resistance in Aedes aegypti in Tapachula, Mexico. MATERIALS AND METHODS: Biochemical assays were undertaken to determine the enzyme levels in mosquitoes from 22 sites collected in 2018 and 2020 in Tapachula. Results of 2018 were correlated with the resistance to insecticides pub-lished. RESULTS: Mosquitoes had higher levels than those of the susceptible strain in 2018 and 2020 respectively of α-esterases in 15 and 12 sites; ß-esterases in 7 and 6 sites; glutathione-S-transferases in 11 and 19 sites; ρNPA-esterases in 21 and 17 sites; and cytochromes P450 in 20 and 22 sites. In mosquitoes of 2018, there was a moderate correlation between previously documented Malathion resistance ratios and the insensitive acetylcholinesterase (r=0.459, p= 0.03). CONCLUSIONS: The elevated enzyme levels found indicate its contribution to the resistance to pyrethroids and organo-phosphates already published in mosquitoes from Tapachula. Bioassays using enzyme inhibitors resulted in greater mor-tality, confirming that metabolism contributes to resistance.

Engineering a nicotinamide mononucleotide redox cofactor system for biocatalysis.

Biological production of chemicals often requires the use of cellular cofactors, such as nicotinamide adenine dinucleotide phosphate (NADP+). These cofactors are expensive to use in vitro and difficult to control in vivo. We demonstrate the development of a noncanonical redox cofactor system based on nicotinamide mononucleotide (NMN+). The key enzyme in the system is a computationally designed glucose dehydrogenase with a 107-fold cofactor specificity switch toward NMN+ over NADP+ based on apparent enzymatic activity. We demonstrate that this system can be used to support diverse redox chemistries in vitro with high total turnover number (~39,000), to channel reducing power in Escherichia coli whole cells specifically from glucose to a pharmaceutical intermediate, levodione, and to sustain the high metabolic flux required for the central carbon metabolism to support growth. Overall, this work demonstrates efficient use of a noncanonical cofactor in biocatalysis and metabolic pathway design.

The Effect of Initial Patient Experiences and Life Stressors on Predicting Lost to Follow-Up in Patients New to an HIV Clinic.

We conducted a prospective cohort study of 450 patients new to an HIV clinic in Houston, TX, to examine the roles of life stressors and initial care experiences in predicting being lost to follow-up in the first year of care. Patients completed a self-administered survey following their initial provider visit. In logistic regression models, patients who reported better experiences with the HIV provider at the first visit were less likely to be lost to follow-up at 6 months (aOR = 0.866, p = 0.038) and 12 months (aOR = 0.825, p = 0.008). Patients with a higher burden of stressful life events were more likely to be lost to follow-up at 6 months (aOR = 1.232, p = 0.037) and 12 months (aOR = 1.263, p = 0.029). Assessments of patient experience and life stressors at the initial visit have potential to predict patients at risk of dropping out of care.

Old Is (Not) Gold: Midazolam Monotherapy versus Midazolam Plus Fentanyl for Sedation during Cardiac Catheterization.

OBJECTIVE: We aimed to study the differences in perception of pain during cardiac catheterization with midazolam monotherapy compared to the current standard of midazolam plus fentanyl. BACKGROUND: Procedural sedation is important to ensure comfort and safety in patients undergoing left heart catheterization. Despite the widespread use of midazolam and fentanyl for procedural sedation, the effectiveness of this dual agent approach to sedation has never been studied in comparison to midazolam monotherapy. METHODS: A total of 129 patients undergoing sedation for outpatient elective cardiac catheterization were randomly assigned to either midazolam monotherapy (n = 69) or combination of midazolam and fentanyl (n = 60). The primary outcome was assessment of pain perception prior to discharge by patient completion of a pain questionnaire. Participants were asked if they experienced any pain during their procedure (yes/no) and, if yes, asked to rate their overall pain level using a 10-point Likert scale that ranged from 1 (minimal pain) to 10 (worst pain imaginable). RESULTS: Most patients (n = 94, 73%) reported no pain during their procedure. Patients sedated with midazolam monotherapy reported similar average pain scores compared to patients sedated with the combination of midazolam and fentanyl (1.1 vs. 1.1, p=0.95). CONCLUSIONS: Among patients undergoing elective cardiac catheterization, no significant differences in pain scores were noted between sedation with midazolam alone compared to midazolam and fentanyl. Due to fentanyl's unfavorable interaction with P2Y12 agents, increased costs, and addiction potential, it is imperative that cardiologists revisit the role of effective procedural sedation with a single agent and avoid the use of fentanyl.

Metabolic engineering of Escherichia coli for optimized biosynthesis of nicotinamide mononucleotide, a noncanonical redox cofactor.

BACKGROUND: Noncanonical redox cofactors are emerging as important tools in cell-free biosynthesis to increase the economic viability, to enable exquisite control, and to expand the range of chemistries accessible. However, these noncanonical redox cofactors need to be biologically synthesized to achieve full integration with renewable biomanufacturing processes. RESULTS: In this work, we engineered Escherichia coli cells to biosynthesize the noncanonical cofactor nicotinamide mononucleotide (NMN+), which has been efficiently used in cell-free biosynthesis. First, we developed a growth-based screening platform to identify effective NMN+ biosynthetic pathways in E. coli. Second, we explored various pathway combinations and host gene disruption to achieve an intracellular level of ~ 1.5 mM NMN+, a 130-fold increase over the cell's basal level, in the best strain, which features a previously uncharacterized nicotinamide phosphoribosyltransferase (NadV) from Ralstonia solanacearum. Last, we revealed mechanisms through which NMN+ accumulation impacts E. coli cell fitness, which sheds light on future work aiming to improve the production of this noncanonical redox cofactor. CONCLUSION: These results further the understanding of effective production and integration of NMN+ into E. coli. This may enable the implementation of NMN+-directed biocatalysis without the need for exogenous cofactor supply.

Initial skin cancer screening for solid organ transplant recipients in the United States: Delphi method development of expert consensus guidelines.

Skin cancer is the most common malignancy affecting solid organ transplant recipients (SOTR), and SOTR experience increased skin cancer-associated morbidity and mortality. There are no formal multidisciplinary guidelines for skin cancer screening after transplant, and current practices are widely variable. We conducted three rounds of Delphi method surveys with a panel of 84 U.S. dermatologists and transplant physicians to establish skin cancer screening recommendations for SOTR. The transplant team should risk stratify SOTR for screening, and dermatologists should perform skin cancer screening by full-body skin examination. SOTR with a history of skin cancer should continue regular follow-up with dermatology for skin cancer surveillance. High-risk transplant patients include thoracic organ recipients, SOTR age 50 and above, and male SOTR. High-risk Caucasian patients should be screened within 2 years after transplant, all Caucasian, Asian, Hispanic, and high-risk African American patients should be screened within 5 years after transplant. No consensus was reached regarding screening for low-risk African American SOTR. We propose a standardized approach to skin cancer screening in SOTR based on multidisciplinary expert consensus. These guidelines prioritize and emphasize the need for screening for SOTR at greatest risk for skin cancer.

Utility of the PROMIS Pediatric Pain Interference Scale in Juvenile Fibromyalgia.

OBJECTIVE: The current study tested the utility of the PROMIS Pediatric Pain Interference (PPI) in relation to the widely-used Functional Disability Inventory (FDI) in a small-scale clinical trial. METHODS: Forty youth with juvenile fibromyalgia (JFM) were randomized to either CBT only or a combined CBT and neuromuscular exercise group (i.e., FIT Teens). Participants completed the PPI and FDI at baseline, post-treatment, and three-month follow-up. RESULTS: The PPI and FDI were significantly correlated at baseline (r = .51) and post treatment (r = .53), and demonstrated similar improvements (d PPI = .87, d FDI = 1.22, p < .05) at post-treatment following FIT Teens. Following CBT only, neither the PPI nor the FDI improved significantly. CONCLUSIONS: The PPI may be appropriate for use in non-pharmacologic interventions for pediatric pain.

Medical Care Costs Were Similar Across the Low-dose Computed Tomography and Chest X-Ray Arms of the National Lung Screening Trial Despite Different Rates of Significant Incidental Findings.

BACKGROUND: The National Lung Screening Trial (NLST) reported lung cancer and all-cause mortality reductions for low-dose computed tomography (LDCT) versus chest x-ray (CXR) screening. Although LDCT lung screening has received a grade B from the United States Preventive Services Task Force and is a covered service under most health plans, concerns remain on the costs engendered by screening, and the impact of the high rate of significant incidental finding (SIF) detection on those costs. METHODS: We linked American College of Radiology Imaging Network NLST and Medicare fee-for-service claims data for participants from 23 sites for 2002-2009. We performed participant-level analyses using generalized linear regression models to estimate the adjusted annual mean of the 3-year total medical costs per person in each study arm and within screen outcome categories (ever positive with abnormalities suspicious for lung cancer, always negative for abnormalities suspicious for lung cancer, but with SIFs, and always negative without SIFs). RESULTS: The adjusted annual mean total per person costs were not significantly different between screening arms [LDCT, $11,029 (95% confidence interval, $10,107-$11,951); CXR, $10,905 (95% confidence interval, $10,059-$11,751)], despite higher proportions of individuals with SIFs in the LDCT versus the CXR arm (18% vs. 4%; P<0.0001). CONCLUSIONS: We found little difference in total annual per person costs between LDCT-screened and CXR-screened Medicare participants, despite the higher number of SIFs in the LDCT arm of the study.

Population genomics reveals that an anthropophilic population of Aedes aegypti mosquitoes in West Africa recently gave rise to American and Asian populations of this major disease vector.

BACKGROUND: The mosquito Aedes aegypti is the main vector of dengue, Zika, chikungunya and yellow fever viruses. This major disease vector is thought to have arisen when the African subspecies Ae. aegypti formosus evolved from being zoophilic and living in forest habitats into a form that specialises on humans and resides near human population centres. The resulting domestic subspecies, Ae. aegypti aegypti, is found throughout the tropics and largely blood-feeds on humans. RESULTS: To understand this transition, we have sequenced the exomes of mosquitoes collected from five populations from around the world. We found that Ae. aegypti specimens from an urban population in Senegal in West Africa were more closely related to populations in Mexico and Sri Lanka than they were to a nearby forest population. We estimate that the populations in Senegal and Mexico split just a few hundred years ago, and we found no evidence of Ae. aegypti aegypti mosquitoes migrating back to Africa from elsewhere in the tropics. The out-of-Africa migration was accompanied by a dramatic reduction in effective population size, resulting in a loss of genetic diversity and rare genetic variants. CONCLUSIONS: We conclude that a domestic population of Ae. aegypti in Senegal and domestic populations on other continents are more closely related to each other than to other African populations. This suggests that an ancestral population of Ae. aegypti evolved to become a human specialist in Africa, giving rise to the subspecies Ae. aegypti aegypti. The descendants of this population are still found in West Africa today, and the rest of the world was colonised when mosquitoes from this population migrated out of Africa. This is the first report of an African population of Ae. aegypti aegypti mosquitoes that is closely related to Asian and American populations. As the two subspecies differ in their ability to vector disease, their existence side by side in West Africa may have important implications for disease transmission.

Alternative patterns of sex chromosome differentiation in Aedes aegypti (L).

BACKGROUND: Some populations of West African Aedes aegypti, the dengue and zika vector, are reproductively incompatible; our earlier study showed that divergence and rearrangements of genes on chromosome 1, which bears the sex locus (M), may be involved. We also previously described a proposed cryptic subspecies SenAae (PK10, Senegal) that had many more high inter-sex FST genes on chromosome 1 than did Ae.aegypti aegypti (Aaa, Pai Lom, Thailand). The current work more thoroughly explores the significance of those findings. RESULTS: Intersex standardized variance (FST) of single nucleotide polymorphisms (SNPs) was characterized from genomic exome capture libraries of both sexes in representative natural populations of Aaa and SenAae. Our goal was to identify SNPs that varied in frequency between males and females, and most were expected to occur on chromosome 1. Use of the assembled AaegL4 reference alleviated the previous problem of unmapped genes. Because the M locus gene nix was not captured and not present in AaegL4, the male-determining locus, per se, was not explored. Sex-associated genes were those with FST values ≥ 0.100 and/or with increased expected heterozygosity (H exp , one-sided T-test, p < 0.05) in males. There were 85 genes common to both collections with high inter-sex FST values; all genes but one were located on chromosome 1. Aaa showed the expected cluster of high inter-sex FST genes proximal to the M locus, whereas SenAae had inter-sex FST genes along the length of chromosome 1. In addition, the Aaa M-locus proximal region showed increased H exp levels in males, whereas SenAae did not. In SenAae, chromosomal rearrangements and subsequent suppressed recombination may have accelerated X-Y differentiation. CONCLUSIONS: The evidence presented here is consistent with differential evolution of proto-Y chromosomes in Aaa and SenAae.

Cost-effectiveness of CT screening in the National Lung Screening Trial.

BACKGROUND: The National Lung Screening Trial (NLST) showed that screening with low-dose computed tomography (CT) as compared with chest radiography reduced lung-cancer mortality. We examined the cost-effectiveness of screening with low-dose CT in the NLST. METHODS: We estimated mean life-years, quality-adjusted life-years (QALYs), costs per person, and incremental cost-effectiveness ratios (ICERs) for three alternative strategies: screening with low-dose CT, screening with radiography, and no screening. Estimations of life-years were based on the number of observed deaths that occurred during the trial and the projected survival of persons who were alive at the end of the trial. Quality adjustments were derived from a subgroup of participants who were selected to complete quality-of-life surveys. Costs were based on utilization rates and Medicare reimbursements. We also performed analyses of subgroups defined according to age, sex, smoking history, and risk of lung cancer and performed sensitivity analyses based on several assumptions. RESULTS: As compared with no screening, screening with low-dose CT cost an additional $1,631 per person (95% confidence interval [CI], 1,557 to 1,709) and provided an additional 0.0316 life-years per person (95% CI, 0.0154 to 0.0478) and 0.0201 QALYs per person (95% CI, 0.0088 to 0.0314). The corresponding ICERs were $52,000 per life-year gained (95% CI, 34,000 to 106,000) and $81,000 per QALY gained (95% CI, 52,000 to 186,000). However, the ICERs varied widely in subgroup and sensitivity analyses. CONCLUSIONS: We estimated that screening for lung cancer with low-dose CT would cost $81,000 per QALY gained, but we also determined that modest changes in our assumptions would greatly alter this figure. The determination of whether screening outside the trial will be cost-effective will depend on how screening is implemented. (Funded by the National Cancer Institute; NLST ClinicalTrials.gov number, NCT00047385.).

Experimental evolution of an RNA virus in wild birds: evidence for host-dependent impacts on population structure and competitive fitness.

Within hosts, RNA viruses form populations that are genetically and phenotypically complex. Heterogeneity in RNA virus genomes arises due to error-prone replication and is reduced by stochastic and selective mechanisms that are incompletely understood. Defining how natural selection shapes RNA virus populations is critical because it can inform treatment paradigms and enhance control efforts. We allowed West Nile virus (WNV) to replicate in wild-caught American crows, house sparrows and American robins to assess how natural selection shapes RNA virus populations in ecologically relevant hosts that differ in susceptibility to virus-induced mortality. After five sequential passages in each bird species, we examined the phenotype and population diversity of WNV through fitness competition assays and next generation sequencing. We demonstrate that fitness gains occur in a species-specific manner, with the greatest replicative fitness gains in robin-passaged WNV and the least in WNV passaged in crows. Sequencing data revealed that intrahost WNV populations were strongly influenced by purifying selection and the overall complexity of the viral populations was similar among passaged hosts. However, the selective pressures that control WNV populations seem to be bird species-dependent. Specifically, crow-passaged WNV populations contained the most unique mutations (~1.7× more than sparrows, ~3.4× more than robins) and defective genomes (~1.4× greater than sparrows, ~2.7× greater than robins), but the lowest average mutation frequency (about equal to sparrows, ~2.6× lower than robins). Therefore, our data suggest that WNV replication in the most disease-susceptible bird species is positively associated with virus mutational tolerance, likely via complementation, and negatively associated with the strength of selection. These differences in genetic composition most likely have distinct phenotypic consequences for the virus populations. Taken together, these results reveal important insights into how different hosts may contribute to the emergence of RNA viruses.

Improving Escherichia coli membrane integrity and fatty acid production by expression tuning of FadL and OmpF.

BACKGROUND: Construction of microbial biocatalysts for the production of biorenewables at economically viable yields and titers is frequently hampered by product toxicity. Membrane damage is often deemed as the principal mechanism of this toxicity, particularly in regards to decreased membrane integrity. Previous studies have attempted to engineer the membrane with the goal of increasing membrane integrity. However, most of these works focused on engineering of phospholipids and efforts to identify membrane proteins that can be targeted to improve fatty acid production have been unsuccessful. RESULTS: Here we show that deletion of outer membrane protein ompF significantly increased membrane integrity, fatty acid tolerance and fatty acid production, possibly due to prevention of re-entry of short chain fatty acids. In contrast, deletion of fadL resulted in significantly decreased membrane integrity and fatty acid production. Consistently, increased expression of fadL remarkably increased membrane integrity and fatty acid tolerance while also increasing the final fatty acid titer. This 34% increase in the final fatty acid titer was possibly due to increased membrane lipid biosynthesis. Tuning of fadL expression showed that there is a positive relationship between fadL abundance and fatty acid production. Combinatorial deletion of ompF and increased expression of fadL were found to have an additive role in increasing membrane integrity, and was associated with a 53% increase the fatty acid titer, to 2.3 g/L. CONCLUSIONS: These results emphasize the importance of membrane proteins for maintaining membrane integrity and production of biorenewables, such as fatty acids, which expands the targets for membrane engineering.