Clinical implementation of deep learning-based automated left breast simultaneous integrated boost radiotherapy treatment planning.

Automation in radiotherapy treatment planning aims to improve both the quality and the efficiency of the process. The aim of this study was to report on a clinical implementation of a Deep Learning (DL) auto-planning model for left-sided breast cancer. The DL model was developed for left-sided breast simultaneous integrated boost treatments under deep-inspiration breath-hold. Eighty manual dose distributions were revised and used for training. Ten patients were used for model validation. The model was then used to design 17 clinical auto-plans. Manual and auto-plans were scored on a list of clinical goals for both targets and organs-at-risk (OARs). For validation, predicted and mimicked dose (PD and MD, respectively) percent error (PE) was calculated with respect to manual dose. Clinical and validation cohorts were compared in terms of MD only. Median values of both PD and MD validation plans fulfilled the evaluation criteria. PE was < 1% for targets for both PD and MD. PD was well aligned to manual dose while MD left lung mean dose was significantly less (median:5.1 Gy vs 6.1 Gy). The left-anterior-descending artery maximum dose was found out of requirements (median values:+5.9 Gy and + 2.9 Gy, for PD and MD respectively) in three validation cases, while it was reduced for clinical cases (median:-1.9 Gy). No other clinically significant differences were observed between clinical and validation cohorts. Small OAR differences observed during the model validation were not found clinically relevant. The clinical implementation outcomes confirmed the robustness of the model

Label-free detection of exosomes using surface plasmon resonance biosensor

The development of a sensitive and specific detection platform for exosomes is highly desirable as they are believed to transmit vital tumour-specific information (mRNAs, microRNAs, and proteins) to remote cells for secondary metastasis. Herein, we report a simple method for the real-time and label-free detection of clinically relevant exosomes using a surface plasmon resonance (SPR) biosensor. Our method shows high specificity in detecting BT474 breast cancer cell-derived exosomes particularly from complex biological samples (e.g. exosome spiked in serum). This approach exhibits high sensitivity by detecting as low as 8280 exosomes/μL which may potentially be suitable for clinical analysis. We believe that this label-free and real-time method along with the high specificity and sensitivity may potentially be useful for clinical settings

Site-specific protein modification using immobilized sortase in batch and continuous-flow systems

Transpeptidation catalyzed by ​sortase A allows the preparation of proteins that are site-specifically and homogeneously modified with a wide variety of functional groups, such as fluorophores, PEG moieties, lipids, glycans, bio-orthogonal reactive groups and affinity handles. This protocol describes immobilization of ​sortase A on a solid support (Sepharose beads). Immobilization of ​sortase A simplifies downstream purification of a protein of interest after labeling of its N or C terminus. Smaller batch and larger-scale continuous-flow reactions require only a limited amount of enzyme. The immobilized enzyme can be reused for multiple cycles of protein modification reactions. The described protocol also works with a Ca²⁺-independent variant of ​sortase A with increased catalytic activity. This heptamutant variant of ​sortase A (7M) was generated by combining previously published mutations, and this immobilized enzyme can be used for the modification of calcium-senstive substrates or in instances in which low temperatures are needed. Preparation of immobilized ​sortase A takes 1–2 d. Batch reactions take 3–12 h and flow reactions proceed at 0.5 ml h⁻¹, depending on the geometry of the reactor used.United States. National Institutes of Health (RO1 AI087879

Global gene disruption in human cells to assign genes to phenotypes

Insertional mutagenesis in a haploid background can disrupt gene function[superscript 1]. We extend our earlier work by using a retroviral gene-trap vector to generate insertions in >98% of the genes expressed in a human cancer cell line that is haploid for all but one of its chromosomes. We apply phenotypic interrogation via tag sequencing (PhITSeq) to examine millions of mutant alleles through selection and parallel sequencing. Analysis of pools of cells, rather than individual clones[superscript 1] enables rapid assessment of the spectrum of genes involved in the phenotypes under study. This facilitates comparative screens as illustrated here for the family of cytolethal distending toxins (CDTs). CDTs are virulence factors secreted by a variety of pathogenic Gram-negative bacteria responsible for tissue damage at distinct anatomical sites[superscript 2]. We identify 743 mutations distributed over 12 human genes important for intoxication by four different CDTs. Although related CDTs may share host factors, they also exploit unique host factors to yield a profile characteristic for each CDT

The Cerebral Microvasculature in Schizophrenia: A Laser Capture Microdissection Study

BACKGROUND: Previous studies of brain and peripheral tissues in schizophrenia patients have indicated impaired energy supply to the brain. A number of studies have also demonstrated dysfunction of the microvasculature in schizophrenia patients. Together these findings are consistent with a hypothesis of blood-brain barrier dysfunction in schizophrenia. In this study, we have investigated the cerebral vascular endothelium of schizophrenia patients at the level of transcriptomics. METHODOLOGY/PRINCIPAL FINDINGS: We used laser capture microdissection to isolate both microvascular endothelial cells and neurons from post mortem brain tissue from schizophrenia patients and healthy controls. RNA was isolated from these cell populations, amplified, and analysed using two independent microarray platforms, Affymetrix HG133plus2.0 GeneChips and CodeLink Whole Human Genome arrays. In the first instance, we used the dataset to compare the neuronal and endothelial data, in order to demonstrate that the predicted differences between cell types could be detected using this methodology. We then compared neuronal and endothelial data separately between schizophrenic subjects and controls. Analysis of the endothelial samples showed differences in gene expression between schizophrenics and controls which were reproducible in a second microarray platform. Functional profiling revealed that these changes were primarily found in genes relating to inflammatory processes. CONCLUSIONS/SIGNIFICANCE: This study provides preliminary evidence of molecular alterations of the cerebral microvasculature in schizophrenia patients, suggestive of a hypo-inflammatory state in this tissue type. Further investigation of the blood-brain barrier in schizophrenia is warranted

Natural regeneration and biodiversity: a global meta-analysis and implications for spatial planning

Natural regeneration offers a cheaper alternative to active reforestation and has the potential to become the predominant way of restoring degraded tropical landscapes at large-scale. We conducted a meta-analysis for tropical regions and quantified the relationships between both ecological and socioeconomic factors and biodiversity responses in naturally regenerating areas. Biogeographic realms, past disturbance, and the human development index (HDI) were used as explanatory variables for biodiversity responses. In addition, we present a case study of large-scale natural regeneration in the Brazilian Atlantic Forest and identify areas where different forms of restoration would be most suitable. Using our dataset for tropical regions, we showed that natural regeneration was predominantly reported within: the Neotropical realm; areas that were intensively disturbed; and countries with medium HDI. We also found that biodiversity in regenerating forests was more similar to the values found in old growth forests in: countries with either low, high, or very high HDI; less biodiverse realms; and areas of less intensive past disturbance. Our case study from Brazil showed that the level of forest gain resulting from environmental legislation, in particular the Brazilian Forest Code, has been reduced, but remains substantial. Complementary market incentives and financial mechanisms to promote large-scale natural regeneration in human-modified agricultural landscapes are also needed. Our analysis provides insights into the factors that promote or limit the recovery of biodiversity in naturally regenerating areas, and aids to identify areas with higher potential for natural regeneration