Cell surface ß-lactamase recruitment: A facile selection to identify protein-protein interactions.

Protein-protein interactions (PPIs) are central to many cellular processes, and the identification of novel PPIs is a critical step in the discovery of protein therapeutics. Simple methods to identify naturally existing or laboratory evolved PPIs are therefore valuable research tools. We have developed a facile selection that links PPI-dependent ß-lactamase recruitment on the surface of Escherichia coli with resistance to ampicillin. Bacteria displaying a protein that forms a complex with a specific protein-ß-lactamase fusion are protected from ampicillin-dependent cell death. In contrast, bacteria that do not recruit ß-lactamase to the cell surface are killed by ampicillin. Given its simplicity and tunability, we anticipate this selection will be a valuable addition to the palette of methods for illuminating and interrogating PPIs.

Laboratory Evolution of Metalloid Reductase Substrate Recognition and Nanoparticle Product Size.

Glutathione reductase-like metalloid reductase (GRLMR) is an enzyme that reduces selenodiglutathione (GS-Se-SG), forming zerovalent Se nanoparticles (SeNPs). Error-prone polymerase chain reaction was used to create a library of ∼10,000 GRLMR variants. The library was expressed in BL21Escherichia coli in liquid culture with 50 mM of SeO32- present, under the hypothesis that the enzyme variants with improved GS-Se-SG reduction kinetics would emerge. The selection resulted in a GRLMR variant with two mutations. One of the mutations (D-E) lacks an obvious functional role, whereas the other mutation is L-H within 5 Šof the enzyme active site. This mutation places a second H residue within 5 Šof an active site dicysteine. This GRLMR variant was characterized for NADPH-dependent reduction of GS-Se-SG, GSSG, SeO32-, SeO42-, GS-Te-SG, and TeO32-. The evolved enzyme demonstrated enhanced reduction of SeO32- and gained the ability to reduce SeO42-. This variant is named selenium reductase (SeR) because of its emergent broad activity for a wide variety of Se substrates, whereas the parent enzyme was specific for GS-Se-SG. This study overall suggests that new biosynthetic routes are possible for inorganic nanomaterials using laboratory-directed evolution methods.

Cloneable inorganic nanoparticles.

When a defined protein/peptide (or combinations thereof) control and define the synthesis of an inorganic nanoparticle, the result is a cloneable NanoParticle (cNP). This is because the protein sequence/structure/function is encoded in DNA, and therefore the physicochemical properties of the nanoparticle are also encoded in DNA. Thus the cloneable nanoparticle paradigm can be considered as an extension of the central dogma of molecular biology (e.g. DNA → mRNA → protein → cNP); modifications to the DNA encoding a cNP can modify the resulting properties of the cNP. Inorganic ion oxidoreductases (e.g., mercuric reductase, tellurite reductase, etc.) can select and reduce specific inorganic oxyanions and coordination complexes, creating zerovalent precipitates. Other proteins/peptides (often genetically concatenated to the parent oxidoreductase) serve as ligands, directing the size, shape, crystal structure and other properties of the nanoparticle. The DNA encoding a cNP can be recombinantly transferred into any organism. Ideally, this enables recombinant production of cNPs with the same defined physiochemical properties. Such cNPs are of interest for applications ranging from molecular imaging, bio-remediation, catalysis, and biomining. In this Feature Article we detail and define the cNP concept, and retrace the story of our creation of a cloneable Se NanoParticle (cSeNP). We also describe our more preliminary work that we expect to result in cloneable semiconductor quantum dots, cloneable Te nanoparticles, and other cNP formulations. We highlight the application of cNPs in cellular electron microscopy and compare this approach to other cloneable imaging contrast approaches.

More than 30 Years of POSSUM: Are Scoring Systems Still Relevant Today for Colorectal Surgery?

BACKGROUND: The Physiological and Operative Severity Score for the enUmeration of Mortality and Morbidity (POSSUM) weights the patient's individual health status and the extent of the surgical procedure to estimate the probability of postoperative complications and death of general surgery patients. The variations Portsmouth-POSSUM (P-POSSUM) and colorectal POSSUM (CR-POSSUM) were developed for estimating mortality in patients with low perioperative risk and for patients with colorectal carcinoma, respectively. The aim of the present study was to evaluate the significance of POSSUM, P-POSSUM, and CR-POSSUM in two independent colorectal cancer cohorts undergoing surgery, with an emphasis on laparoscopic procedures. METHODS: For each patient, an individual physiological score (PS) and operative severity score (OS) was attributed to calculate the predicted morbidity and mortality, respectively. Logistic regression analysis was used to evaluate the possible correlation between the subscores and the probability of postoperative complications and mortality. RESULTS: The POSSUM equation significantly overpredicted postoperative morbidity, and all three scoring systems considerably overpredicted in-hospital mortality. However, the POSSUM score identified patients at risk of anastomotic leakage, sepsis, and the need for reoperation. Logistic regression analysis demonstrated a strong correlation between the subscores and the probability of postoperative complications and mortality, respectively. CONCLUSION: Our results suggest that the three scoring systems are too imprecise for the estimation of perioperative complications and mortality of patients undergoing colorectal surgery in the present day. Since the subscores proved valid, a revision of the scoring systems could increase their reliability in the clinical setting.

Adequate Management of Postoperative Complications after Esophagectomy: A Cornerstone for a Positive Outcome.

BACKGROUND: Esophagectomy for cancer is one of the most complex procedures in visceral surgery. Postoperative complications negatively affect the patient's overall survival. They are not influenced by the histology type (adenocarcinoma (AC)/squamous cell carcinoma (SCC)), or the surgical approach (open, laparoscopic, or robotic-assisted). Among those dreadful complications are anastomotic leak (AL), esophago-respiratory fistula (ERF), and chylothorax (CT). METHODS: In this review, we summarize the methods to avoid these complications, the diagnostic approach, and new therapeutic strategies. RESULTS: In the last 20 years, both centralization of the medical care, and the development of endoscopy and radiology have positively influenced the management of postoperative complications. For the purpose of their prevention, perioperative measures have been applied. The treatment includes conservative, endoscopic, and surgical approaches. CONCLUSIONS: Post-esophagectomy complications are common. Prevention measures should be known. Early recognition and adequate treatment of these complications save lives and lead to better outcomes.

Biomarkers in Liquid Biopsies for Prediction of Early Liver Metastases in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive solid malignancies with poor survival rates. Only 20% of the patients are eligible for R0-surgical resection, presenting with early relapses, mainly in the liver. PDAC patients with hepatic metastases have a worse outcome compared to patients with metastases at other sites. Early detection of hepatic spread bears the potential to improve patient outcomes. Thus, this study sought for serum-based perioperative biomarkers allowing discrimination of early (EHMS ≤ 12 months) and late hepatic metastatic spread (LHMS > 12 months). Serum samples from 83 resectable PDAC patients were divided into EHMS and LHMS and analyzed for levels of inflammatory mediators by LEGENDplexTM, which was validated and extended by Olink® analysis. CA19-9 serum levels served as control. Results were correlated with clinicopathological data. While serum CA19-9 levels were comparable, Olink® analysis confirmed distinct differences between both groups. It revealed significantly elevated levels of factors involved in chemotaxis and migration of immune cells, immune activity, and cell growth in serum of LHMS-patients. Overall, Olink® analysis identified a comprehensive biomarker panel in serum of PDAC patients that could provide the basis for predicting LHMS. However, further studies with larger cohorts are required for its clinical translation.

Paracrine Interaction of Cholangiocellular Carcinoma with Cancer-Associated Fibroblasts and Schwann Cells Impact Cell Migration.

Although the Mitogen-activated protein kinase (MAPK) pathway is enriched in cholangiocarcinoma (CCA), treatment with the multityrosine kinase-inhibitor Sorafenib is disappointing. While cancer-associated fibroblasts (CAF) are known to contribute to treatment resistance in CCA, knowledge is lacking for Schwann cells (SC). We investigated the impact of stromal cells on CCA cells and whether this is affected by Sorafenib. Immunohistochemistry revealed elevated expression of CAF and SC markers significantly correlating with reduced tumor-free survival. In co-culture with CAF, CCA cells mostly migrated, which could be diminished by Sorafenib, while in SC co-cultures, SC predominantly migrated towards CCA cells, unaffected by Sorafenib. Moreover, increased secretion of pro-inflammatory cytokines MCP-1, CXCL-1, IL-6 and IL-8 was determined in CAF mono- and co-cultures, which could be reduced by Sorafenib. Corresponding to migration results, an increased expression of phospho-AKT was measured in CAF co-cultured HuCCT-1 cells, although was unaffected by Sorafenib. Intriguingly, CAF co-cultured TFK-1 cells showed increased activation of STAT3, JNK, ERK and AKT pathways, which was partly reduced by Sorafenib. This study indicates that CAF and SC differentially impact CCA cells and Sorafenib partially reverts these stroma-mediated effects. These findings contribute to a better understanding of the paracrine interplay of CAF and SC with CCA cells.

Microbiota-dependent activation of the myeloid calcineurin-NFAT pathway inhibits B7H3- and B7H4-dependent anti-tumor immunity in colorectal cancer.

Bacterial sensing by intestinal tumor cells contributes to tumor growth through cell-intrinsic activation of the calcineurin-NFAT axis, but the role of this pathway in other intestinal cells remains unclear. Here, we found that myeloid-specific deletion of calcineurin in mice activated protective CD8+ T cell responses and inhibited colorectal cancer (CRC) growth. Microbial sensing by myeloid cells promoted calcineurin- and NFAT-dependent interleukin 6 (IL-6) release, expression of the co-inhibitory molecules B7H3 and B7H4 by tumor cells, and inhibition of CD8+ T cell-dependent anti-tumor immunity. Accordingly, targeting members of this pathway activated protective CD8+ T cell responses and inhibited primary and metastatic CRC growth. B7H3 and B7H4 were expressed by the majority of human primary CRCs and metastases, which was associated with low numbers of tumor-infiltrating CD8+ T cells and poor survival. Therefore, a microbiota-, calcineurin-, and B7H3/B7H4-dependent pathway controls anti-tumor immunity, revealing additional targets for immune checkpoint inhibition in microsatellite-stable CRC.

Eso-Sponge® for anastomotic leakage after oesophageal resection or perforation: outcomes from a national, prospective multicentre registry.

BACKGROUND: Anastomotic leakage (AL) after oesophagectomy and oesophageal perforations are associated with significant morbidity and mortality. Minimally invasive endoscopy is often used as first-line treatment, particularly endoluminal vacuum therapy (EVT). The aim was to assess the performance of the first commercially available endoluminal vacuum device (Eso-Sponge®) in the management of AL and perforation of the upper gastrointestinal tract (GIT). METHODS: The Eso-Sponge® registry was designed in 2014 as a prospective, observational, national, multicentre registry. Patients were recruited with either AL or perforation within the upper GIT. Data were collected with a standardized form and transferred into a web-based platform. Twenty hospitals were enrolled at the beginning of the study (registration number NCT02662777; http://www.clinicaltrials.gov). The primary endpoint was successful closure of the oesophageal defect. RESULTS: Eleven out of 20 centres recruited patients. A total of 102 patients were included in this interim analysis; 69 patients with AL and 33 with a perforation were treated by EVT. In the AL group, a closure of 91 per cent was observed and 76 per cent was observed in the perforation group. The occurrence of mediastinitis (P = 0.002) and the location of the defect (P = 0.008) were identified as significant predictors of defect closure. CONCLUSIONS: The Eso-Sponge® registry offers the opportunity to collate data on EVT with a uniform, commercially available product to improve standardization. Our data show that EVT with the Eso-Sponge® is an option for the management of AL and perforation within the upper GIT.

A novel unconventional T cell population enriched in Crohn's disease.

OBJECTIVE: One of the current hypotheses to explain the proinflammatory immune response in IBD is a dysregulated T cell reaction to yet unknown intestinal antigens. As such, it may be possible to identify disease-associated T cell clonotypes by analysing the peripheral and intestinal T-cell receptor (TCR) repertoire of patients with IBD and controls. DESIGN: We performed bulk TCR repertoire profiling of both the TCR alpha and beta chains using high-throughput sequencing in peripheral blood samples of a total of 244 patients with IBD and healthy controls as well as from matched blood and intestinal tissue of 59 patients with IBD and disease controls. We further characterised specific T cell clonotypes via single-cell RNAseq. RESULTS: We identified a group of clonotypes, characterised by semi-invariant TCR alpha chains, to be significantly enriched in the blood of patients with Crohn's disease (CD) and particularly expanded in the CD8+ T cell population. Single-cell RNAseq data showed an innate-like phenotype of these cells, with a comparable gene expression to unconventional T cells such as mucosal associated invariant T and natural killer T (NKT) cells, but with distinct TCRs. CONCLUSIONS: We identified and characterised a subpopulation of unconventional Crohn-associated invariant T (CAIT) cells. Multiple evidence suggests these cells to be part of the NKT type II population. The potential implications of this population for CD or a subset thereof remain to be elucidated, and the immunophenotype and antigen reactivity of CAIT cells need further investigations in future studies.

Detection of Cancer Mutations by Urine Liquid Biopsy as a Potential Tool in the Clinical Management of Bladder Cancer Patients.

The standard diagnostic and follow-up examination for bladder cancer is diagnostic cystoscopy, an invasive test that requires compliance for a long period. Urine cytology and recent biomarkers come short of replacing cystoscopy. Urine liquid biopsy promises to solve this problem and potentially allows early detection, evaluation of treatment efficacy, and surveillance. A previous study reached 52-68% sensitivity using small-panel sequencing but could increase sensitivity to 68-83% by adding aneuploidy and promoter mutation detection. Here, we explore whether a large 127-gene panel alone is sufficient to detect tumor mutations in urine from bladder cancer patients. We recruited twelve bladder cancer patients, obtained preoperative and postoperative urine samples, and successfully analyzed samples from eleven patients. In ten patients, we found at least one mutation in bladder-cancer-associated genes, i.e., a promising sensitivity of 91%. In total, we identified 114 variants, of which 90 were predicted as nonbenign, 30% were associated with cancer, and 13% were actionable according to the CIViC database. Sanger sequencing of the patients' formalin-fixed, paraffin-embedded (FFPE) tumor tissues confirmed the findings. We concluded that incorporating urine liquid biopsy is a promising strategy in the management of bladder cancer patients.

Temperature profile and residual heat of monopolar laparoscopic and endoscopic dissection instruments.

BACKGROUND: Endoscopic and laparoscopic electrosurgical devices (ED) are of great importance in modern medicine but can cause adverse events such as tissue injuries and burns from residual heat. While laparoscopic tools are well investigated, detailed insights about the temperature profile of endoscopic knives are lacking. Our aim is to investigate the temperature and the residual heat of laparoscopic and endoscopic monopolar instruments to increase the safety in handling ED. METHODS: An infrared camera was used to measure the temperature of laparoscopic and endoscopic instruments during energy application and to determine the cooling time to below 50 °C at a porcine stomach. Different power levels and cutting intervals were studied to investigate their impact on the temperature profile. RESULTS: During activation, the laparoscopic hook exceeded 120 °C regularly for an up to 10 mm shaft length. With regards to endoknives, only the Dual Tip Knife showed a shaft temperature of above 50 °C. The residual heat of the laparoscopic hook remained above 50 °C for at least 15 s after activation. Endoknives cooled to below 50 °C in 4 s. A higher power level and longer cutting duration significantly increased the shaft temperature and prolonged the cooling time (p < 0.001). CONCLUSION: Residual heat and maximum temperature during energy application depend strongly on the chosen effect and cutting duration. To avoid potential injuries, the user should not touch any tissue with the laparoscopic hook for at least 15 s and with the endoknives for at least 4 s after energy application. As the shaft also heats up to over 120 °C, the user should be careful to avoid tissue contact during activation with the shaft. These results should be strongly considered for safety reasons when handling monopolar ED.

Longitudinal Analysis of Circulating Tumor Cells in Colorectal Cancer Patients by a Cytological and Molecular Approach: Feasibility and Clinical Application.

INTRODUCTION: Liquid biopsies allowing for individualized risk stratification of cancer patients have become of high significance in individualized cancer diagnostics and treatment. The detection of circulating tumor cells (CTC) has proven to be highly relevant in risk prediction, e.g., in colorectal cancer (CRC) patients. In this study, we investigate the clinical relevance of longitudinal CTC detection over a course of follow-up after surgical resection of the tumor and correlate these findings with clinico-pathological characteristics. METHODS: In total, 49 patients with histologically proven colorectal carcinoma were recruited for this prospective study. Blood samples were analyzed for CTC presence by two methods: first by marker-dependent immunofluorescence staining combined with automated microscopy with the NYONE® cell imager and additionally, indirectly, by semi-quantitative Cytokeratin-20 (CK20) RT-qPCR. CTC quantification data were compared and correlated with the clinico-pathological parameters. RESULTS: Detection of CTC over a post-operative time course was feasible with both applied methods. In patients who were pre-operatively negative for CTCs with the NYONE® method or below the cut-off for relative CK20 mRNA expression after analysis by PCR, a statistically significant rise in the immediate post-operative CTC detection could be demonstrated. Further, in the cohort analyzed by PCR, we detected a lower CTC load in patients who were adjuvantly treated with chemotherapy compared to patients in the follow-up subgroup. This finding was contrary to the same patient subset analyzed with the NYONE® for CTC detection. CONCLUSION: Our study investigates the occurrence of CTC in CRC patients after surgical resection of the primary tumor and during postoperative follow-up. The resection of the tumor has an impact on the CTC quantity and the longitudinal CTC analysis supports the significance of CTC as a prognostic biomarker. Future investigations with an even more extended follow-up period and larger patient cohorts will have to validate our results and may help to define an optimal longitudinal sampling scheme for liquid biopsies in the post-operative monitoring of cancer patients to enable tailored therapy concepts for precision medicine.

Genome-wide analysis of 944 133 individuals provides insights into the etiology of haemorrhoidal disease.

OBJECTIVE: Haemorrhoidal disease (HEM) affects a large and silently suffering fraction of the population but its aetiology, including suspected genetic predisposition, is poorly understood. We report the first genome-wide association study (GWAS) meta-analysis to identify genetic risk factors for HEM to date. DESIGN: We conducted a GWAS meta-analysis of 218 920 patients with HEM and 725 213 controls of European ancestry. Using GWAS summary statistics, we performed multiple genetic correlation analyses between HEM and other traits as well as calculated HEM polygenic risk scores (PRS) and evaluated their translational potential in independent datasets. Using functional annotation of GWAS results, we identified HEM candidate genes, which differential expression and coexpression in HEM tissues were evaluated employing RNA-seq analyses. The localisation of expressed proteins at selected loci was investigated by immunohistochemistry. RESULTS: We demonstrate modest heritability and genetic correlation of HEM with several other diseases from the GI, neuroaffective and cardiovascular domains. HEM PRS validated in 180 435 individuals from independent datasets allowed the identification of those at risk and correlated with younger age of onset and recurrent surgery. We identified 102 independent HEM risk loci harbouring genes whose expression is enriched in blood vessels and GI tissues, and in pathways associated with smooth muscles, epithelial and endothelial development and morphogenesis. Network transcriptomic analyses highlighted HEM gene coexpression modules that are relevant to the development and integrity of the musculoskeletal and epidermal systems, and the organisation of the extracellular matrix. CONCLUSION: HEM has a genetic component that predisposes to smooth muscle, epithelial and connective tissue dysfunction.

Detection of Marker Associated with CTC in Colorectal Cancer in Mononuclear Cells of Patients with Benign Inflammatory Intestinal Diseases.

Colorectal carcinoma (CRC) belongs to the most common tumor entities in western countries. Circulating tumor cells (CTC) in blood of CRC patients are a powerful prognostic and predictive biomarker. However, whether CTC-associated markers can also be used for early CRC detection and discrimination from benign diseases is not known. This study investigated the presence of CTC-associated markers CK20, PLS3, LAD1, and DEFA5 in blood of patients with benign inflammatory intestinal disease (IID) and their correlation with malignancy. The detection rate of CK20 and DEFA5 significantly differed between diseased patients and healthy controls. LAD1 and PLS3 were detected in all samples with clear differences in gene expression. DEFA5 expression was higher in CRC and IID patients compared to healthy donors, while CK20 and PLS3 were lower in CRC compared to IID patients or healthy controls. Overall, all CTC-associated markers were detectable in blood of IID patients, but not correlating with inflammation severity. Finally, PLS3 emerged as a suitable marker for differentiation between malignant and non-malignant intestinal diseases or healthy controls, however its suitability for early CRC detection needs to be further validated.

Loss of hepatic Mboat7 leads to liver fibrosis.

OBJECTIVE: The rs641738C>T variant located near the membrane-bound O-acyltransferase domain containing 7 (MBOAT7) locus is associated with fibrosis in liver diseases, including non-alcoholic fatty liver disease (NAFLD), alcohol-related liver disease, hepatitis B and C. We aim to understand the mechanism by which the rs641738C>T variant contributes to pathogenesis of NAFLD. DESIGN: Mice with hepatocyte-specific deletion of MBOAT7 (Mboat7Δhep) were generated and livers were characterised by histology, flow cytometry, qPCR, RNA sequencing and lipidomics. We analysed the association of rs641738C>T genotype with liver inflammation and fibrosis in 846 NAFLD patients and obtained genotype-specific liver lipidomes from 280 human biopsies. RESULTS: Allelic imbalance analysis of heterozygous human liver samples pointed to lower expression of the MBOAT7 transcript on the rs641738C>T haplotype. Mboat7Δhep mice showed spontaneous steatosis characterised by increased hepatic cholesterol ester content after 10 weeks. After 6 weeks on a high fat, methionine-low, choline-deficient diet, mice developed increased hepatic fibrosis as measured by picrosirius staining (p<0.05), hydroxyproline content (p<0.05) and transcriptomics, while the inflammatory cell populations and inflammatory mediators were minimally affected. In a human biopsied NAFLD cohort, MBOAT7 rs641738C>T was associated with fibrosis (p=0.004) independent of the presence of histological inflammation. Liver lipidomes of Mboat7Δhep mice and human rs641738TT carriers with fibrosis showed increased total lysophosphatidylinositol levels. The altered lysophosphatidylinositol and phosphatidylinositol subspecies in MBOAT7Δhep livers and human rs641738TT carriers were similar. CONCLUSION: Mboat7 deficiency in mice and human points to an inflammation-independent pathway of liver fibrosis that may be mediated by lipid signalling and a potentially targetable treatment option in NAFLD.

Clinical responses to PD-1 inhibition and their molecular characterization in six patients with mismatch repair-deficient metastatic cancer of the digestive system.

PURPOSE: Immune checkpoint inhibitors have shown efficacy in patients with microsatellite instability-high/mismatch repair-deficient (MSI-H/dMMR) gastrointestinal (GI) cancers. However, depth and duration of clinical response is not uniform. We assessed tumor mutation burden (TMB) as a response marker in patients with GI cancers treated with immune checkpoint inhibitors. METHODS: Detailed clinical and response data were collected from six patients with metastatic MSI-H/dMMR GI cancers treated with immune checkpoint inhibitors. Efficacy was assessed by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Tumors and matched normal tissue were profiled by targeted next generation sequencing (127 gene panel, size 0.8 Mb). Impact of included mutation types, germline filtering methodology and different variant allele frequency thresholds on TMB estimation was assessed. RESULTS: Objective radiographic responses were observed in all six patients, and complete response was achieved in two of the six patients. Responses were durable (minimum 25 months). TMB estimates were clearly above the two recently reported cut-offs for metastatic colorectal cancer of 12 or 37 mutations per megabase for five of six patients, respectively, while one patient had borderline TMB elevation. TMB did not show an association with extent and duration of response but was influenced by included mutation types, germline filtering method and variant allele frequency threshold. CONCLUSION: Our case series confirms the clinical benefit of immune checkpoint blockade in patients with metastatic MSI-H/dMMR GI cancers and illustrates the vulnerability of TMB as predictive marker in a subset of patients.

Identification of a TeO32- reductase/mycothione reductase from Rhodococcus erythropolis PR4.

A Rhodococcus erythropolis bacterium that tolerates normally lethal concentrations of Fe(II), Cu(II), AsO32-, SeO32-, TeO32-, Cd(II) and Zn(II) was identified from an environmental isolate. In characterizing the molecular basis for metal tolerance, a mycothione reductase (Mtr) with remarkable selectivity for TeO32- reduction over SeO32- was identified. In equimolar concentrations of TeO32- and SeO32-, the enzymatic product contains a 7-fold excess of Te. This selectivity is remarkable because the standard reduction potential of SeO32- is 0.20 V more favorable for reduction than TeO32. Selectivity of the enzyme for TeO32- decreases with increasing assay pH. Homology modeling of the enzyme identifies four aromatic residues near the active site, including two histidine residues, that are not present in a related SeO32- preferring reductase. On the basis of more favorable π-interactions for Te than for Se and the pH dependence of the selectivity, the Te-selectivity is attributed in part to these aromatic residues. The resulting Te0 enzymatic product resembles Te nanowires.

Isolation and Enumeration of CTC in Colorectal Cancer Patients: Introduction of a Novel Cell Imaging Approach and Comparison to Cellular and Molecular Detection Techniques.

Circulating tumour cells (CTC) were proven to be prognostically relevant in cancer treatment, e.g., in colorectal cancer (CRC). This study validates a molecular detection technique through using a novel cell imaging approach for CTC detection and enumeration, in comparison to a size-based cellular and correlated the data to clinico-pathological characteristics. Overall, 57 CRC patients were recruited for this prospective study. Blood samples were analysed for CTCs by three methods: (1) Epithelial marker immunofluorescence staining combined with automated microscopy using the NYONE® cell imager; (2) isolation by size using membrane filtration with the ScreenCell® Cyto IS device and immunofluorescence staining; (3) detection by semi-quantitative Cytokeratin-20 RT-qPCR. Enumeration data were compared and correlated with clinic-pathological parameters. CTC were detected by either approach; however, with varying positivity rates: NYONE® 36.4%, ScreenCell® 100%, and PCR 80.5%. All methods revealed a positive correlation of CTC presence and higher tumour burden, which was most striking using the ScreenCell® device. Generally, no intercorrelation of CTC presence emerged amongst the applied techniques. Overall, enumeration of CTC after isolation by size demonstrated to be the most reliable strategy for the detection of CTC in CRC patients. Ongoing studies will have to unravel the prognostic value of this finding, and validate this approach in a larger cohort.

Stage IV Colorectal Cancer Patients with High Risk Mutation Profiles Survived 16 Months Longer with Individualized Therapies.

Personalized treatment vs. standard of care is much debated, especially in clinical practice. Here we investigated whether overall survival differences in metastatic colorectal cancer patients are explained by tumor mutation profiles or by treatment differences in real clinical practice. Our retrospective study of metastatic colorectal cancer patients of confirmed European ancestry comprised 54 Americans and 54 gender-matched Germans. The Americans received standard of care, and on treatment failure, 35 patients received individualized treatments. The German patients received standard of care only. Tumor mutations, tumor mutation burden and microsatellite status were identified by using the FoundationOne assay or the IDT Pan-Cancer assay. High-risk patients were identified according to the mutational classification by Schell and colleagues. Results: Kaplan-Meier estimates show the high-risk patients to survive 16 months longer under individualized treatments than those under only standard of care, in the median (p < 0.001). Tumor mutation profiles stratify patients by risk groups but not by country. Conclusions: High-risk patients appear to survive significantly longer (p < 0.001) if they receive individualized treatments after the exhaustion of standard of care treatments. Secondly, the tumor mutation landscape in Americans and Germans is congruent and thus warrants the transatlantic exchange of successful treatment protocols and the harmonization of guidelines.