Cas12a domain flexibility guides R-loop formation and forces RuvC resetting.

The specific nature of CRISPR-Cas12a makes it a desirable RNA-guided endonuclease for biotechnology and therapeutic applications. To understand how R-loop formation within the compact Cas12a enables target recognition and nuclease activation, we used cryo-electron microscopy to capture wild-type Acidaminococcus sp. Cas12a R-loop intermediates and DNA delivery into the RuvC active site. Stages of Cas12a R-loop formation-starting from a 5-bp seed-are marked by distinct REC domain arrangements. Dramatic domain flexibility limits contacts until nearly complete R-loop formation, when the non-target strand is pulled across the RuvC nuclease and coordinated domain docking promotes efficient cleavage. Next, substantial domain movements enable target strand repositioning into the RuvC active site. Between cleavage events, the RuvC lid conformationally resets to occlude the active site, requiring re-activation. These snapshots build a structural model depicting Cas12a DNA targeting that rationalizes observed specificity and highlights mechanistic comparisons to other class 2 effectors.

Extracellular metabolic energetics can promote cancer progression.

Colorectal cancer primarily metastasizes to the liver and globally kills over 600,000 people annually. By functionally screening 661 microRNAs (miRNAs) in parallel during liver colonization, we have identified miR-551a and miR-483 as robust endogenous suppressors of liver colonization and metastasis. These miRNAs convergently target creatine kinase, brain-type (CKB), which phosphorylates the metabolite creatine, to generate phosphocreatine. CKB is released into the extracellular space by metastatic cells encountering hepatic hypoxia and catalyzes production of phosphocreatine, which is imported through the SLC6A8 transporter and used to generate ATP­fueling metastatic survival. Combinatorial therapeutic viral delivery of miR-551a and miR-483-5p through single-dose adeno-associated viral (AAV) delivery significantly suppressed colon cancer metastasis, as did CKB inhibition with a small-molecule inhibitor. Importantly, human liver metastases express higher CKB and SLC6A8 levels and reduced miR-551a/miR-483 levels relative to primary tumors. We identify the extracellular space as an important compartment for malignant energetic catalysis and therapeutic targeting.

Brap regulates liver morphology and hepatocyte turnover via modulation of the Hippo pathway.

Regulation of hepatocyte proliferation and liver morphology is of critical importance to tissue and whole-body homeostasis. However, the molecular mechanisms that underlie this complex process are incompletely understood. Here, we describe a role for the ubiquitin ligase BRCA1-associated protein (BRAP) in regulation of hepatocyte morphology and turnover via regulation of MST2, a protein kinase in the Hippo pathway. The Hippo pathway has been implicated in the control of liver morphology, inflammation, and fibrosis. We demonstrate here that liver-specific ablation of Brap in mice results in gross and cellular morphological alterations of the liver. Brap-deficient livers exhibit increased hepatocyte proliferation, cell death, and inflammation. We show that loss of BRAP protein alters Hippo pathway signaling, causing a reduction in phosphorylation of YAP and increased expression of YAP target genes, including those regulating cell growth and interactions with the extracellular environment. Finally, increased Hippo signaling in Brap knockout mice alters the pattern of liver lipid accumulation in dietary models of obesity. These studies identify a role for BRAP as a modulator of the hepatic Hippo pathway with relevance to human liver disease.

IL7 in combination with radiotherapy stimulates a memory T-cell response to improve outcomes in HNSCC models.

Clinically approved head and neck squamous cell carcinoma (HNSCC) immunotherapies manipulate the immune checkpoint blockade (ICB) axis but have had limited success outside of recurrent/metastatic disease. Interleukin-7 (IL7) has been shown to be essential for effector T-cell survival, activation, and proliferation. Here, we show that IL7 in combination with radiotherapy (RT) is effective in activating CD8 + T-cells for reducing tumor growth. Our studies were conducted using both human papillomavirus related and unrelated orthotopic HNSCC murine models. Immune populations from the tumor, draining lymph nodes, and blood were compared between treatment groups and controls using flow cytometry, proteomics, immunofluorescence staining, and RNA sequencing. Treatment with RT and IL7 (RT + IL7) resulted in significant tumor growth reduction, high CD8 T-cell tumor infiltration, and increased proliferation of T-cell progenitors in the bone marrow. IL7 also expanded a memory-like subpopulation of CD8 T-cells. These results indicate that IL7 in combination with RT can serve as an effective immunotherapy strategy outside of the conventional ICB axis to drive the antitumor activity of CD8 T-cells.

Optimized genome-wide CRISPR screening enables rapid engineering of growth-based phenotypes in Yarrowia lipolytica.

CRISPR-Cas9 functional genomic screens uncover gene targets linked to various phenotypes for metabolic engineering with remarkable efficiency. However, these genome-wide screens face a number of design challenges, including variable guide RNA activity, ensuring sufficient genome coverage, and maintaining high transformation efficiencies to ensure full library representation. These challenges are prevalent in non-conventional yeast, many of which exhibit traits that are well suited to metabolic engineering and bioprocessing. To address these hurdles in the oleaginous yeast Yarrowia lipolytica, we designed a compact, high-activity genome-wide sgRNA library. The library was designed using DeepGuide, a sgRNA activity prediction algorithm and a large dataset of ∼50,000 sgRNAs with known activity. Three guides per gene enables redundant targeting of 98.8% of genes in the genome in a library of 23,900 sgRNAs. We deployed the optimized library to uncover genes essential to the tolerance of acetate, a promising alternative carbon source, and various hydrocarbons present in many waste streams. Our screens yielded several gene knockouts that improve acetate tolerance on their own and as double knockouts in media containing acetate as the sole carbon source. Analysis of the hydrocarbon screens revealed genes related to fatty acid and alkane metabolism in Y. lipolytica. The optimized CRISPR gRNA library and its successful use in Y. lipolytica led to the discovery of alternative carbon source-related genes and provides a workflow for creating high-activity, compact genome-wide libraries for strain engineering.

Impact of community-based technology training with low-income older adults.

OBJECTIVES: Compared with younger and middle-aged adults, older adults are less likely to adopt new computer technology, potentially limiting access to healthcare and many other important resources available online. This limitation could impact cognitive abilities, well-being, and mental health outcomes of older adults. The aims of the present study were to increase access to online county and healthcare resources, while also assessing the impact of technology access on cognitive functioning and multiple well-being domains. METHODS: A pilot community collaboration provided a two-month tablet training intervention, focused on increasing digital independence via tablet navigation, resources access, and fraud and scam prevention, to 20 low-income older adult participants (75% female, Mage = 70.85). Pre- and post-test phone interviews were conducted to measure any changes in digital independence, cognitive abilities, well-being, mental health, and mindset. RESULTS: Linear mixed effects models revealed no significant changes in outcome measures from pre- to post-test. However, we found effects of digital independence on several well-being measures, providing important information for the impact of technology access and training for low-income older adults. CONCLUSION: This pilot intervention offers limited but promising results, inspiring further investigations that may inform public health and policy services to address barriers to access and potentially improve psychological health.

Morphine induces physiological, structural, and molecular benefits in the diabetic myocardium.

The obesity epidemic has increased type II diabetes mellitus (T2DM) across developed countries. Cardiac T2DM risks include ischemic heart disease, heart failure with preserved ejection fraction, intolerance to ischemia-reperfusion (I-R) injury, and refractoriness to cardioprotection. While opioids are cardioprotective, T2DM causes opioid receptor signaling dysfunction. We tested the hypothesis that sustained opioid receptor stimulus may overcome diabetes mellitus-induced cardiac dysfunction via membrane/mitochondrial-dependent protection. In a murine T2DM model, we investigated effects of morphine on cardiac function, I-R tolerance, ultrastructure, subcellular cholesterol expression, mitochondrial protein abundance, and mitochondrial function. T2DM induced 25% weight gain, hyperglycemia, glucose intolerance, cardiac hypertrophy, moderate cardiac depression, exaggerated postischemic myocardial dysfunction, abnormalities in mitochondrial respiration, ultrastructure and Ca2+ -induced swelling, and cell death were all evident. Morphine administration for 5 days: (1) improved glucose homeostasis; (2) reversed cardiac depression; (3) enhanced I-R tolerance; (4) restored mitochondrial ultrastructure; (5) improved mitochondrial function; (6) upregulated Stat3 protein; and (7) preserved membrane cholesterol homeostasis. These data show that morphine treatment restores contractile function, ischemic tolerance, mitochondrial structure and function, and membrane dynamics in type II diabetic hearts. These findings suggest potential translational value for short-term, but high-dose morphine administration in diabetic patients undergoing or recovering from acute ischemic cardiovascular events.

Paediatric intestinal pseudo-obstruction: a scoping review.

Paediatric intestinal pseudo-obstruction (PIPO) encompasses a group of rare disorders in which patients present with the clinical features of bowel obstruction in the absence of mechanical occlusion. The management of PIPO presents a challenge as evidence remains limited on available medical and surgical therapy. Parenteral nutrition is often the mainstay of therapy. Long-term therapy may culminate in life-threatening complications including intestinal failure-related liver disease, central line thrombosis and sepsis. Intestinal transplantation remains the only definitive cure in PIPO but is a complex and resource-limited solution associated with its own morbidity and mortality. We conducted a scoping review to present a contemporary summary of the epidemiology, aetiology, pathophysiology, diagnosis, management and complications of PIPO.Conclusion: PIPO represents a rare disorder that is difficult to diagnose and challenging to treat, with significant morbitity and mortality. The only known cure is intestinal transplantation. What is Known: • Paediatric intestinal pseudo-obstruction is a rare, heterogeneous disorder that confers a high rate of morbidity and mortality • Complications of paediatric intestinal pseudo-obstruction include chronic pain, small intestine bacterial overgrowth and malrotation. Other complications can occur related to its management, such as line infections with parenteral nutrition or cardiac side effects of prokinetic medications What is New: • Progress in medical and surgical therapy in recent years has led to improved patient outcomes • Enteral autonomy has been reported in most patients at as early as 1 month post-transplantation.

Paediatric chemical burns: a clinical review.

Although they account for a small proportion of burns in paediatrics, injuries from chemicals can be just as devastating as other mechanisms of burn injury. At least 25,000 chemicals exist which can cause burns: in children, they are often caused by household chemicals via accidental exposure. The mechanism by which corrosive substances produce chemical burns highlights the importance of early and plentiful irrigation of the burn area, removal of contaminated clothes and careful clinical assessment. Surgical intervention is uncommon but often follows the principles for thermal burns. This article reviews the aetiology, incidence, clinical presentation, management, complications and prevention of chemical burns. What is Known • Chemical burns in paediatrics are often caused by accidental exposure to chemicals available at home • Differences in the pathophysiology of chemical burns reinforces the need for early irrigation What is New • New irrigation fluids show promise in adults and need further study in children • The nature of chemical cutaneous burns can make assessment of wound depth difficult. Laser Doppler Imaging (LDI) is an accurate technique that can be used clinically to determine burn depth in thermal burns and is an area of future interest in the assessment of chemical burns.

Paediatric adhesive bowel obstruction: a systematic review.

Adhesions following abdominal surgery remain a common cause of bowel obstruction. The incidence is between 1 and 12.6% in children who have had previous abdominal surgery. While conservative management is usually trialled in all patients (including children) suspected of having ASBO, the majority will require surgical intervention. New materials such as Seprafilm® have been studied in the paediatric population, with promising results of its use in index abdominal surgeries to prevent the formation of adhesions. In this article, we conducted a systematic review to present an overview of the current knowledge on the incidence, aetiology, pathophysiology, clinical presentation, and management of ASBO.

Impacting Underserved Communities as a GI Trainee.

Gastroenterology fellowship programs commonly include VA and county hospitals whose patient populations consist of some of the most vulnerable and underserved populations in the country who have a multitude of socioeconomic hurdles that limit their ability to address ongoing medical issues, all while having a restricted political voice and receiving care in under-resourced clinical settings. Since trainees are integral to the care of these patients, they have available two approaches that can affect community and hospital-based change, namely quality improvement (QI) and healthcare advocacy. QI projects focused on optimizing colorectal cancer screening, and Helicobacter pylori testing/eradication can provide value at an institutional level. Healthcare advocacy can be approached through involvement in national gastroenterological associations or locally through means such as establishing a fellowship-based advocacy group similar to a journal club. Both routes enable trainees to positively impact underserved communities.

PowerPoint to the People: The Four Secrets to Delivering a Great Medical Talk.

Medical talks are a staple of post-medical school education, but the effectiveness of these lectures can be quite variable. One significant reason for this is that while physicians and trainees are well trained at presenting information to one another, they have little to no formal training on giving hour-long medical didactics. Focusing on four specific categories including creating a strong first impression, effective use of PowerPoint, impactful delivery of information, and thorough preparation a physician at any stage in training can become a strong presenter.

Balanitis xerotica obliterans: an update for clinicians.

Lichen sclerosus (LS) is a severe, chronic, dermatosis characterised by inflammatory, sclerotic, pruritic lesions that causes significant morbidity in patients of all genders and ages. In boys, the lesions typically affect the foreskin and glans (termed balanitis xerotica obliterans (BXO)), leading to phimosis and potentially meatal stenosis. The incidence of the disease is not well reported but the average age of affected boys is 8 years (range 1-16). Diagnosis can often be made clinically, although histological study remains important to rule out important differential diagnoses. Complications include genital scarring, urinary and sexual dysfunction as well as the development of carcinomas in adult life. Circumcision has been regarded as definitive management of BXO in boys, but this may be supplemented with medical therapies such as topical steroids, immune modulators, intralesional triamcinolone and ozonated olive oil. Supportive measures including emollients, avoidance of irritants, surveillance of complications and recurrence as well as education and counselling remain important.Conclusion: BXO remains an important cause of phimosis in boys. The frequency of this condition appears unclear but seems likely to be less than 1% of males. Treatment generally involves circumcision, with some evidence that topical steroids or immunomodulators may decrease the incidence of recurrent meatal stenosis.What is Known:• Surgical circumcision is considered the definitive management of BXO• Many aspects of BXO are still in contention or require further study including the epidemiology and aetiology.What is New:• There is increasing awareness of non-surgical modalities that may be used in adjunct to surgery including topical corticosteroids, immune modulators, intralesional triamcinolone and ozonated olive oil• Awareness of meatal stenosis-related BXO has led to the development of surgical techniques such as preputioplasty as well as buccal mucosal inlay grafts.

Experience with Eversion Endarterectomy of the Common Femoral Artery with Comparison to Standard Endarterectomy with Patch.

In this retrospective study, we review the outcome of femoral eversion endarterectomy (EE) and standard endarterectomy with patch angioplasty (SEP) over a 10-year period. EE technique involves transection of common femoral artery (CFA), eversion endarterectomy of the distal CFA, and primary end-to-end reconstruction. One hundred forty-two patients underwent femoral endarterectomy, among which 38 (26.8%) endarterectomies were performed using the eversion technique. The cumulative 2-year primary patency of EE with or without a concurrent procedure was greater than 86% and did not statistically differ from SEP. EE is a feasible technique when the use of a patch needs to be avoided.

Cytotoxicity, cellular uptake and apoptotic responses in human coronary artery endothelial cells exposed to ultrasmall superparamagnetic iron oxide nanoparticles.

Ultrasmall superparamagnetic iron oxide nanoparticles (USPION) possess reactive surfaces, are metabolized and exhibit unique magnetic properties. These properties are desirable for designing novel theranostic biomedical products; however, toxicity mechanisms of USPION are not completely elucidated. The goal of this study was to investigate cell interactions (uptake and cytotoxicity) of USPION using human coronary artery endothelial cells as a vascular cell model. Polyvinylpirrolidone-coated USPION were characterized: average diameter 17 nm (transmission electron microscopy [TEM]), average hydrodynamic diameter 44 nm (dynamic light scattering) and zeta potential -38.75 mV. Cells were exposed to 0 (control), 25, 50, 100 or 200 µg/mL USPION. Concentration- and time-dependent cytotoxicity were observed after 3-6 hours through 24 hours of exposure using Alamar Blue and Real-Time Cell Electronic Sensing assays. Cell uptake was evaluated by imaging using live-dead confocal microscopy, actin and nuclear fluorescent staining, and TEM. Phase-contrast, confocal microscopy, and TEM imaging showed significant USPION internalization as early as 3 hours after exposure to 25 µg/mL. TEM imaging demonstrated particle internalization in secondary lysosomes with perinuclear localization. Three orthogonal assays were conducted to assess apoptosis. TUNEL staining demonstrated a marked increase in fragmented DNA, a response pathognomonic of apoptosis, after a 4-hour exposure. Cells subjected to agarose gel electrophoresis exhibited degraded DNA 3 hours after exposure. Caspase-3/7 activity increased after a 3-hour exposure. USPION uptake resulted in cytotoxicity involving apoptosis and these results contribute to further mechanistic understanding of the USPION toxicity in vitro in cardiovascular endothelial cells.

Microneedle-Based Delivery of Amphotericin B for Treatment of Cutaneous Leishmaniasis.

Current therapeutic options against cutaneous leishmaniasis are plagued by several weaknesses. The effective topical delivery of an antileishmanial drug would be useful in treating some forms of cutaneous leishmaniasis. Toward this end, a microneedle based delivery approach for the antileishmanial drug amphotericin B was investigated in murine models of both New World (Leishmania mexicana) and Old World (Leishmania major) infection. In the L. mexicana model, ten days of treatment began on day 35 post infection, when the area of nodules averaged 9-15 mm2. By the end of the experiment, a significant difference in nodule area was observed for all groups receiving topical amphotericin B at 25 mg/kg/day after application of microneedle arrays of 500, 750, and 1000 µM in nominal length compared to the group that received this dose of topical amphotericin B alone. In the L. major model, ten days of treatment began on day 21 post infection when nodule area averaged 51-65 mm2 in the groups. By the end of the experiment, there was no difference in nodule area between the group receiving 25 mg/kg of topical amphotericin B after microneedle application and any of the non-AmBisome groups. These results show the promise of topical delivery of amphotericin B via microneedles in treating relatively small nodules caused by L. mexicana. These data also show the limitations of the approach against a disseminated L. major infection. Further optimization of microneedle delivery is needed to fully exploit this strategy for cutaneous leishmaniasis treatment.

Rising Inpatient Encounters and Economic Burden for Patients with Nonalcoholic Fatty Liver Disease in the USA.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is the fastest-growing chronic liver disease. However, little is known about NAFLD inpatient resource utilization and clinical outcomes. AIMS: The aim of this study was to quantify inpatient NAFLD encounters using patient-level data over time. METHODS: This was a retrospective analysis of de-identified data for NAFLD patients from the California Patient Discharge Database from 2006 to 2013. NAFLD patients were identified by ICD9 codes 571.40, 571.41, 571.49, 571.8, and 571.9. RESULTS: NAFLD patients (n = 91,558) were predominantly female (60%), 45-65 years old (44%), and white (53%). Inpatient encounters increased from 8153 in 2006 to 16,457 in 2013 and were associated with a 207% increase in charges ($686 million in 2006 to $1.42 billion in 2013) and average increase in charges of 9.8% per year adjusting for inflation. Comorbidities (obesity, diabetes, hyperlipidemia, cardiovascular disease, other cancer, and renal disease) increased significantly over time (all P < 0.05). From 2006 to 2011, there were 11,463 deaths (1849 for liver-related hospitalizations) (mean follow-up 4.00 ± 2.13 years). The most significant predictors of death were age > 75 (aHR 3.9, P < 0.0001), male gender (aHR 1.10, P < 0.0001), white race (aHR 1.2, P < 0.0001), decompensated cirrhosis (aHR 2.1, P < 0.0001), and cancer other than HCC (aHR 3.2, P < 0.0001). Within the liver-related hospitalization cohort, mortality predictors were similar, except for Hispanic race (aHR 0.92, P < 0.0096) and renal disease (aHR 1.50, P < 0.0001). CONCLUSIONS: The number of NAFLD inpatient encounters increased significantly from 2006 to 2013, as did the inflation-adjusted inpatient charges. The most significant predictors of death were non-liver cancers (HR 3.11, P < 0.0001, CI 3.06-3.16) and age > 75 years (HR 3.94, P < 0.0001, HR 3.86-4.03).

Engineering Polymer-Binding Bispecific Antibodies for Enhanced Pretargeted Delivery of Nanoparticles to Mucus-Covered Epithelium.

Mucus represents a major barrier to sustained and targeted drug delivery to mucosal epithelium. Ideal drug carriers should not only rapidly diffuse across mucus, but also bind the epithelium. Unfortunately, ligand-conjugated particles often exhibit poor penetration across mucus. In this work, we explored a two-step "pretargeting" approach through engineering a bispecific antibody that binds both cell-surface ICAM-1 and polyethylene glycol (PEG) on the surface of nanoparticles, thereby effectively decoupling cell targeting from particle design and formulation. When tested in a mucus-coated Caco-2 culture model that mimics the physiological process of mucus clearance, pretargeting increased the amount of PEGylated particles binding to cells by around 2-fold or more compared to either non-targeted or actively targeted PEGylated particles. Pretargeting also markedly enhanced particle retention in mouse intestinal tissues. Our work underscores pretargeting as a promising strategy to improve the delivery of therapeutics to mucosal surfaces.

Metastatic renal cell carcinoma initially presenting with hematochezia and subsequently with vaginal bleeding: a case report.

BACKGROUND: We report an unusual case of a synchronous rectal and metachronous vaginal metastatic renal cell carcinoma. CASE PRESENTATION: A 78-year-old woman presented with hematochezia and a colonoscopy revealed a metastatic clear-cell renal cell carcinoma rectal polyp biopsy-proven. Abdominal computed tomography identified a 9.0-cm left renal mass with renal vein thrombosis, for which she underwent a laparoscopic radical nephrectomy. Histopathological examination confirmed a pT3a clear-cell renal cell carcinoma. Seven months later, the patient presented with vaginal bleeding. Physical examination revealed a vaginal polypoid mass and biopsy confirmed a clear-cell renal cell carcinoma metastasis. CONCLUSIONS: This case represents unusual manifestations of metastatic renal cell carcinoma and is a reminder of the wide spectrum of clinical course of this disease.

Long-term outcomes of the minimally invasive free vascularized omental lymphatic flap for the treatment of lymphedema.

BACKGROUND: The free vascularized omental lymphatic flap provides an option without the risk for iatrogenic donor site lymphedema that plagues alternative lymph node transfer donor sites. The omental flap has been associated with significant morbidity in the past; however, with modern techniques and advanced in technology, a minimally invasive approach to flap harvest is feasible. We present the long-term outcomes of the minimally invasive free vascularized omental lymphatic flap for the treatment of lymphedema. METHODS: All consecutive patients with advanced lymphedema undergoing minimally invasive free vascularized omental lymphatic flap transfer were included. Perioperative evaluation included qualitative assessments, lymphoscintigraphy, and volumetric measurements. RESULTS: Overall, 42 patients underwent a free omental lymphatic flap and had a mean follow-up of 14 (3-32) months. Subjective improvements were noted in 83% of patients. Mean volumetric improvement was 22%. Complications occurred in 16% (n = 7) of patients; this included one episode of pancreatitis and one flap loss. Postoperative imaging revealed viable lymphatic transfers. Cellulitis history was present in 74% (n = 31) patients with post-operative cellulitis occurring in 5% (n = 2) patients. CONCLUSIONS: The minimally invasive free vascularized omental lymphatic flap provides a safe donor site, a durable and versatile flap, and an efficacious therapy against lymphedema and lymphedema-related cellulitis. J. Surg. Oncol. 2017;115:84-89. © 2016 Wiley Periodicals, Inc.