Increasing Proteome Coverage Through a Reduction in Analyte Complexity in Single-Cell Equivalent Samples.

The advancement of sophisticated instrumentation in mass spectrometry has catalyzed an in-depth exploration of complex proteomes. This exploration necessitates a nuanced balance in experimental design, particularly between quantitative precision and the enumeration of analytes detected. In bottom-up proteomics, a key challenge is that oversampling of abundant proteins can adversely affect the identification of a diverse array of unique proteins. This issue is especially pronounced in samples with limited analytes, such as small tissue biopsies or single-cell samples. Methods such as depletion and fractionation are suboptimal to reduce oversampling in single cell samples, and other improvements on LC and mass spectrometry technologies and methods have been developed to address the trade-off between precision and enumeration. We demonstrate that by using a monosubstrate protease for proteomic analysis of single-cell equivalent digest samples, an improvement in quantitative accuracy can be achieved, while maintaining high proteome coverage established by trypsin. This improvement is particularly vital for the field of single-cell proteomics, where single-cell samples with limited number of protein copies, especially in the context of low-abundance proteins, can benefit from considering analyte complexity. Considerations about analyte complexity, alongside chromatographic complexity, integration with data acquisition methods, and other factors such as those involving enzyme kinetics, will be crucial in the design of future single-cell workflows.

'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.

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.

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.

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.

SCN VIP Neurons Are Essential for Normal Light-Mediated Resetting of the Circadian System.

The suprachiasmatic nucleus (SCN) synchronizes circadian rhythms in behavior and physiology to the external light cycle, but the mechanisms by which this occurs are unclear. As the neuropeptide vasoactive intestinal peptide (VIP) is important for circadian light responses, we tested the hypothesis that rhythmic VIP-producing SCN neurons mediate circadian light responses in male and female mice. Using in vivo fiber photometry over multiple days, we found daily rhythms in spontaneous calcium events of SCN VIP neurons that peaked during the subjective day and were disrupted by constant light. The light-evoked calcium responses peaked around subjective dusk and were greater during the subjective night. Using novel VIP sensor cells, we found that the activity patterns in SCN VIP neurons correlated tightly with spontaneous and NMDA-evoked VIP release. Finally, in vivo hyperpolarization of VIP neurons attenuated light-induced shifts of daily rhythms in locomotion. We conclude that SCN VIP neurons exhibit circadian rhythms in spontaneous and light-responsive activity and are essential for the normal resetting of daily rhythms by environmental light.SIGNIFICANCE STATEMENT Daily rhythms in behavior and physiology, including sleep/wake and hormone release, are synchronized to local time by the master circadian pacemaker, the suprachiasmatic nucleus (SCN). The advent of artificial lighting and, consequently, light exposure at night, is associated with an increased risk of disease due to disrupted circadian rhythms. However, the mechanisms by which the SCN encodes normal and pathological light information are unclear. Here, we find that vasoactive intestinal peptide (VIP)-producing SCN neurons exhibit daily rhythms in neuronal activity and VIP release, and that blocking the activity of these neurons attenuates light-induced phase shifts. We conclude that rhythmic VIP neurons are an essential component of the circadian light transduction pathway.

Quantifying Syringe Exchange Program Operational Space in the District of Columbia.

Syringe exchange programs (SEPs) are effective structural interventions for HIV prevention among persons who inject drugs. In 2000, a buffer zone policy (the 1000 Foot Rule) was implemented in Washington, DC, that prohibited SEP operations within 1000 feet of schools. We examined changes in the amount of legal SEP operational space over time. We used data pertaining to school operations and their approximate physical property boundaries to quantify the impact of the 1000 Foot Rule on legal SEP operational space from its implementation in 2000-2013. Adherence to the 1000 Foot Rule reduced SEP operational space by more than 50 % annually since its implementation. These findings demonstrate the significant restrictions on the amount of legal SEP operational space in Washington, DC, that are imposed by the 1000 Foot Rule. Changing this policy could have a significant impact on SEP service delivery among injectors.

Assessing Syringe Exchange Program Access among Persons Who Inject Drugs (PWID) in the District of Columbia.

Prior research has explored spatial access to syringe exchange programs (SEPs) among persons who inject drugs (PWID), but these studies have been based on limited data from short periods of time. No research has explored changes in spatial access to SEPs among PWID longitudinally. The purpose of this research is to examine spatial access to SEPs among PWID who accessed services at a SEP in Washington, District of Columbia (DC), from 1996 to 2010. The geometric point distance estimation technique was used to calculate the mean walking distance PWID traveled from the centroid point of their zip code of home residence to the mobile exchange site where they accessed SEP services. Analysis of variance (ANOVA) was used to examine differences in walking distance measures by year. The results of this research suggest that the distance DC PWID traveled to access SEP services remained relatively constant (approximately 2.75 mi) from 2003 to 2008, but increased to just over 4 mi in 2010. This research provides support for expanding SEP operations such that PWID have increased access to their services. Increasing SEP accessibility may help resolve unmet needs among injectors.

Hypocretins, Neural Systems, Physiology, and Psychiatric Disorders.

The hypocretins (Hcrts), also known as orexins, have been among the most intensely studied neuropeptide systems since their discovery about two decades ago. Anatomical evidence shows that the hypothalamic neurons that produce hypocretins/orexins project widely throughout the entire brain, innervating the noradrenergic locus coeruleus, the cholinergic basal forebrain, the dopaminergic ventral tegmental area, the serotonergic raphe nuclei, the histaminergic tuberomammillary nucleus, and many other brain regions. By interacting with other neural systems, the Hcrt system profoundly modulates versatile physiological processes including arousal, food intake, emotion, attention, and reward. Importantly, interruption of the interactions between these systems has the potential to cause neurological and psychiatric diseases. Here, we review the modulation of diverse neural systems by Hcrts and summarize potential therapeutic strategies based on our understanding of the Hcrt system's role in physiology and pathophysiological processes.

Legal space for syringe exchange programs in hot spots of injection drug use-related crime.

BACKGROUND: Copious evidence indicates that syringe exchange programs (SEPs) are effective structural interventions for HIV prevention among persons who inject drugs (PWID). The efficacy of SEPs in supporting the public health needs of PWID populations is partially dependent on their accessibility and consistent utilization among injectors. Research has shown that SEP access is an important predictor of PWID retention at SEPs, yet policies exist that may limit the geographic areas where SEP operations may legally occur. Since 2000 in the District of Columbia (DC), SEP operations have been subject to the 1000 Foot Rule (§48-1121), a policy that prohibits the distribution of "any needle or syringe for the hypodermic injection of any illegal drug in any area of the District of Columbia which is within 1000 feet of a public or private elementary or secondary school (including a public charter school)." The 1000 Foot Rule may impede SEP services in areas that are in urgent need for harm reduction services, such as locations where injections are happening in "real time" or where drugs are purchased or exchanged. We examined the effects of the 1000 Foot Rule on SEP operational space in injection drug use (IDU)-related crime (i.e., heroin possession or distribution) hot spots from 2000 to 2010. METHODS: Data from the DC Metropolitan Police Department were used to identify IDU-related crime hot spots. School operation data were matched to a dataset that described the approximate physical property boundaries of land parcels. A 1000-ft buffer was applied to all school property boundaries. The overlap between the IDU-related crime hot spots and the school buffer zones was calculated by academic year. RESULTS: When overlaying the land space associated with IDU-related crime hot spots on the maps of school boundaries per the 1000-ft buffer zone stipulation, we found that the majority of land space in these locations was ineligible for legal SEP operations. More specifically, the ineligible space in the identified hot spots in each academic year ranged from 51.93 to 88.29 % of the total hot spot area. CONCLUSIONS: The removal of the 1000 Foot Rule could significantly improve the public health of PWID via increased access to harm reduction services. Buffer zone policies that restrict SEP operational space negatively affect the provision of harm reduction services to PWID.

Network-mediated encoding of circadian time: the suprachiasmatic nucleus (SCN) from genes to neurons to circuits, and back.

The transcriptional architecture of intracellular circadian clocks is similar across phyla, but in mammals interneuronal mechanisms confer a higher level of circadian integration. The suprachiasmatic nucleus (SCN) is a unique model to study these mechanisms, as it operates as a ∼24 h clock not only in the living animal, but also when isolated in culture. This "clock in a dish" can be used to address fundamental questions, such as how intraneuronal mechanisms are translated by SCN neurons into circuit-level emergent properties and how the circuit decodes, and responds to, light input. This review addresses recent developments in understanding the relationship between electrical activity, [Ca(2+)]i, and intracellular clocks. Furthermore, optogenetic and chemogenetic approaches to investigate the distinct roles of neurons and glial cells in circuit encoding of circadian time will be discussed, as well as the epigenetic and circuit-level mechanisms that enable the SCN to translate light input into coherent daily rhythms.

Assessing seasonality of travel distance to harm reduction service providers among persons who inject drugs.

BACKGROUND: Prior research has examined access to syringe exchange program (SEP) services among persons who inject drugs (PWID), but no research has been conducted to evaluate variations in SEP access based on season. This is an important gap in the literature given that seasonal weather patterns and inclement weather may affect SEP service utilization. The purpose of this research is to examine differences in access to SEPs by season among PWID in the District of Columbia (DC). FINDINGS: A geometric point distance estimation technique was applied to records from a DC SEP that operated from 1996 to 2011. We calculated the walking distance (via sidewalks) from the centroid point of zip code of home residence to the exchange site where PWID presented for services. Analysis of variance (ANOVA) was used to examine differences in walking distance measures by season. Differences in mean walking distance measures were statistically significant between winter and spring with PWID traveling approximately 2.88 and 2.77 miles, respectively, to access the SEP during these seasons. CONCLUSIONS: The results of this study suggest that seasonal differences in SEP accessibility may exist between winter and spring. PWID may benefit from harm reduction providers adapting their SEP operations to provide a greater diversity of exchange locations during seasons in which inclement weather may negatively influence engagement with SEPs. Increasing the number of exchange locations based on season may help resolve unmet needs among injectors.

Towards evidence-based emergency medicine: best BETs from the Manchester Royal Infirmary. BET 2: Clinical identification of acute thoracic aortic dissection.

A short cut review was carried out to establish whether the risk of thoracic aortic aneurysm can be assessed clinically at the bedside. 393 papers were found using the reported searches, of which two presented the best evidence to answer the clinical question. The author, date and country of publication, patient group studied, study type, relevant outcomes, results and study weaknesses of those best papers are tabulated. It is concluded that there are no prospectively tested rules to risk stratify chest pain for the risk of dissecting aortic aneurysm. The aortic dissection detection score might be useful but requires prospective validation in an emergency department cohort of patients with chest pain.

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.

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.

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.

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.

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.