Reverse metabolomics for the discovery of chemical structures from humans.

Determining the structure and phenotypic context of molecules detected in untargeted metabolomics experiments remains challenging. Here we present reverse metabolomics as a discovery strategy, whereby tandem mass spectrometry spectra acquired from newly synthesized compounds are searched for in public metabolomics datasets to uncover phenotypic associations. To demonstrate the concept, we broadly synthesized and explored multiple classes of metabolites in humans, including N-acyl amides, fatty acid esters of hydroxy fatty acids, bile acid esters and conjugated bile acids. Using repository-scale analysis1,2, we discovered that some conjugated bile acids are associated with inflammatory bowel disease (IBD). Validation using four distinct human IBD cohorts showed that cholic acids conjugated to Glu, Ile/Leu, Phe, Thr, Trp or Tyr are increased in Crohn's disease. Several of these compounds and related structures affected pathways associated with IBD, such as interferon-γ production in CD4+ T cells3 and agonism of the pregnane X receptor4. Culture of bacteria belonging to the Bifidobacterium, Clostridium and Enterococcus genera produced these bile amidates. Because searching repositories with tandem mass spectrometry spectra has only recently become possible, this reverse metabolomics approach can now be used as a general strategy to discover other molecules from human and animal ecosystems.

Self-sacrificial tyrosine cleavage by an Fe:Mn oxygenase for the biosynthesis of para-aminobenzoate in Chlamydia trachomatis.

Chlamydia protein associating with death domains (CADD) is involved in the biosynthesis of para-aminobenzoate (pABA), an essential component of the folate cofactor that is required for the survival and proliferation of the human pathogen Chlamydia trachomatis. The pathway used by Chlamydiae for pABA synthesis differs from the canonical multi-enzyme pathway used by most bacteria that relies on chorismate as a metabolic precursor. Rather, recent work showed pABA formation by CADD derives from l-tyrosine. As a member of the emerging superfamily of heme oxygenase-like diiron oxidases (HDOs), CADD was proposed to use a diiron cofactor for catalysis. However, we report maximal pABA formation by CADD occurs upon the addition of both iron and manganese, which implicates a heterobimetallic Fe:Mn cluster is the catalytically active form. Isotopic labeling experiments and proteomics studies show that CADD generates pABA from a protein-derived tyrosine (Tyr27), a residue that is ∼14 Šfrom the dimetal site. We propose that this self-sacrificial reaction occurs through O2 activation by a probable Fe:Mn cluster through a radical relay mechanism that connects to the "substrate" Tyr, followed by amination and direct oxygen insertion. These results provide the molecular basis for pABA formation in C. trachomatis, which will inform the design of novel therapeutics.

Lactobacillus bile salt hydrolase substrate specificity governs bacterial fitness and host colonization.

Primary bile acids (BAs) are a collection of host-synthesized metabolites that shape physiology and metabolism. BAs transit the gastrointestinal tract and are subjected to a variety of chemical transformations encoded by indigenous bacteria. The resulting microbiota-derived BA pool is a mediator of host-microbiota interactions. Bacterial bile salt hydrolases (BSHs) cleave the conjugated glycine or taurine from BAs, an essential upstream step for the production of deconjugated and secondary BAs. Probiotic lactobacilli harbor a considerable number and diversity of BSHs; however, their contribution to Lactobacillus fitness and colonization remains poorly understood. Here, we define and compare the functions of multiple BSHs encoded by Lactobacillus acidophilus and Lactobacillus gasseri Our genetic and biochemical characterization of lactobacilli BSHs lend to a model of Lactobacillus adaptation to the gut. These findings deviate from previous notions that BSHs generally promote colonization and detoxify bile. Rather, we show that BSH enzymatic preferences and the intrinsic chemical features of various BAs determine the toxicity of these molecules during Lactobacillus growth. BSHs were able to alter the Lactobacillus transcriptome in a BA-dependent manner. Finally, BSHs were able to dictate differences in bacterial competition in vitro and in vivo, defining their impact on BSH-encoding bacteria within the greater gastrointestinal tract ecosystem. This work emphasizes the importance of considering the enzymatic preferences of BSHs alongside the conjugated/deconjugated BA-bacterial interaction. These results deepen our understanding of the BA-microbiome axis and provide a framework to engineer lactobacilli with improved bile resistance and use probiotics as BA-altering therapeutics.

Using Multidimensional Separations to Distinguish Isomeric Amino Acid-Bile Acid Conjugates and Assess Their Presence and Perturbations in Model Systems.

Bile acids play key roles in nutrient uptake, inflammation, signaling, and microbiome composition. While previous bile acid analyses have primarily focused on profiling 5 canonical primary and secondary bile acids and their glycine and taurine amino acid-bile acid (AA-BA) conjugates, recent studies suggest that many other microbial conjugated bile acids (or MCBAs) exist. MCBAs are produced by the gut microbiota and serve as biomarkers, providing information about early disease onset and gut health. Here we analyzed 8 core bile acids synthetically conjugated with 22 proteinogenic and nonproteogenic amino acids totaling 176 MCBAs. Since many of the conjugates were isomeric and only 42 different m/z values resulted from the 176 MCBAs, a platform coupling liquid chromatography, ion mobility spectrometry, and mass spectrometry (LC-IMS-MS) was used for their separation. Their molecular characteristics were then used to create an in-house extended bile acid library for a combined total of 182 unique compounds. Additionally, ∼250 rare bile acid extracts were also assessed to provide additional resources for bile acid profiling and identification. This library was then applied to healthy mice dosed with antibiotics and humans having fecal microbiota transplantation (FMT) to assess the MCBA presence and changes in the gut before and after each perturbation.

Hendra Virus: An Update on Diagnosis, Vaccination, and Biosecurity Protocols for Horses.

Hendra virus (HeV) emerged as a zoonotic pathogen in the 1990s, causing low morbidity but high mortality in humans and horses. Pteropid bats are the natural reservoir of HeV and other important zoonotic viruses such as Nipah and Ebola viruses. Equivac HeV, manufactured by Zoetis (Parkville, Victoria, Australia), is the only commercially available vaccine for horses. There is no commercial vaccine for humans. The epidemiology, clinical features, pathology, diagnosis, management, and prevention of HeV will be reviewed.

"I would rather do it myself": injection initiation and current injection patterns among women who inject drugs in Tijuana, Mexico.

BACKGROUND: Women who inject drugs (WWID) experience unique risks and adverse health outcomes related to injection initiation and patterns of injection drug use. However, there is limited information on injection initiation experiences and injection patterns among women and the protective strategies employed to limit injection-related harms, especially in low- and middle-income settings. Therefore, this study sought to explore injection initiation and current injection patterns (e.g., relying on someone else to inject) among women who inject drugs and engage in sex work in Tijuana, Mexico. METHODS: Semistructured in-depth interviews were conducted with 30 WWID on the following topics: injection initiation, current injection patterns, places where women inject, and protective strategies (i.e., risk reduction). All interviews were audio-recorded, transcribed, and de-identified. An inductive thematic analysis was conducted to identify and compare common themes and patterns across participants. RESULTS: The interviews revealed that the vast majority of study participants were first initiated by another person who injects drugs (PWID), often a male sexual partner. However, the majority of the women transitioned to become self-injectors in order to avoid risks associated with relying on others for injection, including overdose, interpersonal violence, sexual abuse, and wounds. Those who relied on others indicated that they would prefer to inject themselves without assistance from others if they were able to. CONCLUSIONS: The narratives uncovered in this study reveal the importance of multiple risk environments in shaping perceived risks associated with injection drug use among women in Tijuana, Mexico. Specifically, the interviews elucidate the connection between interpersonal relationships with other PWID and protective strategies used to minimize risk and harm. These findings highlight the need for women-centered harm reduction programs to facilitate the development of safer drug use environments among WWID in Tijuana, Mexico.

Metabolomics-Guided Discovery of Microginin Peptides from Cultures of the Cyanobacterium Microcystis aeruginosa.

We report a mass-spectrometry-based metabolomics study of a laboratory-cultured strain of Microcystis aeruginosa (UTEX LB2385), which has led to the discovery of five peptides (1-5) belonging to the microginin class of linear cyanopeptides. The structures and configurations of these peptides were determined by spectroscopic analyses and chemical derivitization. The microginin peptides described herein are the first reported derivatives containing N-methyl methionine (1, 5) and N-methyl methionine sulfoxide (2-4). The two tripeptide microginin analogues (4, 5) identified represent the smallest members of this peptide family. Their angiotensin-converting enzyme (ACE) inhibitory activity was also investigated. Microginin 527 (4) was the most potent of the group, with an IC50 of 31 µM.

Imaging horse tendons using multimodal 2-photon microscopy.

Injuries and damage to tendons plague both human and equine athletes. At the site of injuries, various cells congregate to repair and re-structure the collagen. Treatments for collagen injury range from simple procedures such as icing and pharmaceutical treatments to more complex surgeries and the implantation of stem cells. Regardless of the treatment, the level of mechanical stimulation incurred by the recovering tendon is crucial. However, for a given tendon injury, it is not known precisely how much of a load should be applied for an effective recovery. Both too much and too little loading of the tendon could be detrimental during recovery. A mapping of the complex local environment imparted to any cell present at the site of a tendon injury may however, convey fundamental insights related to their decision making as a function of applied load. Therefore, fundamentally knowing how cells translate mechanical cues from their external environment into signals regulating their functions during repair is crucial to more effectively treat these types of injuries. In this paper, we studied systems of tendons with a variety of 2-photon-based imaging techniques to examine the local mechanical environment of cells in both normal and injured tendons. These tendons were chemically treated to instigate various extents of injury and in some cases, were injected with stem cells. The results related by each imaging technique distinguish with high contrast and resolution multiple morphologies of the cells' nuclei and the alignment of the collagen during injury. The incorporation of 2-photon FLIM into this study probed new features in the local environment of the nuclei that were not apparent with steady-state imaging. Overall, this paper focuses on horse tendon injury pattern and analysis with different 2-photon confocal modalities useful for wide variety of application in damaged tissues.

Monacyclinones, New Angucyclinone Metabolites Isolated from Streptomyces sp. M7_15 Associated with the Puerto Rican Sponge Scopalina ruetzleri.

During an investigation of new actinomycete species from Caribbean sponges for novel bioactive natural products, frigocyclinone (1), dimethyldehydrorabelomycin (3) and six new angucyclinone derivatives were isolated from Streptomyces sp. strain M7_15 associated with the sponge Scopalina ruetzleri. Of these, monacyclinones A-B (4-5) contain the core ring structure of dehydrorabelomycin (2) with the aminodeoxysugar found in frigocyclinone (1). Monacyclinone C (6) is a hydroxylated variant of frigocyclinone (1) and monacyclinone D (7) is a Baeyer Villiger derivative of (6) which also exists as the open chain hydrolysis product monacyclinone E (8). Monacyclinone F (9) contains two unique epoxide rings attached to the angucyclinone moiety and an additional aminodeoxysugar attached through an angular oxygen bond. All structures were confirmed through spectral analyses. Activity against rhabdomycosarcoma cancer cells (SJCRH30) after 48 h of treatment was observed with frigocyclinone (1; EC50 = 5.2 µM), monacyclinone C (6; 160 µM), monacyclinone E (8; 270 µM), and monacyclinone F (9; 0.73 µM). The strongest bioactivity against rhabdomycosarcoma cancer cells and gram-positive bacteria was exhibited by compound 9, suggesting that the extra aminodeoxysugar subunit is important for biological activity.

Update on fungal respiratory disease in horses.

Fungal respiratory disease is a rare occurrence in horses. Fungal organisms are ubiquitous in the equine environment; however, there is a geographic predisposition for disease development, with fungal respiratory infections seen more commonly by practitioners working in tropical or subtropical environments. Diagnosis and treatment of fungal respiratory infections pose a challenge for the equine practitioner, and the prognosis for complete resolution of infection is often guarded; however, new antifungal medications are likely to improve treatment success. This article summarizes the available literature regarding the cause, diagnosis, and treatment of equine fungal respiratory disease.

Surgical treatment of 4 horses for cryptorchidism caused by failure of regression of the cranial suspensory ligament of the testis.

OBJECTIVE: To report surgical management of 4 horses with cryptorchidism caused by failure of regression of the cranial suspensory ligament (CSL). STUDY DESIGN: Retrospective case series. ANIMALS: Cryptorchid horses (n = 4). METHODS: Horses with unilateral or bilateral cryptorchidism caused by failure of regression of the CSL were treated by removing the retained testes through a standing laparoscopic approach (2), flank laparotomy (1), or paramedian celiotomy (1). After identification of the retained testis attached to the caudal pole of the kidney by the CSL, the vascular pedicle and ductus deferens were ligated and removed. RESULTS: Two horses had bilateral cryptorchidism and 2 horses had unilateral cryptorchidism. Standing laparoscopic cryptorchidectomy was performed successfully in 2 horses and in 2, the retained testes were removed using a flank or paramedian celiotomy. All testes were located in the dorsal aspect of the abdomen just caudal to the kidney and had a well-developed CSL. All horses recovered successfully from surgery. CONCLUSIONS: Failure of regression of the CSL is an uncommon cause of cryptorchidism in horses; however, affected horses can be treated using surgical approaches that facilitate exploration of the dorsocaudal aspect of the abdomen.

Assessment of a Teaching Module for Cardiac Auscultation of Horses by Veterinary Students.

Auscultation of heart sounds is an important veterinary skill requiring an understanding of anatomy, physiology, pathophysiology and pattern recognition. This cross-sectional study was developed to evaluate a targeted, audio-visual training resource for veterinary students to improve their understanding and auscultation of common heart conditions in horses. Fourth- and fifth-year 2021 and 2022 Bachelor of Veterinary Science students at the University of Queensland (UQ) were provided the learning resource and surveyed via online pre- and post-intervention surveys. Results were quantitatively analyzed using descriptive statistics and Mann-Whitney U tests. Open-ended survey questions were qualitatively analyzed by thematic analysis and Leximancer™ Version 4 program software analysis. Over the two-year period, 231 fourth-year and 222 fifth-year veterinary students had access to the resource; 89 completed the pre-intervention survey and 57 completed the post-intervention survey. Quantitative results showed the resource helped students prepare for practicals and their perception of competency and confidence when auscultating equine cardiac sounds improved (p < 0.05). Compared to fifth-year students, fourth-year students felt less competent at identifying murmurs and arrythmias prior to accessing the learning resource (p < 0.05). Fourth-year and fifth-year students' familiarity with detection of murmurs improved after completing the learning resource (p < 0.001). Qualitative analysis demonstrated a limited number of opportunities to practice equine cardiac auscultation throughout the veterinary degree, especially during the COVID-19 pandemic, and that integrated audio-visual resources are an effective means of teaching auscultation.

Epidemiological investigation of insulin dysregulation in Shetland and Welsh ponies in Australia.

BACKGROUND: Insulin dysregulation (ID) is central to equine metabolic syndrome. There are limited epidemiological studies investigating dynamic testing of ID in ponies. OBJECTIVES: To evaluate prevalence and risk factors for ID through dynamic testing of hyperinsulinaemia (DHI) and insulin resistance (IR). STUDY DESIGN: Cross-sectional. METHODS: Sex, age, breed, height, cresty neck score (CNS), body condition score (BCS), laminitis, HMGA2:c.83G>A genotype and pituitary pars intermedia dysfunction (PPID) status were documented. Dynamic hyperinsulinaemia was diagnosed with an oral sugar test (OST) and IR with an insulin tolerance test (ITT). Owners completed surveys reporting activity, laminitis history and perception of body condition using a (1-9) visual analogue scale (VASo). Ordinal scores were converted to binary outcomes for CNS (≤2/5 or ≥3/5), BCS and VASo (≤6/9 or ≥7/9). Variables associated with insulin concentrations, glucose reduction after the ITT and laminitis were evaluated with mixed effects regression models accounting for random effects of farms. RESULTS: Among 167 ponies tested, median (range) age was 9 (4-21) years and BCS was 6 (4-8). Prevalence (95% confidence interval [CI]) of ID was 61 (53-68)%. Factors associated with insulin concentrations (estimate [95% CI]; µIU/mL) 60 min post-OST were: age (1.07 [1.02-1.11]), CNS (≥3/5, 1.52 [1.04-2.23]) and VASo (≥7/9, 1.75 [1.09-2.79]); and 90 min post-OST were: age (1.08 [1.03-1.12]), CNS (≥3/5, 1.80 [1.22-2.64]), VASo (≥7/9, 2.49 [1.52-4.08]) and sex (male, 0.64 [0.45-0.91]). Factors associated with glucose reduction after the ITT (estimate [95% CI]; %) were: age (-1.34 [-2.01 to -0.67]), sex (female, -6.21 [-11.68 to -0.74]) and VASo (≥7/9, -1.74 [-18.89 to -4.78]). Factors associated with laminitis (odds ratio [95% CI]) were DHI (4.60 [1.68-12.58]), IR (3.66 [1.26-10.61]) and PPID (11.75 [1.54-89.40]). MAIN LIMITATIONS: Single time-point sampling, laminitis definition and diet analysis. CONCLUSIONS: Ageing, being female and owner-perceived obesity were associated with ID.

Seroprevalence of Anti-SARS-CoV-2 IgG Antibodies in Healthcare Personnel in El Salvador Prior to Vaccination Campaigns.

COVID-19, caused by the SARS-CoV-2 virus, is a highly pathogenic emerging infectious disease. Healthcare personnel (HCP) are presumably at higher risk of acquiring emerging infections because of occupational exposure. The prevalence of COVID-19 in HCP is unknown, particularly in low- to middle-income countries like El Salvador. The goal of this study was to determine the seroprevalence of anti-SARS-CoV-2 antibodies among HCP in El Salvador just prior to vaccine rollout in March 2021. We evaluated 2176 participants from a nationally representative sample of national healthcare institutions. We found 40.4% (n = 880) of the study participants were seropositive for anti-spike protein antibodies. Significant factors associated with infection included younger age; living within the central, more populated zone of the country; living in a larger household (≥7 members); household members with COVID-19 or compatible symptoms; and those who worked in auxiliary services (i.e., housekeeping and food services). These findings provide insight into opportunities to mitigate SARS-CoV-2 risk and other emerging respiratory pathogens in HCP in El Salvador.

Tumor- and circulating-free DNA methylation identifies clinically relevant small cell lung cancer subtypes.

Small cell lung cancer (SCLC) is an aggressive malignancy composed of distinct transcriptional subtypes, but implementing subtyping in the clinic has remained challenging, particularly due to limited tissue availability. Given the known epigenetic regulation of critical SCLC transcriptional programs, we hypothesized that subtype-specific patterns of DNA methylation could be detected in tumor or blood from SCLC patients. Using genomic-wide reduced-representation bisulfite sequencing (RRBS) in two cohorts totaling 179 SCLC patients and using machine learning approaches, we report a highly accurate DNA methylation-based classifier (SCLC-DMC) that can distinguish SCLC subtypes. We further adjust the classifier for circulating-free DNA (cfDNA) to subtype SCLC from plasma. Using the cfDNA classifier (cfDMC), we demonstrate that SCLC phenotypes can evolve during disease progression, highlighting the need for longitudinal tracking of SCLC during clinical treatment. These data establish that tumor and cfDNA methylation can be used to identify SCLC subtypes and might guide precision SCLC therapy.

Soft tissue releases affect the femoral component rotation necessary to create a balanced flexion gap during total knee arthroplasty.

The structures that were released to balance the extension gap were recorded during 1500 consecutive TKA procedures, and the amount of femoral component external rotation (ER) necessary to balance the flexion gap was measured with a tensiometer. The amount of ER necessary to balance the flexion gap significantly decreased as more medial structures were released (1 structure=4.7°, 2=4.1°, 3=2.8°, 4 or more=1.1°, P<0.012), whereas significantly greater ER was necessary when three or more lateral structures were released (1 structure=5.3°, 2=5.5°, 3 or more=8.6°, P<0.03). Soft tissue releases affected the amount of femoral component ER necessary to balance the flexion gap, bringing into question the ability of techniques utilizing bony landmarks to properly align the femoral component in rotation.

Insect Bite Hypersensitivity in Horses: Causes, Diagnosis, Scoring and New Therapies.

Insect Bite Hypersensitivity (IBH, Queensland itch, sweet itch, equine summer eczema) is the most common pruritic disease of horses. It is most often caused by sensitivity to the saliva of Culicoides spp. of biting midges; however, it can also be caused by hypersensitivity to other insect species. The prevalence of IBH in horses is reported to be as high as 60% in some parts of the world. Due to the severe pruritus and effects of secondary self-trauma, IBH has animal welfare concerns, and there is currently no cure. Management of this condition is life-long, time consuming and costly. New grading systems to document disease severity are being validated, which will allow the comparison of clinical trial results of new and existing therapies. Management involves the minimisation of insect bites by use of stabling, fans, rugs and repellents. Symptomatic therapy involves the administration of systemic or topical corticosteroids, systemic antihistamines, and creams and sprays to promote skin healing and decrease inflammation. New immune-mediated therapeutics including vaccines, in addition to desensitisation procedures, show promise at controlling hypersensitivity reactions. This article will review aetiologic agents, pathophysiology, scoring systems and current and new therapies.

Diagnosis of equine pituitary pars intermedia dysfunction.

Equine pituitary pars intermedia dysfunction (PPID) is common in aged horses. The majority of horses respond well to treatment, but treatment is lifelong, meaning accurate diagnosis of PPID is important. Similar to any condition, there is no perfect laboratory test to diagnose PPID and accuracy is affected by the characteristics of the population in which the test is being evaluated. This review details the importance of consideration of clinical factors and diagnostic test accuracy. Basal adrenocorticotrophic hormone (ACTH) concentration is used most frequently in practice and has very good diagnostic accuracy when used in combination with clinical judgement and the correct application of diagnostic thresholds. The thyrotropin-releasing hormone stimulation test can be used in horses with equivocal test results following basal ACTH testing, or to evaluate subtle cases due to its improved accuracy.

Equine Crofton Weed (Ageratina spp.) Pneumotoxicity: What Do We Know and What Do We Need to Know?

Crofton weed (Ageratina adenophora) is a global and highly invasive weed, with ingestion causing severe respiratory disease in horses, leading to irreversible and untreatable pulmonary fibrosis and oedema. While reports of equine pneumotoxicity remain common in Australia and New Zealand, equine pneumotoxicity may be underdiagnosed in other countries where Crofton weed is endemic but poorly differentiated. The pathogenesis of Crofton weed toxicity following ingestion has been well described in a number of different animal models, including rodents, rabbits, and goats. However, induced toxicity is organ-selective across different animal species, and these vastly differ from the pathogenesis described in horses, both clinically and after experimental exposure. Sources of variation may include species-specific susceptibility to different toxins present in the plant, different mechanistic processes of toxicity, and species differences in toxin biotransformation and bioactivation across different organs. Considering disease severity and Crofton weed's invasiveness globally, assessing published toxicological and exposure data is necessary to advance research, identify specific toxins for horses, and possible prophylactic and therapeutic strategies. This review presents an overview of the available literature on equine toxicity, parallels between toxicity in horses and other animal species, and important aspects to be included in the future research agenda.

Sociodemographic Factors Associated with COVID-19 Vaccination among People in Guatemalan Municipalities.

The Republic of Guatemala's reported COVID-19 vaccination coverage is among the lowest in the Americas and there are limited studies describing the disparities in vaccine uptake within the country. We performed a cross-sectional ecological analysis using multi-level modeling to identify sociodemographic characteristics that were associated with low COVID-19 vaccination coverage among Guatemalan municipalities as of 30 November 2022. Municipalities with a higher proportion of people experiencing poverty (ß = -0.25, 95% CI: -0.43--0.07) had lower vaccination coverage. Municipalities with a higher proportion of people who had received at least a primary education (ß = 0.74, 95% CI: 0.38-1.08), children (ß = 1.07, 95% CI: 0.36-1.77), people aged 60 years and older (ß = 2.94, 95% CI: 1.70-4.12), and testing for SARS-CoV-2 infection (ß = 0.25, 95% CI: 0.14-0.36) had higher vaccination coverage. In the simplified multivariable model, these factors explained 59.4% of the variation in COVID-19 vaccination coverage. Poverty remained significantly associated with low COVID-19 vaccination coverage in two subanalyses restricting the data to the time period of the highest national COVID-19-related death rate and to COVID-19 vaccination coverage only among those aged 60 years or older. Poverty is a key factor associated with low COVID-19 vaccination and focusing public health interventions in municipalities most affected by poverty may help address COVID-19 vaccination and health disparities in Guatemala.