The Angiopoietin-2/Angiopoietin-1 ratio increases early in burn patients and predicts mortality.

BACKGROUND: Angiopoietin-2 (Angpt-2) is involved in the pathogenesis of the capillary leak syndrome in sepsis and has been shown to be associated with worse outcomes in diverse critical illnesses. It is however unclear whether Angpt-2 plays a similar role in severely burned patients during the early phase characterized by massive capillary leakage. Our aim was to analyze the Angiopoietin-2/Angiopoietin-1 ratio (Angpt-2/Angpt-1 ratio) over the first two days in critically ill burn patients and examine its association with survival and further clinical parameters. METHODS: Adult burn patients with a total burn surface area (TBSA) ≥ 20% treated in the burn intensive care unit (ICU) of the University Hospital of Zurich, Switzerland, were included. Serum samples were collected prospectively and serum Angpt-1 and Angpt-2 were measured by enzyme-linked immunosorbent assay (ELISA) over the first two days after burn insult and stratified according to survival status, TBSA and the abbreviated burn severity index (ABSI). Due to hemodilution in the initial resuscitation phase, the Angpt-2/Angpt-1 ratio was normalized to albumin. RESULTS: Fifty-six patients were included with a median age of 51.5 years. Overall mortality was 14.3% (8/56 patients). The total amount of infused crystalloids was 12́902 ml (IQR 9́362-16́770 ml) at 24 h and 18́461 ml (IQR 13́024-23́766 ml) at 48 h. The amount of substituted albumin was 20 g (IQR 10-50 g) at 24 h and 50 g (IQR 20-60 g) at 48 h. The albumin-corrected Angpt-2/Angpt-1 ratios increased over the first 48 h after the burn insult (d0: 0.5 pg*l/ml*g [IQR 0.24 - 0.80 pg*l/ml*g]; d1: 0.83 pg*l/ml*g [IQR 0.29 - 1.98 pg*l/ml*g]; d2: 1.76 pg*l/ml*g [IQR 0.70 - 3.23 pg*l/ml*g]; p < 0.001) and were significantly higher in eventual ICU non-survivors (p = 0.005), in patients with a higher TBSA (p = 0.001) and in patients with a higher ABSI (p = 0.001). CONCLUSIONS: In analogy to the pathological host response in sepsis, the Angpt-2/Angpt-1 ratio steadily increases in the first two days in critically ill burn patients, suggesting a putative involvement in the pathogenesis of capillary leakage in burns. A higher Angpt-2/Angpt-1 ratio is associated with mortality, total burn surface area and burn scores.

Methacrylated Gelatin as a Scaffold for Mechanically Isolated Stromal Vascular Fraction for Cutaneous Wound Repair.

Mechanically processed stromal vascular fraction (mSVF) is a highly interesting cell source for regenerative purposes, including wound healing, and a practical alternative to enzymatically isolated SVF. In the clinical context, SVF benefits from scaffolds that facilitate viability and other cellular properties. In the present work, the feasibility of methacrylated gelatin (GelMA), a stiffness-tunable, light-inducible hydrogel with high biocompatibility is investigated as a scaffold for SVF in an in vitro setting. Lipoaspirates from elective surgical procedures were collected and processed to mSVF and mixed with GelMA precursor solutions. Non-encapsulated mSVF served as a control. Viability was measured over 21 days. Secreted basic fibroblast growth factor (bFGF) levels were measured on days 1, 7 and 21 by ELISA. IHC was performed to detect VEGF-A, perilipin-2, and CD73 expression on days 7 and 21. The impact of GelMA-mSVF on human dermal fibroblasts was measured in a co-culture assay by the same viability assay. The viability of cultured GelMA-mSVF was significantly higher after 21 days (p < 0.01) when compared to mSVF alone. Also, GelMA-mSVF secreted stable levels of bFGF over 21 days. While VEGF-A was primarily expressed on day 21, perilipin-2 and CD73-positive cells were observed on days 7 and 21. Finally, GelMA-mSVF significantly improved fibroblast viability as compared with GelMA alone (p < 0.01). GelMA may be a promising scaffold for mSVF as it maintains cell viability and proliferation with the release of growth factors while facilitating adipogenic differentiation, stromal cell marker expression and fibroblast proliferation.

Outcome Comparison of Different Reconstructive Approaches for Axillary Defects Secondary to Radical Excision of Hidradenitis Suppurativa.

BACKGROUND: Radical excision of debilitating hidradenitis suppurativa lesions is the only curative approach in the advanced stages of the disease. Different concepts for axillary reconstruction do exist, but data on their clinical outcome are scarce. METHODS: This is a retrospective cohort study of two reconstructive methods (posterior arm flap vs. vacuum-assisted closure [VAC] + split-thickness skin graft [STSG]) for axillary defects in patients with severe axillary hidradenitis suppurativa treated at the University Hospital Zurich between 2005 and 2020. RESULTS: A total of 35 patients (mean age 36 ± 10 years, mean BMI 29 ± 5 kg/m2, Hurley stage II-III) with 67 operated axillae were stratified according to their type of reconstruction. Median operation time in the flap group was 144 min (IQR 114-207) (cumulative 181 min [IQR 124-300]) and 50 min (IQR 40-81) in the VAC + STSG group (cumulative 151 min [IQR 94-194], p < 0.01; p = 0.20 [cumulative time]). The cumulative length of stay was 6 ± 3 days in the flap group and 14 ± 7 days in the VAC + STSG group (p < 0.01). Time to complete wound healing was 27 days (IQR 20-49) in the flap group and 62 days (IQR 41-75) in the VAC + STSG group (p < 0.01). Vancouver Scar Scale score was 6 (IQR 4-9) in the flap group and 11 (IQR 9-12) in the VAC + STSG group (p < 0.01). Protective sensory recovery was most satisfactory in the flap group (p < 0.01). Forty-four percent of patients of the VAC + STSG group demonstrated functional impairment of arm abduction. Time to return to work was less in group A with 42 days (IQR 27-57) needed as compared to group B with 48 days (IQR 34-55) needed (p = 0.32). The average cost saving was 25% higher for the flap group than for the VAC + STSG group. CONCLUSION: Despite an increased operation time, axillary reconstruction by the posterior arm flap yields a reduced length of stay, less time to complete wound healing along with restoration of a protective sensibility, and less axillary scarring avoiding functional deficits - eventually allowing earlier return to work.

Burn Scar Ectropion Correction: Surgical Technique for Functional Outcomes.

BACKGROUND: Eyelid scarring after severe burn injury of the face is a significant complication endangering vision in addition to the burn scar sequelae. Scar contraction leads to asymmetry and malposition of the eyelid axis, resulting in corneal exposure, eyelid retraction, and incomplete eyelid closure. In consequence, dryness and irritation of the cornea can lead to keratitis, corneal opacity, and vision impairment. In this study, we present our surgical technique for lateral canthopexy in combination with full-thickness skin grafting (FTSGing) in patients with eyelid axis distortion after scar contraction of the periorbital region after severe burn injuries of the face. METHODS: In this retrospective, single-center case study, we present 5 consecutive patients who experienced severe burn injuries to the face between 2014 and 2019. Patients were suffering from ectropion and malposition of the eyelid axis. In all cases, we performed lateral transosseous canthopexy and FTSGing. RESULTS: Improved symmetry and complete eyelid closure were restored in all 5 patients. The following ophthalmological examinations showed resolved corneal erosions, as well as reduction of chemosis and epiphora. Further vision impairment was successfully prohibited. Surgical revision with FTSGing was required in 2 patients because of recurrence of unilateral lower eyelid retraction. CONCLUSIONS: Lateral transosseous canthopexy represents a suitable surgical method to durably correct eyelid malposition, ectropion, and incomplete lid closure in patients with severe scarring of the periorbital region after burns of the face. Early detection of patients at risk and timing of surgical intervention are of great importance.

The Multiple Rib Osteomyocutaneous Split Latissimus Dorsi Flap for Calvarial Reconstruction: Indication, Operative Technique, and Review of Literature.

Major complex cranial defects may be challenging for the reconstructive microsurgeon. Affected patients often present with impaired soft tissues including dura exposure or fistulas. The lacking structural bony support may cause severe neurological issues and in select patients, there is a need for well-vascularized autologous tissue repair. The authors herein elucidate the role of the multiple rib osteomyocutaneous split latissimus dorsi flap for reconstruction of composite skull defects, providing an indication, an exemplary case, operation technique, and literature review. A 40-year-old woman after anaplastic oligodendroglioma resection suffered multiple extrusions and allograft cranioplasty infections. The defect was reconstructed with an osteomyocutaneus split latissimus dorsi flap including costae 3 ribs and a skin island. The included ribs were nourished via the anterior periosteum, while the posterior periosteum was left in place for the protection of the pleura parietalis. A proper amount of craniomedial latissimus dorsi muscle was spared to reduce donor site morbidity. The patient presented after 6 months with stable bony and soft tissue conditions without neurological symptoms, and acceptable donor site morbidity. After failed alloplastic cranioplasties, the free latissimus dorsi flap including vascularized ribs is well suitable for coverage of large compound cranial defects, providing skeletal support, improved contour, and enhanced functional outcome.

PID-1 is a novel factor that operates during 21U-RNA biogenesis in Caenorhabditis elegans.

The Piwi-piRNA pathway represents a small RNA-based mechanism responsible for the recognition and silencing of invading DNA. Biogenesis of piRNAs (21U-RNAs) is poorly understood. In Caenorhabditis elegans, the piRNA-binding Argonaute protein PRG-1 is the only known player acting downstream from precursor transcription. From a screen aimed at the isolation of piRNA-induced silencing-defective (Pid) mutations, we identified, among known Piwi pathway components, PID-1 as a novel player. PID-1 is a mostly cytoplasmic, germline-specific factor essential for 21U-RNA biogenesis, affecting an early step in the processing or transport of 21U precursor transcripts. We also show that maternal 21U-RNAs are essential to initiate silencing.

Pancreatic Stone Protein Predicts Sepsis in Severely Burned Patients Irrespective of Trauma Severity: A Monocentric Observational Study.

OBJECTIVE: The burn victim's inherent state of hyperinflammation frequently camouflages septic events delaying the initiation of targeted intensive care therapy. Accurate biomarkers are urgently needed to support sepsis detection before patients' clinical deterioration. SUMMARY OF BACKGROUND DATA: Evidence on the usefulness of pancreatic stone protein (PSP) as a powerful diagnostic and prognostic marker in critically ill patients has recently accumulated. METHODS: Analysis of biomarker kinetics (PSP, routine markers) was performed on 90 patients admitted to the Zurich Burn Center between May 2015 and October 2018 with burns ≥15% total body surface area with regard to infection and sepsis (Sepsis-3) over a 14-day time course. RESULTS: PSP differentiated between sepsis, infection and sterile inflammation from day 3 onward with an area under the curve of up to 0.89 (P < 0.001), therefore, competing with procalcitonin (area under the curve = 0.86, P < 0.001). Compared to routine inflammatory biomarkers, only PSP demonstrated a significant interaction between time and presence of sepsis - signifying a steeper increase in PSP levels in septic patients as opposed to those exhibiting a nonseptic course (interaction P < 0.001). Event-related analysis demonstrated tripled PSP serum levels within 72 hours and doubled levels within 48 hours before a clinically apparent sepsis. CONCLUSION: PSP is able to differentiate between septic and nonseptic patients during acute burn care. Its steep rise up to 72 hours before clinically overt deterioration has the potential for physicians to timely initiate treatment with reduced mortality and costs.

Accuracy of pancreatic stone protein for the diagnosis of infection in hospitalized adults: a systematic review and individual patient level meta-analysis.

BACKGROUND: Accurate biomarkers to diagnose infection are lacking. Studies reported good performance of pancreatic stone protein (PSP) to detect infection. The objective of the study was to determine the performance of PSP in diagnosing infection across hospitalized patients and calculate a threshold value for that purpose. METHODS: A systematic search across Cochrane Central Register of Controlled Trials and MEDLINE databases (1966-March 2019) for studies on PSP published in English using 'pancreatic stone protein', 'PSP', 'regenerative protein', 'lithostatin' combined with 'infection' and 'sepsis' found 44 records. The search was restricted to the five trials that evaluated PSP for the initial detection of infection in hospitalized adults. Individual patient data were obtained from the investigators of all eligible trials. Data quality and validity was assessed according to PRISMA guidelines. We choose a fixed-effect model to calculate the PSP cut-off value that best discriminates infected from non-infected patients. RESULTS: Infection was confirmed in 371 of 631 patients. The median (IQR) PSP value of infected versus uninfected patients was 81.5 (30.0-237.5) versus 19.2 (12.6-33.57) ng/ml, compared to 150 (82.70-229.55) versus 58.25 (15.85-120) mg/l for C-reactive protein (CRP) and 0.9 (0.29-4.4) versus 0.15 (0.08-0.5) ng/ml for procalcitonin (PCT). Using a PSP cut-off of 44.18 ng/ml, the ROC AUC to detect infection was 0.81 (0.78-0.85) with a sensitivity of 0.66 (0.61-0.71), specificity of 0.83 (0.78-0.88), PPV of 0.85 (0.81-0.89) and NPV of 0.63 (0.58-0.68). When a model combining PSP and CRP was used, the ROC AUC improved to 0.90 (0.87-0.92) with higher sensitivity 0.81 (0.77-0.85) and specificity 0.84 (0.79-0.90) for discriminating infection from non-infection. Adding PCT did not improve the performance further. CONCLUSIONS: PSP is a promising biomarker to diagnose infections in hospitalized patients. Using a cut-off value of 44.18 ng/ml, PSP performs better than CRP or PCT across the considered studies. The combination of PSP with CRP further enhances its accuracy.

Adipose-derived stromal cell therapy combined with a short course nonmyeloablative conditioning promotes long-term graft tolerance in vascularized composite allotransplantation.

The risks of chronic immunosuppression limit the utility of vascularized composite allotransplantation (VCA) as a reconstructive option in complex tissue defects. We evaluated a novel, clinically translatable, radiation-free conditioning protocol that combines anti-lymphocyte serum (ALS), tacrolimus, and cytotoxic T-lymphocyte-associated protein 4 immunoglobulin (CTLA4-Ig) with adipose-derived stromal cells (ASCs) to allow VCA survival without long-term systemic immunosuppression. Full-mismatched rat hind-limb-transplant recipients received tacrolimus (0.5 mg/kg) for 14 days and were assigned to 4 groups: controls (CTRL) received no conditioning; ASC-group received CTLA4-Ig (10 mg/kg body weight i.p. postoperative day [POD] 2, 4, 7) and donor ASCs (1 × 106 iv, POD 2, 4, 7, 15, 28); the ASC-cyclophosphamide (CYP)-group received CTLA4-Ig, ASC plus cyclophosphamide (50 mg/kg ip, POD 3); the ASC-ALS-group received CTLA4-Ig, ASCs plus ALS (500 µL ip, POD 1, 5). Banff grade III or 120 days were endpoints. ASCs suppressed alloresponse in vitro. Median rejection-free VCA survival was 28 days in CTRL (n = 7), 34 in ASC (n = 6), and 27.5 in ASC-CYP (n = 4). In contrast, ASC-ALS achieved significantly longer, rejection-free VCA survival in 6/7 animals (86%), with persistent mixed donor-cell chimerism, and elevated systemic and allograft skin Tregs , with no signs of acute cellular rejection. Taken together, a regimen comprised of short-course tacrolimus, repeated CTLA4-Ig and ASC administration, combined with ALS, promotes long-term VCA survival without chronic immunosuppression.

PingPongPro: a tool for the detection of piRNA-mediated transposon-silencing in small RNA-Seq data.

Summary: Piwi-interacting RNAs (piRNAs) are a class of small non-coding RNAs which guide endonucleases to mRNAs of actively transcribed transposons in order to prevent their translation. The resulting mRNA fragments induce a positive feedback loop (the 'ping-pong cycle'), which reinforces piRNA production and hence the transposon-silencing effect. PingPongPro is a command-line tool to scan small RNA-Seq data for signs of ping-pong cycle activity. It implements a novel algorithm that combines empirical probabilities in a multi-factor model to accurately identify transposons which are suppressed through the ping-pong cycle. Availability and implementation: Source code, a user manual, and binaries for Microsoft Windows and Linux are available at https://github.com/suhrig/pingpongpro under the GPLv3 license. Supplementary information: Supplementary data are available at Bioinformatics online.

Expression of Pancreatic Stone Protein is Unaffected by Trauma and Subsequent Surgery in Burn Patients.

BACKGROUND: Altered levels of pro-inflammatory markers secondary to trauma or surgery present a major problem to physicians in being prone to interfere with the clinical identification of infectious events. METHODS: Patients admitted to Zurich Burn Center between May 2015 and October 2018 with burns ≥10% total body surface area (TBSA) and without infection. Longitudinal analysis of the time course of PSP and routine inflammatory biomarkers [procalcitonin (PCT), C-reactive protein (CRP) and white blood cells (WBC)] over two days after (a) trauma with initial debridement and (b) subsequent burn surgeries was performed. The influence of TBSA, abbreviated burn severity index (ABSI), age and length of operation was investigated using a linear mixed effect regression model. RESULTS: Sixty-six patients (15 female) were included with a mean age of 45.5 ± 18.3 years, median TBSA of 22% (IQR 17) and mean ABSI score 6.8 ± 2.7. PSP was the only biomarker that showed no association with any of the baseline characteristics. Additionally, PSP serum levels did not change over time neither after the burn trauma (p = 0.832) nor after secondary procedures (p = 0.113), while PCT levels increased significantly after the trauma (p < 0.001). Similarly, CRP serum levels were elevated significantly after both trauma and surgery (p < 0.001), whereas WBC values demonstrated a significant decline after the trauma (p < 0.001). CONCLUSION: Established biomarkers (WBC, CRP and PCT) demonstrate decisive alterations after tissue destruction caused by burn injuries and subsequent surgical interventions. The robustness of PSP serum levels toward these inflammatory insults is a quality criterion for an upcoming sepsis biomarker.

The Significance of Vascular Alterations in Acute and Chronic Rejection for Vascularized Composite Allotransplantation.

Vascularized composite allotransplantation (VCA) has emerged as a useful reconstructive option for patients suffering from major tissue defects and functional deficits. While the technical feasibility has been optimized and more than 130 VCAs have been performed during the last two decades, hurdles such as acute and chronic allograft rejection, graft deterioration, and eventual functional impairment need to be addressed. Recently, chronic graft rejection and progressive failure have been linked to vascular alterations observed in the allografts. Graft vasculopathy (GV) may play a pivotal role in long-term graft deterioration. The understanding of the underlying pathophysiological processes and their initial triggers is of utmost importance in the prevention, attenuation, and therapy of GV. While there are reports on the etiology and development of GV in solid organ transplantation, there are limited data with respect to chronic rejection and GV in the realm of VCA. Nevertheless, recent reports from long-term VCA recipients suggest that GV could truly jeopardize allografts in the follow-up evaluation. Chronic rejection and GV include different entities and might have different pathways in distinct organs. Herein, we reviewed the current literature on vascular changes during both acute and chronic allograft rejection, with a focus on their clinical and translational significance for VCA.

Investigation of the Structure of the Modulated Doubly Ordered Perovskite NaLaCoWO6 and Its Reversible Phase Transition with a Colossal Temperature Hysteresis.

Transmission electron microscopy, neutron diffraction, and synchrotron powder X-ray diffraction reveal a complex modulated structure on the doubly ordered perovskite NaLaCoWO6. Electron diffraction patterns as well as high-resolution transmission electron microscopy images clearly show a periodicity of 12 ap, where ap is the cell parameter of the generic perovskite, along either the [100]p or [010]p direction. Annular bright-field scanning transmission electron microscopy of slightly tilted samples shows that there is no chemical origin for the superstructure but that it is caused by geometric rearrangements. An atomic model of the superstructure is proposed on the basis of octahedral tilt twinning. At low temperature, NaLaCoWO6 undergoes a phase transition and the superstructure disappears. The compound takes on the more usual monoclinic P21 structure below the transition. Neutron powder diffraction reveals and electron diffraction confirms an unusually large temperature hysteresis, where the transition takes place at ∼180 K on cooling and at ∼320 K on heating. This hysteresis can be attributed to the necessity of rearranging the oxygen octahedra and the thus induced energy barrier for the transition.

Contemporary Concepts of Primary Dynamic Facial Nerve Reconstruction in the Oncologic Patient.

Transection of the facial nerve and its branches during extensive ablative procedures in the oncologic patient causes loss of control of facial mimetic muscles with severe functional and aesthetic sequelae. In such patients with advanced tumorous disease, copious comorbidities, and poor prognosis, rehabilitation of the facial nerve has long been considered of secondary priority. However, recent advances in primary facial nerve reconstruction after extensive resection demonstrated encouraging results focusing on rapid and reliable restoration of facial functions. The authors summarize 3 innovative approaches of primary dynamic facial nerve reconstruction by using vascularized nerve grafts, dual innervation concepts, and intra-facial nerve transfers.

The International Fellowship Program in Microsurgery at Uppsala University Hospital: A Paragon Throughout Europe.

Personal reports with valuable information on plastic surgery fellowships from all over the world are about to accumulate in recent years. Whereas some institutions have rightly become widely renowned for their excellent microsurgery fellow training in Taiwan, Canada, United States, and Australia, less is known about European fellowships focusing on reconstructive microsurgery. As former fellow at the Department of Plastic and Maxillofacial Surgery at Uppsala University Hospital, Sweden, the author hereby presents a survey on a unique and exemplary 6-month microsurgical fellowship offering hands-on training, academic education, and not last personal development to ambitious candidates who pursue a career in reconstructive microsurgery.

Structural Study of a Doubly Ordered Perovskite Family NaLnCoWO6 (Ln = Y, La, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Yb): Hybrid Improper Ferroelectricity in Nine New Members.

The compounds of the doubly ordered perovskite family NaLnCoWO6 (Ln = Y, La, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, and Yb) were synthesized by solid-state reaction, nine of which (Ln = Y, Sm, Eu, Gd, Tb, Dy, Ho, Er, and Yb) are new phases prepared under high-temperature and high-pressure conditions. Their structural properties were investigated at room temperature by synchrotron X-ray powder diffraction and neutron powder diffraction. All of them crystallize in monoclinic structures, especially the nine new compounds have the polar space group P21 symmetry, as confirmed by second harmonic generation measurements. The P21 polar structures were decomposed and refined in terms of symmetry modes, demonstrating that the polar mode is induced by two nonpolar modes in a manner of Hybrid Improper Ferroelectricity. The amplitudes of these three major modes all increase with decreasing the Ln cation size. The spontaneous ferroelectric polarization is estimated from the neutron diffraction data of three samples (Ln = Y, Tb, and Ho) and can be as large as ∼20 µC/cm2.

Complications After Cosmetic Surgery Tourism.

Background: Cosmetic surgery tourism characterizes a phenomenon of people traveling abroad for aesthetic surgery treatment. Problems arise when patients return with complications or need of follow-up care. Objectives: To investigate the complications of cosmetic surgery tourism treated at our hospital as well as to analyze arising costs for the health system. Methods: Between 2010 and 2014, we retrospectively included all patients presenting with complications arising from cosmetic surgery abroad. We reviewed medical records for patients' characteristics including performed operations, complications, and treatment. Associated cost expenditure and Diagnose Related Groups (DRG)-related reimbursement were analyzed. Results: In total 109 patients were identified. All patients were female with a mean age of 38.5 ± 11.3 years. Most procedures were performed in South America (43%) and Southeast (29.4%) or central Europe (24.8%), respectively. Favored procedures were breast augmentation (39.4%), abdominoplasty (11%), and breast reduction (7.3%). Median time between the initial procedure abroad and presentation was 15 days (interquartile range [IQR], 9) for early, 81.5 days (IQR, 69.5) for midterm, and 4.9 years (IQR, 9.4) for late complications. Main complications were infections (25.7%), wound breakdown (19.3%), and pain/discomfort (14.7%). The majority of patients (63.3%) were treated conservatively; 34.8% became inpatients with a mean hospital stay of 5.2 ± 3.8 days. Overall DRG-related reimbursement premiums approximately covered the total costs. Conclusions: Despite warnings regarding associated risks, cosmetic surgery tourism has become increasingly popular. Efficient patients' referral to secondary/tertiary care centers with standardized evaluation and treatment can limit arising costs without imposing a too large burden on the social healthcare system. Level of Evidence: 4.

dupRadar: a Bioconductor package for the assessment of PCR artifacts in RNA-Seq data.

BACKGROUND: PCR clonal artefacts originating from NGS library preparation can affect both genomic as well as RNA-Seq applications when protocols are pushed to their limits. In RNA-Seq however the artifactual reads are not easy to tell apart from normal read duplication due to natural over-sequencing of highly expressed genes. Especially when working with little input material or single cells assessing the fraction of duplicate reads is an important quality control step for NGS data sets. Up to now there are only tools to calculate the global duplication rates that do not take into account the effect of gene expression levels which leaves them of limited use for RNA-Seq data. RESULTS: Here we present the tool dupRadar, which provides an easy means to distinguish the fraction of reads originating in natural duplication due to high expression from the fraction induced by artefacts. dupRadar assesses the fraction of duplicate reads per gene dependent on the expression level. Apart from the Bioconductor package dupRadar we provide shell scripts for easy integration into processing pipelines. CONCLUSIONS: The Bioconductor package dupRadar offers straight-forward methods to assess RNA-Seq datasets for quality issues with PCR duplicates. It is aimed towards simple integration into standard analysis pipelines as a default QC metric that is especially useful for low-input and single cell RNA-Seq data sets.

Riboswitch-controlled IL-12 gene therapy reduces hepatocellular cancer in mice.

Hepatocellular carcinoma (HCC) and solid cancers with liver metastases are indications with high unmet medical need. Interleukin-12 (IL-12) is a proinflammatory cytokine with substantial anti-tumor properties, but its therapeutic potential has not been realized due to severe toxicity. Here, we show that orthotopic liver tumors in mice can be treated by targeting hepatocytes via systemic delivery of adeno-associated virus (AAV) vectors carrying the murine IL-12 gene. Controlled cytokine production was achieved in vivo by using the tetracycline-inducible K19 riboswitch. AAV-mediated expression of IL-12 led to STAT4 phosphorylation, interferon-γ (IFNγ) production, infiltration of T cells and, ultimately, tumor regression. By detailed analyses of efficacy and tolerability in healthy and tumor-bearing animals, we could define a safe and efficacious vector dose. As a potential clinical candidate, we characterized vectors carrying the human IL-12 (huIL-12) gene. In mice, bioactive human IL-12 was expressed in a vector dose-dependent manner and could be induced by tetracycline, suggesting tissue-specific AAV vectors with riboswitch-controlled expression of highly potent proinflammatory cytokines as an attractive approach for vector-based cancer immunotherapy.

The KRAB-containing zinc-finger transcriptional regulator ZBRK1 activates SCA2 gene transcription through direct interaction with its gene product, ataxin-2.

Gene transcription is controlled by transcriptional regulators acting with specific co-regulators to allow gene activation and repression. Here, we report the identification of the KRAB-containing zinc-finger transcriptional regulator, ZBRK1, as an interaction partner of the SCA2 gene product ataxin-2. Furthermore, we discovered that an elevated ZBRK1 level resulted in increased ataxin-2 levels, whereas interference on transcriptional and protein levels of ZBRK1 yielded reduced ataxin-2 levels, suggesting that a complex comprising ZBRK1 and ataxin-2 regulates SCA2 gene transcription. A bioinformatic analysis utilizing the known ZBRK1 consensus DNA-binding motif revealed ZBRK1-binding sites in the SCA2 promoter. These predicted sites were experimentally validated by chromatin-immunoprecipitation experiments along with luciferase-based promoter analyses corroborating that SCA2 gene transcription is controlled by a ZBRK1/ataxin-2 complex. Finally, we demonstrate that SCA2 gene transcription is significantly reduced in colon tumors possessing low ZBRK1 transcripts. Thus, our results provide first evidence that ataxin-2 acts as a co-regulator of ZBRK1 activating its own transcription, thereby representing the first identified ZBRK1 co-activator.