Hybrid Sequencing Facilitates Robust De Novo Plasmid Assembly.

Despite the wide use of plasmids in research and clinical production, the verification of plasmid sequences is a bottleneck that is too often overlooked in the manufacturing process. Although sequencing platforms continue to improve, the method and assembly pipeline chosen still influence the final plasmid assembly sequence. Furthermore, few dedicated tools exist for plasmid assembly, especially for de novo assembly. Here, we evaluated short-read, long-read, and hybrid (both short and long reads) de novo assembly pipelines across three replicates of a 24-plasmid library. Consistent with previous characterizations of each sequencing technology, short-read assemblies had frequent issues resolving GC-rich regions, and long-read assemblies commonly had small insertions and deletions, especially in repetitive regions. The hybrid approach facilitated the most consistent assembly generation. Although Sanger sequencing can be used to verify specific regions, it requires a reference sequence to design primers, emphasizing the need for accurate de novo plasmid assembly tools. Some GC-rich and repetitive regions were difficult to resolve using any methods, suggesting that easily sequenced genetic parts should be prioritized in the design of new genetic constructs.

Ten simple rules for managing laboratory information.

Information is the cornerstone of research, from experimental (meta)data and computational processes to complex inventories of reagents and equipment. These 10 simple rules discuss best practices for leveraging laboratory information management systems to transform this large information load into useful scientific findings.

Evaluating the Quality-of-Life Effect of Apical Spacing with Hyaluronic Acid Prior to Hypofractionated Prostate Radiation Therapy: A Secondary Analysis.

PURPOSE: Recently, a randomized trial demonstrated that a hyaluronic acid (HA) spacer placed before prostate hypofractionated intensity modulated radiation therapy improved rectal dosimetry and reduced acute grade 2+ gastrointestinal toxicity. However, 26.5% of patients receiving the spacer experienced a minimal clinically important decline (MCID) in bowel quality-of-life (QOL). The purpose of this study is to evaluate whether certain characteristics of the rectal spacer, as determined on postimplant imaging, were associated with change in bowel QOL at 3-months. METHODS AND MATERIALS: This is a secondary analysis of the 136 patients who received the HA spacer on the randomized trial. Postimplant spacer characteristics (ie, prostate-rectum spacing at superior/midgland/inferior/apex planes, symmetry, prostate volume, spacer volume) were systematically analyzed from structure sets using custom software code. Characteristics demonstrating significant associations with rectal V30 on multivariate linear regression were identified. Linear regression models were used to analyze the associations of such characteristics with change (baseline to 3 months) in both bowel and urinary QOL. RESULTS: Apical spacing (mean 9.4 (standard deviation 4.0)) was significantly smaller than spacing measurements at more superior planes. 95.6% of patients had a symmetrical implant. Apical spacing (P < .001) and prostate volume (P = .01) were significantly associated with rectal V30 on multivariate analysis. However, only apical spacing (0.38/mm; P = .01) was associated with change in bowel QOL, even with adjustment of baseline bowel score (-0.33; P < .01). Percentages of patients with bowel MCID were 14.8% for >= 10 mm versus 36.6% for <10 mm apical spacing (P = .01). Apical spacing was not associated with change in urinary QOL (-0.09; P = .72), when adjusted for baseline urinary QOL (-0.52; P < .01). CONCLUSION: Greater apical spacing was associated with improved rectal dosimetry and smaller decline in bowel QOL at 3-months. Further prospective data are needed to fully understand the ramifications of increased apical spacing.

Mastectomy patterns among older women with early invasive breast cancer in England and Wales: A population-based cohort study.

INTRODUCTION: Older women with early invasive breast cancer (EIBC) are more likely to receive a mastectomy compared with younger women. This study assessed factors associated with receiving a mastectomy among older women with EIBC, with a particular focus on comorbidity and frailty. MATERIALS AND METHODS: Women diagnosed with EIBC (stages I-IIIa) aged ≥50 years from 2014 to 2019 in English and Welsh NHS organisations who received breast surgery were identified from cancer registration datasets linked to routine hospital data. Separate multivariable logistic regression models explored factors associated with mastectomy use, within each tumour stage (T1-T3). For each tumour stage, risk-adjusted rates of mastectomy were calculated for each NHS organisation and displayed using funnel plots. RESULTS: We included 106,952 women with EIBC: 23.4% received a mastectomy as their first breast cancer surgery. Receipt of mastectomy was more common among patients with a higher tumour stage (T1: 12.3%; T2: 37.6%; T3: 77.5%), and mastectomy use increased with age within each tumour stage category (50-59 vs 80 + years: 11.8% vs 26.3% for T1; 31.5% vs 56.9% for T2; 73.4% vs 90.3% for T3). Results from a multivariable regression model showed that more severe frailty was associated with mastectomy use for women with T1 (p = 0.002) or T2 (p = 0.003) tumours, but may not be for women with T3 tumours (p = 0.041). There was no association between comorbidity and mastectomy use after accounting for frailty (all p > 0.1). Adjusting for clinical and patient factors only slightly reduced the association between age and mastectomy use. Variation in mastectomy use between NHS organisations was greatest for women with T2 EIBC (unadjusted range: 17.7% to 68.4%). DISCUSSION: Older women with EIBC are more commonly treated with mastectomy. This could not be explained by tumour characteristics or physical fitness, raising questions about whether surgical decision-making inconsistently incorporates information on patient fitness and functional age.

Evidence into practice: a national cohort study of NICE-recommended oncological drug therapy utilisation among women diagnosed with invasive breast cancer in England.

BACKGROUND: Multiple drug treatments are approved for invasive breast cancer (IBC). We investigated uptake of NICE-recommended oncological drugs and variation by age, comorbidity burden and geographical region. METHODS: Women (aged 50+ years) diagnosed with IBC from 2014 to 2019, were identified from England Cancer Registry data and drug utilisation from Systemic Anti-Cancer Therapy data. Interrupted time series analysis assessed national-level changes in drug use after publication of NICE recommendations. Regression models analysed variation in use. RESULTS: This national cohort included 168,449 women. Use of drugs recommended for first-line treatment varied, from 26.6% for CDK 4/6 inhibitors to 63.8% for HER2-targeting therapies. Utilisation of drugs with a NICE recommendation published between 2014 and 2019, increased among patients diagnosed around the time of publication, except in the case of pertuzumab for metastatic breast cancer (MBC) which was previously accessible via the Cancer Drugs Fund (though use of pertuzumab for MBC increased from 34.1% to 75.0% across the study period). Use of trastuzumab and neoadjuvant/adjuvant pertuzumab varied by geographical region. Use was low for ribociclib (2.2%), abemaciclib (2.3%) and for drugs recommended beyond the first-line setting. For all drugs, use after NICE recommendation varied by age at diagnosis and increased as stage increased. CONCLUSIONS: Use of NICE-recommended drugs for IBC in routine care is variable, with lowest use among women aged 70+ years. Improving access to effective treatments is an important step in improving outcomes.

Completeness of endocrine therapy information in the Primary Care Prescription Database (PCPD) and secondary care treatment datasets: A national population-based cohort study using routine healthcare data.

BACKGROUND: Endocrine therapy (ET) is a widely used treatment for breast cancer. In the UK, use is typically initiated in secondary care, with subsequent treatment in primary care. Evaluating use of ET depends on data sources containing accurate and complete information. This study aimed to evaluate the completeness and consistency of ET recorded in primary and secondary care data (SCD) and determine the value of combining data sources in describing use of ET. METHODS: This cohort study included women (50 + years) diagnosed with hormone receptor-positive invasive breast cancer in England, April-2015 to December-2019. Concordance of ET recorded in SCD and the Primary Care Prescription Database (PCPD) was evaluated. Factors associated with recording of ET in each setting were assessed using statistical models. RESULTS: Overall 110,529 women were included. 94% had ET recorded in either SCD or PCPD. ET captured in SCD varied from 3% (in Systemic Anti-Cancer Therapy data) to 52% (in the Cancer Outcomes and Services Dataset; COSD). By contrast, 93% of patients had an ET prescription in PCPD. Among patients with ET recorded, this was not captured in COSD for 45%. Capture in COSD was lowest for younger women, those with no comorbidity/frailty, with lower stage or HER2-positive disease, or with other treatments recorded. Overall concordance between COSD and PCPD was 57%, but varied substantially across NHS trusts (lowest decile≤28%; highest decile≥86%). Among women with ET recorded in both settings, the earliest record was in COSD for 97%; 59% of initial ET prescriptions recorded in COSD were not captured in PCPD. Combining PCPD and COSD data enabled estimation of ET duration. CONCLUSIONS: PCPD is vital for understanding the use of ET within this population. Completeness of SCD could be improved by ensuring information on first ET prescription is recorded. PCPD (linked to SCD) is a valuable resource for examining patterns of care for patients with cancer, including treatment duration and adherence.

A compendium of Androgen Receptor Variant 7 target genes and their role in Castration Resistant Prostate Cancer.

Persistent androgen receptor (AR) signalling is the main driver of prostate cancer (PCa). Truncated isoforms of the AR called androgen receptor variants (AR-Vs) lacking the ligand binding domain often emerge during treatment resistance against AR pathway inhibitors such as Enzalutamide. This review discusses how AR-Vs drive a more aggressive form of PCa through the regulation of some of their target genes involved in oncogenic pathways, enabling disease progression. There is a pressing need for the development of a new generation of AR inhibitors which can repress the activity of both the full-length AR and AR-Vs, for which the knowledge of differentially expressed target genes will allow evaluation of inhibition efficacy. This review provides a detailed account of the most common variant, AR-V7, the AR-V7 regulated genes which have been experimentally validated, endeavours to understand their relevance in aggressive AR-V driven PCa and discusses the utility of the downstream protein products as potential drug targets for PCa treatment.

Concordance of cancer drug therapy information derived from routinely collected hospital admissions data and the Systemic Anti-Cancer Therapy (SACT) dataset, for older women diagnosed with early invasive breast cancer in England.

BACKGROUND: Evaluating uptake of oncological treatments, and subsequent outcomes, depends on data sources containing accurate and complete information about cancer drug therapy (CDT). This study aimed to evaluate the consistency of CDT information in the Hospital Episode Statistics Admitted Patient Care (HES-APC) and Systemic Anti-Cancer Therapy (SACT) datasets for early invasive breast cancer (EIBC). METHODS: The study included women (50 + years) diagnosed with EIBC in England from 2014 to 2019 who had surgery within six months of diagnosis. Concordance of CDT recorded in HES-APC (identified using OPCS codes) and SACT was evaluated at both patient-level and cycle-level. Factors associated with CDT use captured only in HES-APC were assessed using statistical models. RESULTS: The cohort contained 129,326 women with EIBC. Overall concordance between SACT and HES-APC on CDT use was 94 %. Concordance increased over the study period (91-96 %), and there was wide variation across NHS trusts (lowest decile of trusts had concordance≤77 %; highest decile≥99 %). Among women receiving CDT, 9 % (n = 2781/31693) of use was not captured in SACT; incompleteness was worst (18 %=47/259) among women aged 80 + and those diagnosed in 2014 (21%=1121/5401). OPCS codes in HES-APC were good at identifying patient-level and cycle-level use of trastuzumab or FEC chemotherapy (fluorouracil, epirubicin, cyclophosphamide), with 89 % and 93 % concordance with SACT respectively (patient-level agreement). Among cycles of solely oral CDT recorded in SACT, only 24 % were captured in HES-APC, compared to 71 % for intravenous/subcutaneous CDT. CONCLUSIONS: Combining information in HES-APC and SACT provides a more complete picture of CDT treatment in women aged 50 + receiving surgery for EIBC than using either data source alone. HES-APC may have particular value in identifying CDT use among older women, those diagnosed less recently, and in NHS trusts with low SACT data returns.

Hyaluronic Acid Spacer for Hypofractionated Prostate Radiation Therapy: A Randomized Clinical Trial.

Importance: Hypofractionated radiation therapy (RT) for prostate cancer has been associated with greater acute grade 2 gastrointestinal (GI) toxic effects compared with conventionally fractionated RT. Objective: To evaluate whether a hyaluronic acid rectal spacer could (1) improve rectal dosimetry and (2) affect acute grade 2 or higher GI toxic effects for hypofractionated RT. Design, Setting, and Participants: This randomized clinical trial was conducted from March 2020 to June 2021 among 12 centers within the US, Australia, and Spain, with a 6-month follow-up. Adult patients with biopsy-proven, T1 to T2 prostate cancer with a Gleason score 7 or less and prostate-specific antigen level of 20 ng/mL or less (to convert to µg/L, multiply by 1) were blinded to the treatment arms. Of the 260 consented patients, 201 patients (77.3%) were randomized (2:1) to the presence or absence of the spacer. Patients were stratified by intended 4-month androgen deprivation therapy use and erectile quality. Main Outcomes and Measures: For the primary outcome, we hypothesized that more than 70% of patients in the spacer group would achieve a 25% or greater reduction in the rectal volume receiving 54 Gy (V54). For the secondary outcome, we hypothesized that the spacer group would have noninferior acute (within 3 months) grade 2 or higher GI toxic effects compared with the control group, with a margin of 10%. Results: Of the 201 randomized patients, 8 (4.0%) were Asian, 26 (12.9%) Black, 42 (20.9%) Hispanic or Latino, and 153 (76.1%) White; the mean (SD) age for the spacer group was 68.6 (7.2) years and 68.4 (7.3) years for the control group. For the primary outcome, 131 of 133 (98.5%; 95% CI, 94.7%-99.8%) patients in the spacer group experienced a 25% or greater reduction in rectum V54, which was greater than the minimally acceptable 70% (P < .001). The mean (SD) reduction was 85.0% (20.9%). For the secondary outcome, 4 of 136 patients (2.9%) in the spacer group and 9 of 65 patients (13.8%) in the control group experienced acute grade 2 or higher GI toxic effects (difference, -10.9%; 95% 1-sided upper confidence limit, -3.5; P = .01). Conclusions and Relevance: The trial results suggest that rectal spacing with hyaluronic acid improved rectal dosimetry and reduced acute grade 2 or higher GI toxic effects. Rectal spacing should potentially be considered for minimizing the risk of acute grade 2 or higher toxic effects for hypofractionated RT. Trial Registration: ClinicalTrials.gov Identifier: NCT04189913.

Unravelling the Role of Kinases That Underpin Androgen Signalling in Prostate Cancer.

The androgen receptor (AR) signalling pathway is the key driver in most prostate cancers (PCa), and is underpinned by several kinases both upstream and downstream of the AR. Many popular therapies for PCa that target the AR directly, however, have been circumvented by AR mutation, such as androgen receptor variants. Some upstream kinases promote AR signalling, including those which phosphorylate the AR and others that are AR-regulated, and androgen regulated kinase that can also form feed-forward activation circuits to promotes AR function. All of these kinases represent potentially druggable targets for PCa. There has generally been a divide in reviews reporting on pathways upstream of the AR and those reporting on AR-regulated genes despite the overlap that constitutes the promotion of AR signalling and PCa progression. In this review, we aim to elucidate which kinases-both upstream and AR-regulated-may be therapeutic targets and require future investigation and ongoing trials in developing kinase inhibitors for PCa.

Sentinel lymph node biopsy alone compared to systematic lymphadenectomy in patients with uterine carcinosarcoma.

OBJECTIVE: To assess survival among patients diagnosed with uterine carcinosarcoma (CS) who underwent sentinel lymph node (SLN) biopsy alone vs. systematic lymph node dissection (LND). METHODS: We identified newly diagnosed CS patients who underwent primary surgical management from January 1996-December 2019. The SLN cohort underwent SLN biopsy alone with bilateral SLNs identified. The systematic LND cohort did not undergo SLN biopsy. RESULTS: Ninety-nine patients underwent SLN biopsy, and 100 patients underwent systematic LND. There was no difference by age, stage, body mass index, myoinvasion (<50%, ≥50%), lymphovascular space invasion, or positive washings. Eighty-five SLN (85.9%) and 15 LND (15%) underwent minimally invasive surgery (P < 0.001). The median total node count was four (range, 1-13) for SLN and 19 (range, 2-50) for LND (P < 0.001). Nodal metastasis occurred in 23 (23.2%) SLN and in 22 (22%) LND (P = 0.4). Postoperative therapy was administered to 85 (85.9%) SLN and 71 (71%) LND (P = 0.02). Median follow-up was 33 months (range, 1-205) for SLN and 55.3 months (range, 1-269) for LND (P = 0.001). The three-year progression-free survival (PFS) was 62.9% (SE 5.2%) for SLN and 52.3% (SE 5.3%) for LND (P = 0.13). The three-year overall survival (OS) was 72.1% (SE 5.1%) for SLN and 71.6% (SE 4.6%) for LND (P = 0.68). An isolated nodal recurrence occurred in two (2%) SLN and four (4%) LND (P = 0.26). CONCLUSIONS: There is no difference in PFS or OS among CS patients who undergo SLN biopsy vs. systematic LND. SLN biopsy detects nodal metastasis without compromising oncologic outcomes.

The association between guideline adherence, age and overall survival among women with non-metastatic breast cancer: A systematic review.

INTRODUCTION: Conformity with treatment guidelines should benefit patients. Studies have reported variation in adherence to breast cancer (BC) guidelines, particularly among older women. This study investigated (i) whether adherence to treatment guideline recommendations for women with non-metastatic BC improves overall survival (OS), (ii) whether that relationship varies by age. METHODOLOGY: MEDLINE and EMBASE were systematically searched for studies on guideline adherence and OS in women with non-metastatic BC, published after January 2000, which examined recommendations on breast surgery, chemotherapy, radiotherapy or endocrine therapy. Study results were summarised using narrative synthesis. RESULTS: Sixteen studies met the inclusion criteria. The recommendations for each treatment covered were similar, but studies differed in their definitions of adherence. 5-year OS rates among patients having compliant treatment ranged from 91.3% to 93.2%, while rates among patients having non-compliant treatment ranged from 75.9% to 83.4%. Six studies reported an adjusted hazard ratio (aHR) for non-compliant treatment compared with compliant treatment; all concluded OS was worse among patients whose overall treatment was non-compliant (aHR range: 1.52 [1.30-1.82] to 2.57 [1.96-3.37]), but adjustment for potential confounders was limited. Worse adherence among older women was reported in 12/16 studies, but they did not provide consistent evidence on whether OS was associated with treatment adherence and age. CONCLUSIONS: Individual studies reported that better adherence to guidelines improved OS among women with non-metastatic BC, but the evidence base has weaknesses including inconsistent definitions of adherence. More precise and consistent research designs, including the evaluation of barriers to adherence across the spectrum of healthcare practice, are required to fully understand guideline compliance, as well as the relationship between compliance and OS following a BC diagnosis.

Identification of intrinsic primary afferent neurons in mouse jejunum.

BACKGROUND: The gut is the only organ system with intrinsic neural reflexes. Intrinsic primary afferent neurons (IPANs) of the enteric nervous system initiate intrinsic reflexes, form gut-brain connections, and undergo considerable neuroplasticity to cause digestive diseases. They remain inaccessible to study in mice in the absence of a selective marker. Advillin is used as a marker for primary afferent neurons in dorsal root ganglia. The aim of this study was to test the hypothesis that advillin is expressed in IPANs of the mouse jejunum. METHODS: Advillin expression was assessed with immunohistochemistry and using transgenic mice expressing an inducible Cre recombinase under the advillin promoter were used to drive tdTomato and the genetically encoded calcium indicator GCaMP5. These mice were used to characterize the morphology and physiology of advillin-expressing enteric neurons using confocal microscopy, calcium imaging, and whole-cell patch-clamp electrophysiology. KEY RESULTS: Advillin is expressed in about 25% of myenteric neurons of the mouse jejunum, and these neurons demonstrate the requisite properties of IPANs. Functionally, they demonstrate calcium responses following mechanical stimuli of the mucosa and during antidromic action potentials. They have Dogiel type II morphology with neural processes that mostly remain within the myenteric plexus, but also project to the mucosa and express NeuN and calcitonin gene-related peptide (CGRP), but not nNOS. CONCLUSIONS AND INFERENCES: Advillin marks jejunal IPANs providing accessibility to this important neuronal population to study and model digestive disease.

Increasing hay inclusion in silage-based receiving diets and its effects on performance and energy utilization in newly weaned beef steers.

The influence of grass hay (GH) inclusion in replacement of corn silage in receiving diets on growth performance and dietary net energy (NE) utilization was evaluated in newly weaned beef steers (n = 162 Charolais-Red Angus cross steers; initial body weight [BW] = 278 ± 13.4 kg). Treatments were (DM basis): 1) 0% GH, 2) 10% GH, or 3) 20% GH inclusion in replacement of corn silage in receiving diets fed to newly weaned beef steers for 56 d. The study was conducted from October to December of 2019. Data were analyzed as randomized complete block design with pen serving as the experimental unit for all analyses. Increasing dietary inclusion of hay had no influence (P ≥ 0.11) on final BW, ADG, gain:feed or observed/expected dietary NEM and NEG, observed/expected dry matter intake (DMI), or observed/expected ADG. GH inclusion increased (linear effect, P = 0.01) DMI. Observed DMI for all treatments was approximately 15% to 17% less than anticipated based upon steer growth performance and tabular NE values. Evaluation of observed/expected ADG was 31% to 37% greater than expected for the steers in the present study. Particles less than 4 mm increased (linear effect, P = 0.01) and greater than 4 mm decreased (linear effect, P = 0.01) as GH replaced corn silage in the receiving diet. As the proportion of particles greater than 4 mm increased, cumulative ADG was decreased. These data indicate that GH should be considered in corn silage-based receiving diets to improve DMI. In high-risk calves, improved DMI could result in a lesser incidence of morbidity, although no morbidity was observed in any steers from the present study.

Correcting an instance of synthetic lethality with a pro-survival sequence.

A human cDNA encoding the LIM domain containing 194 amino acid cysteine and glycine rich protein 3 (CSRP3) was identified as a BAX suppressor in yeast and a pro-survival sequence that abrogated copper mediated regulated cell death (RCD). Yeast lacks a CSRP3 orthologue but it has four LIM sequences, namely RGA1, RGA2, LRG1 and PXL1. These are known regulators of stress responses yet their roles in RCD remain unknown. Given that LIMs interact with other LIMs, we ruled out the possibility that overexpressed yeast LIMs alone could prevent RCD and that CSRP3 functions by acting as a dominant regulator of yeast LIMs. Of interest was the discovery that even though yeast cells lacking the LIM encoding PXL1 had no overt growth defect, it was nevertheless supersensitive to the effects of sublethal levels of copper. Heterologous expression of human CSPR3 as well as the pro-survival 14-3-3 sequence corrected this copper supersensitivity. These results show that the pxl1∆-copper synthetic lethality is likely due to the induction of RCD. This differs from the prevailing model in which synthetic lethality occurs because of specific defects generated by the combined loss of two overlapping but non-essential functions.

Expectations of Hepato-Pancreato-Biliary Fellows; Do We Meet Them?

OBJECTIVE: There are 16 accredited hepatopancreatobiliary (HPB) fellowships in North America. The purpose of this study is to portray the expectations of the incoming HPB fellows about their training and its implication on their career. DESIGN: A 29-questions survey was sent out to all HPB fellows starting in August 2017. The survey was divided in 3 sections depicting background, in-training and postfellowship expectations. Descriptive statistics were generated for aggregate survey responses. SETTING: This study was performed through an online questionnaire that was sent to the participants via e-mail. The answers were processed in our offices in Methodist Richardson Medical Center, in Richardson, Texas which is a private tertiary medical center part of the Methodist Health System. PARTICIPANTS: Participants were all incoming HPB Fellows (In HPB fellowship programs accredited by the Fellowship Council) starting their fellowship in August 2017. RESULTS: We had a 94% response rate. Forty-six percent of fellows anticipate doing about 150 to 250 HPB cases during the fellowship, and all 15 fellows anticipate having at least 1 publication during fellowship. Despite that >90% of fellows believe that minimally invasive surgery (MIS) approaches will be more frequently utilized in HPB surgery, only 3/15 anticipate being able to apply MIS techniques and only 54% will be robotically trained. Interestingly the majority of fellows believe that the attending should be performing the case the first few months. CONCLUSION: The trainees believe that case volume is the most important factor for choosing a fellowship and for adequate training. Most of the fellows anticipate doing adequate number of cases but only the minority feels they will be adequately trained in MIS-robotic techniques.

Common elements in opioid use disorder guidelines for buprenorphine prescribing.

OBJECTIVES: This study sought to formulate a consolidation of guidelines representing best practices related to office-based opioid treatment (OBOT) of opioid use disorder (OUD) using buprenorphine. It also demonstrates how a set of evidence-based guidelines may be linked with claims data to leverage analytic techniques that drive cost-effective, positive health outcomes. STUDY DESIGN: Literature review of US and international guidelines for OBOT using buprenorphine for OUD. METHODS: The study conducted a review of currently available US and several international guidelines from 2009 to 2018 published on OUD and the use of buprenorphine in OBOT. Guidelines were consolidated based on common elements. The process of correlating common elements with available commercial and state Medicaid claims data is described, including which elements are amenable to analysis along with relative complexity. RESULTS: Seven guidelines met inclusion criteria and are presented as 3 tables, organized by clinical themes and phase of care related to OBOT use of buprenorphine for OUD. Themes included establishing care, monitoring treatment stability and engagement, and nonpharmacologic treatment to improve outcomes. Areas of agreement and divergence between guidelines are highlighted. Specific components are identified as they relate to metrics of interest to public and private payers. CONCLUSIONS: Among US and international guidelines for treatment of OUD, common themes are readily identified and may indicate agreement in regard to interventions. Linking pharmacy and medical billing claims data to evidence-supported best practices provides public and private payers the ability to track individual patients, facilitate high-quality care, and monitor outcomes.

Intracellular second messengers mediate stress inducible hormesis and Programmed Cell Death: A review.

Alterations in the levels of numerous second messengers are ubiquitous responses to all stresses that lead to apoptotic or hormetic responses. The sheer number and vast diversity of different second messenger systems activated in response to stresses belies a complexity that is often overlooked. This negligence is in large part due to the excessive focus on classical stress responsive second messenger mediators of stress especially Reactive Oxygen Species (ROS) but also others like calcium and ceramide. Here we review the many different intracellular second messengers that are involved in stress responses. We further integrate this information to emphasize that initial stress mediated responses consist of increased levels of a multitude of intracellular second messengers that serve to elicit the appropriate cell survival and/or cell death responses. We suggest that a greater focus on second messenger systems may shed more light on the processes that serve in the initiation of stress mediated PCD.

Stress is an agonist for the induction of programmed cell death: A review.

The prevailing models of stress induced Programmed Cell Death (PCD) posit that excess extracellular chemicals interact with or enter cells and disrupts cellular homeostasis. This activates signalling cascades involving the mitochondria, an increase in the steady state levels of Reactive Oxygen Species (ROS) as well as the activation of Bax and caspases. Further, the increased ROS also causes cellular damage that triggers or enhances PCD responses. The models have been modified in a number of ways, for example to include the existence of caspase and Bax independent forms of PCD. More recently, the ubiquity of ROS has also been challenged in part based on the failure of anti-oxidants to protect from diseases with increased intensity of oxidative stress. Here we focus on a number of other, often overlooked, observations regarding stress mediated responses that may further increase our mechanistic understanding of PCD. These include the concept of the "milieu intérieur" which suggests that cells actively protect themselves (adaptive homeostasis) in part by limiting entry to most extracellular chemicals. Of similar importance, stress also increases the levels of other stress inducible second messengers including ceramide, iron and calcium. This review focuses on the concept that stress is an agonist that conveys information that is transduced into the cell to activate the appropriate genetically encoded cell death and survival responses.

Change in Populations of Macrophages Promotes Development of Delayed Gastric Emptying in Mice.

BACKGROUND & AIMS: Muscularis propria macrophages lie close to cells that regulate gastrointestinal motor function, including interstitial cells of Cajal (ICC) and myenteric neurons. In animal models of diabetic gastroparesis, development of delayed gastric emptying has been associated with loss of macrophages that express cytoprotective markers and reduced networks of ICC. Mice with long-term diabetes and normal gastric emptying have macrophages that express anti-inflammatory markers and have normal gastric ICC. Mice homozygous for the osteopetrosis spontaneous mutation in the colony-stimulating factor 1 gene (Csf1op/op) do not have macrophages; when they are given streptozotocin to induce diabetes, they do not develop delayed gastric emptying. We investigated whether population of the gastric muscularis propria of diabetic Csf1op/op mice with macrophages is necessary to change gastric emptying, ICC, and myenteric neurons and investigated the macrophage-derived factors that determine whether diabetic mice do or do not develop delayed gastric emptying. METHODS: Wild-type and Csf1op/op mice were given streptozotocin to induce diabetes. Some Csf1op/op mice were given daily intraperitoneal injections of CSF1 for 7 weeks; gastric tissues were collected and cellular distributions were analyzed by immunohistochemistry. CD45+, CD11b+, F4/80+ macrophages were dissociated from gastric muscularis propria, isolated by flow cytometry and analyzed by quantitative real-time polymerase chain reaction. Cultured gastric muscularis propria from Csf1op/op mice was exposed to medium that was conditioned by culture with bone marrow-derived macrophages from wild-type mice. RESULTS: Gastric muscularis propria from Csf1op/op mice given CSF1 contained macrophages; 11 of 15 diabetic mice given CSF1 developed delayed gastric emptying and had damaged ICC. In non-diabetic Csf1op/op mice, administration of CSF1 reduced numbers of gastric myenteric neurons but did not affect the proportion of nitrergic neurons or ICC. In diabetic Csf1op/op mice given CSF1 that developed delayed gastric emptying, the proportion of nitrergic neurons was the same as in non-diabetic wild-type controls. Medium conditioned by macrophages previously exposed to oxidative injury caused damage to ICC in cultured gastric muscularis propria from Csf1op/op mice; neutralizing antibodies against IL6R or TNF prevented this damage to ICC. CD45+, CD11b+, and F4/80+ macrophages isolated from diabetic wild-type mice with delayed gastric emptying expressed higher levels of messenger RNAs encoding inflammatory markers (IL6 and inducible nitric oxide synthase) and lower levels of messenger RNAs encoding markers of anti-inflammatory cells (heme oxygenase 1, arginase 1, and FIZZ1) than macrophages isolated from diabetic mice with normal gastric emptying. CONCLUSIONS: In studies of Csf1op/op and wild-type mice with diabetes, we found delayed gastric emptying to be associated with increased production of inflammatory factors, and reduced production of anti-inflammatory factors, by macrophages, leading to loss of ICC.