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Abstract Background The Licensing Act 2003 (The Act) was implemented on the 24th November 2005 across England and Wales. The Act allowed more flexible and longer opening hours for licensed premises. We investigated the effect of The Act on alcohol related attendances to an inner city emergency department in Birmingham, UK. Methods We compared the proportion and time of alcohol related emergency department attendances in one week periods in January 2005 and 2006, before and after the implementation of The Licensing Act 2003. An alcohol related attendance was defined as any attendance where there was any documentation of the patient having consumed alcohol before presenting to the emergency department, if they appeared intoxicated on examination, or if alcohol attributed to their final diagnosis. Results The total weekly attendances increased slightly from 1,912 in 2005 to 2,146 in 2006. There was non-significant reduction in the proportion of alcohol related attendances between 2005 (3.6%) and 2006 (2.9%). A significantly greater proportion of attendances occurred at the weekend between 18.00 and 23.59 in 2005 (61.4%) than in 2006 (17.2%). There was a corresponding significant increase in the weekend proportion of attendances occurring between 03.00 to 05.59 in 2006. Conclusion Our findings show that there was a change in the pattern of alcohol related attendances to the emergency department around the time of implementation of the Licensing Act 2003, which has implications for delivery of emergency department services.

Background: Intravenous dihydroergotamine (DHE) has well-established efficacy for the acute treatment of migraine, but its use is limited by the need for in-hospital administration and the nausea/vomiting associated with a high maximum plasma concentration (C max ). Inhalation is an alternative to intravenous dosing. The surface area of the lung allows for rapid absorption of a self-administered dose., Objective: This study evaluated the safety, tolerability, and systemic pharmacokinetics (PK) of a dry powder formulation (PUR3100) DHE when delivered via inhalation compared to intravenous delivery., Methods: In this double-blind, double-dummy Phase 1 study, healthy volunteers (N = 26) were randomized (1:1:1:1) to one of four groups: orally inhaled placebo plus intravenous DHE 1.0 mg or orally inhaled PUR3100 (0.5, 1.0, or 1.5 mg) plus intravenous placebo. Blood samples were drawn pre-dose and at time points post-dose over 48 h. Standard PK and safety parameters were assessed and values for C max and area under plasma concentration time curve (AUC) were used to assess comparative exposures of PUR3100 versus intravenous DHE., Results: All doses of PUR3100 were associated with a lower incidence of nausea (21% vs. 86%), vomiting (0% vs. 29%), and headache (16% vs. 57%) compared to intravenous DHE. The PK profile of PUR3100 versus intravenous DHE was characterized by a similar mean time to C max (5 vs. 5.5 min), with reduced AUC 0-2h (1120-4320 vs. 6340), and a lower C max (3620-14,400 vs. 45,000). Compared to intravenous DHE 1.0 mg, the highest nominal PUR3100 dose (1.5 mg), which delivers a fine-particle dose of approximately 0.9 mg to the lungs, had a geometric mean ratio percentage (90% confidence interval [CI]) for C max of 32% [17.2, 59.6] and AUC 0-inf of 93% (62.9, 138.5), the latter of which was not significantly different., Conclusions: Inhaled PUR3100 is associated with rapid systemic PK within the therapeutic window and an improved safety profile relative to intravenous DHE., (© 2024 Pulmatrix, Inc. Headache: The Journal of Head and Face Pain published by Wiley Periodicals LLC on behalf of American Headache Society.)

Psychosocial assessment is a standard component of patient evaluations for transplant candidacy. The Stanford Integrated Psychosocial Assessment for Transplant (SIPAT) is a widely used measure to assess psychosocial risk for transplant. However, there are questions regarding the SIPAT's reliability and validity. We examined the SIPAT's psychometric performance and its impact on equitable access to transplant in a diverse cohort of 2825 patients seeking liver transplantation between 2014 and 2021 at an urban transplant center. The SIPAT demonstrated good internal consistency reliability at the overall score [Cronbach's α = 0.85, 95% CI (0.83, 0.86)] and domain levels (0.80 > α > 0.70). There was mixed support for structural validity, with poor overall model fit in confirmatory factor analysis and 50% of questions achieving the 0.70-factor loadings threshold. Adjusting for sociodemographic variables, the odds of not being waitlisted for psychosocial reasons were three times higher for patients with Medicaid insurance than patients with private insurance [OR 3.24, 95% CI (2.09, 4.99)] or Medicare [OR 2.89, 95% CI (1.84, 4.53)], mediated by higher SIPAT scores. Black patients had nearly twice the odds of White patients [OR 1.88, 95% CI (1.20, 2.91)], partially mediated by higher social support domain scores. Patients with Medicaid, non-White patients, and those without a college degree scored significantly higher on collinear questions, disproportionately contributing to higher SIPAT scores. The SIPAT did not perform equally across insurance type, race/ethnicity, and education groups, with the lowest subgroup validity associated with patient readiness and psychopathology domains. The SIPAT should be interpreted with caution, especially as a composite score. Future studies should examine validity in other populations., (Copyright © 2023 American Association for the Study of Liver Diseases.)

Leukaemia stem cells (LSCs) in acute myeloid leukaemia present a considerable treatment challenge due to their resistance to chemotherapy and immunosurveillance. The connection between these properties in LSCs remains poorly understood. Here we demonstrate that inhibition of tyrosine phosphatase SHP-1 in LSCs increases their glycolysis and oxidative phosphorylation, enhancing their sensitivity to chemotherapy and vulnerability to immunosurveillance. Mechanistically, SHP-1 inhibition leads to the upregulation of phosphofructokinase platelet (PFKP) through the AKT-β-catenin pathway. The increase in PFKP elevates energy metabolic activities and, as a consequence, enhances the sensitivity of LSCs to chemotherapeutic agents. Moreover, the upregulation of PFKP promotes MYC degradation and, consequently, reduces the immune evasion abilities of LSCs. Overall, our study demonstrates that targeting SHP-1 disrupts the metabolic balance in LSCs, thereby increasing their vulnerability to chemotherapy and immunosurveillance. This approach offers a promising strategy to overcome LSC resistance in acute myeloid leukaemia., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Previous research has argued that a growing multiracial population will blur boundaries between racial groups, reducing racism and improving interracial relations. However, this is unlikely to happen if multiracial groups are judged according to their proximity to Whiteness. We examined how having White ancestry shapes status perceptions of multiracial groups. Studies 1 and 2 showed that multiracial groups with White ancestry (e.g., Black/White) are considered higher status than dual minority multiracial (e.g., Black/Latinx) and monoracial minority (e.g., Black) groups. Study 3 revealed that multiracial groups with White ancestry are perceived as more competent and warmer than monoracial minority and dual minority multiracial groups, leading to higher status perceptions for multiracial groups with White ancestry. Thus, multiracial people, like other racial minorities, may be judged according to White, Eurocentric standards. The results imply that, without anti-racist intervention, the treatment of multiracial people will reinforce, rather than challenge, the existing racial hierarchy.

Trust is an important component of the doctor-patient relationship and is associated with improved patient satisfaction and health outcomes. Previously, we reported that patient feelings of trust and similarity toward their clinician predicted reductions in evoked pain in response to painful heat stimulations. In the present study, we investigated the brain mechanisms underlying this effect. We used face stimuli previously developed using a data-driven computational modeling approach that differ in perceived trustworthiness and superimposed them on bodies dressed in doctors' attire. During functional magnetic resonance imaging, participants (n = 42) underwent a series of virtual medical interactions with these doctors during which they received painful heat stimulation as an analogue of a painful diagnostic procedure. Participants reported increased pain when receiving painful heat stimulations from low-trust doctors, which was accompanied by increased activity in pain-related brain regions and a multivariate pain-predictive neuromarker. Findings suggest that patient trust in their doctor may have tangible impacts on pain and point to a potential brain basis for trust-related reductions in pain through the modulation of brain circuitry associated with the sensory-discriminative and affective-motivational dimensions of pain., (Published by Oxford University Press 2022.)

We present a novel "on-off", cost-effective, rapid electrochemical aptasensor combined with a microfluidics cartridge system for the detection of Δ 9 -THC (Δ 9 -tetrahydrocannabinol) in human saliva via differential pulse voltammetry. The assay relied on the competitive binding between the Δ 9 -THC and a soluble redox indicator methylene blue, using an aptamer selected via FRELEX. We found that the aptasensor can detected 1 nM of Δ 9 -THC in PBS in a three-electrode cell system, while the sensitivity and both the dissociation constant (K d ) and association constant (K b ) were dependent on the aptamer density. The aptamer also showed great affinity towards Δ 9 -THC when tested against cannabinol and cannabidiol. The same limit of detection of 1 nM in PBS was achieved in small volume samples (∼60 μL) using the aptamer-modified gold screen-printed electrodes combined with the microfluidic cartridge setup, however, the presence of 10% raw human saliva had a negative effect which manifested in a 10-fold increase in the LOD due to interfering elements. Filtering the saliva, improved the tested volume to 50% and the LOD to 5 nM of Δ 9 -THC which is lower than the concentrations associated with impairment (6.5-32 nM). The aptasensor showed a good storage capability up to 3 days, however, the reusability significantly dropped from 10 cycles (freshly prepared) to 5 cycles. The results clearly demonstrate the feasibility of the aptasensor platform with the microfluidics chamber towards a point-of-care testing application for the detection of Δ 9 -THC in saliva., 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 © 2022 Elsevier B.V. All rights reserved.)

Hematopoietic stem cells (HSCs) are the source for all blood cells, including immune cells, and they interact dynamically with the immune system. This chapter will explore the nature of stem cells, particularly HSCs, in the context of their immune microenvironment. The dynamic interactions between stem cells and the immune system can have profound implications for current and future therapies, particularly regarding a potential "immune-privileged" HSC microenvironment. Immune/stem cell interactions change during times of stress and injury. Recent advances in cancer immunotherapy have overturned the long-standing belief that, being derived from the self, cancer cells should be immunotolerant. Instead, an immunosurveillance system recognizes and eliminates emergent pre-cancerous cells. Only in the context of a failing immunosurveillance system does cancer fully develop. Combined with the knowledge that stem cells or their unique properties can be critically important for cancer initiation, persistence, and resistance to therapy, understanding the unique immune properties of stem cells will be critical for the development of future cancer therapies. Accordingly, the therapeutic implications for leukemic stem cells (LSCs) inheriting an immune-privileged state from HSCs will be discussed. Through their dynamic interactions with a diverse immune system, stem cells serve as the light and dark root of cancer prevention vs. development., (© 2023. The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd.)

T cell acute lymphoblastic leukaemia (T-ALL) is an aggressive malignancy with poor prognosis, but a decisive marker and effective treatment for leukaemia stem cells (LSCs) remain unclear. Here, using lineage tracing, limiting dilution assays and in vivo live imaging approaches, we identify rare inhibitory receptor programmed cell death 1 (PD-1)-expressing cells that reside at the apex of leukaemia hierarchy for initiation and relapse in T-ALL. Ablation of PD-1-expressing cells, deletion of PD-1 in T-ALL cells or blockade of PD-1 or PD-1 ligand 1 significantly eradicated LSCs and suppressed disease progression. Combination therapy using PD-1 blockade and chemotherapy substantially extended the survival of mice engrafted with mouse or human T-ALL cells. Mechanistically, PD-1 + LSCs had high NOTCH1-MYC activity for disease initiation. Furthermore, PD-1 signalling maintained quiescence and protected LSCs against T cell receptor-signal-induced apoptosis. Overall, our data highlight the hierarchy of leukaemia by identifying PD-1 + LSCs and provide a therapeutic approach for the elimination of LSCs through PD-1 blockade in T-ALL., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Digital microfluidics (DMF) has the signatures of an ideal liquid handling platform - as shown through almost two decades of automated biological and chemical assays. However, in the current state of DMF, we are still limited by the number of parallel biological or chemical assays that can be performed on DMF. Here, we report a new approach that leverages design-of-experiment and numerical methodologies to accelerate experimental optimization on DMF. The integration of the one-factor-at-a-time (OFAT) experimental technique with machine learning algorithms provides a set of recommended optimal conditions without the need to perform a large set of experiments. We applied our approach towards optimizing the radiochemistry synthesis yield given the large number of variables that affect the yield. We believe that this work is the first to combine such techniques which can be readily applied to any other assays that contain many parameters and levels on DMF.

Genome-wide mapping of transcription factors (TFs) is critical to understand their functions. In chromatin immunoprecipitation (ChIP)-seq assay, it's challenging to study recruitment of low-abundant TFs transiently boud to the genome. Here, we present an optimized protocol using ChIP Next-Gen Seq Sepharose (Staph-seq) to efficiently pull down chromatin complexes. The double size selection promotes sensitive capture of genome-wide protein-DNA associations while eliminating potential Staph A contamination, which is a common problem in protocols using Staph A cells. For complete details on the use and execution of this protocol, please refer to Tao et al. (2020). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Biogenic amines (BAs) are toxicological risks present in many food products. Putrescine is the most common foodborne BA and is frequently used as a quality control marker. Currently, there is a lack of regulation concerning safe putrescine limits in food as well as outdated food handling practices leading to unnecessary putrescine intake. Conventional methods used to evaluate BAs in food are generally time-consuming and resource-heavy with few options for on-site analysis. In response to this challenge, we have developed a transcription factor-based biosensor for the quantification of putrescine in beef samples. In this work, we use a naturally occurring putrescine responsive repressor-operator pair (PuuR- puuO ) native to Escherichia coli . Moreover, we demonstrate the use of the cell-free putrescine biosensor on a paper-based device that enables rapid low-cost detection of putrescine in beef samples stored at different temperatures. The results presented demonstrate the potential role of using paper-based biosensors for on-site testing, particularly as an index for determining meat product stability and quality.

Hematopoietic stem cells (HSCs) have reduced capacities to properly maintain and replenish the hematopoietic system during myelosuppressive injury or aging. Expanding and rejuvenating HSCs for therapeutic purposes has been a long-sought goal with limited progress. Here, we show that the enzyme Sphk2 (sphingosine kinase 2), which generates the lipid metabolite sphingosine-1-phosphate, is highly expressed in HSCs. The deletion of Sphk2 markedly promotes self-renewal and increases the regenerative potential of HSCs. More importantly, Sphk2 deletion globally preserves the young HSC gene expression pattern, improves the function, and sustains the multilineage potential of HSCs during aging. Mechanistically, Sphk2 interacts with prolyl hydroxylase 2 and the Von Hippel-Lindau protein to facilitate HIF1α ubiquitination in the nucleus independent of the Sphk2 catalytic activity. Deletion of Sphk2 increases hypoxic responses by stabilizing the HIF1α protein to upregulate PDK3, a glycolysis checkpoint protein for HSC quiescence, which subsequently enhances the function of HSCs by improving their metabolic fitness; specifically, it enhances anaerobic glycolysis but suppresses mitochondrial oxidative phosphorylation and generation of reactive oxygen species. Overall, targeting Sphk2 to enhance the metabolic fitness of HSCs is a promising strategy to expand and rejuvenate functional HSCs., (© 2022 by The American Society of Hematology.)

The objective of this study was to identify factors that predict for sperm granuloma formation and the impact of sperm granuloma presence and quantity on vasectomy reversal (VR) outcomes. A cross sectional retrospective review of prospectively collected data, on the impact of granuloma on VR outcomes from a single academic center was performed. The impact of age, obstructive interval, intraoperative vasal fluid findings, anastomosis type, body mass index, tobacco use and total motile count (TMC) was determined. A total of 1550 men underwent VR between January 2000 and August 2019. Granulomas were present unilaterally in 23.3% (n = 361) and bilaterally in 14.2% (n = 220). On univariate analysis, increasing patient age negatively correlated with a larger number of granulomas (p = .011). Granuloma presence was associated with finding intact and motile sperm from the vasal stump intraoperatively (p = .001), and vasoepididymostomy anastomosis (p < .001). However, granuloma presence (and quantity) did not correlate with obstructive interval or maximum TMC. Tobacco use and body mass index (BMI) were not associated with granuloma presence. On multivariate analysis, granuloma quantity was not associated with TMC. Obstructive interval and vasovasostomy anastomosis were associated with higher TMC, while BMI was negatively associated with TMC. In conclusion, increasing age was negatively correlated with granuloma formation. Granuloma presence was associated with more favourable intraoperative fluid findings and anastomosis type, but not post-VR TMC, suggesting men with and without granulomas undergoing skilled microsurgery will have similar patency rates. Heavier men should be encouraged for weight loss prior to vasectomy reversal as increasing BMI was associated with lower TMC., (© 2022 The Authors. Andrologia published by Wiley-VCH GmbH.)

The surfactant market represents a key sector of the chemical industry and encompasses many diverse applications. Their sustainability in terms of feedstock used, synthetic procedure, biodegradability, and formulation are crucial parameters to assessing the environmental impact of the surfactant. The anionic surfactant linear alkyl benzene sulfonates have proven successful to date because of their high performance, low cost, and extensive studies within formulations to optimize performance, allowing usage in a large variety of applications, especially in cleaning. Due to their advantageous properties and extensive research and development, their substitution with a biobased surfactant such as sodium dodecyl sulfate has struggled to succeed. Furan surfactants have been reported as valuable candidates for the implementation of green alternatives to traditional anionic sulfonated surfactants with a perfect trade-off between performances and green credentials. However, their implementation suffers of scalability and high cost in producing the final product due to feedstock availability and low yields of the final product. Herein, we report a new class of furan surfactants, sulfonated alkyl furoates, which are derived from the esterification of furoic acid and fatty alcohols, followed by a sulfonation step. Compared to traditional surfactants, they showed more favorable behavior in basic proprieties (such as critical micelle concentration, ecotoxicity, hard water resistance, surface tension water/oil), which gives a good prospective for the introduction of a new biobased chemical with superior performances., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Radiation-induced loss of the hematopoietic stem cell progenitor population compromises bone marrow regeneration and development of mature blood cells. Failure to rescue bone marrow functions results in fatal consequences from hematopoietic injury, systemic infections, and sepsis. So far, bone marrow transplant is the only effective option, which partially minimizes radiation-induced hematopoietic toxicities. However, a bone marrow transplant will require HLA matching, which will not be feasible in large casualty settings such as a nuclear accident or an act of terrorism. In this study we demonstrated that human peripheral blood mononuclear cell-derived myeloid committed progenitor cells can mitigate radiation-induced bone marrow toxicity and improve survival in mice. These cells can rescue the recipient's hematopoietic stem cells from radiation toxicity even when administered up to 24 h after radiation exposure and can be subjected to allogenic transplant without GVHD development. Transplanted cells deliver sEVs enriched with regenerative and immune-modulatory paracrine signals to mitigate radiation-induced hematopoietic toxicity. This provides a natural polypharmacy solution against a complex injury process. In summary, myeloid committed progenitor cells can be prepared from blood cells as an off-the-shelf alternative to invasive bone marrow harvesting and can be administered in an allogenic setting to mitigate hematopoietic acute radiation syndrome.

The end of the last decade saw record numbers of refugee arrivals to Italy, straining the existing reception and integration systems. Although significant research attention has focused on the prevalence of posttraumatic stress disorder (PTSD), depression, and suicidal ideation in refugee populations, there are increasing calls to study the interactions between distress, resilience, and positive outcomes of adversity. To fully understand the complexity of the refugee experience, these voices must be heard in dialogue. Studies must be framed within a more nuanced view of refugees, characterizing them not exclusively as victims but also as advocates for their own well-being and the well-being of their communities. This exploratory study performed a thematic analysis of 15 interviews conducted with refugee community leaders in Italy, each a founder or leader of a community association and actively engaged in civil society. In keeping with Papadopoulos' (2007) construct of adversity-activated development, an inductive analysis identified three distinct patterns of positive adversity response: meaning, motivation, and mobilization. A deductive analysis identified the most appropriate theories of resilience to describe the sample, which included resilience as a dynamic positive adaptation and resilience as resistance to change in moral codes and personal value systems. The resulting image of refugee community leaders in Italy is that of individuals who are exposed to adversity and experiences of suffering and distress but safeguard their core values while positively adapting to a new country., (© 2021 International Society for Traumatic Stress Studies.)

Background: Enterobacter spp. are opportunistic pathogens that cause nosocomial infections. Bacteriophages could be used to treat antibiotic-resistant Enterobacter infections. Materials and Methods: We used 10 genetically diverse clinical Enterobacter spp. isolates to identify lytic bacteriophages in hospital and municipal wastewater. Comparative genomics was performed on host bacterial isolates and isolated phages. Activity of each phage against all 10 host isolates was determined. We also tested phage activity against paired isolates from two patients who developed ceftazidime-avibactam resistance. Results: Bacteria belonged to three Enterobacter species and Klebsiella aerogenes. We isolated 12 bacteriophages, most of which belonged to the Myoviridae and Autographiviridae families. Most phages were able to lyse multiple bacterial isolates, and many lysed isolates of different species. Ceftazidime-avibactam-resistant isolates were still phage susceptible, and one isolate showed increased susceptibility compared with the parent isolate. Conclusion: The phages we isolated expand the diversity of Enterobacter- targeting phages, and could be useful for treating antibiotic-resistant Enterobacter infections., Competing Interests: No competing financial interests exist., (© Amanda G. Finney et al., 2022; Published by Mary Ann Liebert, Inc.)

Background: Economic, social, and psychologic stressors are associated with an increased risk for abusive injuries in children. Prolonged physical proximity between adults and children under conditions of severe external stress, such as witnessed during the COVID-19 pandemic with "shelter-in-place orders", may be associated with additional increased risk for child physical abuse. We hypothesized that child physical abuse rates and associated severity of injury would increase during the early months of the pandemic as compared to the prior benchmark period., Methods: We conducted a nine-center retrospective review of suspected child physical abuse admissions across the Western Pediatric Surgery Research Consortium. Cases were identified for the period of April 1-June 30, 2020 (COVID-19) and compared to the identical period in 2019. We collected patient demographics, injury characteristics, and outcome data., Results: There were no significant differences in child physical abuse cases between the time periods in the consortium as a whole or at individual hospitals. There were no differences between the study periods with regard to patient characteristics, injury types or severity, resource utilization, disposition, or mortality., Conclusions: Apparent rates of new injuries related to child physical abuse did not increase early in the COVID-19 pandemic. While this may suggest that pediatric physical abuse was not impacted by pandemic restrictions and stresses, it is possible that under-reporting, under-detection, or delays in presentation of abusive injuries increased during the pandemic. Long-term follow-up of subsequent rates and severity of child abuse is needed to assess for unrecognized injuries that may have occurred., Competing Interests: Declaration of Competing Interest There are no conflicts of interest., (Copyright © 2021. Published by Elsevier Inc.)

Objective: To determine the impact of health care system access on outcomes for Hispanic and Non-Hispanic White patients with renal cell carcinoma (RCC)., Methods: We retrospectively analyzed Hispanic and non-Hispanic White patients diagnosed with localized RCC between 2007 and 2020. We used Health Resources and Services Administration criteria to identify patients living in Medically Underserved Areas (MUA). Primary outcome all-cause mortality and cancer-specific survival using Log Rank test on Kaplan Meier Analysis. Secondary outcome was all-cause mortality and cancer specific survival on Cox Regression when adjusting for risk factors., Results: We analyzed 774 patients, 246 (31.8%) Hispanic patients and 528 (68.2%) Non-Hispanic White patients. Hispanic ethnicity was associated with lower risk of ACM (HR 0.53, P = 0.019) and there was no difference for cancer specific survival (HR 0.57, P = 0.059). Living in a MUA was associated with worse all-cause mortality (P = 0.010) but not cancer specific survival (CSS) (P = 0.169). Comparing Hispanic and Non-Hispanic Whites, KMA revealed no difference in 5-year all-cause mortality (83.1% vs. 78.8%, P = 0.254) and 5-year CSS (85.7% vs. 85.4%, P = 0.403)., Conclusions: Hispanics had lower all-cause mortality risk and no significant differences in 5-year overall survival and CSS compared to non-Hispanic Whites. Our findings indicate that tertiary referral centers may help mitigate inequalities in access to care., (Copyright © 2021. Published by Elsevier Inc.)

DNA assembly and transformation are crucial to the building process in synthetic biology. These steps are significant roadblocks when engineering increasingly complex biological systems. To address this, recent development of widespread 'biofoundry' facilities has employed automation equipment to expedite the synthetic biology workflow. Despite significant progress, there is a clear demand for lower-cost and smaller-footprint automation equipment. The field of microfluidics have emerged to provide automation capabilities to meet this demand. However, we still lack devices capable of building large multi-gene systems in a consolidated process. In response to this challenge, we have developed a digital microfluidic platform that performs "one-pot" Golden Gate DNA assembly of large plasmids and transformation of E coli . The system features a novel electrode geometry and modular design, which make these devices simple to fabricate and use, thus improving the accessibility of microfluidics. This device incorporates an impedance-based adaptive closed loop water replenishment system to compensate for droplet evaporation and maintain constant assembly reaction concentrations, which we found to be crucial to the DNA assembly efficiency. We also showcase a closed-loop temperature control system that generates precise thermodynamic profiles to optimize heat shock transformation. Moreover, we validated the system by assembling and transforming large and complex plasmids conferring a biosynthetic pathway, resulting in performance comparable to those of standard techniques. We propose that the methods described here will contribute to a new generation of accessible automation platforms aimed at speeding up the 'building' process, lowering reagent consumption and removing manual work from synthetic biology.

The central tenet of Place Attachment theory states that an individual has an inborn predisposition to form strong bonds with places as well as with people. Our qualitative study applies this theory to understand how, despite loss and adversity, refugees are able to reconstruct a sense of identity, community, and "home". Participants included 15 forcibly displaced people from different countries of origin. Semistructured interviews explored factors that facilitate participants' integration in a new context and the impact of this context on their sense of identity. Data were analysed using Consensual Qualitative Research Methodology to identify recurrent themes and their frequencies within interview transcripts. Within the relational dimensions of place attachment , affiliation , and seeking help from others , the study explores the factors that facilitate the integration of refugees in a new context and the impact of this context on their sense of identity, identifying recurrent themes and their frequencies within interview transcripts. The most frequent resulting themes were (a) a sense of identity and (b) expectations toward the resettlement country. Additional, though less frequent, themes included: (c) sense of belonging, (d) community integration, (e) trust, (f) opportunity seizing, (g) being a point of reference for others, (h) sense of community, (i) positive memories, (j) refusal. These results begin to describe the ways by which Place Attachment, toward both birth and resettlement countries, contributes to a restructured identity and sense of "feeling at home" for refugees.

Optimization of engineered biological systems requires precise control over the rates and timing of gene expression. Optogenetics is used to dynamically control gene expression as an alternative to conventional chemical-based methods since it provides a more convenient interface between digital control software and microbial culture. Here, we describe the construction of a real-time optogenetics platform, which performs closed-loop control over the CcaR-CcaS two-plasmid system in Escherichia coli . We showed the first model-based design approach by constructing a nonlinear representation of the CcaR-CcaS system, tuned the model through open-loop experimentation to capture the experimental behavior, and applied the model in silico to inform the necessary changes to build a closed-loop optogenetic control system. Our system periodically induces and represses the CcaR-CcaS system while recording optical density and fluorescence using image processing techniques. We highlight the facile nature of constructing our system and how our model-based design approach will potentially be used to model other systems requiring closed-loop optogenetic control.

Introduction: The Veterans Health Administration (VHA) has taken a multifaceted approach to addressing opioid safety and promoting system-wide opioid stewardship., Aim: To provide a comprehensive evaluation of current opioid prescribing practices and implementation of risk mitigation strategies in VHA., Setting: VHA is the largest integrated health care system in the United States., Program Description: VHA prescribing data in conjunction with implementation of opioid risk mitigation strategies are routinely tracked and reviewed by VHA's Pharmacy Benefits Management Services (including Academic Detailing Service) and the Pain Management Program Office. Additional data are derived from the Partnered Evidence-Based Policy Resource Center (PEPReC) and from a 2019 survey of interdisciplinary pain management teams at VHA facilities. Prescribing data are reported quarterly until first quarter fiscal year 2020 (Q1FY2020), ending December 31, 2019., Program Evaluation: VHA opioid dispensing peaked in 2012 with 679,376 Veterans receiving an opioid prescription, and when including tramadol, in 2013 with 869,956 Veterans. Since 2012, the number of Veterans dispensed an opioid decreased 56% and co-prescribed opioid/benzodiazepine decreased 83%. Veterans with high-dose opioids (≥ 100 mg morphine equivalent daily dose) decreased 77%. In Q1FY2020, among Veterans on long-term opioid therapy (LTOT), 91.1% had written informed consent, 90.8% had a urine drug screen, and 89.0% had a prescription drug monitoring program query. Naloxone was issued to 217,469 Veterans and resulted in > 1,000 documented overdose reversals. In 2019, interdisciplinary pain management teams were fully designated at 68%, partially designated at 28%, and not available at 4% of 140 VA parent facilities. Fifty percent of Veterans on opioids at very high risk for overdose/suicide received interdisciplinary team reviews., Implications: VHA clinicians have greatly reduced their volume of opioid prescribing for pain management and expanded implementation of opioid risk mitigation strategies., Impacts: VHA's integrated health care system provides a model for opioid stewardship and interdisciplinary pain care.

Objective: Racial and ethnic minorities in the United States report higher levels of both clinical and experimental pain, yet frequently receive inadequate pain treatment. Although these disparities are well documented, their underlying causes remain largely unknown. Evidence from social psychological and health disparities research suggests that clinician-patient racial/ethnic concordance may improve minority patient health outcomes. Yet whether clinician-patient racial/ethnic concordance influences pain remains poorly understood., Methods: Medical trainees and community members/undergraduates played the role of "clinicians" and "patients," respectively, in simulated clinical interactions. All participants identified as non-Hispanic Black/African American, Hispanic white, or non-Hispanic white. Interactions were randomized to be either racially/ethnically concordant or discordant in a 3 (clinician race/ethnicity) × 2 (clinician-patient racial/ethnic concordance) factorial design. Clinicians took the medical history and vital signs of the patient and administered an analogue of a painful medical procedure., Results: As predicted, clinician-patient racial/ethnic concordance reduced self-reported and physiological indicators of pain for non-Hispanic Black/African American patients and did not influence pain for non-Hispanic white patients. Contrary to our prediction, concordance was associated with increased pain report in Hispanic white patients. Finally, the influence of concordance on pain-induced physiological arousal was largest for patients who reported prior experience with or current worry about racial/ethnic discrimination., Conclusions: Our findings inform our understanding of the sociocultural factors that influence pain within medical contexts and suggest that increasing minority, particularly non-Hispanic Black/African American, physician numbers may help reduce persistent racial/ethnic pain disparities., (© The Author(s) 2020. Published by Oxford University Press on behalf of the American Academy of Pain Medicine. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Mouse embryonic stem cells (ESCs) cultured in defined medium resemble the pre-implantation epiblast in the ground state, with full developmental capacity including the germline. β-Catenin is required to maintain ground state pluripotency in mouse ESCs, but its exact role is controversial. Here, we reveal a Tcf3-independent role of β-catenin in restraining germline and somatic lineage differentiation genes. We show that β-catenin binds target genes with E2F6 and forms a complex with E2F6 and HMGA2 or E2F6 and HP1γ. Our data indicate that these complexes help β-catenin restrain and fine-tune germ cell and neural developmental potential. Overall, our data reveal a previously unappreciated role of β-catenin in preserving lineage differentiation integrity in ground state ESCs., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Cancer stem cells (CSCs) underlie resistance to therapy. Cancer develops only in the context of failing immunosurveillance, and stem cells occupy immune privileged microenvironments. Recent evidence demonstrates that CSCs borrow immune privilege from their normal counterparts. However, low doses of doxorubicin can target CSCs by restoring anticancer immunity., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Leukaemia stem cells (LSCs) underlie cancer therapy resistance but targeting these cells remains difficult. The Wnt-β-catenin and PI3K-Akt pathways cooperate to promote tumorigenesis and resistance to therapy. In a mouse model in which both pathways are activated in stem and progenitor cells, LSCs expanded under chemotherapy-induced stress. Since Akt can activate β-catenin, inhibiting this interaction might target therapy-resistant LSCs. High-throughput screening identified doxorubicin (DXR) as an inhibitor of the Akt-β-catenin interaction at low doses. Here we repurposed DXR as a targeted inhibitor rather than a broadly cytotoxic chemotherapy. Targeted DXR reduced Akt-activated β-catenin levels in chemoresistant LSCs and reduced LSC tumorigenic activity. Mechanistically, β-catenin binds multiple immune-checkpoint gene loci, and targeted DXR treatment inhibited expression of multiple immune checkpoints specifically in LSCs, including PD-L1, TIM3 and CD24. Overall, LSCs exhibit distinct properties of immune resistance that are reduced by inhibiting Akt-activated β-catenin. These findings suggest a strategy for overcoming cancer therapy resistance and immune escape.

Digital microfluidics (DMF) represents an alternative to the conventional microfluidic paradigm of transporting fluids in enclosed channels. One of the major benefits of DMF is that fluid motion and control is achieved without external pumps. The automation component of DMF have pushed the barriers of this "lab-on-chip" technology. However, integration with external components (i.e., "world-to-chip") interfaces have been a challenge. Two common "world-to-chip" challenges are (1) delivering biological samples to DMF devices and (2) accurately controlling temperatures on device. To address these challenges, this work describes two "world-to-chip" interface features that have been integrated on a DMF platform: a reagent delivery system and a thermal control apparatus. This platform enables a variety of biological or chemical experiments to be conducted on-chip while reducing manual intervention. Specifically, our platform increases reagent volumes available to device reservoirs volume by at least 50-fold eliminating the need to manually refill reservoirs while improving droplet dispensing reproducibility. In addition, we have integrated a closed-loop temperature control system that offers precise temperature control on-chip. To validate our "world-to-chip" interface, we have automated bacterial transformation and enzymatic assay protocols, showing that such a system enhances DMF performance. Overall, we propose that this system will improve biological experimentation which requires fluidic and temperature control integrated on DMF platforms.

Regulation of hematopoietic stem cells (HSCs) by bone marrow (BM) niches has been extensively studied; however, whether and how HSC subpopulations are distinctively regulated by BM niches remain unclear. Here, we functionally distinguished reserve HSCs (rHSCs) from primed HSCs (pHSCs) based on their response to chemotherapy and examined how they are dichotomously regulated by BM niches. Both pHSCs and rHSCs supported long-term hematopoiesis in homeostasis; however, pHSCs were sensitive but rHSCs were resistant to chemotherapy. Surviving rHSCs restored the HSC pool and supported hematopoietic regeneration after chemotherapy. The rHSCs were preferentially maintained in the endosteal region that enriches N-cadherin + (N-cad + ) bone-lining cells in homeostasis and post-chemotherapy. N-cad + cells were functional bone and marrow stromal progenitor cells (BMSPCs), giving rise to osteoblasts, adipocytes, and chondrocytes in vitro and in vivo. Finally, ablation of N-cad + niche cells or deletion of SCF from N-cad + niche cells impaired rHSC maintenance during homeostasis and regeneration., (Copyright © 2018 Stowers Institute for Medical Research. Published by Elsevier Inc. All rights reserved.)

In the initial published version of this article, there was an inadvertent omission from the Acknowledgements that this work was supported by Stowers Institute for Medical Research (SIMR-1004) and NIH National Cancer Institute grant to University of Kansas Cancer Center (P30 CA168524). This omission does not affect the description of the results or the conclusions of this work.

Transplantation of hematopoietic stem cells (HSCs) from human umbilical cord blood (hUCB) holds great promise for treating a broad spectrum of hematological disorders including cancer. However, the limited number of HSCs in a single hUCB unit restricts its widespread use. Although extensive efforts have led to multiple methods for ex vivo expansion of human HSCs by targeting single molecules or pathways, it remains unknown whether it is possible to simultaneously manipulate the large number of targets essential for stem cell self-renewal. Recent studies indicate that N 6 -methyladenosine (m 6 A) modulates the expression of a group of mRNAs critical for stem cell-fate determination by influencing their stability. Among several m 6 A readers, YTHDF2 is recognized as promoting targeted mRNA decay. However, the physiological functions of YTHDF2 in adult stem cells are unknown. Here we show that following the conditional knockout (KO) of mouse Ythdf2 the numbers of functional HSC were increased without skewing lineage differentiation or leading to hematopoietic malignancies. Furthermore, knockdown (KD) of human YTHDF2 led to more than a 10-fold increase in the ex vivo expansion of hUCB HSCs, a fivefold increase in colony-forming units (CFUs), and more than an eightfold increase in functional hUCB HSCs in the secondary serial of a limiting dilution transplantation assay. Mapping of m 6 A in RNAs from mouse hematopoietic stem and progenitor cells (HSPCs) as well as from hUCB HSCs revealed its enrichment in mRNAs encoding transcription factors critical for stem cell self-renewal. These m 6 A-marked mRNAs were recognized by Ythdf2 and underwent decay. In Ythdf2 KO HSPCs and YTHDF2 KD hUCB HSCs, these mRNAs were stabilized, facilitating HSC expansion. Knocking down one of YTHDF2's key targets, Tal1 mRNA, partially rescued the phenotype. Our study provides the first demonstration of the function of YTHDF2 in adult stem cell maintenance and identifies its important role in regulating HSC ex vivo expansion by regulating the stability of multiple mRNAs critical for HSC self-renewal, thus identifying potential for future clinical applications.

Hox genes modulate the properties of hematopoietic stem cells (HSCs) and reacquired Hox expression in progenitors contributes to leukemogenesis. Here, our transcriptome and DNA methylome analyses revealed that Hoxb cluster and retinoid signaling genes are predominantly enriched in LT-HSCs, and this coordinate regulation of Hoxb expression is mediated by a retinoid-dependent cis-regulatory element, distal element RARE (DERARE). Deletion of the DERARE reduced Hoxb expression, resulting in changes to many downstream signaling pathways (e.g., non-canonical Wnt signaling) and loss of HSC self-renewal and reconstitution capacity. DNA methyltransferases mediate DNA methylation on the DERARE, leading to reduced Hoxb cluster expression. Acute myeloid leukemia patients with DNMT3A mutations exhibit DERARE hypomethylation, elevated HOXB expression, and adverse outcomes. CRISPR-Cas9-mediated specific DNA methylation at DERARE attenuated HOXB expression and alleviated leukemogenesis. Collectively, these findings demonstrate pivotal roles for retinoid signaling and the DERARE in maintaining HSCs and preventing leukemogenesis by coordinate regulation of Hoxb genes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Access to and characterization of sustained, toroidally confined plasmas with a very high plasma-to-magnetic pressure ratio (β&#95;{t}), low internal inductance, high elongation, and nonsolenoidal current drive is a central goal of present tokamak plasma research. Stable access to this desirable parameter space is demonstrated in plasmas with ultralow aspect ratio and high elongation. Local helicity injection provides nonsolenoidal sustainment, low internal inductance, and ion heating. Equilibrium analyses indicate β&#95;{t} up to ∼100% with a minimum |B| well spanning up to ∼50% of the plasma volume.

Ribosomal DNA is one of the most variable regions in the human genome with respect to copy number. Despite the importance of rDNA for cellular function, we know virtually nothing about what governs its copy number, stability, and sequence in the mammalian genome due to challenges associated with mapping and analysis. We applied computational and droplet digital PCR approaches to measure rDNA copy number in normal and cancer states in human and mouse genomes. We find that copy number and sequence can change in cancer genomes. Counterintuitively, human cancer genomes show a loss of copies, accompanied by global copy number co-variation. The sequence can also be more variable in the cancer genome. Cancer genomes with lower copies have mutational evidence of mTOR hyperactivity. The PTEN phosphatase is a tumor suppressor that is critical for genome stability and a negative regulator of the mTOR kinase pathway. Surprisingly, but consistent with the human cancer genomes, hematopoietic cancer stem cells from a Pten-/- mouse model for leukemia have lower rDNA copy number than normal tissue, despite increased proliferation, rRNA production, and protein synthesis. Loss of copies occurs early and is associated with hypersensitivity to DNA damage. Therefore, copy loss is a recurrent feature in cancers associated with mTOR activation. Ribosomal DNA copy number may be a simple and useful indicator of whether a cancer will be sensitive to DNA damaging treatments.

Background: Ultrasound guidance has been proposed as an alternative imaging modality for sacroiliac (SI) joint injections. Few studies have been published on the accuracy of this modality for the procedure., Objective: The objective of this study was to determine the accuracy of ultrasound-guided SI joint injections using a cadaveric model., Design: Controlled laboratory study., Setting: The study was performed in the Skills Laboratory of the American Sports Medicine Institute in St. Vincent's Hospital, Birmingham, AL., Methods: Seventeen cadaveric SI joints were injected under ultrasound guidance and dissected to determine the accuracy of intra-articular injections., Main Outcome Measurements: The presence of intra-articular spread of a white paint marker in the SI joint after ultrasound-guided injection., Results: Of 17 SI joints, 15 (88.2%) were accurately injected intra-articularly. One of the joints with no intra-articular spread was found to be partially frozen at the time of dissection, and the second joint was considered an unsuccessful injection before dissection due to difficulty entering the joint under ultrasound guidance because of marginal osteophytes at the joint line. Of the 15 joints with intra-articular placement, 5 joints (33.3%) showed partial extra-articular spread at the time of initial injection and required redirection of the needle under ultrasound guidance, and 3 joints (20%) had extra-articular spread that was not seen during ultrasound., Conclusion: Ultrasound allowed intra-articular injection in 88.2% of joints in this cadaveric study. Ultrasound does not expose the patient to radiation, as seen with fluoroscopic guidance, which is currently the gold standard for this injection. In addition, ultrasound may allow visualization of extra-articular spread when caused by extra-articular needle placement, which can allow for redirection of the needle to achieve intra-articular injection., Level of Evidence: IV., (Copyright © 2016 American Academy of Physical Medicine and Rehabilitation. Published by Elsevier Inc. All rights reserved.)

Tokamak experiments at near-unity aspect ratio A≲1.2 offer new insights into the self-organized H-mode plasma confinement regime. In contrast to conventional A∼3 plasmas, the L-H power threshold P&#95;{LH} is ∼15× higher than scaling predictions, and it is insensitive to magnetic topology, consistent with modeling. Edge localized mode (ELM) instabilities shift to lower toroidal mode numbers as A decreases. These ultralow-A operations enable heretofore inaccessible J&#95;{edge}(R,t) measurements through an ELM that show a complex multimodal collapse and the ejection of a current-carrying filament.

During the past 10 years, the sacroiliac (SI) joint has evolved from being barely recognized as a source of pain, to being a joint treated only nonsurgically or with great surgical morbidity, to currently being a joint treated with minimally invasive techniques that are personalized to the individual patient. The complex 3-dimensional anatomy of the SI joint and lack of parallel to traditional imaging planes requires a thorough understanding of the structures within and around the SI joint that may be at risk of injury. Thus, the SI joint is ideally suited for intraoperative 3-dimensional imaging and surgical navigation when being treated minimally invasively., (Copyright 2016, SLACK Incorporated.)

The mammalian imprinted Dlk1-Gtl2 locus produces multiple non-coding RNAs (ncRNAs) from the maternally inherited allele, including the largest miRNA cluster in the mammalian genome. This locus has characterized functions in some types of stem cell, but its role in hematopoietic stem cells (HSCs) is unknown. Here, we show that the Dlk1-Gtl2 locus plays a critical role in preserving long-term repopulating HSCs (LT-HSCs). Through transcriptome profiling in 17 hematopoietic cell types, we found that ncRNAs expressed from the Dlk1-Gtl2 locus are predominantly enriched in fetal liver HSCs and the adult LT-HSC population and sustain long-term HSC functionality. Mechanistically, the miRNA mega-cluster within the Dlk1-Gtl2 locus suppresses the entire PI3K-mTOR pathway. This regulation in turn inhibits mitochondrial biogenesis and metabolic activity and protects LT-HSCs from excessive reactive oxygen species (ROS) production. Our data therefore show that the imprinted Dlk1-Gtl2 locus preserves LT-HSC function by restricting mitochondrial metabolism., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objectives: The craniomandibular morphology of the adapid primates of Europe, especially Adapis and Leptadapis (sensu lato), suggests that they possessed enormous jaw adductor muscles. The goal of this study is to estimate jaw adductor muscle mass, physiological cross-sectional area (PCSA), and fiber length in adapid primates from the Eocene of Europe. We also estimated muscle leverage, bite force, and gape parameters., Materials and Methods: We use bony morphology and osteological correlates of soft tissues in a sample of extant strepsirrhines to estimate these soft-tissue and performance variables in Adapis and Leptadapis., Results: Our results suggest that, compared to a broad sample of extant strepsirrhines, Adapis and Leptadapis had relatively great jaw adductor muscle mass, PCSA, and bite force. They had moderately great jaw adductor leverage but no sign of adaptation for wide gapes. There is no support for the hypothesis that either adapid was a gouger., Discussion: Our estimates support the inference that Adapis and Leptadapis were primarily folivorous, perhaps also consuming small to medium-sized tough fruits, nuts, and seeds. Explanations for the likely extreme development of the jaw adductor muscles in adapids remain speculative. These include (1) foods that were generally tougher and/or of higher yield strength than those eaten by strepsirrhines today, (2) using the muscles "in shifts" to avoid muscle fatigue in the context of an obdurate diet, and (3) potential constraints on reshaping of the skull for more efficient food processing., (© 2015 Wiley Periodicals, Inc.)

The authors present a case of invasive group A Streptococcus (GAS) in a previously healthy 63-year-old male complicated by trans-esophageal echocardiogram negative endocarditis, septic arthritis, and multiple cerebral septic emboli. Despite antibiotics and drainage of his largest brain abscess, the patient expired. This case highlights the potential mortality from invasive GAS disease. Included is a review of current literature regarding invasive GAS that addresses its presentation, prevalence, virulence and treatment.

Objectives: Maximum ingested food size (Vb ) is an empirically tested performance variable that can shed light on feeding energetics and adaptation in the masticatory system. Until now, this variable had been tested in strepsirrhines alone among primates. Here, we present the first data on Vb in a broad sample of anthropoid primates and describe scaling patterns., Materials and Methods: Vb data on anthropoids were collected under captive conditions at the Philadelphia Zoo and compared with published data on strepsirrhines. Data on Vb were scaled against individual body mass and were compared with experimentally determined toughness and stiffness values for the test foods., Results: Unlike in strepsirrhines, where essentially Vb scales isometrically with body mass, Vb in anthropoids scales with negative allometry. There is a significant effect of food material properties on Vb , although bite size in anthropoids varies less based on food properties than in strepsirrhines. Large folivorous strepsirrhines follow the anthropoid trend in bite size scaling, but large frugivorous ones take especially large bites., Discussion: Negative scaling of bite size in the anthropoids sampled could be due to reduced adaptation for gape. Some early anthropoids likely evolved adaptations for maximizing mechanical advantage and fatigue resistance in the chewing muscles, resulting in reduced gape. This might have channeled them toward smaller bites of more-resistant foods and away from taking large bites. This might also be the case for some folivorous strepsirrhines., (© 2015 Wiley Periodicals, Inc.)