Single-molecule DNA sequencing of widely varying GC-content using nucleotide release, capture and detection in microdroplets.

Despite remarkable progress in DNA sequencing technologies there remains a trade-off between short-read platforms, having limited ability to sequence homopolymers, repeated motifs or long-range structural variation, and long-read platforms, which tend to have lower accuracy and/or throughput. Moreover, current methods do not allow direct readout of epigenetic modifications from a single read. With the aim of addressing these limitations, we have developed an optical electrowetting sequencing platform that uses step-wise nucleotide triphosphate (dNTP) release, capture and detection in microdroplets from single DNA molecules. Each microdroplet serves as a reaction vessel that identifies an individual dNTP based on a robust fluorescence signal, with the detection chemistry extended to enable detection of 5-methylcytosine. Our platform uses small reagent volumes and inexpensive equipment, paving the way to cost-effective single-molecule DNA sequencing, capable of handling widely varying GC-bias, and demonstrating direct detection of epigenetic modifications.

Single-molecule detection of deoxyribonucleoside triphosphates in microdroplets.

A new approach to single-molecule DNA sequencing in which dNTPs, released by pyrophosphorolysis from the strand to be sequenced, are captured in microdroplets and read directly could have substantial advantages over current sequence-by-synthesis methods; however, there is no existing method sensitive enough to detect a single nucleotide in a microdroplet. We have developed a method for dNTP detection based on an enzymatic two-stage reaction which produces a robust fluorescent signal that is easy to detect and process. By taking advantage of the inherent specificity of DNA polymerases and ligases, coupled with volume restriction in microdroplets, this method allows us to simultaneously detect the presence of and distinguish between, the four natural dNTPs at the single-molecule level, with negligible cross-talk.

Health and Public Policy to Facilitate Effective Prevention and Treatment of Substance Use Disorders Involving Illicit and Prescription Drugs: An American College of Physicians Position Paper.

Substance use disorders involving illicit and prescription drugs are a serious public health issue. In the United States, millions of individuals need treatment for substance use disorders but few receive it. The rising number of drug overdose deaths and the changing legal status of marijuana pose new challenges. In this position paper, the American College of Physicians maintains that substance use disorder is a treatable chronic medical condition and offers recommendations on expanding treatment options, the legal status of marijuana, addressing the opioid epidemic, insurance coverage of substance use disorders treatment, education and workforce, and public health interventions.

Histopathologic features of melanoma in difficult-to-diagnose lesions: A case-control study.

BACKGROUND: Dermatopathology is considered the gold standard for melanoma diagnosis, but a subset of cases is difficult to diagnose by histopathology. OBJECTIVE: The goals of this study were to measure the accuracy of histopathologic features in difficult-to-diagnose melanocytic tumors and the interobserver agreement of those features. METHODS: This is a case-control study of histopathologic features of melanoma in 100 difficult-to-diagnose melanocytic neoplasms (40 melanomas and 60 nevi). Slides were blindly evaluated by 5 dermatopathologists. Frequencies, predictive values, and interobserver agreement were calculated. Univariate and multivariate logistic regression analyses were performed to identify the most influential features in arriving at a diagnosis of melanoma. RESULTS: Asymmetry, single-cell melanocytosis, solar elastosis, pagetoid melanocytosis, and broad surface diameter were most influential in arriving at a diagnosis of melanoma. Asymmetry and single-cell melanocytosis were most predictive of melanoma. Fleiss kappa was <0.6 for interobserver agreement in 9/10 histopathologic features of melanoma. LIMITATIONS: This study is limited by the small sample size, selection bias, and binary classification of melanocytic lesions. CONCLUSION: Our results indicate histopathologic features of melanoma in difficult-to-diagnose lesions vary in accuracy and reproducibility.

Impact of a Mobile Health Application on User Engagement and Pregnancy Outcomes Among Wyoming Medicaid Members.

BACKGROUND: Pregnancy and birth outcomes are a critical area of healthcare, yet negative outcomes like C-sections and preterm births remain widespread. Studies show that early and ongoing prenatal care can improve outcomes; however, in-person care is difficult to deliver in rural areas. This article examines the impact of mobile health technology on user engagement and birth outcomes in a Wyoming pilot study. The pilot did face some limitations; namely, the small app user group size and scant demographic information collected from users. MATERIALS AND METHODS: Wyoming Medicaid contracted with Xerox State Healthcare to launch WYhealth Due Date Plus, a pregnancy application by Wildflower Health. Pregnant Medicaid members registering for the app and providing a Medicaid ID were assigned to the app user group (N = 85). The non-app user group consisted of other pregnant Medicaid members with delivery outcome records (N = 5,158). Downloads and utilization frequency were tracked to gauge user engagement. Among pregnant Medicaid members, data were collected on app usage and four outcomes of interest-6-month or more prenatal visit, C-section, low birth weight, and Neonatal Intensive Care Unit (NICU) admission-to examine the association between app use and pregnancy/birth outcomes. Chi-square tests were conducted to analyze associations. A Kolmogorov-Smirnov test was used to assess potential confounding. RESULTS: Strong user engagement was observed with over 1,730 downloads. App users had a statistically significant association between app usage and completion of a 6-month or more prenatal visit (p = 0.022). There was a borderline significant association between app use and decreased incidence of low birth weight (p = 0.055). Maternal age was not a possible confounder. CONCLUSIONS: Preliminary data indicate that Due Date Plus attracted an engaged user base and that app usage was associated with improvements in prenatal visit completion and reduced incidence of low-birth weight delivery. These promising results suggest broader implementation and further study of mobile applications for prenatal support.

Cutaneous histiocytoid Sweet syndrome and its relationship to hematological diseases.

Sweet syndrome (SS) was described over 50 years ago as a distinctive form of neutrophilic dermatosis. It may be idiopathic, drug-induced or paraneoplastic, and in the last of those subtypes, myeloproliferative diseases are prominently represented. A peculiar variant of SS is termed 'histiocytoid' SS (HSS), and early accounts of that condition asserted that it showed no linkage to hematological disorders. We herein report two additional cases of HSS--both of which were associated with myeloid dyscrasias--together with a review of the pertinent literature. Along with our observations, the latter process appears to contradict the contention that HSS has no relationship to hematopoietic diseases; between 35 and 55% of reported cases have indeed shown such an association, usually with myelogenous leukemia or myelodysplastic syndromes.

BAM! Utilizing the frequency of Benign, Atypical and Malignant diagnoses for quality improvement in the histopathologic diagnosis of melanocytic neoplasms.

BACKGROUND: Healthcare quality measures are metrics that quantify medical outcomes. There is a need for performance indicators in the diagnosis of melanocytic neoplasms. We sought to determine whether the atypical to malignant diagnosis ratio (AMR) and benign to malignant diagnosis ratio (BMR) could identify differences between practitioners within a group as well as individual diagnostic drift. METHODS: Diagnoses of melanocytic neoplasms from 2013 and 2014 by two dermatopathologists were prospectively subclassified as benign, atypical or malignant and reported on a monthly basis to the pathologists. Case diagnoses of melanocytic neoplasms from 2012 and 2009 were retrospectively subclassified as benign, atypical or malignant for comparison. RESULTS: A total of 33,169 cases were used in this study. Interpathologist AMR and BMR were significantly different. One pathologist demonstrated a significant increase in AMR over time. CONCLUSION: AMR and BMR metrics are potential performance indicators that can measure pathologist uncertainty, identify diagnostic drift and provide a surrogate measure of the relative risk level of laboratory patient populations. If applied to multiple laboratories, AMR and BMR metrics could help inform physicians' choice of dermatopathology laboratory and provide a method for comparative analysis between laboratories.

A Statewide Child Telepsychiatry Consult System Yields Desired Health System Changes and Savings.

BACKGROUND: Telepsychiatry has clinical efficacy with children, but questions remain about cost-effectiveness. State agencies and health systems need to know if a child telepsychiatry consult system can address system concerns and improve care quality while lowering costs. MATERIALS AND METHODS: To assist care in a rural state with few child and adolescent psychiatrists, an academic center coordinated a consult system of (1) televideo consults for high-needs children with Medicaid and state Multidisciplinary Team (MDT)/foster care involvement, (2) remote medication reviews for beyond guidelines prescribing, and (3) elective community provider telephone-based consults. Consult service data were collected and analyzed with Wyoming's Medicaid and Foster Care Divisions between the program start in January 2011 until March 2013. RESULTS: There were 229 televideo MDT/foster care consults, 125 mandatory medication reviews, and 277 elective phone consultations supporting community providers during this period. Following implementation, the number of Medicaid children ≤5 years of age using psychotropic medications decreased by 42% (p<0.001), and the number of children using psychotropic doses >150% of the Food and Drug Administration maximum decreased by 52% (p<0.001). Televideo consults redirected 60% of children slated by caseworkers for a psychiatric residential treatment facility admission into alternative community treatment and placements. A financial return on investment was 1.82 to 1 for combined services. CONCLUSIONS: This coordinated child telepsychiatry consult system for a state Medicaid division reduced outlier pediatric psychiatric medication prescribing, supported local community-delivered treatments, and reduced unnecessary hospitalizations in a financially advantageous manner that was well received by the practice community.

Interleukin-10 reduces scar formation in both animal and human cutaneous wounds: results of two preclinical and phase II randomized control studies.

Cutaneous scarring affects up to 100 million people per annum. There is no effective scar reducing/preventing therapeutic developed to date. Interleukin (IL)-10 is an anti-inflammatory and antifibrotic cytokine. In the embryo it is important for scarless wound repair. We investigated the effect on wound healing and scarring of a double deletion of the IL-10 and IL-4 genes in a knockout (KO) mouse model, and also the effect of exogenous addition of recombinant human (rh) IL-10 into rat and human cutaneous incisions. Mouse study: Two incisions were made on the dorsal skin of 20 double IL-4/IL-10 KO mice and 20 wild-type (WT) controls. Rat study: Three concentrations of rhIL-10 were investigated. Four incisions were made on the dorsal skin of 30 rats. Each rat received two concentrations. Each incision receiving a concentration of rhIL-10 was matched with a control incision, which received either placebo or standard care. Human study: Eight concentrations of rhIL-10 were investigated. Four incisions were made on each arm of 175 healthy volunteers. Four incisions received four different concentrations, which were matched with four control incisions that received either standard care or placebo. KO mice healed with poor scar histology and increased inflammation. rhIL-10-treated rat incisions healed with decreased inflammation, better scar histology, and better macroscopic scar appearance. rhIL-10-treated human incisions at low concentrations healed with better macroscopic scar appearance and less red scars. IL-10 is an important cytokine in wound healing and its suppression of inflammation and scarring is demonstrated in mice and rats with a translational effect in humans.

Association for Human Pharmacology in the Pharmaceutical Industry conference 2022: impending change, innovations and future challenges.

The Association for Human Pharmacology in the Pharmaceutical Industry's annual meeting focused on current and impending challenges facing the United Kingdom's (UK) pharmaceutical industry and how these opportunities can inspire innovation and best practice. The UK pharmaceutical landscape is still evolving following Brexit and learnings from the coronavirus disease 2019 (COVID-19) pandemic. As such, the UK's clinical community is in a unique position to steer innovation in a meaningful direction. With the continuation of remote forms of working, further opportunities have arisen to support novel practices away from the clinic. The keynote speaker reflected on clinical development over the past 40 years and how the industry must continue to concentrate on patient welfare. The future of drug development was discussed regarding challenges associated with developing translational gene therapies, and the status of investment markets analyzed from a business strategy and consulting perspective. The patient viewpoint was a core theme throughout the conference with patient-centric blood sampling and decentralized clinical trials providing suggestions for how the industry can save costs and increase efficiency. Moreover, the patient perspective was central to a debate over whether ethics requirements should be the same for oncology patients taking part in first-in-human studies as those for healthy subjects. Discussions continued around the changing roles of the Qualified Person and Principal Investigators which underpins how sponsors may want to run future trials in the UK. Lessons learned from conducting challenge trials in healthy volunteers and patients were discussed following a presentation from the serving Chair of the COVID-19 challenge ethics committee. The current state of interactions with the Medicines and Healthcare products Regulatory Agency were also explored. It was considered how the immediate future for the UK clinical trials community is inevitably still linked with Europe; the newly implemented European Medicines Agency Clinical Trials Information System has been met with lukewarm responses, providing a promising opportunity to ensure UK Phase I units continue to play a vital role in global research.

Recommendations on clinical proof of efficacy for potential scar prevention and reduction therapies.

Cutaneous scarring is an enormous medical problem with approximately 100 million patients acquiring scars each year. Scar prevention/reduction represents a significant, and largely unmet, clinical need. Research into the prophylactic modulation of scar outcome differs from research into other disease processes as the scar is not present at the start of the study; measurements of changes from baseline are impossible. Final scar morphology is influenced by many variables. A fundamental principle that should be observed in the prospective evaluation of scar prevention/reduction therapies is that, if left untreated, wounds in treatment and control groups should have healed with identical scars. Observation of this principle will allow the detection of true treatment effects. The many variables that influence scar morphology mean that the evaluation of potential pharmaceutical products for this indication favors the use of self-controlled designs in clinical trials. In this article, we review variables that affect scar morphology and recommend the self-controlled design for clinical trials aiming to establish proof of efficacy of scar prevention and reduction pharmaceuticals. With no pharmaceutical products currently licensed for this indication, this represents a new therapeutic area. The principles discussed will also have direct relevance to the wider fields of wound healing and regenerative medicine.

Prophylactic administration of avotermin for improvement of skin scarring: three double-blind, placebo-controlled, phase I/II studies.

BACKGROUND: Research into mechanisms of skin scarring identified transforming growth factor beta3 (TGFbeta3) as a potential antiscarring therapy. We assessed scar improvement with avotermin (recombinant, active, human TGFbeta3). METHODS: In three double-blind, placebo-controlled studies, intradermal avotermin (concentrations ranging from 0.25 to 500 ng/100 microL per linear cm wound margin) was administered to both margins of 1 cm, full-thickness skin incisions, before wounding and 24 h later, in healthy men and women. Treatments (avotermin and placebo or standard wound care) were randomly allocated to wound sites by a computer generated randomisation scheme, and within-participant controls compared avotermin versus placebo or standard wound care alone. Primary endpoints were visual assessment of scar formation at 6 months and 12 months after wounding in two studies, and from week 6 to month 7 after wounding in the third. Investigators, participants, and scar assessors were blinded to treatment. Efficacy analyses were intention to treat. These studies are registered with ClinicalTrials.gov, numbers NCT00847925, NCT00847795, and NCT00629811. RESULTS: In two studies, avotermin 50 ng/100 microL per linear cm significantly improved median score on a 100 mm visual analogue scale (VAS) by 5 mm (range -2 to 14; p=0.001) at month 6 and 8 mm (-29 to 18; p=0.0230) at month 12. In the third, avotermin significantly improved total scar scores at all concentrations versus placebo (mean improvement: from 14.84 mm [95 % CI 5.5-24.2] at 5 ng/100 microL per linear cm to 64.25 mm [49.4-79.1] at 500 ng/100 microL per linear cm). Nine [60%] scars treated with avotermin 50 ng/100 microL per linear cm showed 25% or less abnormal orientation of collagen fibres in the reticular dermis versus five [33%] placebo scars. After only 6 weeks from wounding, avotermin 500 ng/100 microL per linear cm improved VAS score by 16.12 mm (95% CI 10.61-21.63). Adverse events at wound sites were similar for avotermin and controls. Erythema and oedema were more frequent with avotermin than with placebo, but were transient and deemed to be consistent with normal wound healing. INTERPRETATION: Avotermin has potential to provide an accelerated and permanent improvement in scarring.

The Association for Human Pharmacology in the Pharmaceutical Industry London Meeting October 2019: Impending Change, Innovation, and Future Challenges.

The Association for Human Pharmacology in the Pharmaceutical Industry (AHPPI) annual meeting focused on impending change, innovation, and future challenges facing early phase drug development as we move into the second decade of the 21th century. The meeting opened with discussion around the technical revolution in pharmaceutical medicine over the 4 decades since the AHPPI was founded and how transformative technologies have accompanied the introduction of processes such as physiologically based pharmacokinetic modeling. During the meeting examples were presented of how in terms of the development of new therapies, the classic phases of clinical drug development are becoming a thing of the past and the lines between the phases have begun to blur, particularly in the field of oncology. The contribution that monoclonal antibodies have made to medicine and the next chapter in their design and use was also discussed. A representative of the UK's Medicine and Healthcare Products Regulatory Agency discussed the increasing numbers of requests to approve complex innovative design trials, how novel trial designs are impacting on the traditional linear "phase" approach to drug development and the common pitfalls associated with them. Guidance was provided from a regulator's viewpoint on what was meant by the term "novel design" and how to submit successful trial applications for such complex trials. In an Oxford-style debate, the audience discussed the motion that "there is no longer a need to include placebo subjects in early clinical trials." The keynote speaker focused on delivering change in complex environments such as the field of drug development. The afternoon session included presentations on the challenges associated with drug product design, the complexities within non-oral dosage forms and proposed new methods of formulations for drug delivery. Presentations were also given on advances in mechanistic and computational pharmacokinetic modeling and how they have proved to be valuable tools to rationalize and facilitate the process of drug development.

Dynamic skin tension in the forearm: effects of pronation and supination.

PURPOSE: Longitudinal scars on the radial quadrant of the distal forearm skin envelope are typically observed to be wider than those on the ulnar quadrant and have an increased incidence of hypertrophic change. Forearm rotation movements may produce differential skin tensions within the forearm skin envelope, and this may lead to differential scarring patterns. This study was designed to measure skin tension changes in the forearm as a result of rotational position to see if these would be consistent with the hypothesis that greater tension changes are observed on the radial aspect of the forearm. METHODS: The effect of forearm position on the magnitude and direction of skin tension was measured on human volunteers. Standardized circles were marked in circumferential fashion at specified intervals on forearm skin, and the angular and dimensional distortion of these circles that occurred with forearm rotation was measured with caliper and goniometer. Data were analyzed for statistical significance using paired t-test. RESULTS: Pronation and supination resulted in marked angular rotation of the lines of maximal skin tension at all sites on the forearm. Supination resulted in a greater angular deviation of the lines of maximal skin tension from the longitudinal line of usual surgical incision, particularly on the radial aspect of the forearm. In supination, the magnitude of ellipsoid deformation at the distal forearm was greater on the radial aspect compared with that of the ulnar. Similar significant changes were also demonstrated at the mid-forearm and proximal forearm levels. CONCLUSIONS: This study systematically maps the effects of pronation and supination on skin tension within the forearm skin envelope. The significant changes occurring in both the ellipsoid deformation and ellipsoid orientation support our hypothesis that the magnitude of skin tension changes significantly with forearm rotation. The radial aspect of the distal forearm experiences the greatest changes, particularly as the forearm supinates.

Live cell imaging of Plasmodiophora brassicae-host plant interactions based on a two-step axenic culture system.

Plasmodiophora brassicae, a parasitic protist, induces club-shaped tumor-like growth of host Brassicas roots and hypocotyls after infection. Due to its soil-borne nature and intracellular, biotrophic parasitism the infection biology and early pathogenesis remains in doubt. In this study, we have established a new protocol, based on a two-step axenic culture of P. brassicae with its host tissues, for easy and in planta observation of cellular interactions between P. brassicae and host plants: first, coculture of P. brassicae with infected canola root tissues, on growth-medium plates, enables the propagation of P. brassicae that serves as pure inoculum for pathogenicity assays, and second, the pure inoculum is subsequently used for pathogenicity tests on both canola and Arabidopsis seedlings grown on medium plates in Petri dishes. During the first axenic culture, we established a staining protocol by which the pathogen was fluorescently labeled with Nile red and calcofluor white, thus allowing in planta observation of pathogen development. In the pathogenicity assays, our results showed that axenic cultures of P. brassicae, in calli, remains fully virulent and completes its life cycle in both canola and Arabidopsis roots grown in Petri dishes. Combining visualization of fluorescent probe-labeled P. brassicae structures with fluorescent protein tagging of Arabidopsis cellular components, further revealed dynamic responses of host cells at the early stages of P. brassicae infection. Thus, established protocols for in planta detection of P. brassicae structures and the live cell imaging of P. brassicae-Arabidopsis interactions provide a novel strategy for improving our detailed knowledge of P. brassicae infection in host tissues.

Immunoreactivity for Sox10 in Basaloid Neoplasms of The Skin.

Basaloid tumors of the skin pose a diagnostic challenge to pathologists, because the differential diagnosis is broad, sometimes with subtle differentiating features. We evaluated SOX10 expression in 120 primary cutaneous tumors with epidermal, sweat glandular, neuroendocrine/neuroectodermal, follicular, and sebaceous lineages. Our findings were compared with those of previous studies that evaluated SOX10 in tumors of the skin. SOX10 staining was seen in the majority of sweat gland tumors with the exception of syringoma and microcystic adnexal carcinoma. There were no immunoreactive cases among epidermal, neuroendocrine/neuroectodermal, follicular, or sebaceous tumors. These findings are comparable to reported in previous studies, and show SOX10 can be a useful adjunct in the differential diagnosis of nodular basaloid skin tumors. That marker has less utility in the assessment of sclerosing basaloid cutaneous neoplasms, because such tumors are almost uniformly nonreactive for it.

The Association for Human Pharmacology in the Pharmaceutical Industry London Meeting 2018: Brexit and Other Challenges in Early Phase Drug Development.

The Association for Human Pharmacology in the Pharmaceutical Industry (AHPPI) annual meeting focused on the changing face of early phase drug development and opened with a keynote speech concerning the revolution in pharmaceutical medicine over the last 30 years and the impact this has had on the way patients are treated. Examples were presented of how translational pharmaceutics is being used to tackle the high drug candidate failure rate and is improving productivity when moving drug candidates from the laboratory through to clinical proof of concept. The European Medicines Agency revised 2007 Risk Mitigation guideline on first in human (FIH) clinical trials was discussed. The focus of the revised guideline, which came into force in February 2018, is on risk mitigation and promotion of safety and will assist drug sponsors with the design and performance of early clinical studies. The use of integrated adaptive protocol designs in early clinical development was discussed in relation to the challenges involved when running early phase clinical trials in patients. The Health Regulatory Authority presented its strategies to ensure that following Brexit, the United Kingdom remains an attractive place to conduct Phase I clinical trials. The Medicines and Healthcare products Regulatory Agency confirmed that in the event of a "no deal" Brexit, it is well placed to implement and influence many provisions of the new EU CTR. The meeting provided an opportunity to discuss the changing regulatory environment and the opportunities and challenges facing the United Kingdom following Brexit with invited speakers from a range of disciplines including drug development, clinical trials and research organizations, government science policy and regulatory agencies.

Optimal plaster conformation derived using a custom-made jig to obtain maximum strength of protective plaster of Paris for hand surgery.

BACKGROUND: Surgical repair of soft tissue trauma in the upper limb frequently requires postoperative immobilization to prevent tension across repaired tendons, vessels, or nerves. A plaster of Paris backslab, placed on either the volar or the dorsal surface, is frequently used, as it is inexpensive and easy to apply. METHODS: In an effort to improve upon the strength of plaster of Paris backslabs, we designed a custom-made jig to subject angled backslabs (n = 5 per group) of various designs to a torsional force. We tested 10-layered backslabs with 0, 1, or 2 ridges and 15-layered backslabs without ridges. RESULTS: We demonstrate that the addition of longitudinal reinforcing ridges on the convex surface of backslabs can improve simple backslabs in terms of breaking strength, plaster stiffness, and failure load. The optimal design employs a single, centrally placed ridge, which provides the maximum support in the axis of the net force acting across the angle, the fulcrum of angle backslabs. The formation of two parallel longitudinal ridges does not offer any additional support. CONCLUSIONS: Backslabs can be strengthened significantly by placement of a single reinforcing ridge. This allows plaster weight and thickness to be kept to a minimum, reducing the setting time of the plaster and contributing to patient comfort. These findings demonstrate the ideal construction of angled plaster of Paris backslabs for the protection of soft tissue repairs.