Identification of pathological transcription in autosomal dominant polycystic kidney disease epithelia.

Autosomal dominant polycystic kidney disease (ADPKD) affects more than 12 million people worldwide. Mutations in PKD1 and PKD2 cause cyst formation through unknown mechanisms. To unravel the pathogenic mechanisms in ADPKD, multiple studies have investigated transcriptional mis-regulation in cystic kidneys from patients and mouse models, and numerous dysregulated genes and pathways have been described. Yet, the concordance between studies has been rather limited. Furthermore, the cellular and genetic diversity in cystic kidneys has hampered the identification of mis-expressed genes in kidney epithelial cells with homozygous PKD mutations, which are critical to identify polycystin-dependent pathways. Here we performed transcriptomic analyses of Pkd1- and Pkd2-deficient mIMCD3 kidney epithelial cells followed by a meta-analysis to integrate all published ADPKD transcriptomic data sets. Based on the hypothesis that Pkd1 and Pkd2 operate in a common pathway, we first determined transcripts that are differentially regulated by both genes. RNA sequencing of genome-edited ADPKD kidney epithelial cells identified 178 genes that are concordantly regulated by Pkd1 and Pkd2. Subsequent integration of existing transcriptomic studies confirmed 31 previously described genes and identified 61 novel genes regulated by Pkd1 and Pkd2. Cluster analyses then linked Pkd1 and Pkd2 to mRNA splicing, specific factors of epithelial mesenchymal transition, post-translational protein modification and epithelial cell differentiation, including CD34, CDH2, CSF2RA, DLX5, HOXC9, PIK3R1, PLCB1 and TLR6. Taken together, this model-based integrative analysis of transcriptomic alterations in ADPKD annotated a conserved core transcriptomic profile and identified novel candidate genes for further experimental studies.

Safety and Clinical Evaluation of Dual Inhibition with Pertuzumab and Trastuzumab Biosimilar SB3 in HER2-Positive Breast Cancer Patients.

BACKGROUND: The addition of trastuzumab to standard chemotherapy has improved survival in patients with HER2-positive breast cancer in neoadjuvant, adjuvant, and metastatic settings. In higher tumor stages, the addition of pertuzumab is now a standard of care and associated with a favorable toxicity profile. We evaluated the safety and efficacy of the trastuzumab biosimilar SB3 in combination with pertuzumab in HER2-positive breast cancer patients. METHODS: Seventy-eight patients with HER2-positive breast cancer treated at the Division of Oncology at the Medical University of Graz were included. Summary measures are reported as medians (25th to 75th percentile) for continuous variables and as absolute frequencies (%) for count data. RESULTS: Thirty-five patients received a median of 4 (3-7) cycles of trastuzumab biosimilar SB3 plus pertuzumab. All patients had a normal baseline left ventricular ejection fraction (LVEF; >50%) prior to the initiation of SB3 plus pertuzumab treatment with a median LVEF of 60% (60-65). Twenty-one patients had a median absolute LVEF decline of 1% (-5 to 0). Two patients (5.7%) had a LVEF reduction ≤50%, but none ≥10%. There were no unexpected adverse events. Twenty-two of 35 patients (63%) were treated with trastuzumab biosimilar SB3 and pertuzumab in the neoadjuvant setting and 11 patients (50%) achieved a pathological complete response. The safety and the efficacy in this setting was comparable to the trastuzumab plus pertuzumab combination in neoadjuvantly treated matched samples. CONCLUSION: In this series of HER2-positive breast cancer patients, the combination of SB3 plus pertuzumab was consistent with the known safety and efficacy profile of trastuzumab and pertuzumab combination.

Longitudinal tumor fraction trajectories predict risk of progression in metastatic HR+ breast cancer patients undergoing CDK4/6 treatment.

Despite improved clinical outcomes, intrinsic or acquired resistance to CDK4/6 inhibitor treatment has limited the success of this treatment in HR+ HER2- metastatic breast cancer patients. Biomarkers are urgently needed, and longitudinal biomarker measurements may harbor more dynamic predictive and prognostic information compared to single time point measurements. The aim of this study was to explore the longitudinal evolution of circulating tumor fractions within cell-free DNA assessed by an untargeted sequencing approach during CDK4/6 therapy and to quantify the potential association between longitudinal z-score measurements and clinical outcome by using joint models. Forty-nine HR+ HER2- metastatic breast cancer patients were enrolled, and z-score levels were measured at baseline and during 132 follow-up visits (median number of measurements per patient = 3, 25th -75th percentile: 3-5, range: 1-8). We observed higher baseline z-score levels (estimated difference 0.57, 95% CI: 0.147-0.983, P-value = 0.008) and a constant increase of z-score levels over follow-up time (overall P-value for difference in log z-score over time = 0.024) in patients who developed progressive disease. Importantly, the joint model revealed that elevated z-score trajectories were significantly associated with higher progression risk (HR of log z-score at any time of follow-up = 3.3, 95% CI, 1.44-7.55, P = 0.005). In contrast, single z-score measurement at CDK4/6 inhibitor treatment start did not predict risk of progression. In this prospective study, we demonstrate proof-of-concept that longitudinal z-score trajectories rather than single time point measurements may harbor important dynamic information on the development of disease progression in HR+ HER2- breast cancer patients undergoing CDK4/6 inhibitor treatment.

Single Blinded Study on the Feasibility of Decontaminating LA-MRSA in Pig Compartments under Routine Conditions.

： In countries with intensive pig husbandry in stables, the prevalence of livestock-associated (LA) methicillin-resistant Staphylococcus aureus (MRSA) on such farms has remained high in the last few years or has also further increased. Simple measures to reduce the LA-MRSA among pigs have not yet been successfully implemented. Earlier publications showed a decontamination of LA-MRSA was only possible with great effort. The aim of this study is to determine the suitability of routine cleaning and disinfection (C&D) for adequate LA-MRSA decontamination. For this purpose, at least 115 locations in a piglet-rearing compartment were examined before and after cleaning and disinfection. The sample locations were stratified according to accessibility for pigs and the difficulty of cleaning. The cleaning work was carried out routinely by farm employees, who were not informed about the sampling (single blinded). While before cleaning and disinfection, 85% of the samples from the surfaces were LA-MRSA positive, while only 2% were positive thereafter. All LA-MRSA-positive samples after cleaning and disinfection were outside the animal area. Air samples also showed no LA-MRSA after cleaning and disinfection. Conclusion: In well-managed livestock farms, decontamination of the LA-MRSA barn is quite possible; after C&D no LA-MRSA was detectable at animal height.

Validation of Residual Cancer Burden as Prognostic Factor for Breast Cancer Patients After Neoadjuvant Therapy.

BACKGROUND: Assessing the residual cancer burden (RCB) predictive performance, the potential subgroup effects, and time-dependent impact on breast cancer patients who underwent neoadjuvant therapy in a developer's independent cohort is essential for its usage in clinical routine. METHODS: Between 2011 and 2016, the RCB scores of 184 female breast cancer patients were prospectively collected, and subsequent clinicopathological and follow-up data were obtained retrospectively. Recurrence-free survival (RFS), overall survival (OS), as well as subgroup analysis, and time-dependent variables were calculated with multivariate, complex, or linear statistical models. RESULTS: A total of 184 patients (HER2 33%, TNBC 27%), with a mean follow-up time of 4 years, treated with neoadjuvant systemic therapy (92% anthracycline-taxane based) were analyzed revealing 43 events (38 recurrences, 28 deaths). High RCB scores were associated with recurrence (median index: 2.34 vs. 1.39 points, rank-sum p < 0.0001), decreased RFS (hazard ratio [HR] = 1.80, 95% confidence interval [CI] 1.44-2.24, p < 0.0001) and reduced OS (HR 1.96, 95% CI 1.49-2.59, p < 0.0001). The RCB score showed proportionality of hazards (interaction HR with linear follow-up time = 1.00, p = 0.896) and good discriminating power (Harrell's c index 0.7). CONCLUSIONS: Our results confirm the RCB score as externally valid prognostic marker and being independent of molecular subtype for RFS and OS in a clinical setting.

Analyzing candidemia guideline adherence identifies opportunities for antifungal stewardship.

Candidemia epidemiology varies significantly by region; thus, local data are essential for evidence-based decision-making in prophylaxis and treatment. Current management strategies are derived from large randomized controlled trials mostly executed in large high-volume tertiary care centers. Results may not be entirely transferable to smaller hospitals. This study investigates epidemiology, diagnosis, and treatment standards in six hospitals in the Cologne metropolitan area (number of inhabitants approx. one million). We assessed adherence to the current guideline of the European Society for Clinical Microbiology and Infectious Diseases (ESCMID) and the Infectious Diseases Society of America (IDSA) using the EQUAL Candida Score of the European Confederation of Medical Mycology (ECMM). Data were documented by trained medical students as part of an integrated research and teaching concept at the University of Cologne. Between January 2014 and June 2017, 77 patients had candidemia, corresponding to an incidence of 0.2 cases/1000 admissions. While 55 patients were enrolled, 22 patients were excluded due to incompletely retrievable health records. Fluconazole monotherapy was the preferred first-line treatment in cases with Candida albicans infection (21/29). A central vascular catheter was present in 40 patients and was removed in 17 (43%) during treatment. Overall mortality at 30 days was 44%. Patients reached a mean EQUAL Candida Score of 9.9 (range 8-14), which was well below the maximum score of 22 for perfect guideline adherence. In summary, management of candidemia differed from current European recommendations. It remains unclear to what extent enhanced adherence would improve patient outcome. Larger prospective studies need to answer that question.

Controlled assembly of single colloidal crystals using electro-osmotic micro-pumps.

We assemble charged colloidal spheres at deliberately chosen locations on a charged unstructured glass substrate utilizing ion exchange based electro-osmotic micro-pumps. Using microscopy, a simple scaling theory and Brownian dynamics computer simulations, we systematically explore the control parameters of crystal assembly and the mechanisms through which they depend on the experimental boundary conditions. We demonstrate that crystal quality depends crucially on the assembly distance of the colloids. This is understood as resulting from the competition between inward transport by the electro-osmotic pump flow and the electro-phoretic outward motion of the colloids. Optimized conditions include substrates of low and colloids of large electro-kinetic mobility. Then a sorting of colloids by size is observed in binary mixtures with larger particles assembling closer to the ion exchanger beads. Moreover, mono-sized colloids form defect free single domain crystals which grow outside a colloid-free void with facetted inner crystal boundaries centered on the ion exchange particle. This works remarkably well, even with irregularly formed ion exchange resin splinters.

LGALS3BP regulates centriole biogenesis and centrosome hypertrophy in cancer cells.

Centrosome morphology and number are frequently deregulated in cancer cells. Here, to identify factors that are functionally relevant for centrosome abnormalities in cancer cells, we established a protein-interaction network around 23 centrosomal and cell-cycle regulatory proteins, selecting the interacting proteins that are deregulated in cancer for further studies. One of these components, LGALS3BP, is a centriole- and basal body-associated protein with a dual role, triggering centrosome hypertrophy when overexpressed and causing accumulation of centriolar substructures when downregulated. The cancer cell line SK-BR-3 that overexpresses LGALS3BP exhibits hypertrophic centrosomes, whereas in seminoma tissues with low expression of LGALS3BP, supernumerary centriole-like structures are present. Centrosome hypertrophy is reversed by depleting LGALS3BP in cells endogenously overexpressing this protein, supporting a direct role in centrosome aberration. We propose that LGALS3BP suppresses assembly of centriolar substructures, and when depleted, causes accumulation of centriolar complexes comprising CPAP, acetylated tubulin and centrin.

Functional analysis of centrosomal kinase substrates in Drosophila melanogaster reveals a new function of the nuclear envelope component otefin in cell cycle progression.

Phosphorylation is one of the key mechanisms that regulate centrosome biogenesis, spindle assembly, and cell cycle progression. However, little is known about centrosome-specific phosphorylation sites and their functional relevance. Here, we identified phosphoproteins of intact Drosophila melanogaster centrosomes and found previously unknown phosphorylation sites in known and unexpected centrosomal components. We functionally characterized phosphoproteins and integrated them into regulatory signaling networks with the 3 important mitotic kinases, cdc2, polo, and aur, as well as the kinase CkIIß. Using a combinatorial RNA interference (RNAi) strategy, we demonstrated novel functions for P granule, nuclear envelope (NE), and nuclear proteins in centrosome duplication, maturation, and separation. Peptide microarrays confirmed phosphorylation of identified residues by centrosome-associated kinases. For a subset of phosphoproteins, we identified previously unknown centrosome and/or spindle localization via expression of tagged fusion proteins in Drosophila SL2 cells. Among those was otefin (Ote), an NE protein that we found to localize to centrosomes. Furthermore, we provide evidence that it is phosphorylated in vitro at threonine 63 (T63) through Aurora-A kinase. We propose that phosphorylation of this site plays a dual role in controlling mitotic exit when phosphorylated while dephosphorylation promotes G(2)/M transition in Drosophila SL2 cells.

Treatment satisfaction and health status in patients with systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is a rare connective tissue disease. Few data are available on treatment satisfaction, determinants of quality of life, and the health status of dermatology patients with SSc. PATIENTS AND METHODS: Cross-sectional study based on 72 consecutive dermatological patients with SSc. Objective clinical data were collected with a physician's questionnaire and subjective data were collected with a patients' questionnaire on disease characteristics, treatment satisfaction, quality of life, depressive symptoms, and Antonovsky's sense of coherence (SOC). We also tested the significance of possible determinants of treatment satisfaction. RESULTS: Treatment satisfaction was 72.0 (± 22.2; VAS 1-100). The assessment of professional competence of the treating physician was the most important determinant of treatment satisfaction and was independent of the patient's age and sex. The assessment of physician empathy, information about the disease, and the patient's own evaluation of the severity of disease were also associated with treatment satisfaction. The mean health-related quality of life (QoL; EQ-5D) was 0.74 (± 0.28) and the mean SOC was 72.6 (± 10.6). 58 % of patients reported moderate to severe pain and 13 % were treated for pain symptoms. In 69 % there was evidence of probable depression (CES-D $ 22); 8 % were on antidepressants. CONCLUSIONS: Treatment satisfaction was average and correlated especially with the sense of professional competence of the treating physician. In SSc patients, a diminished health-related quality of life as well as pain and evidence of depression are common and seem to be inadequately treated. However, the SOC indicates a lower general vulnerability. In the future, screening for pain and symptoms of depression should part of routine practice in SSc patients and, if necessary, interdisciplinary care should be initiated.

Gene ontology analysis of the centrosome proteomes of Drosophila and human.

The centrosome is a complex cell organelle in higher eukaryotic cells that functions in microtubule organization and is integrated into major cellular signaling pathways.1-3 For example, a tight link exists between cell cycle regulation and centrosome duplication, as centrosome numbers must be precisely controlled to ensure high fidelity of chromosome segregation.4 The analysis of the centrosome's protein composition provides the opportunity for a better understanding of centrosome function and to identify possible links to cellular signaling pathways.5,6 Our proteomics study of the Drosophila centrosome recently identified 251 centrosome candidate proteins that we subsequently characterized by RNAi in Drosophila SL2 cells and classified according to their function in centrosome duplication/segregation, structure maintenance and cell cycle regulation.7 Interestingly, functional characterization of their human orthologous proteins revealed the highest functional conservation in the process of centrosome duplication and separation. To analyze functional and biochemical interdependencies further, we carried out an analysis of the gene ontology (GO) annotation of the identified Drosophila centrosome proteins, as well as of the human centrosome proteome.5 The GO analysis of the group of proteins that did not show a centrosome, chromosome segregation or cell cycle related phenotype in our RNAi assays suggests that these molecules may constitute linker proteins to other cellular signaling pathways. Furthermore, the results of our GO analysis of components of the human and of the Drosophila centrosome reflect the somatic and embryonic origin, respectively, of the isolated centrosomes, implicating the Drosophila centrosome proteins in developmental signaling and cell differentiation.

Proteomic and functional analysis of the mitotic Drosophila centrosome.

Regulation of centrosome structure, duplication and segregation is integrated into cellular pathways that control cell cycle progression and growth. As part of these pathways, numerous proteins with well-established non-centrosomal localization and function associate with the centrosome to fulfill regulatory functions. In turn, classical centrosomal components take up functional and structural roles as part of other cellular organelles and compartments. Thus, although a comprehensive inventory of centrosome components is missing, emerging evidence indicates that its molecular composition reflects the complexity of its functions. We analysed the Drosophila embryonic centrosomal proteome using immunoisolation in combination with mass spectrometry. The 251 identified components were functionally characterized by RNA interference. Among those, a core group of 11 proteins was critical for centrosome structure maintenance. Depletion of any of these proteins in Drosophila SL2 cells resulted in centrosome disintegration, revealing a molecular dependency of centrosome structure on components of the protein translation machinery, actin- and RNA-binding proteins. In total, we assigned novel centrosome-related functions to 24 proteins and confirmed 13 of these in human cells.

A centrosome-independent role for gamma-TuRC proteins in the spindle assembly checkpoint.

The spindle assembly checkpoint guards the fidelity of chromosome segregation. It requires the close cooperation of cell cycle regulatory proteins and cytoskeletal elements to sense spindle integrity. The role of the centrosome, the organizing center of the microtubule cytoskeleton, in the spindle checkpoint is unclear. We found that the molecular requirements for a functional spindle checkpoint included components of the large gamma-tubulin ring complex (gamma-TuRC). However, their localization at the centrosome and centrosome integrity were not essential for this function. Thus, the spindle checkpoint can be activated at the level of microtubule nucleation.

Immunoisolation of centrosomes from Drosophila melanogaster.

Classical protocols for the isolation of centrosomes from higher eukaryotic cells are based on enrichment of cell organelles by density gradient centrifugation. Various successful protocols have been described that isolate centrosomes from mammalian tissue culture cells, tissue, clam oocytes, Drosophila, and yeast, to mention only some of the more frequently used sources. The material produced is subsequently used in various assays. These include functional tests such as the microtubule nucleation assay, electron microscopic study of centrosome morphology, and antigen localization; the organelles may also be used for the generation of antibodies. Furthermore, centrosomal preparations have been used for the characterization of their protein composition. The method described here focuses on the isolation of centrosomes from the syncytial stages of the early Drosophila embryo. This is a particularly attractive system because these organelles are not bounded by cellular membranes. Moreover, the abundance of pericentriolar material of these centrosomes produces excellent total protein yields.