Genome-wide profiling of Hfq-bound RNAs reveals the iron-responsive small RNA RusT in Caulobacter crescentus.

The alphaproteobacterium Caulobacter crescentus thrives in oligotrophic environments and is able to optimally exploit minimal resources by entertaining an intricate network of gene expression control mechanisms. Numerous transcriptional activators and repressors have been reported to contribute to these processes, but only few studies have focused on regulation at the post-transcriptional level in C. crescentus. Small RNAs (sRNAs) are a prominent class of regulators of bacterial gene expression, and most sRNAs characterized today engage in direct base-pairing interactions to modulate the translation and/or stability of target mRNAs. In many cases, the ubiquitous RNA chaperone, Hfq, contributes to the establishment of RNA-RNA interactions. Although the deletion of the hfq gene is associated with a severe loss of fitness in C. crescentus, the RNA ligands of the chaperone have remained largely unexplored. Here we report on the identification of coding and non-coding transcripts associated with Hfq in C. crescentus and demonstrate Hfq-dependent post-transcriptional regulation in this organism. We show that the Hfq-bound sRNA RusT is transcriptionally controlled by the NtrYX two-component system and induced in response to iron starvation. By combining RusT pulse expression with whole-genome transcriptome analysis, we determine 16 candidate target transcripts that are deregulated, many of which encode outer membrane transporters. We hence suggest RusT to support remodeling of the C. crescentus cell surface when iron supplies are limited.IMPORTANCEThe conserved RNA-binding protein Hfq contributes significantly to the adaptation of bacteria to different environmental conditions. Hfq not only stabilizes associated sRNAs but also promotes inter-molecular base-pairing interactions with target transcripts. Hfq plays a pivotal role for growth and survival, controlling central metabolism and cell wall synthesis in the oligotroph Caulobacter crescentus. However, direct evidence for Hfq-dependent post-transcriptional regulation and potential oligotrophy in C. crescentus has been lacking. Here, we identified sRNAs and mRNAs associated with Hfq in vivo, and demonstrated the requirement of Hfq for sRNA-mediated regulation, particularly of outer membrane transporters in C. crescentus.

An RNA sponge controls quorum sensing dynamics and biofilm formation in Vibrio cholerae.

Small regulatory RNAs (sRNAs) acting in concert with the RNA chaperone Hfq are prevalent in many bacteria and typically act by base-pairing with multiple target transcripts. In the human pathogen Vibrio cholerae, sRNAs play roles in various processes including antibiotic tolerance, competence, and quorum sensing (QS). Here, we use RIL-seq (RNA-interaction-by-ligation-and-sequencing) to identify Hfq-interacting sRNAs and their targets in V. cholerae. We find hundreds of sRNA-mRNA interactions, as well as RNA duplexes formed between two sRNA regulators. Further analysis of these duplexes identifies an RNA sponge, termed QrrX, that base-pairs with and inactivates the Qrr1-4 sRNAs, which are known to modulate the QS pathway. Transcription of qrrX is activated by QrrT, a previously uncharacterized LysR-type transcriptional regulator. Our results indicate that QrrX and QrrT are required for rapid conversion from individual to community behaviours in V. cholerae.

Towards maximum acceleration of monoclonal antibody development: Leveraging transposase-mediated cell line generation to enable GMP manufacturing within 3 months using a stable pool.

In recent years, acceleration of development timelines has become a major focus within the biopharmaceutical industry to bring innovative therapies faster to patients. However, in order to address a high unmet medical need even faster further acceleration potential has to be identified to transform "speed-to-clinic" concepts into "warp-speed" development programs. Recombinant Chinese hamster ovary (CHO) cell lines are the predominant expression system for monoclonal antibodies (mAbs) and are routinely generated by random transgene integration (RTI) of the genetic information into the host cell genome. This process, however, exhibits considerable challenges such as the requirement for a time-consuming clone screening process to identify a suitable clonally derived manufacturing cell line. Hence, RTI represents an error prone and tedious method leading to long development timelines until availability of Good Manufacturing Practice (GMP)-grade drug substance (DS). Transposase-mediated semi-targeted transgene integration (STI) has been recently identified as a promising alternative to RTI as it allows for a more rapid generation of high-performing and stable production cell lines. In this report, we demonstrate how a STI technology was leveraged to develop a very robust DS manufacturing process based on a stable pool cell line at unprecedented pace. Application of the novel strategy resulted in the manufacturing of GMP-grade DS at 2,000 L scale in less than three months paving the way for a start of Phase I clinical trials only six months after transfection. Finally, using a clonally derived production cell line, which was established from the parental stable pool, we were able to successfully implement a process with an increased mAb titer of up to 5 g per liter at the envisioned commercial scale (12,000 L) within eight months.

Evaluation of a New Virtual Reality Concept Teaching K-Wire Drilling With Force Feedback Simulated Haptic in Orthopedic Skills Training.

PURPOSE: Surgical simulations are becoming increasingly relevant in musculoskeletal training. They provide the opportunity to develop surgical skills in a controlled environment while reducing the risks for patients. For K-wire internal fixation in musculoskeletal surgery, a force feedback virtual reality (VR) simulator was developed. The aim of this study was to evaluate training results using this technology and compare the results with that of standard teaching on cadavers. METHODS: Twenty participants attending an AO Trauma Course during 2020 were randomly allocated in 2 groups. On day 1, group A was trained by senior surgeons using a cadaver and group B was trained by the VR simulator for K-wire insertion in the distal radius. On day 2, all participants performed K-wire insertion on the cadaver model, without assistance, to validate the training effect. RESULTS: On a surgical skills test, group B performed better than group A. In group B, the entry point of the first K-wire was closer to the targeted styloid process of the radius, and the protrusion of the K-wires into soft tissue was less than that in group A. CONCLUSIONS: Training with the VR simulator for K-wire insertion resulted in better surgical skills than training by a surgeon and cadaver model. CLINICAL RELEVANCE: Training with the VR simulator provides the opportunity to improve and refine surgical skills without the risk of harming patients. It offers easier access, unlimited repetitions, and is more cost-effective compared with training sessions with cadavers.

Symbiosis, virulence and natural-product biosynthesis in entomopathogenic bacteria are regulated by a small RNA.

Photorhabdus and Xenorhabdus species have mutualistic associations with nematodes and an entomopathogenic stage1,2 in their life cycles. In both stages, numerous specialized metabolites are produced that have roles in symbiosis and virulence3,4. Although regulators have been implicated in the regulation of these specialized metabolites3,4, how small regulatory RNAs (sRNAs) are involved in this process is not clear. Here, we show that the Hfq-dependent sRNA, ArcZ, is required for specialized metabolite production in Photorhabdus and Xenorhabdus. We discovered that ArcZ directly base-pairs with the mRNA encoding HexA, which represses the expression of specialized metabolite gene clusters. In addition to specialized metabolite genes, we show that the ArcZ regulon affects approximately 15% of all transcripts in Photorhabdus and Xenorhabdus. Thus, the ArcZ sRNA is crucial for specialized metabolite production in Photorhabdus and Xenorhabdus species and could become a useful tool for metabolic engineering and identification of commercially relevant natural products.

Gene autoregulation by 3' UTR-derived bacterial small RNAs.

Negative feedback regulation, that is the ability of a gene to repress its own synthesis, is the most abundant regulatory motif known to biology. Frequently reported for transcriptional regulators, negative feedback control relies on binding of a transcription factor to its own promoter. Here, we report a novel mechanism for gene autoregulation in bacteria relying on small regulatory RNA (sRNA) and the major endoribonuclease, RNase E. TIER-seq analysis (transiently-inactivating-an-endoribonuclease-followed-by-RNA-seq) revealed ~25,000 RNase E-dependent cleavage sites in Vibrio cholerae, several of which resulted in the accumulation of stable sRNAs. Focusing on two examples, OppZ and CarZ, we discovered that these sRNAs are processed from the 3' untranslated region (3' UTR) of the oppABCDF and carAB operons, respectively, and base-pair with their own transcripts to inhibit translation. For OppZ, this process also triggers Rho-dependent transcription termination. Our data show that sRNAs from 3' UTRs serve as autoregulatory elements allowing negative feedback control at the post-transcriptional level.

Switching fatty acid metabolism by an RNA-controlled feed forward loop.

Hfq (host factor for phage Q beta) is key for posttranscriptional gene regulation in many bacteria. Hfq's function is to stabilize sRNAs and to facilitate base-pairing with trans-encoded target mRNAs. Loss of Hfq typically results in pleiotropic phenotypes, and, in the major human pathogen Vibrio cholerae, Hfq inactivation has been linked to reduced virulence, failure to produce biofilms, and impaired intercellular communication. However, the RNA ligands of Hfq in V. cholerae are currently unknown. Here, we used RIP-seq (RNA immunoprecipitation followed by high-throughput sequencing) analysis to identify Hfq-bound RNAs in V. cholerae Our work revealed 603 coding and 85 noncoding transcripts associated with Hfq, including 44 sRNAs originating from the 3' end of mRNAs. Detailed investigation of one of these latter transcripts, named FarS (fatty acid regulated sRNA), showed that this sRNA is produced by RNase E-mediated maturation of the fabB 3'UTR, and, together with Hfq, inhibits the expression of two paralogous fadE mRNAs. The fabB and fadE genes are antagonistically regulated by the major fatty acid transcription factor, FadR, and we show that, together, FadR, FarS, and FadE constitute a mixed feed-forward loop regulating the transition between fatty acid biosynthesis and degradation in V. cholerae Our results provide the molecular basis for studies on Hfq in V. cholerae and highlight the importance of a previously unrecognized sRNA for fatty acid metabolism in this major human pathogen.

Optically tracked and 3D printed haptic phantom hand for surgical training system.

BACKGROUND: For surgical fixation of bone fractures of the human hand, so-called Kirschner-wires (K-wires) are drilled through bone fragments. Due to the minimally invasive drilling procedures without a view of risk structures like vessels and nerves, a thorough training of young surgeons is necessary. For the development of a virtual reality (VR) based training system, a three-dimensional (3D) printed phantom hand is required. To ensure an intuitive operation, this phantom hand has to be realistic in both, its position relative to the driller as well as in its haptic features. The softest 3D printing material available on the market, however, is too hard to imitate human soft tissue. Therefore, a support-material (SUP) filled metamaterial is used to soften the raw material. Realistic haptic features are important to palpate protrusions of the bone to determine the drilling starting point and angle. An optical real-time tracking is used to transfer position and rotation to the training system. METHODS: A metamaterial already developed in previous work is further improved by use of a new unit cell. Thus, the amount of SUP within the volume can be increased and the tissue is softened further. In addition, the human anatomy is transferred to the entire hand model. A subcutaneous fat layer and penetration of air through pores into the volume simulate shiftability of skin layers. For optical tracking, a rotationally symmetrical marker attached to the phantom hand with corresponding reference marker is developed. In order to ensure trouble-free position transmission, various types of marker point applications are tested. RESULTS: Several cuboid and forearm sample prints lead to a final 30 centimeter long hand model. The whole haptic phantom could be printed faultless within about 17 hours. The metamaterial consisting of the new unit cell results in an increased SUP share of 4.32%. Validated by an expert surgeon study, this allows in combination with a displacement of the uppermost skin layer a good palpability of the bones. Tracking of the hand marker in dodecahedron design works trouble-free in conjunction with a reference marker attached to the worktop of the training system. CONCLUSIONS: In this work, an optically tracked and haptically correct phantom hand was developed using dual-material 3D printing, which can be easily integrated into a surgical training system.

Quantitative Analysis of Surface Contouring with Pulsed Bipolar Radiofrequency on Thin Chondromalacic Cartilage.

The purpose of this study was to evaluate the quality of surface contouring of chondromalacic cartilage by bipolar radio frequency energy using different treatment patterns in an animal model, as well as examining the impact of the treatment onto chondrocyte viability by two different methods. Our experiments were conducted on 36 fresh osteochondral sections from the tibia plateau of slaughtered 6-month-old pigs, where the thickness of the cartilage is similar to that of human wrist cartilage. An area of 1 cm2 was first treated with emery paper to simulate the chondromalacic cartilage. Then, the treatment with RFE followed in 6 different patterns. The osteochondral sections were assessed for cellular viability (live/dead assay, caspase (cell apoptosis marker) staining, and quantitative analysed images obtained by fluorescent microscopy). For a quantitative characterization of none or treated cartilage surfaces, various roughness parameters were measured using confocal laser scanning microscopy (Olympus LEXT OLS 4000 3D). To describe the roughness, the Root-Mean-Square parameter (Sq) was calculated. A smoothing effect of the cartilage surface was detectable upon each pattern of RFE treatment. The Sq for native cartilage was Sq = 3.8 ± 1.1 µm. The best smoothing pattern was seen for two RFE passes and a 2-second pulsed mode (B2p2) with an Sq = 27.3 ± 4.9 µm. However, with increased smoothing, an augmentation in chondrocyte death up to 95% was detected. Using bipolar RFE treatment in arthroscopy for small joints like the wrist or MCP joints should be used with caution. In the case of chondroplasty, there is a high chance to destroy the joint cartilage.

Force-feedback assisted and virtual fixtures based K-wire drilling simulation.

One common method to fix fractures of the human hand after an accident is an osteosynthesis with Kirschner wires (K-wires) to stabilize the bone fragments. The insertion of K-wires is a delicate minimally invasive surgery, because surgeons operate almost without a sight. Since realistic training methods are time consuming, costly and insufficient, a virtual-reality (VR) based training system for the placement of K-wires was developed. As part of this, the current work deals with the real-time bone drilling simulation using a haptic force-feedback device. To simulate the drilling, we introduce a virtual fixture based force-feedback drilling approach. By decomposition of the drilling task into individual phases, each phase can be handled individually to perfectly control the drilling procedure. We report about the related finite state machine (FSM), describe the haptic feedback of each state and explain, how to avoid jerking of the haptic force-feedback during state transition. The usage of the virtual fixture approach results in a good haptic performance and a stable drilling behavior. This was confirmed by 26 expert surgeons, who evaluated the virtual drilling on the simulator and rated it as very realistic. To make the system even more convincing, we determined real drilling feed rates through experimental pig bone drilling and transferred them to our system. Due to a constant simulation thread we can guarantee a precise drilling motion. Virtual fixtures based force-feedback calculation is able to simulate force-feedback assisted bone drilling with high quality and, thus, will have a great potential in developing medical applications.

Imitating human soft tissue on basis of a dual-material 3D print using a support-filled metamaterial to provide bimanual haptic for a hand surgery training system.

BACKGROUND: Currently, it is common practice to use three-dimensional (3D) printers not only for rapid prototyping in the industry, but also in the medical area to create medical applications for training inexperienced surgeons. In a clinical training simulator for minimally invasive bone drilling to fix hand fractures with Kirschner-wires (K-wires), a 3D-printed hand phantom must not only be geometrically but also haptically correct. Due to a limited view during an operation, surgeons need to perfectly localize underlying risk structures only by feeling of specific bony protrusions of the human hand. METHODS: The goal of this experiment is to imitate human soft tissue with its haptic and elasticity for a realistic hand phantom fabrication, using only a dual-material 3D printer and support-material-filled metamaterial between skin and bone. We present our workflow to generate lattice structures between hard bone and soft skin with iterative cube edge (CE) or cube face (CF) unit cells. Cuboid and finger shaped sample prints with and without inner hard bone in different lattice thickness are constructed and 3D printed. RESULTS: The most elastic available rubber-like material is too firm to imitate soft tissue. By reducing the amount of rubber in the inner volume through support material (SUP), objects become significantly softer. Without metamaterial, after disintegration, the SUP can be shifted through the volume and thus the body loses its original shape. Although the CE design increases the elasticity, it cannot restore the fabric form. In contrast to CE, the CF design increases not only the elasticity but also guarantees a local limitation of the SUP. Therefore, the body retains its shape and internal bones remain in its intended place. Various unit cell sizes, lattice thickening and skin thickness regulate the rubber material and SUP ratio. Test prints with higher SUP and lower rubber material percentage appear softer and vice versa. This was confirmed by an expert surgeon evaluation. Subjects adjudged pure rubber-like material as too firm and samples only filled with SUP or lattice structure in CE design as not suitable for imitating tissue. 3D-printed finger samples in CF design were rated as realistic compared to the haptic of human tissue with a good palpable bone structure. CONCLUSIONS: We developed a new dual-material 3D print technique to imitate soft tissue of the human hand with its haptic properties. Blowy SUP is trapped within a lattice structure to soften rubber-like 3D print material, which makes it possible to reproduce a realistic replica of human hand soft tissue.

3D analysis of Osteosyntheses material using semi-automated CT segmentation: a case series of a 4 corner fusion plate.

BACKROUND: Scaphoidectomy and midcarpal fusion can be performed using traditional fixation methods like K-wires, staples, screws or different dorsal (non)locking arthrodesis systems. The aim of this study is to test the Aptus four corner locking plate and to compare the clinical findings to the data revealed by CT scans and semi-automated segmentation. METHODS: This is a retrospective review of eleven patients suffering from scapholunate advanced collapse (SLAC) or scaphoid non-union advanced collapse (SNAC) wrist, who received a four corner fusion between August 2011 and July 2014. The clinical evaluation consisted of measuring the range of motion (ROM), strength and pain on a visual analogue scale (VAS). Additionally, the Disabilities of the Arm, Shoulder and Hand (QuickDASH) and the Mayo Wrist Score were assessed. A computerized tomography (CT) of the wrist was obtained six weeks postoperatively. After semi-automated segmentation of the CT scans, the models were post processed and surveyed. RESULTS: During the six-month follow-up mean range of motion (ROM) of the operated wrist was 60°, consisting of 30° extension and 30° flexion. While pain levels decreased significantly, 54% of grip strength and 89% of pinch strength were preserved compared to the contralateral healthy wrist. Union could be detected in all CT scans of the wrist. While X-ray pictures obtained postoperatively revealed no pathology, two user related technical complications were found through the 3D analysis, which correlated to the clinical outcome. CONCLUSION: Due to semi-automated segmentation and 3D analysis it has been proved that the plate design can keep up to the manufacturers' promises. Over all, this case series confirmed that the plate can compete with the coexisting techniques concerning clinical outcome, union and complication rate.

Effects on the Distal Radioulnar Joint of Ablation of Triangular Fibrocartilage Complex Tears With Radiofrequency Energy.

PURPOSE: This cadaver study investigated the temperature profile in the wrist joint and distal radioulnar joint (DRUJ) during radiofrequency energy (RFE) application for triangular fibrocartilage complex resection. METHODS: An arthroscopic partial resection of the triangular fibrocartilage complex using monopolar and bipolar RFE was simulated in 14 cadaver limbs. The temperature was recorded simultaneously in the DRUJ and at 6 other anatomic locations of the wrist during RFE application. RESULTS: The mean temperature in the DRUJ was 43.3 ± 8.2°C for the bipolar system in the ablation mode (60 W) and 30.4 ± 3.4°C for the monopolar system in the cut mode (20 W) after 30 seconds. The highest measured temperature in the DRUJ was 54.3°C for the bipolar system and 68.1°C for the monopolar system. CONCLUSIONS: The application of RFE for debridement or resection of the triangular fibrocartilage complex in a clinical setting can induce peak temperatures that might cause damage to the cartilage of the DRUJ. Bipolar systems produce higher mean temperatures than monopolar devices. CLINICAL RELEVANCE: RFE application increases the mean temperature in the DRUJ after 30 seconds to a level that may jeopardize cartilage tissue.

Leukocyte-Reduced Platelet-Rich Plasma Treatment of Basal Thumb Arthritis: A Pilot Study.

A positive effect of intra-articular platelet-rich plasma (PRP) injection has been discussed for osteoarthritic joint conditions in the last years. The purpose of this study was to evaluate PRP injection into the trapeziometacarpal (TMC) joint. We report about ten patients with TMC joint osteoarthritis (OA) that were treated with 2 intra-articular PRP injections 4 weeks apart. PRP was produced using the Double Syringe System (Arthrex Inc., Naples, Florida, USA). A total volume of 1.47 ± 0.25 mL PRP was injected at the first injection and 1.5 ± 0.41 mL at the second injection, depending on the volume capacity of the joint. Patients were evaluated using VAS, strength measures, and the Mayo Wrist score and DASH score after 3 and 6 months. VAS significantly decreased from 6.2 ± 1.6 to 5.4 ± 2.2 at six-month follow-up (P < 0.05). The DASH score was unaffected; however, the Mayo Wrist score significantly improved from 46.5 ± 18.6 to 67.5 ± 19.0 at six-month follow-up (P = 0.05). Grip was unaffected, whereas pinch declined from 6.02 ± 2.99 to 3.96 ± 1.77 at six-month follow-up (P < 0.05). We did not observe adverse events after the injection of PRP, except one occurrence of a palmar wrist ganglion, which resolved without treatment. PRP injection for symptomatic TMC OA is a reasonable therapeutic option in early stages TMC OA and can be performed with little to no morbidity.

uPAR enhances malignant potential of triple-negative breast cancer by directly interacting with uPA and IGF1R.

BACKGROUND: Due to lack of a targeted therapy for the triple-negative breast cancer (TNBC) patients, it is important to explore this aggressive breast cancer type in more detail and to establish novel therapeutic approaches. TNBC is defined negative for the protein expression of oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). One prominent feature of this cancer type is the frequent overexpression of major components of the urokinase-type plasminogen activator system (uPAS) including uPA, its receptor uPAR and the inhibitor PAI-1, which may be valuable as therapeutic targets. METHODS: Direct interactions of uPAR with interactors were demonstrated by immunoprecipitations and proximity ligation assays. For stable knockdowns of target proteins, lentiviral vectors were used and the effects were analysed by immunoblottings and using in vitro cell viability, migration and invasion assays. Immunohistochemical and statistical analyses of biomarkers and clinical parameters were conducted in a TNBC cohort (n = 174). RESULTS: Direct tumour-promoting interactions of uPAR with uPA and the insulin-like growth factor receptor 1 (IGF1R) were shown in TNBC cells and these interactions were significantly reduced (p = 0.001) when uPAR was downregulated. The combined knockdown of uPAR and uPA or IGF1R additively and significantly reduced cell viability, migration and invasion of the model cell lines. In TNBC tissue, the complexes formed by uPAR with uPA or with IGF1R significantly correlated with the histological grade (p = 0.0019) as well as with cathepsin B and D (p ≤ 0.0001) that are implicated in cell invasion and metastasis. CONCLUSIONS: Our outcomes show that not only overexpressed biomarkers promote tumourigenesis, but rather their interactions further potentiate tumour progression. This study emphasises the potential of combined approaches targeting uPAR and its interactors with regard to an improved therapy of TNBC.

Cyr61 and YB-1 are novel interacting partners of uPAR and elevate the malignancy of triple-negative breast cancer.

The triple-negative breast cancer (TNBC) is a very aggressive tumor type often occurring in young women and is associated with a bad prognosis for the patients. TNBC lacks established targets for breast cancer therapy, such as the estrogen receptor (ER), progesterone receptor (PR) and the human epidermal growth factor receptor 2 (HER2). Therefore, novel therapeutic targets and strategies are needed for an improved treatment of this breast cancer subtype. TNBC and respective cell lines often overexpress proteins of the urokinase plasminogen activator system (uPAS) including uPA, its receptor uPAR and inhibitor PAI-1, which together with co-factors contribute to the malignancy of TNBC. Here, two novel interacting partners of uPAR, the cysteine-rich angiogenic inducer 61 (Cyr61) and the Y-box-binding protein 1 (YB-1) were identified and their differential expression demonstrated in TNBC cells as well as in tumors. In the TNBC cohort, both interactors significantly correlated with expression levels of cathepsin B, c-Met and the tumor grade. In addition, expression levels of Cyr61 significantly correlated with cathepsin D (p=0.03), insulin receptor (p≤0.001), insulin-like growth factor receptor 1 (IGF1R, p=0.015) and also with YB-1 (p=0.0004) levels. The interactions of uPAR with Cyr61 significantly correlated with expression levels of tumor-promoting biomarkers including plasminogen (p=0.0014), cathepsin B (p=0.032), c-Met (p=0.0192) as well as with the tumor grade (p=0.02). In multivariate survival analysis, YB-1 showed independent prognostic value (p=0.01). As the novel interacting partners, also together with uPAR, contribute to tumor progression and metastasis, both may be potential therapeutic targets in breast cancer.

Leukocyte-Reduced Platelet-Rich Plasma Alters Protein Expression of Adipose Tissue-Derived Mesenchymal Stem Cells.

BACKGROUND: Application of platelet-rich plasma and stem cells has become important in regenerative medicine. Recent literature supports the use of platelet-rich plasma as a cell culture media supplement to stimulate proliferation of adipose tissue-derived mesenchymal stem cells. The underlying mechanism of proliferation stimulation by platelet-rich plasma has not been investigated so far. METHODS: Adipose tissue-derived mesenchymal stem cells were cultured in α-minimal essential medium supplemented with platelet-rich plasma or fetal calf serum. Cell proliferation was assessed with cell cycle kinetics using flow cytometric analyses after 48 hours. Differences in proteome expression of the adipose tissue-derived mesenchymal stem cells were analyzed using a reverse-phase protein array to quantify 214 proteins. Complementary Ingenuity Pathways Analysis and gene set enrichment analysis were performed using protein data, and confirmed by Western blot analysis. RESULTS: A higher percentage of adipose tissue-derived mesenchymal stem cells in the S phase in the presence of platelet-rich plasma advocates the proliferation stimulation. Ingenuity Pathways Analysis and gene set enrichment analysis confirm the involvement of the selected proteins in the process of cell growth and proliferation. Ingenuity Pathways Analysis revealed a participation in the top-ranked canonical pathways PI3K/AKT, PTEN, ILK, and IGF-1. Gene set enrichment analysis identified the authors' protein set as being part of significantly regulated protein sets with the focus on cell cycle, metabolism, and the Kyoto Encyclopedia of Genes and Genomes transforming growth factor-ß signaling pathway. CONCLUSIONS: The present study provides evidence that platelet-rich plasma stimulates proliferation and induces a unique change in the proteomic profile of adipose tissue-derived mesenchymal stem cells. The interpretation of altered expression of regulatory proteins represents a step forward toward achieving good manufacturing practice-compliant criteria for cell-based strategies.

The effect of leukocyte-reduced platelet-rich plasma on the proliferation of autologous adipose-tissue derived mesenchymal stem cells.

Clinical application of platelet-rich plasma (PRP) and stem cells has become more and more important in regenerative medicine during the last decade. However, differences in PRP preparations may contribute to variable PRP compositions with unpredictable effects on a cellular level. In the present study, we modified the centrifugation settings in order to provide a leukocyte-reduced PRP and evaluated the interactions between PRP and adipose-tissue derived mesenchymal stem cells (ASCs).PRP was obtained after modification of three different centrifugation settings and investigated by hemogram analysis, quantification of protein content and growth factor concentration. ASCs were cultured in serum-free α-MEM supplemented with autologous 10% or 20% leukocyte-reduced PRP. Cell cycle kinetics of ASCs were analyzed using flow cytometric analyses after 48 hours.Thrombocytes in PRP were concentrated, whereas erythrocytes, and white blood cells (WBC) were reduced, independent of centrifugation settings. Disabling the brake further reduced the number of WBCs. A higher percentage of cells in the S-phase in the presence of 20% PRP in comparison to 10% PRP and 20% fetal calf serum (FCS) advocates the proliferation stimulation of ASCs.These findings clearly demonstrate considerable differences between three PRP separation settings and assist in safeguarding the combination of leukocyte-reduced PRP and stem cells for regenerative therapies.

Seeing Red: Anger Increases How Much Republican Identification Predicts Partisan Attitudes and Perceived Polarization.

We examined the effects of incidental anger on perceived and actual polarization between Democrats and Republicans in the context of two national tragedies, Hurricane Katrina (Study 1) and the mass shooting that targeted Representative Gabrielle Giffords in Arizona (Study 2). We hypothesized that because of its relevance to intergroup conflict, incidental anger exacerbates the political polarization effects of issue partisanship (the correlation between partisan identification and partisan attitudes), and, separately, the correlation between conservative partisan identification and perceived polarization between Democrats and Republicans. We further hypothesized that these effects would be strongest for Republican identification because Republican leaders were targets of public criticism in both tragedies and because conservative (Republican) ideology tends to be more sensitive to threat. In the studies, participants first completed an emotion induction procedure by recalling autobiographical events that made them angry (Studies 1 & 2), sad (Studies 1 & 2), or that involved recalling emotionally neutral events (Study 2). Participants later reported their attitudes regarding the two tragedies, their perceptions of the typical Democrat's and Republican's attitudes on those issues, and their identification with the Democratic and Republican parties. Compared with incidental sadness (Studies 1 and 2) and a neutral condition (Study 2), incidental anger exacerbated the associations between Republican identification and partisan attitudes, and, separately between Republican identification and perceived polarization between the attitudes of Democrats and Republicans. We discuss implications for anger's influence on political attitude formation and perceptions of group differences in political attitudes.