Search

Standard methods for mitochondrial DNA (mtDNA) extraction do not provide the level of enrichment for mtDNA sufficient for direct sequencing and must be followed by long-range-PCR amplification, which can bias the sequencing results. Here, we describe a fast, cost-effective, and reliable method for preparation of mtDNA enriched samples from eukaryotic cells ready for direct sequencing. Our protocol utilizes a conventional miniprep kit, paramagnetic bead-based purification, and an optional, limited PCR amplification of mtDNA. The first two steps alone provide more than 2000-fold enrichment for mtDNA when compared with total cellular DNA (∼200-fold in comparison with current commercially available kits) as demonstrated by real-time PCR. The percentage of sequencing reads aligned to mtDNA was about 22% for non-amplified samples and greater than 99% for samples subjected to 10 cycles of long-range-PCR with mtDNA specific primers.

Homologous recombination (HR) is essential for accurate genome duplication and maintenance of genome stability. In eukaryotes, chromosomal double strand breaks (DSBs) are central to HR during specialized developmental programs of meiosis and antigen receptor gene rearrangements, and form at unusual DNA structures and stalled replication forks. DSBs also result from exposure to ionizing radiation, reactive oxygen species, some anti-cancer agents, or inhibitors of topoisomerase II. Literature predicts that repair of such breaks normally will occur by non-homologous end-joining (in G1), intrachromosomal HR (all phases), or sister chromatid HR (in S/G(2)). However, no in vivo model is in place to directly determine the potential for DSB repair in somatic cells of mammals to occur by HR between repeated sequences on heterologs (i.e., interchromosomal HR). To test this, we developed a mouse model with three transgenes-two nonfunctional green fluorescent protein (GFP) transgenes each containing a recognition site for the I-SceI endonuclease, and a tetracycline-inducible I-SceI endonuclease transgene. If interchromosomal HR can be utilized for DSB repair in somatic cells, then I-SceI expression and induction of DSBs within the GFP reporters may result in a functional GFP+ gene. Strikingly, GFP+ recombinant cells were observed in multiple organs with highest numbers in thymus, kidney, and lung. Additionally, bone marrow cultures demonstrated interchromosomal HR within multiple hematopoietic subpopulations including multi-lineage colony forming unit-granulocyte-erythrocyte-monocyte-megakaryocte (CFU-GEMM) colonies. This is a direct demonstration that somatic cells in vivo search genome-wide for homologous sequences suitable for DSB repair, and this type of repair can occur within early developmental populations capable of multi-lineage differentiation.

The aim of this study was to investigate the difference in head acceleration event (HAE) incidence between training and match-play in women's and men's players competing at the highest level of domestic rugby union globally. Players from Women's (Premiership Women's Rugby, Farah Palmer Cup) and Men's (Premiership Rugby, Currie Cup) rugby union competitions wore instrumented mouthguards during matches and training sessions during the 2022/2023 seasons. Peak linear (PLA) and angular (PAA) acceleration were calculated from each HAE and included within generalized linear mixed-effects models. The incidence of HAEs was significantly greater in match-play compared to training for all magnitude thresholds in both forwards and backs, despite players spending approximately 1.75-2.5 times more time in training. For all HAEs (PLA > 5 g and PAA > 400 rad/s 2 ), incidence rate ratios (IRRs) for match versus training ranged from 2.80 (95% CI: 2.38-3.30; men's forwards) to 4.00 (3.31-4.84; women's forwards). At higher magnitude thresholds (PLA > 25 g; PAA > 2000 rad/s 2 ), IRRs ranged from 3.64 (2.02-6.55; PAA > 2000 rad/s 2 in men's backs) to 11.70 (6.50-21.08; PAA > 2000 rad/s 2 in women's forwards). Similar trends were observed in each competition. Players experienced significantly more HAEs during match-play than training, particularly at higher magnitude thresholds. Where feasible, HAE mitigation strategies may have more scope for HAE reduction if targeted at match-play, particularly where higher magnitude HAEs are the primary concern. However, the number of HAEs associated with different training drills requires exploration to understand if HAEs can be reduced in training, alongside optimizing match performance (e.g., enhancing contact technique)., (© 2024 The Author(s). Scandinavian Journal of Medicine & Science In Sports published by John Wiley & Sons Ltd.)

This study examined the association between a dental implant and changes in adjacent teeth over time. Electronic health records of 1818 patients who received a dental implant were retrospectively evaluated over 14 years (2005-2019) in a university setting. The status of the adjacent tooth and vertical and horizontal distance from the implant platform to adjacent teeth were determined using digital intraoral radiographs taken at baseline and the last follow-up visit (1-14 years, median 4 years). In total, 1085 dental implants were evaluated. There were 234 instances of a change in the adjacent tooth. Decay was observed in 83 (7.6%) of adjacent teeth; the mean time to development was 4 years (range 1-14 years). Approximately 9% of adjacent teeth received direct restorations, 4.8% received indirect restorations, 1% received endodontic root canal treatment, and 5.6% were extracted. The mean horizontal distance between the implant platform and the adjacent teeth was 3.56 mm; the mean vertical distance from the contact point to the alveolar crest on the tooth side was 6.2 mm at the first time of the reported decay on X ray. These distances did not significantly influence the occurrence of caries. The prevalence of interproximal contact loss was higher on the mesial of the implant crown at 63% compared with 20% on the distal side. This large retrospective analysis identified that teeth adjacent to a dental implant were at risk of decay and changes in their condition. In addition, the implant-to-tooth distance and inadequate emergence profile may contribute as caries risk factors in addition to hygiene and a high sugar level diet. These findings appear essential for clinicians when making treatment decisions and discussing outcomes with patients.

Metallosis is a known yet rare late complication of unicompartmental and total knee arthroplasty (TKA), usually secondary to either metal-backed patellar component failure, mobile-bearing polyethylene dislocation, or catastrophic polyethylene failure and wear through. The majority of literature surrounding metallosis has been published in relation to total hip arthroplasty (THA) metal on metal bearing wear or mechanically assisted crevice corrosion.This case report describes the development of metallosis in a 77-year-old male patient with advanced (Kellgren-Lawrence Grade 4) osteoarthritis with associated valgus deformity, who underwent index TKA with a semiconstrained revision knee system due to intraoperative medial collateral ligament laxity. The taper junction between the titanium alloy stem and cobalt chromium femoral component was the source of diffuse intra-articular metallosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Solid organ transplants (SOT) have evolved into life-saving interventions for end-stage diseases affecting vital organs. Advances in transplantation techniques, donor selection, and immunosuppressive therapies have enhanced outcomes, leading to a growing demand for SOT. Patients with a solid organ transplant are living long enough to develop the same pathologies which are indicated for joint replacement surgery in the general population. SOT patients who undergo a total hip, knee, or shoulder arthroplasty do similarly in the context of clinical outcomes and implant survival when compared to the general population. These immunosuppressed patients tend to have higher complication rates in the short-term following surgery. Prudent management of these patients in the short-term may be necessary, but patients can expect to do well otherwise., (© 2024 Professor P K Surendran Memorial Education Foundation. Published by Elsevier B.V. All rights reserved.)

Background: The COVID-19 pandemic created complex challenges regarding the timing and appropriateness of do-not-attempt cardiopulmonary resuscitation (DNACPR) and/or Do Not Intubate (DNI) code status orders. This paper sought to determine differences in utilization of DNACPR and/or DNI orders during different time periods of the COVID-19 pandemic, including prevalence, predictors, timing, and outcomes associated with having a documented DNACPR and/or DNI order in hospitalized patients with COVID-19., Methods: A cohort study of hospitalized patients with COVID-19 at two hospitals located in the Midwest. DNACPR code status orders including, DNI orders, demographics, labs, COVID-19 treatments, clinical interventions during hospitalization, and outcome measures including mortality, discharge disposition, and hospice utilization were collected. Patients were divided into two time periods (early and late) by timing of hospitalization during the first wave of the pandemic (March-October 2020)., Results: Among 1375 hospitalized patients with COVID-19, 19% ( n  = 258) of all patients had a documented DNACPR and/or DNI order. In multivariable analysis, age (older) p  =< 0.01, OR 1.12 and hospitalization early in the pandemic p  = 0.01, OR 2.08, were associated with having a DNACPR order. Median day from DNACPR order to death varied between cohorts p  => 0.01 (early cohort 5 days versus late cohort 2 days). In-hospital mortality did not differ between cohorts among patients with DNACPR orders, p  = 0.80., Conclusions: There was a higher prevalence of DNACPR and/or DNI orders and these orders were written earlier in the hospital course for patients hospitalized early in the pandemic versus later despite similarities in clinical characteristics and medical interventions. Changes in clinical care between cohorts may be due to fear of resource shortages and changes in knowledge about COVID-19., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Conventional wisdom suggests that widely utilized self-assembled alkylthiolate monolayers on gold are too unstable to last more than several days when exposed to complex fluids such as raw serum at body temperature. Demonstrated here is that these monolayers can not only last at least 1 week under such harsh conditions but that significant applied value can be captured for continuous electrochemical aptamer biosensors. Electrochemical aptamer biosensors provide an ideal tool to investigate monolayer degradation, as aptamer sensors require a tightly packed monolayer to preserve sensor signal vs background current and readily reveal fouling by albumin and other solutes when operating in biofluids. Week-long operation in serum at 37 °C is achieved by (1) increasing van der Waals interactions between adjacent monolayer molecules to increase the activation energy required for desorption, (2) optimizing electrochemical measurement to decrease both alkylthiolate oxidation and electric-field-induced desorption, and (3) mitigating fouling using protective zwitterionic membranes and zwitterion-based blocking layers with antifouling properties. This work further proposes origins and mechanisms of monolayer degradation in a logical stepwise manner that was previously unobservable over multiday time scales. Several of the observed results are surprising, revealing that short-term improvements to sensor longevity (i.e., hours) actually increase sensor degradation in the longer term (i.e., days). The results and underlying insights on mechanisms not only push forward fundamental understanding of stability for self-assembled monolayers but also demonstrate an important milestone for continuous electrochemical aptamer biosensors.

This study aims to (a) quantify the movement patterns during rugby league match-play and (b) identify if differences exist by levels of competition within the movement patterns and units through the sequential movement pattern (SMP) algorithm. Global Positioning System data were analysed from three competition levels; four Super League regular ( regular-SL ), three Super League (semi-)Finals ( final-SL ) and four international rugby league ( international ) matches. The SMP framework extracted movement pattern data for each athlete within the dataset. Between competition levels, differences were analysed using linear discriminant analysis (LDA). Movement patterns were decomposed into their composite movement units; then Kruskal-Wallis rank-sum and Dunn post-hoc were used to show differences. The SMP algorithm found 121 movement patterns comprised mainly of "walk" and "jog" based movement units. The LDA had an accuracy score of 0.81, showing good separation between competition levels. Linear discriminant 1 and 2 explained 86% and 14% of the variance. The Kruskal-Wallis found differences between competition levels for 9 of 17 movement units. Differences were primarily present between regular-SL and international with other combinations showing less differences. Movement units which showed significant differences between competition levels were mainly composed of low velocities with mixed acceleration and turning angles. The SMP algorithm found 121 movement patterns across all levels of rugby league match-play, of which, 9 were found to show significant differences between competition levels. Of these nine, all showed significant differences present between international and domestic, whereas only four found differences present within the domestic levels. This study shows the SMP algorithm can be used to differentiate between levels of rugby league and that higher levels of competition may have greater velocity demands. Highlights This study shows that movement patterns and movement units can be used to investigate team sports through the application of the SMP frameworkOne hundred and twenty-one movement patterns were found to be present within rugby league match-play, with the walk- and jog-based movement units most prevalent. No movement pattern was unique to a single competition level.Further analysis revealed that the majority of movement units analysed had significant differences between international and domestic rugby league, whereas only four movement units (i.e. f,m,n,q) had significant differences within the two domestic rugby league levels.International rugby league had higher occurrences of the movement patterns consisting of higher velocity movement units (ie. T,S,y). This suggests that international rugby league players may need greater high velocity exposure in training.

Purpose: The COVID-19 pandemic significantly impacted United States (US) health professions educational programs. Physician assistant (PA) programs were forced to respond to rapidly changing circumstances early in the pandemic. This study describes the impact that the early phase of the COVID-19 pandemic had on PA programs in the United States., Methods: This cross-sectional study is based on data from the COVID-19 Rapid Response Report 1, conducted by the Physician Assistant Education Association in April 2020. The survey sample included 254 US PA program directors with a response rate of 64.2%. Our outcome measures included temporary changes to PA program operations and clinical training, and current and upcoming budget and tuition changes, as well as the impact of the pandemic on faculty and staff employment. We used descriptive statistics to summarize these outcomes, stratified by 4 categories characterizing features of PA programs, including geographical location, academic housing, funding model, and academic health center status., Results: The COVID-19 pandemic impact on programs varied geographically. A majority of programs reported making numerous temporary changes to their operations. Most programs moved both didactic and clinical education to an online format. Clinical training was temporarily suspended at almost all programs., Conclusions: PA programs continued training despite the pandemic. The long-term impact of the pandemic may be the instability of the PA education workforce., Competing Interests: The authors declare no conflict of interest., (Copyright © 2022 Physician Assistant Education Association.)

Athlete external load is typically quantified as volumes or discretised threshold values using distance, speed and time. A framework accounting for the movement sequences of athletes has previously been proposed using radio frequency data. This study developed a framework to identify sequential movement sequences using GPS-derived spatiotemporal data in team-sports and establish its stability. Thirteen rugby league players during one match were analysed to demonstrate the application of the framework. The framework (Sequential Movement Pattern-mining [SMP]) applies techniques to analyse i) geospatial data (i.e., decimal degree latitude and longitude), ii) determine players turning angles, iii) improve movement descriptor assignment, thus improving movement unit formation and iv) improve the classification and identification of players' frequent SMP. The SMP framework allows for sub-sequences of movement units to be condensed, removing repeated elements, which offers a novel technique for the quantification of similarities or dis-similarities between players and playing positions. The SMP framework provides a robust and stable method that allows, for the first time the analysis of GPS-derived data and identifies the frequent SMP of field-based team-sport athletes. The application of the SMP framework in practice could optimise the outcomes of training of field-based team-sport athletes by improving training specificity.

Objective: Primary care workforce projections continue to predict significant physician shortages. An oversupply of primary care physician assistants (PAs) and NPs also is predicted. This paradox calls into question the assumptions that underlie workforce projection models, which likely underestimate the primary care contributions of PAs and NPs., Methods: Federally qualified health center data from the 2016-2019 Uniform Data System were used to calculate the number of clinic visits per full-time equivalent (FTE) physician, PA, and NP. Visits per FTE were compared across provider type to determine provider-specific productivity ratios., Results: The combined PA and NP productivity ratio increased relative to physicians in each year, ranging from 0.85 in 2016 to 0.88 in 2019. Clinic visits per FTE for PAs and family physicians were nearly equivalent., Conclusions: Primary care workforce projection models should be reexamined to more accurately capture the productivity of PAs and NPs., (Copyright © 2021 American Academy of Physician Assistants.)

Background: ACL reconstruction often results in an extended period of muscle atrophy and weakness. Blood flow restriction (BFR) training is a technique that has been shown to decrease muscle atrophy in a variety of populations., Purpose: The purpose of this systematic review was to analyze the research presented on the effect of blood flow restriction training on quadriceps muscle atrophy and circumference post ACL reconstruction., Study Design: Systematic Review., Methods: Articles were reviewed using the databases Google Scholar, PubMed, and EBSCO. Keywords included blood flow restriction training, ACL reconstruction, and quadriceps., Inclusion Criteria Included: English language, peer-reviewed journals; randomized control trials; and articles including blood flow restriction and measurement of quadriceps atrophy and circumference post ACL reconstruction. Exclusion criteria included non-English language publications; studies without a control group; and articles without sufficient data to evaluate the methodology. Four studies met the selection criteria and were assessed using the GRADE scale, which analyzes the strength of a study based on study limitations, precision, consistency, directness, and publication bias. After a GRADE designation was assigned, the following information was extracted from and compared across the studies: participant demographics, cuff used, graft used during ACL reconstruction, tool used to assess muscle atrophy, protocol used, and conclusions., Results: Three out of four studies showed some amount of an increase in femoral muscle cross sectional area after the use of BFR combined with low-intensity resistance training (LIRT). The strength of all four studies was moderate when assessed using the GRADE scale., Conclusion: This review of the available evidence yields promising results regarding the use of BFR and LIRT in the remediation of femoral muscle atrophy after an ACL reconstruction. Further research is necessary before BFR can be recommended for use in clinical settings., Level of Evidence: 3a., (© 2020 by the Sports Physical Therapy Section.)

Transient receptor potential canonical (TRPC) proteins form nonselective cation channels that play physiological roles in a wide variety of cells. Despite growing evidence supporting the therapeutic potential of TRPC6 inhibition in treating pathological cardiac and renal conditions, mechanistic understanding of TRPC6 function and modulation remains obscure. Here we report cryo-EM structures of TRPC6 in both antagonist-bound and agonist-bound states. The structures reveal two novel recognition sites for the small-molecule modulators corroborated by mutagenesis data. The antagonist binds to a cytoplasm-facing pocket formed by S1-S4 and the TRP helix, whereas the agonist wedges at the subunit interface between S6 and the pore helix. Conformational changes upon ligand binding illuminate a mechanistic rationale for understanding TRPC6 modulation. Furthermore, structural and mutagenesis analyses suggest several disease-related mutations enhance channel activity by disrupting interfacial interactions. Our results provide principles of drug action that may facilitate future design of small molecules to ameliorate TRPC6-mediated diseases., Competing Interests: YB At the time of the study YB was affiliated with Amgen Research, Amgen Inc and has no financial interests to declare. The author has no other competing interests to declare. XY XY is affiliated with Amgen Research, Amgen Inc and has no financial interests to declare. The author has no other competing interests to declare. HC At the time of the study HC was affiliated with Amgen Research, Amgen Inc and has no financial interests to declare. The author has no other competing interests to declare. DH At the time of the study DH was affiliated with Amgen Research, Amgen Inc and has no financial interests to declare. The author has no other competing interests to declare. RW At the time of the study RW was affiliated with Amgen Research, Amgen Inc and has no financial interests to declare. The author has no other competing interests to declare. XW XW is affiliated with Amgen Research, Amgen Inc and has no financial interests to declare. The author has no other competing interests to declare. PL At the time of the study PL was affiliated with Amgen Research, Amgen Inc and has no financial interests to declare. The author has no other competing interests to declare. YG At the time of the study YG was affiliated with Amgen Research, Amgen Inc and has no financial interests to declare. The author has no other competing interests to declare. SG SGR is affiliated with Amgen Research, Amgen Inc and has no financial interests to declare. The author has no other competing interests to declare. DL DCL is affiliated with Amgen Research, Amgen Inc and has no financial interests to declare. The author has no other competing interests to declare. XH At the time of the study XH was affiliated with Amgen Research, Amgen Inc and has no financial interests to declare. The author has no other competing interests to declare., (© 2020, Bai et al.)

Study Objective: We assess the feasibility of using our community hospital emergency department (ED) as an immediate portal to medication-assisted treatment for patients in opioid withdrawal., Methods: This was a prospective observational cohort study. In collaboration with an outpatient substance abuse treatment center, we alerted the public through media outlets that individuals could receive immediate buprenorphine treatment for opioid withdrawal in the ED, with rapid referral for medication-assisted treatment. If medication-assisted treatment intake was delayed, patients could return for up to 2 more days for buprenorphine administration to treat their withdrawal symptoms. We measured compliance with initial follow-up and continued treatment engagement at 30 and 90 days., Results: The study was conducted during 12 months. A total of 62 patients were enrolled, evaluated for buprenorphine criteria, and referred for medication-assisted treatment. Fifty subjects were compliant with their first medication-assisted treatment follow-up visit (81% [95% confidence interval 71% to 91%]), and 43 of these 50 patients were still engaged in medication-assisted treatment at 30 days (86% [95% confidence interval 76% to 96%]), with 33 of the 50 still engaged at 90 days (66% [95% confidence interval 53% to 79%]). We observed no instances of precipitated withdrawal or other adverse reactions in the ED., Conclusion: A substantial number of patients responded to this program and received accelerated engagement in medication-assisted treatment. Such a program is feasible in the community hospital ED and may prevent some individuals from relapsing into high-risk illicit drug use when immediate medication-assisted treatment is not otherwise available., (Copyright © 2019 American College of Emergency Physicians. Published by Elsevier Inc. All rights reserved.)

Hot isostatic pressing (HIP) treatments are traditionally used to seal internal porosity, because defects exist in as-built Ti-6Al-4V parts produced by electron-beam melting powder-bed fusion. Standard HIP treatment of Ti-6Al-4V parts results in decreased strength due to coarsening of the microstructure. We present a new HIP strategy with the following steps: hold above the β-transus, rapid quenching, and tempering. This new HIP treatment seals internal porosity, causes a columnar-to-equiaxed transition in morphology of prior-β grains, changes the α lath aspect ratio, removes microstructural heterogeneities and matches the yield and ultimate tensile strength of the as-built condition., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Generation of renewable energy is one of the grand challenges facing our society. We present a new bio-electric technology driven by chemical gradients generated by photosynthesis and respiration. The system does not require pure cultures nor particular species as it works with the core metabolic principles that define phototrophs and heterotrophs. The biology is interfaced with electrochemistry with an alkaline aluminum oxide cell design. In field trials we show the system is robust and can work with an undefined natural microbial community. Power generated is light and photosynthesis dependent. It achieved a peak power output of 33 watts/m(2) electrode. The design is simple, low cost and works with the biological processes driving the system by removing waste products that can impede growth. This system is a new class of bio-electric device and may have practical implications for algal biofuel production and powering remote sensing devices.

Aberrant activation of the Wnt pathway is believed to drive the development and growth of some cancers. The central role of CK1γ in Wnt signal transduction makes it an attractive target for the treatment of Wnt-pathway dependent cancers. We describe a structure-based approach that led to the discovery of a series of pyridyl pyrrolopyridinones as potent and selective CK1γ inhibitors. These compounds exhibited good enzyme and cell potency, as well as selectivity against other CK1 isoforms. A single oral dose of compound 13 resulted in significant inhibition of LRP6 phosphorylation in a mouse tumor PD model.

A series of biphenyl-based N(3)O ligands, 2, 4, 6, and 8 were synthesized and their Cu(II) complexes prepared. These complexes were characterized by a combination of elemental analysis, FAB-MS, UV-vis spectroscopy and electrochemistry. The structure of [Cu(N(3)O-mpy-NO2)Cl2], 12 [N(3)O-mpy = 2-(3-pyridylmethylimino)-2'-(2-methylaminophenol)biphenyl], was solved and showed that the ligand coordinates through the three nitrogens with the phenol oxygen uncoordinated. Titration of azide anion into solutions of the complexes in methanol resulted in the appearance of a new band between 485-495 nm at the expense of the starting peak at 380 nm. Cyclic voltammetry studies indicated that the complexes undergo quasi-reversible one-electron reductions in acetonitrile at potentials between 0.13-0.58 V vs. Ag/AgCl. The complexes were found to be weakly active for the oxidation of di-tert-butyl catechol (DTBC).