Insights into docking in megasynthases from the investigation of the toblerol trans-AT polyketide synthase: many α-helical means to an end.

The fidelity of biosynthesis by modular polyketide synthases (PKSs) depends on specific moderate affinity interactions between successive polypeptide subunits mediated by docking domains (DDs). These sequence elements are notably portable, allowing their transplantation into alternative biosynthetic and metabolic contexts. Herein, we use integrative structural biology to characterize a pair of DDs from the toblerol trans-AT PKS. Both are intrinsically disordered regions (IDRs) that fold into a 3 α-helix docking complex of unprecedented topology. The C-terminal docking domain (CDD) resembles the 4 α-helix type (4HB) CDDs, which shows that the same type of DD can be redeployed to form complexes of distinct geometry. By carefully re-examining known DD structures, we further extend this observation to type 2 docking domains, establishing previously unsuspected structural relations between DD types. Taken together, these data illustrate the plasticity of α-helical DDs, which allow the formation of a diverse topological spectrum of docked complexes. The newly identified DDs should also find utility in modular PKS genetic engineering.

Point-of-need mastitis pathogen biosensing in bovine milk: From academic sample preparation novelty to industry prototype field testing.

Bovine mastitis is an inflammation of the mammary gland, and it is the most common infectious disease in dairy cattle. Mastitis reduces milk yield and quality, costing dairy farmers millions of dollars each year. The aim of this study was to develop a point-of-need test for identifying mastitis pathogens that is field portable, cost-effective and can be used with minimal training. Using a proprietary polymer-based milk sample preparation method to rapidly extract pathogen DNA in milk samples, we demonstrated quantitative Polymerase Chain Reaction (qPCR) assays for six common bovine bacterial mastitis pathogens: Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Mycoplasma bovis and Escherichia coli. We also implemented this sample preparation method on a prototype point-of-need system in a proof-of-concept field trial to evaluate user experience. Importantly, the protype system enabled a sample-to-result turnaround time of within 70 min to quantitatively detect all six target pathogens. The key advantage of our point-of-need prototype system is being culture-independent yet providing automated milk sample preparation for molecular identification of key mastitis pathogens by non-expert users. Our point-of-need prototype system showed a good correlation to laboratory-based qPCR for target pathogen detection outcomes, thus potentially removing the need for milk samples to be transported off-site for laboratory testing. Above all, we successfully achieved our objective of developing a point-of-need biosensor technology for mastitis and increased its readiness level with industry partners towards technology commercialization.

Exposure to the antineoplastic ifosfamide alters molecular pathways related to cardiovascular function, increases heart rate, and induces hyperactivity in zebrafish (Danio rerio).

Ifosfamide is an alkylating antineoplastic drug used in chemotherapy, but it is also detected in wastewater. Here, the objectives were to (1) determine teratogenic, cardiotoxic, and mitochondrial toxicity potential of ifosfamide exposure; (2) elucidate mechanisms of toxicity; (3) characterize exposure effects on larval behavior. Survival rate, hatch rate, and morphological deformity incidence were not different amongst treatments following exposure levels up to 1000 µg/L ifosfamide over 7 days. RNA-seq reveled 231 and 93 differentially expressed transcripts in larvae exposed to 1 µg/L and 100 µg/L ifosfamide, respectively. Several gene networks related to vascular resistance, cardiovascular response, and heart rate were affected, consistent with tachycardia observed in exposed embryonic fish. Hyperactivity in larval zebrafish was observed with ifosfamide exposure, potentially associated with dopamine-related gene networks. This study improves ecological risk assessment of antineoplastics by elucidating molecular mechanisms related to ifosfamide toxicity, and to alkylating agents in general.

Temporal Variation and Persistence of Methane Emissions from Shallow Water Oil and Gas Production in the Gulf of Mexico.

Methane emissions from the oil and gas supply chain can be intermittent, posing challenges for monitoring and mitigation efforts. This study examines shallow water facilities in the US Gulf of Mexico with repeat atmospheric observations to evaluate temporal variation in site-specific methane emissions. We combine new and previous observations to develop a longitudinal study, spanning from days to months to almost five years, evaluating the emissions behavior of sites over time. We also define and determine the chance of subsequent detection (CSD): the likelihood that an emitting site will be observed emitting again. The average emitting central hub in the Gulf has a 74% CSD at any time interval. Eight facilities contribute 50% of total emissions and are over 80% persistent with a 96% CSD above 100 kg/h and 46% persistent with a 42% CSD above 1000 kg/h, indicating that large emissions are persistent at certain sites. Forward-looking infrared (FLIR) footage shows many of these sites exhibiting cold venting. This suggests that for offshore, a low sampling frequency over large spatial coverage can capture typical site emissions behavior and identify targets for mitigation. We further demonstrate the preliminary use of space-based observations to monitor offshore emissions over time.

Biosynthesis of novel desferrioxamine derivatives requires unprecedented crosstalk between separate NRPS-independent siderophore pathways.

Iron is essential to many biological processes but its poor solubility in aerobic environments restricts its bioavailability. To overcome this limitation, bacteria have evolved a variety of strategies, including the production and secretion of iron-chelating siderophores. Here, we describe the discovery of four series of siderophores from Streptomyces ambofaciens ATCC23877, three of which are unprecedented. MS/MS-based molecular networking revealed that one of these series corresponds to acylated desferrioxamines (acyl-DFOs) recently identified from S. coelicolor. The remaining sets include tetra- and penta-hydroxamate acyl-DFO derivatives, all of which incorporate a previously undescribed building block. Stable isotope labeling and gene deletion experiments provide evidence that biosynthesis of the acyl-DFO congeners requires unprecedented crosstalk between two separate non-ribosomal peptide synthetase (NRPS)-independent siderophore (NIS) pathways in the producing organism. Although the biological role(s) of these new derivatives remain to be elucidated, they may confer advantages in terms of metal chelation in the competitive soil environment due to the additional bidentate hydroxamic functional groups. The metabolites may also find application in various fields including biotechnology, bioremediation, and immuno-PET imaging.IMPORTANCEIron-chelating siderophores play important roles for their bacterial producers in the environment, but they have also found application in human medicine both in iron chelation therapy to prevent iron overload and in diagnostic imaging, as well as in biotechnology, including as agents for biocontrol of pathogens and bioremediation. In this study, we report the discovery of three novel series of related siderophores, whose biosynthesis depends on the interplay between two NRPS-independent (NIS) pathways in the producing organism S. ambofaciens-the first example to our knowledge of such functional cross-talk. We further reveal that two of these series correspond to acyl-desferrioxamines which incorporate four or five hydroxamate units. Although the biological importance of these novel derivatives is unknown, the increased chelating capacity of these metabolites may find utility in diagnostic imaging (for instance, 89Zr-based immuno-PET imaging) and other applications of metal chelators.

How does parasite environmental transmission stage concentration change before, during, and after disease outbreaks?

Outbreaks of environmentally transmitted parasites require that susceptible hosts encounter transmission stages in the environment and become infected, but we also know that transmission stages can be in the environment without triggering disease outbreaks. One challenge in understanding the relationship between environmental transmission stages and disease outbreaks is that the distribution and abundance of transmission stages outside of their hosts have been difficult to quantify. Thus, we have limited data about how changes in transmission stage abundance influence disease dynamics; moreover, we do not know whether the relationship between transmission stages and outbreaks differs among parasite species. We used digital PCR to quantify the environmental transmission stages of five parasites in six lakes in southeastern Michigan every 2 weeks from June to November 2021. At the same time, we quantified infection prevalence in hosts and host density. Our study focused on eight zooplankton host species (Daphnia spp. and Ceriodaphnia dubia) and five of their parasites from diverse taxonomic groups (bacteria, yeast, microsporidia, and oomycete) with different infection mechanisms. We found that parasite transmission stage concentration increased prior to disease outbreaks for all parasites. However, parasites differed significantly in the relative timing of peaks in transmission stage concentration and infection outbreaks. The "continuous shedder" parasites had transmission stage peaks at the same time as or slightly after the outbreak peaks. In contrast, parasites relying on host death for transmission ("obligate killers") had transmission stage peaks before outbreak peaks. For most parasites, lakes with outbreaks had higher spore concentrations than those without outbreaks, especially once an outbreak began; the exception was for a parasite, Pasteuria ramosa, with very strong genotypic specificity of infection. Overall, our results show that disease outbreaks are tightly linked to transmission stage concentration; outbreaks were preceded by increases in transmission stage concentration in the environment and then were fueled by the production of more transmission stages during the outbreak itself, with concentrations decreasing to pre-outbreak levels as outbreaks waned. Thus, tracking transmission stages in the environment improves our understanding of the drivers of disease outbreaks and reveals how parasite traits may affect these dynamics.

Resilient responses to stressful events among chiropractic students: A cross-sectional survey.

OBJECTIVE: Resilient students may better navigate the challenges of chiropractic training. This study explored the relationships between response to stressful experiences, perceived stress, and quality of life among students at 1 US chiropractic college campus. METHODS: A cross-sectional survey was conducted with 873 students. The anonymous online questionnaire included demographics, quality of life, perceived stress, and the Response to Stressful Events Scale (RSES). Hierarchical multiple regression analyses assessed for significant relationships among variables. RESULTS: A sample of 221 students (60% female) completed the survey (25% response rate). Male respondents reported greater psychological health. Participants reporting high quality of life exhibited higher resiliency on the RSES flat score and many RSES domains. Respondents who rated greater psychological health and social relationships exhibited greater spiritual resiliency. Respondents reporting higher psychological health and lower levels of perceived stress exhibited higher self-efficacy resilience. Psychological health was the most important predictor for RSES flat score and domains, except for spiritual resilience, for which social relationships were most important. Male gender was predictive of 3 RSES domains: meaning making, active coping, and cognitive flexibility. CONCLUSION: More resilient responses to stressful events were reported by male chiropractic students and those who reported greater psychological health, higher quality of life, or lower perceived stress. Female students and those experiencing psychological challenges or lower quality of life might consider resilience training to increase the use of protective coping strategies. These findings may permit academic institutions to identify students at highest risk and employ interventions to prevent program withdrawal.

Data Gap: Air Quality Networks Miss Air Pollution from Concentrated Animal Feeding Operations.

In the U.S., the agricultural sector is the largest controllable source of several air pollutants, including ammonia (NH3), which is a key precursor to PM2.5 formation. Livestock waste is the dominant contributor to ammonia emissions. In contrast to most controllable air pollutants, satellite records show ammonia mixing ratios are rising. The number of confined animal feeding operations (CAFOs) that generate considerable livestock waste is also increasing. Spatial and temporal trends in USDA-reported animal numbers normalized by county area at medium and large CAFOs provide plausible explanations for patterns in satellite-derived NH3 over the contiguous U.S. (CONUS). The correlation between summertime ammonia derived from the European Space Agency's (ESA) Infrared Atmospheric Sounding Interferometer (IASI) and CAFO animal unit density in 2017 is positive and significant (r = 0.642; p ≈ 0). The temporal changes from 2002 to 2017 in animal unit density and NH3 derived from NASA's Atmospheric Infrared Sounder (AIRS) are spatially similar. Trends and ambient concentrations of PM2.5 mass in agricultural regions are difficult to assess relative to those of urban population centers given the sparseness of rural monitors in regulatory surface networks. Results suggest that in agricultural areas where ammonia concentrations and animal density are highest, air quality improvement lags behind the national average.

A Case of a Serum Sickness-Like Reaction After Postexposure Rabies Prophylaxis.

BACKGROUND: Serum sickness secondary to rabies postexposure prophylaxis is not well documented in the medical literature. Our case describes serum sickness after exposure to human-derived rabies immunoglobulin (HRIG) and three human diploid rabies vaccines (HDCV) in a young adult male. CASE REPORT: A 30-year-old previously healthy male patient presented to the Emergency Department with complaints of fever, rash, and jaundice, and had a hospital course complicated by biliary stenosis likely secondary to reactive periportal lymphadenopathy. His initial laboratory values demonstrated obstructive jaundice and slightly elevated complement component 4 levels. These symptoms likely are due to the course of HRIG and HDCV vaccines the patient completed after being exposed to a rabies-positive bat in his home. The patient was hospitalized for 8 days, during which he underwent an endoscopic retrograde cholangiopancreatography with sphincterotomy and biliary stenting. He had one repeat hospitalization for acute blood loss anemia attributed to sphincterotomy, which did not require transfusion or further intervention. Liver biopsy showed cholestatic hepatitis. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Medical literature describing serum sickness or a serum sickness-like reaction occurring from exposure to HRIG or HDCV is sparse despite the commonality of postexposure rabies prophylaxis in health care. It is important to educate practitioners on this potential complication and highlight next potential consultations and treatments.

KidneyGPS: a user-friendly web application to help prioritize kidney function genes and variants based on evidence from genome-wide association studies.

BACKGROUND: Genome-wide association studies (GWAS) have identified hundreds of genetic loci associated with kidney function. By combining these findings with post-GWAS information (e.g., statistical fine-mapping to identify independent association signals and to narrow down signals to causal variants; or different sources of annotation data), new hypotheses regarding physiology and disease aetiology can be obtained. These hypotheses need to be tested in laboratory experiments, for example, to identify new therapeutic targets. For this purpose, the evidence obtained from GWAS and post-GWAS analyses must be processed and presented in a way that they are easily accessible to kidney researchers without specific GWAS expertise. MAIN: Here we present KidneyGPS, a user-friendly web-application that combines genetic variant association for estimated glomerular filtration rate (eGFR) from the Chronic Kidney Disease Genetics consortium with annotation of (i) genetic variants with functional or regulatory effects ("SNP-to-gene" mapping), (ii) genes with kidney phenotypes in mice or human ("gene-to-phenotype"), and (iii) drugability of genes (to support re-purposing). KidneyGPS adopts a comprehensive approach summarizing evidence for all 5906 genes in the 424 GWAS loci for eGFR identified previously and the 35,885 variants in the 99% credible sets of 594 independent signals. KidneyGPS enables user-friendly access to the abundance of information by search functions for genes, variants, and regions. KidneyGPS also provides a function ("GPS tab") to generate lists of genes with specific characteristics thus enabling customizable Gene Prioritisation (GPS). These specific characteristics can be as broad as any gene in the 424 loci with a known kidney phenotype in mice or human; or they can be highly focussed on genes mapping to genetic variants or signals with particularly with high statistical support. KidneyGPS is implemented with RShiny in a modularized fashion to facilitate update of input data ( https://kidneygps.ur.de/gps/ ). CONCLUSION: With the focus on kidney function related evidence, KidneyGPS fills a gap between large general platforms for accessing GWAS and post-GWAS results and the specific needs of the kidney research community. This makes KidneyGPS an important platform for kidney researchers to help translate in silico research results into in vitro or in vivo research.

Factors Associated with Utilization of Teleretinal Imaging in a Hospital-Based Primary Care Setting.

Regular eye examinations to screen for the initial signs of diabetic retinopathy (DR) are crucial for preventing vision loss. Teleretinal imaging (TRI) offered in a primary care setting provides a means to improve adherence to DR screening, particularly for patients who face challenges in visiting eye care providers regularly. The present study evaluates the utilization of TRI to screen for DR in an outpatient, hospital-based primary care clinic. Patients with diabetes mellitus (DM) but without DR were eligible for point-of-care screening facilitated by their primary care provider, utilizing a non-mydriatic, handheld fundus camera. Patient demographics and clinical characteristics were extracted from the electronic medical record. Patients who underwent TRI were more likely to be male, non-White, and have up-to-date monitoring and treatment measures, including hemoglobin A1c (HbA1c), microalbumin, and low-density lipoprotein (LDL) levels, in accordance with Healthcare Effectiveness Data and Information Set (HEDIS) guidelines. Our findings demonstrate that TRI can reduce screening costs compared to a strategy where all patients are referred for in-person eye examinations. A net present value (NPV) analysis indicates that a screening site reaches the break-even point of operation within one year if an average of two patients are screened per workday.

Adverse childhood experiences and trauma informed care for chiropractors: a call to awareness and action.

BACKGROUND: Trauma is an emotional response to distressing events where coping and subsequent recovery are absent. Adverse Childhood Experiences (ACEs) are traumas, occurring before the age of 18 years, such as child abuse or neglect, caregiver instability, and household dysfunction. Sixty-four percent of the U.S. population report experiencing at least one ACE, with over 1 billion children experiencing abuse and neglect annually worldwide. Chronic exposure to stressful circumstances or multiple traumatic events has negative physiologic impacts. Persons who experience 3 or more ACEs in childhood are at greater risk of poor mental health outcomes and may be more likely to engage in high-risk behaviors, predisposing them to long-term health impacts, such as metabolic diseases, anxiety, depression, substance use, and chronic pain. Trauma informed care (TIC) is a recommended approach to healthcare delivery across professions, especially when a trauma history is suspected. This commentary aims to increase awareness of the impact of ACEs on health outcomes and introduce TIC concepts as they may apply to chiropractic care for adults with a history of ACEs. DISCUSSION: This commentary reviews an introductory model (4R's: realize, recognize, respond, resist re-traumatization) as one TIC framework used by healthcare practitioners. Prior trauma can lessen trust, alter perceptions of physical touch, and hands-on examinations and chiropractic treatments may trigger stress responses. Using TIC after appropriate training, includes referrals to multidisciplinary providers to address trauma-related concerns outside the scope of chiropractic, and screening for ACEs if deemed appropriate. Creating safe spaces, communicating clearly, avoiding victimizing language, explaining procedures, asking for consent before physical contact, and giving patients choice and control in their own care may avoid triggering prior traumas. CONCLUSION: Given the high worldwide prevalence of persons experiencing 3 or more ACEs, TIC principles are practical adaptations to chiropractic care for use with many patient populations. As TIC and ACEs are emerging concepts within chiropractic, students and practitioners are encouraged to undertake additional training to better understand these complex and sensitive topics. Exploratory research on the incidence, presentation, and impacts of various trauma types, including ACEs, to support adoption of TIC in chiropractic settings is essential.

Polygenic scores for estimated glomerular filtration rate in a population of general adults and elderly - comparative results from the KORA and AugUR study.

BACKGROUND: Polygenic scores (PGSs) combining genetic variants found to be associated with creatinine-based estimated glomerular filtration rate (eGFRcrea) have been applied in various study populations with different age ranges. This has shown that PGS explain less eGFRcrea variance in the elderly. Our aim was to understand how differences in eGFR variance and the percentage explained by PGS varies between population of general adults and elderly. RESULTS: We derived a PGS for cystatin-based eGFR (eGFRcys) from published genome-wide association studies. We used the 634 variants known for eGFRcrea and the 204 variants identified for eGFRcys to calculate the PGS in two comparable studies capturing a general adult and an elderly population, KORA S4 (n = 2,900; age 24-69 years) and AugUR (n = 2,272, age ≥ 70 years). To identify potential factors determining age-dependent differences on the PGS-explained variance, we evaluated the PGS variance, the eGFR variance, and the beta estimates of PGS association on eGFR. Specifically, we compared frequencies of eGFR-lowering alleles between general adult and elderly individuals and analyzed the influence of comorbidities and medication intake. The PGS for eGFRcrea explained almost twice as much (R2 = 9.6%) of age-/sex adjusted eGFR variance in the general adults compared to the elderly (4.6%). This difference was less pronounced for the PGS for eGFRcys (4.7% or 3.6%, respectively). The beta-estimate of the PGS on eGFRcrea was higher in the general adults compared to the elderly, but similar for the PGS on eGFRcys. The eGFR variance in the elderly was reduced by accounting for comorbidities and medication intake, but this did not explain the difference in R2-values. Allele frequencies between general adult and elderly individuals showed no significant differences except for one variant near APOE (rs429358). We found no enrichment of eGFR-protective alleles in the elderly compared to general adults. CONCLUSIONS: We concluded that the difference in explained variance by PGS was due to the higher age- and sex-adjusted eGFR variance in the elderly and, for eGFRcrea, also by a lower PGS association beta-estimate. Our results provide little evidence for survival or selection bias.

Modular Oxime Formation by a trans-AT Polyketide Synthase.

Modular trans-acyltransferase polyketide synthases (trans-AT PKSs) are enzymatic assembly lines that biosynthesize complex polyketide natural products. Relative to their better studied cis-AT counterparts, the trans-AT PKSs introduce remarkable chemical diversity into their polyketide products. A notable example is the lobatamide A PKS, which incorporates a methylated oxime. Here we demonstrate biochemically that this functionality is installed on-line by an unusual oxygenase-containing bimodule. Furthermore, analysis of the oxygenase crystal structure coupled with site-directed mutagenesis allows us to propose a model for catalysis, as well as identifying key protein-protein interactions that support this chemistry. Overall, our work adds oxime-forming machinery to the biomolecular toolbox available for trans-AT PKS engineering, opening the way to introducing such masked aldehyde functionalities into diverse polyketides.

In silico microRNA network data in zebrafish after antineoplastic ifosfamide exposure.

Ifosfamide is a cancer-fighting chemotherapeutic that has been detected in aquatic ecosystems. Zebrafish larvae were exposed to either 0, 1 or 100 µg/L ifosfamide in the water for 7 days, and fish were subjected to total RNA extraction and RNA-seq analysis with the Illumina NovoSeq 6000 instrument. Raw sequence data were processed through fastp and clean reads obtained by removing adapter and poly-N sequences, as well as low quality reads. Differential gene expression was performed using the abundance of transcripts that mapped to the zebrafish genome. To uncover putative targets regulated by microRNAs, Pathway Studio 12.0 was used to conduct a subnetwork enrichment analysis. Expression data were used to predict which microRNAs were important for the response to ifosfamide exposure. There were 21 common microRNAs identified in both the "IFOS1" and "IFOS100" datasets. These were MIR150, MIR6515, MIR657, MIR216A, m_Mir741, MIRLET7E, miR-let-7, MIR2392, r_Mir3551, MIR181B1, MIR33A, MIR502, MIR193B, MIR146A, MIR431, MIR647, m_Mir1192, MIR297, MIR328, and MIR4717. Data can be re-used to advance adverse outcome pathways in regulatory toxicology and to refine biomarker discovery for antineoplastics in aquatic environments.

The Relationship Between Directly Observed Therapy for Chlamydia Infections and Retesting Rates in an Adolescent Population.

BACKGROUND: Chlamydia trachomatis is the most common reportable sexually transmitted infection in the United States, with >60% of reported cases occurring in individuals aged 15 to 24 years. US practice guidelines recommend directly observed therapy (DOT) for the treatment of chlamydia in adolescents, but almost no research has been done to evaluate whether DOT results in improved outcomes. METHODS: We conducted a retrospective cohort study of adolescents who sought care at 1 of 3 clinics within a large academic pediatric health system for a chlamydia infection. The study outcome was return for retesting within 6 months. Unadjusted analyses were performed using χ2 , Mann-Whitney U , and t tests, and adjusted analyses were performed using multivariable logistic regression. RESULTS: Of the 1970 individuals included in the analysis, 1660 (84.3%) received DOT and 310 (15.7%) had a prescription sent to a pharmacy. The population was primarily Black/African American (95.7%) and female (78.2%). After controlling for confounders, individuals who had a prescription sent to a pharmacy were 49% (95% confidence interval, 31%-62%) less likely than individuals who received DOT to return for retesting within 6 months. CONCLUSIONS: Despite clinical guidelines recommending the use of DOT for chlamydia treatment in adolescents, this is the first study to describe the association between DOT and an increase in the number of adolescents and young adults who return for sexually transmitted infection retesting within 6 months. Further research is needed to confirm this finding in diverse populations and explore nontraditional settings for the provision of DOT.

Decrypting the programming of ß-methylation in virginiamycin M biosynthesis.

During biosynthesis by multi-modular trans-AT polyketide synthases, polyketide structural space can be expanded by conversion of initially-formed electrophilic ß-ketones into ß-alkyl groups. These multi-step transformations are catalysed by 3-hydroxy-3-methylgluratryl synthase cassettes of enzymes. While mechanistic aspects of these reactions have been delineated, little information is available concerning how the cassettes select the specific polyketide intermediate(s) to target. Here we use integrative structural biology to identify the basis for substrate choice in module 5 of the virginiamycin M trans-AT polyketide synthase. Additionally, we show in vitro that module 7, at minimum, is a potential additional site for ß-methylation. Indeed, analysis by HPLC-MS coupled with isotopic labelling and pathway inactivation identifies a metabolite bearing a second ß-methyl at the expected position. Collectively, our results demonstrate that several control mechanisms acting in concert underpin ß-branching programming. Furthermore, variations in this control - whether natural or by design - open up avenues for diversifying polyketide structures towards high-value derivatives.

Metacarpophalangeal Joint Pathology and Bone Mineral Density Increase with Exercise but Not with Incidence of Proximal Sesamoid Bone Fracture in Thoroughbred Racehorses.

Proximal sesamoid bone (PSB) fracture is the leading cause of fatal musculoskeletal injury in Thoroughbred racehorses in Hong Kong and the US. Efforts are underway to investigate diagnostic modalities that could help identify racehorses at increased risk of fracture; however, features associated with PSB fracture risk are still poorly understood. The objectives of this study were to (1) investigate third metacarpal (MC3) and PSB density and mineral content using dual-energy X-ray absorptiometry (DXA), computed tomography (CT), Raman spectroscopy, and ash fraction measurements, and (2) investigate PSB quality and metacarpophalangeal joint (MCPJ) pathology using Raman spectroscopy and CT. Forelimbs were collected from 29 Thoroughbred racehorse cadavers (n = 14 PSB fracture, n = 15 control) for DXA and CT imaging, and PSBs were sectioned for Raman spectroscopy and ash fraction measurements. Bone mineral density (BMD) was greater in MC3 condyles and PSBs of horses with more high-speed furlongs. MCPJ pathology, including palmar osteochondral disease (POD), MC3 condylar sclerosis, and MC3 subchondral lysis were greater in horses with more high-speed furlongs. There were no differences in BMD or Raman parameters between fracture and control groups; however, Raman spectroscopy and ash fraction measurements revealed regional differences in PSB BMD and tissue composition. Many parameters, including MC3 and PSB bone mineral density, were strongly correlated with total high-speed furlongs.

DNA extraction from clotted blood in genotyping quality.

DNA extraction from frozen blood clots is challenging. Here, the authors applied QIAGEN Clotspin Baskets and the Gentra Puregene Blood Kit for DNA extraction to cellular fraction of 5.5 ml whole blood without anticoagulating additives. The amount and quality of extracted DNA were assessed via spectrophotometer and gel electrophoresis. Results from array-based genotyping were analyzed. All steps were compared with DNA isolated from anticoagulated blood samples from a separate study. The quality and concentration of DNA extracted from clotted blood were comparable to those of DNA extracted from anticoagulated blood. DNA yield was on average 27 µg per ml clotted blood, with an average purity of 1.87 (A260/A280). Genotyping quality was similar for both DNA sources (call rate: 99.56% from clotted vs 99.49% from anticoagulated blood).