Racial Comparisons in Treatment of Rectal Adenocarcinoma and Survival in the Military Health System.

BACKGROUND: Racial disparities in treatment and outcomes of rectal cancer have been attributed to patients' differential access to care. We aimed to study treatment and outcomes of rectal cancer in the equal access Military Health System (MHS) to better understand potential racial disparities. METHODS: We accessed the MilCanEpi database to study a cohort of patients aged 18 and older who were diagnosed with rectal adenocarcinoma between 1998 and 2014. Receipt of guideline recommended treatment per tumor stage, cancer recurrence, and all-cause death were compared between Non-Hispanic White and Black patients using multivariable regression models with associations expressed as odds (AORs) or hazard ratios (AHRs) and their 95% confidence intervals (CIs). RESULTS: The study included 171 Black and 845 White patients with rectal adenocarcinoma. Overall, there were no differences in receipt of guideline concordant treatment (AOR = 0.76, 95% CI = 0.45, 1.29), recurrence (AHR = 1.34, 95% CI = 0.85, 2.12), or survival (AHR = 1.08, 95% CI = 0.77, 1.54) for Black patients compared to White patients. However, Black patients aged <50 years at diagnosis (AOR = 0.34, 95% CI 0.13, 0.90) or with stage III or IV tumors (AOR = 0.28, 95% CI = 0.12, 0.64) were less likely to receive guideline recommended treatment than White patients in stratified analysis. CONCLUSIONS: In the equal access MHS, while there were no overall racial disparities in rectal cancer treatment or clinical outcomes between Black and White patients, disparities among those with early-onset or late-stage rectal cancers were noted. This suggests factors other than access to care may play a role in the observed disparities and warrants further research.

Structural basis of transcription: RNA polymerase II substrate binding and metal coordination using a free-electron laser.

Catalysis and translocation of multisubunit DNA-directed RNA polymerases underlie all cellular mRNA synthesis. RNA polymerase II (Pol II) synthesizes eukaryotic pre-mRNAs from a DNA template strand buried in its active site. Structural details of catalysis at near-atomic resolution and precise arrangement of key active site components have been elusive. Here, we present the free-electron laser (FEL) structures of a matched ATP-bound Pol II and the hyperactive Rpb1 T834P bridge helix (BH) mutant at the highest resolution to date. The radiation-damage-free FEL structures reveal the full active site interaction network, including the trigger loop (TL) in the closed conformation, bonafide occupancy of both site A and B Mg2+, and, more importantly, a putative third (site C) Mg2+ analogous to that described for some DNA polymerases but not observed previously for cellular RNA polymerases. Molecular dynamics (MD) simulations of the structures indicate that the third Mg2+ is coordinated and stabilized at its observed position. TL residues provide half of the substrate binding pocket while multiple TL/BH interactions induce conformational changes that could allow translocation upon substrate hydrolysis. Consistent with TL/BH communication, a FEL structure and MD simulations of the T834P mutant reveal rearrangement of some active site interactions supporting potential plasticity in active site function and long-distance effects on both the width of the central channel and TL conformation, likely underlying its increased elongation rate at the expense of fidelity.

The LTAR Grazing Land Common Experiment at the Great Basin.

The Long-Term Agroecosystem Research Network (LTAR), through its Common Experiment (CE) framework, contrasts prevailing and alternative agricultural practices for efficacy and sustainability within the indicator domains of environment, productivity, economics, and society. Invasive species, wildfire, and climate change are principal threats to Great Basin agroecosystems. Prescribed grazing may be an effective tool for restoring lands degraded by these disturbances. At the Great Basin (GB) LTAR site headquartered in Boise, ID, our contribution to the CE contrasts a prevailing (PRV), cattle grazing practice of fixed moderate stocking and duration with an alternative (ALT), prescribed grazing practice called high-intensity low-frequency (HILF) grazing where stocking and duration are tailored to suppress invasive annual grass competition with native or desirable plant species and thus promote recovery of rangelands degraded by annual grass invasion and recurrent wildfire. Preliminary results indicate cheatgrass density and fuel height have been reduced in ALT-treated paddocks compared to PRV paddocks. Since its inception in 2014, our GB CE has been a research co-production effort among ranchers, public land managers, and researchers. Future directions for this research will center on expanding the experiment to multiple study areas to better address the scope of the annual grass/wildfire problem. We expect this research will lead to effective and sustainable grazing practices for restoring >41 million hectares of degraded rangelands in the Great Basin and other areas of the western United States.

Task-sharing spinal anaesthesia care in three rural Indian hospitals: a non-inferiority randomised controlled clinical trial.

BACKGROUND: Task-sharing of spinal anaesthesia care by non-specialist graduate physicians, termed medical officers (MOs), is commonly practised in rural Indian healthcare facilities to mitigate workforce constraints. We sought to assess whether spinal anaesthesia failure rates of MOs were non-inferior to those of consultant anaesthesiologists (CA) following a standardised educational curriculum. METHODS: We performed a randomised, non-inferiority trial in three rural hospitals in Tamil Nadu and Chhattisgarh, India. Patients aged over 18 years with low perioperative risk (ASA I & II) were randomised to receive MO or CA care. Prior to the trial, MOs underwent task-based anaesthesia training, inclusive of remotely accessed lectures, simulation-based training and directly observed anaesthetic procedures and intraoperative care. The primary outcome measure was spinal anaesthesia failure with a non-inferiority margin of 5%. Secondary outcome measures consisted of incidence of perioperative and postoperative complications. FINDINGS: Between 12 July 2019 and 8 June 2020, a total of 422 patients undergoing surgical procedures amenable to spinal anaesthesia care were randomised to receive either MO (231, 54.7%) or CA care (191, 45.2%). Spinal anaesthesia failure rate for MOs (7, 3.0%) was non-inferior to those of CA (5, 2.6%); difference in success rate of 0.4% (95% CI=0.36-0.43%; p=0.80). Additionally, there were no statistically significant differences observed between the two groups for intraoperative or postoperative complications, or patients' experience of pain during the procedure. INTERPRETATION: This study demonstrates that failure rates of spinal anaesthesia care provided by trained MOs are non-inferior to care provided by CAs in low-risk surgical patients. This may support policy measures that use task-sharing as a means of expanding anaesthesia care capacity in rural Indian hospitals. TRIAL REGISTRATION NUMBER: NCT04438811.

Gender Differences in Defibrillator Practices in Out-of-Hospital Cardiac Arrest.

OBJECTIVES: Disparities remain in survival after out-of-hospital cardiac arrest (OHCA) for women compared to men. Our objective was to evaluate differences in automated external defibrillator (AED) use before Emergency Medical Services (EMS) arrival and time from arrival to initial EMS defibrillation by EMS-assessed gender (women or men). METHODS: This was a secondary analysis of adult non-traumatic, EMS-treated OHCA cases in the Portland Cardiac Arrest Epidemiologic Registry from 2018 to 2021. Emergency Medical Services-witnessed cardiac arrests were excluded and the primary outcomes were pre-EMS AED application and the time from EMS arrival to first defibrillation among patients in a shockable rhythm at first rhythm assessment without pre-EMS AED application. We examined pre-EMS AED application rates overall and separately for law enforcement, in cases where they were on-scene before EMS without a lay bystander AED applied, and lay responders, in cases where law enforcement had not applied an AED. We used multivariable logistic and linear regressions to adjust for potential confounders, including age, arrest location, witness status, bystander CPR, year, and time from dispatch to EMS arrival. We accounted for clustering by county of arrest using a mixed-effects approach. RESULTS: Of the 3,135 adult, EMS-treated non-traumatic OHCAs that were not witnessed by EMS, 3,049 had all variables for analysis, of which 1,011 (33.2%) were women. The adjusted odds (adjusted odds ratio [95% CI]) for any pre-EMS placement of an AED was significantly higher for men compared to women (1.40 [1.05-1.86]). These odds favoring men remained when examining law enforcement AED application (1.89 [1.16-3.07]), but not lay bystander AED application (1.19 [0.83-1.71]). Among patients still in arrest on EMS arrival, with a shockable initial EMS rhythm, and without pre-EMS AED application, the time from EMS arrival on-scene to initial defibrillation was significantly longer for women compared to men (+0.81 min [0.22-1.41 min]). CONCLUSIONS: Women with OHCA received lower rates of pre-EMS AED application and delays in initial EMS defibrillation compared to men.

RNA Polymerase II activity control of gene expression and involvement in disease.

Gene expression is dependent on RNA Polymerase II (Pol II) activity in eukaryotes. In addition to determining the rate of RNA synthesis for all protein coding genes, Pol II serves as a platform for the recruitment of factors and regulation of co-transcriptional events, from RNA processing to chromatin modification and remodeling. The transcriptome can be shaped by changes in Pol II kinetics affecting RNA synthesis itself or because of alterations to co-transcriptional events that are responsive to or coupled with transcription. Genetic, biochemical, and structural approaches to Pol II in model organisms have revealed critical insights into how Pol II works and the types of factors that regulate it. The complexity of Pol II regulation generally increases with organismal complexity. In this review, we describe fundamental aspects of how Pol II activity can shape gene expression, discuss recent advances in how Pol II elongation is regulated on genes, and how altered Pol II function is linked to human disease and aging.

Identification of a family of peptidoglycan transpeptidases reveals that Clostridioides difficile requires noncanonical cross-links for viability.

Most bacteria are surrounded by a cell wall that contains peptidoglycan (PG), a large polymer composed of glycan strands held together by short peptide cross-links. There are two major types of cross-links, termed 4-3 and 3-3 based on the amino acids involved. 4-3 cross-links are created by penicillin-binding proteins, while 3-3 cross-links are created by L,D-transpeptidases (LDTs). In most bacteria, the predominant mode of cross-linking is 4-3, and these cross-links are essential for viability, while 3-3 cross-links comprise only a minor fraction and are not essential. However, in the opportunistic intestinal pathogen Clostridioides difficile, about 70% of the cross-links are 3-3. We show here that 3-3 cross-links and LDTs are essential for viability in C. difficile. We also show that C. difficile has five LDTs, three with a YkuD catalytic domain as in all previously known LDTs and two with a VanW catalytic domain, whose function was until now unknown. The five LDTs exhibit extensive functional redundancy. VanW domain proteins are found in many gram-positive bacteria but scarce in other lineages. We tested seven non-C. difficile VanW domain proteins and confirmed LDT activity in three cases. In summary, our findings uncover a previously unrecognized family of PG cross-linking enzymes, assign a catalytic function to VanW domains, and demonstrate that 3-3 cross-linking is essential for viability in C. difficile, the first time this has been shown in any bacterial species. The essentiality of LDTs in C. difficile makes them potential targets for antibiotics that kill C. difficile selectively.

Flow Cytometry-based Immune Phenotyping of T and B Lymphocytes in the Evaluation of Immunodeficiency and Immune Dysregulation.

There are approximately 500 congenital disorders that impair immune cell development and/or function. Patients with these disorders may present with a wide range of symptoms, including increased susceptibility to infection, autoimmunity, autoinflammation, lymphoproliferation, and/or atopy. Flow cytometry-based immune phenotyping of T and B lymphocytes plays an essential role in the evaluation of patients with these presentations. In this review, we describe the clinical utility of flow cytometry as part of a comprehensive evaluation of immune function and how this testing may be used as a diagnostic tool to identify underlying aberrant immune pathways, monitor disease activity, and assess infection risk.

Cytokine levels in breast cancer are highly dependent on cytomegalovirus (CMV) status.

PURPOSE: Breast cancer accounts for 30% of all female cancers in the US. Cytomegalovirus (CMV), a herpesvirus that establishes lifelong infection, may play a role in breast cancer. CMV is not oncogenic, yet viral DNA and proteins have been detected in breast tumors, indicating possible contribution to tumor development. CMV encodes cmvIL-10, a homolog of human cellular IL-10 (cIL-10) with potent immunosuppressive activities. We investigated the relationship between CMV infection, cytokines, and breast cancer. METHODS: We evaluated CMV serostatus and cytokine levels in plasma of women with benign breast disease (n = 38), in situ carcinoma (n = 41), invasive carcinoma, no lymph node involvement (Inv/LN-; n = 41), and invasive with lymph node involvement (Inv/LN+; n = 37). RESULTS: Fifty percent of the patient samples (n = 79) were CMV seropositive. There was no correlation between CMV status and diagnosis (p = 0.75). For CMV+ patients, there was a trend toward higher CMV IgG levels in invasive disease (p = 0.172). CmvIL-10 levels were higher in CMV+ in situ patients compared to the Inv/LN- and Inv/LN+ groups (p = 0.020). Similarly, cIL-10 levels were higher in CMV+ in situ patients compared to the Inv/LN- and Inv/LN+ groups (p = 0.043). The results were quite different in CMV- patients where cIL-10 levels were highest in Inv/LN- compared to benign, in situ, or Inv/LN+ (p = 0.019). African American patients were significantly associated with CMV+ status (p = 0.001) and had lower cmvIL-10 levels than Caucasian patients (p = 0.046). CONCLUSION: No association was observed between CMV IgG and diagnosis, but CMV infection influences cytokine production and contributes to altered cytokine profiles in breast cancer.

Multidecadal vegetation transformations of a New Mexico ponderosa pine landscape after severe fires and aerial seeding.

Wildfires and climate change increasingly are transforming vegetation composition and structure, and postfire management may have long-lasting effects on ecosystem reorganization. Postfire aerial seeding treatments are commonly used to reduce runoff and soil erosion, but little is known about how seeding treatments affect native vegetation recovery over long periods of time, particularly in type-converted forests that have been dramatically transformed by the effects of repeated, high-severity fire. In this study, we analyze and report on a rare long-term (23-year) dataset that documents vegetation dynamics following a 1996 post-fire aerial seeding treatment and a subsequent 2011 high-severity reburn in a dry conifer landscape of northern New Mexico, USA. Repeated surveys between 1997 and 2019 of 49 permanent transects were analyzed for differences in vegetation cover, richness, and diversity between seeded and unseeded areas, and to characterize the development of seeded and unseeded vegetation communities through time and across gradients of burn severity, elevation, and soil-available water capacity. Seeded plots showed no significant difference in bare ground cover during the initial years postfire relative to unseeded plots. Postfire seeding led to a clear and sustained divergence in herbaceous community composition. Seeded plots had a much higher cover of non-native graminoids, primarily Bromus inermis, a likely contaminant in the seed mix. High-severity reburning of all plots in 2011 reduced native graminoid cover by half at seeded plots compared with both prefire levels and with plots that were unseeded following the initial 1996 fire. In addition, higher fire severity was associated with increased non-native graminoid cover and reduced native graminoid cover. This study documents fire-driven ecosystem transformation from conifer forest into a shrub-and-grass-dominated system, reinforced by aerial seeding of grasses and high-severity reburning. This unique long-term dataset illustrates that post-fire seeding carries significant risks of unwanted non-native species invasions that persist through subsequent fires-thus alternative postfire management actions merit consideration to better support native ecosystem resilience given emergent climate change and increasing disturbance. This study also highlights the importance of long-term monitoring of postfire vegetation dynamics, as short-term assessments miss key elements of complex ecosystem responses to fire and postfire management actions.

Changes in Emergency Department Pediatric Readiness and Mortality.

Importance: High emergency department (ED) pediatric readiness is associated with improved survival, but the impact of changes to ED readiness is unknown. Objective: To evaluate the association of changes in ED pediatric readiness at US trauma centers between 2013 and 2021 with pediatric mortality. Design, Setting, and Participants: This retrospective cohort study was performed from January 1, 2012, through December 31, 2021, at EDs of trauma centers in 48 states and the District of Columbia. Participants included injured children younger than 18 years with admission or injury-related death at a participating trauma center, including transfers to other trauma centers. Data analysis was performed from May 2023 to January 2024. Exposure: Change in ED pediatric readiness, measured using the weighted Pediatric Readiness Score (wPRS, range 0-100, with higher scores denoting greater readiness) from national assessments in 2013 and 2021. Change groups included high-high (wPRS ≥93 on both assessments), low-high (wPRS <93 in 2013 and wPRS ≥93 in 2021), high-low (wPRS ≥93 in 2013 and wPRS <93 in 2021), and low-low (wPRS <93 on both assessments). Main Outcomes and Measures: The primary outcome was lives saved vs lost, according to ED and in-hospital mortality. The risk-adjusted association between changes in ED readiness and mortality was evaluated using a hierarchical, mixed-effects logistic regression model based on a standardized risk-adjustment model for trauma, with a random slope-random intercept to account for clustering by the initial ED. Results: The primary sample included 467 932 children (300 024 boys [64.1%]; median [IQR] age, 10 [4 to 15] years; median [IQR] Injury Severity Score, 4 [4 to 15]) at 417 trauma centers. Observed mortality by ED readiness change group was 3838 deaths of 144 136 children (2.7%) in the low-low ED group, 1804 deaths of 103 767 children (1.7%) in the high-low ED group, 1288 deaths of 64 544 children (2.0%) in the low-high ED group, and 2614 deaths of 155 485 children (1.7%) in the high-high ED group. After risk adjustment, high-readiness EDs (persistent or change to) had 643 additional lives saved (95% CI, -328 to 1599 additional lives saved). Low-readiness EDs (persistent or change to) had 729 additional preventable deaths (95% CI, -373 to 1831 preventable deaths). Secondary analysis suggested that a threshold of wPRS 90 or higher may optimize the number of lives saved. Among 716 trauma centers that took both assessments, the median (IQR) wPRS decreased from 81 (63 to 94) in 2013 to 77 (64 to 93) in 2021 because of reductions in care coordination and quality improvement. Conclusions and Relevance: Although the findings of this study of injured children in US trauma centers were not statistically significant, they suggest that trauma centers should increase their level of ED pediatric readiness to reduce mortality and increase the number of pediatric lives saved after injury.

Blood Glucose During High Altitude Trekking in Young Healthy Adults.

Reid, Ly-Anh, Jordan L Rees, Miranda Kimber, Marina James, Graeme M Purdy, Megan Smorschok, Lauren E Maier, Normand G. Boulé, Trevor A. Day, Margie H. Davenport, and Craig D. Steinback. Blood glucose during high altitude trekking in young healthy adults. High Alt Med Biol. 00:00-00, 2024. Introduction: High altitude trekking is becoming more popular and accessible to an increased number of people. Simultaneously, there is a worldwide rise in the prevalence of metabolic diseases. The purpose of this study was to examine the impact of a gradual trekking ascent to high altitude on continuous glucose monitoring outcomes including fasting, mean 24-hour, postprandial, and post-75 g modified oral glucose tolerance test. This study also investigated the relationship between physical activity intensity, high altitude, and glucose concentrations. Methods: Individuals (n = 9) from Alberta, Canada participated in a 2-week trek in the Khumbu Valley in Nepal, ascending by foot from 2,860 m to 5,300 m (∼65 km) over 10 days. A standardized 75 g oral glucose load was given to participants at four different altitudes (1,130 m, 3,440 m, 3,820 m, 5,160 m). Physical activity (Actigraph accelerometry) and interstitial glucose (iPro2, Medtronic) were measured continuously during the trek. Results: Fasting and mean 24-hour glucose concentrations were not different between altitudes. However, 2-hour post dinner glucose and 2-hour post lunch glucose, AUC concentrations were different between altitudes. The relationship between physical activity intensity and glucose was not influenced by increasing altitudes. Conclusion: Our findings suggest that glucose regulation is largely preserved at high altitude; however, inconsistency in our postprandial glucose concentrations at altitude warrants further investigation.

Racial-Ethnic Comparison of Treatment for Papillary Thyroid Cancer in the Military Health System.

PURPOSE: We aimed to compare Asian or Pacific Islander, Black, Hispanic, and non-Hispanic White patients in treatment for papillary thyroid cancer (PTC) in the equal access Military Health System to better understand racial-ethnic cancer health disparities observed in the United States. METHODS: We used the MilCanEpi database to identify a cohort of men and women aged 18 or older who were diagnosed with PTC between 1998 and 2014. Low- or high-risk status was assigned using tumor size and lymph node involvement. Treatment with surgery (e.g., thyroidectomy) overall and treatment by risk status [active surveillance (low-risk) or adjuvant radioactive iodine (RAI) (high-risk)] was compared between racial-ethnic groups using multivariable logistic regression and expressed as adjusted odds ratios (AOR) with 95% confidence intervals (CIs). RESULTS: The study included 598 Asian, 553 Black, 340 Hispanic, and 2958 non-Hispanic White patients with PTC. Asian (AOR = 1.21, 95% CI 0.98, 1.49), Black (AOR = 1.07, 95% CI 0.87, 1.32), and Hispanic (AOR = 0.92, 95% CI 0.71, 1.19) patients were as likely as White patients to receive surgery. By risk status, there were no significant racial-ethnic differences in receipt of active surveillance or thyroidectomy for low-risk PTC or in thyroidectomy or total thyroidectomy with adjuvant RAI for high-risk PTC. CONCLUSIONS: In the Military Health System, where patients have equal access to care, there were no overall racial-ethnic differences in surgical treatment for PTC. As American Thyroid Association guidelines evolve to include more conservative treatment, further research is warranted to understand potential disparities in active surveillance and surgical management in U.S. healthcare settings.

Allometry and ecology shape eye size evolution in spiders.

Eye size affects many aspects of visual function, but eyes are costly to grow and maintain. The allometry of eyes can provide insight into this trade-off, but this has mainly been explored in species that have two eyes of equal size. By contrast, animals possessing larger visual systems can exhibit variable eye sizes within individuals. Spiders have up to four pairs of eyes whose sizes vary dramatically, but their ontogenetic, static, and evolutionary allometry has not yet been studied in a comparative context. We report variable dynamics in eye size across 1,098 individuals in 39 species and 8 families, indicating selective pressures and constraints driving the evolution of different eye pairs and lineages. Supplementing our sampling with a recently published phylogenetically comprehensive dataset, we confirmed these findings across more than 400 species; found that ecological factors such as visual hunting, web building, and circadian activity correlate with eye diameter; and identified significant allometric shifts across spider phylogeny using an unbiased approach, many of which coincide with visual hunting strategies. The modular nature of the spider visual system provides additional degrees of freedom and is apparent in the strong correlations between maximum/minimum investment and interocular variance and three key ecological factors. Our analyses suggest an antagonistic relationship between the anterior and posterior eye pairs. These findings shed light on the relationship between spider visual systems and their diverse ecologies and how spiders exploit their modular visual systems to balance selective pressures and optical and energetic constraints.

Adapting the Zero Suicide framework to the adult emergency department setting: Strategies for nurse leaders.

The ED has been increasingly recognized as a key setting for suicide prevention. Zero Suicide (ZS) is an aspirational goal to eliminate suicide for all patients within a health care system through utilization of best practices. However, there has been limited exploration of ZS implementation within the ED. As ED nurses play an important role in suicide prevention through their close contact with patients at risk for suicide, ZS implementation would benefit from tailored strategies for ED nurse leadership. We describe the ZS framework and provides strategies for nurse leaders to adapt each ZS component in the adult ED.

Development and external validation of a head and neck cancer risk prediction model.

BACKGROUND: Head and neck cancer (HNC) incidence is on the rise, often diagnosed at late stage and associated with poor prognoses. Risk prediction tools have a potential role in prevention and early detection. METHODS: The IARC-ARCAGE European case-control study was used as the model development dataset. A clinical HNC risk prediction model using behavioral and demographic predictors was developed via multivariable logistic regression analyses. The model was then externally validated in the UK Biobank cohort. Model performance was tested using discrimination and calibration metrics. RESULTS: 1926 HNC cases and 2043 controls were used for the development of the model. The development dataset model including sociodemographic, smoking, and alcohol variables had moderate discrimination, with an area under curve (AUC) value of 0.75 (95% CI, 0.74-0.77); the calibration slope (0.75) and tests were suggestive of good calibration. 384 616 UK Biobank participants (with 1177 HNC cases) were available for external validation of the model. Upon external validation, the model had an AUC of 0.62 (95% CI, 0.61-0.64). CONCLUSION: We developed and externally validated a HNC risk prediction model using the ARCAGE and UK Biobank studies, respectively. This model had moderate performance in the development population and acceptable performance in the validation dataset. Demographics and risk behaviors are strong predictors of HNC, and this model may be a helpful tool in primary dental care settings to promote prevention and determine recall intervals for dental examination. Future addition of HPV serology or genetic factors could further enhance individual risk prediction.

University teaching of mass spectrometry as a key practical technique within the context of a fully integrated, spiral curriculum.

RATIONALE: Mass spectrometry (MS) is introduced to high school students in the UK in many pre-university course syllabi. As such, we have identified the use of MS as a key technique that should be taught practically to undergraduates from the outset of their studies. This mini-review describes how we introduce and develop students' use of MS throughout our three-year undergraduate spiral curriculum practical programme, using atmospheric pressure chemical ionization MS (APCI-MS). METHODS: We have used an Advion ExpressionL spectrometer, fitted with an atmospheric solids analysis probe or a Plate Express TLC sampler for sample introduction. RESULTS: We have successfully demonstrated the use of APCI-MS in a range of practicals and experiments covering organic and organometallic chemistries, with large cohorts of students gaining hands-on instrumental experience in authentic research settings. CONCLUSIONS: APCI-MS has proven to be an easy-to-use and valuable addition to our undergraduate practical course. The robustness of the spectrometer enables routine use by large cohorts of students with minimal supervision, and routine maintenance can be carried out by non-specialist technicians. Students can readily process and interpret results for a series of routine analyses, as well as demonstrate uses in problem-solving exercises.

Assessing eDNA capture method from aquatic environment to optimise recovery of human mt-eDNA.

Previous studies have shown that environmental DNA (eDNA) from human sources can be recovered from natural bodies of water, and the generation of DNA profiles from such environmental samples may assist in forensic investigations. However, fundamental knowledge gaps exist around the factors influencing the probability of detecting human eDNA and the design of optimal sampling protocols. One of these is understanding the particle sizes eDNA signals are most strongly associated with and the most appropriate filter size needed for efficiently capturing eDNA particles. This study assessed the amount of mitochondrial eDNA associated with different particle sizes from human blood and skin cells recovered from freshwater samples. Samples (300 mL) were taken from experimental 10 L tanks of freshwater spiked with 50 µL of human blood or skin cells deposited by vigorously rubbing hands together for two minutes in freshwater. Subsamples were collected by passing 250 mL of experimental water sample through six different filter pore sizes (from 0.1 to 8 µm). This process was repeated at four time intervals after spiking over 72 hours to assess if the particle size of the amount of eDNA recovered changes as the eDNA degrades. Using a human-specific quantitative polymerase chain reaction (qPCR) assay targeting the HV1 mitochondrial gene region, the total amount of mitochondrial eDNA associated with different particle size fractions was determined. In the case of human blood, at 0 h, the 0.45 µm filter pore size captured the greatest amount of mitochondrial eDNA, capturing 42 % of the eDNA detected. The pattern then changed after 48 h, with the 5 µm filter pore size capturing the greatest amount of eDNA (67 %), and 81 % of eDNA at 72 h. Notably, a ten-fold dilution proved to be a valuable strategy for enhancing eDNA recovery from the 8 µm filter at all time points, primarily due to the PCR inhibition observed in hemoglobin. For human skin cells, the greatest amounts of eDNA were recovered from the 8 µm filter pore size and were consistent through time (capturing 37 %, 56 %, and 88 % of eDNA at 0 hours, 48 hours, and 72 hours respectively). There is a clear variation in the amount of eDNA recovered between different cell types, and in some forensic scenarios, there is likely to be a mix of cell types present. These results suggest it would be best to use a 5 µm filter pore size to capture human blood and an 8 µm filter pore size to capture human skin cells to maximize DNA recovery from freshwater samples. Depending on the cell type contributing to the eDNA, a combination of different filter pore sizes may be employed to optimize the recovery of human DNA from water samples. This study provides the groundwork for optimizing a strategy for the efficient recovery of human eDNA from aquatic environments, paving the way for its broader application in forensic and environmental sciences.