[Treatment strategies for traumatic anterior shoulder dislocation]. / Therapiestrategien bei traumatischer ventraler Schultererstluxation.

Anterior glenohumeral instability is the most frequent type of shoulder instability. This is often associated with labral and osseous lesions leading to recurrent instability. A detailed medical history, a physical examination and targeted diagnostic imaging are necessary to assess possible pathological soft tissue alterations as well as bony lesions of the humeral head and the glenoid bone. Early surgical treatment has been shown to reduce the risk of recurrence, especially in young active athletes, and can avoid secondary damage. Shoulder dislocations in older patients also require a detailed assessment and selection of treatment as persisting pain and limitation of movement can occur due to rotator cuff lesions and nerve injuries. The purpose of this article is to provide an overview of the currently available evidence and results regarding diagnostic considerations and conservative vs. surgical treatment and time to return to sport after treatment of a primary anterior shoulder dislocation.

Patient education on subacromial impingement syndrome : Reliability and educational quality of content available on Google and YouTube.

OBJECTIVE: The purpose of this study was to assess the reliability and educational quality of content available on Google and YouTube regarding subacromial impingement syndrome (SAIS). METHODS: Google and YouTube were queried for English and German results on SAIS using the search terms "shoulder impingement" and the German equivalent "Schulter Impingement". The analysis was restricted to the first 30 results of each query performed. Number of views and likes as well as upload source and length of content were recorded. Each result was evaluated by two independent reviewers using the Journal of the American Medical Association (JAMA) benchmark criteria (score range, 0-5) to assess reliability and the DISCERN score (score range, 16-80) and a SAIS-specific score (SAISS, score range, 0-100) to evaluate educational content. RESULTS: The 58 websites found on Google and 48 videos found on YouTube were included in the analysis. The average number of views per video was 220,180 ± 415,966. The average text length was 1375 ± 997 words and the average video duration 456 ± 318 s. The upload sources were mostly non-physician based (74.1% of Google results and 79.2% of YouTube videos). Overall, there were poor results in reliability and educational quality, with sources from doctors having a significantly higher mean reliability measured in the JAMA score (p < 0.001) and educational quality in DISCERN (p < 0.001) and SAISS (p = 0.021). There was no significant difference between German and English results but texts performed significantly better than videos in terms of reliability (p = 0.002) and educational quality (p < 0.001). CONCLUSION: Information on SAIS found on Google and YouTube is of low reliability and quality. Therefore, orthopedic health practitioners and healthcare providers should inform patients that this source of information may be unreliable and make efforts to provide patients with higher quality alternatives. LEVEL OF EVIDENCE: IV, case series.

Minced Cartilage Procedure for One-Stage Arthroscopic Repair of Chondral Defects at the Glenohumeral Joint.

Chondral defects of the glenohumeral joint are common but still remain a diagnostic and management challenge. Whereas arthroplasty is a reasonable treatment option in the elderly and low-demand population, joint preservation should be aimed for the remaining patients. For larger defects the current gold standard of treatment is autologous chondrocyte implantation. However, disadvantages such as high cost, the restriction in availability of specialized laboratories, and the 2-stage surgical design need to be accounted for if choosing this option. Showing first good clinical results for the knee joint, minced cartilage implantation is moreover a cost-effective procedure bringing autologous cartilage chips harvested from the defect walls and bringing them into the area of damage in a single-step open or arthroscopic approach. We describe an arthroscopic strategy of this technique to treat chondral defects at the glenohumeral joint.

mRNA-based vaccines synergize with radiation therapy to eradicate established tumors.

BACKGROUND: The eradication of large, established tumors by active immunotherapy is a major challenge because of the numerous cancer evasion mechanisms that exist. This study aimed to establish a novel combination therapy consisting of messenger RNA (mRNA)-based cancer vaccines and radiation, which would facilitate the effective treatment of established tumors with aggressive growth kinetics. METHODS: The combination of a tumor-specific mRNA-based vaccination with radiation was tested in two syngeneic tumor models, a highly immunogenic E.G7-OVA and a low immunogenic Lewis lung cancer (LLC). The molecular mechanism induced by the combination therapy was evaluated via gene expression arrays as well as flow cytometry analyses of tumor infiltrating cells. RESULTS: In both tumor models we demonstrated that a combination of mRNA-based immunotherapy with radiation results in a strong synergistic anti-tumor effect. This was manifested as either complete tumor eradication or delay in tumor growth. Gene expression analysis of mouse tumors revealed a variety of substantial changes at the tumor site following radiation. Genes associated with antigen presentation, infiltration of immune cells, adhesion, and activation of the innate immune system were upregulated. A combination of radiation and immunotherapy induced significant downregulation of tumor associated factors and upregulation of tumor suppressors. Moreover, combination therapy significantly increased CD4+, CD8+ and NKT cell infiltration of mouse tumors. CONCLUSION: Our data provide a scientific rationale for combining immunotherapy with radiation and provide a basis for the development of more potent anti-cancer therapies.

Highly potent mRNA based cancer vaccines represent an attractive platform for combination therapies supporting an improved therapeutic effect.

Direct vaccination with mRNA encoding tumor antigens is a novel and promising approach in cancer immunotherapy. CureVac's mRNA vaccines contain free and protamine-complexed mRNA. Such two-component mRNA vaccines support both antigen expression and immune stimulation. These self-adjuvanting RNA vaccines, administered intradermally without any additional adjuvant, induce a comprehensive balanced immune response, comprising antigen specific CD4+ T cells, CD8+ T cells and B cells. The balanced immune response results in a strong anti-tumor effect and complete protection against antigen positive tumor cells. This tumor inhibition elicited by mRNA vaccines is a result of the concerted action of different players. After just two intradermal vaccinations, we observe multiple changes at the tumor site, including the up-regulation of many genes connected to T and natural killer cell activation, as well as genes responsible for improved infiltration of immune cells into the tumor via chemotaxis. The two-component mRNA vaccines induce a very fast and boostable immune response. Therefore, the vaccination schedules can be adjusted to suit the clinical situation. Moreover, by combining the mRNA vaccines with therapies in clinical use (chemotherapy or anti-CTLA-4 antibody therapy), an even more effective anti-tumor response can be elicited. The first clinical data obtained from two separate Phase I/IIa trials conducted in PCA (prostate cancer) and NSCLC (non-small cell lung carcinoma) patients have shown that the two-component mRNA vaccines are safe, well tolerated and highly immunogenic in humans.

Protein expression from exogenous mRNA: uptake by receptor-mediated endocytosis and trafficking via the lysosomal pathway.

Insertional mutagenesis and the inherent risk of malignancy compromise the clinical use of DNA-based therapies. Being a transient copy of genetic material, mRNA is a safe alternative, overcoming this limitation. As a prerequisite for the development of efficient mRNA-based therapies, we investigated the cellular uptake and intracellular fate of mRNA for the first time. To this end we determined cell-type, dose and energy dependence of mRNA internalisation. Moreover, we employed markers for uptake pathways and cellular compartments to analyse the route of mRNA internalisation and its intracellular destination. Finally, we addressed the involvement of receptors and their nature using a competitor-based approach. We found that all cell types tested were amenable to uptake and expression of naked mRNA. Internalisation mainly occurred via caveolae/lipid raft-rich membrane domains and involved scavenger-receptor(s). Following endocytosis, mRNA eventually accumulated in lysosomes, while part of it escaped into the cytosol giving rise to protein synthesis. Taken together, our findings provide unprecedented insights into the internalisation and trafficking of exogenous mRNA, greatly facilitating the development of effective mRNA-based therapies in the future.

Messenger RNA-based vaccines with dual activity induce balanced TLR-7 dependent adaptive immune responses and provide antitumor activity.

Direct vaccination with messenger RNA (mRNA) molecules encoding tumor-associated antigens is a novel and promising approach in cancer immunotherapy. The main advantage of using mRNA for vaccination is that the same molecule not only provides an antigen source for adaptive immunity, but can simultaneously bind to pattern recognition receptors, thus stimulating innate immunity. However, achieving both features remains challenging, as the complexation of mRNA required for immune-stimulating activity may inhibit its translatability. In this study, we present a new and more effective vaccine design: a two-component mRNA-based tumor vaccine that supports both: antigen expression and immune stimulation, mediated by Toll like receptor 7 (TLR7). The two-component mRNA vaccines, containing free and protamine-complexed mRNA, induce balanced adaptive immune responses providing humoral as well as T cell mediated immunity. This balanced immune response is based on the induction of antigen-specific CD4(+) T helper cells and cytotoxic CD8(+) T cells. Once activated, these CD4(+) and CD8(+) T cells secrete a wide set of cytokines, which drive a TH1 response. Immunization with the two-component vaccines induces sustained memory responses, mediated by antigen-specific memory T cells. Moreover, treatment of mice with the two-component mRNA vaccine mediates a strong antitumor response against OVA-expressing tumor cells, not only in a prophylactic but also in a therapeutic setting. In conclusion, two-component mRNA vaccines with self-adjuvanting activity induce balanced adaptive immune responses and mediate sustained antitumor activity.

Genomic SELEX: a discovery tool for genomic aptamers.

Genomic SELEX is a discovery tool for genomic aptamers, which are genomically encoded functional domains in nucleic acid molecules that recognize and bind specific ligands. When combined with genomic libraries and using RNA-binding proteins as baits, Genomic SELEX used with high-throughput sequencing enables the discovery of genomic RNA aptamers and the identification of RNA-protein interaction networks. Here we describe how to construct and analyze genomic libraries, how to choose baits for selections, how to perform the selection procedure and finally how to analyze the enriched sequences derived from deep sequencing. As a control procedure, we recommend performing a "Neutral" SELEX experiment in parallel to the selection, omitting the selection step. This control experiment provides a background signal for comparison with the positively selected pool. We also recommend deep sequencing the initial library in order to facilitate the final in silico analysis of enrichment with respect to the initial levels. Counter selection procedures, using modified or inactive baits, allow strengthening the binding specificity of the winning selected sequences.

Monitoring genomic sequences during SELEX using high-throughput sequencing: neutral SELEX.

BACKGROUND: SELEX is a well established in vitro selection tool to analyze the structure of ligand-binding nucleic acid sequences called aptamers. Genomic SELEX transforms SELEX into a tool to discover novel, genomically encoded RNA or DNA sequences binding a ligand of interest, called genomic aptamers. Concerns have been raised regarding requirements imposed on RNA sequences undergoing SELEX selection. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate SELEX and assess the extent of these effects, we designed and performed a Neutral SELEX experiment omitting the selection step, such that the sequences are under the sole selective pressure of SELEX's amplification steps. Using high-throughput sequencing, we obtained thousands of full-length sequences from the initial genomic library and the pools after each of the 10 rounds of Neutral SELEX. We compared these to sequences obtained from a Genomic SELEX experiment deriving from the same initial library, but screening for RNAs binding with high affinity to the E. coli regulator protein Hfq. With each round of Neutral SELEX, sequences became less stable and changed in nucleotide content, but no sequences were enriched. In contrast, we detected substantial enrichment in the Hfq-selected set with enriched sequences having structural stability similar to the neutral sequences but with significantly different nucleotide selection. CONCLUSIONS/SIGNIFICANCE: Our data indicate that positive selection in SELEX acts independently of the neutral selective requirements imposed on the sequences. We conclude that Genomic SELEX, when combined with high-throughput sequencing of positively and neutrally selected pools, as well as the gnomic library, is a powerful method to identify genomic aptamers.

RNA chaperone activity and RNA-binding properties of the E. coli protein StpA.

The E. coli protein StpA has RNA annealing and strand displacement activities and it promotes folding of RNAs by loosening their structures. To understand the mode of action of StpA, we analysed the relationship of its RNA chaperone activity to its RNA-binding properties. For acceleration of annealing of two short RNAs, StpA binds both molecules simultaneously, showing that annealing is promoted by crowding. StpA binds weakly to RNA with a preference for unstructured molecules. Binding of StpA to RNA is strongly dependent on the ionic strength, suggesting that the interactions are mainly electrostatic. A mutant variant of the protein, with a glycine to valine change in the nucleic-acid-binding domain, displays weaker RNA binding but higher RNA chaperone activity. This suggests that the RNA chaperone activity of StpA results from weak and transient interactions rather than from tight binding to RNA. We further discuss the role that structural disorder in proteins may play in chaperoning RNA folding, using bioinformatic sequence analysis tools, and provide evidence for the importance of conformational disorder and local structural preformation of chaperone nucleic-acid-binding sites.

Stabilities of HIV-1 DIS type RNA loop-loop interactions in vitro and in vivo.

RNA loop-loop interactions are a prevalent motif in the formation of tertiary structure and are well suited to trigger molecular recognition between RNA molecules. We determined the stabilities of several loop-loop interactions with a constant 6 bp core sequence and varying unpaired flanking nucleotides and found that the flanking bases have a strong influence on the stability and ion dependence of the kissing complex. In general, the stabilities determined in 1 M Na+ are equivalent to those in the presence of near physiological Mg2+ concentrations. Therefore we further tested whether the stabilities determined in vitro and within yeast cells correlate, using a recently developed yeast RNA-hybrid system. For the majority of the loop types analyzed here, the melting temperatures determined in vitro are in good agreement with the relative beta-galactosidase activity in yeast cells, showing that data derived from in vitro measurements reflect in vivo properties. The most stable interactions are the naturally occurring HIV-1 DIS MAL and LAI derived loops with the motif (5' A(A)/(G)N6A 3'), emphasizing the crucial role of stable kissing complexes in HIV genome dimerization.

Genomic systematic evolution of ligands by exponential enrichment (Genomic SELEX) for the identification of protein-binding RNAs independent of their expression levels.

Genomic systematic evolution of ligands by exponential enrichment (Genomic SELEX) is an experimental procedure for the expression condition-independent identification of protein-binding RNAs. RNA libraries derived from genomic DNA are generated via random priming, PCR amplification and in vitro transcription. Libraries consist of genomic sequences of selected size, and fragments are flanked by constant sequences required for amplification and transcription. This RNA pool is then subjected to several rounds of selection and amplification to enrich for RNAs meeting the selection criteria. Various selection criteria are possible. Here we describe selection by affinity to a protein of interest. High-affinity ligands can then be cloned and sequenced to allow their identification. With this method, protein-binding RNAs can be discovered, nucleic acid-protein interactions can be identified, and whole protein-nucleic acid networks can be defined. This method is also suitable for discovering novel genes, including non-protein-coding RNAs, and it complements in silico approaches. It is better suited to detect protein-binding RNAs that are differentially expressed (and therefore absent from many tissues) and low-abundance RNAs than experimental procedures that start from the isolation of expressed RNAs. The protocol takes approximately 3 months to complete.

Steroid and lipid conjugates of siRNAs to enhance cellular uptake and gene silencing in liver cells.

Double-stranded short interfering RNAs (siRNAs) mediate post-transcriptional inhibition of gene expression in a variety of biological systems. However, human liver cells show poor uptake of these nucleic acids. In order to improve the delivery of siRNA into these cells without transfection agents, we have synthesized two series of lipophilic siRNAs conjugated with derivatives of cholesterol, lithocholic acid or lauric acid. The lipid moieties were covalently linked to the 5'-ends of the RNAs using phosphoramidite chemistry. The potency of these chemically modified siRNAs to inhibit reporter gene expression was further investigated in vitro with beta-galactosidase expressing liver cells.