Separation of mycolic acid isomers by cyclic ion mobility-mass spectrometry.

RATIONALE: Mycobacterial species contain high concentrations of mycolic acids in their cell wall. Mycobacteria can pose a threat to both human health and the environment. Mass spectrometry lipidomic characterization can identify bacterial species and suggest targets for microbiological interventions. Due to the complex structures of mycolic acids and the possibility of isobaric isomers, multiple levels of separation are required for complete characterization. In this study, cyclic ion mobility (cIM) mass spectrometry (MS) was used for the analysis, separation and fragmentation of mycolic acids isomers from the bacterial species Gordonia amarae and Mycobacterium bovis. METHODS: Mycolic acid isomers were interrogated from cultured G. amarae biomass and commercially available M. bovis mycolic acid extracts. These were infused into a cIM-enabled quadrupole time-of-flight MS. Ions of interest were non-simultaneously selected with the quadrupole and passed around the cyclic ion mobility device multiple times. Fragment ion analysis was then performed for the resolved isomers of the quadrupole-selected ions. RESULTS: Repeated passes of the cIM device successfully resolved otherwise overlapping MA isomers, allowing isomer isolation and producing an ion-specific post-mobility fragmentation spectrum without isomeric interference. CONCLUSIONS: Mycolic acids (MA) isomers from G. amarae and M. bovis were resolved, resulting in a high mobility resolution and low interference fragmentation analysis. These revealed varying patterns of MA isomers in the two species: G. amarae's most abundant ion of each set of MA has 1-2 conformations, while the MA + 2 m/z the most abundant ion of each set has 3-6 conformations. These were resolved after 70 passes of the cyclic device. M. bovis' most abundant ion of each keto-MA set has 2 conformations, while the keto-MA + 2 m/z has 1-2 conformations. These were resolved after 75 passes.

Feline friendly POCUS: how to implement it into your daily practice.

PRACTICAL RELEVANCE: Cats are great pretenders; they often hide illness until they are critical. This makes patients of this species challenging to assess and manage in the emergency setting where quick and stress-free diagnosis and treatment are necessary. Veterinary point-of-care ultrasound (POCUS) is a rapid, evidence-based, non-invasive, repeatable, cage-side ultrasonographic examination designed to answer clinically driven questions without compromising feline wellbeing. Integrating feline friendly POCUS as an extension of the physical examination to streamline diagnostic and therapeutic interventions, thereby limiting stress and improving overall patient care, is advocated by the authors of this article. EQUIPMENT: Given the multitude of ultrasound machines and probes available that are portable, meaning they can be moved around the clinic and used patient-side, it should be possible for most practitioners to integrate POCUS into daily practice. The authors' preferred equipment for feline POCUS is a microconvex probe and a portable machine with a fixed pre-set. This set-up allows the clinician to complete all POCUS (abdominal, lung and pleural space, and heart) without needing to move the patient, change probes or restrain the patient in a particular position, ultimately saving time, personnel and cost while maintaining patient comfort and safety. AIM: This review aims to serve as a valuable resource for veterinarians seeking to improve their feline patient care through the judicious utilisation of POCUS. In this article, the complex challenges posed by cats are addressed, and the different POCUS techniques, applications and clinical recommendations are discussed. EVIDENCE BASE: This review draws on the published literature, as well as the authors' own collective experience when providing recommendations.

Volatile organic compound analysis of malignant pleural mesothelioma chorioallantoic membrane xenografts.

Malignant pleural mesothelioma (MPM) is an aggressive cancer associated with asbestos exposure. MPM is often diagnosed late, at a point where limited treatment options are available, but early intervention could improve the chances of successful treatment for MPM patients. Biomarkers to detect MPM in at-risk individuals are needed to implement early diagnosis technologies. Volatile organic compounds (VOCs) have previously shown diagnostic potential as biomarkers when analysed in MPM patient breath. In this study, chorioallantoic membrane (CAM) xenografts of MPM cell lines were used as models of MPM tumour development for VOC biomarker discovery with the aim of generating targets for investigation in breath, biopsies or other complex matrices. VOC headspace analysis of biphasic or epithelioid MPM CAM xenografts was performed using solid-phase microextraction and gas chromatography-mass spectrometry. We successfully demonstrated the capture, analysis and separation of VOC signatures from CAM xenografts and controls. A panel of VOCs was identified that showed discrimination between MPM xenografts generated from biphasic and epithelioid cells and CAM controls. This is the first application of the CAM xenograft model for the discovery of VOC biomarkers associated with MPM histological subtypes. These findings support the potential utility of non-invasive VOC profiling from breath or headspace analysis of tissues for detection and monitoring of MPM.

Clinical features and outcome of dogs and cats with gastrointestinal pneumatosis: 30 cases (2010-2021).

OBJECTIVES: To describe the presentation, etiology, and outcome of dogs and cats diagnosed with gastrointestinal pneumatosis (GP). DESIGN: Retrospective study. SETTING: Three referral institutions. ANIMALS: Twenty-six dogs and 4 cats. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The most common sites of GP were the stomach (n = 19), followed by the colon (n = 8) and small intestine (n = 2). One case had pneumatosis of both the stomach and the colon. GP was most commonly associated with gastrointestinal disease in dogs (18/26 [69%]) and cats (3/4 [75%]), with common diagnoses including gastric dilatation and volvulus (n = 5), acute hemorrhagic diarrhea syndrome (n = 4), and gastrointestinal ulceration (n = 4). Of the 4 cases of gastrointestinal ulceration, 3 were dogs with a history of glucocorticosteroid or nonsteroidal anti-inflammatory drug administration and vomiting and diarrhea. Six of 30 cases (20%), all of which were dogs, were determined to have a surgical indication for exploratory celiotomy, although not solely on the basis of diagnosis of GP. Five cases underwent exploratory celiotomy, of which 1 (20%) survived to hospital discharge. Of the medically managed cases, 13 of 24 (54%) survived to hospital discharge. Overall, 14 of 30 cases (47%) survived to hospital discharge. CONCLUSIONS: GP is an uncommon diagnostic imaging finding that is associated with a variety of disease processes. Its development is often related to primary gastrointestinal diseases. In the absence of other surgical disease, exploratory celiotomy based solely on the diagnosis of GP is unlikely to be indicated.

Multimodal Mass Spectrometry Imaging of an Osteosarcoma Multicellular Tumour Spheroid Model to Investigate Drug-Induced Response.

A multimodal mass spectrometry imaging (MSI) approach was used to investigate the chemotherapy drug-induced response of a Multicellular Tumour Spheroid (MCTS) 3D cell culture model of osteosarcoma (OS). The work addresses the critical demand for enhanced translatable early drug discovery approaches by demonstrating a robust spatially resolved molecular distribution analysis in tumour models following chemotherapeutic intervention. Advanced high-resolution techniques were employed, including desorption electrospray ionisation (DESI) mass spectrometry imaging (MSI), to assess the interplay between metabolic and cellular pathways in response to chemotherapeutic intervention. Endogenous metabolite distributions of the human OS tumour models were complemented with subcellularly resolved protein localisation by the detection of metal-tagged antibodies using Imaging Mass Cytometry (IMC). The first application of matrix-assisted laser desorption ionization-immunohistochemistry (MALDI-IHC) of 3D cell culture models is reported here. Protein localisation and expression following an acute dosage of the chemotherapy drug doxorubicin demonstrated novel indications for mechanisms of region-specific tumour survival and cell-cycle-specific drug-induced responses. Previously unknown doxorubicin-induced metabolite upregulation was revealed by DESI-MSI of MCTSs, which may be used to inform mechanisms of chemotherapeutic resistance. The demonstration of specific tumour survival mechanisms that are characteristic of those reported for in vivo tumours has underscored the increasing value of this approach as a tool to investigate drug resistance.

Do NSm Virulence Factors in the Bunyavirales Viral Order Originate from Gn Gene Duplication?

One-third of the nine WHO shortlisted pathogens prioritized for research and development in public health emergencies belong to the Bunyavirales order. Several Bunyavirales species carry an NSm protein that acts as a virulence factor. We predicted the structures of these NSm proteins and unexpectedly found that in two families, their cytosolic domain was inferred to have a similar fold to that of the cytosolic domain of the viral envelope-forming glycoprotein N (Gncyto) encoded on the same genome fragment. We show that although the sequence identity between the NSmcyto and the Gncyto domains is low, the conservation of the two zinc finger-forming CysCysHisCys motifs explains the predicted structural conservation. Importantly, our predictions provide a first glimpse into the long-unknown structure of NSm. Also, these predictions suggest that NSm is the result of a gene duplication event in the Bunyavirales Nairoviridae and Peribunyaviridae families and that such events may be common in the recent evolutionary history of RNA viruses.

A Novel Home-Based Medication Management Program and Its Influence on Hospitalization Rates among Home Health Care Recipients.

Patients do not take their medicine as prescribed 50% of the time, and of medication-related hospital admissions in the United States, 33% to 69% are due to poor medication adherence, at a cost of approximately $100 billion a year. Continue CareRx (CCRx) is a novel home-based medication management program that includes adherence packaging alongside medication reconciliation, review, and education by clinicians. We hypothesized that home health patients receiving the CCRx service may have a lower hospitalization rate than control home health patients. Between May 1, 2021, and March 31, 2023, 113 home health patients whose insurance covered the program were enrolled in CCRx. Home health patients not eligible for the program due to a noncovered pharmacy insurance benefit made up the control group (n = 21,304), which was matched with the CCRx group on age range (45-99 years old) and gender (67% women). Hospitalization rate was calculated in both groups and compared using generalized estimating equations analysis. The control group had a total of 7015 hospitalizations during the study period during 2,128,738 total managed days, whereas the CCRx group had 21 hospitalizations during 23,622 total managed days. These translated into rates of 1203 hospitalizations per 1000 per year for the control group, and 324 hospitalizations per 1000 per year for the CCRx group. The results showed that there was a significant main effect of group in predicting individual annual hospitalization rate (Wald χ2 = 56.415, P < .01). Specifically, being in the control group was associated with a 43.42-fold increase in the likelihood of a higher hospitalization rate (95% Wald CI for odds ratios: 7.24-230.44). Home health recipients enrolled in CCRx experienced a 73.1% lower hospitalization rate than controls. Making the program more widely available to patients receiving home health care may present a significant opportunity to reduce hospitalizations in this group.

Cellular Responses to Extracellular Vesicles as Potential Markers of Colorectal Cancer Progression.

The development of novel screening tests aims to support early asymptomatic diagnosis and subtyping patients according to similar traits in the heterogeneous cancer cohort. Extracellular vesicles (EVs) are promising candidates for the detection of disease markers from bodily fluids, but limitations in the standardisation of isolation methods and the intrinsic EV heterogeneity obtained from liquid biopsies are currently obstacles to clinical adoption. Here, cellular responses to cancer EVs were initially explored as potential complementary biomarkers for stage separation using colorectal cancer (CRC) SW480 and SW620 cell line models. A pilot study on a small cohort of CRC patients and controls was then developed by performing a multivariate analysis of cellular responses to plasma-derived EVs. Several cell activities and markers involved in tumour microenvironment pathways were influenced by the treatment of cell line EVs in a stage-dependent manner. The multivariate analysis combining plasma EV markers and cellular responses to plasma EVs was able to separate patients according to disease stage. This preliminary study offers the potential of considering cellular responses to EVs in combination with EV biomarkers in the development of screening methods.

Anomalous peroxidase activity of cytochrome c is the primary pathogenic target in Barth syndrome.

Barth syndrome (BTHS) is a life-threatening genetic disorder with unknown pathogenicity caused by mutations in TAFAZZIN (TAZ) that affect remodeling of mitochondrial cardiolipin (CL). TAZ deficiency leads to accumulation of mono-lyso-CL (MLCL), which forms a peroxidase complex with cytochrome c (cyt c) capable of oxidizing polyunsaturated fatty acid-containing lipids. We hypothesized that accumulation of MLCL facilitates formation of anomalous MLCL-cyt c peroxidase complexes and peroxidation of polyunsaturated fatty acid phospholipids as the primary BTHS pathogenic mechanism. Using genetic, biochemical/biophysical, redox lipidomic and computational approaches, we reveal mechanisms of peroxidase-competent MLCL-cyt c complexation and increased phospholipid peroxidation in different TAZ-deficient cells and animal models and in pre-transplant biopsies from hearts of patients with BTHS. A specific mitochondria-targeted anti-peroxidase agent inhibited MLCL-cyt c peroxidase activity, prevented phospholipid peroxidation, improved mitochondrial respiration of TAZ-deficient C2C12 myoblasts and restored exercise endurance in a BTHS Drosophila model. Targeting MLCL-cyt c peroxidase offers therapeutic approaches to BTHS treatment.

Assessing primary care veterinarians' use of and confidence in performing point-of-care ultrasound.

BACKGROUND: Point-of-care ultrasound (POCUS) is gaining popularity in the veterinary field, but there is little information on operator confidence. METHODS: A survey was distributed to primary care veterinarians (PCVs) via social media between May and July 2020. Details of participants' training in and use of POCUS were recorded. Participants' confidence in using thoracic and abdominal POCUS was also assessed using a five-point Likert scale. RESULTS: Two hundred and one PCVs used POCUS, of which 32% reported using a non-standardised protocol. Fifty percent of PCVs were self-taught and 17.4% had attended a specific practical course. The median confidence score was 4 out of 5 (interquartile range [IQR] 2-5) for identifying abdominal abnormalities, irrespective of the training method. The median confidence score for thoracic abnormalities was 3 out of 5 (IQR 1-4) for those taught by a colleague or who were self-taught using journal articles or videos. LIMITATIONS: The survey-based nature of the study relies on self-reporting and is therefore liable to recall bias. CONCLUSIONS: PCVs' confidence in using POCUS is lacking, particularly with thoracic POCUS. Standardised practical training for PCVs, particularly in thoracic POCUS, would be beneficial. Future studies should explore how best to deliver this training.

MALDI and Trace Metal Analysis in Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) remains one of the most prevalent causes of blindness throughout the world. Key to prevention of AMD is furthering the understanding of its pathology. In recent years, both the proteins within the innate immune system and essential and non-essential metals have been implicated in the pathology of AMD. Herein, a multidisciplinary and multimodal methodology has been taken to further our understanding of the role of the innate immune proteins and the essential metals within mouse ocular tissue.

Perspective: Mass Spectrometry Imaging - The Next 5 Years.

In order to achieve even more widespread adoption over the next 5 years, a number of issues in mass spectrometry imaging need to be addressed. These are non-observation of compounds (due to ionization suppression), sample throughput, imaging of low-abundant species, and how to extract information from the large volumes of data generated. In this article, how current research indicates that these issues will be resolved along with potential application areas that MSI could look to exploit is discussed.

Understanding factors influencing residential respite service use by carers of people living with dementia using Andersen's behavioural model of health services use: A qualitative study.

OBJECTIVES: Residential respite (RR) provides a valuable break for family carers, but little known about its offer, take-up or experiences of carers of people living with dementia. This paper aims to further understandings of factors influencing RR use. DESIGN: RR stakeholder workshop and qualitative interviews. SETTING: Stakeholder or living in the community in own home. PARTICIPANTS: RR stakeholders (13); family carers with experience of RR, or had declined it, or were planning to use it for the first time (n = 36). METHODS: Stakeholders participated in a workshop to discuss provision, models and funding of RR. Family carer interviews focused on expectations, experiences and outcomes of use of RR. Data were analysed thematically and mapped against Andersen's model of health service use. RESULTS: Identifying need for RR does not necessarily transpire into use. Planning and ease of booking were crucial for carers, but many felt there was little support with this. Systemic factors concerning funding, planning and booking RR act as barriers to its use. CONCLUSION: Findings highlight how systemic factors influence RR use. Discussing respite need in routine care planning or reviews may support carers and people living with dementia to consider RR, but system changes are needed to address barriers.

A novel chemogenomic discovery platform identifies bioactive hits with rapid bactericidal activity against Mycobacteroides Abscessus.

Mycobacteroides abscessus (M. ab) infections are innately resistant to most currently available antibiotics and present a growing, poorly addressed medical need. The existing treatment regimens are lengthy and produce inadequate outcomes for many patients. Importantly, most clinically used drugs and drug candidates against M. ab are either bacteriostatic, or only weakly bactericidal. New strategies exploring a broader chemical space are urgently needed, as innovative agents in development are scarce and hit rates in large unbiased screens against the mycobacterium have been discouragingly low. Here we present a computational chemogenomics-driven approach to discovery of novel antibacterials that effectively reveals drug-like compounds active against M. ab, paired with small sets of predicted molecular targets for the compounds. Several of the bioactive hits identified exhibited rapid bactericidal, including sterilizing, activity against the mycobacterium, indicating that there are currently unexploited chemically tractable molecular mechanisms for rapid sterilization of M. ab. Interestingly, starvation, which typically induces drug tolerance, sensitized M. ab to some of the compounds, resulting in potencies similar to those of drugs in clinical use. The presented drug discovery platform has potential to identify highly differentiated prototype anti-infective molecules and thereby contribute to development of regimens for shorter treatment and improved outcomes for non-tuberculous mycobacterial infections.

Feeling WELL: COVID-19 and the Adoption of Wellness Themes in Interior Design Curricula.

Interior design (ID) pedagogy should include theories and applications, which advance human health and wellness in the built environment. Design for wellness and well-being is referenced in 5 of the 13 Council for Interior Design Accreditation (CIDA) knowledge application standards. This focus on wellness was amplified during the coronavirus disease 2019 (COVID-19) pandemic experience, which elevated our collective understanding of contagions, disease transmission, sanitation practices, vaccination efficacy, and immunity, as well as the devastating turmoil-social, economic, and psychological-of the pandemic's tragic spread. Thus, the purpose of this study was to examine ID educators' attitudes, intentions, and behaviors related to teaching wellness and to determine if the pandemic impacted wellness pedagogy. For this mixed methods study, we employed an online survey (n = 86) followed by participant interviews (n = 11). Data from the questionnaire and interviews suggested that peer attitudes toward wellness were a significant factor that influenced teaching behavior. Teaching intentions were predicted by prior teaching, attitudes, and perceived behavioral control (ability to teach wellness). Although experience with the pandemic was not a predictor of teaching behavior, it did surface as a strong motivator for future teaching adjustments. Findings also showed wide-ranging understandings and definitions of wellness-related themes. Implications for design programs seeking to equip faculty with the tools needed to integrate wellness more deeply into ID curricula are discussed.

Multiomic Mass Spectrometry Imaging to Advance Future Pathological Understanding of Ocular Disease.

Determining the locations of proteins within the eye thought to be involved in ocular pathogenesis is important to determine how best to target them for therapeutic benefits. However, immunohistochemistry is limited by the availability and specificity of antibodies. Additionally, the perceived role of both essential and non-essential metals within ocular tissue has been at the forefront of age-related macular degeneration (AMD) pathology for decades, yet even key metals such as copper and zinc have yet to have their roles deconvoluted. Here, mass spectrometry imaging (MSI) is employed to identify and spatially characterize both proteomic and metallomic species within ocular tissue to advance the application of a multiomic imaging methodology for the investigation of ocular diseases.

In depth investigation of the capabilities and limitations of combined proteomic-MALDI MS based approach for the forensic detection of blood.

For the past 7 years, Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI MS) based methods have been developed and published for the forensic detection of blood in stains and fingermarks. However, in the view of adoption in an operational context, further investigation into the capabilities and limitations of this approach must be conducted. The refinement and testing of this approach must also be tailored to the requirements of the end users, enabling them to address the specific circumstances most encountered in a forensic scenario. The present study delves deeper into the assessment of the applicability of MALDI MS based strategy for the reliable and robust detection of human blood through: (i) a semi-qualitative assessment of the sensitivity of the method, (ii) a wider investigation of the compatibility of the method with the prior application of commonly used presumptive tests and (iii) assessment of the specificity of the method (when blood is present in mixture with other biofluids) and of its robustness, by assessing blood detection from a range of porous materials. The findings strengthen the evidence supporting the adoption of MALDI MS based approaches as a confirmatory test for the forensic detection of human blood in an operational context.

Tafazzin deficiency attenuates anti-cluster of differentiation 40 and interleukin-4 activation of mouse B lymphocytes.

Barth syndrome (BTHS) is a rare X-linked genetic disease caused by mutations in TAFAZZIN. The tafazzin (Taz) protein is a cardiolipin remodeling enzyme required for maintaining mitochondrial function. Patients with BTHS exhibit impaired mitochondrial respiratory chain and metabolic function and are susceptible to serious infections. B lymphocytes (B cells) play a vital role in humoral immunity required to eradicate circulating antigens from pathogens. Intact mitochondrial respiration is required for proper B-cell function. We investigated whether Taz deficiency in mouse B cells altered their response to activation by anti-cluster of differentiation 40 (anti-CD40) + interleukin-4 (IL-4). B cells were isolated from 3-4-month-old wild type (WT) or tafazzin knockdown (TazKD) mice and were stimulated with anti-CD40 + IL-4 for 24 h and cellular bioenergetics, surface marker expression, proliferation, antibody production, and proteasome and immunoproteasome activities determined. TazKD B cells exhibited reduced mRNA expression of Taz, lowered levels of cardiolipin, and impairment in both oxidative phosphorylation and glycolysis compared to WT B cells. In addition, anti-CD40 + IL-4 stimulated TazKD B cells expressed lower levels of the immunogenic surface markers, cluster of differentiation 86 (CD86) and cluster of differentiation 69 (CD69), exhibited a lower proliferation rate, reduced production of immunoglobulin M and immunoglobulin G, and reduced proteasome and immunoproteasome proteolytic activities compared to WT B cells stimulated with anti-CD40 + IL-4. The results indicate that Taz is required to support T-cell-dependent signaling activation of mouse B cells.

Tonic and phasic effects of reward on the pupil: implications for locus coeruleus function.

The locus coeruleus (LC), a nucleus in the pons of the brainstem, plays a significant role in attention and cognitive control. Here, we use an adapted auditory oddball paradigm and measured the pupil dilation response, to provide a marker of LC activity in humans. In Experiment 1, we show event-related pupil responses to rare auditory events which were further elevated by task relevant. In Experiment 2, by asking participants to silently count the number of oddballs, we demonstrated that the task-relevance elevation was not a result of the generation or execution of the manual response. In Experiment 3, we observed two separate effects of reward on the pupil response. First, we found an overall increase in pupil area in the high compared to the low-reward blocks: a sustained effect reminiscent of the tonic changes that occur in LC. Second, we found elevated event-related pupil responses to behaviourally relevant stimuli in the high-reward condition compared with the low-reward condition, consistent with phasic changes in LC in response to a stimulus. These results highlight the complexity of the relationship between the pupil response and reward, and the inferred role of LC in both top-down and bottom-up cognitive control.

Tafazzin deficiency in mouse mesenchymal stem cells promote reprogramming of activated B lymphocytes toward immunosuppressive phenotypes.

Barth Syndrome (BTHS) is a rare X-linked genetic disorder caused by mutation in the TAFAZZIN gene. Tafazzin (Taz) deficiency in BTHS patients results in an increased risk of infections. Mesenchymal stem cells (MSCs) are well known for their immune-inhibitory function. We examined how Taz-deficiency in murine MSCs impact their ability to modulate the function of lipopolysaccharide (LPS)-activated wild type (WT) B lymphocytes. MSCs from tafazzin knockdown (TazKD) mice exhibited a reduction in mitochondrial cardiolipin compared to wild type (WT) MSCs. However, mitochondrial bioenergetics and membrane potential were unaltered. In contrast, TazKD MSCs exhibited increased reactive oxygen species generation and increased glycolysis. The increased glycolysis was associated with an elevated proliferation, phosphatidylinositol-3-kinase expression and expression of the immunosuppressive markers indoleamine-2,3-dioxygenase, cytotoxic T-lymphocyte-associated protein 4, interleukin-10, and cluster of differentiation 59 compared to controls. Inhibition of glycolysis with 2-deoxyglucose attenuated the TazKD-mediated increased expression of cytotoxic T-lymphocyte-associated protein 4 and interleukin-10. When co-cultured with LPS-activated WT B cells, TazKD MSCs inhibited B cell proliferation and growth rate and reduced B cell secretion of immunoglobulin M compared to controls. In addition, co-culture of LPS-activated WT B cells with TazKD MSCs promoted B cell differentiation toward interleukin-10 secreting plasma cells and B regulatory cells compared to controls. The results indicate that Taz deficiency in MSCs promote reprogramming of activated B lymphocytes toward immunosuppressive phenotypes.