Automated 3D light-sheet screening with high spatiotemporal resolution reveals mitotic phenotypes.

3D cell cultures enable the in vitro study of dynamic biological processes such as the cell cycle, but their use in high-throughput screens remains impractical with conventional fluorescent microscopy. Here, we present a screening workflow for the automated evaluation of mitotic phenotypes in 3D cell cultures by light-sheet microscopy. After sample preparation by a liquid handling robot, cell spheroids are imaged for 24 h in toto with a dual-view inverted selective plane illumination microscope (diSPIM) with a much improved signal-to-noise ratio, higher imaging speed, isotropic resolution and reduced light exposure compared to a spinning disc confocal microscope. A dedicated high-content image processing pipeline implements convolutional neural network-based phenotype classification. We illustrate the potential of our approach using siRNA knockdown and epigenetic modification of 28 mitotic target genes for assessing their phenotypic role in mitosis. By rendering light-sheet microscopy operational for high-throughput screening applications, this workflow enables target gene characterization or drug candidate evaluation in tissue-like 3D cell culture models.

Predictive factors for response to salvage stereotactic body radiotherapy in oligorecurrent prostate cancer limited to lymph nodes: a single institution experience.

BACKGROUND: In patients presenting with limited nodal recurrence following radical prostatectomy (RP), stereotactic body radiotherapy (SBRT) results might improve with a better case selection. METHODS: Single-institution retrospective analysis of patients presenting with 1-3 lymph node (LN) recurrences (N1 or M1a) on 18F-Choline PET/CT. Prior therapy included radical prostatectomy (RP) ± salvage radiotherapy (RT), in absence of any systemic therapy. Outcome parameters were biochemical response (BR), time to biochemical recurrence (TBR) and time interval between SBRT and androgen deprivation therapy start (TADT). Time to event endpoints was analysed using Kaplan-Meier method. Potential prognostic factors were examined using univariate proportional hazards regression for TADT and logistic regression for BR. The optimal cut-off point for LN size was calculated using the Contal and O'Quigley method. RESULTS: 25 patients fulfilling study criteria were treated with SBRT from January 2010 to January 2015 and retrospectively analysed. Median follow up was 18 months and median LN diameter 10.5 mm. SBRT was delivered to a median dose of 36 Gy in three fractions (range: 30-45 Gy). BR was reached in 52% of cases. Median TBR was 11.9 months and significantly longer in patients with larger LN (Hazard ratio [HR] = 0.87, P = 0.03). Using 14 mm as cut off for LN, median TBR was 10.8 months for patients with small LN (18 patients), and 21.2 months for patients with large LN (6 patients) (P unadjusted = 0.009; P adjusted = 0.099). ADT was started in 32% of patients after a median follow-up of 18 months. CONCLUSIONS: For PCa patients with 1-3 LN recurrence after RP (± salvage RT), SBRT might result in a better biochemical control when delivered to larger sized (≥ 14 mm) LN metastases. This study is hypothesis generating and results should be tested in a larger prospective trial.

Deep inspirational breast hold (DIBH) for right breast irradiation: Improved sparing of liver and lung tissue.

Objective: To reduce liver and lung dose during right breast irradiation while maintaining optimal dose to the target volume. This dose reduction has the potential to decrease acute side effects and long-term toxicity. Materials and Methods: 16 patients treated with radiation therapy for localized carcinoma of the right breast were included retrospectively. For the planning CT, each patient was immobilised on an indexed board with the arms placed above the head. CT scans were acquired in free-breathing (FB) as well as with deep inspiration breath hold (DIBH). Both scans were acquired with the same length. Planning target volumes (PTV's) were created with a 5 mm margin from the respective clinical target volumes (CTV's) on both CT datasets. The liver was outlined as scanned. Dose metrics evaluated were as follows: differences in PTV coverage, dose to the liver (max, mean, V90%, V50%, V30%), dose to lung (mean, V20Gy, relative electron density) and dose to heart (Dmax). The p-values were calculated using Wilcoxon signed-rank tests. A p-value was significant when <0.05. Results: Differences in PTV coverage between plans using FB and DIBH were less than 2 %. Maximum liver dose was significantly less using DIBH: 17.5 Gy versus FB: 40.3 Gy (p < 0.001). The volume of the liver receiving 10 % of the dose was significantly less using DIBH with 1.88 cm3 versus 72.2 cm3 under FB (p < 0.001). The absolute volume receiving 20 Gy in the right lung was larger using DIBH: 291 cm3 versus 230 cm3 under FB (p < 0.001) and the relative volume of lung receiving dose greater than 20 Gy was smaller with DIBH: 11.5 % versus 14 % in FB (p = 0.007). The relative electron density of lung was significantly less with DIBH: 0.59 versus 0.62 with FB, (p < 0.001). This suggests that the lung receives less dose due to its lower density when using DIBH. Conclusion: Radiation of the right breast using DIBH spares liver and lung tissue significantly and thus carries the potential of best practice for right sided breast cancer.

Transcriptomics-inferred dynamics of SARS-CoV-2 interactions with host epithelial cells.

Virus-host interactions can reveal potentially effective and selective therapeutic targets for treating infection. Here, we performed an integrated analysis of the dynamics of virus replication and the host cell transcriptional response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection using human Caco-2 colon cancer cells as a model. Time-resolved RNA sequencing revealed that, upon infection, cells immediately transcriptionally activated genes associated with inflammatory pathways that mediate the antiviral response, which was followed by an increase in the expression of genes involved in ribosome and mitochondria function, thus suggesting rapid alterations in protein production and cellular energy supply. At later stages, between 24 and 48 hours after infection, the expression of genes involved in metabolic processes-in particular, those related to xenobiotic metabolism-was decreased. Mathematical modeling incorporating SARS-CoV-2 replication suggested that SARS-CoV-2 proteins inhibited the host antiviral response and that virus transcripts exceeded the translation capacity of the host cells. Targeting kinase-dependent pathways that exhibited increases in transcription in host cells was as effective as a virus-targeted inhibitor at repressing viral replication. Our findings in this model system delineate a sequence of SARS-CoV-2 virus-host interactions that may facilitate the identification of druggable host pathways to suppress infection.

RASPD+: Fast Protein-Ligand Binding Free Energy Prediction Using Simplified Physicochemical Features.

The virtual screening of large numbers of compounds against target protein binding sites has become an integral component of drug discovery workflows. This screening is often done by computationally docking ligands into a protein binding site of interest, but this has the drawback of a large number of poses that must be evaluated to obtain accurate estimates of protein-ligand binding affinity. We here introduce a fast pre-filtering method for ligand prioritization that is based on a set of machine learning models and uses simple pose-invariant physicochemical descriptors of the ligands and the protein binding pocket. Our method, Rapid Screening with Physicochemical Descriptors + machine learning (RASPD+), is trained on PDBbind data and achieves a regression performance that is better than that of the original RASPD method and traditional scoring functions on a range of different test sets without the need for generating ligand poses. Additionally, we use RASPD+ to identify molecular features important for binding affinity and assess the ability of RASPD+ to enrich active molecules from decoys.

Influencing Factors on Radiotherapy Outcome in Stage I-II Glottic Larynx Cancer-A Multicenter Study.

Background and Purpose: Larynx cancer represents one of the most frequently diagnosed head and neck malignancies, which is most often confined to the glottic area. The aim of this study was to report the oncological outcome and identify prognostic factors in early-stage glottic squamous cell carcinoma treated with radiotherapy. Material and Methods: Patients (n = 761) diagnosed and treated in 10 centers between 1990 and 2015 were retrospectively analyzed. Probabilities of loco-regional control (LRC) and overall survival (OS) were calculated and possible prognostic factors were analyzed using Cox proportional hazards models. Results: The median follow-up was 63 months (range: 2-243). Three hundred and sixty-four, 148 and 249 patients had cT1a, cT1b, and cT2 stage I-II disease, respectively. Five and 10-years LRC/OS rates in the whole cohort were 83/82% and 80/68%, respectively. Three patients developed distant recurrences. In univariate analysis, male sex (HR: 3.49; 95% CI: 1.47-11.37; p < 0.01), T2 vs. T1a (HR: 1.62; 95% CI: 1.08-2.43; p = 0.02) and anterior commissure involvement (ACI) (HR: 1.66; 95% CI: 1.38-2.45; p < 0.01) were associated with impaired LRC. In multivariate analysis, male sex (HR: 3.42; 95% CI: 1.44-11.17; p < 0.01) and ACI (HR: 1.51; 95% CI: 1.01-2.28; p = 0.047) remained poor prognostic factors. No relation of treatment technique and biologically equivalent dose (BED) to oncological outcome was identified except for higher BED10(L = 25; T = 1) yielding better LRC in T1a tumors (p = 0.04) in univariate analyses. Conclusion: Our results highlight the negative impact of ACI on tumor control. A less-expected finding was the impact of sex on tumor control. Further research is needed to validate its prognostic value and investigate any related biologic or behavioral factors, which may be modified to improve oncologic outcome.