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Recent documents in eScholarship@UMMS
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Brief Report on Obstetricians'/Gynecologists' Distribution of Scarce Resources

Tue, 02/02/2016 - 1:11pm

On a day-to-day basis, doctors must decide which treatments are most beneficial for their patients, and which make the most sense in terms of costs. In medical decision making, factors such as efficiency and cost-effectiveness can be particularly challenging to navigate because many of the most expensive procedures encountered in medical practice are also high-stake treatments for patients. One-hundred-six obstetricians-gynecologists (Obs/Gyns) completed a survey asking them to allocate the following resources in scenarios in which they are scarce: human papilloma virus (HPV) vaccinations, mammograms, and in vitro fertilization (IVF) treatments. Additional questions focused on how fairness and cost-effectiveness factored into the allocation decisions of each group. Results indicated that Obs/Gyns were more efficient in their distribution of HPV vaccinations and mammograms than in their distribution of IVF treatments. More efficient responding was associated with placing less emphasis on fairness in decision making. This study demonstrates the differences that exist in the emphasis that physicians place on medical evidence, cost, outcomes, and perceptions of fair (equal) allocation when faced with different costs and health impacts.

Obstetric Ultrasound Simulator With Task-Based Training and Assessment

Tue, 02/02/2016 - 1:11pm

The increasing use of point-of-care (POC) ultrasound presents a challenge in providing efficient training to POC ultrasound users for whom formal training is not readily available. In response to this need, we developed an affordable compact laptop-based obstetric ultrasound training simulator. It offers a realistic scanning experience, task-based training, and performance assessment. The position and orientation of the sham transducer are tracked with 5 DoF on an abdomen-sized scan surface with the shape of a cylindrical segment. On the simulator, user interface is rendered a virtual torso whose body surface models the abdomen of the pregnant scan subject. A virtual transducer scans the virtual torso by following the sham transducer movements on the scan surface. A given 3-D training image volume is generated by combining several overlapping 3-D ultrasound sweeps acquired from the pregnant scan subject using a Markov random field-based approach. Obstetric ultrasound training is completed through a series of tasks, guided by the simulator and focused on three aspects: basic medical ultrasound, orientation to obstetric space, and fetal biometry. The scanning performance is automatically evaluated by comparing user-identified anatomical landmarks with reference landmarks preinserted by sonographers. The simulator renders 2-D ultrasound images in real time with 30 frames/s or higher with good image quality; the training procedure follows standard obstetric ultrasound protocol. Thus, for learners without access to formal sonography programs, the simulator is intended to provide structured training in basic obstetrics ultrasound.

Absence or Presence of High-Grade Squamous Intraepithelial Lesion in Cervical Conization Specimens: A Clinicopathologic Study of 540 Cases

Tue, 02/02/2016 - 1:11pm

OBJECTIVES: To explore the implications of cervical conization specimens lacking the targeted high-grade squamous intraepithelial lesions (negative cone).

METHODS: We studied 540 conization procedures: 400 positive cones and 140 negative cones. Clinicopathologic features and 2-year follow-up results were reported.

RESULTS: Negative cones comprised 22% of procedures triggered by CIN2 or higher biopsies. Procedures triggered by cytology produced much higher percentages of negative cones (37% high-grade squamous intraepithelial lesion [HSIL], 46% atypical squamous cells-cannot exclude high-grade squamous intraepithelial lesion [ASC-H], and 76% low-grade squamous intraepithelial lesion-cannot exclude high-grade squamous intraepithelial lesion [LSIL-H]). Upon reviewing negative excision-triggering biopsy and cytology, we downgraded 24 (24%) CIN2 biopsies, three (14%) HSIL, five (83%) ASC-H, and 12 (92%) LSIL-H. One-third of our negative cones can be attributed to overdiagnosis either on biopsy or cytology. Patients with negative cones were older and had smaller excisions, negative colposcopic findings, and negative/equivocal high-risk human papillomavirus (HR-HPV). Within 2 years, 35 (25%) women with negative cones experienced ASCUS or LSIL. Only one (0.7%) recurred as CIN3, a significantly lower percentage than women with positive cones (13%).

CONCLUSIONS: We advocate careful review of all excision-triggering biopsy and cytology, especially in cases of LSIL-H. Patients with negative cones should be surveyed with cytology and HR-HPV testing.

FGF2 and insulin signaling converge to regulate cyclin D expression in multipotent neural stem cells

Mon, 02/01/2016 - 3:54pm

The ex vivo expansion of stem cells is making major contribution to biomedical research. The multipotent nature of neural precursors acutely isolated from the developing central nervous system has been established in a series of studies. Understanding the mechanisms regulating cell expansion in tissue culture would support their expanded use either in cell therapies or to define disease mechanisms. Basic fibroblast growth factor (FGF2) and insulin, ligands for tyrosine kinase receptors, are sufficient to sustain neural stem cells (NSCs) in culture. Interestingly, real-time imaging shows that these cells become multipotent every time they are passaged. Here, we analyze the role of FGF2 and insulin in the brief period when multipotent cells are present. FGF2 signaling results in the phosphorylation of Erk1/2, and activation of c-Fos and c-Jun that lead to elevated cyclin D mRNA levels. Insulin signals through the PI3k/Akt pathway to regulate cyclins at the post-transcriptional level. This precise Boolean regulation extends our understanding of the proliferation of multipotent NSCs and provides a basis for further analysis of proliferation control in the cell states defined by real-time mapping of the cell lineages that form the central nervous system.

Effect of an office-based surgical safety system on patient outcomes

Mon, 02/01/2016 - 3:54pm

OBJECTIVE: To implement a customizable checklist in an interdisciplinary, team-based plastic surgery setting to reduce surgical complications.

METHODS: We examined the effects on patient outcomes and documentation of a customizable, office-based surgical safety checklist. On the basis of the World Health Organization Surgical Safety Checklist, we developed a 28-element, perioperative checklist for use in the office-based surgical setting. The checklist was implemented in an office-based plastic surgery practice with an already high standard of care. We recorded baseline, prechecklist rates for each checklist item and postoperative adverse outcomes via a retrospective chart review of 219 cases. After an education program and 30-day run-in period, a prospective, post-checklist implementation chart review was initiated (n = 184), with outcome data compared to the baseline.

RESULTS: The total number of complications per 100 patients decreased from 15.1 to 2.72 after checklist implementation (P < .0001), for an absolute risk reduction of 12.4. The proportion of patients with one or more complications decreased from 11.9% to 2.72% (P = .0006). Site and side marking increased from 69.9% prechecklist to 97.8% (P < .0001). Medical optimization increased from 90.9% to 99.5% (P < .0001). Emergency medical services (EMS) policy confirmation, case-specific equipment availability, anticipation of estimated blood loss, and verbal confirmation of local anesthetic toxicity precautions increased from 0% to 90.0% (P < .0001), 92.4% (P < .0001), 82.1% (P < .0001), and 91.3% (P < .0001), respectively. Assessment of patient satisfaction increased from 57.1% to 90.8% (P < .0001).

CONCLUSIONS: Implementation of a customizable checklist was associated with a reduction in surgical complications in an office-based plastic surgery practice with an already high standard of care.

Patterns in immunohistochemical usage in extended core prostate biopsies: comparisons among genitourinary pathologists and nongenitourinary pathologists

Mon, 02/01/2016 - 3:54pm

CONTEXT: Immunohistochemical (IHC) stains have known utility in prostate biopsies and are widely used to augment routine staining in difficult cases. Patterns in IHC utilization and differences based on pathologist training and experience is understudied in the peer-reviewed literature.

OBJECTIVES: To compare the rates of IHC usage between specialized (genitourinary; [GU]) and nonspecialized (non-GU) pathologists in extended core prostate biopsies (ECPBs) and the effects of diagnosis; and in cancer cases Gleason grade, disease extent, and perineural invasion on the rate.

DESIGN: Consecutive ECPBs from 2009-2011 were identified and billing data were used to determine the number of biopsies and IHC stains per case. Diagnoses were mapped and in cancer cases, Gleason grade, extent of disease, and perineural invasion were recorded. Pathologists were classified as GU or non-GU on the basis of training and experience.

RESULTS: A total of 618 ECPBs were included in the study. Genitourinary pathologists ordered significantly fewer IHC tests per case and per biopsy than non-GU pathologists. The rate of ordering was most disparate for biopsies of cancerous and benign lesions. For biopsies of cancerous lesions, high-grade cancer, bilateral disease, and perineural invasion decreased the rate of ordering in both groups. In cancer cases, GU pathologists ordered significantly fewer stain tests for highest Gleason grade of 3 + 3 = 6, for patients with focal disease and for patients with multiple positive bilateral cores. The effect of the various predictors on IHC ordering rates was similar in both groups.

CONCLUSIONS: Genitourinary pathologists ordered significantly fewer IHC stain tests than non-GU pathologists in ECPBs. Guidelines to define when IHC workup is necessary and not necessary may be helpful to guide workups.

Systolic and diastolic mechanics in stress cardiomyopathy

Mon, 02/01/2016 - 3:53pm

BACKGROUND: Stress cardiomyopathy (SCM) is a peculiar form of reversible left ventricular dysfunction seen predominantly in women and occurs in response to emotional or physical stress. Because dysfunction in SCM is reversible and that of acute myocardial infarction (MI) is not, we hypothesized that these fundamental mechanistic differences between SCM and MI would be associated with different systolic and diastolic properties.

METHODS AND RESULTS: We examined 3 groups, all women: patients with SCM (n=24; mean age, 63+/-12 years), those with left anterior (LAD) ST-segment-elevation MI (n=36; mean age, 63+/-10 years), and referent control subjects (n=30; mean age, 62+/-8 years). All underwent angiography, ventriculography, and pressure measurements within 48 hours of presentation. Left ventricular volumes, diastolic pressures, and diastolic stiffness were higher in SCM and LAD MI patients than in control subjects but no different from each other. Similarly, left ventricular diastolic pressures and diastolic stiffness were elevated in the SCM and LAD MI groups compared with the control group. Left ventricular ejection fraction in SCM and LAD MI were 40.8+/-12.3% and 49.6+/-5.6%, respectively, versus 70.4+/-9.4% in control subjects (P < 0.001), and stroke work less than half the value of control subjects. Indexes of contractility and ventricular-arterial coupling were similarly abnormal in SCM and LAD MI.

CONCLUSIONS: SCM and LAD MI show severe diastolic dysfunction. At similar left ventricular volumes, their diastolic pressures are more than twice as high as in control subjects, and systolic dysfunction is equally reduced in SCM and LAD MI. Despite a completely different pathophysiology in terms of systolic and diastolic function, SCM is indistinguishable from acute LAD-territory MI.

The use of acellular dermal regeneration template for recalcitrant pilonidal disease

Mon, 02/01/2016 - 3:53pm

Numerous techniques have been described for the treatment of pilonidal disease, yet there remains no consensus on the optimal management of recurrent pilonidal disease. Pilonidal wounds often lack the structural integrity to heal over and Integra provides a scaffold for the regrowth of an autogenous dermis from the patient's own fibroblasts and collagen. Postoperative negative pressure wound therapy (NPWT) may speed vascularisation of Integra, re-epithelialisation, and wound closure. This case report concerns two patients with chronic pilonidal sinuses who underwent wide excision and placement of Integra with postoperative NPWT. Postoperatively, the patients were assessed for complications and recurrence. Both patients went on to heal and did not require further surgical treatment after a median follow-up of 29 months. Integra may help prevent pocket or cyst formation during the closure process and provides a neodermis, allowing for full re-epithelialisation. More research and a longer follow-up are needed to evaluate the role of Integra and NPWT in recurrent pilonidal disease.

Determining cochlear implant users' true noise tolerance: use of speech reception threshold in noise testing

Mon, 02/01/2016 - 3:53pm

OBJECTIVE: The speech perception abilities of cochlear implant (CI) recipients have significantly improved over the past decade. At the same time, clinical test batteries to measure their performance in noise remain mostly unchanged, resulting in ceiling-level performance for the most successful recipients. The goal of this study is to determine the true noise tolerance abilities of CI recipients using adaptive speech reception threshold (SRT) in noise testing.

STUDY DESIGN: Prospective clinical study.

SETTING: Tertiary care hospital; CI program.

PATIENTS: Ten CI users, either unilateral or bilateral, with HINT scores that equaled or exceeded 80% when administered with a fixed +10 dB signal-to-noise (SNR) ratio (i.e., HINT(+10dB)).

INTERVENTION: The HINT with adaptive SNR levels and QuickSIN test were administered to measure noise tolerance at speech thresholds where 50% of the stimuli were correctly perceived.

MAIN OUTCOME MEASURE(S): SRTs were measured for both the adaptive SNR HINT (i.e., HINT(50%)) and the QuickSIN test. These SRTs were compared with the fixed noise level HINT(+10dB) scores as well as to CNC monosyllable word perception scores.

RESULTS: Despite small variance in performance levels on the HINT(+10dB), results of the HINT(50%) ( approximately 16 dB range) and QuickSIN ( approximately 12 dB range) tests demonstrate significant differences in noise tolerance levels among these CI recipients.

CONCLUSION: For excellent CI users, use of adaptive speech threshold tests in noise better defines a user's actual ability to perceive speech than do fixed SNR level tests. SRT-in-noise tests have the advantage of being quick to administer, and the same stimuli can be used over a very wide range of performance levels. The use of adaptive SRT-in-noise tests should be considered a viable and valuable replacement of fixed SNR tests in the CI clinical test battery.

Consumer-oriented interventions for evidence-based prescribing and medicines use: an overview of systematic reviews

Mon, 02/01/2016 - 3:53pm

BACKGROUND: Numerous systematic reviews exist on interventions to improve consumers' medicines use, but this research is distributed across diseases, populations and settings. The scope and focus of reviews on consumers' medicines use also varies widely. Such differences create challenges for decision makers seeking review-level evidence to inform decisions about medicines use. OBJECTIVES: To synthesise the evidence from systematic reviews on the effects of interventions which target healthcare consumers to promote evidence-based prescribing for, and medicines use, by consumers. We sought evidence on the effects on health and other outcomes for healthcare consumers, professionals and services. METHODS: We included systematic reviews published on the Cochrane Database of Systematic Reviews and the Database of Abstracts of Reviews of Effects. We identified relevant reviews by handsearching both databases from start date to Issue 3 2008. We screened and ranked reviews based on relevance to consumers' medicines use, using criteria developed for this overview. Standardised forms were used to extract data, and reviews were assessed for methodological quality using the AMSTAR instrument. We used standardised language to summarise results within and across reviews; and a further synthesis step was used to give bottom-line statements about intervention effectiveness. Two review authors selected reviews, extracted and analysed data. We used a taxonomy of interventions to categorise reviews. MAIN RESULTS: We included 37 reviews (18 Cochrane, 19 non-Cochrane), of varied methodological quality.Reviews assessed interventions with diverse aims including support for behaviour change, risk minimisation, skills acquisition and information provision. No reviews aimed to promote systems-level consumer participation in medicines-related activities. Medicines adherence was the most commonly reported outcome, but others such as clinical (health and wellbeing), service use and knowledge outcomes were also reported. Reviews rarely reported adverse events or harms, and the evidence was sparse for several populations, including children and young people, carers, and people with multimorbidity.Promising interventions to improve adherence and other key medicines use outcomes (eg adverse events, knowledge) included self-monitoring and self-management, simplified dosing and interventions directly involving pharmacists. Other strategies showed promise in relation to adherence but their effects were less consistent. These included reminders; education combined with self-management skills training, counselling or support; financial incentives; and lay health worker interventions.No interventions were effective to improve all medicines use outcomes across all diseases, populations or settings. For some interventions, such as information or education provided alone, the evidence suggests ineffectiveness; for many others there is insufficient evidence to determine effects on medicines use outcomes. AUTHORS' CONCLUSIONS: Systematically assembling the evidence across reviews allows identification of effective or promising interventions to improve consumers' medicines use, as well as those for which the evidence indicates ineffectiveness or uncertainty.Decision makers faced with implementing interventions to improve consumers' medicines use can use this overview to inform these decisions and also to consider the range of interventions available; while researchers and funders can use this overview to determine where research is needed. However, the limitations of the literature relating to the lack of evidence for important outcomes and specific populations, such as people with multimorbidity, should also be considered.

Semi-jailing technique for coil embolization of complex, wide-necked intracranial aneurysms

Mon, 02/01/2016 - 3:53pm

OBJECTIVE: Stent-assisted coiling of intracranial aneurysms is performed by placing a microcatheter through a stent's interstices or jailing the microcatheter between the stent and the artery. Both approaches impede manipulation of the microcatheter during coiling. We describe a modified jailing technique that improves catheter maneuverability and report the safety and efficacy of the method for the treatment of complex, wide-necked aneurysms.

METHODS: The semi-jailing technique involves the partial deployment of a retrievable stent, bridging part of the aneurysm neck while leaving space to maneuver the microcatheter. Twenty-two complex, wide-necked aneurysms, including 3 ruptured and 5 dissecting, were treated using the semi-jailing technique (15 women; mean age, 55.2 years).

RESULTS: The semi-jailing technique was successfully applied in all cases. Immediate posttreatment angiograms showed total occlusion of the aneurysm in 17 cases (77%), neck remnant in 3 cases (14%), and aneurysm dome filling in 2 cases (9%). Follow-up angiography available in 10 patients at an average of 8.5 months showed progressive occlusion in 1 aneurysm and 7 remained occluded. In 2 cases of dissecting aneurysms, retreatment was required. No permanent periprocedural morbidity was encountered. One patient died of complications secondary to intracranial hemorrhage 6 days after treatment. In 2 cases (9%), thromboembolic events after final stent placement were successfully treated with intraarterial thrombolysis. No delayed stent migration was seen.

CONCLUSION: Semi-jailing is a safe and effective stent-assisted coiling technique that facilitates treatment of complex, wide-necked aneurysms.

Evidence-based review of therapeutic plasma exchange in neurological disorders

Sat, 01/30/2016 - 9:55pm

Several neurologic disorders have been treated with therapeutic plasma exchange (TPE). Case reports, case series, and clinical trials have published results regarding the outcomes in such patients. The data gathered have been used to formulate evidence-based guidelines, which can be used to guide therapy in patients with these neurological disorders. Adequately designed and powered randomized controlled trials have proven the efficacy of TPE in some disease entities, while other diseases are lacking such data. In the latter, decisions for the use of TPE must be made using the limited published data available. In this review, we discuss the published evidence regarding the use of TPE in neurological disorders, focusing on the most recent guidelines published by the American Society of Apheresis in 2010 and the American Academy of Neurology in 2011.

Automated red blood cell exchange for acute drug removal in a patient with sirolimus toxicity

Sat, 01/30/2016 - 9:55pm

Sirolimus is an immunosuppressant used to prevent graft versus host disease in allogeneic hematopoietic stem cell transplant recipients. It has a large volume of distribution (12 +/- 7.5 l/kg) and within the intravascular space approximately 95% of it is bound to red blood cells. Because of potential toxic effects at high trough levels, therapeutic drug monitoring is recommended for sirolimus. We present a case of severe hepatic dysfunction due to Hepatitis B and sirolimus toxicity, in a 51-year-old male stem cell transplant recipient. An automated red cell exchange decreased his blood sirolimus level from 22.6 to 10.3 ng/ml (55% reduction) and improved his liver enzymes. Re-equilibration of sirolimus from other compartments to the blood necessitated a series of four red cell exchanges, after which the sirolimus level was 4.7 ng/ml. Although the patient ultimately succumbed to multiorgan failure, red cell exchange may be considered for acute removal of sirolimus in selected patients.

Prospective surveillance of D- recipients of D+ apheresis platelets: alloimmunization against D is not detected

Sat, 01/30/2016 - 9:55pm

BACKGROUND: Recent retrospective studies indicate that D- recipients of D+ apheresis platelets (PLTs) are not alloimmunized to D. Our hospital policy is to offer RhIG to D- women of childbearing age who received D+ apheresis PLTs but not to other D- recipients of D+ apheresis PLTs. We instituted prospective surveillance of the D- recipients who were not given RhIG.

STUDY DESIGN AND METHODS: All apheresis PLT recipients were prospectively entered into a database that recorded the patient's age, sex, diagnosis, D status, apheresis PLT transfusions, and antibody screen results from before and after PLT transfusions. Data are reported for PLTs transfused between October 16, 2012, and April 16, 2014, and antibody screens obtained through June 16, 2014. The analysis excludes neonates; women not more than 50 years of age; and patients who also received D+ red blood cells, received only D- PLTs, received RhIG, were previously alloimmunized to D, and did not have a follow-up antibody screen after the first D-incompatible apheresis PLT transfusion.

RESULTS: A total of 158 of 1107 apheresis PLT recipients were D-. Seventy-nine were eligible for analysis based on the exclusion criteria listed above. None became alloimmunized to D during the observation interval. In 45 (57%) cases the last follow-up antibody screen was obtained at least 4 weeks after the first D-incompatible apheresis PLT transfusion.

CONCLUSION: Prospective surveillance confirms prior retrospective observations that D- patients do not appear to risk D alloimmunization after receiving D+ apheresis PLTs.

Physiological measurements corroborate symptomatic improvement after therapeutic leukapheresis in a pregnant woman with chronic myelogenous leukemia

Sat, 01/30/2016 - 9:55pm

Therapeutic leukapheresis can control the white blood cell count (WBC) of pregnant women with chronic myelogenous leukemia (CML) who have hyperleukocytosis without leukostasis. The medical justification for this treatment has not been objectively documented. We report a 27-year-old woman, diagnosed with CML at 10-week gestation, who developed severe dyspnea on exertion. A workup that included chest CT and echocardiography with a bubble study detected no cardiopulmonary pathology to explain her symptoms, and thus she was referred for leukapheresis. Prior to her first leukapheresis, which lowered her WBC from 154 x 103 /muL to 133 x 103 /muL, her oxygen saturation (SpO2 ) on room air decreased from 98 to 93% during 100 feet of slow ambulation and she was dyspneic. Just after the leukapheresis, her dyspnea on exertion was much improved and her SpO2 remained at 98% with repeat ambulation. Spirometry and lung volume studies obtained before and after her first leukapheresis demonstrated 32 and 31% improvements in forced vital capacity and forced expiratory volume in 1 s respectively, a 25% increase in functional residual capacity, and a 142% improvement in expiratory reserve volume. Residual volume decreased by almost 20%. Three times in a week, leukapheresis was continued until her WBC was controlled with interferon alpha-2b approximately 4 weeks later. Her dyspnea had completely resolved. She gave birth by elective caesarean section to a healthy boy at 32 weeks. Corroboration of symptom relief by leukapheresis with physiological data may justify such treatment in pregnant patients with CML.

Therapeutic leukocytapheresis for improvement in respiratory function in a woman with hyperleukocytosis and mantle cell lymphoma with a circulating small lymphocyte phenotype

Sat, 01/30/2016 - 9:55pm

Mantle cell lymphoma is an aggressive malignant B-cell disorder that often presents with a leukemic picture. Circulating lymphoma cell morphology may vary from small round mature-appearing lymphocytes resembling the lymphocytes of chronic lymphocytic leukemia to large prolymphocytoid or blastoid cells. Rare reports of hyperleukocytosis with leukostasis, treated with leukocytapheresis, are described in patients with prolymphocytoid or blastoid morphology. We report an 88 year old woman with mantle cell lymphoma, hyperleukocytosis (WBC > 400 x 103 /microL) with severe respiratory compromise but without interstitial or alveolar infiltrates on radiograph or computerized tomography of the chest. She was afebrile and had no central nervous system signs. Circulating lymphoma cell morphology was predominantly of the small lymphocyte type. A two-whole-blood-volume leukocytapheresis reduced her WBC from 465 to 221 x 103 /microL in 150 min. Her respiratory rate decreased from 28/min to 18/min and her arterial oxygen saturation (SpO2 ) rose from 91% to 97% on 6 L/min of oxygen by nasal cannula. Severe breathlessness before the procedure abated completely by the end of the procedure. Respiratory compromise may occur in mantle cell lymphoma with hyperleukocytosis with a mature lymphoma cell phenotype, even without a clear picture of leukostasis. Although the ultimate survival of the patient depends on treatment with chemotherapy, leukocytapheresis for alleviation of symptoms may be warranted and should be considered. Respiratory status and response to leukocytapheresis should be documented with physiological measurements.

Simultaneous generation of many RNA-seq libraries in a single reaction

Sat, 01/30/2016 - 9:11pm

Although RNA-seq is a powerful tool, the considerable time and cost associated with library construction has limited its utilization for various applications. RNAtag-Seq, an approach to generate multiple RNA-seq libraries in a single reaction, lowers time and cost per sample, and it produces data on prokaryotic and eukaryotic samples that are comparable to those generated by traditional strand-specific RNA-seq approaches.

Functional annotation of native enhancers with a Cas9-histone demethylase fusion

Sat, 01/30/2016 - 9:11pm

Understanding of mammalian enhancers is limited by the lack of a technology to rapidly and thoroughly test the cell type-specific function. Here, we use a nuclease-deficient Cas9 (dCas9)-histone demethylase fusion to functionally characterize previously described and new enhancer elements for their roles in the embryonic stem cell state. Further, we distinguish the mechanism of action of dCas9-LSD1 at enhancers from previous dCas9-effectors.

Adenovirus-Mediated Somatic Genome Editing of Pten by CRISPR/Cas9 in Mouse Liver in Spite of Cas9-Specific Immune Responses

Sat, 01/30/2016 - 9:11pm

CRISPR/Cas9 derived from the bacterial adaptive immunity pathway is a powerful tool for genome editing, but the safety profiles of in vivo delivered Cas9 (including host immune responses to the bacterial Cas9 protein) have not been comprehensively investigated in model organisms. Nonalcoholic steatohepatitis (NASH) is a prevalent human liver disease characterized by excessive fat accumulation in the liver. In this study, we used adenovirus (Ad) vector to deliver a Streptococcus pyogenes-derived Cas9 system (SpCas9) targeting Pten, a gene involved in NASH and a negative regulator of the PI3K-AKT pathway, in mouse liver. We found that the Ad vector mediated efficient Pten gene editing even in the presence of typical Ad vector-associated immunotoxicity in the liver. Four months after vector infusion, mice receiving the Pten gene-editing Ad vector showed massive hepatomegaly and features of NASH, consistent with the phenotypes following Cre-loxP-induced Pten deficiency in mouse liver. We also detected induction of humoral immunity against SpCas9 and the potential presence of an SpCas9-specific cellular immune response. Our findings provide a strategy to model human liver diseases in mice and highlight the importance considering Cas9-specific immune responses in future translational studies involving in vivo delivery of CRISPR/Cas9.

Massively parallel sampling of lattice proteins reveals foundations of thermal adaptation

Sat, 01/30/2016 - 9:11pm

Evolution of proteins in bacteria and archaea living in different conditions leads to significant correlations between amino acid usage and environmental temperature. The origins of these correlations are poorly understood, and an important question of protein theory, physics-based prediction of types of amino acids overrepresented in highly thermostable proteins, remains largely unsolved. Here, we extend the random energy model of protein folding by weighting the interaction energies of amino acids by their frequencies in protein sequences and predict the energy gap of proteins designed to fold well at elevated temperatures. To test the model, we present a novel scalable algorithm for simultaneous energy calculation for many sequences in many structures, targeting massively parallel computing architectures such as graphics processing unit. The energy calculation is performed by multiplying two matrices, one representing the complete set of sequences, and the other describing the contact maps of all structural templates. An implementation of the algorithm for the CUDA platform is available at and calculates protein folding energies over 250 times faster than a single central processing unit. Analysis of amino acid usage in 64-mer cubic lattice proteins designed to fold well at different temperatures demonstrates an excellent agreement between theoretical and simulated values of energy gap. The theoretical predictions of temperature trends of amino acid frequencies are significantly correlated with bioinformatics data on 191 bacteria and archaea, and highlight protein folding constraints as a fundamental selection pressure during thermal adaptation in biological evolution.