Dark Matter Annihilation inside Large-Volume Neutrino Detectors.

New particles in theories beyond the standard model can manifest as stable relics that interact strongly with visible matter and make up a small fraction of the total dark matter abundance. Such particles represent an interesting physics target since they can evade existing bounds from direct detection due to their rapid thermalization in high-density environments. In this work we point out that their annihilation to visible matter inside large-volume neutrino telescopes can provide a new way to constrain or discover such particles. The signal is the most pronounced for relic masses in the GeV range, and can be efficiently constrained by existing Super-Kamiokande searches for dinucleon annihilation. We also provide an explicit realization of this scenario in the form of secluded dark matter coupled to a dark photon, and we show that the present method implies novel and stringent bounds on the model that are complementary to direct constraints from beam dumps, colliders, and direct detection experiments.

Highly complementary target RNAs promote release of guide RNAs from human Argonaute2.

Argonaute proteins use small RNAs to guide the silencing of complementary target RNAs in many eukaryotes. Although small RNA biogenesis pathways are well studied, mechanisms for removal of guide RNAs from Argonaute are poorly understood. Here we show that the Argonaute2 (Ago2) guide RNA complex is extremely stable, with a half-life on the order of days. However, highly complementary target RNAs destabilize the complex and significantly accelerate release of the guide RNA from Ago2. This "unloading" activity can be enhanced by mismatches between the target and the guide 5' end and attenuated by mismatches to the guide 3' end. The introduction of 3' mismatches leads to more potent silencing of abundant mRNAs in mammalian cells. These findings help to explain why the 3' ends of mammalian microRNAs (miRNAs) rarely match their targets, suggest a mechanism for sequence-specific small RNA turnover, and offer insights for controlling small RNAs in mammalian cells.

Akt-mediated phosphorylation of argonaute 2 downregulates cleavage and upregulates translational repression of MicroRNA targets.

A high-throughput RNA interference (RNAi) screen targeting 542 genes of the human kinome was used to discover regulators of RNAi. Here we report that the proto-oncogene Akt-3/PKBγ (Akt3) phosphorylates Argonaute 2 (Ago2) at S387, which downregulates cleavage and upregulates translational repression of endogenous microRNA (miRNA)-targeted messenger RNAs (mRNAs). We further demonstrate that Akt3 coimmunoprecipitates with Ago2 and phosphorylation of Ago2 at S387 facilitates its interaction with GW182 and localization to cytoplasmic processing bodies (P bodies), where miRNA-targeted mRNAs are thought to be stored and degraded. Therefore, Akt3-mediated phosphorylation of Ago2 is a molecular switch between target mRNA cleavage and translational repression activities of Ago2.

Incidence of avascular necrosis following biceps tenodesis during proximal humerus open reduction and internal fixation.

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.

Gravitational Wave Bursts as Harbingers of Cosmic Strings Diluted by Inflation.

A standard expectation of primordial cosmological inflation is that it dilutes all relics created before its onset to unobservable levels. We present a counterexample to this expectation by demonstrating that a network of cosmic strings diluted by inflation can regrow to a level that is potentially observable today in gravitational waves (GWs). In contrast to undiluted cosmic strings, whose primary GW signals are typically in the form of a stochastic GW background, the leading signal from a diluted cosmic string network can be distinctive bursts of GWs within the sensitivity reach of current and future GW observatories.

Comparison of partially and fully chemically-modified siRNA in conjugate-mediated delivery in vivo.

Small interfering RNA (siRNA)-based drugs require chemical modifications or formulation to promote stability, minimize innate immunity, and enable delivery to target tissues. Partially modified siRNAs (up to 70% of the nucleotides) provide significant stabilization in vitro and are commercially available; thus are commonly used to evaluate efficacy of bio-conjugates for in vivo delivery. In contrast, most clinically-advanced non-formulated compounds, using conjugation as a delivery strategy, are fully chemically modified (100% of nucleotides). Here, we compare partially and fully chemically modified siRNAs in conjugate mediated delivery. We show that fully modified siRNAs are retained at 100x greater levels in various tissues, independently of the nature of the conjugate or siRNA sequence, and support productive mRNA silencing. Thus, fully chemically stabilized siRNAs may provide a better platform to identify novel moieties (peptides, aptamers, small molecules) for targeted RNAi delivery.

The rough endoplasmatic reticulum is a central nucleation site of siRNA-mediated RNA silencing.

Despite progress in mechanistic understanding of the RNA interference (RNAi) pathways, the subcellular sites of RNA silencing remain under debate. Here we show that loading of lipid-transfected siRNAs and endogenous microRNAs (miRNA) into RISC (RNA-induced silencing complexes), encounter of the target mRNA, and Ago2-mediated mRNA slicing in mammalian cells are nucleated at the rough endoplasmic reticulum (rER). Although the major RNAi pathway proteins are found in most subcellular compartments, the miRNA- and siRNA-loaded Ago2 populations co-sediment almost exclusively with the rER membranes, together with the RISC loading complex (RLC) factors Dicer, TAR RNA binding protein (TRBP) and protein activator of the interferon-induced protein kinase (PACT). Fractionation and membrane co-immune precipitations further confirm that siRNA-loaded Ago2 physically associates with the cytosolic side of the rER membrane. Additionally, RLC-associated double-stranded siRNA, diagnostic of RISC loading, and RISC-mediated mRNA cleavage products exclusively co-sediment with rER. Finally, we identify TRBP and PACT as key factors anchoring RISC to ER membranes in an RNA-independent manner. Together, our findings demonstrate that the outer rER membrane is a central nucleation site of siRNA-mediated RNA silencing.

Biofabrication of soft tissue templates for engineering the bone-ligament interface.

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.

Exosome-mediated Delivery of Hydrophobically Modified siRNA for Huntingtin mRNA Silencing.

Delivery represents a significant barrier to the clinical advancement of oligonucleotide therapeutics for the treatment of neurological disorders, such as Huntington's disease. Small, endogenous vesicles known as exosomes have the potential to act as oligonucleotide delivery vehicles, but robust and scalable methods for loading RNA therapeutic cargo into exosomes are lacking. Here, we show that hydrophobically modified small interfering RNAs (hsiRNAs) efficiently load into exosomes upon co-incubation, without altering vesicle size distribution or integrity. Exosomes loaded with hsiRNAs targeting Huntingtin mRNA were efficiently internalized by mouse primary cortical neurons and promoted dose-dependent silencing of Huntingtin mRNA and protein. Unilateral infusion of hsiRNA-loaded exosomes, but not hsiRNAs alone, into mouse striatum resulted in bilateral oligonucleotide distribution and statistically significant bilateral silencing of up to 35% of Huntingtin mRNA. The broad distribution and efficacy of hsiRNA-loaded exosomes delivered to brain is expected to advance the development of therapies for the treatment of Huntington's disease and other neurodegenerative disorders.

Arthroscopic Sternoclavicular Joint Diskectomy for Acute and Chronic Tears.

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.

Harvesting (67)Cu from the Collection of a Secondary Beam Cocktail at the National Superconducting Cyclotron Laboratory.

Isotope harvesting is a promising new method to obtain isotopes for which there is no reliable continuous supply at present. To determine the possibility of obtaining radiochemically pure radioisotopes from an aqueous beam dump at a heavy-ion fragmentation facility, preliminary experiments were performed to chemically extract a copper isotope from a large mixture of projectile fragmentation products in an aqueous medium. In this work a 93 MeV/u secondary beam cocktail was collected in an aqueous beam stop at the National Superconducting Cyclotron Laboratory (NSCL) located on the Michigan State University (MSU) campus. The beam cocktail consisted of ∼2.9% (67)Cu in a large mixture of co-produced isotopes ranging in atomic number from ∼19 to 34. The chemical extraction of (67)Cu was achieved via a two-step process: primary extraction using a divalent metal chelation disk followed by anion-exchange chromatography. A significant fraction (74 ± 4%) of the (67)Cu collected in the aqueous beam stop was recovered with >99% radiochemical purity. To illustrate the utility of this product, the purified (67)Cu material was then used to radiolabel an anti-EGFR antibody, Panitumumab, and injected into mice bearing colon cancer xenografts. The tumor uptake at 5 days postinjection was found to be 12.5 ± 0.7% which was in very good agreement with previously reported studies with this radiolabeled antibody. The present results demonstrate that harvesting isotopes from a heavy-ion fragmentation facility could be a promising new method for obtaining high-quality isotopes that are not currently available by traditional methods.

Whole-body scanning PCR; a highly sensitive method to study the biodistribution of mRNAs, noncoding RNAs and therapeutic oligonucleotides.

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.

Feasibility of the hydrogen sulfide test for the assessment of drinking water quality in post-earthquake Haiti.

In 2010, a magnitude 7.0 earthquake struck Haiti, severely damaging the drinking and wastewater infrastructure and leaving millions homeless. Compounding this problem, the introduction of Vibrio cholerae 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 ml. 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.

Definitions of serious injury in long-term residential care: a systematic review protocol.

Background: Evidence indicates that the reporting of serious injury in long-term residential care has increased substantially over the past decade. However, what constitutes a serious injury in residential care is poorly and inconsistently defined. This may result in incidences being unnecessarily reported as a serious injury. It is therefore, crucial to develop a consistent definition of serious injury to reduce reporting burden and to facilitate comparison between different residential care settings and across jurisdictions. This protocol describes the methods for a systematic review of existing definitions from the literature to inform the development of a consistent definition of serious injury in long-term residential care. Methods: A wide range of published peer-reviewed and grey literature will be sought for this review, including guidance and policy documents. Searches will be conducted of databases including MEDLINE, CINAHL, SocINDEX, Academic Search Ultimate, and Westlaw International. Grey literature database searches will include Trip and Social Care Online. Country specific searches of government and health and social care websites will be conducted. Quality appraisal will be facilitated using the Quality Assessment for Diverse Studies (QuADS) tool and Tyndall's checklist. The level of confidence in the findings will be assessed using the GRADE CERQual approach. A customised data extraction form will be used to extract data to reduce the risk of bias. Conceptual content analysis of data will facilitate identification of definitions of serious injury and their frequency within texts. Conclusions: The findings will inform the development of a consistent definition of serious injury in long-term residential care that will reduce reporting burden, facilitate the accuracy of data collected and allow for comparison across jurisdictions. A more universal and consistent definition will enable regulators, policy makers, service providers and researchers to develop policy and practical interventions to prevent the occurrence of serious injury in long-term residential care.

Individualized approach to elexacaftor/tezacaftor/ivacaftor dosing in cystic fibrosis, in response to self-reported anxiety and neurocognitive adverse events: A case series.

The prevalence of mental health disorders is high among people with Cystic Fibrosis. The psychological symptoms in CF are associated with poor adherence, worse treatment outcomes, and greater health utilization/cost. Mental health and neurocognitive Adverse Events (AEs) have been reported with all available Cystic Fibrosis Transmembrane conductance Regulator (CFTR) modulators in small groups of patients. We report our experience with a dose reduction strategy in 10 of our patients on elexacaftor/tezacaftor/ivacaftor (7.9% of total number of patients) who self-reported developing intense anxiety, irritability, sleep disturbance and/or mental slowness after initiation of full dose treatment. Standard dose elexacaftor/tezacaftor/ivacaftor resulted in 14.3 points improvement in mean Percent Predicted Forced Expiratory Volume in 1 s (ppFEV1), and a mean difference in sweat chloride of -39.3 mmol/L. We initially discontinued and/or reduced therapy according to the AEs severity, with a subsequent planned dose escalation every 4-6 weeks guided by sustainability of clinical effectiveness, absence of AEs recurrence, and patients' preferences. Clinical parameters including lung function and sweat chloride were monitored for up to 12 weeks to assess ongoing clinical response to the reduced dose regimen. Dose reduction resulted in resolution of self-reported mental/psychological AEs, without loss of clinical effectiveness (ppFEV1 was 80.7% on standard dose, and 83.4% at 12 weeks on reduced dose; sweat chloride was 33.4 and 34 mmol/L on standard and reduced dose, respectively). Furthermore, in a subgroup of patients who completed 24 weeks of the reduced dose regimen, repeat low dose Computed Tomography imaging showed a significant response when compared to pre-initiation of elexacaftor/tezacaftor/ivacaftor.

Control and augmentation of long-term plasmid transgene expression in vivo in murine muscle tissue and ex vivo in patient mesenchymal tissue.

PURPOSE: In vivo gene therapy directed at tissues of mesenchymal origin could potentially augment healing. We aimed to assess the duration and magnitude of transene expression in vivo in mice and ex vivo in human tissues. METHODS: Using bioluminescence imaging, plasmid and adenoviral vector-based transgene expression in murine quadriceps in vivo was examined. Temporal control was assessed using a doxycycline-inducible system. An ex vivo model was developed and optimised using murine tissue, and applied in ex vivo human tissue. RESULTS: In vivo plasmid-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 detected ex vivo in human muscle and tendon. CONCLUSIONS: Plasmid constructs resulted in long-term in vivo gene expression in skeletal muscle, in a controllable fashion utilising an inducible promoter in combination with oral agents. Successful plasmid gene transfection in human ex vivo mesenchymal tissue was demonstrated for the first time.

Repair of sternoclavicular joint ligament: a novel approach.

BACKGROUND: Posterior sternoclavicular dislocations are common in younger patients and are frequently due to high energy sporting incidents. AIM: We aim to demonstrate a novel technique that is safe and aims to provide good functional post-operative outcomes for patients with this injury. METHODS: 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. RESULTS: 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. DISCUSSION: 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. CONCLUSIONS: We believe this novel technique is safer than the existing described techniques, and it does not compromise on functional outcomes.

Posterior (Boyd) approach to terrible triad injuries.

BACKGROUND: A 'terrible triad injury of the elbow' (TTIE) refers to an injury pattern involving posterior dislocation of the ulnohumeral joint, fracture of the radial head, and fracture of the coronoid process of the ulna. It is a complex injury to the elbow joint and can result in long-term elbow instability, pain, stiffness, and arthritis. In specific cases, it may be treated conservatively, but in most circumstances, surgical stabilization is advised.The 'drop sign' is an objective static radiographically measured ulnohumeral distance of ≥4 mm seen intraoperatively and postoperatively. Although controversial, it may portend postoperative instability and arthritis. The senior author repairs these injuries in a standardized fashion through a modified Boyd rather than a lateral approach. Our aim was to assess the number of cases demonstrating an intraoperative drop sign after surgical treatment of a TTIE with this approach. METHODS: We retrospectively analyzed 22 consecutive patients with 23 acute TTIEs. These injuries were treated by a single surgeon using a modified Boyd (posterior) approach to the elbow. Intraoperative image intensifier x-rays were analyzed by the two authors to assess for a 'drop sign'. RESULTS: None of the 23 cases had 'drop signs' on intraoperative imaging after stabilization. No patient has redislocated, underwent reoperation, or had symptoms of instability at follow-up. CONCLUSION: None of our patient cohort had an intraoperative 'drop sign' after standardized stabilization for a TTIE injury using a modified Boyd approach.

Orally administered bifidobacteria as vehicles for delivery of agents to systemic tumors.

Certain bacteria have emerged as biological gene vectors with natural tumor specificity, capable of specifically delivering genes or gene products to the tumor environment when intravenously (i.v.) administered to rodent models. We show for the first time that oral administration of bacteria to mice resulted in their translocation from the gastrointestinal tract (GIT) with subsequent homing to and replication specifically in tumors. The commensal, nonpathogenic Bifidobacterium breve UCC2003 harboring a plasmid expressing lux fed to mice bearing subcutaneous (s.c.) tumors were readily detected specifically in tumors, by live whole-body imaging, at levels similar to i.v. administration. Reporter gene expression was visible for >2 weeks in tumors. Mice remained healthy throughout experiments. Cytokine analyses indicated a significant upregulation of interferon-gamma (IFN-gamma) in the GIT of bifidobacteria-fed mice, which is associated with increases in epithelial permeability. However, B. breve feeding did not increase systemic levels of other commensal bacteria. The presence of tumor was not necessary for translocation to systemic organs to occur. These findings indicate potential for safe and efficient gene-based treatment and/or detection of tumors via ingestion of nonpathogenic bacteria expressing therapeutic or reporter genes.