Early rhombic lip Protogenin+ve stem cells in a human-specific neurovascular niche initiate and maintain group 3 medulloblastoma.

We identify a population of Protogenin-positive (PRTG+ve) MYChigh NESTINlow stem cells in the four-week-old human embryonic hindbrain that subsequently localizes to the ventricular zone of the rhombic lip (RLVZ). Oncogenic transformation of early Prtg+ve rhombic lip stem cells initiates group 3 medulloblastoma (Gr3-MB)-like tumors. PRTG+ve stem cells grow adjacent to a human-specific interposed vascular plexus in the RLVZ, a phenotype that is recapitulated in Gr3-MB but not in other types of medulloblastoma. Co-culture of Gr3-MB with endothelial cells promotes tumor stem cell growth, with the endothelial cells adopting an immature phenotype. Targeting the PRTGhigh compartment of Gr3-MB in vivo using either the diphtheria toxin system or chimeric antigen receptor T cells constitutes effective therapy. Human Gr3-MBs likely arise from early embryonic RLVZ PRTG+ve stem cells inhabiting a specific perivascular niche. Targeting the PRTGhigh compartment and/or the perivascular niche represents an approach to treat children with Gr3-MB.

Epigenetic mechanisms of osteoarthritis risk in human skeletal development.

The epigenome, including the methylation of cytosine bases at CG dinucleotides, is intrinsically linked to transcriptional regulation. The tight regulation of gene expression during skeletal development is essential, with ~1/500 individuals born with skeletal abnormalities. Furthermore, increasing evidence is emerging to link age-associated complex genetic musculoskeletal diseases, including osteoarthritis (OA), to developmental factors including joint shape. Multiple studies have shown a functional role for DNA methylation in the genetic mechanisms of OA risk using articular cartilage samples taken from aged patients. Despite this, our knowledge of temporal changes to the methylome during human cartilage development has been limited. We quantified DNA methylation at ~700,000 individual CpGs across the epigenome of developing human articular cartilage in 72 samples ranging from 7-21 post-conception weeks, a time period that includes cavitation of the developing knee joint. We identified significant changes in 8% of all CpGs, and >9400 developmental differentially methylated regions (dDMRs). The largest hypermethylated dDMRs mapped to transcriptional regulators of early skeletal patterning including MEIS1 and IRX1. Conversely, the largest hypomethylated dDMRs mapped to genes encoding extracellular matrix proteins including SPON2 and TNXB and were enriched in chondrocyte enhancers. Significant correlations were identified between the expression of these genes and methylation within the hypomethylated dDMRs. We further identified 811 CpGs at which significant dimorphism was present between the male and female samples, with the majority (68%) being hypermethylated in female samples. Following imputation, we captured the genotype of these samples at >5 million variants and performed epigenome-wide methylation quantitative trait locus (mQTL) analysis. Colocalization analysis identified 26 loci at which genetic variants exhibited shared impacts upon methylation and OA genetic risk. This included loci which have been previously reported to harbour OA-mQTLs (including GDF5 and ALDH1A2), yet the majority (73%) were novel (including those mapping to CHST3, FGF1 and TEAD1). To our knowledge, this is the first extensive study of DNA methylation across human articular cartilage development. We identify considerable methylomic plasticity within the development of knee cartilage and report active epigenomic mediators of OA risk operating in prenatal joint tissues.

Roles of RNA scaffolding in nanoscale Gag multimerization and selective protein sorting at HIV membranes.

HIV-1 Gag proteins can multimerize upon the viral genomic RNA or multiple random cellular messenger RNAs to form a virus particle or a virus-like particle, respectively. To date, whether the two types of particles form via the same Gag multimerization process has remained unclarified. Using photoactivated localization microscopy to illuminate Gag organizations and dynamics at the nanoscale, here, we showed that genomic RNA mediates Gag multimerization in a more cluster-centric, cooperative, and spatiotemporally coordinated fashion, with the ability to drive dense Gag clustering dependent on its ability to act as a long-stranded scaffold not easily attainable by cellular messenger RNAs. These differences in Gag multimerization were further shown to affect downstream selective protein sorting into HIV membranes, indicating that the choice of RNA for packaging can modulate viral membrane compositions. These findings should advance the understanding of HIV assembly and further benefit the development of virus-like particle-based therapeutics.

Spatially resolved expression landscape and gene-regulatory network of human gastric corpus epithelium.

Molecular knowledge of human gastric corpus epithelium remains incomplete. Here, by integrated analyses using single-cell RNA sequencing (scRNA-seq), spatial transcriptomics, and single-cell assay for transposase accessible chromatin sequencing (scATAC-seq) techniques, we uncovered the spatially resolved expression landscape and gene-regulatory network of human gastric corpus epithelium. Specifically, we identified a stem/progenitor cell population in the isthmus of human gastric corpus, where EGF and WNT signaling pathways were activated. Meanwhile, LGR4, but not LGR5, was responsible for the activation of WNT signaling pathway. Importantly, FABP5 and NME1 were identified and validated as crucial for both normal gastric stem/progenitor cells and gastric cancer cells. Finally, we explored the epigenetic regulation of critical genes for gastric corpus epithelium at chromatin state level, and identified several important cell-type-specific transcription factors. In summary, our work provides novel insights to systematically understand the cellular diversity and homeostasis of human gastric corpus epithelium in vivo.

Using pre-fracture mobility to augment prediction of post-operative outcomes in hip fracture.

PURPOSE: Pre-operative scores based on patient characteristics are commonly used to predict hip fracture outcomes. Mobility, an indicator of pre-operative function, has been neglected as a potential predictor. We assessed the ability of pre-fracture mobility to predict post-operative outcomes following hip fracture. METHODS: We analysed prospectively collected data from hip fracture surgery patients at a large-volume trauma unit. Mobility was classified into four groups. Post-operative outcomes studied were mortality and residence at 30 days, medical complications within 30- or 60-days post-operatively, and prolonged length of stay (LOS, ≥ 28 days). We performed multivariate regression analyses adjusting for age and sex to assess the discriminative ability of the Nottingham Hip Fracture Score (NHFS), with and without mobility, for predicting outcomes using the area under the receiver operating characteristic curve (AUROC). RESULTS: 1919 patients were included, mean age 82.6 (SD 8.2); 1357 (70.7%) were women. Multivariate analysis demonstrated patients with worse mobility had a 1.7-5.5-fold higher 30-day mortality (p ≤ 0.001), and 1.9-3.2-fold higher likelihood of prolonged LOS (p ≤ 0.001). Worse mobility was associated with a 2.3-3.8-fold higher likelihood of living in a care home at 30-days post-operatively (p < 0.001) and a 1.3-2.0-fold higher likelihood of complications within 30 days (p ≤ 0.001). Addition of mobility improved NHFS discrimination for discharge location, AUROC NHFS 0.755 [0.733-0.777] to NHFS + mobility 0.808 [0.789-0.828], and LOS, AUROC NHFS 0.584 [0.557-0.611] to NHFS + mobility 0.616 [0.590-0.643]. CONCLUSION: Incorporating mobility assessment into risk scores may improve casemix adjustment, prognostication following hip fracture, and identify high-risk patient groups requiring enhanced post-operative care at admission.

Pharmacological assessment of Ru(II) complex with GidA protein- A novel topoisomerase II inhibitor towards cancer therapeutics.

The interactions of ruthenium(II) complex with Glucose inhibited division protein A (GidA protein) was studied through various spectroscopic techniques with the ultimate goal of preparing adducts with good selectivity for cancer cells. In all the cases, formation of a tight metal-protein conjugate was observed. The influence of pH, reducing agents and chelators on the formation of adduct was analysed by UV- visible spectroscopy. While there was no effect on the addition of sodium ascorbate, some alterations on some selected bands were seen on the UV-visible spectra on the addition of EDTA. The adduct was stable in the pH range of 5-8. Addition of ruthenium(II) complex effectively quenched the intrinsic fluorescence of GidA and it occurred through static quenching. The effect of ruthenium(II) complex on the conformation of GidA has been examined by analyzing CD spectrum. Though, there was some conformational changes observed in the presence of ruthenium(II) complex, α- helix in the secondary structure of GidA retained its identity. Molecular docking of ruthenium(II) complex with GidA also indicated that GidA docks through hydrophobic interaction. The stable semisynthetic complex (ruthenium(II) complex with GidA) was checked for topoisomerase II inhibition. Relaxation and decatenation assay proved topoisomerase II inhibition of semisynthetic complex.Communicated by Ramaswamy H. Sarma.

Spatially resolved expression landscape and gene-regulatory network of human gastric corpus epithelium

Molecular knowledge of human gastric corpus epithelium remains incomplete. Here, by integrated analyses using single-cell RNA sequencing (scRNA-seq), spatial transcriptomics, and single-cell assay for transposase accessible chromatin sequencing (scATAC-seq) techniques, we uncovered the spatially resolved expression landscape and gene-regulatory network of human gastric corpus epithelium. Specifically, we identified a stem/progenitor cell population in the isthmus of human gastric corpus, where EGF and WNT signaling pathways were activated. Meanwhile, LGR4, but not LGR5, was responsible for the activation of WNT signaling pathway. Importantly, FABP5 and NME1 were identified and validated as crucial for both normal gastric stem/progenitor cells and gastric cancer cells. Finally, we explored the epigenetic regulation of critical genes for gastric corpus epithelium at chromatin state level, and identified several important cell-type-specific transcription factors. In summary, our work provides novel insights to systematically understand the cellular diversity and homeostasis of human gastric corpus epithelium in vivo.

Recent advances in electrochemical sensor developments for detecting emerging pollutant in water environment.

In the latest times, considerable studies have been performed closer to detecting emerging pollutant such as paracetamol in wastewater. Electrochemical sensor developments have recently started to determine in fewer concentrations effectively. The detection of paracetamol using standard protocols corresponding to electroanalytical techniques has a greater impact noticed in directing the detecting process toward biosensors. Non-enzymatic sensors are the peak of all electro analysis approaches. Functionalized materials, such as metal oxide nanoparticles, conducting polymers, and carbon-based materials for electrode surface functionalization have been used to create a fortification for distributing passive enzyme-free biosensors. Synergic effects are possible by enhancing loading capacity and mass transfer of reactants for attaining high analytical sensitivity using a variety of nanomaterials with large surface areas. The main focus of this study is to address the prevailing issues in the identification of paracetamol with the tasks in the non-enzymatic sensors field, followed by the useful methods of electro analysis studies.

MyoD is a 3D genome structure organizer for muscle cell identity.

The genome exists as an organized, three-dimensional (3D) dynamic architecture, and each cell type has a unique 3D genome organization that determines its cell identity. An unresolved question is how cell type-specific 3D genome structures are established during development. Here, we analyzed 3D genome structures in muscle cells from mice lacking the muscle lineage transcription factor (TF), MyoD, versus wild-type mice. We show that MyoD functions as a "genome organizer" that specifies 3D genome architecture unique to muscle cell development, and that H3K27ac is insufficient for the establishment of MyoD-induced chromatin loops in muscle cells. Moreover, we present evidence that other cell lineage-specific TFs might also exert functional roles in orchestrating lineage-specific 3D genome organization during development.

Rational design of self-assembled RNA nanostructures for HIV-1 virus assembly blockade.

Many pathological processes are driven by RNA-protein interactions, making such interactions promising targets for molecular interventions. HIV-1 assembly is one such process, in which the viral genomic RNA interacts with the viral Gag protein and serves as a scaffold to drive Gag multimerization that ultimately leads to formation of a virus particle. Here, we develop self-assembled RNA nanostructures that can inhibit HIV-1 virus assembly, achieved through hybridization of multiple artificial small RNAs with a stem-loop structure (STL) that we identify as a prominent ligand of Gag that can inhibit virus particle production via STL-Gag interactions. The resulting STL-decorated nanostructures (double and triple stem-loop structures denoted as Dumbbell and Tribell, respectively) can elicit more pronounced viral blockade than their building blocks, with the inhibition arising as a result of nanostructures interfering with Gag multimerization. These findings could open up new avenues for RNA-based therapy.

A Background Assessable and Correctable Bimolecular Fluorescence Complementation System for Nanoscopic Single-Molecule Imaging of Intracellular Protein-Protein Interactions.

Bimolecular Fluorescence Complementation (BiFC) is a versatile approach for intracellular analysis of protein-protein interactions (PPIs), but the tendency of the split fluorescent protein (FP) fragments to self-assemble when brought into close proximity of each other by random collision can lead to generation of false-positive signals that hamper high-definition imaging of PPIs occurring on the nanoscopic level. While it is thought that expressing the fusion proteins at a low level can remove false positives without impacting specific signals, there has been no effective strategy to test this possibility. Here, we present a system capable of assessing and removing BiFC false positives, termed Background Assessable and Correctable-BiFC (BAC-BiFC), in which one of the split FP fragments is fused with an optically distinct FP that serves as a reference marker, and the single-cell fluorescence ratio of the BiFC signal to the reference signal is used to gauge an optimal transfection condition. We showed that when BAC-BiFC is designed to image PPIs regulating Human Immunodeficiency Virus type 1 (HIV-1) assembly, the fluorescence ratio could decrease with decreasing probe quantity, and ratios approaching the limit of detection could allow physiologically relevant characterization of the assembly process on the nanoscale by single-molecule localization microscopy (SMLM). With much improved clarity, previously undescribed features of HIV-1 assembly were revealed.

Implementing grip strength assessment in hip fracture patients: a feasibility project.

OBJECTIVES: Risk stratification scores are used in hip fracture surgery, but none incorporate objective tests for low muscle strength. Grip strength testing is simple and cheap but not routinely assessed for patients with hip fracture. This project aimed to assess the feasibility of implementing grip strength testing into admission assessment of patients with hip fracture. METHODS: A scalable protocol and a corresponding training programme of instructional presentations and practical assessments were designed and delivered by and for physiotherapy staff. Grip strength values were collected pre-surgery on patients with hip fracture at a single centre whilst supine in bed. Implementation of the process was evaluated using narrative, quantitative and cost measures. RESULTS: 53 hip fracture patients with a mean age 80.6 (SD 10.4), of which 36 (67.9%) were female, were included. Testing was offered to 42/52 (81%) patients. Cognitive impairment prevented 14/42 (33%) of patients from completing testing; one patient declined testing. Of the 27 patients who completed testing, 14/27 (52%) had low grip strength as defined by EWGSOP2 criteria. The projected cost of testing for one year was £2.68-£2.82 per patient. Fidelity to the protocol was high using multiple criteria. CONCLUSIONS: Grip strength assessment is acceptable to physiotherapy staff and can be rapidly and cost-effectively implemented into hip fracture admission assessment.

1/f-noise-free optical sensing with an integrated heterodyne interferometer.

Optical evanescent sensors can non-invasively detect unlabeled nanoscale objects in real time with unprecedented sensitivity, enabling a variety of advances in fundamental physics and biological applications. However, the intrinsic low-frequency noise therein with an approximately 1/f-shaped spectral density imposes an ultimate detection limit for monitoring many paramount processes, such as antigen-antibody reactions, cell motions and DNA hybridizations. Here, we propose and demonstrate a 1/f-noise-free optical sensor through an up-converted detection system. Experimentally, in a CMOS-compatible heterodyne interferometer, the sampling noise amplitude is suppressed by two orders of magnitude. It pushes the label-free single-nanoparticle detection limit down to the attogram level without exploiting cavity resonances, plasmonic effects, or surface charges on the analytes. Single polystyrene nanobeads and HIV-1 virus-like particles are detected as a proof-of-concept demonstration for airborne biosensing. Based on integrated waveguide arrays, our devices hold great potentials for multiplexed and rapid sensing of diverse viruses or molecules.

The impact of research intercalation during medical school on post-graduate career progression.

BACKGROUND: Medical students at The University of Manchester have the option of research intercalation on the Master of Research programme. There is a paucity of evidence for the benefits of research intercalation. However, we hypothesised that research intercalation would accelerate post-graduate career progression and aimed to objectively measure the career enhancing impact, quantify the benefits and determine the alumni perception of research intercalation. METHODS: Data was collected retrospectively by electronic questionnaire (in 2018) from those commencing research intercalation between 2005 and 2012. RESULTS: Participants (n=52) returned questionnaires (68% response), demonstrating that the cohort had completed 67 postgraduate qualifications, published 304 manuscripts (median 3 publications per person (PP); range: 0-53) and made 430 presentations (median 7 PP; range: 0-37). Alumni had been awarded 49 research grants; funding disclosed on 43% totalled £823,000. Career progression of 73% of alumni had taken the minimum number of years; 27% took longer due to time spent working abroad or to gain additional experience prior to specialty training. Fifty-five publications and 71 presentations were directly related to MRes projects. CONCLUSION: Research intercalation provides graduates with an opportunity to learn valuable transferrable skills, contribute to translational research, and objectively enhances medical career progression.

Prediction of Postoperative Outcomes Following Hip Fracture Surgery: Independent Validation and Recalibration of the Nottingham Hip Fracture Score.

OBJECTIVES: Independent validation of risk scores after hip fracture is uncommon, particularly for evaluation of outcomes other than death. We aimed to assess the Nottingham Hip Fracture Score (NHFS) for prediction of mortality, physical function, length of stay, and postoperative complications. DESIGN: Analysis of routinely collected prospective data partly collected by follow-up interviews. SETTING AND PARTICIPANTS: Consecutive hip fracture patients were identified from the Northumbria hip fracture database between 2014 and 2018. Patients were excluded if they were not surgically managed or if scores for predictive variables were missing. METHODS: C statistics were calculated to test the discriminant ability of the NHFS, Abbreviated Mental Test Score (AMTS), and American Society of Anesthesiologists (ASA) grade for in-hospital, 30-day, and 120-day mortality; functional independence at discharge, 30 days, and 120 days; length of stay; and postoperative complications. RESULTS: We analyzed data from 3208 individuals, mean age 82.6 (standard deviation 8.6). 2192 (70.9%) were female. 194 (6.3%) died during the first 30 days, 1686 (54.5%) were discharged to their own home, 211 (6.8%) had no mobility at 120 days, 141 (4.6%) experienced a postoperative complication. The median length of stay was 18 days (interquartile range 8-28). For mortality, C statistics for the NHFS ranged from 0.68 to 0.69, similar to ASA and AMTS. For postoperative mobility, the C statistics for the NHFS ranged from 0.74 to 0.83, similar to AMTS (0.61-0.82) and better than the ASA grade (0.68-0.71). Length of stay was significantly correlated with each score (P < .001 by Jonckheere-Terpstra test); NHFS and AMTS showed inverted U-shaped relationships with length of stay. For postoperative complications, C statistics for NHFS (0.54-0.59) were similar to ASA grade (0.53-0.61) and AMTS (0.50-0.58). CONCLUSIONS AND IMPLICATIONS: The NHFS performed consistently well in predicting functional outcomes, moderately in predicting mortality, but less well in predicting length of stay and complications. There remains room for improvement by adding further predictors such as measures of physical performance in future analyses.

Recent Advances in the Molecular Beacon Technology for Live-Cell Single-Molecule Imaging.

Nucleic acids, aside from being best known as the carrier of genetic information, are versatile biomaterials for constructing nanoscopic devices for biointerfacing, owing to their unique properties such as specific base pairing and predictable structure. For live-cell analysis of native RNA transcripts, the most widely used nucleic acid-based nanodevice has been the molecular beacon (MB), a class of stem-loop-forming probes that is activated to fluoresce upon hybridization with target RNA. Here, we overview efforts that have been made in developing MB-based bioassays for sensitive intracellular analysis, particularly at the single-molecule level. We also describe challenges that are currently limiting the widespread use of MBs and provide possible solutions. With continued refinement of MBs in terms of labeling specificity and detection accuracy, accompanied by new development in imaging platforms with unprecedented sensitivity, the application of MBs is envisioned to expand in various biological research fields.

Live-Cell Imaging of Genomic Loci Using CRISPR/Molecular Beacon Hybrid Systems.

The ability to monitor the behavior of specific genomic loci in living cells can offer tremendous opportunities for deciphering the molecular basis driving cellular physiology and disease evolution. Toward this goal, clustered regularly interspersed short palindromic repeat (CRISPR)-based imaging systems have been developed, with tagging of either the nuclease-deactivated mutant of the CRISPR-associated protein 9 (dCas9) or the CRISPR single-guide RNA (sgRNA) with fluorescent protein (FP) molecules currently the major strategies for labeling. Recently, we have demonstrated the feasibility of tagging the sgRNA with molecular beacons, a class of small molecule dye-based, fluorogenic oligonucleotide probes, and demonstrated that the resulting system, termed CRISPR/MB, could be more sensitive and quantitative than conventional approaches employing FP reporters in detecting single telomere loci. In this chapter, we describe detailed protocols for the synthesis of CRISPR/MB, as well as its applications for imaging single telomere and centromere loci in live mammalian cells.

A new metagenome binning method based on gene uniqueness.

BACKGROUND: The human gut microbiome contains millions of genes and many undetected bacteria species. Recovering bacterial genomes from large complex metagenomes remains highly challenging, and current binning methods show insufficient recall rates. OBJECTIVE: This study was performed to put forward a new metagenome binning method with promising recall rate and accuracy. METHODS: We found that more than 85% of the genes could be aligned to only one bacteria species by using strict BLAST parameters (identity > 90% and aligning length > 100 bp). This phenomenon was called "the gene uniqueness", which indicated that the most bacterial genes could be exclusive to the species' taxonomy. In our new metagenome binning method, we could cluster contigs based on gene similarity via a graph model. Any contig shared with same gene under Strict Blast parameters would be clustered into one bin. RESULTS: we obtained 1,131 bins and reconstructed the genomes of 12 unknown species for MetaHIT data Our method exhibited a more promising recall rate, faster running speed and lower time complexity than the current methods. CONCLUSIONS: The present new metagenome binning method based on gene uniqueness had high recall rate and low error, which could be applied to assemble the bacterial genomes efficiently in complex metagenome.

Species Composition, Phenotypic and Genotypic Resistance Levels in Major Malaria Vectors in Teso North and Teso South Subcounties in Busia County, Western Kenya.

INTRODUCTION: Knockdown resistance (kdr) is strongly linked to pyrethroid insecticide resistance in Anopheles gambiae in Africa, which may have vital significance to the current increased use of pyrethroid-treated bed net programmes. The study is aimed at determining species composition, levels of insecticide resistance, and knockdown patterns in Anopheles gambiae sensu lato in areas with and areas without insecticide resistance in Teso North and Teso South subcounties, Western Kenya. MATERIALS AND METHODS: For WHO vulnerability tests, mosquito larvae were sampled using a dipper, reared into 3-5-day-old female mosquitoes (4944 at 100 mosquitoes per insecticide) which were exposed to 0.75% permethrin, 0.05% deltamethrin, and 0.1% bendiocarb using the WHO tube assay method. Species identification and kdr East gene PCRs were also performed on randomly selected mosquitoes from the collections; including adult mosquitoes (3448) sampled using standard collection methods. RESULTS: Anopheles gambiae sensu stricto were the majority in terms of species composition at 78.9%. Bendiocarb caused 100% mortality while deltamethrin had higher insecticidal effects (77%) on female mosquitoes than permethrin (71%). Susceptible Kengatunyi cluster had higher proportion of An. arabiensis (20.9%) than resistant Rwatama (10.7%). Kengatunyi mosquitoes exposed to deltamethrin had the highest KDT50 R of 8.2. Both Anopheles gambiae sensu stricto and Anopheles arabiensis had equal S allelic frequency of 0.84. Indoor resting mosquitoes had 100% mortality rate after 24 h since exposure. Overall SS genotypic frequency in Teso North and Teso South subcounties was 79.4% against 13.7% homozygous LL genotype and 6.9% heterozygous LS genotype. There was a significant difference (ρ < 0.05) in S allele frequencies between Kengatunyi (0.61) and Rwatama (0.95). Mosquito samples collected in 2013 had the highest S allelic frequency of 0.87. Discussion. Most likely, the higher the selection pressure exerted indoors by insecticidal nets, the higher were the resistance alleles. Use of pyrethroid impregnated nets and agrochemicals may have caused female mosquitoes to select for pyrethroid resistance. Different modes of action and chemical properties in different types of pyrethroids aggravated by a variety of edaphic and climatic factors may have caused different levels of susceptibility in both indoor and outdoor vectors to pyrethroids and carbamate. Species composition and populations in each collection method may have been influenced by insecticide resistance capacity in different species. Conclusions and Recommendations. Both phenotypic and genotypic insecticide resistance levels have been confirmed in Teso North and Teso South subcounties in Western Kenya. Insecticide resistance management practices in Kenya should be fast tracked and harmonized with agricultural sector agrochemical-based activities and legislation, and possibly switch to carbamate use in order to ease selection pressure on pyrethroids which are useable in insecticidal nets and indoor residual spray due to their low human toxicity. The implication of such high resistance levels in mosquitoes collected in Teso subcounties is that resistance is likely to persist and or even increase if monomolecules of permethrin and deltamethrin or both continue to be used in all net- and nonnet-based mosquito control purposes. Usage of mutually reinforcing piperonyl butoxide (PBO) that prohibits particular enzymes vital in metabolic activities inside mosquito systems and has been integrated into pyrethroid-LLINs to create pyrethroid-PBO nets is an extremely viable option.

Impact of Insecticide Resistance on P. falciparum Vectors' Biting, Feeding, and Resting Behaviour in Selected Clusters in Teso North and South Subcounties in Busia County, Western Kenya.

INTRODUCTION: Behavioural resistance to insecticides restrains the efficacy of vector control tools against mosquito-transmitted diseases. The current study is aimed at determining the impact of insecticide resistance on major malaria vectors' biting, feeding, and resting behaviour in areas with and areas without insecticide resistance in Teso North and Teso South, Busia County, Western Kenya. METHODS: Mosquito larvae were sampled using a dipper, reared into 3-5-day-old female mosquitoes [4944] which were exposed to 0.75% permethrin and 0.05% deltamethrin using World Health Organization tube assay method. Blood meal, species identification, and kdr Eastgene PCRs were also performed on adult mosquitoes sampled using mosquito collection methods [3448]. Biting, feeding, resting, and exiting behaviours of field-collected mosquitoes from five selected clusters were analysed. RESULTS: The lowest Kdr genotypic frequency (SS) proportion was found in female Anophelines collected in Kengatunyi at 58% while Rwatama had the highest genotypic frequency at 93%, thus susceptible and resistant clusters, respectively. The peak hour for mosquito seeking a human bite was between 0300 and 0400 hrs in the resistant cluster and 0400-0500 hrs in the susceptible cluster. The heterozygous mosquitoes maintained the known 2100-2200 hrs peak hour. There was a higher proportion of homozygous susceptible vectors (86.4%) seeking humans indoor than outdoor bitters (78.3%). Mosquito blood meals of human origin were 60% and 87% in susceptible Kengatunyi and resistant Rwatama cluster, respectively. There was significant difference between homozygous-resistant vectors feeding on human blood compared to homozygous susceptible mosquitoes (p ≤ 0.05). The proportion of bovine blood was highest in the susceptible cluster. A higher proportion of homozygous-resistant anophelines were feeding and resting indoors. No heterozygous mosquito was found resting indoor while 4.2% of the mosquitoes were caught while exiting the house through the window. Discussion. A shift in resistant Anopheles gambiae sl highest peak hour of aggressiveness from 2100-2200 hrs to 0300-0400 hrs is a key change in its biting pattern. Due to the development of resistance, mosquitoes no longer have to compete against the time the human host enters into the formerly lethal chemical and or physical barrier in the form of long-lasting insecticide-treated net. No heterozygous LS mosquito rested indoors possibly due to disadvantages of heterozygosity which could have increased their fitness costs as well as energy costs in the presence of the insecticidal agents in the treated nets. Conclusions and recommendations. Out of bed biting by female mosquitoes and partial susceptibility may contribute to residual malaria transmission. Insecticide-resistant vectors have become more endophagic and anthropophillic. Hence, insecticidal nets, zooprophylaxis, and novel repellents are still useful chemical, biological, and physical barriers against human blood questing female mosquitoes. Further studies should be done on genetic changes in mosquitoes and their effects on changing mosquito behaviour.