Implementing L-DNA analogs as mirrors of PCR reactant hybridization state: theoretical and practical guidelines for PCR cycle control.

In previous reports, we described a PCR cycle control approach in which the hybridization state of optically labeled L-DNA enantiomers of the D-DNA primers and targets determined when the thermal cycle was switched from cooling to heating and heating to cooling. A consequence of this approach is that it also "adapts" the cycling conditions to compensate for factors that affect the hybridization kinetics of primers and targets. It assumes, however, that the hybridization state of the labeled L-DNA analogs accurately reflects the hybridization state of the D-DNA primers and targets. In this report, the Van't Hoff equation is applied to determine the L-DNA concentration and ratio of L-DNA strands required by this assumption. Simultaneous fluorescence and temperature measurements were taken during L-DNA controlled cycling, and the optical and thermal switch points compared as a function of both total L-DNA concentration and ratio of strands. Based on the Van't Hoff relationship and these experimental results, L-DNA best mirrors the hybridization of PCR primers and targets when total L-DNA concentration is set equal to the initial concentration of the D-DNA primer of interest. In terms of strand ratios, L-DNA hybridization behavior most closely matches the behavior of their D-DNA counterparts throughout the reaction when one of the L-DNA strands is far in excess of the other. The L-DNA control algorithm was then applied to the practical case of the SARS-CoV-2 N2 reaction, which has been shown to fail or have a delayed Cq when PCR was performed without nucleic acid extraction. PCR Cq values for simulated "unextracted" PCR samples in a nasopharyngeal background and in an NaCl concentration similar to that of viral transport media were determined using either the L-DNA control algorithm (N = 6) or preset cycling conditions (N = 3) and compared to water background controls run in parallel. For preset cycling conditions, the presence of nasopharyngeal background or a high salt background concentration significantly increased Cq, but the L-DNA control algorithm had no significant delay. This suggests that a carefully designed L-DNA-based control algorithm "adapts" the cycling conditions to compensate for hybridization errors of the PCR D-DNA reactants that produce false negatives.

Energy dissipation during homogeneous wetting of surfaces with randomly and periodically distributed cylindrical pillars.

HYPOTHESIS: Understanding contact angle hysteresis on rough surfaces is important as most industrially relevant and naturally occurring surfaces possess some form of random or structured roughness. We hypothesise that hysteresis can be described by the dilute defect model of Joanny & de Gennes [1] and that the energy dissipation occurring during the stick-slip motion of the contact line is key to developing a predictive equation for hysteresis. EXPERIMENTS: We measured hysteresis on surfaces with randomly distributed and periodically arranged microscopic cylindrical pillars for a variety of different liquids in air. The inherent (flat surface) contact angles tested range from lyophilic (θe=33.8°) to lyophobic (θe=112.0°). FINDINGS: A methodology for averaging the measured advancing and receding contact angles on random surfaces is presented. Based on these results correlations for roughness-induced energy dissipation are derived, and an equation for predicting the advancing and receding contact angles during homogeneous (Wenzel) wetting on random surfaces is presented. Equations that predict the onset of the alternate wetting conditions of hemiwicking, split-advancing, split-receding and heterogeneous (Cassie) wetting are also derived, thus defining the range of validity for the homogeneous wetting equation. A 'cluster' concept is proposed to explain the measurably higher hysteresis exhibited by structured surfaces compared to random surfaces.

A safer framework to evaluate characterization technologies of exhaled biologic materials using electrospun nanofibers.

Exhaled biologic material is the source for the spread of many respiratory tract infections. To avoid the high-level of biosafety required to manage dangerous pathogens, we developed a safer framework using the endogenous surrogate targets RNase P and Streptococcus mitis as a means to sample exhaled biologics. Our exhalation collection scheme uses nanoscale fibrous poly(vinyl alcohol) substrates as facemask inserts. After a period of breathing or speaking, the inserts are removed and dissolved. RNase P RNA and S. mitis DNA are extracted for quantification by multiplexed RT-qPCR. Both surrogate biomarkers were detected in all samples obtained during breathing for at least five minutes or speaking for one minute. Phrases repeated 30 times had the most copies with 375 ± 247 of S. mitis and 54 ± 33 of RNase P. When the phrases were repeated just 5 times, the S. mitis copies collected were still detectable but at a significantly lower level of 11 ± 5 for S. mitis and 12 ± 9 for RNase P. These results demonstrate a collection and quantification framework that can be readily adapted to further characterize the exhalation of nanoscale biologic materials from healthy individuals, explore new collection designs safely, and serve as a method to incorporate sample controls for future pathogen exhalation studies.

Longitudinal Assessment of Prevalence and Incidence of Salmonella and Escherichia coli O157 Resistance to Antimicrobials in Feedlot Cattle Sourced and Finished in Two Different Regions of the United States.

The objective was to investigate the influence of cattle origin and region of finishing on the prevalence of Salmonella, Escherichia coli O157:H7, and select antimicrobial resistance in E. coli populations. Yearling heifers (n = 190) were utilized in a 2 × 2 factorial arrangement. After determining fecal Salmonella prevalence, heifers were sorted into one of four treatments: heifers originating from South Dakota (SD) and finished in SD (SD-SD); heifers originating from SD and finished in Texas (SD-TX); heifers originating from TX and finished in SD (TX-SD); and heifers originating from TX and finished in TX (TX-TX). Fecal, pen, and water scum line samples were collected longitudinally throughout the study; hide swab and subiliac lymph node (SLN) samples were collected at study end. A treatment × time interaction was observed (p ≤ 0.01) for fecal Salmonella prevalence, with prevalence being greatest for TX-TX and TX-SD heifers before transport. From day (d) 14 through study end, prevalence was greatest for TX-TX and SD-TX heifers compared with SD-SD and TX-SD heifers. Salmonella prevalence on hides were greater (p ≤ 0.01) for heifers finished in TX compared with SD. Salmonella prevalence in SLN tended (p = 0.06) to be greater in TX-TX and SD-TX heifers compared with TX-SD and SD-SD. Fecal E. coli O157:H7 prevalence had a treatment × time interaction (p = 0.04), with SD-TX prevalence being greater than TX-SD on d 56 and SD-SD and TX-TX being intermediate. A treatment × time interaction was observed for fecal trimethoprim-sulfamethoxazole-resistant and cefotaxime-resistant E. coli O157:H7 prevalence (p ≤ 0.01). Overall, these data suggest that the region of finishing influences pathogenic bacterial shedding patterns, with the initial 14 d after feedlot arrival being critical for pathogen carriage.

Intravenous heterologous prime-boost vaccination activates innate and adaptive immunity to promote tumor regression.

Therapeutic neoantigen cancer vaccines have limited clinical efficacy to date. Here, we identify a heterologous prime-boost vaccination strategy using a self-assembling peptide nanoparticle TLR-7/8 agonist (SNP) vaccine prime and a chimp adenovirus (ChAdOx1) vaccine boost that elicits potent CD8 T cells and tumor regression. ChAdOx1 administered intravenously (i.v.) had 4-fold higher antigen-specific CD8 T cell responses than mice boosted by the intramuscular (i.m.) route. In the therapeutic MC38 tumor model, i.v. heterologous prime-boost vaccination enhances regression compared with ChAdOx1 alone. Remarkably, i.v. boosting with a ChAdOx1 vector encoding an irrelevant antigen also mediates tumor regression, which is dependent on type I IFN signaling. Single-cell RNA sequencing of the tumor myeloid compartment shows that i.v. ChAdOx1 reduces the frequency of immunosuppressive Chil3 monocytes and activates cross-presenting type 1 conventional dendritic cells (cDC1s). The dual effect of i.v. ChAdOx1 vaccination enhancing CD8 T cells and modulating the TME represents a translatable paradigm for enhancing anti-tumor immunity in humans.

Perspectives of dairy employees at the beginning of the COVID-19 pandemic: A survey of health risks and educational needs.

Our objective was to understand dairy employees' perceptions and educational needs at the onset of the COVID-19 pandemic. A bilingual (English and Spanish), anonymous survey targeted at dairy employees was circulated nationwide via university and allied industry media outlets. Responses (n = 63) from 11 states were received (May-Sep. 2020). Respondents worked in herds ranging from 50 to 40,000 animals in size. Dairy managers (33%) responded mostly to the English survey (52%), whereas entry-level workers (67%) chose the Spanish format (76%). Survey results highlighted different perspectives, educational needs, and preferred sources of information between English- and Spanish-speaking dairy workers. Overall, 83% of the respondents were somewhat concerned or very concerned about the COVID-19 pandemic. Respondents (51%) indicated that their main concern was "to bring the virus from work to home and make my family sick." Most dairy employees (83%) perceived that their employers were somewhat or very concerned about the pandemic. Respondents (65%) indicated that COVID-19 informative training was provided at the workplace, but training was more frequently undertaken among dairy managers (86%) than entry-level workers (53%). Most trainings (72%) were limited to posters on walls. The preferred means of information delivery was through in-person meetings at work (35%), with YouTube (29%) and on-demand videos (27%) as second and third options. The main source of information regarding the pandemic was social media (52%). Frequent handwashing (81%), limiting on-farm visits (70%), limiting agglomeration in break rooms (65%), hand sanitizer use (60%), and social distancing (60%) were the most common safety measures implemented at the workplace among the options given to respondents. Few respondents (38%) indicated that face-covering was required at work. Successful emergency plans on dairies should consider the outreach needs and preferences of dairy workers.

Automated Variable Electric-Field DFT Application for Evaluation of Optimally Oriented Electric Fields on Chemical Reactivity.

Recent theoretical work and experiments at molecular junctions have provided a strong conceptualization for the effects of oriented electric fields (OEFs) on organic reactions. Depending on the axis of application, OEFs can increase (or decrease) the reaction rate or distinguish between isomeric pathways. Despite the conceptual elegance of OEFs, which may be applied externally or induced locally, as tools for catalyzing organic reactions, implementation in synthetically relevant systems has been hampered by inefficiencies in evaluating reaction sensitivity to field effects. Herein, we describe the development of the Automated Variable Electric-Field DFT Application (A.V.E.D.A.) for streamlined evaluation of a reaction's susceptibility to OEFs. This open-source software was designed to be accessible for nonexpert users of computational and programming tools. Following initiation by a single command (and with no subsequent intervention) the Linux workflow manages a series of density functional theory calculations and mathematical manipulations to optimize local-minimum and transition-state structures in oriented electric fields of increasing magnitude. The resulting molecular and reaction dipole moments, field-perturbed geometries, and net effective activation energies are compiled for user interpretation. Ten representative pericyclic reactions that showcase the development and evaluation of A.V.E.D.A. are described.

Ligation-based assay for variant typing without sequencing: Application to SARS-CoV-2 variants of concern.

BACKGROUND: COVID-19 prevalence has remained high throughout the pandemic with intermittent surges, due largely to the emergence of genetic variants, demonstrating the need for more accessible sequencing technologies for strain typing. METHODS: A ligation-based typing assay was developed to detect known variants of severe acute respiratory syndrome virus 2 (SARS-CoV-2) by identifying the presence of characteristic single-nucleotide polymorphisms (SNPs). General principles for extending the strategy to new variants and alternate diseases with SNPs of interest are described. Of note, this strategy leverages commercially available reagents for assay preparation, as well as standard real-time polymerase chain reaction (PCR) instrumentation for assay performance. RESULTS: The assay demonstrated a combined sensitivity and specificity of 96.6% and 99.5%, respectively, for the classification of 88 clinical samples of the Alpha, Delta, and Omicron variants relative to the gold standard of viral genome sequencing. It achieved an average limit of detection of 7.4 × 104 genome copies/mL in contrived nasopharyngeal samples. The ligation-based strategy performed robustly in the presence of additional polymorphisms in the targeted regions of interest as shown by the sequence alignment of clinical samples. CONCLUSIONS: The assay demonstrates the potential for robust variant typing with performance comparable with next-generation sequencing without the need for the time delays and resources required for sequencing. The reduced resource dependency and generalizability could expand access to variant classification information for pandemic surveillance.

Plasma microrna quantification protocol.

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate translation and are involved in many pathological processes. They have emerged as promising biomarkers for diagnosis of conditions such as aortic aneurysm disease. Quantifying miRNAs in plasma is uniquely challenging because of the lack of standardized reproducible protocols. To facilitate the independent verification of conclusions, it is necessary to provide a thorough disclosure of all pertinent experimental details. In this technical note, we present a comprehensive protocol for quantifying plasma miRNAs using droplet digital PCR. We detail the entire workflow, including blood collection, plasma processing, cryo-storage, miRNA isolation, reverse transcription, droplet generation, PCR amplification, fluorescence reading, and data analysis. We offer comprehensive guidance regarding optimization, assay conditions, expected results, and insight into the troubleshooting of common issues. The stepwise normalization and detailed methodological guide enhance reproducibility. Moreover, multiple portions of this protocol may be automated. The data provided in this technical note is demonstrative of the values typically obtained when following its steps. To facilitate standardization in data reporting, we include a table of expected aortic aneurysm-related miRNA levels in healthy human plasma. This versatile protocol can be easily adapted to quantify most circulating miRNAs in plasma, making it a valuable resource for diagnostic development.

Magnetic Bead Processing Enables Sensitive Ligation-Based Detection of HIV Drug Resistance Mutations.

HIV develops single nucleotide polymorphisms (SNPs), some of which lead to drug resistance mutations (DRMs) that prevent therapeutic viral suppression. Genomic sequencing enables healthcare professionals to select effective combination antiretroviral therapy (ART) to achieve and maintain viral suppression. However, sequencing technologies, which are resource-intensive, are limited in their availability. This report describes the first step toward a highly specific ligation-based SNP discrimination method with endpoint PCR detection, which is more suitable for resource-limited clinics. The approach is based on magnetic bead processing to maximize reaction product transfer and minimize the carryover of incompatible buffer for three consecutive enzymatic reactions─reverse transcription (RT), oligonucleotide ligation assay (OLA), and PCR. The method improved PCR detection following RT → OLA by 8.06 cycles (∼250-fold) compared to direct pipette processing and detected between 103 and 104 RNA copies per reaction. In studies with synthesized nucleic acids based on the well-studied HIV mutation, K103N, the assay successfully differentiated between wild-type and mutant for RNA targets with high specificity. With further development, this design provides a pathway for SNP detection with more accessible PCR instrumentation and is a step toward a self-contained processing approach that incorporates the SNP specificity of the ligation reaction for more effective clinical management of DRMs in resource-constrained settings.

Baseball Pitching Arm Three-Dimensional Inertial Parameter Calculations From Body Composition Imaging and a Novel Overweight Measure for Youth Pitching Arm Kinetics.

Many baseball pitching studies have used inverse dynamics to assess throwing arm kinetics as high and repetitive kinetics are thought to be linked to pitching injuries. However, prior studies have not used participant-specific body segment inertial parameters (BSIPs), which are thought to improve analysis of high-acceleration motions and overweight participants. This study's objectives were to (1) calculate participant-specific BSIPs using dual energy X-ray absorptiometry (DXA) measures, (2) compare inverse dynamic calculations of kinetics determined by DXA-calculated BSIPs (full DXA-driven inverse dynamics) against kinetics using the standard inverse dynamics approach with scaled BSIPs (scaled inverse dynamics), and (3) examine associations between full DXA-driven kinetics and overweight indices: body mass index (BMI) and segment mass index (SMI). Eighteen participants (10-11 years old) threw 10 fastballs that were recorded for motion analysis. DXA scans were used to calculate participant-specific BSIPs (mass, center of mass, radii of gyration) for each pitching arm segment (upper arm, forearm, hand), BMI, and SMI. The hypotheses were addressed with t-tests and linear regression analyses. The major results were that (1) DXA-calculated BSIPs differed from scaled BSIPs for each pitching arm segment; (2) calculations for shoulder, but not elbow, kinetics differed between the full DXA-driven and scaled inverse dynamics analyses; and (3) full DXA-driven inverse dynamics calculations for shoulder kinetics were more often associated with SMI than BMI. Results suggest that using participant-specific BSIPs and pitching arm, SMIs may improve evidence-based injury prevention guidelines for youth pitchers.

Perspective of dairy producers from California, Idaho, South Dakota, and Washington: Health and business implications of the COVID-19 pandemic during the second wave.

The aim of this study was to document California, Idaho, South Dakota, and Washington dairy producers' perspectives on the health and business implications of COVID-19 during the pandemic second wave. Dairy producers were reached by a 14-question anonymous mail survey during June and July of 2020. We obtained 226 responses (response rate: 9.3% CA, 8.6% ID, 31.4%, and 10.0% WA). Responses were grouped by state (CA: 48.7%, ID: 15.9%, SD: 21.7%, or WA: 13.7%) and dairy size [based on number of cows; small (<100): 14.1%, medium (100-499): 27.7%, medium-large (500-1,999): 33.2%, or large (≥2,000): 25.0%]. Survey responses were summarized, and multiple correspondence analysis was used to map responses and identify data clusters. At the time of the survey, some respondents suspected (3%) or had confirmed (9%) COVID-19 cases on their premises. Respondents were somewhat or very concerned about the health (75%) and business (92%) implications associated with COVID-19. Producers were concerned about the health of their families, employees, and employees' families; having to reduce production; workforce shortage; limited goods availability; and lack of services. Producers perceived that their employees were somewhat or very concerned (74%) with the pandemic. There was COVID-19 safety information or training provided (78%) or intended (4%) in English (22%), Spanish (23%), or both (55%). The focus of training was as follows: how to remain healthy at work (91%) and at home (60%), what to do if a worker gets sick (77%), and sick leave information (54%). Nevertheless, 18% of the respondents answered that training was not going to be provided. The following control measures implemented: providing hand sanitizer or disinfectant wipes (78%), instruction on frequent hand-washing (76%), social distancing (59%), prevention of employee gatherings (54%), providing and requiring the use of face masks (49%), and limiting on-farm visitors (44%). Multiple correspondence analysis showed that concern with health risk and business impact of the COVID-19 pandemic, as well as mitigation and response strategies implemented, varied based on respondent's dairy size and state. The greatest concerns were reported by producers from California and large and medium-large dairies, whereas the fewest concerns were reported by producers from small dairies. Results from this survey highlighted the health and business concerns of dairy producers from California, Idaho, South Dakota, and Washington during the COVID-19 pandemic and the mitigation efforts adopted. In conclusion, mitigation measures were adopted by most dairy producers but in various degrees. Dairy industry service providers and educators were secondary educational resources during the pandemic; thus, future efforts toward centralized access to dairy specific bilingual educational materials are suggested.

Seeking progress in disease modification in Parkinson disease.

OBJECTIVE: Disease modification in Parkinson disease (PD) has remained an elusive goal, in spite of large investments over several decades. Following a large meeting of experts, this review article discusses the state of the science, possible reasons for past PD trials' failures to demonstrate disease-modifying benefit, and potential solutions. METHODS: The National Institute of Neurological Disorders and Stroke (NINDS) convened a meeting including leaders in the field and representatives of key stakeholder groups to discuss drug therapy with the goal of disease modification in PD. RESULTS: Important lessons can be learned from previous attempts, as well as from other fields. The selection process for therapeutic targets and agents differs among various organizations committed to therapeutic development. The areas identified as critical to target in future research include the development of relevant biomarkers, refinements of the targeted patient populations, considerations of novel trial designs, and improving collaborations between all stakeholders. CONCLUSIONS: We identify potential barriers to progress in disease modification for Parkinson's and propose a set of research priorities that may improve the likelihood of success.

Ct Values Do Not Predict Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Transmissibility in College Students.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly contagious and has caused significant medical/socioeconomic impacts. Other than vaccination, effective public health measures, including contact tracing, isolation, and quarantine, is critical for deterring viral transmission, preventing infection progression and resuming normal activities. Viral transmission is affected by many factors, but the viral load and vitality could be among the most important ones. Although in vitro studies have indicated that the amount of virus isolated from infected individuals affects the successful rate of virus isolation, whether the viral load carried at the individual level would determine the transmissibility was unknown. We examined whether the cycle threshold (Ct) value, a measurement of viral load by RT-PCR assay, could differentiate the spreaders from the non-spreaders in a population of college students. Our results indicate that while at the population level the Ct value is lower, suggesting a higher viral load, in the symptomatic spreaders than that in the asymptomatic non-spreaders, there is a significant overlap in the Ct values between the two groups. Thus, Ct value, or the viral load, at the individual level could not predict the transmissibility. Instead, a sensitive method to detect the presence of virus is needed to identify asymptomatic individuals who may carry a low viral load but can still be infectious.

Ultrapermeable Composite Membranes Enhanced Via Doping with Amorphous MOF Nanosheets.

Thin-film composite (TFC) polymeric membranes have attracted increasing interest to meet the demands of industrial gas separation. However, the development of high-performance TFC membranes within their current configuration faces two key challenges: (i) the thickness-dependent gas permeability of polymeric materials (mainly poly(dimethylsiloxane) (PDMS)) and (ii) the geometric restriction effect due to the limited pore accessibility of the underlying porous substrate. Here we demonstrate that the incorporation of trace amounts (∼1.8 wt %) of amorphous metal-organic framework (MOF) nanosheets into the gutter layer of TFC assemblies can simultaneously address these two limitations by the creation of rapid, transmembrane gas diffusion pathways. The resultant PDMS&MOF membrane displayed excellent CO2 permeance of 10450 GPU and CO2/N2 selectivity of 9.1. Leveraging this strategy, we successfully fabricate a novel TFC membrane, consisting of a PDMS&MOF gutter and an ultrathin (∼54 nm) poly(ethylene glycol) top selective layer via surface-initiated atom transfer radical polymerization. The complete TFC membrane exhibits excellent processability and remarkable CO2/N2 separation performance (1990 GPU with a CO2/N2 ideal selectivity of 39). This study reveals a strategy for the design and fabrication of a new TFC membrane system with unprecedented gas-separation performance.

An Analysis of the External Validity of EEG Spectral Power in an Uncontrolled Outdoor Environment during Default and Complex Neurocognitive States.

Traditionally, quantitative electroencephalography (QEEG) studies collect data within controlled laboratory environments that limit the external validity of scientific conclusions. To probe these validity limits, we used a mobile EEG system to record electrophysiological signals from human participants while they were located within a controlled laboratory environment and an uncontrolled outdoor environment exhibiting several moderate background influences. Participants performed two tasks during these recordings, one engaging brain activity related to several complex cognitive functions (number sense, attention, memory, executive function) and the other engaging two default brain states. We computed EEG spectral power over three frequency bands (theta: 4-7 Hz, alpha: 8-13 Hz, low beta: 14-20 Hz) where EEG oscillatory activity is known to correlate with the neurocognitive states engaged by these tasks. Null hypothesis significance testing yielded significant EEG power effects typical of the neurocognitive states engaged by each task, but only a beta-band power difference between the two background recording environments during the default brain state. Bayesian analysis showed that the remaining environment null effects were unlikely to reflect measurement insensitivities. This overall pattern of results supports the external validity of laboratory EEG power findings for complex and default neurocognitive states engaged within moderately uncontrolled environments.

Identifying resting locations of a small elusive forest carnivore using a two-stage model accounting for GPS measurement error and hidden behavioral states.

BACKGROUND: Studies of animal movement using location data are often faced with two challenges. First, time series of animal locations are likely to arise from multiple behavioral states (e.g., directed movement, resting) that cannot be observed directly. Second, location data can be affected by measurement error, including failed location fixes. Simultaneously addressing both problems in a single statistical model is analytically and computationally challenging. To both separate behavioral states and account for measurement error, we used a two-stage modeling approach to identify resting locations of fishers (Pekania pennanti) based on GPS and accelerometer data. METHODS: We developed a two-stage modelling approach to estimate when and where GPS-collared fishers were resting for 21 separate collar deployments on 9 individuals in southern Oregon. For each deployment, we first fit independent hidden Markov models (HMMs) to the time series of accelerometer-derived activity measurements and apparent step lengths to identify periods of movement and resting. Treating the state assignments as given, we next fit a set of linear Gaussian state space models (SSMs) to estimate the location of each resting event. RESULTS: Parameter estimates were similar across collar deployments. The HMMs successfully identified periods of resting and movement with posterior state assignment probabilities greater than 0.95 for 97% of all observations. On average, fishers were in the resting state 63% of the time. Rest events averaged 5 h (4.3 SD) and occurred most often at night. The SSMs allowed us to estimate the 95% credible ellipses with a median area of 0.12 ha for 3772 unique rest events. We identified 1176 geographically distinct rest locations; 13% of locations were used on > 1 occasion and 5% were used by > 1 fisher. Females and males traveled an average of 6.7 (3.5 SD) and 7.7 (6.8 SD) km/day, respectively. CONCLUSIONS: We demonstrated that if auxiliary data are available (e.g., accelerometer data), a two-stage approach can successfully resolve both problems of latent behavioral states and GPS measurement error. Our relatively simple two-stage method is repeatable, computationally efficient, and yields directly interpretable estimates of resting site locations that can be used to guide conservation decisions.

Artificial insemination of cattle: Description and assessment of a training program for veterinary students.

The objective was to assess the effectiveness of a comprehensive artificial insemination (AI) training program designed to facilitate an understanding of the breadth of the AI process, including AI skill acquisition, for preclinical veterinary students. Participants (n = 303) were enrolled at the Ross University School of Veterinary Medicine (Basseterre, St. Kitts, West Indies). The 2-d AI training program (n = 20) consisted of ∼8 h of instruction and ∼8 h of demonstration and hands-on activity. Oral presentations were used to deliver educational content, followed by video clips, discussion, demonstrations, and hands-on activity. Reproductive anatomy and physiology of the estrous cycle, AI sire acquisition, collection, evaluation, cryopreservation and distribution of conventional and sexed semen, storage and handling of frozen semen, use of synchronization protocols, accurate and efficient detection of estrus, and correct AI technique were discussed. True or false pre- and posttests were used to determine the level of knowledge gained by participants during the AI training program. Preclinical veterinary students were required to complete a semen handling and AI technique practical exam to achieve a certificate of completion. Participant program evaluations conducted at the conclusion of the program indicated that veterinary students found the content, structure, discussion, demonstrations, and hands-on activities to be appropriate and useful. No negative comments were offered about the training program, instructor, or activity coinstructors. The AI training program increased the posttest knowledge scores of veterinary students by 22 percentage points. Only 1 participant was unable to achieve a certificate of completion due to failure of the semen handling and AI technique practical exam. These results provide evidence that the AI training program was relevant and effective and that it offered information and skill acquisition with immediate field application.