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Avascular necrosis (AVN) may occur in up to 77% of proximal humeral fractures and can cause fixation failure. Risk factors include fracture position, calcar length and medial hinge integrity. We routinely perform intra-articular biceps tenotomy with tenodesis at the level of pectoralis major to facilitate fragment identification and potentially ameliorate post-operative pain relief. Concern exists that tenotomising the biceps damages the adjacent arcuate artery, potentially increasing the rate of AVN. The purpose of this study was to evaluate whether biceps tenodesis is associated with an increased risk of radiographically evident humeral head AVN. 61 fractures surgically treated over a 52-month period were retrospectively reviewed and radiographically assessed in accordance with Neer's classification, calcar-length and medial hinge integrity. 40, 20 and 1 were four-, three- and two-part fractures respectively. 37 had a calcar-length less than 8mm and 26 suffered loss of the medial hinge. The median radiographic follow-up was 23 months. There was radiographic evidence of humeral head AVN in only one case, comparing favourably to rates quoted in current literature. In our experience, intra-articular biceps tenotomy with the deltopectoral approach was thus not associated with a significantly increased risk of humeral head AVN, even in complex four-part fractures.

Posterior sternoclavicular dislocations are common in younger patients and are frequently due to high energy sporting incidents.We aim to demonstrate a novel technique that is safe and aims to provide good functional post-operative outcomes for patients with this injury.This was a single-surgeon case series of four young patients from October 2017 to July 2019. The operative technique involved relocating the joint and holding it in situ with nylon suture tape. The tape was anchored in holes drilled in the sternum and passed through tunnels drilled into the medial clavicle. All of the patients were contacted retrospectively and a Nottingham Clavicle Score (NCS) was performed for each patient on a post-operative basis.No intra-operative or post-operative complications were noted. All of the patients demonstrated a significant improvement in their functional outcomes after the operation. The average NCS for the four patients was 82/100.There are a variety of techniques described in the literature to repair this ligament using either plates or tendon grafts. All of these techniques describe the drilling of anteroposterior holes in the manubrium and clavicle which run the intra-operative risk of perforating a major vessel. This paper is the first one to describe a technique which uses superior inferior holes which minimizes the risks, making the procedure safer for the patient.We believe this novel technique is safer than the existing described techniques, and it does not compromise on functional outcomes.

Regenerating damaged tissue interfaces remains a significant clinical challenge, requiring recapitulation of the structure, composition and function of the native enthesis. In the ligament-to-bone interface this region transitions from ligament to fibrocartilage, to calcified cartilage and then to bone. This gradation in tissue types facilitates the transfer of load between soft and hard structures while minimizing stress concentrations at the interface. Previous attempts to engineer the ligament-bone interface have utilized various scaffold materials with an array of various cell types and/or biological cues. The primary goal of this study was to engineer a multiphased construct mimicking the ligament-bone interface by driving differentiation of a single population of mesenchymal stem cells (MSCs), seeded within blended fibrin-alginate hydrogels, down an endochondral, fibrocartilaginous or ligamentous pathway through spatial presentation of growth factors along the length of the construct within a custom-developed, dual-chamber culture system. MSCs within these engineered constructs demonstrated spatially distinct regions of differentiation, adopting either a cartilaginous or ligamentous phenotype depending on their local environment. Furthermore, there was also evidence of spatially defined progression towards an endochondral phenotype when chondrogenically primed MSCs within this construct were additionally exposed to hypertrophic cues. The study demonstrates the feasibility of engineering spatially complex soft tissues within a single MSC laden hydrogel through the defined presentation of biochemical cues. This novel approach represents a new strategy for engineering the ligament-bone interface. This article is protected by copyright. All rights reserved

The sternoclavicular joint (SCJ) has a complete intra-articular disk that can be damaged either as a result of trauma or as part of ongoing degenerative joint disease. Although often asymptomatic, SCJ disk tears may lead to mechanical symptoms and pain. Previously, isolated symptomatic SCJ disk tears have only occasionally been mentioned in the literature with a few associated case reports of diskectomy by open arthrotomy. With improved imaging and availability of magnetic resonance imaging scans and the advent of SCJ arthroscopy it is now possible to treat symptomatic SCJ disk tears by arthroscopic excision. In this Technical Note, we describe the diagnosis of a torn SCJ disk and the technique of arthroscopic excision of a torn SCJ disk.

The double-stranded RNA-binding protein Staufen has been implicated in various posttranscriptional gene regulatory processes. Here, we demonstrate that the Caenorhabditis elegans homolog of Staufen, STAU-1, functionally interacts with microRNAs. Loss-of-function mutations of stau-1 significantly suppress phenotypes of let-7 family microRNA mutants, a hypomorphic allele of dicer, and a lsy-6 microRNA partial loss-of-function mutant. Furthermore, STAU-1 modulates the activity of lin-14, a target of lin-4 and let-7 family microRNAs, and this modulation is abolished when the 3′ untranslated region of lin-14 is removed. Deep sequencing of small RNA cDNA libraries reveals no dramatic change in the levels of microRNAs or other small RNA populations between wild-type and stau-1 mutants, with the exception of certain endogenous siRNAs in the WAGO pathway. The modulation of microRNA activity by STAU-1 does not seem to be associated with the previously reported enhanced exogenous RNAi (Eri) phenotype of stau-1 mutants, since eri-1 exhibits the opposite effect on microRNA activity. Altogether, our results suggest that STAU-1 negatively modulates microRNA activity downstream of microRNA biogenesis, possibly by competing with microRNAs for binding on the 3′ untranslated region of target mRNAs.

Purpose To describe the results and functional scores in a group of patients who underwent arthroscopic excision of a symptomatic sternoclavicular joint (SCJ) disk tear with a minimum follow-up period of 2 years. Methods Between April 2010 and December 2014, 14 patients underwent arthroscopic excision of a torn SCJ disk. Patients whose intended surgery was an isolated diskectomy and underwent that surgery only, with no additional procedure, were included. The minimum follow-up period was 24 months. All patients underwent an arthroscopic SCJ diskectomy. Postoperatively, no immobilization was required, and the patients were encouraged to mobilize as pain permitted. The patients were assessed preoperatively and at final follow-up with the visual analog scale score for pain, Rockwood score, and QuickDASH (short version of the Disabilities of the Arm, Shoulder and Hand questionnaire) score. Results The average age at surgery was 29.4 years (range, 19-39 years). Ten of the patients had been symptom free before a specific incident, after which SCJ symptoms developed. The other 4 patients reported a gradual onset of symptoms and were considered to have chronic tears. The average duration of symptoms was 22.8 months (range, 6-48 months). At a mean follow-up of 33.4 months (range, 24-59 months), a significant improvement in the Rockwood score was noted, from 7 (range, 5-9; standard deviation [SD], 1.4) to 13.6 (range, 9-15; SD, 1.9) ( P = .001) (minimal clinically important difference not described). The mean QuickDASH score improved from 23.7 points (range, 6.8-40.9 points; SD, 11.8 points) to 8 points (range, 0-29.5 points; SD, 9 points) ( P = .0024) (minimal clinically important difference, 13.4 points). There were no reported complications and specifically no instability. Conclusions The results of this series suggest that arthroscopic SCJ diskectomy is a safe and reproducible procedure for the treatment of patients with symptomatic SCJ disk tears. Level of Evidence Level IV, therapeutic case series.

The study's goal was to determine if differences in weight misperception by race and/or gender occur within a sample of economically disadvantaged rural patients with diabetes and/or hypertension. Diabetic and hypertensive patients were enrolled in the study from a network of federally qualified health centers (FQHCs) in the rural South. Multivari-ate logistic regression analysis suggests that, even when controlling for age, education level, employment status, and poverty, rural African American patients with chronic disease are more likely than their White counterparts to misperceive their weight status (OR = 1.709, p = .037). This difference in perceived weight occurred despite the absence of an underlying difference in actual weight status between African American and White patients (p = .171). In addition, rural men were much more likely than rural women to misperceive their weight status (OR = 2.688, plt; .001). Implications for intervention development and implementation are discussed.

Regenerating damaged tissue interfaces remains a significant clinical challenge, requiring recapitulation of the structure, composition, and function of the native enthesis. In the ligament-to-bone interface, this region transitions from ligament to fibrocartilage, to calcified cartilage and then to bone. This gradation in tissue types facilitates the transfer of load between soft and hard structures while minimizing stress concentrations at the interface. Previous attempts to engineer the ligament-bone interface have utilized various scaffold materials with an array of various cell types and/or biological cues. The primary goal of this study was to engineer a multiphased construct mimicking the ligament-bone interface by driving differentiation of a single population of mesenchymal stem cells (MSCs), seeded within blended fibrin-alginate hydrogels, down an endochondral, fibrocartilaginous, or ligamentous pathway through spatial presentation of growth factors along the length of the construct within a custom-developed, dual-chamber culture system. MSCs within these engineered constructs demonstrated spatially distinct regions of differentiation, adopting either a cartilaginous or ligamentous phenotype depending on their local environment. Furthermore, there was also evidence of spatially defined progression toward an endochondral phenotype when chondrogenically primed MSCs within this construct were additionally exposed to hypertrophic cues. The study demonstrates the feasibility of engineering spatially complex soft tissues within a single MSC laden hydrogel through the defined presentation of biochemical cues. This novel approach represents a new strategy for engineering the ligament-bone interface. Biotechnol. Bioeng. 2017;114: 2400-2411. © 2017 Wiley Periodicals, Inc.

We inventoried non-surface water sources in the Leogane and Gressier region of Haiti (approximately 270 km(2)) in 2012 and 2013 and screened water from 345 sites for fecal coliforms and Vibrio cholerae. An international organization/non-governmental organization responsible for construction could be identified for only 56% of water points evaluated. Sixteen percent of water points were non-functional at any given time; 37% had evidence of fecal contamination, with spatial clustering of contaminated sites. Among improved water sources (76% of sites), 24.6% had fecal coliforms versus 80.9% in unimproved sources. Fecal contamination levels increased significantly from 36% to 51% immediately after the passage of Tropical Storm Sandy in October of 2012, with a return to 34% contamination in March of 2013. Long-term sustainability of potable water delivery at a regional scale requires ongoing assessment of water quality, functionality, and development of community-based management schemes supported by a national plan for the management of potable water.

In 2010 a magnitude 7.0 earthquake struck Haiti, severely damaging the drinking and waste water infrastructure and leaving millions homeless. Compounding this problem, the introduction of Vibrio cholera resulted in a massive cholera outbreak that infected over 700,000 people and threatened the safety of Haiti’s drinking water. To mitigate this public health crisis, non-government organizations installed thousands of wells to provide communities with safe drinking water. However, despite increased access, Haiti currently lacks the monitoring capacity to assure the microbial safety of any of its water resources. For these reasons, this study was designed to assess the feasibility of using a simple, low cost method to detect indicators of fecal contamination of drinking water that could be implemented at the community level. Water samples from 358 sources of drinking water in the Léogâne flood basin were screened with a commercially available hydrogen sulfide test and a standard membrane method for the enumeration of thermotolerant coliforms. When compared with the gold standard method, the hydrogen sulfide test had a sensitivity of 65% and a specificity of 93%. While the sensitivity of the assay increased at higher fecal coliform concentrations, it never exceeded 88%, even with fecal coliform concentrations greater than 100 colony forming units per 100 milliliters. While its simplicity makes the hydrogen sulfide test attractive for assessing water quality in low resource settings, the low sensitivity raises concerns about its use as the sole indicator of the presence or absence of fecal coliforms in individual or community water sources.

Towards the end of my first year of training, I was on placement on a hospital ward when I heard the alarm bell go off in a bathroom.

Preclinical results with various gene therapy strategies indicate significant potential for new cancer treatments. However, many therapeutics fail at clinical trial, often due to differences in tissue physiology between animal models and humans, and tumor phenotype variation. Clinical data relevant to treatment strategies may be generated prior to clinical trial through experimentation using intact patient tissue ex vivo. We developed a novel tumor slice model culture system that is universally applicable to gene delivery methods, using a realtime luminescence detection method to assess gene delivery. Methods investigated include viruses (adenovirus [Ad] and adeno-associated virus), lipofection, ultrasound (US), electroporation and naked DNA. Viability and tumor populations within the slices were well maintained for seven days, and gene delivery was qualitatively and quantitatively examinable for all vectors. Ad was the most efficient gene delivery vector with transduction efficiency >50%. US proved the optimal non-viral gene delivery method in human tumor slices. The nature of the ex vivo culture system permitted examination of specific elements. Parameters shown to diminish Ad gene delivery included blood, regions of low viability and secondary disease. US gene delivery was significantly reduced by blood and skin, while tissue hyperthermia improved gene delivery. US achieved improved efficacy for secondary disease. The ex vivo model was also suitable for examination of tissue-specific effects on vector expression, with Ad expression mediated by the CXCR4 promoter shown to provide a tumor selective advantage over the ubiquitously active cytomegalovirus promoter. In conclusion, this is the first study incorporating patient tissue models in comparing gene delivery from various vectors, providing knowledge on cell-type specificity and examining the crucial biological factors determining successful gene delivery. The results highlight the importance of in-depth preclinical assessment of novel therapeutics and may serve as a platform for further testing of current, novel gene delivery approaches.

Development of gene-based therapies for the treatment of inherited and acquired diseases, including cancer, has seen renewed interest in the use of nonviral vectors coupled to physical delivery modalities. Low-frequency ultrasound (US), with a well-established record in a clinical setting, has the potential to deliver DNA efficiently, accurately and safely. Optimal in vivo parameters for US-mediated delivery of naked plasmid DNA were established using the firefly luciferase reporter gene construct. Optimized parameters were used to administer a therapeutic gene construct, coding for granulocyte-macrophage colony-stimulating factor (GM-CSF) and B7-1 costimulatory molecule, to growing murine fibrosarcoma tumors. Tumor progression and animal survival was monitored throughout the study and the efficacy of the US-mediated gene therapy determined and compared with an electroporation-based approach. Optimal parameters for US-mediated delivery of plasmid DNA to tumors were deduced to be 1.0 W/cm 2 at 20% duty cycle for 5 min (60 J/cm 2 ). In vivo US-mediated gene therapy resulted in a 55% cure rate in tumor-bearing animals. The immunological response invoked was cell mediated, conferring resistance against re-challenge and resistance to tumor challenge after transfer of splenocytes to naive animals. US treatment was noninjurious to treated tissue, whereas therapeutic efficacy was comparable to an electroporation-based approach. US-mediated delivery of an immune-gene construct to growing tumors was therapeutically effective. Sonoporation has the potential to be a major factor in the development of nonviral gene delivery approaches. (E-mail: geraldc@iol.ie )

Challenges for oncology practitioners and researchers include specific treatment and detection of tumours. The ideal anti-cancer therapy would selectively eradicate tumour cells, whilst minimising side effects to normal tissue. Bacteria have emerged as biological gene vectors with natural tumour specificity, capable of homing to tumours and replicating locally to high levels when systemically administered. This property enables targeting of both the primary tumour and secondary metastases. In the case of invasive pathogenic species, this targeting strategy can be used to deliver genes intracellularly for tumour cell expression, while non-invasive species transformed with plasmids suitable for bacterial expression of heterologous genes can secrete therapeutic proteins locally within the tumour environment (cell therapy approach). Many bacterial genera have been demonstrated to localise to and replicate to high levels within tumour tissue when intravenously (IV) administered in rodent models and reporter gene tagging of bacteria has permitted real-time visualisation of this phenomenon. Live imaging of tumour colonising bacteria also presents diagnostic potential for this approach. The nature of tumour selective bacterial colonisation appears to be tumour origin- and bacterial species- independent. While originally a correlation was drawn between anaerobic bacterial colonisation and the hypoxic nature of solid tumours, it is recently becoming apparent that other elements of the unique microenvironment within solid tumours, including aberrant neovasculature and local immune suppression, may be responsible. Here, we consider the pre-clinical and clinical data supporting the use of bacteria as a tumour-targeting tool, recent advances in the area, and future work required to develop a beneficial clinical treatment.

An improved system for luciferase tagging Listeria monocytogenes was developed by constructing a highly active, constitutive promoter. This construct gave 100-fold-higher activity in broth than any native promoter tested and allowed for imaging of lux -tagged L. monocytogenes in food products, during murine infections, and in tumor targeting studies.

A cholera outbreak began in Haiti during October, 2010. Spatiotemporal patterns of household-level cholera in Ouest Department showed that the initial clusters tended to follow major roadways; subsequent clusters occurred further inland. Our data highlight transmission pathway complexities and the need for case and household-level analysis to understand disease spread and optimize interventions.

The Drosophila Paired (Prd) transcription factor has homeodomain (HD) and paired domain (PD) DNA-binding activities required for in vivo function. Correspondingly, Prd activation of late even-skipped (eve) expression occurs through a conserved target sequence (PTE) with HD and PD half sites, both of which are required for activation. To investigate the relationship between the HD and PD, and their roles in conferring specificity to Prd function, we tested altered versions of the Prd protein and of the PTE target site using in vivo assays in embryos. We found that function through PTE was constrained by the targeting specifications of both the HD and PD as well as the spatial relationship between these two domains. PTE function was also constrained by the spacing between the target half sites for the PD and HD, although surprisingly, late eve activation was retained when PTE was replaced by in vitro optimized binding sites for either the PD alone or for an HD dimer. In contrast to late eve regulation, other Prd targets tolerated more changes in the Prd protein, suggesting that their target sequences may be qualitatively different from PTE.

Both President Clinton and Congress have attempted to increase access to on-line information, but most government agencies have resisted these efforts.

Osteoradionecrosis of the temporal bone is a serious, late complication of radiotherapy to the temporal bone region. The aim of this study was to determine the incidence, risk and protective factors relating to the development of osteoradionecrosis of the temporal bone.This is a retrospective review of prospectively collected data. A single surgeon collated a database over a 10-year period regarding patients undergoing surgery and subsequent radiotherapy for head and neck malignancies. This was reviewed, and information pertaining to demographics, nature of disease, radiation treatment regime and complications including osteoradionecrosis was extracted. Statistical analysis was then completed independently.A total of seven patients from the cohort of 82 developed osteoradionecrosis of the temporal bone within the period of the data collection. This represents an incidence of 8.5% (95% confidence interval = 3.6-13.4%). All who developed osteoradionecrosis had a metastatic parotid squamous cell carcinoma treated operatively with subsequent radiotherapy. There were no statistically significant risks or prognostic factors identified. Mean duration of follow-up was 32 months.The incidence of osteoradionecrosis of the temporal bone was 8.5% (95% confidence interval = 3.6-13.4%). Osteoradionecrosis of the temporal bone can present in excess of 10 years after treatment completion. This result may actually under -represent the true incidence of the complication. It has significant morbidity associated with its development and, occasionally, mortality. Those people undergoing radiotherapy to the parotid region may be at higher risk of osteoradionecrosis. A new classification system for osteoradionecrosis of the temporal bone has been proposed.

Purpose. In vivogene therapy directed at tissues of mesenchymal origin could potentially augment healing. We aimed to assess the duration and magnitude of transene expressionin vivoin mice andex vivoin human tissues.Methods.Using bioluminescence imaging, plasmid and adenoviral vector-based transgene expression in murine quadricepsin vivowas examined. Temporal control was assessed using a doxycycline-inducible system. Anex vivomodel was developed and optimised using murine tissue, and applied inex vivohuman tissue.Results. In vivoplasmid-based transgene expression did not silence in murine muscle, unlike in liver. Although maximum luciferase expression was higher in muscle with adenoviral delivery compared with plasmid, expression reduced over time. The inducible promoter cassette successfully regulated gene expression with maximum levels a factor of 11 greater than baseline. Expression was re-induced to a similar level on a temporal basis. Luciferase expression was readily detectedex vivoin human muscle and tendon.Conclusions.Plasmid constructs resulted in long-termin vivogene expression in skeletal muscle, in a controllable fashion utilising an inducible promoter in combination with oral agents. Successful plasmid gene transfection in humanex vivomesenchymal tissue was demonstrated for the first time.

Sara A. Collins1,2, David Morrissey1, Simon Rajendran1, Garrett Casey1, Martina F. Scallan2, Patrick T. Harrison3, Gerald C. O’Sullivan1 and Mark Tangney1 1Cork Cancer Research Centre, Mercy University Hospital and Leslie C. Quick Jnr. Laboratory, University College Cork, Cork, 2Department of Microbiology, University College Cork, Cork, 3Department of Physiology and Biosciences Institute, University College Cork, Cork, Ireland

Electroweak baryogenesis in the MSSM can account for the cosmological baryon asymmetry, but only with a very light scalar top and a SM‐like Higgs boson. We investigate the effects of this light scalar top on Higgs boson production and decay. Relative to the standard model Higgs boson, we find a large enhancement of the Higgs production rate through gluon fusion and a suppression of the Higgs branching fraction into photon pairs. These modifications in the properties of the Higgs boson are large enough that they can potentially be tested at the Tevatron and the LHC.

An ectopic expression assay in Drosophila embryos was used to investigate the roles of pair-rule segmentation genes in the spatial regulation of the segment-polarity gene, engrailed (en). It is hypothesized that the regions of overlap in expression of two genes, paired (prd) and even-skipped (eve), define the odd-numbered en expression stripes. Consistent with this combinatorial model, ectopic expression of prd caused these en stripes to be expanded posteriorly. Surprisingly, however, ectopic expression of a prd gene with a deletion of the conserved paired box resulted in loss of these odd-numbered en stripes. This dominant negative effect is a phenocopy of en expression in prd embryos and suggests that the paired box is necessary for normal prd- function. A similar deletion of odd-numbered en stripes was also observed after ectopic expression of a chimeric fushi tarazu (ftz) gene containing a substituted prd gene homeo box; in addition, in these embryos, the even-numbered en stripes were expanded anteriorly, as observed when the unaltered ftz gene is ectopically expressed. These effects suggest that the chimeric protein may have DNA or protein targets of both the normal Ftz and Prd proteins.

To develop synthetic short interfering RNA (siRNA) molecules as therapeutic agents for systemic administration in vivo, chemical modifications were introduced into siRNAs targeted to conserved sites in hepatitis B virus (HBV) RNA. These modifications conferred significantly prolonged stability in human serum compared with unmodified siRNAs. Cell culture studies revealed a high degree of gene silencing after treatment with the chemically modified siRNAs. To assess activity of the stabilized siRNAs in vivo initially, an HBV vector-based model was used in which the siRNA and the HBV vector were codelivered via high-volume tail vein injection. More than a 3 log10 decrease in levels of serum HBV DNA and hepatitis B surface antigen, as well as liver HBV RNA, were observed in the siRNA-treated groups compared with the control siRNA-treated and saline groups. Furthermore, the observed decrease in serum HBV DNA was 1.5 log10 more with stabilized siRNA compared with unmodified siRNA, indicating the value of chemical modification in therapeutic applications of siRNA. In subsequent experiments, standard systemic intravenous dosing of stabilized siRNA 72 hours after injection of the HBV vector resulted a 0.9 log10 reduction of serum HBV DNA levels after 2 days of dosing. In conclusion, these experiments establish the strong impact that siRNAs can have on the extent of HBV infection and underscore the importance of stabilization of siRNA against nuclease degradation. (HEPATOLOGY 2005.)

The efficacy of lipid-encapsulated, chemically modified short interfering RNA (siRNA) targeted to hepatitis B virus (HBV) was examined in an in vivo mouse model of HBV replication. Stabilized siRNA targeted to the HBV RNA was incorporated into a specialized liposome to form a stable nucleic-acid-lipid particle (SNALP) and administered by intravenous injection into mice carrying replicating HBV. The improved efficacy of siRNA-SNALP compared to unformulated siRNA correlates with a longer half-life in plasma and liver. Three daily intravenous injections of 3 mg/kg/day reduced serum HBV DNA >1.0 log(10). The reduction in HBV DNA was specific, dose-dependent and lasted for up to 7 d after dosing. Furthermore, reductions were seen in serum HBV DNA for up to 6 weeks with weekly dosing. The advances demonstrated here, including persistence of in vivo activity, use of lower doses and reduced dosing frequency are important steps in making siRNA a clinically viable therapeutic approach.

Activation of CDK4 is regulated, in part, by its association with a D-type cyclin. Conversely, CDK4 activity is inhibited when it is bound to the cyclin-dependent kinase inhibitor, p16(INK4A). To investigate the molecular basis of the interactions between CDK4 and cyclin D1 or p16(INK4A) we performed site-directed mutagenesis of CDK4. The interaction was examined using in vitro translated wild type and mutant CDK4 proteins and bacterially expressed cyclin D1 and p16 fusion proteins. As mutational analysis of CDC2 suggests that its cyclin binding domain is primarily located near its amino terminus, the majority of the mutations constructed in CDK4 were located near its amino terminus. In addition, CDK4 residues homologous to CDC2 sites involved in Suc1 binding were also mutated. Our analysis indicates that cyclin D1 and p16 binding sites are overlapping and located primarily near the amino terminus. All CDK4 mutations that resulted in decreased p16 binding capability also diminished cyclin D1 binding. In contrast, amino-terminal sequences were identified, including the PSTAIRE region, that are important for cyclin D1 binding but are not involved in p16 binding.

Efficient tissue-specific delivery is a crucial factor in the successful development of therapeutic oligonucleotides. Screening for novel delivery methods with unique tissue-homing properties requires a rapid, sensitive, flexible and unbiased technique able to visualize the in vivo biodistribution of these oligonucleotides. Here, we present whole body scanning PCR, a platform that relies on the local extraction of tissues from a mouse whole body section followed by the conversion of target-specific qPCR signals into an image. This platform was designed to be compatible with a novel RT-qPCR assay for the detection of siRNAs and with an assay suitable for the detection of heavily chemically modified oligonucleotides, which we termed Chemical-Ligation qPCR (CL-qPCR). In addition to this, the platform can also be used to investigate the global expression of endogenous mRNAs and non-coding RNAs. Incorporation of other detection systems, such as aptamers, could even further expand the use of this technology.

RNA interference (RNAi) represents a powerful, naturally occurring biological strategy for inhibition of gene expression that has demonstrated utility in the inhibition of viral replication. However, the challenges associated with the effective in vivo delivery of siRNAs has been a major obstacle to their use. Here we describe the development of an effective siRNA based therapy for hepatitis virus infection.

This report, based on the Dark Sectors workshop at SLAC in April 2016, summarizes the scientific importance of searches for dark sector dark matter and forces at masses beneath the weak-scale, the status of this broad international field, the important milestones motivating future exploration, and promising experimental opportunities to reach these milestones over the next 5-10 years., 66 pages, 15 figures, 3 tables. Workshop website and agenda: http://www-conf.slac.stanford.edu/darksectors2016/ https://indico.cern.ch/event/507783/ Editors: J. Alexander, M. Battaglieri, B. Echenard, R. Essig, M. Graham, E. Izaguirre, J. Jaros, G. Krnjaic, J. Mardon, D. Morrissey, T. Nelson, M. Perelstein, M. Pyle, A. Ritz, P. Schuster, B. Shuve, N. Toro, R. Van De Water

Drosophila paired, a homolog of mammalian Pax-3, is key to the coordinated regulation of segment-polarity genes during embryogenesis. The paired gene and its homologs are unusual in encoding proteins with two DNA-binding domains, a paired domain and a homeodomain. We are using an in vivo assay to dissect the functions of the domains of this type of molecule. In particular, we are interested in determining whether one or both DNAbinding activities are required for individual in vivo functions of Paired. We constructed point mutants in each domain designed to disrupt DNA binding and tested the mutants with ectopic expression assays in Drosophila embryos. Mutations in either domain abolished the normal regulation of the target genes engrailed, hedgehog, gooseberry and even-skipped, suggesting that these in vivo functions of Paired require DNA binding through both domains rather than either domain alone. However, when the two mutant proteins were placed in the same embryo, Paired function was restored, indicating that the two DNAbinding activities need not be present in the same molecule. Quantitation of this effect shows that the paired domain mutant has a dominant-negative effect consistent with the observations that Paired protein can bind DNA as a dimer.