Stereospecific radical coupling with a non-natural photodecarboxylase.

Photoenzymes are light-powered biocatalysts that typically rely on the excitation of cofactors or unnatural amino acids for their catalytic activities1,2. A notable natural example is the fatty acid photodecarboxylase, which uses light energy to convert aliphatic carboxylic acids to achiral hydrocarbons3. Here we report a method for the design of a non-natural photodecarboxylase based on the excitation of enzyme-bound catalytic intermediates, rather than reliance on cofactor excitation4. Iminium ions5, transiently generated from enals within the active site of an engineered class I aldolase6, can absorb violet light and function as single-electron oxidants. Activation of chiral carboxylic acids, followed by decarboxylation, generates two radicals that undergo stereospecific cross-coupling, yielding products with two stereocentres. Using the appropriate enantiopure chiral substrate, the desired diastereoisomeric product is selectively obtained with complete enantiocontrol. This finding underscores the ability of the active site to transfer stereochemical information from the chiral radical precursor into the product, effectively addressing the long-standing problem of rapid racemization of chiral radicals. The resulting 'memory of chirality' scenario7 is a rarity in enantioselective radical chemistry.

The Complexity of Shapes: How the Circularity of Tumor Nodules Affects Prognosis in Colorectal Cancer.

The current stratification of tumor nodules in colorectal cancer (CRC) staging is subjective and leads to high interobserver variability. In this study, the objective assessment of the shape of lymph node metastases (LNMs), extranodal extension (ENE), and tumor deposits (TDs) was correlated with outcomes. A test cohort and a validation cohort were included from 2 different institutions. The test cohort consisted of 190 cases of stage III CRC. Slides with LNMs and TDs were annotated and processed using a segmentation algorithm to determine their shape. The complexity ratio was calculated for every shape and correlated with outcomes. A cohort of 160 stage III CRC cases was used to validate findings. TDs showed significantly more complex shapes than LNMs with ENE, which were more complex than LNMs without ENE (P < .001). In the test cohort, patients with the highest sum of complexity ratios had significantly lower disease-free survival (P < .01). When only the nodule with the highest complexity was considered, this effect was even stronger (P < .001). This maximum complexity ratio per patient was identified as an independent prognostic factor in the multivariate analysis (hazard ratio, 2.47; P < .05). The trends in the validation cohort confirmed the results. More complex nodules in stage III CRC were correlated with significantly worse disease-free survival, even if only based on the most complex nodule. These results suggest that more complex nodules reflect more invasive tumor biology. As most of the more complex nodules were diagnosed as TDs, we suggest providing a more prominent role for TDs in the nodal stage and include an objective complexity measure in their definition.

Cancer and left atrial enlargement in patients with ischemic stroke.

BACKGROUND: Cancer is associated with an increased risk of atrial fibrillation. Whether cancer is also associated with atrial cardiopathy, another atrial pathology associated with heightened ischemic stroke risk, is uncertain. METHODS: We conducted a retrospective cross-sectional study among consecutive patients hospitalized with acute ischemic stroke at a quaternary care center in New York, United States from 2011 through 2016. The study exposure was active cancer. The study outcome was atrial cardiopathy, defined as a left atrial volume index ≥35 mL/m2 on echocardiography. We used multivariable logistic regression, adjusting for baseline characteristics, to evaluate the relationship between cancer (active or historical) and atrial cardiopathy. We performed a subgroup analysis among patients with embolic stroke of undetermined source (ESUS). RESULTS: The final cohort included 1104 patients with acute ischemic stroke, of whom 10 % had active cancer and 47 % had atrial cardiopathy. Patients with atrial cardiopathy, compared to those without, were older (median age, 77 versus 68 years), and more frequently had hypertension, coronary disease, and atrial fibrillation. Active cancer was present in 9.6 % of patients with atrial cardiopathy (n = 50/520) and 10.4 % of patients without (n = 61/584). There was no association between active cancer and atrial cardiopathy among the overall ischemic stroke cohort (adjusted odds ratio [OR], 0.91; 95 % confidence interval [CI], 0.60-1.37) nor in patients with ESUS (aOR, 0.64; 95 % CI, 0.30-1.36). When the cancer exposure was broadened to include any history of cancer (n = 236, 21.4 %), there still was no significant association with atrial cardiopathy (aOR, 0.93; 95 % CI, 0.68-1.25). CONCLUSIONS: When defining atrial cardiopathy by left atrial volume, we did not find an association between cancer and atrial cardiopathy in patients with ischemic stroke, including among those with ESUS. Future studies, evaluating other atrial cardiopathy biomarkers and settings, are needed to further investigate any potential link between cancer and atrial cardiopathy.

Approach for Phased Sequence-Based Genotyping of the Critical Pharmacogene Dihydropyrimidine Dehydrogenase (DPYD).

Pre-treatment genotyping of four well-characterized toxicity risk-variants in the dihydropyrimidine dehydrogenase gene (DPYD) has been widely implemented in Europe to prevent serious adverse effects in cancer patients treated with fluoropyrimidines. Current genotyping practices are largely limited to selected commonly studied variants and are unable to determine phasing when more than one variant allele is detected. Recent evidence indicates that common DPYD variants modulate the functional impact of deleterious variants in a phase-dependent manner, where a cis- or a trans-configuration translates into different toxicity risks and dosing recommendations. DPYD is a large gene with 23 exons spanning nearly a mega-base of DNA, making it a challenging candidate for full-gene sequencing in the diagnostic setting. Herein, we present a time- and cost-efficient long-read sequencing approach for capturing the complete coding region of DPYD. We demonstrate that this method can reliably produce phased genotypes, overcoming a major limitation with current methods. This method was validated using 21 subjects, including two cancer patients, each of whom carried multiple DPYD variants. Genotype assignments showed complete concordance with conventional approaches. Furthermore, we demonstrate that the method is robust to technical challenges inherent in long-range sequencing of PCR products, including reference alignment bias and PCR chimerism.

Mortality in acute ischemic stroke patients with new cancer diagnosed during the index hospitalization versus after discharge.

BACKGROUND: Early diagnosis of previously unknown cancer (i.e., occult cancer) after an acute ischemic stroke (AIS) could result in faster initiation of cancer therapy and potentially improve clinical outcomes. Our study aimed to compare mortality rates between AIS patients with occult cancer diagnosed during the index stroke hospitalization versus those diagnosed after hospital discharge. METHODS: Among consecutive AIS patients treated at our stroke center from 2015 through 2020, we identified new cancer diagnoses made within the year after the AIS. We used multivariable Cox regression analyses to evaluate the association between the timing of occult cancer diagnosis (during the AIS hospitalization versus after discharge) and long-term survival. RESULTS: Of 3894 AIS patients with available long-term follow-up data, 59 (1.5 %) were diagnosed with a new cancer within one year after index stroke. Of these, 27 (46 %) were diagnosed during the index hospitalization and 32 (54 %) were diagnosed after discharge. During a median follow-up of 406 days (interquartile range, 89-1073), 70 % (n = 19) of patients whose cancer was diagnosed during hospitalization had died, compared to 63 % (n = 20) of patients whose cancer was diagnosed after discharge (p= 0.58). In our main multivariable model, there was no difference in long-term mortality between patient groups (adjusted hazard ratio, 1.16; 95 % confidence interval, 0.53-2.52; p= 0.71). CONCLUSIONS: In this analysis, timing of a new cancer diagnosis after AIS did not seem to influence patients' long-term survival. Given the fairly small number of included patients with previously occult cancer, larger multicenter studies are needed to confirm our results.

Thrombospondin-1 promotes hemostasis through modulation of cAMP signaling in blood platelets.

Thrombospondin-1 (TSP-1) is released by platelets upon activation and can increase platelet activation, but its role in hemostasis in vivo is unclear. We show that TSP-1 is a critical mediator of hemostasis that promotes platelet activation by modulating inhibitory cyclic adenosine monophosphate (cAMP) signaling. Genetic deletion of TSP-1 did not affect platelet activation in vitro, but in vivo models of hemostasis and thrombosis showed that TSP-1-deficient mice had prolonged bleeding, defective thrombosis, and increased sensitivity to the prostacyclin mimetic iloprost. Adoptive transfer of wild-type (WT) but not TSP-1-/- platelets ameliorated the thrombotic phenotype, suggesting a key role for platelet-derived TSP-1. In functional assays, TSP-1-deficient platelets showed an increased sensitivity to cAMP signaling, inhibition of platelet aggregation, and arrest under flow by prostacyclin (PGI2). Plasma swap experiments showed that plasma TSP-1 did not correct PGI2 hypersensitivity in TSP-1-/- platelets. By contrast, incubation of TSP-1-/- platelets with releasates from WT platelets or purified TSP-1, but not releasates from TSP-1-/- platelets, reduced the inhibitory effects of PGI2. Activation of WT platelets resulted in diminished cAMP accumulation and downstream signaling, which was associated with increased activity of the cAMP hydrolyzing enzyme phosphodiesterase 3A (PDE3A). PDE3A activity and cAMP accumulation were unaffected in platelets from TSP-1-/- mice. Platelets deficient in CD36, a TSP-1 receptor, showed increased sensitivity to PGI2/cAMP signaling and diminished PDE3A activity, which was unaffected by platelet-derived or purified TSP-1. This scenario suggests that the release of TSP-1 regulates hemostasis in vivo through modulation of platelet cAMP signaling at sites of vascular injury.

Anticancer genes (NOXA, PAR-4, TRAIL) are de-regulated in breast cancer patients and can be targeted by using a ribosomal inactivating plant protein (riproximin).

BACKGROUND: Anticancer genes are an endogenous defense against transformed cells as they impose antineoplastic effects upon ectopic expression. Profiling the expression of these genes is fundamental for exploring their prognostic and therapeutic relevance in cancers. Natural compounds can upregulate anticancer genes in malignant cells and thus be useful for therapeutic purposes. In this study, we identified the expression levels of anticancer genes in breast cancer clinical isolates. In addition, the purified and sequenced plant protein (riproximin) was evaluated for its potential to induce anticancer genes in two breast cancer cell lines. METHODOLOGY: Expression profiles of three anticancer genes (NOXA, PAR-4, TRAIL) were identified by immunohistochemistry in 45 breast cancer clinical isolates. Breast cancer cells were exposed to riproximin and expression of the anticancer genes was determined by microarray, real-time PCR and western blot methodologies. Lastly, a bioinformatic approach was adopted to highlight the molecular/functional significance of the anticancer genes. RESULTS: NOXA expression was evenly de-regulated among the clinical isolates, while PAR-4 was significantly down-regulated in majority of the breast cancer tissues. In contrast, TRAIL expression was increased in most of the clinical samples. Expression levels of the anticancer genes followed a distinct trend in accordance with the disease severity. Riproximin showed a substantial potential of inducing expression of the anticancer genes in breast cancer cells at transcriptomic and protein levels. The bioinformatic approach revealed involvement of anticancer genes in multiple cellular functions and signaling cascades. CONCLUSION: Anticancer genes were de-regulated and showed discrete expression patterns in breast cancer patient samples. Riproximin effectively induced the expression of selected anticancer genes in breast cancer cells.

Free water diffusion MRI and executive function with a speed component in healthy aging.

Extracellular free water (FW) increases are suggested to better provide pathophysiological information in brain aging than conventional biomarkers such as fractional anisotropy. The aim of the present study was to determine the relationship between conventional biomarkers, FW in white matter hyperintensities (WMH), FW in normal appearing white matter (NAWM) and in white matter tracts and executive functions (EF) with a speed component in elderly persons. We examined 226 healthy elderly participants (median age 69.83 years, IQR: 56.99-74.42) who underwent brain MRI and neuropsychological examination. FW in WMH and in NAWM as well as FW corrected diffusion metrics and measures derived from conventional MRI (white matter hyperintensities, brain volume, lacunes) were used in partial correlation (adjusted for age) to assess their correlation with EF with a speed component. Random forest analysis was used to assess the relative importance of these variables as determinants. Lastly, linear regression analyses of FW in white matter tracts corrected for risk factors of cognitive and white matter deterioration, were used to examine the role of specific tracts on EF with a speed component, which were then ranked with random forest regression. Partial correlation analyses revealed that almost all imaging metrics showed a significant association with EF with a speed component (r = -0.213 - 0.266). Random forest regression highlighted FW in WMH and in NAWM as most important among all diffusion and structural MRI metrics. The fornix (R2=0.421, p = 0.018) and the corpus callosum (genu (R2 = 0.418, p = 0.021), prefrontal (R2 = 0.416, p = 0.026), premotor (R2 = 0.418, p = 0.021)) were associated with EF with a speed component in tract based regression analyses and had highest variables importance. In a normal aging population FW in WMH and NAWM is more closely related to EF with a speed component than standard DTI and brain structural measures. Higher amounts of FW in the fornix and the frontal part of the corpus callosum leads to deteriorating EF with a speed component.

A combined spatial score of granzyme B and CD68 surpasses CD8 as an independent prognostic factor in TNM stage II colorectal cancer.

BACKGROUND: Previous assessments of peritumoral inflammatory infiltrate in colorectal cancer (CRC) have focused on the role of CD8+ T lymphocytes. We sought to compare the prognostic value of CD8 with downstream indicators of active immune cell function, specifically granzyme B (GZMB) and CD68 in the tumour microenvironment. METHODS: Immunohistochemical (IHC) staining was performed for CD8, GZMB, CD68 and CD163 on next-generation tissue microarrays (ngTMAs) in a primary cohort (n = 107) and a TNM stage II validation cohort (n = 151). Using digital image analysis, frequency of distinct immune cell types was calculated for tumour proximity (TP) zones with varying radii (10 µm-100 µm) around tumour cells. RESULTS: Associations notably of advanced TNM stage were observed for low density of CD8 (p = 0.002), GZMB (p < 0.001), CD68 (p = 0.034) and CD163 (p = 0.011) in the primary cohort. In the validation cohort only low GZMB (p = 0.036) was associated with pT4 stage. Survival analysis showed strongest prognostic effects in the TP25µm zone at the tumour centre for CD8, GZMB and CD68 (all p < 0.001) in the primary cohort and for CD8 (p = 0.072), GZMB (p = 0.035) and CD68 (p = 0.004) in the validation cohort with inferior prognostic effects observed at the tumour invasive margin. In a multivariate survival analysis, joint analysis of GZMB and CD68 was similarly prognostic to CD8 in the primary cohort (p = 0.007 vs. p = 0.002) and superior to CD8 in the validation cohort (p = 0.005 vs. p = 0.142). CONCLUSION: Combined high expression of GZMB and CD68 within 25 µm to tumour cells is an independent prognostic factor in CRC and of superior prognostic value to the well-established CD8 in TNM stage II cancers. Thus, assessment of antitumoral effect should consider the quality of immune activation in peritumoral inflammatory cells and their actual proximity to tumour cells.

[Heart and diabetes : Platelet function and antiplatelet therapy in chronic kidney disease]. / Herz und Diabetes : Thrombozytenfunktion und antithrombozytäre Therapie bei chronischer Nierenerkrankung.

Patients with chronic kidney disease (CKD) have an increased risk of thrombosis and approximately 50% of patients with advanced CKD die because of a cardiovascular disease. In addition to an increased risk of thrombosis, patients with CKD and particularly with advanced CKD, have an increased risk of hemorrhage, which increases parallel to the decline of kidney function. Due to this parallel existence of the prohemorrhagic and prothrombotic phenotype, antiplatelet treatment is difficult in the daily routine and data show that CKD patients with acute coronary syndrome (ACS) are less likely to receive guideline-conform treatment. The underlying mechanisms are currently insufficiently understood and both platelet-dependent mechanisms and also platelet-independent mechanisms are under discussion. Accordingly, there is currently no specific treatment or treatment strategy for patients with CKD. In addition, CKD patients are underrepresented in registration studies on antiplatelet treatment and there are no data from randomized trials for patients with advanced CKD (CKD ≥ 4). Current guideline recommendations are therefore based on subgroup analyses and observational studies. In addition, questions on the duration of treatment, on risk scores for estimation of the risk of hemorrhage and on potential benefits of escalation and de-escalation strategies remain largely unanswered and should therefore be the focus of future studies.

Oxidised Low-Density Lipoprotein-Induced Platelet Hyperactivity-Receptors and Signalling Mechanisms.

Dyslipidaemia leads to proatherogenic oxidative lipid stress that promotes vascular inflammation and thrombosis, the pathologies that underpin myocardial infarction, stroke, and deep vein thrombosis. These prothrombotic states are driven, at least in part, by platelet hyperactivity, and they are concurrent with the appearancxe of oxidatively modified low-density lipoproteins (LDL) in the circulation. Modified LDL are heterogenous in nature but, in a general sense, constitute a prototype circulating transporter for a plethora of oxidised lipid epitopes that act as danger-associated molecular patterns. It is well-established that oxidatively modified LDL promote platelet activation and arterial thrombosis through a number of constitutively expressed scavenger receptors, which transduce atherogenic lipid stress to a complex array of proactivatory signalling pathways in the platelets. Stimulation of these signalling events underlie the ability of modified LDL to induce platelet activation and blunt platelet inhibitory pathways, as well as promote platelet-mediated coagulation. Accumulating evidence from patients at risk of arterial thrombosis and experimental animal models of disease suggest that oxidised LDL represents a tangible link between the dyslipidaemic environment and increased platelet activation. The aim of this review is to summarise recent advances in our understanding of the pro-thrombotic signalling events induced in platelets by modified LDL ligation, describe the contribution of individual platelet scavenger receptors, and highlight potential future challenges of targeting these pathways.

Development of a Score for Prediction of Occult Malignancy in Stroke Patients (Occult-5 Score).

BACKGROUND AND PURPOSE: Malignancy associated acute ischemic stroke (AIS) requires specific diagnostic work-up, treatment and prevention to improve outcome. This study aimed to develop a biomarker-based score for prediction of occult malignancy in AIS patients. METHODS: Single-center cross-sectional study including consecutive AIS patients treated between July 2017 and November 2018. Patients with active malignancy at presentation, or diagnosed within 1 year thereafter and patients free of malignancy, were included and malignancy associated biomarkers were assessed. LASSO analyses of logistic regression were performed to determine biomarkers predictive of active malignancy. Predictors were derived from a predictive model for active malignancy. A comparison between known and unknown (=occult) malignancies when the index stroke occurred was used to eliminate variables not associated with occult malignancy. A predictive score (OCCULT-5 score) for occult malignancy was developed based on the remaining variables. RESULTS: From 1001 AIS patients, 61 (6%) presented an active malignancy. Thirty-nine (64%) were known and 22 (36%) occult. Five variables were included in the final OCCULT-5 score: age ≥ 77 years, embolic stroke of undetermined source, multi-territorial infarcts, D-dimer levels ≥ 820 µ/gL, and female sex. A score of ≥ 3 predicted an underlying occult malignancy with a sensitivity of 64%, specificity of 73%, positive likelihood ratio of 2.35 and a negative likelihood ratio of 0.50. CONCLUSIONS: The OCCULT-5 score might be useful to identify patients with occult malignancy. It may thus contribute to a more effective and timely treatment and thus lead to a positive impact on overall outcome.

Direct Synthesis of Enamides via Electrophilic Activation of Amides.

A novel, one-step N-dehydrogenation of amides to enamides is reported. This reaction employs the unlikely combination of LiHMDS and triflic anhydride, which serves as both the electrophilic activator and the oxidant, and is characterized by its simple setup and broad substrate scope. The synthetic utility of the formed enamides was readily demonstrated in a range of downstream transformations.

Role of enterocyte-specific gene polymorphisms in response to adjuvant treatment for stage III colorectal cancer.

OBJECTIVES: The enterocyte subtype of colorectal cancer (CRC) responds favorably to oxaliplatin-based adjuvant treatment for stage III CRC. We examined the clinical significance of single-nucleotide polymorphisms (SNPs) in enterocyte-related genes MS4A12 and CDX2 in response to adjuvant treatment for stage III CRC. PATIENTS AND METHODS: A total of 350 patients with stage III CRC were included: 274 received adjuvant treatment with surgical resection (discovery cohort) and 76 received surgery alone (control cohort). In the discovery cohort, 68 patients received FOLFOX and 206 received oral fluoropyrimidine. SNPs were analyzed by PCR-based direct sequencing. RESULTS: In the discovery cohort, the MS4A12 rs4939378 G/G variant was associated with lower 5-year survival than any A allele [70% vs. 90%, univariate: hazard ratio (HR) 2.29, 95% confidence interval (CI) 1.03-5.06, P = 0.035; multivariate: HR 2.58, 95% CI 1.15-5.76, P = 0.021]. Patients with the CDX2 rs3812863 G/G variant had better overall survival than those with any A allele, although this was not significant in multivariate analysis (5 year-survival: 95% vs. 82%, univariate: HR 0.34, 95% CI 0.12-0.97, P = 0.034; multivariate: HR 0.39, 95% CI 0.13-1.11, P = 0.078). The SNPs did not show significant association with overall survival in the control cohort, and significant interaction was observed between MS4A12 genotypes and groups (P = 0.007). CONCLUSIONS: Our findings suggest that MS4A12 and CDX2 gene polymorphisms may predict outcome in stage III CRC. However, the clinical significance of SNPs for response to oxaliplatin may differ by tumor stage.

Clinical significance of enterocyte-specific gene polymorphisms as candidate markers of oxaliplatin-based treatment for metastatic colorectal cancer.

Colorectal cancer (CRC) can be classified into subtypes based on gene expression signatures. Patients with stage III enterocyte subtype of the CRC Assigner classifier have been shown to benefit from oxaliplatin adjuvant therapy. Here, we investigated whether single nucleotide polymorphisms (SNPs) in two enterocyte subtype-related genes, MS4A12 and CDX2, could predict the efficacy of oxaliplatin in first-line treatment for patients with metastatic CRC (mCRC). Three cohorts of patients were included: a discovery cohort receiving FOLFOX ± bevacizumab (BEV) (n = 146), a validation cohort receiving FOLFOXIRI + BEV (n = 230), and a control cohort receiving FOLFIRI + BEV (n = 228). SNPs were analyzed by PCR-based direct sequencing. In the discovery cohort, MS4A12 rs4939378 and CDX2 rs3812863 were identified as potential markers of efficacy. In the validation cohort, any G allele of MS4A12 rs4939378 was associated with longer progression-free survival (PFS) than the A/A variant in both univariate analysis (12.4 vs. 10.9 months, hazard ratio [HR] 0.70, 95% confidence interval [CI] 0.49-0.99, P = 0.033) and multivariable analysis (HR 0.65, 95%CI 0.44-0.97, P = 0.035) in patients expressing wild-type KRAS, but not mutant KRAS. In contrast, longer PFS was observed for patients expressing the CDX2 rs3812863 G/G variant than any A allele in univariate analysis (32.3 vs. 10.3 months, HR 0.39, 95%CI 0.19-0.81, P = 0.004) only in patients expressing mutant KRAS. These findings were not observed in the control cohort. Thus, MS4A12 and CDX2 SNPs may have utility as predictive biomarkers of response to oxaliplatin-based treatment in mCRC patients.

Impact on survival through consolidation radiotherapy for diffuse large B-cell lymphoma: a comprehensive meta-analysis.

Rituximab has improved response rates and overall survival in B-cell lymphoma (DLBCL). Radiotherapy is an effective treatment modality for lymphomas, but there is uncertainty on its use as consolidation after chemo-immunotherapy mainly in advanced stages. We evaluated its efficacy with a comprehensive meta-analysis and a systematic search of Pubmed, Embase, Cochrane, and abstracts from ASCO, ASH, ESMO and ASTRO published from June 1966 and December 2018. We identified 11 trials that evaluated consolidation radiotherapy following chemotherapy in a randomized fashion in 4'584 patients. The primary endpoint of this meta-analysis was PFS. As three of the eleven trials were retracted, this data is based on 2414 patients. For the primary endpoint (PFS), we found a hazard ratio (HR) of 0.77 (0.51 to 1.17, pooled (tau2: 0.25; I2: 85%), and a HR of 0.80 (0.53 to 1.21, pooled (bivariate meta-analysis). For overall survival, the HR is 0.93 (0.61 to 1.40; pooled (tau2: 0.25; I2: 74%) and 0.86 (0.58 to 1.27) in a bivariate meta-analysis. The lack of benefit did not change over time (p-value: 0.95 (tau2: 0.32; I2: 88%), and was also absent for PFS when stratifying for chemotherapy, the use of Rituximab, age, the dose of radiotherapy, application to patients in complete remission and with bulky disease. None of the trials used a PET-guided approach. This meta-analysis revealed no survival benefit when consolidation radiotherapy is given to unselected DLBCL patients following chemotherapy. These results need to be considered in future trials in the PET-CT era.

Sniffer dogs can identify lung cancer patients from breath and urine samples.

BACKGROUND: Lung cancer is the most common oncological cause of death in the Western world. Early diagnosis is critical for successful treatment. However, no effective screening methods exist. A promising approach could be the use of volatile organic compounds as diagnostic biomarkers. To date there are several studies, in which dogs were trained to discriminate cancer samples from controls. In this study we evaluated the abilities of specifically trained dogs to distinguish samples derived from lung cancer patients of various tumor stages from matched healthy controls. METHODS: This single center, double-blind clinical trial was approved by the local ethics committee, project no FF20/2016. The dog was conditioned with urine and breath samples of 36 cancer patients and 150 controls; afterwards, further 246 patients were included: 41 lung cancer patients comprising all stages and 205 healthy controls. From each patient two breath and urine samples were collected and shock frozen. Only samples from new subjects were presented to the dog during study phase randomized, double-blinded. This resulted in a specific conditioned reaction pointing to the cancer sample. RESULTS: Using a combination of urine and breath samples, the dog correctly predicted 40 out of 41 cancer samples, corresponding to an overall detection rate of cancer samples of 97.6% (95% CI [87.1, 99.9%]). Using urine samples only the dog achieved a detection rate of 87.8% (95% CI [73.8, 95.9%]). With breath samples, the dog correctly identified cancer in 32 of 41 samples, resulting in a detection rate of 78% (95% CI [62.4, 89.4%]). CONCLUSIONS: It is known from current literature that breath and urine samples carry VOCs pointing to cancer growth. We conclude that olfactory detection of lung cancer by specifically trained dogs is highly suggestive to be a simple and non-invasive tool to detect lung cancer. To translate this approach into practice further target compounds need to be identified.

The Tangential Extraperitoneal Retrorenal Approach in Kidney Transplant Biopsy: An Observational Study to Assess Complication and Adequacy Rates.

Introduction: Ultrasound-guided percutaneous kidney allograft biopsy is the gold-standard for pathology work-up. Recent studies postulate better safety and efficacy for tangential approaches, however, there is no recommendation regarding biopsy needle path. In this context, we previously described the unified tangential extraperitoneal retrorenal (TER) approach for standard allograft biopsy. Methods: A single-center retrospective observational study evaluated safety and efficacy of the TER biopsy approach among 250 patients that underwent 330 ultrasound-guided kidney transplant biopsies between January 2011 and May 2020. Results: The overall major complication rate was 0.56% per biopsy attempt (1.21% per biopsy) including blood transfusion, arterial embolization and bladder catheterization for gross hematuria in 0.28, 0.14 and 0.14% of biopsy attempts, respectively (0.61, 0.30 and 0.30% of biopsies, respectively). Minor complications included subcapsular and/or perinephric hematoma, superficial bleeding, arteriovenous fistula and gross hematuria in 12.6, 3.0, 2.5 and 1.4% of biopsy attempts, respectively (27.0, 6.4, 5.5 and 3.0% of biopsies, respectively). Sample adequacy rate was 86.7%, ranging from 82.2 to 94.1% if one or ≥two cores were analyzed, respectively. Residents and consultants yielded similar complication and adequacy rates. Conclusion: According to current literature, ultrasound-guided TER kidney transplant biopsy is a safe and efficient approach eligible for nephrology training.

Dual Akt and Bcl-2 inhibition induces cell-type specific modulation of apoptotic and autophagic signaling in castration resistant prostate cancer cell lines.

BACKGROUND: Cancer cell survival depends on the cross-regulation between apoptosis and autophagy which share common signaling pathways including PI3K/Akt/mTOR and Bcl-2. The aim of this study was to elucidate the modulation patterns between apoptosis and autophagy following dual inhibition by Akt inhibitor erufosine and Bcl-2 inhibitor ABT-737 in castration-resistant prostate cancer (CRPC) cell lines, PC-3 (Bax+) and DU-145 (Bax-). METHODS AND RESULTS: Cell cycle progression, apoptotic and autophagic signaling were examined by flow cytometry, multi-caspase assay, Hoechst staining, acridine orange staining of acidic vesicular organelles (AVOs), qRT-PCR and Western Blot. Dual inhibition increased G2/M arrest in PC-3 and DU-145, but not in the healthy prostate epithelium cells, PNT-1A. Only in PC-3, dual inhibition induced synergistic apoptotic and additive autophagic effects. In DU-145 and PNT-1A cells, ABT-737 did not display any remarkable effect on multicaspase activity and erufosine and ABT-737, neither alone nor in combination induced AVOs. By dual inhibition, AKT, BCL-2 and NF-κB gene expressions were downregulated in PC-3, both ATG-5 and BECLIN-1 gene expressions were upregulated in DU-145 but Beclin-1 protein expression was substantially reduced in both CRPC cells. Dual inhibition-induced synergistic multicaspase activation in PC-3 degrades and disrupts autophagic activity of Beclin-1, enhancing caspase-dependent apoptosis. However, in DU-145, following dual inhibition, rate of multicaspase induction and apoptosis are lower but autophagy is completely abolished despite markedly increased BECLIN-1 gene expression. CONCLUSION: In conclusion, antineoplastic drug combinations may display cell-type specific modulation of apoptotic and autophagic signaling and lack of protective autophagy may not necessarily indicate increased chemotherapeutic sensitivity in heterogenous tumor subpopulations.

The Treatment of Capsular Contracture Around Breast Implants Induced by Fractionated Irradiation: The Collagenase of the Bacterium Clostridium Histolyticum as a Novel Therapeutic Approach.

BACKGROUND: Irradiation therapy limits the utilization of silicone implants for breast reconstruction due to a significant risk for capsular contracture. The injection of the collagenase of the bacterium Clostridium histolyticum (CCH) might trivialize this risk by providing a minimal-invasive treatment option by capsular contracture degradation. However, efficacy in degrading breast implant capsules induced by fractionated irradiation remains unclear. METHODS: Twenty-four rats in three groups received miniature silicone implants in a submuscular pocket. After 3D dose calculation and treatment field definition, rats of two groups underwent fractionated radiotherapy (6 × 8 Gy) using a linear accelerator. A third group served as control. On day 120, one irradiated group received injections of 0.3 mg/ml collagenase. Administration of plain solvent solution served as control in the two other groups. Outcome parameters included CT-imaging, histology, vessel wall analysis, immunohistochemistry, chemical collagen quantification and gene expression analysis. RESULTS: Fractioned irradiation leads to a significant increase in collagen deposition around silicone implants with higher capsule thickness and collagen density when comparing all groups. Additionally, significant alterations of collagen fiber deposition were evident. Vessel wall thickness was significantly increased after radiotherapy. The injection of collagenase led to a significant reduction of capsule thickness, collagen density and content. However, the collagenase application induced a significant overexpression of TGFß1. No side effects were monitored. CONCLUSIONS: The CCH proved to be a safe and effective approach to degrade capsule tissue induced by fractionated irradiation in an animal model. This may pave its way for clinical application in implant-based breast reconstruction patients. LEVEL OF EVIDENCE: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .