Icenticaftor, a CFTR Potentiator, in COPD: A Multicenter, Parallel-Group, Double-Blind Clinical Trial.

Rationale: CFTR (cystic fibrosis transmembrane conductance regulator) dysfunction is associated with mucus accumulation and worsening chronic obstructive pulmonary disease (COPD) symptoms. Objectives: The aim of this phase IIb dose-finding study was to compare a CFTR potentiator, icenticaftor (QBW251), with placebo in patients with COPD and chronic bronchitis. Methods: Patients with COPD on triple therapy for at least three months were randomized to six treatment arms (icenticaftor 450, 300, 150, 75, or 25 mg or placebo twice daily [b.i.d.]) in a 24-week, multicenter, parallel-group, double-blind study. The primary endpoint was change from baseline in trough FEV1 after 12 weeks. Secondary endpoints included change from baseline in trough FEV1 and Evaluating Respiratory Symptoms in COPD (E-RS) total and cough and sputum scores after 24 weeks. Multiple comparison procedure-modeling was conducted to characterize dose-response relationship. Rescue medication use, exacerbations, and change in serum fibrinogen concentration after 24 weeks were assessed in exploratory and post hoc analyses, respectively. Measurements and Main Results: Nine hundred seventy-four patients were randomized. After 12 weeks of icenticaftor treatment, no dose-response relationship for change from baseline in trough FEV1 was observed; however, it was observed for E-RS cough and sputum score. A dose-response relationship was observed after 24 weeks for trough FEV1, E-RS cough and sputum and total scores, rescue medication use, and fibrinogen. A dose of 300 mg b.i.d. was consistently the most effective. Improvements for 300 mg b.i.d. versus placebo were also seen in pairwise comparisons of these endpoints. All treatments were well tolerated. Conclusions: The primary endpoint was negative, as icenticaftor did not improve trough FEV1 over 12 weeks. Although the findings must be interpreted with caution, icenticaftor improved trough FEV1; reduced cough, sputum, and rescue medication use; and lowered fibrinogen concentrations at 24 weeks. Clinical trial registered with www.clinicaltrials.gov (NCT04072887).

Rapid, Label-Free Screening of Diverse Biotransformations by Flow-Injection Mass Spectrometry.

Mass spectrometry-based high-throughput screening methods combine the advantages of photometric or fluorometric assays and analytical chromatography, as they are reasonably fast (throughput ≥1 sample/min) and broadly applicable, with no need for labelled substrates or products. However, the established MS-based screening approaches require specialised and expensive hardware, which limits their broad use throughout the research community. We show that a more common instrumental platform, a single-quadrupole HPLC-MS, can be used to rapidly analyse diverse biotransformations by flow-injection mass spectrometry (FIA-MS), that is, by automated infusion of samples to the ESI-MS detector without prior chromatographic separation. Common organic buffers can be employed as internal standard for quantification, and the method provides readily validated activity and selectivity information with an analytical run time of one minute per sample. We report four application examples that cover a broad range of analyte structures and concentrations (0.1-50 mM before dilution) and diverse biocatalyst preparations (crude cell lysates and whole microbial cells). Our results establish FIA-MS as a versatile and reliable alternative to more traditional methods for screening enzymatic reactions.

Injectable autologous chondrocyte implantation in acetabular cartilage defects: 2-year minimum clinical and MRI results.

INTRODUCTION: Early results using injectable autologous chondrocyte implantation (ACI) for the treatment of full thickness acetabular cartilage defects have been promising. However, so far there is no information on radiological results after injectable ACI using spheroids. The purpose of this sturdy was to (1) investigate the quality of tissue repair on MRI and (2) investigate the correlation between the MRI results and clinical results at a minimum follow-up of 24 months after third generation ACI in full thickness acetabular cartilage defects. It was hypothesized that ACI shows good MRI results in patients with large full thickness acetabular cartilage defects 24 months after surgery. It was also hypothesized that there is a correlation between postoperative clinical and MRI morphological results at a minimum follow-up of 24 months. STUDY DESIGN: Retrospective case series. MATERIALS AND METHODS: Patients with ACI for full thickness acetabular cartilage defects > 2 cm2 were evaluated by preoperative and postoperative clinical scoring tools including the modified Harris Hip Score (mHHS), the International Hip Outcome Tool (iHOT-33), and the Subjective Hip Value (SHV) as well as a high resolution indirect arthro-MRI 24 months after surgery utilizing an identical imaging protocol for all patients. The magnetic resonance observation of cartilage repair tissue (MOCART) scoring system was used to classify the repair tissue on MRI. Demographic patient data was evaluated for influencing factors for pre- and postoperative clinical as well as radiological results. RESULTS: Thirty six consecutive patients (5 women/31 men, average age 32.9 years) had undergone two stage ACI procedure. The average size of the cartilage defect was 5.0 (2-6) cm2. The average follow-up was 29.9 (24-42) months. Four patients were not available for the final follow-up (follow-up rate 89%). The postoperative average MOCART score was 82.2 (± 14.2). MOCART score showed medium correlation of the item defect fill and the postoperative mHHS (r = 0.384, p = 0.043). There was no correlation of the other items or the total score with postoperative results. The patients showed significant improvement in the outcome measurements between preoperative and postoperative in the mHHS, the iHOT-33, and the SHV. CONCLUSIONS: Despite the large acetabular cartilage defects included in this study, ACI showed good MRI results with complete defect fill in 87.5% after a minimum 24-month follow-up. Statistically significant correlation of MRI and clinical results could only be seen with the item defect fill. Further research with longer follow-up is needed to evaluate the long-term results of ACI in acetabular cartilage defects.

Effectiveness and safety of tocilizumab in patients with systemic sclerosis: a propensity score matched controlled observational study of the EUSTAR cohort.

OBJECTIVES: Tocilizumab showed trends for improving skin fibrosis and prevented progression of lung fibrosis in systemic sclerosis (SSc) in randomised controlled clinical trials. We aimed to assess safety and effectiveness of tocilizumab in a real-life setting using the European Scleroderma Trial and Research (EUSTAR) database. METHODS: Patients with SSc fulfilling the American College of Rheumatology (ACR)/EULAR 2013 classification criteria, with baseline and follow-up visits at 12±3 months, receiving tocilizumab or standard of care as the control group, were selected. Propensity score matching was applied. Primary endpoints were the modified Rodnan skin score (mRSS) and FVC at 12±3 months compared between the groups. Secondary endpoints were the percentage of progressive/regressive patients for skin and lung at 12±3 months. RESULTS: Ninety-three patients with SSc treated with tocilizumab and 3180 patients with SSc with standard of care fulfilled the inclusion criteria. Comparison between groups did not show significant differences, but favoured tocilizumab across all predefined primary and secondary endpoints: mRSS was lower in the tocilizumab group (difference -1.0, 95% CI -3.7 to 1.8, p=0.48). Similarly, FVC % predicted was higher in the tocilizumab group (difference 1.5 (-6.1 to 9.1), p=0.70). The percentage of progressive/regressive patients favoured tocilizumab over controls. These results were robust regarding the sensitivity analyses. Safety analysis confirmed previously reported adverse event profiles. CONCLUSION: Although this large, observational, controlled, real-life EUSTAR study did not show significant effectiveness of tocilizumab on skin and lung fibrosis, the consistency of direction of all predefined endpoints generates hypothesis for potential effectiveness in a broader SSc population.

Regioselective Biocatalytic C4-Prenylation of Unprotected Tryptophan Derivatives.

Regioselective carbon-carbon bond formation belongs to the challenging tasks in organic synthesis. In this context, C-C bond formation catalyzed by 4-dimethylallyltryptophan synthases (4-DMATSs) represents a possible tool to regioselectively synthesize C4-prenylated indole derivatives without site-specific preactivation and circumventing the need of protection groups as used in chemical synthetic approaches. In this study, a toolbox of 4-DMATSs to produce a set of 4-dimethylallyl tryptophan and indole derivatives was identified. Using three wild-type enzymes as well as variants, various C5-substituted tryptophan derivatives as well as N-methyl tryptophan were successfully prenylated with conversions up to 90 %. Even truncated tryptophan derivatives like tryptamine and 3-indole propanoic acid were regioselectively prenylated in position C4. The acceptance of C5-substituted tryptophan derivatives was improved up to 5-fold by generating variants (e. g. T108S). The feasibility of semi-preparative prenylation of selected tryptophan derivatives was successfully demonstrated on 100 mg scale at 15 mM substrate concentration, allowing to reduce the previously published multistep chemical synthetic sequence to just a single step.

Power of Biocatalysis for Organic Synthesis.

Biocatalysis, using defined enzymes for organic transformations, has become a common tool in organic synthesis, which is also frequently applied in industry. The generally high activity and outstanding stereo-, regio-, and chemoselectivity observed in many biotransformations are the result of a precise control of the reaction in the active site of the biocatalyst. This control is achieved by exact positioning of the reagents relative to each other in a fine-tuned 3D environment, by specific activating interactions between reagents and the protein, and by subtle movements of the catalyst. Enzyme engineering enables one to adapt the catalyst to the desired reaction and process. A well-filled biocatalytic toolbox is ready to be used for various reactions. Providing nonnatural reagents and conditions and evolving biocatalysts enables one to play with the myriad of options for creating novel transformations and thereby opening new, short pathways to desired target molecules. Combining several biocatalysts in one pot to perform several reactions concurrently increases the efficiency of biocatalysis even further.

The role of biocatalysis in the asymmetric synthesis of alkaloids - an update.

Alkaloids are a group of natural products with interesting pharmacological properties and a long history of medicinal application. Their complex molecular structures have fascinated chemists for decades, and their total synthesis still poses a considerable challenge. In a previous review, we have illustrated how biocatalysis can make valuable contributions to the asymmetric synthesis of alkaloids. The chemo-enzymatic strategies discussed therein have been further explored and improved in recent years, and advances in amine biocatalysis have vastly expanded the opportunities for incorporating enzymes into synthetic routes towards these important natural products. The present review summarises modern developments in chemo-enzymatic alkaloid synthesis since 2013, in which the biocatalytic transformations continue to take an increasingly 'central' role.

Asymmetric Biocatalytic Synthesis of 1-Aryltetrahydro-ß-carbolines Enabled by "Substrate Walking".

Stereoselective catalysts for the Pictet-Spengler reaction of tryptamines and aldehydes may allow a simple and fast approach to chiral 1-substituted tetrahydro-ß-carbolines. Although biocatalysts have previously been employed for the Pictet-Spengler reaction, not a single one accepts benzaldehyde and its substituted derivatives. To address this challenge, a combination of substrate walking and transfer of beneficial mutations between different wild-type backbones was used to develop a strictosidine synthase from Rauvolfia serpentina (RsSTR) into a suitable enzyme for the asymmetric Pictet-Spengler condensation of tryptamine and benzaldehyde derivatives. The double variant RsSTR V176L/V208A accepted various ortho-, meta- and para-substituted benzaldehydes and produced the corresponding chiral 1-aryl-tetrahydro-ß-carbolines with up to 99 % enantiomeric excess.

Correlation of the Subjective Hip Value with Validated Patient-Reported Outcome Measurements for the Hip.

BACKGROUND: The subjective hip value (SHV) was developed as a patient-reported outcome measurement (PROM) that is easily and quickly performed and interpreted. The SHV is defined as a patient's subjective hip measurement tool expressed as a percentage of an entirely normal hip joint, which would score 100%. The hypothesis is that results of the subjective hip value correlate with the results of the modified Harris hip score and the International Hip Outcome Tool in patients with hip-related diseases. METHODS: 302 patients completed the modified Harris hip score (mHHS), the International Hip Outcome Tool (iHot-33) as well as the SHV. The SHV consist of only one question: "What is the overall percent value of your hip if a completely normal hip represents 100%?". The patients were divided into five different groups depending on the diagnosis. Pearson correlation was used to evaluate the correlation between the different PROMs and linear regression analysis was used to calculate R2. RESULTS: 302 complete datasets were available for evaluation. There was a high correlation between the SHV and the iHOT-33 (r = 0.847; r2 = 0.692, p < 0.001) and the mHHS (r = 0.832; r2 = 0.717, p < 0.001). The SHV showed a medium (r = 0.653) to high (r = 0.758) correlation with the mHHS and the iHOT-33 in all diagnosis groups. CONCLUSION: The SHV offers a useful adjunct to established hip outcome measurements, as it is easily and quickly performed and interpreted. The SHV reflects the view of the patient and is independent of the diagnosis. Further research with prospective studies is needed to test the psychometric properties of the score.

The lncRNA lincNMR regulates nucleotide metabolism via a YBX1 - RRM2 axis in cancer.

Long intergenic non-coding RNA-Nucleotide Metabolism Regulator (lincNMR) is a long non-coding RNA (lncRNA) which is induced in hepatocellular carcinoma. Its depletion invokes a proliferation defect, triggers senescence and inhibits colony formation in liver, but also breast and lung cancer cells. Triple-label SILAC proteomics profiles reveal a deregulation of key cell cycle regulators in lincNMR-depleted cells like the key dNTP synthesizing enzymes RRM2, TYMS and TK1, implicating lincNMR in regulating nucleotide metabolism. LincNMR silencing decreases dNTP levels, while exogenous dNTPs rescues the proliferation defect induced by lincNMR depletion. In vivo RNA Antisense Purification (RAP-MS) identifies YBX1 as a direct interaction partner of lincNMR which regulates RRM2, TYMS and TK1 expression and binds to their promoter regions. In a Chick Chorioallantoic Membrane (CAM) in vivo model, lincNMR-depleted tumors are significantly smaller. In summary, we discover a lincRNA, lincNMR, which regulates tumor cell proliferation through a YBX1-RRM2-TYMS-TK1 axis governing nucleotide metabolism.

Contrast Induced Acute Kidney Injury and its Impact on Mid-Term Kidney Function, Cardiovascular Events and Mortality.

The existence and clinical relevance of contrast induced acute kidney injury (CI-AKI) is still heavily debated and angiographic procedures are often withheld in fear of CI-AKI, especially in CKD-patients. We investigated the incidence of CI-AKI in cardiovascular high risk patients undergoing intra-arterial angiography and its impact on mid-term kidney function, cardiovascular events and mortality. We conducted a prospective observational trial on patients undergoing planned intra-arterial angiographic procedures. All subjects received standardized intravenous hydration prior to contrast application. CI-AKI was defined according to a ≥25% increase of creatinine from baseline to either 24hrs or 48hrs after angiography. Plasma creatinine and eGFR were recorded from the institutional medical record system one and three months after hospital discharge. Patients were followed up for two years to investigate the long term effects of CI-AKI on cardiovascular events and mortality. We studied 706 (317 female) patients with a mean eGFR of 52.0 ± 15 ml·min-1·1.73 m-2. The incidence of CI-AKI was 10.2% (72 patients). In 94 (13.3%) patients serum creatinine decreased ≥25% either 24 or 48 hours after angiography. Patients with CI-AKI had a lower creatinine and a higher eGFR at baseline, but no other independent predictors of CI-AKI could be identified. Kidney function was not different between both groups one and three months after discharge. After a two year follow up the overall incidence of cardiovascular events was 56.5% in the CI-AKI group and 58.8% in the Non CI-AKI group (p = 0.8), the incidence of myocardial infarctions, however, was higher in CI-AKI-patients. Overall survival was also not different between patients with CI-AKI (88.6%) and without CI-AKI (84.7%, p = 0.48). The occurrence of CI-AKI did not have any negative impact on mid-term kidney function, the incidence of cardiovascular events and mortality. Considerable fluctuations of serum creatinine interfere with the presumed diagnosis of CI-AKI. Necessary angiographic procedures should not be withheld in fear of CI-AKI.

In-hospital mortality of pelvic ring fractures in older adults now and then: A pelvic registry study.

AIM: With the predicted demographic change, the treatment of geriatric patients will become a major issue for health systems worldwide. The majority of pelvic ring fractures occur in older adults, and their treatment might be associated with a distinct mortality. Herein, we analyzed the data of 5665 patients with pelvic ring fractures aged ≥60 years included in the German Pelvic Trauma Registry from 1991 to 2013. METHODS: The data were collected prospectively, multicentrically in hospitals participating in the German Pelvic Trauma Registry. Demographic data were retrospectively analyzed, stratified for age, sex, type of injury, mode of therapy, injury severity score (ISS) and mortality. RESULTS: The overall mortality decreased over the 22-year study period from 9.3% to 3.8% (P < 0.05), whereas the median ISS significantly increased. During the observation period, mortality was higher in patients with type B and, particularly, type C fractures when compared with patients with type A fractures. Mortality rates of patients aged >80 years did not significantly differ from those aged >60 or >70 years. Male patients showed a significantly higher mortality compared with female patients, as well as a significantly higher median ISS. The mortality rate of patients with surgically-treated type C fractures decreased over the study period from 35.7% to 6.9% (P < 0.05). CONCLUSIONS: Over the past two decades, the mortality of older patients after pelvic ring fractures has significantly decreased. The higher overall mortality rate of male patients might mainly be accounted for by the relatively higher fraction of type C fractures and a higher ISS. Geriatr Gerontol Int 2019; 19: 24-29.

Sequence-Based In-silico Discovery, Characterisation, and Biocatalytic Application of a Set of Imine Reductases.

Imine reductases (IREDs) have recently become a primary focus of research in biocatalysis, complementing other classes of amine-forming enzymes such as transaminases and amine dehydrogenases. Following in the footsteps of other research groups, we have established a set of IRED biocatalysts by sequence-based in silico enzyme discovery. In this study, we present basic characterisation data for these novel IREDs and explore their activity and stereoselectivity using a panel of structurally diverse cyclic imines as substrates. Specific activities of >1 U/mg and excellent stereoselectivities (ee>99 %) were observed in many cases, and the enzymes proved surprisingly tolerant towards elevated substrate loadings. Co-expression of the IREDs with an alcohol dehydrogenase for cofactor regeneration led to whole-cell biocatalysts capable of efficiently reducing imines at 100 mM initial concentration with no need for the addition of extracellular nicotinamide cofactor. Preparative biotransformations on gram scale using these 'designer cells' afforded chiral amines in good yield and excellent optical purity.

The non-neuronal cyclin-dependent kinase 5 is a fibrotic mediator potentially implicated in systemic sclerosis and a novel therapeutic target.

The mechanisms underlying persistent fibroblast activation and myofibroblast phenoconversion in underlying multi-organ fibrosis in systemic sclerosis (SSc) remain incompletely understood, hindering effective therapies to slow or reverse the process. Cyclin-dependent kinase 5 (CDK5) is a pleiotropic member of the CDK family originally identified in neuronal cells. In contrast to other CDKs, CDK5 activity depends on its CDK5R1 subunit p35. Here we demonstrate that expression of p35 and CDK5 activity are induced by TGF-ß in fibroblasts and adipocytic cell types. Levels of p35 are markedly elevated in both SSc skin biopsies and explanted SSc fibroblasts, as well as in fibrotic skin in mice. Ectopic p35 and CDK5 suppressed adipogenic markers while stimulating collagen production and myofibroblast markers, whereas RNAi-mediated CDK5 knockdown abrogated TGF-ß fibrotic responses in a Smad-independent manner. Pharmacological inhibitors of CDK5 likewise prevented and reversed TGF-ß responses in fibroblast monolayers and in ex vivo human skin organ cultures, ameliorated collagen overproduction in SSc fibroblasts, and prevented and reversed skin fibrosis in two distinct mouse models of SSc. Together, these results reveal a previously unrecognized key function for p35/CDK5 as a mediator of mesenchymal cell fibrotic responses. The results suggest a potential pathogenic role for elevated p35 expression and CDK5 activity in SSc, and raise the possibility that their selective pharmacological targeting might represent a novel treatment approach in fibrosis.

Artificial Biocatalytic Linear Cascades for Preparation of Organic Molecules.

The review compiles artificial cascades involving enzymes with a focus on the last 10 years. A cascade is defined as the combination of at least two reaction steps in a single reaction vessel without isolation of the intermediates, whereby at least one step is catalyzed by an enzyme. Additionally, cascades performed in vivo and in vitro are discussed separately, whereby in vivo cascades are defined here as cascades relying on cofactor recycling by the metabolism or on a metabolite from the living organism. The review introduces a systematic classification of the cascades according to the number of enzymes in the linear sequence and differentiates between cascades involving exclusively enzymes and combinations of enzymes with non-natural catalysts or chemical steps. Since the number of examples involving two enzymes is predominant, the two enzyme cascades are further subdivided according to the number, order, and type of redox steps. Furthermore, this classification differentiates between cascades where all reaction steps are performed simultaneously, sequentially, or in flow.

Injectable autologous chondrocyte implantation (ACI) in acetabular cartilage defects-three-year results.

To evaluate the clinical outcome after arthroscopic matrix-associated injectable autologous chondrocyte implantation (ACI) in patients with large full-thickness acetabular cartilage defects. ACI was performed in young patients with full-thickness acetabular cartilage defects ≥2 cm2 in a two-step arthroscopic procedure. The patients were followed closely with clinical examinations and pre- and postoperative scores. The modified Harris Hip Score (mHHS), iHOT33 questionnaire (iHOT33) and the Subjective Hip Value (SHV) were surveyed. Demographic patient data was evaluated for influencing factors for the pre- and postoperative results. Thirty-two consecutive cases (4 female, 28 male, mean age 33 years) were included. The average defect size was 4.9 (range: 2-6) cm2. They were followed at 6, 12, 24 and 36 months postoperatively. Patients had improved significantly from 64 to 91 points (P < 0.001) in the mHHS, from 44% to 86% (P < 0.001) in the iHOT33 and from 54% to 87% (P < 0.001) in the SHV. No surgery related complications were noted. Cell cultivation failed in two cases (7%) and the patients decided for a repeated harvesting of cartilage cylinders followed by a successful ACI. Patients age and size of the cartilage defect showed no significant correlation with the pre- or postoperative results. Injectable ACI is a reliable procedure treating full-thickness acetabular cartilage defects leading to promising results 3 years postoperatively with a significant increase in all scores despite large acetabular cartilage defects in the weight-bearing zone.

P53-induced miR-30e-5p inhibits colorectal cancer invasion and metastasis by targeting ITGA6 and ITGB1.

The tumor suppressor P53 is a critical regulator of normal cellular homeostasis whose function is either distorted or lost in several cancer types including colorectal cancer (CRC). A small group of microRNAs have come to be recognized as essential mediators of P53 function. In a genome-wide systematic approach, we explored miRNAs that are substantially altered by P53 loss and found miR-30e to be the most significantly deregulated miRNA in P53-knockout human CRC cells. We identified miR-30e-5p to be a novel direct transcriptional target of P53 with gain and loss of function experiments revealing miR-30e-5p to be a significant regulator of tumor cell migration, invasion and in vivo metastasis mediated in part by integrins alpha-6 and beta-1 as novel targets. MiR-30e-5p also significantly reduced tumor cell proliferation by causing G1/S cell cycle arrest, which was achieved by inducing P21 and P27 expression. Finally, we found miR-30e-5p to be lost in resected CRC tumors as compared to normal colon tissues. Taken together, miR-30e-5p is a novel effector of P53-induced suppression of migration, invasion and metastasis.

Is the evaluation of the anterior inferior iliac spine (AIIS) in the AP pelvis possible? Analysis of conventional X-rays and 3D-CT reconstructions.

INTRODUCTION: A hypertrophic AIIS has been identified as a cause for extraarticular hip impingement and is classified according to Hetsroni using 3D-CT reconstructions. The role of the conventional AP pelvis X-ray, which is the first standard imaging step for the evaluation of hip pain, has not been investigated yet. MATERIALS AND METHODS: AP pelvis X-rays and 3D-CT reconstructions of patients were evaluated regarding their morphology of the AIIS. The conventional X-rays were categorized into three groups according to the projection of the AIIS: above (A) or below (B) the acetabular sourcil or even exceeding the anterior acetabular rim (C). They were compared to the morphologic types in the 3D-CT reconstruction (Hetsroni type I-III). RESULTS: Ninety patients with an equal distribution of type A, B or C projection in the AP pelvis were evaluated and compared to the morphology in the 3D-CT reconstruction. The projection of the AIIS below the acetabular sourcil (B + C) showed only moderate sensitivity (0.76) and specificity (0.64) for a hypertrophic AIIS (Hetsroni type II + III), but if the AIIS exceeds the anterior rim, all cases showed a hypertrophic AIIS in the 3D-CT reconstructions (Hetsroni type II + III). CONCLUSIONS: Distinct differentiation of the AIIS morphology in the AP pelvis is not possible, but the projection of the AIIS below the anterior acetabular rim represented a hypertrophic AIIS in all cases and should, therefore, be critically investigated for a relevant AIIS impingement.