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UNLABELLED: Patients with H3K27M-mutant diffuse midline glioma (DMG) have no proven effective therapies. ONC201 has recently demonstrated efficacy in these patients, but the mechanism behind this finding remains unknown. We assessed clinical outcomes, tumor sequencing, and tissue/cerebrospinal fluid (CSF) correlate samples from patients treated in two completed multisite clinical studies. Patients treated with ONC201 following initial radiation but prior to recurrence demonstrated a median overall survival of 21.7 months, whereas those treated after recurrence had a median overall survival of 9.3 months. Radiographic response was associated with increased expression of key tricarboxylic acid cycle-related genes in baseline tumor sequencing. ONC201 treatment increased 2-hydroxyglutarate levels in cultured H3K27M-DMG cells and patient CSF samples. This corresponded with increases in repressive H3K27me3 in vitro and in human tumors accompanied by epigenetic downregulation of cell cycle regulation and neuroglial differentiation genes. Overall, ONC201 demonstrates efficacy in H3K27M-DMG by disrupting integrated metabolic and epigenetic pathways and reversing pathognomonic H3K27me3 reduction. SIGNIFICANCE: The clinical, radiographic, and molecular analyses included in this study demonstrate the efficacy of ONC201 in H3K27M-mutant DMG and support ONC201 as the first monotherapy to improve outcomes in H3K27M-mutant DMG beyond radiation. Mechanistically, ONC201 disrupts integrated metabolic and epigenetic pathways and reverses pathognomonic H3K27me3 reduction. This article is featured in Selected Articles from This Issue, p. 2293.

Abstract Background Incarceration provides an opportunity for health interventions, including opioid use disorder (OUD) treatment and prevention of opioid-related overdoses post-release. All FDA-approved forms of medication for OUD (MOUD) treatment were mandated in several Massachusetts jails in 2019, with some jails offering extended-release buprenorphine (XR-Bup). Little is known about patient perspectives on and experiences with XR-Bup in carceral settings. Methods We conducted semi-structured interviews in 2022 with community-dwelling people who received MOUD during a recent incarceration in a Massachusetts jail. We asked participants about their experiences with and perspectives on XR-Bup while in jail. Qualitative data were double-coded deductively and reviewed inductively to identify emergent themes, which were structured using the Theoretical Framework of Acceptability (TFA). Results Participants (n = 38) had a mean age of 41.5 years, were 86% male, 84% White, 24% Hispanic, and 95% continued to receive MOUD at the time of their interview, including 11% receiving XR-Bup. Participants who viewed XR-Bup favorably appreciated avoiding the taste of sublingual buprenorphine; avoiding procedural difficulties and indignities associated with daily dosing in carceral settings (e.g., mouth checks, stigmatizing treatment from correctional staff); avoiding daily reminders of their addiction; experiencing less withdrawal; having extra time for other activities, such as work; and reduction of diversion of MOUD within the jail setting. Participants who viewed XR-Bup less favorably preferred to maintain their daily dosing routine; liked daily time out of their housing unit; wanted to know what was “going into my body everyday”; and feared needles and adverse events. Participants also reported that jail clinicians used XR-Bup for patients who were previously caught diverting sublingual buprenorphine, suggesting limited patient participation in decision-making around XR-Bup initiation in some jails. Conclusion People who received MOUD in Massachusetts jails had both favorable and unfavorable views and experiences with XR-Bup. Understanding these preferences can inform protocols in jails that are considering implementation of XR-Bup treatment.

Abstract Aim Illicitly manufactured fentanyl and its analogs are the primary drivers of opioid overdose deaths in the United States (U.S.). People who use drugs may be exposed to fentanyl or its analogs intentionally or unintentionally. This study sought to identify strategies used by rural people who use drugs to reduce harms associated with unintentional fentanyl exposure. Methods This analysis focused on 349 semi-structured qualitative interviews across 10 states and 58 rural counties in the U.S conducted between 2018 and 2020. Interview guides were collaboratively standardized across sites and included questions about drug use history (including drugs currently used, frequency of use, mode of administration) and questions specific to fentanyl. Deductive coding was used to code all data, then inductive coding of overdose and fentanyl codes was conducted by an interdisciplinary writing team. Results Participants described being concerned that fentanyl had saturated the drug market, in both stimulant and opioid supplies. Participants utilized strategies including: (1) avoiding drugs that were perceived to contain fentanyl, (2) buying drugs from trusted sources, (3) using fentanyl test strips, 4) using small doses and non-injection routes, (5) using with other people, (6) tasting, smelling, and looking at drugs before use, and (7) carrying and using naloxone. Most people who used drugs used a combination of these strategies as there was an overwhelming fear of fatal overdose. Conclusion People who use drugs living in rural areas of the U.S. are aware that fentanyl is in their drug supply and use several strategies to prevent associated harms, including fatal overdose. Increasing access to harm reduction tools (e.g., fentanyl test strips, naloxone) and services (e.g., community drug checking, syringe services programs, overdose prevention centers) should be prioritized to address the polysubstance-involved overdose crisis. These efforts should target persons who use opioids and other drugs that may contain fentanyl.

Abstract Background Fatal opioid-involved overdose rates increased precipitously from 5.0 per 100,000 population to 33.5 in Massachusetts between 1999 and 2022. Methods We used spatial rate smoothing techniques to identify persistent opioid overdose-involved fatality clusters at the ZIP Code Tabulation Area (ZCTA) level. Rate smoothing techniques were employed to identify locations of high fatal opioid overdose rates where population counts were low. In Massachusetts, this included areas with both sparse data and low population density. We used Local Indicators of Spatial Association (LISA) cluster analyses with the raw incidence rates, and the Empirical Bayes smoothed rates to identify clusters from 2011 to 2021. We also estimated Empirical Bayes LISA cluster estimates to identify clusters during the same period. We constructed measures of the socio-built environment and potentially inappropriate prescribing using principal components analysis. The resulting measures were used as covariates in Conditional Autoregressive Bayesian models that acknowledge spatial autocorrelation to predict both, if a ZCTA was part of an opioid-involved cluster for fatal overdose rates, as well as the number of times that it was part of a cluster of high incidence rates. Results LISA clusters for smoothed data were able to identify whether a ZCTA was part of a opioid involved fatality incidence cluster earlier in the study period, when compared to LISA clusters based on raw rates. PCA helped in identifying unique socio-environmental factors, such as minoritized populations and poverty, potentially inappropriate prescribing, access to amenities, and rurality by combining socioeconomic, built environment and prescription variables that were highly correlated with each other. In all models except for those that used raw rates to estimate whether a ZCTA was part of a high fatality cluster, opioid overdose fatality clusters in Massachusetts had high percentages of Black and Hispanic residents, and households experiencing poverty. The models that were fitted on Empirical Bayes LISA identified this phenomenon earlier in the study period than the raw rate LISA. However, all the models identified minoritized populations and poverty as significant factors in predicting the persistence of a ZCTA being part of a high opioid overdose cluster during this time period. Conclusion Conducting spatially robust analyses may help inform policies to identify community-level risks for opioid-involved overdose deaths sooner than depending on raw incidence rates alone. The results can help inform policy makers and planners about locations of persistent risk.

BackgroundLuspatercept, an inhibitor of the transforming growth factor beta (TGF-β) pathway, is a novel treatment for anemic patients with lower-risk myelodysplastic syndromes (MDS) with transfusion dependence (TD) who do not respond to erythropoiesis-stimulating agents (ESA) therapy or are not suitable candidates for this treatment. We present real-world experience with luspatercept therapy from two hematology centers in the Czech Republic.MethodsBy January 2024, 54 MDS patients (33 men, 21 women) with a median age of 74 years (range, 55–95) were treated with luspatercept ± ESA at two Charles University hematology centers in Prague and Hradec Králové. According to the WHO 2016 classification, the cohort included 32 MDS-RS-MLD, seven MDS-MLD, two patients with 5q- + ring sideroblasts (RS), 12 RARS-T, and 1 patient with CMML-0 + RS. SF3B1 mutation data were available for 45 patients. All patients were in the IPSS-R and IPSS-M lower-risk groups (except four IPSS-M high). The median follow-up was 17 months (range, 1–54). All patients were transfusion-dependent. Thirty-five (64.8%) patients had a high transfusion burden (HTB) with ≥ 4 transfusion units (TU)/8 weeks, and 19 (35.2%) had a low transfusion burden (LTB) (< 4 TU/8 weeks). The median time between diagnosis and initiation of luspatercept was 27 months (range, 4–156). ESA were used prior to luspatercept in 45 patients, and luspatercept was used as first-line treatment in nine patients. Thirty-one (61%) patients were treated simultaneously with ESA.ResultsOnly patients who received luspatercept for ≥ 8 weeks (51 patients) were assessed. We evaluated the achievement of transfusion independence (TI) lasting 8, 12, 16, and 24 weeks. Thirty-two (62.7%) patients achieved TI for ≥ 8 weeks, 31 (60.7%) for ≥ 12 weeks, 29 (56.8%) for ≥ 16 weeks, and 25 (49%) for ≥ 24 weeks. Hematologic improvement (HI) without TI was achieved in six patients (11.7%). Overall, HI + TI was achieved in 38 patients (74.5%). Epoetin alfa was used simultaneously in 31 patients (60.7%). In 21 (55.2%) of all responding patients, concomitant therapy with epoetin alfa led to an improved response, with 16 reaching TI. Thirteen (25.5%) patients were nonresponders. Eight (21%) patients experienced therapy failure and became transfusion-dependent again. Optimal response required a gradual increase in the luspatercept dose to 1.75 mg/kg in up to 35 patients, with 23 responders (TI + HI). Response rates varied by transfusion burden: 79% in LTB and 50% in HTB reached TI. Of RS+ patients, 70% reached TI, while only one out of five RS− patients achieved TI. Among 39 SF3B1-positive patients, 61.6% achieved TI. In the low and very low IPSS-M groups, 86% of patients responded (TI + HI), compared to 62% in the moderate-low group. Luspatercept was well-tolerated, with no adverse events higher than grade II toxicity.ConclusionWe have demonstrated in real-world clinical practice that luspatercept is a very effective agent, even in an unselected, pretreated, significantly TD MDS population. The effect was particularly high in the IPSS-M low and very low groups. We believe that the relatively high response rate in our patients was influenced by the frequent use of a higher dose (1.75 mg/kg) and especially by adding ESA to luspatercept in poorly responding patients.

Abstract Cryptic female choice (CFC) is a component of postcopulatory sexual selection that allows females to influence the fertilization success of sperm from different males. While its precise mechanisms remain unclear, they may involve the influence of the protein composition of the female reproductive fluids on sperm functionality. This study maps the protein composition of the cloacal fluid across different phases of female reproductive cycle in a sexually promiscuous passerine, the barn swallow. Similar to mammals, the protein composition in the female reproductive tract differed between receptive (when females copulate) and nonreceptive phases. With the change in the protein background, the enriched gene ontology terms also shifted. Within the receptive phase, distinctions were observed between proteomes sampled just before and during egg laying. However, three proteins exhibited increased abundance during the entire receptive phase compared to nonreceptive phases. These proteins are candidates in cryptic female choice, as all of them can influence the functionality of sperm or sperm-egg interaction. Our study demonstrates dynamic changes in the cloacal environment throughout the avian breeding cycle, emphasizing the importance of considering these fluctuations in studies of cryptic female choice.

Abstract Background Efforts to distribute naloxone have equipped more people with the ability to reverse opioid overdoses but people who use drugs are often reluctant to call 911 due to concerns for legal repercussions. Rural communities face unique challenges in reducing overdose deaths compared to urban communities, including limited access to harm reduction services as well as greater concerns about stigma and privacy. Methods The Rural Opioid Initiative was funded in 2017 to better understand the health-related harms associated with the opioid crisis in rural US communities and consists of eight studies spanning ten states and 65 counties. Each study conducted semi-structured qualitative interviews with people who use drugs to understand contextual factors influencing drug use and health behaviors. We analyzed qualitative data from seven studies with data available at the time of analysis to understand peer response to overdose. Results Of the 304 participants interviewed, 55% were men, 70% were white, 80% reported current injection drug use, and 60% reported methamphetamine use. Similar to what has been found in studies focused on urban settings, people who use drugs in rural communities use a range of strategies to reverse overdoses, including non-evidence-based approaches. Several reported that multiple doses of naloxone are needed to reverse overdose. Three themes emerged around the willingness to call 911, including (1) hesitancy to call 911 for fear of legal consequences, (2) negative perceptions or experiences with law enforcement officers, and (3) efforts to obtain medical intervention while avoiding identification/law enforcement involvement. Conclusion People who use drugs employ multiple strategies to attempt overdose reversal, including non-evidence-based approaches. Greater education about the most effective and least harmful strategies is needed. Reluctance to call 911 is rooted in concerns about potential legal consequences as well as perceptions about law enforcement officers, which may be heightened in rural communities where people who use drugs are more easily identified by law enforcement. People who use drugs will go to great strides to connect their peers to needed medical services, suggesting that comprehensive interventions to reduce interactions with law enforcement officers and eliminate legal consequences for reporting overdoses are critical.

Abstract Background Accurate prevalence estimates of drug use and its harms are important to characterize burden and develop interventions to reduce negative health outcomes and disparities. Lack of a sampling frame for marginalized/stigmatized populations, including persons who use drugs (PWUD) in rural settings, makes this challenging. Respondent-driven sampling (RDS) is frequently used to recruit PWUD. However, the validity of RDS-generated population-level prevalence estimates relies on assumptions that should be evaluated. Methods RDS was used to recruit PWUD across seven Rural Opioid Initiative studies between 2018-2020. To evaluate RDS assumptions, we computed recruitment homophily and design effects, generated convergence and bottleneck plots, and tested for recruitment and degree differences. We compared sample proportions with three RDS-adjusted estimators (two variations of RDS-I and RDS-II) for five variables of interest (past 30-day use of heroin, fentanyl, and methamphetamine; past 6-month homelessness; and being positive for hepatitis C virus (HCV) antibody) using linear regression with robust confidence intervals. We compared regression estimates for the associations between HCV positive antibody status and (a) heroin use, (b) fentanyl use, and (c) age using RDS-1 and RDS-II probability weights and no weights using logistic and modified Poisson regression and random-effects meta-analyses. Results Among 2,842 PWUD, median age was 34 years and 43% were female. Most participants (54%) reported opioids as their drug of choice, however regional differences were present (e.g., methamphetamine range: 4-52%). Many recruitment chains were not long enough to achieve sample equilibrium. Recruitment homophily was present for some variables. Differences with respect to recruitment and degree varied across studies. Prevalence estimates varied only slightly with different RDS weighting approaches, most confidence intervals overlapped. Variations in measures of association varied little based on weighting approach. Conclusions RDS was a useful recruitment tool for PWUD in rural settings. However, several violations of key RDS assumptions were observed which slightly impacts estimation of proportion although not associations.

Abstract Background In recent years, stimulant use has increased among persons who use opioids in the rural U.S., leading to high rates of overdose and death. We sought to understand motivations and contexts for stimulant use among persons who use opioids in a large, geographically diverse sample of persons who use drugs (PWUD) in the rural settings. Methods We conducted semi-structured individual interviews with PWUD at 8 U.S. sites spanning 10 states and 65 counties. Content areas included general substance use, injection drug use, changes in drug use, and harm reduction practices. We used an iterative open-coding process to comprehensively itemize and categorize content shared by participants related to concurrent use. Results We interviewed 349 PWUD (64% male, mean age 36). Of those discussing current use of stimulants in the context of opioid use (n = 137, 39%), the stimulant most used was methamphetamine (78%) followed by cocaine/crack (26%). Motivations for co-use included: 1) change in drug markets and cost considerations; 2) recreational goals, e.g., seeking stronger effects after heightened opioid tolerance; 3) practical goals, such as a desire to balance or alleviate the effects of the other drug, including the use of stimulants to avoid/reverse opioid overdose, and/or control symptoms of opioid withdrawal; and 4) functional goals, such as being simultaneously energized and pain-free in order to remain productive for employment. Conclusion In a rural U.S. cohort of PWUD, use of both stimulants and opioids was highly prevalent. Reasons for dual use found in the rural context compared to urban studies included changes in drug availability, functional/productivity goals, and the use of methamphetamine to offset opioid overdose. Education efforts and harm reduction services and treatment, such as access to naloxone, fentanyl test strips, and accessible drug treatment for combined opioid and stimulant use, are urgently needed in the rural U.S. to reduce overdose and other adverse outcomes.

Abstract This is a comprehensive retrospective study to characterize female dogs with canine mammary tumors (CMTs) using a dataset retrieved from the archives of the Division of Animal Pathology, Institute of Veterinary Medicine in Warsaw, and to identify prognostic factors. Clinical and histopathological data of 1447 dogs with CMTs were included. Malignant tumours were found in 83.3% (n = 1206), benign tumours in 11.7% (n = 169), and non-neoplastic lesions in 5.0% (n = 72) of dogs. Dogs most often had grade II carcinomas (38.2%, 215/562) of a single histological subtype (88.5%, 1281/1447), mostly simple carcinoma (35.3%, 510/1447). Dogs with a median age of 10 years significantly often had larger (≥ 3 cm) and malignant CMTs, whereas intact females had smaller tumours (median size 2.0 cm). However, the threshold value for the age of the dog in the differentiation of malignant and non-neoplastic/benign masses could not be determined. Most females were hormonally active (76.4%, 372/487). Hormonally active dogs significantly more often had multiple tumours. Multiple tumours were significantly smaller (median 2.5 cm) than single ones. Among pedigree dogs, small-breed dogs were mostly recorded (43%, 428/1006). Twelve breeds had an increased risk of CMTs, regardless of tumour behaviour, compared with the theoretical distribution of pedigree dogs in Poland. Four breeds were often affected only by malignant and other four breeds only by non-neoplastic/benign CMT. Large-breed dogs were significantly younger and affected by larger CMT (median 4 cm) compared with small- and medium-breed dogs. Ninety dogs with a malignant CMT and complete records were included in the full analysis of CMT-specific survival (CMT-SS) with a median follow-up time of 20.0 months. We showed that the timing of ovariohysterectomy in relation to mastectomy was significantly associated with grade, CMT-SS, and CMT-related death. We indicated the low diagnostic accuracy of palpation of regional lymph nodes (RLN) in the prediction of their metastatic involvement. By multivariable analysis, dogs with neoplastic emboli, tumour ulceration, and simple or complex carcinoma had a significantly higher risk of local recurrence. Tumour size > 3 cm was as a strong independent predictor of lung metastases. Compared with dogs with an easily separated localized tumour, dogs with a multiple/diffuse malignant CMT pattern had a fivefold higher risk of death. The risk of death was significantly higher in the presence of neoplastic emboli (~ fivefold) and tumour ulceration (~ fourfold). Furthermore, the presence of neoplastic emboli and large tumour size were independent predictors of CMT-related death.

Abstract Background Dirofilariasis is a vector-borne disease caused by parasitic nematodes of the genus Dirofilaria spp., considered an emerging concern in both veterinary and human medicine. Climate changes and human activities, such as pet travel, contribute to the spread of diseases to new non-endemic regions. Poland is dominated by subcutaneous dirofilariasis caused by D. repens infections. Cardiopulmonary dirofilariasis, also known as a heartworm disease is much more rare with only single autochthonous cases reported so far. Also, imported infections are observed sporadically in dogs traveling to endemic countries. In this study, we report the first case of a dog in Poland, never having traveled abroad, co-infected with Dirofilaria repens and Dirofilaria immitis. Case presentation A 14-year-old mixed breed, an intact male dog with fever, lightly pale mucosal membranes, moderate abdominal pain, and a mild cough was presented in a veterinary clinic in Warsaw, Poland. The examination of the blood sample collected for complete morphology and biochemistry revealed the presence of live microfilariae. Presence of the DNA of both microfilariae species was detected using Real-Time PCR with species-specific primers. Conclusions Since the remaining diagnostic methods like Knott’s test, antigen test or echocardiography did not reveal the presence of D. immitis, we discussed the impact of microfilariae periodicity and low worm burden infections on the limited efficiency of these techniques. We strongly recommend using a mixed diagnostic approach for the most sensitive and specific diagnosis since the ideal diagnostic method does not exist, and several factors may contribute to misdiagnosis. Furthermore, we considered factors that contribute to the uncontrolled spread of dirofilariasis such as climate changes, introduction of new species of mosquitoes competent for the transmission of the disease, and wildlife animals as an important reservoir of this parasitosis. Given that Poland shares borders with countries classified as endemic and pre-endemic for D. immitis, such as Slovakia and Ukraine, it is reasonable to anticipate a rise in autochthonous heartworm infections and shifts in the epidemiological pattern of dirofilariasis in the coming years.

Abstract The formation of hematopoietic cells relies on the chromatin remodeling activities of ISWI ATPase SMARCA5 (SNF2H) and its complexes. The Smarca5 null and conditional alleles have been used to study its functions in embryonic and organ development in mice. These mouse model phenotypes vary from embryonic lethality of constitutive knockout to less severe phenotypes observed in tissue-specific Smarca5 deletions, e.g., in the hematopoietic system. Here we show that, in a gene dosage-dependent manner, the hypomorphic allele of SMARCA5 (S5 tg ) can rescue not only the developmental arrest in hematopoiesis in the hCD2iCre model but also the lethal phenotypes associated with constitutive Smarca5 deletion or Vav1iCre-driven conditional knockout in hematopoietic progenitor cells. Interestingly, the latter model also provided evidence for the role of SMARCA5 expression level in hematopoietic stem cells, as the Vav1iCre S5 tg animals accumulate stem and progenitor cells. Furthermore, their hematopoietic stem cells exhibited impaired lymphoid lineage entry and differentiation. This observation contrasts with the myeloid lineage which is developing without significant disturbances. Our findings indicate that animals with low expression of SMARCA5 exhibit normal embryonic development with altered lymphoid entry within the hematopoietic stem cell compartment.

Abstract Background Hepatitis C virus (HCV) disproportionately affects rural communities, where health services are geographically dispersed. It remains unknown whether proximity to a syringe services program (SSP) is associated with HCV infection among rural people who inject drugs (PWID). Methods Data are from a cross-sectional sample of adults who reported injecting drugs in the past 30 days recruited from rural counties in New Hampshire, Vermont, and Massachusetts (2018–2019). We calculated the road network distance between each participant’s address and the nearest fixed-site SSP, categorized as ≤ 1 mile, 1–3 miles, 3–10 miles, and > 10 miles. Staff performed HCV antibody tests and a survey assessed past 30-day injection equipment sharing practices: borrowing used syringes, borrowing other used injection equipment, and backloading. Mixed effects modified Poisson regression estimated prevalence ratios (aPR) and 95% confidence intervals (95% CI). Analyses were also stratified by means of transportation. Results Among 330 PWID, 25% lived ≤ 1 mile of the nearest SSP, 17% lived 1–3 miles of an SSP, 12% lived 3–10 miles of an SSP, and 46% lived > 10 miles from an SSP. In multivariable models, compared to PWID who lived within 1 mile of an SSP, those who lived 3 to 10 miles away had a higher prevalence of HCV seropositivity (aPR: 1.25, 95% CI 1.06–1.46), borrowing other used injection equipment (aPR: 1.23, 95% CI 1.04–1.46), and backloading (aPR: 1.48, 95% CI 1.17–1.88). Similar results were observed for PWID living > 10 miles from an SSP: aPR [HCV]: 1.19, 95% CI 1.01–1.40; aPR [borrowing other used equipment]:1.45, 95% CI 1.29–1.63; and aPR [backloading]: 1.59, 95% CI 1.13–2.24. Associations between living 1 to 3 miles of an SSP and each outcome did not reach statistical significance. When stratified by means of transportation, associations between distance to SSP and each outcome (except borrowing other used injection equipment) were only observed among PWID who traveled by other means (versus traveled by automobile). Conclusions Among PWID in rural New England, living farther from a fixed-site SSP was associated with a higher prevalence of HCV seropositivity, borrowing other used injection equipment, and backloading, reinforcing the need to increase SSP accessibility in rural areas. Means of transportation may modify this relationship.

In most mammals, conspecific chemosensory communication relies on semiochemical release within complex bodily secretions and subsequent stimulus detection by the vomeronasal organ (VNO). Urine, a rich source of ethologically relevant chemosignals, conveys detailed information about sex, social hierarchy, health, and reproductive state, which becomes accessible to a conspecific via vomeronasal sampling. So far, however, numerous aspects of social chemosignaling along the vomeronasal pathway remain unclear. Moreover, since virtually all research on vomeronasal physiology is based on secretions derived from inbred laboratory mice, it remains uncertain whether such stimuli provide a true representation of potentially more relevant cues found in the wild. Here, we combine a robust low-noise VNO activity assay with comparative molecular profiling of sex- and strain-specific mouse urine samples from two inbred laboratory strains as well as from wild mice. With comprehensive molecular portraits of these secretions, VNO activity analysis now enables us to (i) assess whether and, if so, how much sex/strain-selective ‘raw’ chemical information in urine is accessible via vomeronasal sampling; (ii) identify which chemicals exhibit sufficient discriminatory power to signal an animal’s sex, strain, or both; (iii) determine the extent to which wild mouse secretions are unique; and (iv) analyze whether vomeronasal response profiles differ between strains. We report both sex- and, in particular, strain-selective VNO representations of chemical information. Within the urinary ‘secretome’, both volatile compounds and proteins exhibit sufficient discriminative power to provide sex- and strain-specific molecular fingerprints. While total protein amount is substantially enriched in male urine, females secrete a larger variety at overall comparatively low concentrations. Surprisingly, the molecular spectrum of wild mouse urine does not dramatically exceed that of inbred strains. Finally, vomeronasal response profiles differ between C57BL/6 and BALB/c animals, with particularly disparate representations of female semiochemicals.

The paper deals with the implementation of research concerning the collection routes of a selected company and subsequent streamlining processes in the field of distribution logistics. Specifically, the paper is focused on the optimization of collection routes of textile waste for the customer, i.e., a contractual partner of the company under investigation. The objective of the paper is to analyse step by step the current state of logistics of supplying a specific warehouse, followed by the application of the centre of gravity method for proposing a warehouse relocation. Finally, the individual routes to collection points are optimized. In general, optimization is done for two basic reasons: profit maximization and logistics costs minimization, which ultimately has a positive impact on earnings. The outcome is to determine the optimal routes with respect to costs and traffic while considering complications that may occur in a given transport territory.

Abstract Background Research conducted in urban areas has highlighted the impact of housing instability on people who inject drugs (PWID), revealing that it exacerbates vulnerability to drug-related harms and impedes syringe service program (SSP) use. However, few studies have explored the effects of houselessness on SSP use among rural PWID. This study examines the relationship between houselessness and SSP utilization among PWID in eight rural areas across 10 states. Methods PWID were recruited using respondent-driven sampling for a cross-sectional survey that queried self-reported drug use and SSP utilization in the prior 30 days, houselessness in the prior 6 months and sociodemographic characteristics. Using binomial logistic regression, we examined the relationship between experiencing houselessness and any SSP use. To assess the relationship between houselessness and the frequency of SSP use, we conducted multinomial logistic regression analyses among participants reporting any past 30-day SSP use. Results Among 2394 rural PWID, 56.5% had experienced houselessness in the prior 6 months, and 43.5% reported past 30-day SSP use. PWID who had experienced houselessness were more likely to report using an SSP compared to their housed counterparts (adjusted odds ratio [aOR] = 1.24 [95% confidence intervals [CI] 1.01, 1.52]). Among those who had used an SSP at least once (n = 972), those who experienced houselessness were just as likely to report SSP use two (aOR = 0.90 [95% CI 0.60, 1.36]) and three times (aOR = 1.18 [95% CI 0.77, 1.98]) compared to once. However, they were less likely to visit an SSP four or more times compared to once in the prior 30 days (aOR = 0.59 [95% CI 0.40, 0.85]). Conclusion This study provides evidence that rural PWID who experience houselessness utilize SSPs at similar or higher rates as their housed counterparts. However, housing instability may pose barriers to more frequent SSP use. These findings are significant as people who experience houselessness are at increased risk for drug-related harms and encounter additional challenges when attempting to access SSPs.

The ongoing hepatitis C virus (HCV) epidemic in the United States disproportionately affects rural people who inject drugs (PWID). This study explores the HCV risk environment in rural northern New England by examining PWID experiences and perceptions of HCV and injection equipment-sharing practices. We performed a thematic analysis on semi-structured interviews conducted with 21 adults with a history of injection drug use from rural New Hampshire, Vermont, and Massachusetts between April 2018 and August 2019. Salient themes included: (1) limited and varied access to sterile syringe sources; (2) syringe scarcity contributing to the use of informal syringe sources (e.g., secondary syringe exchange or syringe sellers who purchased syringes from out-of-state pharmacies); (3) syringe scarcity contributing to syringe sharing; (4) linkages among decisions about syringe sharing and perceptions of HCV risk, HCV status, and interpersonal trust; and (5) confusion and misconceptions about HCV, including difficulty learning one’s HCV status, inadequate HCV education, and misconceptions regarding HCV transmission and treatment. Efforts to prevent and eliminate HCV among rural PWID should expand syringe access, increase awareness of HCV as a serious but preventable risk, and acknowledge social connections as potential influences on syringe access and syringe-sharing decisions.

Hepatitis C virus (HCV) disproportionately affects people who inject drugs (PWID). Although HCV has become universally curable since the arrival of direct-acting antivirals, barriers exist to facilitating care and cure in this historically hard-to-reach population, including limited testing and healthcare services and healthcare stigma, issues that are compounded in rural areas. Telehealth is effective in increasing access to HCV care and cure, but innovative approaches of testing and care are required to fully address the need among rural PWID, which led to our study examining a mobile telehealth model for treating HCV. In this commentary, we discuss lessons learned delivering telehealth on a mobile unit, important factors for consideration when designing a mobile intervention, and we suggest an ideal model to increase access to HCV testing and treatment and other services for rural PWID.

We have investigated how connexin 46 (Cx46) regulates lens stiffness by studying different Cx46 knockout (Cx46KO) mice. A modified muscle lever system was used to determine the lens stiffness of wild-type (WT) and Cx46KO mice at the C57BL/6J (B6) and the 129SvJae (129) strain backgrounds according to total lens displacement at the point of maximum force when fresh lenses were compressed with a maximum of 2 mN of force. In comparison to B6-WT controls, young and old B6-Cx46KO lenses showed 23% and 28% reductions in lens displacement, respectively. Comparing to 129-WT controls, old 129-Cx46KO lenses showed 50% reduction in the lens displacement while young 129-Cx46KO lenses displayed similar displacement. Old B6-Cx46KO and old 129-Cx46KO lenses showed almost identical lens displacement, 128 μm versus 127 μm. Morphological data revealed unique changes of peripheral fiber cell shapes in young B6-WT lenses but not in young B6-Cx46KO, 129-WT and 129-Cx46KO lenses. This work reveals Cx46 deletion increases the lens stiffness in both young and old mice at B6 strain background but only in old mice at 129 strain background which contains intermediate filament CP49 gene deletion. Cx46 impairment increases old mouse lens stiffness and may contribute to the development of presbyopia.

ABSTRACTSymbiotic microbial communities affect the host immune system and produce molecules contributing to the odor of an individual. In many mammalian species, saliva and vaginal fluids are important sources of chemical signals that originate from bacterial metabolism and may act as honest signals of health and reproductive status. In this study, we aimed to define oral and vaginal microbiomes and their dynamics throughout the estrous cycle in wild house mice. In addition, we analyzed a subset of vaginal proteomes and metabolomes to detect potential interactions with microbiomes. 16S rRNA sequencing revealed that both saliva and vagina are dominated by Firmicutes and Proteobacteria but differ at the genus level. The oral microbiome is more stable during the estrous cycle and most abundant bacteria belong to the genera Gemella and Streptococcus, while the vaginal microbiome shows higher bacterial diversity and dynamics during the reproductive cycle and is characterized by the dominance of Muribacter and Rodentibacter. These two genera cover around 50% of the bacterial community during estrus. Proteomic profiling of vaginal fluids revealed specific protein patterns associated with different estrous phases. Highly expressed proteins in estrus involve the keratinization process thus providing estrus markers (e.g., Hrnr) while some proteins are downregulated such as immune-related proteins that limit bacterial growth (Camp, Clu, Elane, Lyz2, and Ngp). The vaginal metabolome contains volatile compounds potentially involved in chemical communication, for example, ketones, aldehydes, and esters of carboxylic acids. Data integration of all three OMICs data sets revealed high correlations, thus providing evidence that microbiomes, host proteomes, and metabolomes may interact.IMPORTANCEOur data revealed dynamic changes in vaginal, but not salivary, microbiome composition during the reproductive cycle of wild mice. With multiple OMICs platforms, we provide evidence that changes in microbiota in the vaginal environment are accompanied by changes in the proteomic and metabolomics profiles of the host. This study describes the natural microbiota of wild mice and may contribute to a better understanding of microbiome-host immune system interactions during the hormonal and cellular changes in the female reproductive tract. Moreover, analysis of volatiles in the vaginal fluid shows particular substances that can be involved in chemical communication and reproductive behavior.

The sustainability of international transport remains a critical topic of concern in both theory and practice. Ideally, scientific literature should align with practical needs, but such concurrence is not always present. This disparity is also evident in the specific issue of consignment delivery, which is currently of a great practical importance but lacks sufficient coverage in scientific literature. This paper aims to analyse current trends in maritime transport and their relevance to achieving global sustainability in international transportation. Additionally, it seeks to compare these findings with the practical needs and requirements of the industry, in order to provide constructive recommendations for further research. To accomplish this objective, a bibliometric analysis was conducted using scientific databases, including Web of Science and/or SCOPUS.

Abstract Background Chemical communication in mammals involves globular lipocalins that protect and transport pheromones during their passage out of the body. Efficient communication via this protein - pheromone complex is essential for triggering multiple responses including aggression, mate choice, copulatory behaviour, and onset and synchronization of oestrus. The roles of lipocalins in communication were studied in many organisms and especially in mice (i.e. Mus musculus domesticus) which excrete Major Urinary Proteins (Mup) in excessive amounts in saliva and urine. Other mammals, however, often lack the genes for Mups or their expression is very low. Therefore, we aimed at characterization of candidate lipocalins in Myodes glareolus which are potentially linked to chemical communication. One of them is Aphrodisin which is a unique lipocalin that was previously described from hamster vaginal discharge and is known to carry pheromones stimulating copulatory behaviour in males. Results Here we show that Aphrodisin-like proteins exist in other species, belong to a group of Odorant Binding Proteins (Obp), and contrary to the expression of Aphrodisin only in hamster genital tract and parotid glands of females, we have detected these transcripts in both sexes of M. glareolus with the expression confirmed in various tissues including prostate, prepucial and salivary glands, liver and uterus. On the level of mRNA, we have detected three different gene variants. To assess their relevance for chemical communication we investigated the occurrence of particular proteins in saliva, urine and vaginal discharge. On the protein level we confirmed the presence of Obp2 and Obp3 in both saliva and urine. Appropriate bands in the range of 17-20 kDa from vaginal discharge were, however, beyond the MS detection limits. Conclusion Our results demonstrate that three novel Obps (Obp1, Obp2, and Obp3) are predominant lipocalins in Myodes urine and saliva. On the protein level we have detected further variants and thus we assume that similarly as Major Urinary Proteins in mice, these proteins may be important in chemical communication in this Cricetid rodent.

Abstract Background Medications for opioid use disorder (MOUD; methadone, buprenorphine, naltrexone) are the most effective treatments for OUD, and MOUD is protective against fatal overdoses. However, continued illegal drug use can increase the risk of treatment discontinuation. Given the widespread presence of fentanyl in the drug supply, research is needed to understand who is at greatest risk for concurrent MOUD and drug use and the contexts shaping use and treatment discontinuation. Methods From 2017 to 2020, Massachusetts residents with past-30-day illegal drug use completed surveys (N = 284) and interviews (N = 99) about MOUD and drug use. An age-adjusted multinomial logistic regression model tested associations between past-30-day drug use and MOUD use (current/past/never). Among those on methadone or buprenorphine (N = 108), multivariable logistic regression models examined the association between socio-demographics, MOUD type; and past-30-day use of heroin/fentanyl; crack; benzodiazepines; and pain medications. Qualitative interviews explored drivers of concurrent drug and MOUD use. Results Most (79.9%) participants had used MOUD (38.7% currently; 41.2% past), and past 30-day drug use was high: 74.4% heroin/fentanyl; 51.4% crack cocaine; 31.3% benzodiazepines, and 18% pain medications. In exploring drug use by MOUD history, multinomial regression analyses found that crack use was positively associated with past and current MOUD use (outcome referent: never used MOUD); whereas benzodiazepine use was not associated with past MOUD use but was positively associated with current use. Conversely, pain medication use was associated with reduced odds of past and current MOUD use. Among those on methadone or buprenorphine, separate multivariable logistic regression models found that benzodiazepine and methadone use were positively associated with heroin/fentanyl use; living in a medium-sized city and sex work were positively associated with crack use; heroin/fentanyl use was positively associated with benzodiazepine use; and witnessing an overdose was inversely associated with pain medication use. Many participants qualitatively reported reducing illegal opioid use while on MOUD, yet inadequate dosage, trauma, psychological cravings, and environmental triggers drove their continued drug use, which increased their risk of treatment discontinuation and overdose. Conclusions Findings highlight variations in continued drug use by MOUD use history, reasons for concurrent use, and implications for MOUD treatment delivery and continuity.

Abstract In most mammals and particularly in mice, chemical communication relies on the detection of ethologically relevant fitness-related cues from other individuals. In mice, urine is the primary source of these signals, so we employed proteomics and metabolomics to identify key components of chemical signalling. We show that there is a correspondence between urinary volatiles and proteins in the representation of genetic background, sex and environment in two house mouse subspecies Mus musculus musculus and M. m. domesticus. We found that environment has a strong influence upon proteomic and metabolomic variation and that volatile mixtures better represent males while females have surprisingly more sex-biased proteins. Using machine learning and combined-omics techniques, we identified mixtures of metabolites and proteins that are associated with biological features.

Abstract Background In rodents, the cell surface complement regulatory protein CD46 is expressed solely on the spermatozoal acrosome membrane. Ablation of the CD46 gene is associated with a faster acrosome reaction. Sperm from Apodemus flavicollis (yellow-necked field mice), A. microps (pygmy field mice) and A. sylvaticus (European wood mice) fail to express CD46 protein and exhibit a more rapid acrosome reaction rate than Mus (house mice) or BALB/c mice. A. agrarius (striped field mice) belong to a different Apodemus subgenus and have pronounced promiscuity and large relative testis size. The aim of this study was to determine whether A. agrarius sperm fail to express CD46 protein and, if so, whether A. agrarius have a faster acrosome reaction than Mus. Methods Reverse transcription polymerase chain reaction (RT-PCR) was used to assess whether A. agrarius transcribe testicular CD46 mRNA. RT-PCR was supplemented with 3'- and 5'-rapid amplification of cDNA ends to determine the complete nucleotide sequence of A. agrarius CD46. Fluorescence microscopy was used to assess whether CD46 protein is expressed by A. agrarius sperm. The acrosome status of A. agrarius sperm was calculated over time by immunocytochemistry using peanut agglutinin lectin. Results We demonstrate that A. agrarius mice transcribe two unique alternatively spliced testicular CD46 mRNA transcripts, both lacking exon 7, which differ from those described previously in other Apodemus species. The larger A. agrarius CD46 transcript has an insert between exons 10 and 11 which, if translated, would result in a novel cytoplasmic tail. In addition, A. agrarius CD46 transcripts have an extended AU-rich 3'-untranslated region (UTR) and a truncated 5'-UTR, resulting in failure to express spermatozoal CD46 protein. We show that A. agrarius has a significantly faster spontaneous acrosome reaction rate than A. sylvaticus and Mus. Conclusion Absence of CD46 protein expression is associated with acrosomal instability in rodents. A. agrarius mice express novel CD46 transcripts, resulting in the trade of spermatozoal CD46 protein expression for a rapid acrosome reaction rate, in common with other species of field mice. This provides a strategy to increase competitive sperm advantage for individuals, leading to faster fertilisation in this highly promiscuous genus.

This article presents the results of dynamic tests of sandstone samples differing in strength parameters and porosity, which were carried out with the use of the split Hopkinson pressure bar (SHPB). For this study, three types of sandstones were considered: two from the region of India (Kandla Grey and Apricot Pink) and one from Central Europe (Barwald). The strength parameters of the samples were identified in static tests (UCS, BTS tests), whereas the porosity was measured using computed tomography. The performed scanning allowed the volume of the pores and their distribution in the samples to be identified. Dynamic tests involved loading the cylindrical samples with a diameter of 23 m in the range of high strain rates, i.e., ε˙ = 102 ÷ 103/s, using the SHPB (split Hopkinson pressure bar) method. Samples with three different values of slenderness were used for testing (L/D = 1, 0.75 and 0.5). Based on the dynamic characteristics of the samples, the maximum dynamic stresses, Dynamic Increase Factor (DIF) and the amount of energy absorbed by the samples were determined. The conducted research indicates a significant impact of material porosity on the amount of dissipated energy under conditions of high strain rates. The research indicates that the values of this parameter for Apricot Pink and Kandla Grey sandstones (slenderness L/D = ¾ and L/D = ½) are similar, although the uniaxial compressive strength (UCS) of Kandla Grey sandstone is approximately 60% higher than that of Apricot Pink sandstone. As a result of the sample destruction process, various forms of sample destruction were obtained. The performed grain analysis indicates a significant increase in the smallest fraction (