Design and Selection of Heterodimerizing Helical Hairpins for Synthetic Biology.

Synthetic biology applications would benefit from protein modules of reduced complexity that function orthogonally to cellular components. As many subcellular processes depend on peptide-protein or protein-protein interactions, de novo designed polypeptides that can bring together other proteins controllably are particularly useful. Thanks to established sequence-to-structure relationships, helical bundles provide good starting points for such designs. Typically, however, such designs are tested in vitro and function in cells is not guaranteed. Here, we describe the design, characterization, and application of de novo helical hairpins that heterodimerize to form 4-helix bundles in cells. Starting from a rationally designed homodimer, we construct a library of helical hairpins and identify complementary pairs using bimolecular fluorescence complementation in E. coli. We characterize some of the pairs using biophysics and X-ray crystallography to confirm heterodimeric 4-helix bundles. Finally, we demonstrate the function of an exemplar pair in regulating transcription in both E. coli and mammalian cells.

Rotator Cuff Injury and Repair.

Rotator cuff pathology is a commonly encountered clinical and radiologic entity that can manifest as tendinopathy or tearing. Magnetic resonance imaging (MRI) and ultrasonography offer similar sensitivity and specificity for the evaluation of the native rotator cuff, and the chosen modality may vary, depending on local practice and accessibility. MR arthrography is frequently used in the postoperative setting as a problem-solving tool. Key findings to include in the preoperative MRI report include the size and location of the tear, thickness of the tendon involved (partial versus full thickness), and overall tendon quality. The report should also address features associated with poor surgical outcomes, such as fatty atrophy, a decreased acromiohumeral interval, and evidence of rotator cuff arthropathy. Musculoskeletal radiologists should be familiar with the various surgical techniques and expected postoperative imaging appearance of rotator cuff repairs. Imaging also plays a role in identifying recurrent tearing, graft failure, hardware loosening, infection, and other complications.

Public perceptions of pharmacogenomic services in Ireland - Are people with chronic disease more likely to want service availability than those without? A questionnaire study.

Background: As pharmacogenomic services begin to emerge in primary care, the insight of the public is crucial for its integration into clinical practice. Objectives: To establish perceptions of pharmacogenomics (awareness, understanding, openness to availability, perceived benefits and concerns, willingness to pay, and service setting) and investigate if they differ between those with and without chronic disease(s). Methods: An anonymous, online questionnaire generated using Qualtrics® and circulated via social media and posters placed in eight participating community pharmacies was conducted with Irish adults. The questions were designed to consider existing literature on patient perceptions of pharmacogenomics. Descriptive statistics were used to summarize questionnaire responses. Chi-square test was used to compare categorical variables, while independent sample t-test and one-way ANOVA were used to compare the mean values of two (with and without chronic disease) and three groups (multimorbidity (two or more chronic conditions) and polypharmacy (prescribed four or more regular medicines) (MMPP), a single chronic disease, and those without existing medical conditions) respectively Logistic regression was used to evaluate age and gender adjusted associations of chronic disease(s) with responses. A p-value <0.05 was considered statistically significant. Results: A total of 421 responses were received, 30% (n = 120) of whom reported having a chronic disease. Overall, respondents reported low awareness (44%, n = 166) and poor knowledge (55%, n = 212) of pharmacogenomics. After explaining pharmacogenomics to respondents, patients with chronic disease(s) were 2.17 times more likely (p < 0.001) to want pharmacogenomic services availability than those without existing conditions, adjusted for age and gender (driven by preferences of those with MMPP than those with single chronic disease). Respondents demonstrated a high level of interest and noted both the potential benefits and downsides of pharmacogenomic testing. Willingness-to-pay was not associated with having a chronic disease and respondents were more positive about primary care (community pharmacy or general practice) rather than hospital-based pharmacogenomics implementation. Conclusion: The Irish public in general and those with chronic disease in particular are strongly supportive of pharmacogenomic testing, highlighting an unmet need for its incorporation in medicines optimization. These data underline the need for more research on the implementation of community-based pharmacogenomics services for MMPP patients and ubiquitous pharmacogenomics education programs.

Adverse pregnancy and maternal outcomes in women with hidradenitis suppurativa.

BACKGROUND: Hidradenitis suppurativa (HS) disproportionately affects women of childbearing age. However, pregnancy and maternal outcomes for women with HS are unknown. OBJECTIVE: To compare risk of adverse pregnancy and maternal outcomes among women with and without HS and to evaluate the influence of comorbid conditions. METHODS: Retrospective cohort analysis between January 1, 2011, and September 30, 2015. RESULTS: Compared to control pregnancies (n = 64,218), HS pregnancies (n = 1862) had a higher risk of spontaneous abortion (15.5% vs 11.3%), preterm birth (9.1% vs 6.7%), gestational diabetes mellitus (11.6% vs 8.4%), gestational hypertension (6.1% vs 4.4%), preeclampsia (6.6% vs 3.8%), and cesarean section (32.4% vs 27.1%). Relative risk of some pregnancy and maternal outcomes were attenuated after comorbidity adjustment. In the fully adjusted model, HS pregnancies were independently associated with spontaneous abortion (odds ratio, 1.20; 95% CI, 1.04-1.38), gestational diabetes mellitus (odds ratio, 1.26; 95% CI, 1.07-1.48), and cesarean section (odds ratio, 1.09; 95% CI, 1.004-1.17). LIMITATIONS: We could not evaluate potential influences of disease duration, activity, or severity. Newborn outcomes could not be evaluated. CONCLUSION: HS appears to be an independent risk factor for adverse pregnancy and maternal outcomes. This risk is influenced by comorbidities that may be modifiable with early identification and management.

Genetic therapies for neurological disorders.

In recent years, it has become increasingly apparent that many neurological disorders are underpinned by a genetic aetiology. This has resulted in considerable efforts to develop therapeutic strategies which can treat the disease-causing mutation, either by supplying a functional copy of the mutated gene or editing the genomic sequence. In this review, we will discuss the main genetic strategies which are currently being explored for the treatment of monogenic neurological disorders, as well as some of the challenges they face. In addition, we will address some of the ethical difficulties which may arise.

Three-year review of a capacity building pilot for a sustainable regional network on food, nutrition and health systems education in India.

BACKGROUND: In Kolkata (India), there are high rates of malnourished children (45.9%) under the age of three, impacting growth, organ development, function, and cognition. Mothers have a major role to play during this crucial development stage, with research showing nutrition knowledge, attitudes and practices (KAP) of mothers are important determinants of childhood malnutrition. AIMS: To document 3 years of capacity building towards a sustainable nutrition education network in Kolkata, India, while assessing the ability to perform data collection in the form of needs assessments, impact assessments and capacity reviews. METHODS: Descriptive review and analysis of engagement and impact from 3 years of work by the NNEdPro Global Centre for Nutrition and Health, initiating locally led nutrition education interventions. Mapping to the Indian National Nutrition Strategy was also performed to review adherence to nationwide priorities surrounding nutrition and determine the wider application potential of the network. RESULTS: Two simultaneous projects were taken forward by a team of local healthcare professionals and student champions. Project 1-medical college workshops for medical student nutrition education with added focus on underserved populations, Project 2-preparation for a 'Mobile Teaching Kitchen' (MTK) in marginalised communities to empower local women as nutrition educators.Data collection methods used for analysing markers of impact and sustainability were semi-structured interviews of the community members, and KAP questionnaires to assess response to educational sessions. CONCLUSION: With local support it is possible to create and sustain fieldwork for an extended period with meaningful outputs and impact. This initiative demonstrates that it is possible to use healthcare professionals, students and volunteers with low-intensity training and a low-cost approach to produce action research with considerable impact and results in rapid, reliable and robust manner.

Pearls, Pitfalls, and Conditions that Mimic Mesenteric Ischemia at CT.

Acute mesenteric ischemia (AMI) is a life-threatening condition with a high mortality rate. The diagnosis of AMI is challenging because patient symptoms and laboratory test results are often nonspecific. A high degree of clinical and radiologic suspicion is required for accurate and timely diagnosis. CT angiography of the abdomen and pelvis is the first-line imaging test for suspected AMI and should be expedited. A systematic "inside-out" approach to interpreting CT angiographic images, beginning with the bowel lumen and proceeding outward to the bowel wall, mesentery, vasculature, and extraintestinal viscera, provides radiologists with a practical framework to improve detection and synthesis of imaging findings. The subtypes of AMI are arterial and venoocclusive disease, nonocclusive ischemia, and strangulating bowel obstruction; each may demonstrate specific imaging findings. Chronic mesenteric ischemia is more insidious at onset and almost always secondary to atherosclerosis. Potential pitfalls in the diagnosis of AMI include mistaking pneumatosis as a sign that is specific for AMI and not an imaging finding, misinterpretation of adynamic ileus as a benign finding, and pseudopneumatosis. Several enterocolitides can mimic AMI at CT angiography, such as inflammatory bowel disease, infections, angioedema, and radiation-induced enterocolitis. Awareness of pitfalls, conditions that mimic AMI, and potential distinguishing clinical and imaging features can assist radiologists in making an early and accurate diagnosis of AMI. ©RSNA, 2020.

Toxicity of overexpressed MeCP2 is independent of HDAC3 activity.

Duplication of the X-linked MECP2 gene causes a severe neurological syndrome whose molecular basis is poorly understood. To determine the contribution of known functional domains to overexpression toxicity, we engineered a mouse model that expresses wild-type or mutated MeCP2 from the Mapt (Tau) locus in addition to the endogenous protein. Animals that expressed approximately four times the wild-type level of MeCP2 failed to survive to weaning. Strikingly, a single amino acid substitution that prevents MeCP2 from binding to the TBL1X(R1) subunit of nuclear receptor corepressor 1/2 (NCoR1/2) complexes, when expressed at equivalent high levels, was phenotypically indistinguishable from wild type, suggesting that excessive corepressor recruitment underlies toxicity. In contrast, mutations affecting the DNA-binding domain were toxic when overexpressed. As the NCoR1/2 corepressors are thought to act through histone deacetylation by histone deacetylase 3 (HDAC3), we asked whether mutations in NCoR1 and NCoR2 that drastically reduced their ability to activate this enzyme would relieve the MeCP2 overexpression phenotype. Surprisingly, severity was unaffected, indicating that the catalytic activity of HDAC3 is not the mediator of toxicity. Our findings shed light on the molecular mechanisms underlying MECP2 duplication syndrome and call for a re-evaluation of the precise biological role played by corepressor recruitment.

High affinity single-chain variable fragments are specific and versatile targeting motifs for extracellular vesicles.

Exosomes are extracellular vesicles that mediate cell-to-cell communication by transferring biological cargo, such as DNA, RNA and proteins. Through genetic engineering of exosome-producing cells or manipulation of purified exosomes, it is possible to load exosomes with therapeutic molecules and target them to specific cells via the display of targeting moieties on their surface. This provides an opportunity to exploit a naturally-occurring biological process for therapeutic purposes. In this study, we explored the potential of single chain variable fragments (scFv) as targeting domains to achieve delivery of exosomes to cells expressing a cognate antigen. We generated exosomes targeting the Her2 receptor and, by varying the affinity of the scFvs and the Her2 expression level on recipient cells, we determined that both a high-affinity anti-Her2-scFv (KD≤ 1 nM) and cells expressing a high level (≥106 copies per cell) of Her2 were optimally required to enable selective uptake. We also demonstrate that targeting exosomes to cells via a specific cell surface receptor can alter their intracellular trafficking route, providing opportunities to influence the efficiency of delivery and fate of intracellular cargo. These experiments provide solid data to support the wider application of exosomes displaying antibody fragments as vehicles for the targeted delivery of therapeutic molecules.

A mutation-led search for novel functional domains in MeCP2.

Most missense mutations causing Rett syndrome (RTT) affect domains of MeCP2 that have been shown to either bind methylated DNA or interact with a transcriptional co-repressor complex. Several mutations, however, including the C-terminal truncations that account for ∼10% of cases, fall outside these characterized domains. We studied the molecular consequences of four of these 'non-canonical' mutations in cultured neurons and mice to see if they reveal additional essential domains without affecting known properties of MeCP2. The results show that the mutations partially or strongly deplete the protein and also in some cases interfere with co-repressor recruitment. These mutations therefore impact the activity of known functional domains and do not invoke new molecular causes of RTT. The finding that a stable C-terminal truncation does not compromise MeCP2 function raises the possibility that small molecules which stabilize these mutant proteins may be of therapeutic value.

Oral contraceptives for acne treatment: US Dermatologists' knowledge, comfort, and prescribing practices.

The use of oral contraceptive pills (OCPs), which can be an effective treatment of acne in women, is poorly understood among many dermatologists. In this study, we surveyed 116 US dermatologists about their knowledge, comfort, and prescribing practices pertaining to the use of OCPs. The majority of respondents had previously prescribed OCPs and believed they were an effective treatment of acne in women. Despite adverse effects such as increased risk for venous thromboembolism (VTE) associated with OCPs, especially those containing drospirenone, our study indicated that many dermatologists believe the benefits of increased treatment efficacy may outweigh the risks.

Vulnerable carotid plaque imaging and histopathology without a dedicated MRI receiver coil.

Stroke is associated with vulnerable carotid artery plaques showing specific histopathologic features, namely a lipid-rich necrotic core, intraplaque hemorrhage, ulceration, and thin fibrous cap. While ultrasound and computed tomography (CT) can identify carotid plaques and determine the extent of stenosis, magnetic resonance imaging (MRI) provides further information regarding plaque composition and morphology. In this feasibility study, three patients with symptomatic, moderately stenosed plaques were imaged with CT angiography (CTA) and MRI (3T and 1.5T) without a dedicated receiver coil. The patients subsequently underwent carotid endarterectomy with en-bloc excision of the plaque. The CT and MR images were analyzed independently by three neuroradiologists to identify vulnerable plaque features. The images were correlated with the histopathology to confirm the findings. All three patients had one or more vulnerable plaque features on histopathology. MRI allowed for better characterization of these features when compared to CTA. The pre- and post-contrast T1-weighted (T1W) images were most helpful for identifying the lipid-rich necrotic core and thin fibrous cap, while the time of flight-magnetic resonance angiography (TOF-MRA) and contrast-enhanced (CE)-MRA were excellent for detecting plaque hemorrhage and ulceration, respectively. The 3T images showed superior spatial and contrast resolution compared to the 1.5T images for all sequences. By providing direct correlation between imaging and histopathology, this study demonstrates that 3T MRI without a dedicated surface coil is an excellent tool for assessing plaque vulnerability. In smaller hospitals or those with limited resources, it is reasonable to consider conventional MRI for patient risk stratification. Further studies are needed to determine how MRI and plaque vulnerability can be incorporated into routine clinical practice.

Clinical features and response to systemic therapy in a historical cohort of advanced or unresectable mucosal melanoma.

There are very few data available regarding the pattern of first metastases in resected mucosal melanomas (MMs) as well as the response of advanced MM to cytotoxic therapy. A retrospective, single-institution cohort was assembled of all patients with advanced/unresectable MM between 1995 and 2012 who had received systemic therapy with available imaging (N=81). Responses to first-line and second-line systemic therapy were assessed using Response Evaluation Criteria in Solid Tumors (RECIST) 1.1. The relationship between response, overall survival, and clinical covariates was investigated using Cox proportional hazards regression. Primary sites included anorectal (N=31, 38%), vulvovaginal (N=28, 35%), head and neck (N=21, 26%), and gallbladder (N=1, 1%) mucosa. Seven percent of patients had their first relapse in the brain. Cytotoxic therapy represented 82 and 51% of first-line and second-line regimens. The best response achieved in the first-line setting was similar for single-agent [10%; 95% confidence interval (CI): 1-32%] and combination alkylator therapy (8%; 95% CI: 2-21%). Median overall survival from first-line treatment was 10.3 months (95% CI: 8.7-13.9 months). Patients with elevated lactic dehydrogenase [hazard ratio (HR): 1.87, 95% CI: 1.10-3.19, P=0.020] and Eastern Cooperative Oncology Group performance status 1-2 (HR: 1.69, 95% CI: 1.05-2.72, P=0.030) had a higher risk of death, whereas patients with 12-week objective responses had a lower risk of death (HR: 0.12, 95% CI: 0.04-0.41, P<0.001). Cytotoxic systemic therapy has modest activity in advanced/unresectable MM, belying its adjuvant benefit. Patients whose tumors have an objective response to therapy have a lower probability of death. Brain imaging should be considered in routine surveillance.

Response to clozapine in a clinically identifiable subtype of schizophrenia.

BACKGROUND: Genetic testing in psychiatry promises to improve patient care through advances in personalised medicine. However, there are few clinically relevant examples. AIMS: To determine whether patients with a well-established genetic subtype of schizophrenia show a different response profile to the antipsychotic clozapine than those with idiopathic schizophrenia. METHOD: We retrospectively studied the long-term safety and efficacy of clozapine in 40 adults with schizophrenia, half with a 22q11.2 deletion (22q11.2DS group) and half matched for age and clinical severity but molecularly confirmed to have no pathogenic copy number variant (idiopathic group). RESULTS: Both groups showed similar clinical improvement and significant reductions in hospitalisations, achieved at a lower median dose for those in the 22q11.2DS group. Most common side-effects were similarly prevalent between the two groups, however, half of the 22q11.2DS group experienced at least one rare serious adverse event compared with none of the idiopathic group. Many were successfully retried on clozapine. CONCLUSIONS: Individuals with 22q11.2DS-schizophrenia respond as well to clozapine treatment as those with other forms of schizophrenia, but may represent a disproportionate number of those with serious adverse events, primarily seizures. Lower doses and prophylactic (for example anticonvulsant) management strategies can help ameliorate side-effect risks. This first systematic evaluation of antipsychotic response in a genetic subtype of schizophrenia provides a proof-of-principle for personalised medicine and supports the utility of clinical genetic testing in schizophrenia.

Nonoccupational post-exposure prophylaxis for HIV in New York State Emergency Departments.

New York State (NYS) established guidelines for nonoccupational post-exposure prophylaxis (nPEP) to HIV in 1997. To assess current nPEP practices in NYS Emergency Departments (EDs), we electronically surveyed all ED directors in NYS, excluding Veterans' Affairs hospitals, about nPEP and linkage-to-care protocols in the EDs. Basic descriptive statistics were used for analysis. The response rate was 96% (184/191). Of respondents, 88% reported evaluating any patient with a possible nonoccupational exposure to HIV, in accordance with NYS guidelines. Of these, 83% provided the patient with a starter pack of medications, while 4% neither supplied nor prescribed antiretroviral drugs in the ED. Sexually transmitted infection screening, risk reduction counseling, and education about symptoms of acute HIV seroconversion were performed inconsistently, despite NYS guidelines recommendations. Only 22% of EDs confirmed whether linkage to follow-up care was successful. Most NYS EDs prescribe nPEP to appropriate patients but full implementation of guidelines remains incomplete.

Accuracy of Canadian food labels for sodium content of food.

The accuracy of the Nutrition Facts table (NFt) has a significant impact on Canadian efforts to reduce dietary sodium and monitor sodium content in foods. This study assessed the accuracy of sodium (and calories, trans fat, saturated fat, sugar) reported on the NFt for selected foods and beverages in Canada. The Canadian Food Inspection Agency (CFIA) sampled over 1000 foods and beverages from supermarkets, bakeries, and restaurants across Canada between January 2006 and December 2010. The samples were analyzed in CFIA laboratories. Results were requested for products with ≥1 of the following nutrients tested: sodium, calories, saturated fat, trans fat, and sugar. Differences between the label and laboratory values were calculated for each product. Overall, 16.7% (n = 169) of products were "unsatisfactory" with laboratory values exceeding ±20% of the NFt value. Sodium had the highest number of unsatisfactory products (n = 49, 18.4%) and trans fat had the lowest number of unsatisfactory products (n = 16, 4.3%). The proportion of unsatisfactory products for saturated fat, calories, and sugar was 15.8%, 14.2%, and 12.9%, respectively. All of the unsatisfactory products had excess nutrient content relative to the NFt. Sodium and calories were consistently underreported (p < 0.05), while NFt values for the other nutrients were not statistically different than laboratory values. Increased monitoring of NFt sodium values is recommended in order to increase consumer confidence in this nutrition tool, to encourage industry to accurately report nutrient content and to continue using the NFt to guide research, education, and policy development.

Who uses the emergency department for dermatologic care? A statewide analysis.

BACKGROUND: Emergency department (ED) visits for skin symptoms are on the rise. The nature of these visits is not yet well characterized. OBJECTIVE: We sought to conduct a longitudinal analysis comparing patients discharged from the ED with dermatologic primary diagnoses to those with nondermatologic primary diagnoses. METHODS: Using the California State ED Database, we compared demographic variables and visit characteristics of patients discharged with dermatologic primary diagnoses (International Classification of Diseases, Ninth Revision, Clinical Modification codes 680-709) to those of patients discharged with nondermatologic primary diagnoses from 2005 to 2011. RESULTS: Patients given dermatologic primary diagnoses in the ED were more likely to be male, aged 18 to 54 years, white or Native American, and low income. They tended to be self-pay patients or have Medicaid, to live in less populous areas, and to visit the ED on the weekend. LIMITATIONS: Results from California may not be generalizable nationally. The databases we used were based on administrative records, which have limited clinical detail. CONCLUSION: The population of patients discharged home from the ED with dermatologic primary diagnoses appears to differ significantly from the population receiving nondermatologic primary diagnoses.