Mitochondrial and microbial diversity of the invasive mosquito vector species Culex tritaeniorhynchus across its extensive inter-continental geographic range.

Background: Culex (Cx.) tritaeniorhynchus is an invasive mosquito species with an extensive and expanding inter-continental distribution, currently reported across Asia, Africa, the Middle East, Europe and now Australia. It is an important vector of medical and veterinary pathogens which cause significant morbidity and mortality in human and animal populations. Across regions endemic for Japanese encephalitis virus (JEV), Cx. tritaeniorhynchus is considered the major vector and has also been shown to contribute to the transmission of several other zoonotic arboviruses including Rift Valley fever virus (RVFV) and West Nile virus (WNV). Methods: In this study, we used laboratory vector competence experiments to determine if Cx. tritaeniorhynchus from a Southern European population were competent JEV vectors. We also obtained samples from multiple geographically dispersed Cx. tritaeniorhynchus populations from countries within Europe, Africa, Eurasia and Asia to perform phylogenetic analysis to measure the level of mitochondrial divergence using the cytochrome oxidase subunit 1 ( CO1) gene. We also undertook bacterial 16S rRNA gene amplicon sequencing to determine microbial diversity and used multi-locus sequence typing (MLST) to determine any evidence for the presence of strains of the naturally occurring endosymbiotic bacterium Wolbachia. Results: Cx. tritaeniorhynchus from a Greek population were shown be be competent vectors of JEV with high levels of virus present in saliva. We found a signficant level of mitochondrial genetic diversity using the mosquito CO1 gene between geographically dispersed populations. Furthermore, we report diverse microbiomes identified by 16S rRNA gene amplicon sequencing within and between geographical populations. Evidence for the detection of the endosymbiotic bacteria Wolbachia was confirmed using Wolbachia-specific PCR and MLST. Conclusions: This study enhances our understanding of the diversity of Cx. tritaeniorhynchus and the associated microbiome across its inter-continental range and highlights the need for greater surveillance of this invasive vector species in Europe.

The mosquito species Culex (Cx.) tritaeniorhynchus is expanding its range and is now present in over 50 countries across Asia, Africa, the Middle East, Europe and now Australasia. It can transmit human and animal pathogens, resulting in significant morbidity and mortality. This species transmits Japanese encephalitis virus in endemic areas of Asia, and it has also been shown to contribute to the transmission of several other viruses that can infect humans, including Rift Valley fever virus and West Nile virus. In this study, we firstly undertook some lab experiments to show that Cx. tritaeniorhynchus from a Southern European population are competent vectors of Japanese encephalitis virus. We also obtained field mosquitoes from countries within Europe, Africa, Eurasia and Asia and used phylogenetic analysis to demonstrate a high level of mitochondrial divergence within and between populations. In addition, we analysed the bacteria present within mosquitoes and found a high level of microbial diversity. Finally, we present evidence for the presence of Wolbachia endosymbiotic bacteria in some populations of this mosquito species. This study highlights the need for greater surveillance of this invasive vector species ­ particularly in Europe.

Clinical relevance of subthreshold PTSD versus full criteria PTSD following traumatic brain injury in U.S. service members and veterans.

BACKGROUND: The purpose of this cross-sectional study was to examine the influence of subthreshold posttraumatic stress disorder (PTSD) and full PTSD on quality of life following mild traumatic brain injury (mTBI). METHODS: Participants were 734 service members and veterans (SMV) classified into two injury groups: uncomplicated mild TBI (MTBI; n = 596) and injured controls (IC, n = 139). Participants completed a battery of neurobehavioral measures, 12-or-more months post-injury, that included the PTSD Checklist Civilian version, Neurobehavioral Symptom Inventory, and select scales from the TBI-QOL and MPAI. The MTBI group was divided into three PTSD subgroups: No-PTSD (n = 266), Subthreshold PTSD (n = 139), and Full-PTSD (n = 190). RESULTS: There was a linear relationship between PTSD severity and neurobehavioral functioning/quality of life in the MTBI sample. As PTSD severity increased, significantly worse scores were found on 11 of the 12 measures (i.e. , MTBI: Full-PTSD > Sub-PTSD > No-PTSD). When considering the number of clinically elevated scores, a linear relationship between PTSD severity and neurobehavioral functioning/quality of life was again observed in the MTBI sample (e.g., 3-or-more elevated scores: Full-PTSD = 92.1 %, Sub-PTSD = 61.9 %, No-PTSD = 19.9 %). LIMITATIONS: Limitations included the use of a self-report measure to determine diagnostic status that may under/overcount or mischaracterize individuals. CONCLUSION: PTSD symptoms, whether at the level of diagnosable PTSD, or falling short of that because of the intensity or characterization of symptoms, have a significant negative impact on one's quality of life following MTBI. Clinicians' treatment targets should focus on the symptoms that are most troubling for an individual and the individual's perception of quality of life, regardless of the diagnosis itself.

Retrospective evaluation of a right heart parameter score in the assessment of precapillary pulmonary hypertension in dogs (2017-2021): 135 cases.

OBJECTIVE: To determine if emergency and critical care residents can identify moderate to severe precapillary pulmonary hypertension on cardiologist-obtained cineloops using a pulmonary hypertension score (PHS) and report the interobserver variability of the PHS. DESIGN: Multicenter, retrospective, case-control study from 2017 to 2021. SETTING: Private referral center and veterinary teaching hospital. ANIMALS: One hundred and thirty-five client-owned dogs that underwent diagnostic echocardiography. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Medical records of dogs with stage B1 myxomatous mitral valve disease (MMVD) and dogs diagnosed with precapillary pulmonary hypertension (PCPH) via echocardiograms were reviewed. Dogs were categorized by a cardiologist into 5 groups (normal, B1 MMVD, mild, moderate, and severe PCPH) based on Doppler pulmonary pressure gradients and right heart morphology. Cineloops from each case were subjectively evaluated by emergency and critical care residents for the presence of right atrial and ventricular enlargement, right ventricular hypertrophy, interventricular septal flattening, and pulmonary artery and trunk enlargement to form a composite pulmonary hypertension score out of 8 (PHS8). When available, signs of peritoneal effusion and distention of the caudal vena cava were subjectively assessed to generate a pulmonary hypertension score out of 10 (PHS10). There was excellent discrimination of moderate to severe PCPH versus grouped absent to mild PCPH using PHS8 (area under the receiver operator curve [AUC] [95% confidence interval, CI] = 0.90 [0.84-0.95], P < 0.0001) and PHS10 (AUC [95% CI] = 0.89 [0.81-0.97], P < 0.0001). PHS8 ≥3 was 64% sensitive and 98% specific for moderate to severe PCPH (positive likelihood ratio [LR+] 32, negative likelihood ration [LR-] 0.37). PHS10 ≥ 3.3 was 64% sensitive and 92% specific for moderate to severe PCPH (LR+ 8, LR- 0.39). Interobserver agreement was good to excellent (intraclass correlation coefficient [ICC] = 0.74 [95% CI: 0.66-0.80], n = 135). CONCLUSIONS: Residents identified moderate to severe PCPH in dogs using PHS on cineloops previously obtained by a cardiologist. The interrater agreement was good to excellent with limited training. Prospective studies to determine if residents can obtain diagnostic images for PHS are warranted.

Trapping Ion Coulomb Crystals in an Optical Lattice.

We report the optical trapping of multiple ions localized at individual lattice sites of a one-dimensional optical lattice. We observe a fivefold increased range of axial dc-electric field strength for which ions can be optically trapped with high probability and an increase of the axial eigenfrequency by 2 orders of magnitude compared to an optical dipole trap without interference but of similar intensity. Our findings motivate an alternative pathway to extend arrays of trapped ions in size and dimension, enabling quantum simulations with particles interacting at long range.

Postintervention Immunological and Entomological Survey of Lymphatic Filariasis in the City of Olinda, Brazil, 2015-2016.

Lymphatic filariasis (LF) is a leading cause of disability due to infectious disease worldwide. The Recife Metropolitan Region (RMR) is the only remaining focus of LF in Brazil, where the parasite Wuchereria bancrofti is transmitted solely by the mosquito Culex quinquefasciatus. This study reports the results of transmission assessment surveys and molecular xenomonitoring in the city of Olinda, RMR, after nearly 15 years (2015-2016) of interventions for LF elimination. Participants were screened for W. bancrofti antigen via immunochromatographic card tests (ICT) in: 1) door-to-door surveys conducted for all children aged 5-7 years from 4 out of 17 intervention areas treated with at least five annual doses of mass drug administration (MDA), and 2) a two-stage cluster sampling survey of residents aged 5 years and older in non-MDA areas. Mosquitoes were collected via handheld aspirators in four MDA areas, differentiated by species, sex, and physiological status, pooled into groups of up to 10 blood-fed, semigravid, and gravid mosquitoes, and screened for W. bancrofti infection by real-time quantitative polymerase chain reaction (RT-qPCR). All 1,170 children from MDA areas and the entire population sample of 990 residents in non-MDA areas were ICT negative. In MDA areas, a total of 3,152 female Cx. quinquefasciatus mosquitoes in 277 households (range, 0-296 mosquitoes per house) were collected via aspiration. RT-qPCR of 233 pools of mosquitos were negative for W. bancrofti RNA; an independent reference laboratory confirmed these results. These results provide evidence that LF transmission has been halted in this setting.

Nonlinear Frequency Modulation for Fourier Transform Ion Mobility Mass Spectrometry Improves Experimental Efficiency.

Through optimization of terminal frequencies and effective sampling rates, we have developed nonlinear sawtooth-shaped frequency sweeps for efficient Fourier transform ion mobility mass spectrometry (FT-IM-MS) experiments. This is in contrast to conventional FT-IM-MS experiments where ion gates are modulated according to a linear frequency sweep. Linear frequency sweeps are effective but can be hindered by the amount of useful signal obtained using a single sweep over a large frequency range imposed by ion gating inefficiencies, particularly small ion packets, and gate depletion. These negative factors are direct consequences of the inherently low gate pulse widths of high-frequency ion gating events, placing an upper bound on FT-IM-MS performance. Here, we report alternative ion modulation strategies. Sawtooth frequency sweeps may be constructed for the purpose of either extending high-SNR transients or conducting efficient signal-averaging experiments for low-SNR transients. The data obtained using this approach show high-SNR signals for a set of low-mass tetraalkylammonium salts (<1000 m/z) where resolving powers in excess of 500 are achieved. Data for low-SNR obtained for multimeric protein complexes streptavidin (53 kDa) and GroEL (800 kDa) also reveal large increases in the signal-to-noise ratio for reconstructed arrival time distributions.

Mid-infrared spectroscopy of 1-cyanonaphthalene cation for astrochemical consideration.

We present the low temperature gas-phase vibrational spectrum of ionised 1-cyanonaphthalene (1-CNN+) in the mid-infrared region. Experimentally, 1-CNN+ ions are cooled below 10 K in a cryogenic ion trapping apparatus, tagged with He atoms and probed with tuneable radiation. Quantum-chemical calculations are carried out at a density functional theory level. The spectrum is dominated by the CN-stretch at 4.516 µm, with weaker CH modes near 3.2 µm.

Diverse novel Wolbachia bacteria strains and genera-specific co-infections with Asaia bacteria in Culicine mosquitoes from ecologically diverse regions of Cameroon.

Background: The endosymbiotic bacterium Wolbachia infects numerous species of insects and Wolbachia transinfection of Aedes mosquito species is now being used for biocontrol programs as Wolbachia strains can both inhibit arboviruses and invade wild mosquito populations. The discovery of novel, resident Wolbachia strains in mosquito species warrants further investigation as potential candidate strains for biocontrol strategies. Methods: We obtained mosquito specimens from diverse Culicine mosquitoes from Cameroon including ecologically diverse locations in the Central and West Regions. Wolbachia prevalence rates were assessed in addition to the environmentally acquired bacterial species Asaia in major Culicine genera. PCR-based methods were also used with phylogenetic analysis to confirm identities of host mosquito species and Wolbachia strains were classified using multi-locus sequence typing (MLST). Results: We report high Wolbachia prevalence rates for Culicine species, including in a large cohort of Aedes africanus collected from west Cameroon in which 100% of mono-specific pools were infected. Furthermore, co-infections with Asaia bacteria were observed across multiple genera, demonstrating that these two bacteria can co-exist in wild mosquito populations.  Wolbachia strain MLST and phylogenetic analysis provided evidence for diverse Wolbachia strains in 13 different mosquito species across seven different genera.  Full or partial MLST profiles were generated from resident Wolbachia strains in six Culex species ( quinquefasciatus, watti, cinerus, nigripalpus, perexiguus and rima), two Aedes species (africanus and denderensis) and in Mansonia uniformis, Catageiomyia argenteopunctata, Lutzia tigripes, Eretmapodites chrysogaster and Uranotaenia bilineata. Conclusions: Our study provides further evidence that Wolbachia is widespread within wild mosquito populations of diverse Culicine species and provides further candidate strains that could be investigated as future options for Wolbachia-based biocontrol to inhibit arbovirus transmission.

Detection of insecticide resistance markers in Anopheles funestus from the Democratic Republic of the Congo using a targeted amplicon sequencing panel.

Vector control strategies have been successful in reducing the number of malaria cases and deaths globally, but the spread of insecticide resistance represents a significant threat to disease control. Insecticide resistance has been reported across Anopheles (An.) vector populations, including species within the An. funestus group. These mosquitoes are responsible for intense malaria transmission across sub-Saharan Africa, including in the Democratic Republic of the Congo (DRC), a country contributing > 12% of global malaria infections and mortality events. To support the continuous efficacy of vector control strategies, it is essential to monitor insecticide resistance using molecular surveillance tools. In this study, we developed an amplicon sequencing ("Amp-seq") approach targeting An. funestus, and using multiplex PCR, dual index barcoding, and next-generation sequencing for high throughput and low-cost applications. Using our Amp-seq approach, we screened 80 An. funestus field isolates from the DRC across a panel of nine genes with mutations linked to insecticide resistance (ace-1, CYP6P4, CYP6P9a, GSTe2, vgsc, and rdl) and mosquito speciation (cox-1, mtND5, and ITS2). Amongst the 18 non-synonymous mutations detected, was N485I, in the ace-1 gene associated with carbamate resistance. Overall, our panel represents an extendable and much-needed method for the molecular surveillance of insecticide resistance in An. funestus populations.

Effects of next-generation, dual-active-ingredient, long-lasting insecticidal net deployment on insecticide resistance in malaria vectors in Tanzania: an analysis of a 3-year, cluster-randomised controlled trial.

BACKGROUND: Insecticide resistance among malaria-vector species is a pervasive problem that might jeopardise global disease-control efforts. Novel vector-control tools with different modes of action, including long-lasting insecticidal nets (LLINs) incorporating new active ingredients, are urgently needed to delay the evolution and spread of insecticide resistance. We aimed to measure phenotypic and genotypic insecticide-resistance profiles among wild Anopheles collected over 3 years to assess the longitudinal effects of dual-active-ingredient LLINs on insecticide resistance. METHODS: For this analysis, data nested in a 3-year, four parallel-arm, superiority cluster-randomised controlled trial (cRCT) in Tanzania, collected from 84 clusters (39 307 households) formed of 72 villages in the Misungwi district, were used to measure insecticide-resistance profiles among female Anopheles mosquitoes via insecticide-resistance bioassays and quantitative RT-PCR of metabolic-resistance genes. Wild, blood-fed, indoor-resting mosquitoes were collected annually during the rainy seasons from house walls in clusters from all four trial groups. Mosquitoes were morphologically identified as An gambiae sensu lato (SL) or An funestus SL before separate bioassay testing. The primary outcomes were lethal-dose values for α-cypermethrin, permethrin, and piperonyl butoxide pre-exposure plus permethrin-resistance intensity bioassays, mortality 72 h after insecticidal exposure for chlorfenapyr bioassays, fertility reduction 72 h after insecticidal exposure for pyriproxyfen bioassays, and fold change in metabolic-enzyme expression relative to an insecticide-susceptible laboratory strain. All primary outcomes were measured in An funestus SL 1 year, 2 years, and 3 years after LLIN distribution. Primary outcomes were also assessed in An gambiae SL if enough mosquitoes were collected. The cRCT is registered with ClinicalTrials.gov (NCT03554616). FINDINGS: Between May 24, 2019, and Oct 25, 2021, 47 224 female Anopheles were collected for resistance monitoring. In the pyrethroid (PY)-LLIN group, there were significant increases in α-cypermethrin-resistance intensity (year 1 LD50=9·52 vs year 2 76·20, p<0·0001) and permethrin-resistance intensity (year 1 13·27 vs year 2 35·83, p=0·0019) in An funestus SL. In the pyriproxyfen PY-LLIN group, there was similar increase in α-cypermethrin-resistance intensity (year 1 0·71 vs year 2 81·56, p<0·0001) and permethrin-resistance intensity (year 1 5·68 vs year 2 50·14, p<0·0001). In the piperonyl butoxide PY-LLIN group, α-cypermethrin-resistance intensity (year 1 33·26 vs year 3 70·22, p=0·0071) and permethrin-resistance intensity (year 1 47·09 vs year 3 2635·29, p<0·0001) also increased over time. In the chlorfenapyr PY-LLIN group, there were no effects on α-cypermethrin-resistance intensity (year 1 0·42 vs year 3 0·99, p=0·54) or permethrin-resistance intensity (data were not estimable due to nearly 100% mortality). There were also minimal reductions in chlorfenapyr susceptibility. However, in the chlorfenapyr PY-LLIN group, a significant decline in piperonyl-butoxide synergy was seen by year 3 (year 1 0·02 vs year 3 0·26, p=0·020). Highly over-expressed detoxification enzymes showed dynamic patterns of selection throughout the trial. INTERPRETATION: Our phenotypic data supports trial epidemiological findings; chlorfenapyr PY-LLINs provided superior protection from malaria across multiple transmission seasons, with few effects on insecticide-resistance selection. Rapid pyrethroid-resistance intensification in the piperonyl butoxide PY-LLIN group and pre-existing tolerance of pyriproxyfen in vector populations might explain the poorer performance of these two interventions regarding malaria outcomes. Further work is required to elucidate the potential mechanisms driving cross-resistance between pyrethroids and novel active ingredients to better inform the design of pre-emptive resistance-management strategies. FUNDING: UK Department for International Development; UK Medical Research Council; Wellcome Trust; UK Department of Health and Social Care; UK Foreign, Commonwealth and Development Office; and The Bill and Melinda Gates Foundation via the Innovative Vector Control Consortium.

Elevated Serum Tau and UCHL-1 Concentrations Within 12 Months of Injury Predict Neurobehavioral Functioning 2 or More Years Following Traumatic Brain Injury: A Longitudinal Study.

OBJECTIVE: Blood-based biomarkers have received considerable attention for their diagnostic and prognostic value in the acute and postacute period following traumatic brain injury (TBI). The purpose of this study was to examine whether blood-based biomarker concentrations within the first 12 months of TBI can predict neurobehavioral outcome in the chronic phase of the recovery trajectory. SETTING: Inpatient and outpatient wards from 3 military medical treatment facilities. PARTICIPANTS: A total of 161 service members and veterans classified into 3 groups: (a) uncomplicated mild TBI (MTBI; n = 37), (b) complicated mild, moderate, severe, penetrating TBI combined (STBI; n = 46), and (c) controls (CTRL; n = 78). DESIGN: Prospective longitudinal. MAIN MEASURES: Participants completed 6 scales from the Traumatic Brain Injury Quality of Life (ie, Anger, Anxiety, Depression, Fatigue, Headaches, and Cognitive Concerns) within 12 months (baseline) and at 2 or more years (follow-up) post-injury. Serum concentrations of tau, neurofilament light, glial fibrillary acidic protein, and UCHL-1 at baseline were measured using SIMOA. RESULTS: Baseline tau was associated with worse anger, anxiety, and depression in the STBI group at follow-up (R2 = 0.101-0.127), and worse anxiety in the MTBI group (R2 = 0.210). Baseline ubiquitin carboxyl-terminal hydrolase L1 (UCHL-1) was associated with worse anxiety and depression at follow-up in both the MTBI and STBI groups (R2Δ = 0.143-0.207), and worse cognitive concerns in the MTBI group (R2Δ = 0.223). CONCLUSIONS: A blood-based panel including these biomarkers could be a useful tool for identifying individuals at risk of poor outcome following TBI.

To plate, or not to plate? A systematic review of functional outcomes and complications of plate fixation in patellar fractures.

PURPOSE: Poor outcomes and high complication and reoperation rates have been reported with tension-band wiring (TBW) in the management of patellar fractures and particularly the comminuted ones. The purpose of this study was to investigate the functional outcomes and complication rates of patellar fractures managed with open reduction and internal fixation (ORIF) with a plate. METHODS: MEDLINE, EMCare, CINAHL, AMED and HMIC were searched, and the PRISMA guidelines were followed. Two independent reviewers extracted the data from the included studies and assessed them for the risk of bias. RESULTS: Plating of patellar fractures is associated with satisfactory range of movement (ROM) and postoperative function and low pain levels. We found a 10.44% complication rate and a low reoperation rate. Reoperations were mainly performed for metalwork removal. CONCLUSION: ORIF with plating of patellar fractures is a safe alternative in the management of patellar fractures and may be associated with a lower complication and reoperation rate compared to TBW. Future randomized prospective studies are needed to validated the results of the present systematic review.

Water risk modeling: A framework for finance.

Climate change has intensified water risk events, threatening water security for societies and ecosystems. Although current water risk models focus on geophysical and business-related effects, they do not quantify the monetary dimensions of water-related challenges and opportunities. This study aims to address this gap by exploring the objectives and directions for modeling water risk in finance. We identify requirements for adequately modeling financial water risk, assess existing water risk approaches in finance, outline their benefits and shortcomings, and develop directions for future modeling. Recognizing the interplay between climate and water as well as the systemic dimension of water risk, we emphasize the need for forward-looking, diversification-based, and mitigation-adjusted modeling approaches.

Immune checkpoint inhibitors efficacy across solid cancers and the utility of PD-L1 as a biomarker of response: a systematic review and meta-analysis.

Background: Immune checkpoint inhibitors (ICPI) are a tumor agnostic treatment. However, trials of their use have been site specific. Here we summarize the trial data and explore the utility of programmed death-ligand 1 (PD-L1) expression as a biomarker to direct their pan-cancer use. Method: A systematic review of literature, following PRISMA guidelines, was performed. Medline, Embase, Cochrane CENTRAL, NHS Health and Technology, and Web of Science were searched from their conception to June 2022 limited to the English language. The search terms and method were devised by a specialist medical librarian. Studies were limited to adults with solid cancers (excluding melanomas) treated with ICPIs. Only phase III randomized control trials (RCT) were included. The primary outcome was overall survival and secondary outcomes were progression free survival, PD-L1 expression, quality of life outcomes and adverse event data. Where present in eligible clinical trials, hazard ratios (HR), risk ratios (RR), standard error (SE) and 95% confidence intervals (CI) were extracted or calculated. Heterogeneity across studies was described with the use of an I2 score (Low: 25, 50%: moderate, 75% low heterogeneity). HR pools inverse variance methods were adopted by Random Effects (RE). Means were standardized across any heterogenous scale limits. Results: In total 46,510 participants were included in the meta-analysis. Overall, meta-analysis favored the use of ICPIs with an overall survival (OS) HR of 0.74 (95% CI 0.71 to 0.78). Lung cancers showed the most benefit in OS [HR 0.72 (95% 0.66-0.78)] followed by head and neck cancers [HR 0.75 (95% CI 0.66-0.84)] and gastroesophageal junction cancers [HR 0.75 (95% CI 0.61-0.92)]. ICPIs seem to be efficacious at both primary presentation and recurrence [OS HR 0.73 (95% CI 0.68-0.77)] vs. [OS HR 0.79 (95% CI 0.72 to 0.87)] respectively. Interestingly, subgroup analysis comparing studies in which most cancers demonstrated PD-L1 expression vs. those studies in which a minority of cancer demonstrated PD-L1 expression reported similar effect of ICPI use on OS; oddly the data favored ICPI use in studies with a minority of PD-L1 expression. Specifically, studies with minority PD-L1 expression had an HR 0.73 (95% CI 0.68-0.78) vs. studies with majority PD-L1 expression HR 0.76 (95% CI 0.70-0.84). This was maintained even when studies exploring the same cancer site were directly compared. Subgroup analysis was performed comparing the impact on OS subdivided by the specific ICPI used. Where meta-analysis was performed, Nivolumab led to the greatest impact [HR 0.70 (95% CI 0.64-0.77)] with Avelumab failing to reach significance [HR 0.93 (95% CI 0.80-1.06)]. However, overall heterogenicity was high (I2 = 95%). Finally, the use of ICPIs led to an improved side effect profile when compared with standard chemotherapy [RR 0.85 (95% CI 0.73-0.98)]. Conclusion: ICPIs improve survival outcomes in all cancer types. These effects are seen in the primary, recurrent, chemotherapy sensitive, chemotherapy resistant disease. These data support their use as a tumor agnostic therapy. Furthermore, they are well tolerated. However, PD-L1 as a biomarker for the targeting of ICPI use seems problematic. Other biomarkers such as mismatch repair or tumor mutational burden should be explored in randomized trials. In addition, there are still limited trials looking at ICPI use outside of lung cancer.

Identification of two insecticide resistance markers in Ethiopian Anopheles stephensi mosquitoes using a multiplex amplicon sequencing assay.

Since its first detection in 2012 in Djibouti, Anopheles stephensi has invaded and established in the Horn of Africa, and more recently Nigeria. The expansion of this vector poses a significant threat to malaria control and elimination efforts. Integrated vector management is the primary strategy used to interrupt disease transmission; however, growing insecticide resistance is threatening to reverse gains in global malaria control. We present a next-generation amplicon-sequencing approach, for high-throughput monitoring of insecticide resistance genes (ace1, GSTe2, vgsc and rdl), species identification and characterization of genetic diversity (its2 and cox1) in An. stephensi. Ninety-five An. stephensi mosquitoes, collected in Ethiopia, were screened, identifying 104 SNPs, including the knock-down mutation L958F (L1014F in Musca domestica), and for the first time in this vector species, the A296S substitution (A301S in Drosophila melanogaster) in the rdl locus. Two other amino acid substitutions (ace1-N177D, GSTe2-V189L) were also identified but have not been previously implicated in insecticide resistance. Genetic diversity in the mitochondrial cox1 gene revealed shared haplotypes between Ethiopian An. stephensi with samples from Pakistan, Sudan, and Djibouti. Overall, we present a reliable, cost-effective strategy using amplicon-sequencing to monitor known insecticide resistance mutations, with the potential to identify new genetic variants, to assist in the high-throughput surveillance of insecticide resistance in An. stephensi populations.

Modified Ion Source for the Improved Collisional Activation of Protein Complexes.

The analysis of large molecules is challenging, as they often have salts and adducts retained through the electrospray process, which increase the observed mass and compromise the achievable mass resolution. Mild collisional activation has been shown to be very effective for the removal of adducts and increases both measurement accuracy and mass resolution of large (>100 kDa) protein complexes. Collisionally activated protein ions are more completely desolvated due to the increased number of collisions when trapped following activation. A short square quadrupole maintained at 300 mTorr by a mechanical pump was added between the ion funnel and transmission quadrupole. This configuration and operation effectively removed adducts from the 800 kDa tetradecamer GroEL as well as fragmented smaller protein complexes like C-reactive protein. Due to the gas high pressure, ions of low size-to-charge ratio, such as those in charge reducing buffers, had low ejection efficiency. We show that segmenting the quadrupole rods greatly improves signal intensity for charge reduced GroEL D398A mutant compared to nonsegmented rods when operating at high pressure.

Dissecting the Thermodynamics of ATP Binding to GroEL One Nucleotide at a Time.

Variable-temperature electrospray ionization (vT-ESI) native mass spectrometry (nMS) is used to determine the thermodynamics for stepwise binding of up to 14 ATP molecules to the 801 kDa GroEL tetradecamer chaperonin complex. Detailed analysis reveals strong enthalpy-entropy compensation (EEC) for the ATP binding events leading to formation of GroEL-ATP7 and GroEL-ATP14 complexes. The observed variations in EEC and stepwise free energy changes of specific ATP binding are consistent with the well-established nested cooperativity model describing GroEL-ATP interactions, viz., intraring positive cooperativity and inter-ring negative cooperativity (Dyachenko A.; Proc. Natl. Acad. Sci. U.S.A.2013, 110, 7235-7239). Entropy-driven ATP binding is to be expected for ligand-induced conformational changes of the GroEL tetradecamer, though the magnitude of the entropy change suggests that reorganization of GroEL-hydrating water molecules and/or expulsion of water from the GroEL cavity may also play key roles. The capability for determining complete thermodynamic signatures (ΔG, ΔH, and -TΔS) for individual ligand binding reactions for the large, nearly megadalton GroEL complex expands our fundamental view of chaperonin functional chemistry. Moreover, this work and related studies of protein-ligand interactions illustrate important new capabilities of vT-ESI-nMS for thermodynamic studies of protein interactions with ligands and other molecules such as proteins and drugs.

The DNA damage response in advanced ovarian cancer: functional analysis combined with machine learning identifies signatures that correlate with chemotherapy sensitivity and patient outcome.

BACKGROUND: Ovarian cancers are hallmarked by chromosomal instability. New therapies deliver improved patient outcomes in relevant phenotypes, however therapy resistance and poor long-term survival signal requirements for better patient preselection. An impaired DNA damage response (DDR) is a major chemosensitivity determinant. Comprising five pathways, DDR redundancy is complex and rarely studied alongside chemoresistance influence from mitochondrial dysfunction. We developed functional assays to monitor DDR and mitochondrial states and trialled this suite on patient explants. METHODS: We profiled DDR and mitochondrial signatures in cultures from 16 primary-setting ovarian cancer patients receiving platinum chemotherapy. Explant signature relationships to patient progression-free (PFS) and overall survival (OS) were assessed by multiple statistical and machine-learning methods. RESULTS: DR dysregulation was wide-ranging. Defective HR (HRD) and NHEJ were near-mutually exclusive. HRD patients (44%) had increased SSB abrogation. HR competence was associated with perturbed mitochondria (78% vs 57% HRD) while every relapse patient harboured dysfunctional mitochondria. DDR signatures classified explant platinum cytotoxicity and mitochondrial dysregulation. Importantly, explant signatures classified patient PFS and OS. CONCLUSIONS: Whilst individual pathway scores are mechanistically insufficient to describe resistance, holistic DDR and mitochondrial states accurately predict patient survival. Our assay suite demonstrates promise for translational chemosensitivity prediction.

Towards environmental detection, quantification, and molecular characterization of Anopheles stephensi and Aedes aegypti from experimental larval breeding sites.

The invasion and establishment of An. stephensi mosquitoes in the Horn of Africa represents a significant regional threat, which may jeopardise malaria control, particularly in urban areas which were formally free from disease transmission. Novel vector surveillance methods are urgently needed, both agnostic to mosquito larval morphology, and simple to implement at the sampling stage. Using new multiplex TaqMan assays, specifically targeting An. stephensi and Ae. aegypti, we validated the use of environmental DNA (eDNA) for simultaneous vector detection in shared artificial breeding sites. Study findings demonstrated that An. stephensi and Ae. aegypti eDNA deposited by as few as one second instar larva in 1L of water was detectable. Characterization of molecular insecticide resistance mechanisms, using novel amplicon-sequencing panels for both vector species, was possible from eDNA shed by as few as 16-32 s instar larvae in 50 ml of water. An. stephensi eDNA, derived from emergent pupae for 24 h, was remarkably stable, and still detectable ~ 2 weeks later. eDNA surveillance has the potential to be implemented in local endemic communities and at points of country entry, to monitor the spread of invasive vector species. Further studies are required to validate the feasibility of this technique under field conditions.

Optimization of a Digital Mass Filter for the Isolation of Intact Protein Complexes in Stability Zone 1,1.

Digital mass filters are advantageous for the analysis of large molecules due to the ability to perform ion isolation of high-m/z ions without the generation of very high radio frequency (RF) and DC voltages. Experimentally determined Mathieu stability diagrams of stability zone 1,1 for capacitively coupled digital waveforms show a voltage offset between the quadrupole rod pairs is introduced by the capacitors which is dependent on the voltage magnitude of the waveform and the duty cycle. This changes the ion's a value from a = 0 to a < 0. These effects are illustrated for isolation for single-charge states for various protein complexes up to 800 kDa (GroEL) for stability zone 1,1. Isolation resolving power (m/Δm) of approximately 280 was achieved for an ion of m/z 12,315 (z = 65+ for 800.5 kDa GroEL D398A), which corresponds to an m/z window of 44.