The Effect of Surrounding Vegetation on Basal Stem Measurements Acquired Using Low-Cost Depth Sensors in Urban and Native Forest Environments.

Three colour and depth (RGB-D) devices were compared, to assess the effect of depth image misalignment, resulting from simultaneous localisation and mapping (SLAM) error, due to forest structure complexity. Urban parkland (S1) was used to assess stem density, and understory vegetation (≤1.3 m) was assessed in native woodland (S2). Individual stem and continuous capture approaches were used, with stem diameter at breast height (DBH) estimated. Misalignment was present within point clouds; however, no significant differences in DBH were observed for stems captured at S1 with either approach (Kinect p = 0.16; iPad p = 0.27; Zed p = 0.79). Using continuous capture, the iPad was the only RGB-D device to maintain SLAM in all S2 plots. There was significant correlation between DBH error and surrounding understory vegetation with the Kinect device (p = 0.04). Conversely, there was no significant relationship between DBH error and understory vegetation for the iPad (p = 0.55) and Zed (p = 0.86). The iPad had the lowest DBH root-mean-square error (RMSE) across both individual stem (RMSE = 2.16cm) and continuous (RMSE = 3.23cm) capture approaches. The results suggest that the assessed RGB-D devices are more capable of operation within complex forest environments than previous generations.

Reversal of viral and epigenetic HLA class I repression in Merkel cell carcinoma.

Cancers avoid immune surveillance through an array of mechanisms, including perturbation of HLA class I antigen presentation. Merkel cell carcinoma (MCC) is an aggressive, HLA-I-low, neuroendocrine carcinoma of the skin often caused by the Merkel cell polyomavirus (MCPyV). Through the characterization of 11 newly generated MCC patient-derived cell lines, we identified transcriptional suppression of several class I antigen presentation genes. To systematically identify regulators of HLA-I loss in MCC, we performed parallel, genome-scale, gain- and loss-of-function screens in a patient-derived MCPyV-positive cell line and identified MYCL and the non-canonical Polycomb repressive complex 1.1 (PRC1.1) as HLA-I repressors. We observed physical interaction of MYCL with the MCPyV small T viral antigen, supporting a mechanism of virally mediated HLA-I suppression. We further identify the PRC1.1 component USP7 as a pharmacologic target to restore HLA-I expression in MCC.

A highly multiplexed quantitative phosphosite assay for biology and preclinical studies.

Reliable methods to quantify dynamic signaling changes across diverse pathways are needed to better understand the effects of disease and drug treatment in cells and tissues but are presently lacking. Here, we present SigPath, a targeted mass spectrometry (MS) assay that measures 284 phosphosites in 200 phosphoproteins of biological interest. SigPath probes a broad swath of signaling biology with high throughput and quantitative precision. We applied the assay to investigate changes in phospho-signaling in drug-treated cancer cell lines, breast cancer preclinical models, and human medulloblastoma tumors. In addition to validating previous findings, SigPath detected and quantified a large number of differentially regulated phosphosites newly associated with disease models and human tumors at baseline or with drug perturbation. Our results highlight the potential of SigPath to monitor phosphoproteomic signaling events and to nominate mechanistic hypotheses regarding oncogenesis, response, and resistance to therapy.

The conserved translation factor LepA is required for optimal synthesis of a porin family in Mycobacterium smegmatis.

The recalcitrance of mycobacteria to antibiotic therapy is in part due to its ability to build proteins into a multi-layer cell wall. Proper synthesis of both cell wall constituents and associated proteins is crucial to maintaining cell integrity, and intimately tied to antibiotic susceptibility. How mycobacteria properly synthesize the membrane-associated proteome, however, remains poorly understood. Recently, we found that loss of lepA in Mycobacterium smegmatis (Msm) altered tolerance to rifampin, a drug that targets a non-ribosomal cellular process. LepA is a ribosome-associated GTPase found in bacteria, mitochondria, and chloroplasts, yet its physiological contribution to cellular processes is not clear. To uncover the determinants of LepA-mediated drug tolerance, we characterized the whole-cell proteomes and transcriptomes of a lepA deletion mutant relative to strains with lepA We find that LepA is important for the steady-state abundance of a number of membrane-associated proteins, including an outer membrane porin, MspA, which is integral to nutrient uptake and drug susceptibility. Loss of LepA leads to a decreased amount of porin in the membrane which leads to the drug tolerance phenotype of the lepA mutant. In mycobacteria, the translation factor LepA modulates mycobacterial membrane homeostasis, which in turn affects antibiotic tolerance.ImportanceThe mycobacterial cell wall is a promising target for new antibiotics due to the abundance of important membrane-associated proteins. Defining mechanisms of synthesis of the membrane proteome will be critical to uncovering and validating drug targets. We found that LepA, a universally conserved translation factor, controls the synthesis of a number of major membrane proteins in M. smegmatis LepA primarily controls synthesis of the major porin MspA. Loss of LepA results in decreased permeability through the loss of this porin, including permeability to antibiotics like rifampin and vancomycin. In mycobacteria, regulation from the ribosome is critical for the maintenance of membrane homeostasis and, importantly, antibiotic susceptibility.

Quantitative, multiplexed workflow for deep analysis of human blood plasma and biomarker discovery by mass spectrometry.

Proteomic characterization of blood plasma is of central importance to clinical proteomics and particularly to biomarker discovery studies. The vast dynamic range and high complexity of the plasma proteome have, however, proven to be serious challenges and have often led to unacceptable tradeoffs between depth of coverage and sample throughput. We present an optimized sample-processing pipeline for analysis of the human plasma proteome that provides greatly increased depth of detection, improved quantitative precision and much higher sample analysis throughput as compared with prior methods. The process includes abundant protein depletion, isobaric labeling at the peptide level for multiplexed relative quantification and ultra-high-performance liquid chromatography coupled to accurate-mass, high-resolution tandem mass spectrometry analysis of peptides fractionated off-line by basic pH reversed-phase (bRP) chromatography. The overall reproducibility of the process, including immunoaffinity depletion, is high, with a process replicate coefficient of variation (CV) of <12%. Using isobaric tags for relative and absolute quantitation (iTRAQ) 4-plex, >4,500 proteins are detected and quantified per patient sample on average, with two or more peptides per protein and starting from as little as 200 µl of plasma. The approach can be multiplexed up to 10-plex using tandem mass tags (TMT) reagents, further increasing throughput, albeit with some decrease in the number of proteins quantified. In addition, we provide a rapid protocol for analysis of nonfractionated depleted plasma samples analyzed in 10-plex. This provides ∼600 quantified proteins for each of the ten samples in ∼5 h of instrument time.

Investigating Surface and Near-Surface Bushfire Fuel Attributes: A Comparison between Visual Assessments and Image-Based Point Clouds.

Visual assessment, following guides such as the Overall Fuel Hazard Assessment Guide (OFHAG), is a common approach for assessing the structure and hazard of varying bushfire fuel layers. Visual assessments can be vulnerable to imprecision due to subjectivity between assessors, while emerging techniques such as image-based point clouds can offer land managers potentially more repeatable descriptions of fuel structure. This study compared the variability of estimates of surface and near-surface fuel attributes generated by eight assessment teams using the OFHAG and Fuels3D, a smartphone method utilising image-based point clouds, within three assessment plots in an Australian lowland forest. Surface fuel hazard scores derived from underpinning attributes were also assessed. Overall, this study found considerable variability between teams on most visually assessed variables, resulting in inconsistent hazard scores. Variability was observed within point cloud estimates but was, however, on average two to eight times less than that seen in visual estimates, indicating greater consistency and repeatability of this method. It is proposed that while variability within the Fuels3D method may be overcome through improved methods and equipment, inconsistencies in the OFHAG are likely due to the inherent subjectivity between assessors, which may be more difficult to overcome. This study demonstrates the capability of the Fuels3D method to efficiently and consistently collect data on fuel hazard and structure, and, as such, this method shows potential for use in fire management practices where accurate and reliable data is essential.