Advances in targeted liquid chromatography-tandem mass spectrometry methods for endocannabinoid and N-acylethanolamine quantification in biological matrices: A systematic review.

Liquid chromatography paired with tandem mass spectrometry (LC-MS/MS) is the gold standard in measurement of endocannabinoid concentrations in biomatrices. We conducted a systematic review of literature to identify advances in targeted LC-MS/MS methods in the period 2017-2024. We found that LC-MS/MS methods for endocannabinoid quantification are relatively consistent both across time and across biomatrices. Recent advances have primarily been in three areas: (1) sample preparation techniques, specific to the chosen biomatrix; (2) the range of biomatrices tested, recently favoring blood matrices; and (3) the breadth of endocannabinoid and endocannabinoid-like analytes incorporated into assays. This review provides a summary of the recent literature and a guide for researchers looking to establish the best methods for quantifying endocannabinoids in a range of biomatrices.

Playing pin-the-tail-on-the-protein in extracellular vesicle (EV) proteomics.

Extracellular vesicles (EVs) are anucleate particles enclosed by a lipid bilayer that are released from cells via exocytosis or direct budding from the plasma membrane. They contain an array of important molecular cargo such as proteins, nucleic acids, and lipids, and can transfer these cargoes to recipient cells as a means of intercellular communication. One of the overarching paradigms in the field of EV research is that EV cargo should reflect the biological state of the cell of origin. The true relationship or extent of this correlation is confounded by many factors, including the numerous ways one can isolate or enrich EVs, overlap in the biophysical properties of different classes of EVs, and analytical limitations. This presents a challenge to research aimed at detecting low-abundant EV-encapsulated nucleic acids or proteins in biofluids for biomarker research and underpins technical obstacles in the confident assessment of the proteomic landscape of EVs that may be affected by sample-type specific or disease-associated proteoforms. Improving our understanding of EV biogenesis, cargo loading, and developments in top-down proteomics may guide us towards advanced approaches for selective EV and molecular cargo enrichment, which could aid EV diagnostics and therapeutics research.

SWATH-MS Analysis of Blood Plasma and Circulating Small Extracellular Vesicles Enables Detection of Putative Protein Biomarkers of Fertility in Young and Aged Dairy Cows.

The development of biomarkers of fertility could provide benefits for the genetic improvement of dairy cows. Circulating small extracellular vesicles (sEVs) show promise as diagnostic or prognostic markers since their cargo reflects the metabolic state of the cell of origin; thus, they mirror the physiological status of the host. Here, we employed data-independent acquisition mass spectrometry to survey the plasma and plasma sEV proteomes of two different cohorts of Young (Peripubertal; n = 30) and Aged (Primiparous; n = 20) dairy cows (Bos taurus) of high- and low-genetic merit of fertility and known pregnancy outcomes (ProteomeXchange data set identifier PXD042891). We established predictive models of fertility status with an area under the curve of 0.97 (sEV; p value = 3.302e-07) and 0.95 (plasma; p value = 6.405e-08). Biomarker candidates unique to high-fertility Young cattle had a sensitivity of 0.77 and specificity of 0.67 (*p = 0.0287). Low-fertility biomarker candidates uniquely identified in sEVs from Young and Aged cattle had a sensitivity and specificity of 0.69 and 1.0, respectively (***p = 0.0005). Our bioinformatics pipeline enabled quantification of plasma and circulating sEV proteins associated with fertility phenotype. Further investigations are warranted to validate this research in a larger population, which may lead to improved classification of fertility status in cattle.

Repairing small type I hiatal hernias at the time of RYGB is not necessary to achieve resolution of reflux symptoms.

INTRODUCTION: Roux-en-Y gastric bypass (RYGB) is the gold standard operation for gastroesophageal reflux disease (GERD) in patients with severe obesity, but there is variability in surgeon opinion regarding whether small type I hiatal hernias (HH) require routine repair concurrently with RYGB. We sought to examine whether leaving small type I HHs unrepaired during RYGB affected GERD outcomes. METHODS: Pre-operatively our patients all receive endoscopy, and select patients with reflux symptoms receive esophagram based on attending surgeon practice and preference. We routinely repair paraesophageal hernias (PEH) concurrently with RYGB, but refrain from repairing small type I HH if, intra-operatively, the gastric fat pad and cardia are below the diaphragm with no evidence of retraction into the mediastinum. Records from 268 consecutive patients undergoing primary RYGB between January 2016 and February 2021 who completed pre-operative GERD-HRQL assessments were reviewed for presence of type I HH or PEH. Mann-Whitney U tests examined the pre-operative to post-operative change in GERD-HRQL in patients with type I HH left unrepaired at the time of RYGB (HH group) and patients with no hernia (NH group). RESULTS: Pre-operatively, GERD-HRQL scores were not statistically different between HH group (median = 7, mean = 8.5, n = 100) and NH group (median = 6.5, mean = 7.2, n = 141) (p > 0.05). Post-operatively, there was no increase in GERD-HRQL scores patients whose hernias were left unrepaired. Neither group had clinically pathologic post-operative GERD-HRQL scores, with median 6 months scores of 1 for HH group (n = 68) versus 1.5 for NH group (n = 90) (p > 0.05), and median 12 months scores of 1.5 for HH group (n = 40) versus 1 for NH group (n = 56) (p > 0.05). CONCLUSION: Repair of small type I HH is not necessary to achieve effective, durable resolution of reflux symptoms with RYGB. Omitting repair reduces operative time, cost, and potential risk without adverse impact on post-operative reflux symptoms.

The Role of Trust in Older Adult Service Provision at the Onset of the COVID-19 Pandemic.

The COVID-19 pandemic caused near immediate service delivery adaptation among social service and healthcare sectors. Findings from in-depth interviews with 45 senior leaders of social services and healthcare organizations serving older adults in Washington State elucidate the role of trust in service provision at the onset of the pandemic. First, a history of trust facilitated service adaptation. Intentional outreach, programs, and culturally responsive services sustained trust with service recipients. Providing services and information in an older adult's preferred language built trust. Community networks facilitated and reflected trust between organizations and older adults. Finally, mistrust was prevalent for clients who perceived a high risk of experiencing negative consequences from accessing services. Our findings support the need for culturally and linguistically diverse services. To improve trust, the aging network should strengthen and expand partnerships with community-based organizations who have established trust through history, intentionality, and relevance to their service populations..

Exosomal Cargo May Hold the Key to Improving Reproductive Outcomes in Dairy Cows.

The reproductive status of dairy cows remains a challenge for dairy farmers worldwide, with impaired fertility linked to a significant reduction in herd profitability, due in part to impaired immunity, increased metabolic pressure, and longer postpartum anestrous interval (PPAI). Exosomes are nanovesicles released from a variety of cell types and end up in circulation, and carry proteins, bioactive peptides, lipids, and nucleic acids specific to the place of origin. As such, their role in health and disease has been investigated in humans and animals. This review discusses research into exosomes in the context of reproduction in dairy herds and introduces recent advances in mass-spectrometry (MS) based proteomics that have a potential to advance quantitative profiling of exosomal protein cargo in a search for early biomarkers of cattle fertility.

Developmental constraints on fin diversity.

The fish fin is a breathtaking repository full of evolutionary diversity, novelty, and convergence. Over 500 million years, the adaptation to novel habitats has provided landscapes of fin diversity. Although comparative anatomy of evolutionarily divergent patterns over centuries has highlighted the fundamental architectures and evolutionary trends of fins, including convergent evolution, the developmental constraints on fin evolution, which bias the evolutionary trajectories of fin morphology, largely remain elusive. Here, we review the evolutionary history, developmental mechanisms, and evolutionary underpinnings of paired fins, illuminating possible developmental constraints on fin evolution. Our compilation of anatomical and genetic knowledge of fin development sheds light on the canalized and the unpredictable aspects of fin shape in evolution. Leveraged by an arsenal of genomic and genetic tools within the working arena of spectacular fin diversity, evolutionary developmental biology embarks on the establishment of conceptual framework for developmental constraints, previously enigmatic properties of evolution.

The Role of Exosomal Epigenetic Modifiers in Cell Communication and Fertility of Dairy Cows.

Abnormal uterine function affects conception rate and embryo development, thereby leading to poor fertility and reproduction failure. Exosomes are a nanosized subclass of extracellular vesicles (EV) that have important functions as intercellular communicators. They contain and carry transferable bioactive substances including micro RNA (miRNA) for target cells. Elements of the cargo can provide epigenetic modifications of the recipient cells and may have crucial roles in mechanisms of reproduction. The dairy industry accounts for a substantial portion of the economy of many agricultural countries. Exosomes can enhance the expression of inflammatory mediators in the endometrium, which contribute to various inflammatory diseases in transition dairy cows. This results in reduced fertility which leads to reduced milk production and increased cow maintenance costs. Thus, gaining a clear knowledge of exosomal epigenetic modifiers is critical to improving the breeding success and profitability of dairy farms. This review provides a brief overview of how exosomal miRNA contributes to inflammatory diseases and hence to poor fertility, particularly in dairy cows.

The evolutionary origins and diversity of the neuromuscular system of paired appendages in batoids.

Appendage patterning and evolution have been active areas of inquiry for the past two centuries. While most work has centred on the skeleton, particularly that of amniotes, the evolutionary origins and molecular underpinnings of the neuromuscular diversity of fish appendages have remained enigmatic. The fundamental pattern of segmentation in amniotes, for example, is that all muscle precursors and spinal nerves enter either the paired appendages or body wall at the same spinal level. The condition in finned vertebrates is not understood. To address this gap in knowledge, we investigated the development of muscles and nerves in unpaired and paired fins of skates and compared them to those of chain catsharks. During skate and shark embryogenesis, cell populations of muscle precursors and associated spinal nerves at the same axial level contribute to both appendages and body wall, perhaps representing an ancestral condition of gnathostome appendicular neuromuscular systems. Remarkably in skates, this neuromuscular bifurcation as well as colinear Hox expression extend posteriorly to pattern a broad paired fin domain. In addition, we identified migratory muscle precursors (MMPs), which are known to develop into paired appendage muscles with Pax3 and Lbx1 gene expression, in the dorsal fins of skates. Our results suggest that muscles of paired fins have evolved via redeployment of the genetic programme of MMPs that were already involved in dorsal fin development. Appendicular neuromuscular systems most likely have emerged as side branches of body wall neuromusculature and have been modified to adapt to distinct aquatic and terrestrial habitats.

Mental health care treatment seeking among African Americans and Caribbean Blacks: what is the role of religiosity/spirituality?

OBJECTIVES: As adults increase in age, the likelihood for using mental health care services decrease. Underutilization, expecially among racial/ethnic minorities such as African American and Caribbean Blacks, can result in a decrease in quality of life, as well as significant costs to families, employers, and health systems. METHODS: The study explored the differences in relationships between mental health care usage and strength of religious/spiritual beliefs between African American and Caribbean Black older adults (54 years or older) and adults (18-53 years) using data from the National Survey of American Life (NSAL). Descriptive statistics and logistic regression analyses were conducted using Stata version 13.1. RESULTS: Subjective ratings about the strength of religious/spiritual beliefs (OR = 1.26; 95 CI: 0.99, 1.61), age (OR = 0.62; 95 CI: 0.48, 0.81), and sex (OR = 1.59; 95 CI: 1.25, 2.02) were significantly associated with the odds of seeking mental health care. Additionally, persons living in the South were less likely to seek mental health care services (OR = 0.47; 95 CI: 0.37, 0.60). CONCLUSION: Strong religious/spiritual beliefs may promote mental health care usage. Future studies should examine the strength of religious/spiritual beliefs on mental health care usage among different demographic groups.

Examining the impact of regular aspirin use and PIK3CA mutations on survival in stage 2 colon cancer.

BACKGROUND/AIM: Data suggest aspirin improves survival in colorectal cancer (CRC) harbouring PIK3CA mutations. The impact of aspirin is thought predominantly to be through an anti-inflammatory effect. The aim of this study is to explore the effect of aspirin use on survival in a real-world cohort of stage 2 colon cancer (CC) patients. METHODS: A prospective CRC database identified patients diagnosed with stage 2 CC between 2000 and 2011. PIK3CA mutation status was determined by next generation sequencing. Neutrophil-lymphocyte ratio greater than 5 at diagnosis represented systemic inflammation. Chart review was used to record regular aspirin use at diagnosis. Clinico-pathological features and survival data were available. Survival analyses used the Cox proportional hazards method. RESULTS: Of 488 patients with stage 2 CC, 95 patients were aspirin users and 70 patients had PIK3CA mutations. Aspirin users were more likely to be older (median: 76.4 years vs 68.3 years, P < 0.001), to be less fit (American Society of Anaesthetists Score 3-4: 58% vs 31%, P < 0.001) and to have systemic inflammation (neutrophil-lymphocyte ratio > 5: 39% vs 27%, P = 0.027). Regular aspirin use did not significantly improve recurrence-free survival. In the PIK3CA mutated group, there was a trend towards improved recurrence-free survival (hazard ratio: 0.45, P = 0.42). CONCLUSIONS: Our study did not demonstrate a significant survival advantage from aspirin use in stage 2 PIK3CA mutated CC. The 'real-world' nature of our cohort and the subsequent uncontrolled differences in age and fitness in aspirin users are likely to have contributed to this result. Defining the true impact of aspirin in CRC requires prospective randomised clinical trials.

Analysis of local chronic inflammatory cell infiltrate combined with systemic inflammation improves prognostication in stage II colon cancer independent of standard clinicopathologic criteria.

In Stage II colon cancer, multiple independent studies have shown that a dense intratumoural immune infiltrate (local inflammation) is associated with improved outcomes, while systemic inflammation, measured by various markers, has been associated with poorer outcomes. However, previous studies have not considered the interaction between local and systemic inflammation, nor have they assessed the type of inflammatory response compared with standard clinicopathologic criteria. In order to evaluate the potential clinical utility of inflammatory markers in Stage II colon cancer, we examined local and systemic inflammation in a consecutive series of patients with resected Stage II colon cancer between 2000 and 2010 who were identified from a prospective clinical database. Increased intratumoural chronic inflammatory cell (CIC) density, as assessed by pathologist review of hematoxylin and eosin stained slides, was used to represent local inflammation. Neutrophil-to-lymphocyte ratio (NLR) >5, as calculated from pre-operative full blood counts, was used to represent systemic inflammation. In 396 eligible patients identified, there was a non-significant inverse relationship between local and systemic inflammation. Increased CIC density was significantly associated with improved overall (HR 0.45, p = 0.001) and recurrence-free survival (HR 0.37, p = 0.003). High NLR was significantly associated with poorer overall survival (HR 2.56, p < 0.001). The combination of these markers further stratified prognosis independent of standard high-risk criteria, with a dominant systemic inflammatory response (low CIC/high NLR) associated with the worst outcome (5-year overall survival 55.8%). With further validation this simple, inexpensive combined inflammatory biomarker might assist in patient selection for adjuvant chemotherapy in Stage II colon cancer.

Continued value of adjuvant anthracyclines as treatment for early breast cancer.

Anthracyclines are frequently used in the adjuvant treatment of early-stage breast cancer. However, with the increasing use of other active drugs--mainly taxanes and trastuzumab in HER2-positive disease--coupled with concerns about anthracycline-associated toxic effects, there is debate about whether anthracyclines are still needed. Three major factors should be taken into consideration with the investigation of the role of anthracyclines in management of early breast cancer; specifically, the proven efficacy of anthracyclines in breast cancer, the absence of superiority of non-anthracycline-based chemotherapy over anthracycline-taxane regimens, and the low risk of toxic effects associated with the cumulative doses of anthracyclines used in contemporary regimens. The risks remain substantially outweighed by the benefits of treatment with anthracyclines, and thus, they maintain an important role in adjuvant treatment of breast cancer, particularly in women with high-risk disease.

Food-derived extracellular vesicles in the human gastrointestinal tract: Opportunities for personalised nutrition and targeted therapeutics.

Food-derived extracellular vesicles (FDEVs) such as those found in mammalian milk and plants are of great interest for both their health benefits and ability to act as biological nanocarriers. While the extracellular vesicle (EV) field is expanding rapidly to perform characterisation studies on FDEVs from plants, yeasts and bacteria, species-specific differences in EV uptake and function in the human gastrointestinal (GI) tract are poorly understood. Moreover, the effects of food processing on the EV surfaceome and intraluminal content also raises questions surrounding biological viability once consumed. Here, I present a case for increasing community-wide focus on understanding the cellular uptake of FDEVs from different animal, plant, yeast, and bacterial species and how this may impact their function in the human, which will have implications for human health and therapeutic strategies alike.

A test of pre-exposure spacing and multiple context pre-exposure on the mechanisms of latent inhibition of dental fear: A study protocol.

BACKGROUND: Latent inhibition occurs when exposure to a stimulus prior its direct associative conditioning impairs learning. Results from naturalistic studies suggest that latent inhibition disrupts the learning of dental fear from aversive associative conditioning and thereby reduces the development of dental phobia. Although theory suggests latent inhibition occurs because pre-exposure changes the expected relevance and attention directed to the pre-exposed stimulus, evidence supporting these mechanisms in humans is limited. The aim of this study is to determine if two variables, pre-exposure session spacing and multiple context pre-exposure, potentiate the hypothesized mechanisms of expected relevance and attention and, in turn, increase latent inhibition of dental fear. METHODS: In a virtual reality simulation, child and adult community members (ages 6 to 35) will take part in pre-exposure and conditioning trials, followed by short- and long-term tests of learning. A 100ms puff of 60 psi air to a maxillary anterior tooth will serve as the unconditioned stimulus. Pre-exposure session spacing (no spacing vs. sessions spaced) and multiple context pre-exposure (single context vs. multiple contexts) will be between-subject factors. Stimulus type (pre-exposed to-be conditioned stimulus, a non-pre-exposed conditioned stimulus, and an unpaired control stimulus) and trial will serve as within-subject factors. Baseline pain sensitivity will also be measured as a potential moderator. DISCUSSION: It is hypothesized that spaced pre-exposure and pre-exposure in multiple contexts will increase the engagement of the mechanisms of expected relevance and attention and increase the latent inhibition of dental fear. It is expected that the findings will add to theory on fear learning and provide information to aid the design of future interventions that leverage latent inhibition to reduce dental phobia.

A study protocol testing pre-exposure dose and compound pre-exposure on the mechanisms of latent inhibition of dental fear.

BACKGROUND: Dental stimuli can evoke fear after being paired - or conditioned - with aversive outcomes (e.g., pain). Pre-exposing the stimuli before conditioning can impair dental fear learning via a phenomenon known as latent inhibition. Theory suggests changes in expected relevance and attention are two mechanisms responsible for latent inhibition. In the proposed research, we test whether pre-exposure dose and degree of pre-exposure novelty potentiate changes in expected relevance and attention to a pre-exposed stimulus. We also assess if the manipulations alter latent inhibition and explore the possible moderating role of individual differences in pain sensitivity. METHODS: Participants will be healthy individuals across a wide range of ages (6 to 35 years), from two study sites. Participants will undergo pre-exposure and conditioning followed by both a short-term and long-term test of learning, all in a novel virtual reality environment. The unconditioned stimulus will be a brief pressurized puff of air to a maxillary anterior tooth. Pre-exposure dose (low vs. high) and pre-exposure novelty (element stimulus vs. compound stimuli) will be between-subject factors, with stimulus type (pre-exposed to-be conditioned stimulus, a non-pre-exposed conditioned stimulus, and an unpaired control stimulus) and trial as within-subject factors. Pain sensitivity will be measured through self-report and a cold pressor test. It is hypothesized that a larger dose of pre-exposure and compound pre-exposure will potentiate the engagement of the target mechanisms and thereby result in greater latent inhibition in the form of reduced fear learning. Further, it is hypothesized that larger effects will be observed in participants with greater baseline pain sensitivity. DISCUSSION: The proposed study will test whether pre-exposure dose and compound stimulus presentation change expected relevance and attention to the pre-exposed stimulus, and thereby enhance latent inhibition of dental fear. If found, the results will add to our theoretical understanding of the latent inhibition of dental fear and inform future interventions for dental phobia prevention.

Breast cancer in the elderly: which lessons have we learned?

Management of older breast cancer patients is challenging due to a lack of good quality evidence regarding the role of adjuvant chemotherapy. Older women can benefit as much from adjuvant chemotherapy as younger women, although they have an increased risk of toxicities. Decisions regarding adjuvant chemotherapy should be made based on tumor biology and biological age, rather than chronological age. Geriatrician assessment can detect subtle functional deficits that may impact on the ability of the patient to tolerate chemotherapy; however, implementation of comprehensive geriatric assessment in the oncology setting is challenging. Instead, numerous frailty screening tools are in development. Future advances should incorporate more accurate and efficient means for determining the biological age of elderly breast cancer patients, which will better define the risk:benefit ratio of adjuvant chemotherapy.

How I want technology used in my care: Learning from documented choices of people living with dementia using a dyadic decision making tool.

While technologies for aging in place are promoted to support care partners and people living with dementia, perspectives of people living with dementia are underrepresented in both use decisions among families and discussions within academia and industry. This mixed-methods study examined the use preferences of twenty-nine people living with mild Alzheimer's disease (AD) for four categories of technologies: location tracking, in-home sensors, web-cameras, and virtual companion robots. Participants completed a novel dyadic intervention, Let's Talk Tech, where they documented their preferences of the four technology categories for care planning purposes. Post-test interviews were thematically analyzed and provide insight into selection processes. Technology preferences varied considerably by and within participant living with mild AD. Excepting location tracking, non-technology and low-technology options were more desirable than the featured technologies. Control over technology use was of great importance to people living with AD. Considerations given to technology preference selection imperfectly fit within the new Health Technology Acceptance Model (H-TAM) developed for older adults. These findings underscore the importance of including people living with dementia in decision making about technologies to support care at home and the need for further personalization and tailorable technological devices to accommodate and align with their preferences.

Dynamics of inflammatory cytokine expression in bovine endometrial cells exposed to cow blood plasma small extracellular vesicles (sEV) may reflect high fertility.

Aberrant inflammation in the endometrium impairs reproduction and leads to poor fertility. Small extracellular vesicles (sEV) are nanoparticles 30-200 nm in-size and contain transferable bioactive molecules that reflect the parent cell. Holstein-Friesian dairy cows with divergent genetic merit, high- (n = 10) and low-fertile (n = 10), were identified based on fertility breeding value (FBV), cow ovulation synchronization and postpartum anovulatory intervals (PPAI). In this study, we evaluated the effects of sEVs enriched from plasma of high-fertile (HF-EXO) and low-fertile (LF-EXO) dairy cows on inflammatory mediator expression by bovine endometrial epithelial (bEEL) and stromal (bCSC) cells. Exposure to HF-EXO in bCSC and bEEL cells yielded lower expression of PTGS1 and PTGS2 compared to the control. In bCSC cells exposed to HF-EXO, pro-inflammatory cytokine IL1-α was downregulated compared to the untreated control, IL-12α and IL-8 were downregulated compared to the LF-EXO treatment. Our findings demonstrate that sEVs interact with both endometrial epithelial and stromal cells to initiate differential gene expression, specifically genes relate to inflammation. Therefore, even subtle changes on the inflammatory gene cascade in the endometrium via sEV may affect reproductive performance and/or outcomes. Further, sEV from high-fertile animals acts in a unique direction to deactivate prostaglandin synthases in both bCSC and bEEL cells and deactivate pro-inflammatory cytokines in the endometrial stroma. The results suggest that circulating sEV may serve as a potential biomarker of fertility.

Omics Analysis of Extracellular Vesicles Recovered from Infant Formula Products and Milk: Towards Personalized Infant Nutrition.

SCOPE: Milk and milk products such as infant formula (IF) play a fundamental role in serving the nutritional needs of the developing infant. Extracellular vesicles (EVs) in human (HM) and cow milk (CM) contain molecular cargo such as proteins and micro(mi)RNAs that serve as functional messengers between cells and may be of importance to infant health. Most IF is derived from a CM protein base, however differences between HM and CM EV molecular cargo have not been extensively studied. METHODS AND RESULTS: This study develops a pipeline using advanced proteomics and transcriptomics to enable cross-species comparison of milk and IF EVs. The number of nanoparticles per mL of IF is significantly reduced compared to unprocessed CM. 130 proteins and 514 miRNAs are differentially abundant between HM and CM EVs. While 90% of CM EV miRNAs are also identified in IF EVs, only 20% of CM EV proteins are identified in IF EVs. CONCLUSIONS: This workflow identifies key species-specific differences that can be used to optimize IF recipes and enhance infant nutrition. Improved preservation of EV functional molecular cargo in IF products is of critical importance to retaining molecular drivers of good health and should be the focus of future investigations.