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Insecticide resistance (IR) is an increasing problem globally, making control of vector-borne diseases more difficult. Reduced susceptibility to permethrin in Culex pipiens , an important vector for West Nile virus, has been reported across the US based on a standardized laboratory method: the CDC bottle bioassay. This bioassay uses a rapid phenotypic outcome to reveal evidence for IR, but how this translates to the effectiveness of formulated products used in an operational setting is unclear. Therefore, other methods for IR monitoring are recommended to quantify IR or evaluate formulated products against field populations in real-world conditions. To compare some of the available methods, we collected populations of Cx. pipiens from six sites in the Northwest Mosquito Abatement District (Cook Co., Illinois), and used a susceptible laboratory strain of Cx. pipiens as a control, to test for IR to pyrethroids using CDC bottle bioassays, caged field trials, and topical applications. CDC bottle bioassays suggested that Cx. pipiens from this area exhibit IR to both etofenprox and Sumithrin®. Caged field trials with ultra-low volume Anvil® 10 + 10 (Sumithrin®) demonstrated resistance to the product and underscored the need for inclusion of a susceptible control to differentiate IR from inadequate product distribution. Topical applications revealed low to high levels of resistance to synergized and unsynergized pyrethroids (etofenprox, Sumithrin®, and deltamethrin) in all field populations. Based on these data, we provide a new decision-making tree for mosquito control professionals which will guide selection of the most optimal assay for IR surveillance based on their goals, needs, and resources.

Variation in gene regulation is ubiquitous, yet identifying the mechanisms producing such variation, especially for complex traits, is challenging. Snake venoms provide a model system for studying the phenotypic impacts of regulatory variation in complex traits because of their genetic tractability. Here, we sequence the genome of the Tiger Rattlesnake, which possesses the simplest and most toxic venom of any rattlesnake species, to determine whether the simple venom phenotype is the result of a simple genotype through gene loss or a complex genotype mediated through regulatory mechanisms. We generate the most contiguous snake-genome assembly to date and use this genome to show that gene loss, chromatin accessibility, and methylation levels all contribute to the production of the simplest, most toxic rattlesnake venom. We provide the most complete characterization of the venom gene-regulatory network to date and identify key mechanisms mediating phenotypic variation across a polygenic regulatory network.

The operating length ranges of mammalian jaw muscles have been estimated using twitch contractions or force measurements at the bite point, prompting a consensus that jaw muscles operate at short lengths on their force-length (FL) curve. However, since activation intensity truncates muscle optimal length ( L O ), we hypothesized that L O of rat superficial masseter (SM) would decrease with activation intensity, with high-force biting involving muscle shortening from long lengths on the FL curve. We measured muscle activation, strain, and force in vivo during biting on food with varying hardness and mapped the in vivo data from each muscle ( N  = 6) onto its FL relationship, measured in situ . Submaximal L O was approx. 12% shorter than twitch L O , and SM bite forces averaged 4.1 ± 3.9 N/cm 2 (mean ± S.D.) and reached 10.6 N/cm 2 , corresponding to muscle activation and food hardness. Length operation ranged from 7% below L O (ascending FL plateau), to 27% beyond (descending limb). The finding that jaw muscles operate at long, potentially unstable lengths, particularly during hard food biting significantly expands our understanding of skeletal muscle function, with broad implications for craniofacial evolution, muscle mechanics and control, and healthy as well as pathological function of the jaw musculoskeletal system.

Tuberculosis (TB) infection was responsible for an estimated 1.3 million deaths in 2017. Better diagnostic tools are urgently needed. We sought to determine whether accurate TB antigen detection in blood or urine has the potential to meet the WHO target product profiles for detection of active TB. We developed Electrochemiluminescence (ECL) immunoassays for Lipoarabinomannan (LAM) and ESAT-6 detection with detection limits in the pg/ml range and used them to compare the concentrations of the two antigens in the urine and serum of 81 HIV-negative and -positive individuals with presumptive TB enrolled across diverse geographic sites. LAM and ESAT-6 overall sensitivities in urine were 93% and 65% respectively. LAM and ESAT-6 overall sensitivities in serum were 55% and 46% respectively. Overall specificity was ≥97% in all assays. Sensitivities were higher in HIV-positive compared to HIV-negative patients for both antigens and both sample types, with signals roughly 10-fold higher on average in urine than in serum. The two antigens showed similar concentration ranges within the same sample type and correlated. LAM and ESAT-6 can be detected in the urine and serum of TB patients, regardless of the HIV status and further gains in clinical sensitivity may be achievable through assay and reagent optimization. Accuracy in urine was higher with current methods and has the potential to meet the WHO accuracy target if the findings can be transferred to a point-of-care TB test.

GGGGCC repeat expansion in C9ORF72 , which can be translated in both sense and antisense directions into five dipeptide repeat (DPR) proteins, including poly(GP), poly(GR), and poly(GA), is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we developed sensitive assays that can detect poly(GA) and poly(GR) in the cerebrospinal fluid (CSF) of patients with C9ORF72 mutations. CSF poly(GA) and poly(GR) levels did not correlate with age at disease onset, disease duration, or rate of decline of ALS Functional Rating Scale, and the average levels of these DPR proteins were similar in symptomatic and pre-symptomatic patients with C9ORF72 mutations. However, in a patient with C9ORF72 -ALS who was treated with antisense oligonucleotide (ASO) targeting the aberrant C9ORF72 transcript, CSF poly(GA) and poly(GR) levels decreased approximately 50% within 6 weeks, indicating they may serve as sensitive fluid-based biomarkers in studies directed against the production of GGGGCC repeat RNAs or DPR proteins. The GGGGCC repeat expansion in C9ORF72 -ALS/FTD can be translated into five dipeptide repeat (DPR) proteins, including poly(GR) and poly(GA). Here, the authors develop assays to detect the levels of these DPR proteins in the CSF of individuals with ALS/FTD.

Insecticide resistance (IR) is an increasing problem globally, making control of vector-borne diseases more difficult. Reduced susceptibility to permethrin in Culex pipiens, an important vector for West Nile virus, has been reported across the US based on a standardized laboratory method: the CDC bottle bioassay. This bioassay uses a rapid phenotypic outcome to reveal evidence for IR, but how this translates to the effectiveness of formulated products used in an operational setting is unclear. Therefore, other methods for IR monitoring are recommended to quantify IR or evaluate formulated products against field populations in real-world conditions. To compare some of the available methods, we collected populations of Cx. pipiens from six sites in the Northwest Mosquito Abatement District (Cook Co., Illinois), and used a susceptible laboratory strain of Cx. pipiens as a control, to test for IR to pyrethroids using CDC bottle bioassays, caged field trials, and topical applications. CDC bottle bioassays suggested that Cx. pipiens from this area exhibit IR to both etofenprox and Sumithrin®. Caged field trials with ultra-low volume Anvil® 10 + 10 (Sumithrin®) demonstrated resistance to the product and underscored the need for inclusion of a susceptible control to differentiate IR from inadequate product distribution. Topical applications revealed low to high levels of resistance to synergized and unsynergized pyrethroids (etofenprox, Sumithrin®, and deltamethrin) in all field populations. Based on these data, we provide a new decision-making tree for mosquito control professionals which will guide selection of the most optimal assay for IR surveillance based on their goals, needs, and resources.

The 4D Nucleome Network aims to develop and apply approaches to map the structure and dynamics of the human and mouse genomes in space and time with the goal of gaining deeper mechanistic insights into how the nucleus is organized and functions. The project will develop and benchmark experimental and computational approaches for measuring genome conformation and nuclear organization, and investigate how these contribute to gene regulation and other genome functions. Validated experimental technologies will be combined with biophysical approaches to generate quantitative models of spatial genome organization in different biological states, both in cell populations and in single cells. The 4D Nucleome Network aims to map the spatial and dynamic organization of the human and mouse genomes to gain insight into the structure and biological functions of the nucleus. The 4D nucleome project In this Perspective, Job Dekker et al . outline the goals and strategies of the 4D Nucleome Network, a consortium of researchers that aims to map the spatial organization and dynamics of the human and mouse genomes to gain insight into the structure and biological functions of the nucleus.

Recent advances in long-read transcriptome sequencing enable high-throughput profiling of full-length RNA isoforms in bulk, single-cell, and single-nucleus samples. However, long-read datasets typically contain a mixture of complete and partial transcripts, leading to pervasive ambiguity in read-to-isoform assignment and complicating accurate isoform identification and quantification, particularly in the absence of reliable reference annotations. These challenges are further amplified in single-cell and single-nucleus samples, where coverage is sparse and transcriptional heterogeneity is high. Here, we present the Long Read Alignment Assembler (LRAA), a unified and versatile computational framework for isoform identification and quantification from long-read RNA sequencing data across bulk, single-cell, and single-nucleus transcriptomic samples. LRAA combines splice-graph based structural modeling with expectation maximization based optimization to probabilistically resolve ambiguous read assignments and improve isoform abundance estimation. The framework supports quantification-only, reference-guided, and fully reference-free (de novo) modes of analysis within a single methodological paradigm. We benchmarked LRAA using both simulated and genuine long-read datasets spanning sequencing standards and whole transcriptomes. Central to this evaluation is a novel benchmarking strategy based on Multiplexed Overexpression of Regulatory Factors (MORFs), which provides biologically expressed, barcoded isoforms with unambiguous read-level ground truth. Across all benchmarks, including MORFs, synthetic spike-ins, and whole-transcriptome datasets, LRAA consistently outperformed state-of-the-art methods in isoform identification accuracy, sensitivity, and expression quantification. Finally, we demonstrate the biological utility of LRAA by resolving cell-type-specific isoform usage across peripheral blood immune cell populations and by detecting a pathogenic cryptic isoform of with associated transcriptional changes in single-nucleus RNA-seq data from frontal cortex tissue of an individual with frontotemporal dementia (FTD). Together, these results establish LRAA as a robust and general solution for resolving transcript diversity in complex biological systems, from development to disease.