Disruption of Circadian Clock Induces Abnormal Mammary Morphology and Aggressive Basal Tumorigenesis by Enhancing LILRB4 Signaling.

Epidemiological studies have shown that circadian rhythm disruption (CRD) is associated with the risk of breast cancer. However, the role of CRD in mammary gland morphology and aggressive basal mammary tumorigenesis and the molecular mechanisms underlying CRD and cancer risk remain unknown. To investigate the effect of CRD on aggressive tumorigenesis, a genetically engineered mouse model that recapitulates the human basal type of breast cancer was used for this study. The effect of CRD on mammary gland morphology was investigated using wild-type mice model. The impact of CRD on the tumor microenvironment was investigated using the tumors from LD12:12 and CRD mice via scRNA seq. ScRNA seq was substantiated by multiplexing immunostaining, flow cytometry, and realtime PCR. The effect of LILRB4 immunotherapy on CRD-induced tumorigenesis was also investigated. Here we identified the impact of CRD on basal tumorigenesis and mammary gland morphology and identified the role of LILRB4 on CRD-induced lung metastasis. We found that chronic CRD disrupted mouse mammary gland morphology and increased tumor burden, and lung metastasis and induced an immunosuppressive tumor microenvironment by enhancing LILRB4a expression. Moreover, CRD increased the M2-macrophage and regulatory T-cell populations but decreased the M1-macrophage populations. Furthermore, targeted immunotherapy against LILRB4 reduced CRD-induced immunosuppressive microenvironment and lung metastasis. These findings identify and implicate LILRB4a as a link between CRD and aggressive mammary tumorigenesis. This study also establishes the potential role of the targeted LILRB4a immunotherapy as an inhibitor of CRD-induced lung metastasis.

'Staying in the lane' of public health? Boundary-work in the roles of state health officials and experts in COVID-19 policymaking.

The state-level COVID-19 response in the United States necessitated collaboration between governor' offices, health departments and numerous other departments and outside experts. To gain insight into how health officials and experts contributed to advising on COVID-19 policies, we conducted semi-structured interviews with 25 individuals with a health specialisation who were involved in COVID-19 policymaking, taking place between February and December 2022. We found two diverging understandings of the role of health officials and experts in COVID-19 policymaking: the role of 'staying in the lane' of public health in terms of the information that they collected, their advocacy for policies and their area of expertise and the role of engaging in the balancing of multiple considerations, such as public health, feasibility and competing objectives (such as the economy) in the crafting of pandemic policy. We draw on the concept of boundary-work to examine how these roles were constructed. We conclude by considering the appropriateness as well as the ethical implications of these two approaches to public health policymaking.

Insecticidal roof barriers mounted on untreated bed nets can be as effective against Anopheles gambiae as regular insecticide-treated bed nets.

Barrier bednets (BBnets), regular bednets with a vertical insecticidal panel to target mosquitoes above the bednet roof, where they are most active, have the potential to improve existing Insecticidal Treated Bednets (ITNs), by reducing the quantity of insecticide required per net, reducing the toxic risks to those using the net, potentially increasing insecticide choice. We evaluated the performance of PermaNet 3.0 (P3) and untreated (Ut) bed nets with and without pyrethroid and piperonyl butoxide roof barriers in killing pyrethroid-resistant and susceptible Anopheles gambiae, simultaneously video-recording mosquito flight tracks. Bioassay results showed that treated roof barriers, particularly the longitudinal P3 barrier (P3L) could be an effective addition to a bed net: P3 + P3L were consistently significantly more effective than the reference P3 bednet while performance of untreated nets could be raised to equal that of the reference P3 following the addition of a P3 barrier. The BBnet's potential to augment existing bednets and enhance their performance is considered.

Video augmentation of the WHO cone assay to quantify mosquito behavioural responses to insecticide-treated nets.

BACKGROUND: Insecticide-treated nets (ITNs) using pyrethroids have been the main vector control tools deployed in malaria endemic countries and are responsible for the dramatic reduction in African malaria cases in the early 2000s. The World Health Organization (WHO) cone test was designed to assess the rapid toxicity effects of pyrethroid exposure on mosquito vectors but has yielded no insights beyond 60-min knockdown and 24-h mortality. As dual-active-ingredient (AI) ITNs become more widespread, bioassays that can provide realistic assessment of single- and dual-treated ITNs (i.e. nets with more than one active ingredient) are urgently needed. METHODS: We present an augmentation of the cone test that enables accurate quantification of vector behavioural responses (specifically movement, spatial and temporal occupancy) to ITNs using video recording and bespoke software that uses background segmentation methods to detect spatial changes in the movement of mosquitoes within the cone. Four strains of Anopheles gambiae sensu lato (s.l.) were exposed to four ITNs (PermaNet 2.0, PermaNet 3.0, Olyset Net, Interceptor G2) and untreated nets in these modified cone tests. Life history data (post-exposure blood-feeding, blood meal weight, longevity) for individual mosquitoes were recorded. RESULTS: All mosquitoes responded to the presence of ITNs, spending from 1.48 to 3.67 times more time in the upper region of the cone, depending on the ITN type. Of all ITNs, PermaNet 2.0 provoked the smallest change in behavioural response. Activity in the cone influenced observed post-exposure longevity, and in resistant strains exposed to Interceptor G2, the higher the activity, the greater the risk of dying, as long as the proportion of activity at the net surface was less than 50%. All ITNs inhibited blood-feeding, and smaller blood meals were taken when mosquitoes fed. CONCLUSIONS: The additional mosquito behaviour data obtained by using this modification to the WHO cone test provides unique insight into the innate responses of different mosquito strains on untreated nets and the entomological mode of action of ITNs, important evidence when evaluating ITN characteristics.

Acquired resistance to KRAS G12C small-molecule inhibitors via genetic/nongenetic mechanisms in lung cancer.

Inherent or acquired resistance to sotorasib poses a substantialt challenge for NSCLC treatment. Here, we demonstrate that acquired resistance to sotorasib in isogenic cells correlated with increased expression of integrin ß4 (ITGB4), a component of the focal adhesion complex. Silencing ITGB4 in tolerant cells improved sotorasib sensitivity, while overexpressing ITGB4 enhanced tolerance to sotorasib by supporting AKT-mTOR bypass signaling. Chronic treatment with sotorasib induced WNT expression and activated the WNT/ß-catenin signaling pathway. Thus, silencing both ITGB4 and ß-catenin significantly improved sotorasib sensitivity in tolerant, acquired, and inherently resistant cells. In addition, the proteasome inhibitor carfilzomib (CFZ) exhibited synergism with sotorasib by down-regulating ITGB4 and ß-catenin expression. Furthermore, adagrasib phenocopies the combination effect of sotorasib and CFZ by suppressing KRAS activity and inhibiting cell cycle progression in inherently resistant cells. Overall, our findings unveil previously unrecognized nongenetic mechanisms underlying resistance to sotorasib and propose a promising treatment strategy to overcome resistance.

Mapping Aedes aegypti indoor resting behavior reveals a preference vulnerable to householder-led vector control.

Many mosquito vectors rest inside human habitations, a behavioral trait that is exploited for vector control by indoor residual spraying (IRS) of interior walls with insecticide. Although IRS and its refined version targeted IRS are very effective against Aedes aegypti, they are expensive and logistically challenging to deliver in densely populated urban areas where outbreaks of dengue and other arboviruses are the greatest challenge. In experiments in Recife, Brazil, we set out to quantify the indoor resting behavior of Ae. aegypti at a level beyond that previously reported. We found that significantly more Ae. aegypti males, unfed and fed females visited the base of walls (height 0-20 cm, corresponding to 12.3% of the total wall surface) more frequently than upper wall areas, with the difference more pronounced at higher temperatures. When the lowest 20 cm of the walls was treated with an appropriate insecticide and colored black, we recorded up to 85% cumulative mortality after 24-h exposure in the experimental room. The findings are significant because feasibly, householders could treat this small and accessible target zone manually, without the need for visits by costly IRS teams or equipment, reducing insecticide use and enabling communities to actively protect their own indoor environment.

Tidyproteomics: an open-source R package and data object for quantitative proteomics post analysis and visualization.

BACKGROUND: The analysis of mass spectrometry-based quantitative proteomics data can be challenging given the variety of established analysis platforms, the differences in reporting formats, and a general lack of approachable standardized post-processing analyses such as sample group statistics, quantitative variation and even data filtering. We developed tidyproteomics to facilitate basic analysis, improve data interoperability and potentially ease the integration of new processing algorithms, mainly through the use of a simplified data-object. RESULTS: The R package tidyproteomics was developed as both a framework for standardizing quantitative proteomics data and a platform for analysis workflows, containing discrete functions that can be connected end-to-end, thus making it easier to define complex analyses by breaking them into small stepwise units. Additionally, as with any analysis workflow, choices made during analysis can have large impacts on the results and as such, tidyproteomics allows researchers to string each function together in any order, select from a variety of options and in some cases develop and incorporate custom algorithms. CONCLUSIONS: Tidyproteomics aims to simplify data exploration from multiple platforms, provide control over individual functions and analysis order, and serve as a tool to assemble complex repeatable processing workflows in a logical flow. Datasets in tidyproteomics are easy to work with, have a structure that allows for biological annotations to be added, and come with a framework for developing additional analysis tools. The consistent data structure and accessible analysis and plotting tools also offers a way for researchers to save time on mundane data manipulation tasks.

Inputs and Outputs of the Mammalian Circadian Clock.

Circadian rhythms in mammals are coordinated by the central circadian pacemaker, the suprachiasmatic nucleus (SCN). Light and other environmental inputs change the timing of the SCN neural network oscillator, which, in turn, sends output signals that entrain daily behavioral and physiological rhythms. While much is known about the molecular, neuronal, and network properties of the SCN itself, the circuits linking the outside world to the SCN and the SCN to rhythmic outputs are understudied. In this article, we review our current understanding of the synaptic and non-synaptic inputs onto and outputs from the SCN. We propose that a more complete description of SCN connectivity is needed to better explain how rhythms in nearly all behaviors and physiological processes are generated and to determine how, mechanistically, these rhythms are disrupted by disease or lifestyle.

The Widening Gap between the Digital Capability of the Care Workforce and Technology-Enabled Healthcare Delivery: A Nursing and Allied Health Analysis.

There is a need to ensure that healthcare organisations enable their workforces to use digital methods in service delivery. This study aimed to evaluate the current level of digital understanding and ability in nursing, midwifery, and allied health workforces and identify some of the training requirements to improve digital literacy in these health professionals. Representatives from eight healthcare organizations in Victoria, Australia participated in focus groups. Three digital frameworks informed the focus group topic guide that sought to examine the barriers and enablers to adopting digital healthcare along with training requirements to improve digital literacy. Twenty-three participants self-rated digital knowledge and skills using Likert scales and attended the focus groups. Mid-range scores were given for digital ability in nursing, midwifery, and allied health professionals. Focus group participants expressed concern over the gap between their organizations' adoption of digital methods relative to their digital ability, and there were concerns about cyber security. Participants also saw a need for the inclusion of consumers in digital design. Given the widening gap between digital innovation and health workforce digital capability, there is a need to accelerate digital literacy by rapidly deploying education and training and policies and procedures for digital service delivery.

From Housewives to Employees, the Mental Benefits of Employment across Women with Different Gender Role Attitudes and Parenthood Status.

Previous studies suggest that paid employment can improve workers' mental health status by offering a series of manifest and latent benefits (i.e., income, self-achievement and social engagement), which motivates policymakers' ongoing promotion of labour force participation as an approach to protect women's mental health status. This study extends the literature by investigating the mental health consequences of housewives' transition into paid employment across different gender role attitude groups. In addition, the study also tests the potential moderating role of the presence of children in relationships. This study yields two major findings by using nationally representative data (N = 1222) from the United Kingdom Longitudinal Household Study (2010-2014) and OLS regressions. First, from the first wave to the next, housewives who transitioned into paid employment reported better mental health status than those who remained housewives. Second, the presence of children can moderate such associations, but only among housewives with more traditional gender role attitudes. Specifically, among the traditional group, the mental benefits of transition into paid employment are more pronounced among those without children. Therefore, policymakers should develop more innovative approaches to promote housewives' mental health by considering a more gender-role-attitudes-sensitive design of future labour market policies.

MTCH2 is a mitochondrial outer membrane protein insertase.

In the mitochondrial outer membrane, α-helical transmembrane proteins play critical roles in cytoplasmic-mitochondrial communication. Using genome-wide CRISPR screens, we identified mitochondrial carrier homolog 2 (MTCH2), and its paralog MTCH1, and showed that it is required for insertion of biophysically diverse tail-anchored (TA), signal-anchored, and multipass proteins, but not outer membrane ß-barrel proteins. Purified MTCH2 was sufficient to mediate insertion into reconstituted proteoliposomes. Functional and mutational studies suggested that MTCH2 has evolved from a solute carrier transporter. MTCH2 uses membrane-embedded hydrophilic residues to function as a gatekeeper for the outer membrane, controlling mislocalization of TAs into the endoplasmic reticulum and modulating the sensitivity of leukemia cells to apoptosis. Our identification of MTCH2 as an insertase provides a mechanistic explanation for the diverse phenotypes and disease states associated with MTCH2 dysfunction.

Mechanisms of chlorate toxicity and resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that often encounters hypoxic/anoxic environments within the host, which increases its tolerance to many conventional antibiotics. Toward identifying novel treatments, we explored the therapeutic potential of chlorate, a pro-drug that kills hypoxic/anoxic, antibiotic-tolerant P. aeruginosa populations. While chlorate itself is relatively nontoxic, it is enzymatically reduced to the toxic oxidizing agent, chlorite, by hypoxically induced nitrate reductase. To better assess chlorate's therapeutic potential, we investigated mechanisms of chlorate toxicity and resistance in P. aeruginosa. We used transposon mutagenesis to identify genes that alter P. aeruginosa fitness during chlorate treatment, finding that methionine sulfoxide reductases (Msr), which repair oxidized methionine residues, support survival during chlorate stress. Chlorate treatment leads to proteome-wide methionine oxidation, which is exacerbated in a ∆msrA∆msrB strain. In response to chlorate, P. aeruginosa upregulates proteins involved in a wide range of functions, including metabolism, DNA replication/repair, protein repair, transcription, and translation, and these newly synthesized proteins are particularly vulnerable to methionine oxidation. The addition of exogenous methionine partially rescues P. aeruginosa survival during chlorate treatment, suggesting that widespread methionine oxidation contributes to death. Finally, we found that mutations that decrease nitrate reductase activity are a common mechanism of chlorate resistance.

Surface cysteines could protect the SARS-CoV-2 main protease from oxidative damage.

The SARS-CoV-2 main protease (Mpro) is responsible for cleaving twelve nonstructural proteins from the viral polyprotein. Mpro, a cysteine protease, is characterized by a large number of noncatalytic cysteine (Cys) residues, none involved in disulfide bonds. In the absence of a tertiary-structure stabilizing role for these residues, a possible alternative is that they are involved in redox processes. We report experimental work in support of a proposal that surface cysteines on Mpro can protect the active-site Cys145 from oxidation by reactive oxygen species (ROS). In investigations of enzyme kinetics, we found that mutating three surface cysteines to serines did not greatly affect activity, which in turn indicates that these cysteines could protect Cys145 from oxidative damage.

The Social Bridging Project: Intergenerational Phone-Based Connections With Older Adults During the COVID-19 Pandemic.

Millions of Americans aged 65+ are socially isolated and millions more report feeling lonely. Social isolation and loneliness in older adults were compounded by stay-at-home orders and other COVID-19 prevention measures. Although many Americans experienced no difficulties transitioning to the use of electronic devices as their primary means of communication and connection, some older adults were not similarly able to espouse this shift. Our aim was to reduce the impact of social isolation on older adults, increase their comfort in expressing feelings of loneliness, and assist them in acquiring technology skills and accessing telehealth and community supports. Participants received wellness calls for conversation, resource access and technology-based support. Most participants reported decreased loneliness and increased connectedness after the calls; half reported increased ease in expressing their feelings. Programs that provide phone-based support for older adults may reduce loneliness and increase social connectedness.

Circadian neurons in the paraventricular nucleus entrain and sustain daily rhythms in glucocorticoids.

Signals from the central circadian pacemaker, the suprachiasmatic nucleus (SCN), must be decoded to generate daily rhythms in hormone release. Here, we hypothesized that the SCN entrains rhythms in the paraventricular nucleus (PVN) to time the daily release of corticosterone. In vivo recording revealed a critical circuit from SCN vasoactive intestinal peptide (SCNVIP)-producing neurons to PVN corticotropin-releasing hormone (PVNCRH)-producing neurons. PVNCRH neurons peak in clock gene expression around midday and in calcium activity about three hours later. Loss of the clock gene Bmal1 in CRH neurons results in arrhythmic PVNCRH calcium activity and dramatically reduces the amplitude and precision of daily corticosterone release. SCNVIP activation reduces (and inactivation increases) corticosterone release and PVNCRH calcium activity, and daily SCNVIP activation entrains PVN clock gene rhythms by inhibiting PVNCRH neurons. We conclude that daily corticosterone release depends on coordinated clock gene and neuronal activity rhythms in both SCNVIP and PVNCRH neurons.

A minimal 3D model of mosquito flight behaviour around the human baited bed net.

BACKGROUND: Advances in digitized video-tracking and behavioural analysis have enabled accurate recording and quantification of mosquito flight and host-seeking behaviours, facilitating development of individual (agent) based models at much finer spatial scales than previously possible. METHODS: Quantified behavioural parameters were used to create a novel virtual testing model, capable of accurately simulating indoor flight behaviour by a virtual population of host-seeking mosquitoes as they interact with and respond to simulated stimuli from a human-occupied bed net. The model is described, including base mosquito behaviour, state transitions, environmental representation and host stimulus representation. RESULTS: In the absence of a bed net and human host bait, flight distribution of the model population was relatively uniform throughout the arena. Introducing an unbaited untreated bed net induced a change in distribution with an increase in landing events on the net surface, predominantly on the sides of the net. Adding the presence of a simulated human bait dramatically impacted flight distribution patterns, exploratory foraging and, the number and distribution of landing positions on the net, which were determined largely by the orientation of the human within. The model replicates experimental results with free-flying living mosquitoes at human-occupied bed nets, where contact occurs predominantly on the top surface of the net. This accuracy is important as it quantifies exposure to the lethal insecticide residues that may be unique to the net roof (or theoretically any other surface). Number of net contacts and height of contacts decreased with increasing attractant dispersal noise. CONCLUSIONS: Results generated by the model are an accurate representation of actual mosquito behaviour recorded at and around a human-occupied bed net in untreated and insecticide-treated nets. This fine-grained model is highly flexible and has significant potential for in silico screening of novel bed net designs, potentially reducing time and cost and accelerating the deployment of new and more effective tools for protecting against malaria in sub-Saharan Africa.

Optogenetic Methods for the Study of Circadian Rhythms.

A fundamental feature of circadian clock neurons across species is that they express circadian rhythms in spontaneous spike frequency. Spike frequency rhythms serve as both output timing signals of clock neurons as well as resonant elements of rhythms generation. Importantly, optogenetics, as applied to clock neurons, can enable investigation of the roles of clock neuron electrical activity in circadian timing. Here we describe protocols for using both in vitro and in vivo optogenetics directed to mammalian clock neurons in the suprachiasmatic nucleus to study circadian physiology and behavior. Optogenetic stimulation via channelrhodopsin, or inhibition via halorhodopsin, allows for the precise manipulation of neuronal firing rates across the SCN, and within specific neuronal subpopulations thereof, and can be combined with actigraphy and gene expression analysis.

Temporal and spatial Mycobacterium bovis prevalence patterns as evidenced in the All Wales Badgers Found Dead (AWBFD) survey of infection 2014-2016.

In order to better understand the spatial spread of bovine tuberculosis (bTB) in Wales, an All Wales Badgers Found Dead (AWBFD) survey was carried out from 2014-2016. For Wales, as a whole, there was a significant decrease (p < 0.001) in prevalence of bTB in badgers since a similar survey was carried out in 2005-2006, with a drop from 13.3% to 7.3%. The highest prevalence was observed for the High TB Area East (18.6%), which shares its border with England, and differed significantly (p < 0.001) from the High TB Area West (7.4%). The lowest proportion of carcases diagnosed with the disease (0.7%) was in the Low TB Area, followed by the two Intermediate TB Areas of Wales (2.7%). The M. bovis isolates from badgers tended to be similar to the genotypes of cattle in the same area, except in the Low TB Area. The direction of any cross species transmission and the drivers for this cannot be determined from this study. The spatial variations described here support the need for regionally adapted surveillance and control measures for bovine tuberculosis in Wales.