A comparison of RNA extraction and sequencing protocols for detection of small RNAs in plasma.

BACKGROUND: Circulating microRNAs (miRNAs) are attractive non-invasive biomarkers for a variety of conditions due to their stability and altered pathophysiological expression levels. Reliable detection of global expression profiles is required to maximise miRNA biomarker discovery. Although developments in small RNA-Seq technology have improved detection of plasma-based miRNAs, the low RNA content and sequencing bias introduced during library preparation remain challenging. In this study we compare commercially available RNA extraction methods using MagnaZol (Bioo Scientific) or miRNeasy (QIAGEN) and three library preparation methods - CleanTag (TriLink), NEXTflex (Bioo Scientific) and QIAseq (QIAGEN) - which aim to address one or both of these issues. RESULTS: Different RNA extractions and library preparation protocols result in differential detection of miRNAs. A greater proportion of reads mapped to miRNAs in libraries prepared with MagnaZol RNA than with miRNeasy RNA. Libraries prepared using QIAseq demonstrated the greatest miRNA diversity with many more very low abundance miRNAs detected (~ 2-3 fold more with < 10 reads), whilst CleanTag detected the fewest individual miRNAs and considerably over-represented miR-486-5p. Libraries prepared with QIAseq had the strongest correlation with RT-qPCR quantification. Analysis of unique molecular indices (UMIs) incorporated in the QIAseq protocol indicate that little PCR bias is introduced during small RNA library preparation. CONCLUSIONS: Small RNAs were consistently detected using all RNA extraction and library preparation protocols tested, but with some miRNAs at significantly different levels. Choice of the most suitable protocol should be informed by the relative importance of minimising the total sequencing required, detection of rare miRNAs or absolute quantification.

Influence of lifestyle choices on risks of CYP1B1 polymorphisms for prostate cancer.

Cytochrome P450 1B1 (CYP1B1) converts xenobiotics to carcinogens and how lifestyle choices may interact with CYP1B1 polymorphisms and affect prostate cancer risk was assessed. Blood genomic DNA from a Caucasian population was analysed at polymorphic sites of the 5' untranslated region of CYP1B1 using TaqMan genotyping assays. Overall, drinker status and minor alleles at rs2551188, rs2567206 and rs10175368 were associated with prostate cancer. Linkage was observed between rs2551188, rs2567206, rs2567207 and rs10175368, and the G-C-T-G haplotype (major allele at respective sites) was decreased in cancer. Interestingly when classified by lifestyle factors, no associations of genotypes were found for non-smokers and non-drinkers, whereas on the contrary, minor type at rs2567206 and rs10175368 increased and major G-C-T-G decreased risk for cancer among smokers and drinkers. Interestingly, rs2551188, rs2567206 and rs10175368 minor genotypes correlated with increased tissue CYP1B1 as determined by immunohistochemistry. Further, rs10175368 enhanced luciferase activity and mobility shift show stronger binding of nuclear factor for the minor allele. These results demonstrate smoking and alcohol consumption to modify the risks of CYP1B1 polymorphisms for prostate cancer which may be through rs10175368, and this is of importance in understanding their role in the pathogenesis and as a biomarker for this disease.

Reactivation of retinopathy of prematurity after ranibizumab treatment.

INTRODUCTION: Retinopathy of prematurity (ROP) is a vasoproliferative disorder of the developing retina and a significant cause of childhood blindness around the world. Vascular endothelial growth factor (VEGF) plays an important role in the neovascular phase of ROP, and treatment with an anti-VEGF agent is justified in select cases. Bevacizumab is the most commonly used anti-VEGF agent in ROP, but ranibizumab has a shorter half-life with the potential for decreased systemic toxicity. The purpose of this study is to report our experience with anti-VEGF agents for the treatment of ROP. METHODS: A retrospective chart review was performed on consecutive infants screened for ROP. Infants treated with peripheral retinal ablation, bevacizumab 0.625 mg/0.025 mL, or ranibizumab 0.25 mg/0.025 mL were specifically identified for review of their clinical outcomes. All treated infants had at least 6 months of follow-up with the treating team and were examined until total regression of ROP. RESULTS: One hundred and forty-two infants were screened over a two-year period. Six infants received anti-VEGF agents, with a mean gestational age of 23.48 weeks and mean birth weight of 620 g. Ten eyes from the six infants received anti-VEGF treatment. All ten eyes demonstrated initial regression of ROP. However, ROP reactivation occurred in 5/6 (83%) eyes treated with ranibizumab, on average 5.9 weeks after treatment; whereas none of the 4 eyes treated with bevacizumab experienced reactivation (P < 0.05). One infant who received a unilateral injection of ranibizumab demonstrated bilateral regression of ROP. CONCLUSION: The role of anti-VEGF treatment for ROP is still being evaluated. Although the shorter half-life of ranibizumab makes it an attractive option, reactivation of ROP is possible. Physicians and families should be aware of this to follow infants closely for an extended period of time.

Providing prescheduled appointments as a strategy for improving follow-up compliance after community-based glaucoma screening: results from an urban underserved population.

To determine if receiving a prescheduled appointment is associated with an increased likelihood of complying with follow-up eye care among individuals identified as at risk for glaucoma during community-based glaucoma screening in an urban underserved population. This study sampled 362 individuals aged ≥30 years without known glaucoma from low-income, predominantly black/Hispanic neighborhoods in New Haven, Connecticut presenting to one of twelve community-based glaucoma screening events from May 2010 to October 2012. A quasi-experimental design systematically assigned 63 individuals identified as at risk for glaucoma into either intervention or control group with a 1:2 ratio. Individuals in the control group (n = 41) received counseling on glaucoma and a recommendation for obtaining a follow-up appointment at the eye department of a local community health center, which offers affordable health services with income-adjusted fee discounts to uninsured, low-income patients. Those in the intervention group (n = 22) received the same counseling and a prescheduled appointment at the community health center. The overall rate of follow-up compliance within 3 months of screening was 30 % (41 % in the intervention group; 24 % in the control group). Multivariate logistic regression analysis adjusting for sex, age, ethnicity, health insurance status, car access, living situation, and smoking status found that follow-up compliance was significantly associated with intervention (adjusted odds ratio 4.8; 95 % confidence interval 1.1-20.9). Providing prescheduled appointments can improve follow-up compliance after community-based glaucoma screening. This finding may be potentially applicable to community-based health screening for other preventable diseases.

Outcomes of breast reconstruction in breast cancer patients with a history of mantle radiation for Hodgkin lymphoma.

BACKGROUND: Although mantle radiation (ie, extended field radiation) represented the standard of care in the past for Hodgkin disease, contemporary treatment of lymphoma consists of a multimodal approach with chemotherapy. Patients who were exposed to mantle radiation have a higher risk of breast cancer and are more susceptible to postoperative complications after breast reconstruction due to radiation. In this study, we present postoperative outcomes in patients with a history of mantle radiation who underwent mastectomy and breast reconstruction. METHODS: All patients at Stanford University Medical Center between January 2006 and December 2012 with a history of Hodgkin lymphoma treated with mantle radiation who received breast reconstruction were identified. A retrospective chart review was conducted analyzing patient demographics, history of Hodgkin treatment, type of reconstruction, follow-up, and complications. Complications were further classified into medical complications, donor-site complications, and recipient-site complications. RESULTS: Sixteen patients with a history of Hodgkin disease and mantle radiation received breast reconstruction. The average age of the patients at their mastectomy was 46 (33-60) years, with the average age at the time of their mantle radiation of 20.5 (10-33) years with an average interval of radiation to breast cancer treatment of 24.8 (16-38) years. There were five unilateral and 11 bilateral reconstructions. All patients had immediate reconstruction with tissue expanders (14 patients) or autologous tissue (one muscle-sparing transverse rectus abdominis myocutaneous and one transverse upper gracilis flap). Eleven (69%) patients had postoperative complications. In the patients who had tissue expander reconstruction, there was an overall complication rate of 64%, which included capsular contracture (n=5, 56%), mastectomy flap necrosis (n=5, 56%), cellulitis (n=4, 44%), seroma (n=3, 33%), hematoma (n=1, 11%), and chronic pain (n=1, 11%). Three (two unilateral and one bilateral) tissue expander infections required removal of the expander and delayed reconstruction with a latissimus dorsi flap, whereas one patient with chronic pain and capsular contracture required a muscle-sparing transverse rectus abdominis myocutaneous for a unilateral implant failure. CONCLUSIONS: Although the risk of complications associated with preoperative radiation is well documented, physicians and patients should be cognizant of the increased risk of complications after mantle radiation as it represents a unique modality of radiation exposure.

Endoscopic delivery of calcium phosphate cement for secondary craniofacial reconstruction.

Contour defects are common following primary craniofacial procedures including cranial vault remodeling, fronto-orbital and midface advancements, and complex posttraumatic reconstructions. When onlayed as fast-setting pastes, calcium phosphate cements (CPCs) have been used to effectively correct contour defects in open secondary reconstruction procedures. Here, we describe an endoscopic procedure using an injectable CPC and compare surgical outcomes with the open technique. A retrospective review was conducted for 36 consecutive patients aged 3.0-28.9 years (mean, 10.1 years) who underwent secondary craniofacial reconstruction over a 3-year period. Patients were stratified into endoscopic or open groups depending on the surgical approach utilized. Mean operative time was significantly shorter (P < 0.001) for the endoscopic group (64 minutes) than for the open group (131 minutes). Similarly, hospital stay was significantly shorter (P = 0.005) in the endoscopic group than in the open group. There was also a significant difference with respect to cost (P < 0.001), with the endoscopic approach resulting in a per-patient cost savings of $2208.05. In conclusion, endoscopic delivery of CPC appears to be a safe, efficacious, and cost-effective method of performing secondary craniofacial reconstruction, with the additional benefits of decreased operative time and shorter postoperative hospital stay when compared with an open procedure.

Nonsyndromic craniosynostosis: the Rady Children's Hospital approach.

ABSTRACT: Surgical correction for nonsyndromic craniosynostosis has continued to evolve over the last century. The criterion standard has remained open correction of the cranial deformities, and many techniques have been described that yield satisfactory results. However, technology has allowed for minimally invasive techniques to be developed with the aid of endoscopic visualization. With proper patient selection and the aid of postoperative helmet therapy, there is increasing evidence that supports these techniques' safety and efficacy. In this article, our purpose was to describe our algorithm for treating nonsyndromic craniosynostosis at Rady Children's Hospital.

Huperzine a as potential treatment of Alzheimer's disease: an assessment on chemistry, pharmacology, and clinical studies.

Alzheimer's disease (AD) is the fourth leading cause of death in adults, characterized by hallmark neuritic plaques and neurofibrillary tangles. Current treatments focus only on symptom relief. As a possible new treatment option for AD, huperzine A's chemistry, pharmacology, and clinical effectiveness are assessed. The chemical synthesis of huperzine A has been optimized, while an in vitro technique has provided a renewable plant source. Pharmacological studies showed that the drug inhibits the enzyme acetylcholinesterase reversibly and selectively. Huperzine A also displayed good pharmacokinetics with a rapid absorption and a wide distribution in the body at a low to moderate rate of elimination. Presently, inadequate toxicity data in human have been reported, yet animal studies demonstrated mild to moderate cholinergic side effects at therapeutic doses. Previous clinical trials have shown improvement in memory function using MMSE, MQ, ADAS-COG, and ADL tests. In an unpublished phase II clinical trial, the ADAS-COG and MMSE tests indicated cognitive enhancement at a dose of 0.4 mg, yet no improvement was observed at a dose of 0.2 mg. The MMSE scores indicated cognitive enhancement at 0.4 mg. Promising data suggested that huperzine A is well tolerated at doses up to 0.4 mg for 24 weeks. Therefore, huperzine A seems to be a potential treatment option for AD.

Complications of hydroxyapatite bone cement in secondary pediatric craniofacial reconstruction.

Hydroxyapatite cement has become a popular alternative to bone grafts in reconstructing the calvarium. Although animal studies have shown promising results with use of hydroxyapatite, human clinical studies have shown mixed results including significant rates of infection. This is a retrospective chart review during a 7-year period (1997-2003) of 20 patients who underwent secondary forehead cranioplasty with hydroxyapatite cement (Norian Craniofacial Reconstruction System). Basic demographics including age, sex, and diagnosis were identified. Characteristics of the defects were recorded including size, location, and depth (full versus partial thickness). The volume of hydroxyapatite and any adjunctive procedures were identified. The postoperative course was analyzed for length of follow-up and the presence of infections.Twenty patients were identified in which Norian had been used and 3 patients were lost to follow-up. Secondary forehead asymmetry was the most common presentation. The mean volume of hydroxyapatite used was 24.4 mL. All patients had initially acceptable aesthetic results. Of the 17 patients, 10 (59%) ultimately had infectious complications. Infection occurred on a mean of 17.3 months after surgery (range, 4 mo to 4 y), and the mean amount of hydroxyapatite used was 32.5 mL (infections) versus 14.3 mL (no infections). Of the 10 patients with complications, 9 required surgical debridement and subsequent delayed reconstruction. Although hydroxyapatite cement can yield excellent aesthetic results, its use in secondary reconstruction has yielded unacceptably high infection rates leading to discontinuation of its use in this patient population.

Upregulation of miR-130b Contributes to Risk of Poor Prognosis and Racial Disparity in African-American Prostate Cancer.

Prostate cancer incidence and mortality rates are higher in African-American (AA) than in European-American (EA) men. The main objective of this study was to elucidate the role of miR-130b as a contributor to prostate cancer health disparity in AA patients. We also determined whether miR-130b is a prognostic biomarker and a new therapeutic candidate for AA prostate cancer. A comprehensive approach of using cell lines, tissue samples, and the TCGA database was employed. We performed a series of functional assays such as cell proliferation, migration, invasion, RT2-PCR array, qRT-PCR, cell cycle, luciferase reporter, immunoblot, and IHC. Various statistical approaches such as Kaplan-Meier, uni-, and multivariate analyses were utilized to determine the clinical significance of miR-130b. Our results showed that elevated levels of miR-130b correlated with race disparity and PSA levels/failure and acted as an independent prognostic biomarker for AA patients. Two tumor suppressor genes, CDKN1B and FHIT, were validated as direct functional targets of miR-130b. We also found race-specific cell-cycle pathway activation in AA patients with prostate cancer. Functionally, miR-130b inhibition reduced cell proliferation, colony formation, migration/invasion, and induced cell-cycle arrest. Inhibition of miR-130b modulated critical prostate cancer-related biological pathways in AA compared with EA prostate cancer patients. In conclusion, attenuation of miR-130b expression has tumor suppressor effects in AA prostate cancer. miR-130b is a significant contributor to prostate cancer racial disparity as its overexpression is a risk factor for poor prognosis in AA patients with prostate cancer. Thus, regulation of miR-130b may provide a novel therapeutic approach for the management of prostate cancer in AA patients.

Skin sparing nipple reconstruction with polytetrafluoroethylene implant.

This paper describes a technique in which implanted polytetrafluoroethylene is used to reconstruct the nipple without having to perform a local flap or graft from a distant site. This procedure offers the advantage of not taking skin to reconstruct the nipple. In addition, the projection of the nipple is provided by the implanted material and is not obligated to blood supplying a local tissue flap. For these reasons, this technique offers an attractive alternative for patients who have failed past techniques or have compromised vascular supply due to radiation therapy.

Leucine regulation of glucokinase and ATP synthase sensitizes glucose-induced insulin secretion in pancreatic beta-cells.

We have recently shown that leucine culture upregulates ATP synthase beta-subunit (ATPSbeta) and increases ATP level, cytosolic Ca(2+), and glucose-induced insulin secretion in rat islets. The aim is to test whether glucokinase expression is also affected in rat islets and its role in glucose sensitization during leucine culture. Leucine culture increased glucose-induced NAD(P)H level at 1 and 2 days but not at 1 week. The half-maximal effective concentration of the glucose response curve for NAD(P)H was left-shifted from 5-7 to 2-3 mmol/l. The effect was dose dependent and rapamycin insensitive. Leucine culture did not affect glyceraldehyde effects on NAD(P)H. Leucine pretreatment for 30 min had no effects on NAD(P)H levels. Leucine culture for 2 days also increased glucose-induced cytosolic Ca(2+) elevation, ATP level, and insulin secretion. Leucine increase of glucokinase mRNA levels occurred as early as day 1 and lasted through 1 week. That of ATPSbeta did not occur until day 2 and lasted through 1 week. Leucine effects on both mRNAs were dose dependent. The upregulation of both genes was confirmed by Western blotting. Leucine culture also increased glucose-induced insulin secretion, ATP level, glucokinase, and ATPSbeta levels of type 2 diabetic human islets. In conclusion, leucine culture upregulates glucokinase, which increases NAD(P)H level, and ATPSbeta, which increases oxidation of NADH and production of ATP. The combined upregulation of both genes increases glucose-induced cytosolic Ca(2+) and insulin secretion.

PANDER-induced cell-death genetic networks in islets reveal central role for caspase-3 and cyclin-dependent kinase inhibitor 1A (p21).

PANcreatic DERived factor is an islet-specific cytokine that promotes apoptosis in primary islets and islet cell lines. To elucidate the genetic mechanisms of PANDER-induced cell death we performed expression profiling using the mouse PancChip version 5.0 in conjunction with Ingenuity Pathway Analysis. Murine islets were treated with PANDER and differentially expressed genes were identified at 48 and 72 h post-treatment. 64 genes were differentially expressed in response to PANDER treatment. 22 genes are associated with cell death. In addition, the genes with the highest fold change were linked with cell death or apoptosis. The most significantly affected gene at 48 h was the downregulated cyclin-dependent kinase inhibitor 1A (CDKN1A or p21). Approximately half of the genes impacted at 72 h were linked to cell death. Cell death differentially expressed genes were confirmed by quantitative RT-PCR. Further analysis identified cell death genetic networks at both time points with 21 of the 22 cell death genes related in various biological pathways. Caspase-3 (CASP3) was biologically linked to CDKN1A in several genetic networks and these two genes were further examined. Elevated cleaved CASP3 levels in PANDER-treated beta-TC3 insulinoma cells were found to abrogate CDKN1A expression. Levels of CDKN1A were not affected in the absence of cleaved CASP3. PANDER-induced downregulation of CDKN1A expression coupled with induced CASP3-activation may serve a central role in islet cell death and offers further insight into the mechanisms of cytokine-induced beta-cell apoptosis.

Colorado retinopathy of prematurity model: a multi-institutional validation study.

PURPOSE: The Colorado retinopathy of prematurity (ROP) prediction model (CO-ROP), developed using a cohort of infants from Colorado, calls for ROP examination of infants meeting all of the following criteria: gestational age of ≤30 weeks, birth weight of ≤1500 g, and a net weight gain of ≤650 g between birth and 4 weeks of age. The purpose of this study was to perform an external validation to assess the sensitivity and specificity of the CO-ROP model in a larger cohort of babies screened for ROP from four academic institutions in the United States. METHODS: The medical records of neonates screened for ROP according current national guidelines was conducted at 4 US academic centers were retrospectively reviewed. Sensitivity, specificity, and respective 95% confidence intervals in detecting ROP using CO-ROP were calculated for type 1, type 2, and any grade of ROP. RESULTS: A total of 858 cases were included. The CO-ROP algorithm had a sensitivity of 98.1% (95% CI, 93.3%-99.8%) for type 1 ROP, 95.6% (95% CI 78.0-99.9%) for type 2 ROP, and 95.0% (95% CI, 93.1-97.4%) for all grades of ROP. The CO-ROP model would have reduced the total number of infants screened by 23.9% compared to current 2013 screening guidelines. CONCLUSIONS: CO-ROP demonstrated high sensitivity in predicting ROP and would have greatly reduced the number of infants needing examination.

Distinguishing features of leucine and alpha-ketoisocaproate sensing in pancreatic beta-cells.

Culturing rat islets in high glucose (HG) increased 1-(14)C-alpha-ketoisocaproate (KIC) oxidation compared with culturing them in low glucose. Leucine caused insulin secretion (IS) in low glucose but not in HG rat islets, whereas KIC did so in both. Pretreatment with HG for 40 min abolished leucine stimulation of IS by mouse islets and prevented the cytosolic Ca(2+) rise without inhibiting IS and Ca(2+) increments caused by KIC. When islets were pretreated without glucose and glutamine, aminooxyacetic acid (AOA) markedly decreased KIC effects. When islets were pretreated without glucose and with glutamine, AOA potentiated leucine effects but attenuated KIC effects. AOA stimulated glutamine oxidation in the presence but not the absence of +/-2-amino-2-norbornane-carboxylic acid, a nonmetabolized leucine analog. Pretreatment with HG and glutamine partially reversed AOA inhibition of KIC effects. Glucose increased intracellular ATP and GTP, whereas it decreased ADP and GDP in beta HC9 cells. Glutamate dehydrogenase activity of beta HC9 cell extracts was increased by leucine and attenuated by GTP, but it was potentiated by ADP. In conclusion, leucine and KIC stimulated beta-cells via distinct mechanisms. Glutamate dehydrogenase is the sensor of leucine, whereas transamination plays an important role in KIC stimulation of pancreatic beta-cells.

5-Fluorouracil for epithelial downgrowth after Descemet stripping automated endothelial keratoplasty.

PURPOSE: To report the use of intracameral 5-fluorouracil (5-FU) to treat epithelial downgrowth after performing a Descemet-stripping automated endothelial keratoplasty (DSAEK). METHODS: We describe the case of a 79-year-old woman who underwent a DSAEK for pseudophakic Fuchs endothelial corneal dystrophy. The patient required 2 repeat DSAEKs for graft failure and persistent, visually significant interface haze. Six months after the third and most recent DSAEK, the patient was followed up and found to have a visual acuity of 20/100. On examination, the patient continued to have a persistent interface haze. However, the patient also had a cellular anterior chamber reaction despite the administration of frequent topical corticosteroids, and subtle findings of a translucent, membranous growth over the iris. Argon laser photocoagulation of the area resulted in a whitening response, characteristic of epithelial growth. RESULTS: Epithelial downgrowth was diagnosed, and intracameral 5-FU was administered. One month after the injection was given, the patient's examination results and vision improved to 20/60. Six months after the single injection, the patient had a clear DSAEK graft without interface haze, a quiet chamber, and 20/30 visual acuity. CONCLUSIONS: Epithelial downgrowth that occurs after performing a DSAEK can be treated successfully using intracameral 5-FU.

Iris cyst after iris-sutured intraocular lens implantation in a child.

Iris cysts are uncommon in childhood and can present diagnostic and therapeutic challenges. We present the case of a 5-year-old boy with Marfan syndrome who, 9 months after an uncomplicated lensectomy with iris-sutured intraocular lens (IOL), developed a translucent epithelial inclusion cyst. The cyst was observed without treatment and no complications developed. To our knowledge, this is the first case of an iris cyst in a child after implantation of an iris-sutured IOL. This potential complication should be considered when implanting IOLs in children.

Bilateral drug (ipilimumab)-induced vitritis, choroiditis, and serous retinal detachments suggestive of vogt-koyanagi-harada syndrome.

PURPOSE: To report a case of bilateral drug (ipilimumab)-induced vitritis, choroiditis, and serous retinal detachments suggestive of Vogt-Koyanagi-Harada syndrome. METHODS: This is a single case report. RESULTS: A 43-year-old woman with metastatic melanoma reported a 1-day history of blurry vision after starting a phase 1 clinical trial of ipilimumab, a cytotoxic T lymphocyte-associated antigen 4 blocker, 2 weeks before presentation. Fundus examination of both eyes showed bilateral serous detachments with associated vitritis and choroiditis. Optical coherence tomography confirmed the presence of serous detachments, and fluorescein angiography demonstrated multiple areas of pinpoint leakage at the level of the retinal pigment epithelium. Treatment with high-dose corticosteroids led to a rapid improvement in vision and resolution of her serous detachments. Ten weeks after initial presentation, the patient developed vitiligo and poliosis. CONCLUSION: Vogt-Koyanagi-Harada syndrome is a bilateral granulomatous panuveitis associated with dermatologic, neurologic, and audiologic manifestations of yet an unclear pathogenesis. To our knowledge, we report the first case of ipilimumab treatment resulting in the acute presentation of bilateral vitritis, choroiditis, and serous detachments suggestive of Vogt-Koyanagi-Harada syndrome, which has particular significance because it may corroborate previous research attempting to elucidate the underlying mechanism of the disease.