Type VI secretion system is widely used in Gram-negative bacteria for injecting toxic effectors into neighboring prokaryotic or eukaryotic cells. Various effectors can be loaded onto the T6SS ...delivery tube via its core components: Hcp, VgrG, or PAAR. Here, we report 2.8-Å resolution cryo-EM structure of intact T6SS Hcp5-VgrG-PAAR cargo delivery system and crystal structure of unbound Hcp5 from B. fragilis NCTC 9343. Loading of Hcp5 hexameric ring onto VgrG causes expansion of its inner cavity and external surface, explaining how structural changes could be propagated to regulate co-polymerization and surrounding contractile sheath. High-affinity binding between Hcp and VgrG causes entropically unfavorable structuring of long loops. Furthermore, interactions between VgrG trimer and Hcp hexamer are asymmetric, with three of the six Hcp monomers exhibiting a major loop flip. Our study provides insights into the assembly, loading, and firing of T6SS nanomachine that contributes to bacterial inter-species competition and host interactions.
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•Cryo-EM structure of T6SS effector cargo delivery vehicle Hcp-VgrG-PAAR is resolved at 2.8 Å•Loading of Hcp hexamer to VgrG-PAAR causes expansion of the ring and conformational change•VgrG-Hcp interaction is asymmetric with three of Hcp monomers exhibiting a major loop flip
He et al. elucidate the assembly and loading mechanism of the intact T6SS cargo delivery vehicle with cryo-EM structure of Hcp-VgrG-PAAR complex at 2.8 Å and unbound Hcp crystal structure. The loading of Hcp hexamer onto trimer VgrG causes expansion of the inner cavity in an asymmetric way with loop flipping.
Despite advances in treatment approaches for head and neck squamous cell carcinoma (HNSCC), survival rates have remained stagnant due to the paucity of preclinical models that accurately reflect the ...human tumor. Patient-derived xenografts (PDX) are an emerging model system where patient tumors are implanted directly into mice. Increased understanding of the application and limitations of PDXs will facilitate their rational use. Studies to date have not reported protein profiles of PDXs. Therefore, we developed a large cohort of HNSCC PDXs and found that tumor take rate was not influenced by the clinical, pathologic, or processing features. Protein expression profiles, from a subset of the PDXs, were characterized by reverse-phase protein array and the data was compared with The Cancer Genome Atlas HNSCC data. Cluster analysis revealed that HNSCC PDXs were more similar to primary HNSCC than to any other tumor type. Interestingly, while a significant fraction of proteins were expressed similarly in both primary HNSCC and PDXs, a subset of proteins/phosphoproteins were expressed at higher (or lower) levels in PDXs compared with primary HNSCC. These findings indicate that the proteome is generally conserved in PDXs, but mechanisms for both positive and negative model selection and/or differences in the stromal components exist.
Proteomic characterization of HNSCC PDXs demonstrates potential drivers for model selection and provides a framework for improved utilization of this expanding model system.
In endometrioid endometrial cancer (EEC), current clinical algorithms do not accurately predict patients with lymph node metastasis (LNM), leading to both under- and over-treatment. We aimed to ...develop models that integrate protein data with clinical information to identify patients requiring more aggressive surgery, including lymphadenectomy.
Protein expression profiles were generated for 399 patients using reverse-phase protein array. Three generalised linear models were built on proteins and clinical information (model 1), also with magnetic resonance imaging included (model 2), and on proteins only (model 3), using a training set, and tested in independent sets. Gene expression data from the tumours were used for confirmatory testing.
LNM was predicted with area under the curve 0.72-0.89 and cyclin D1; fibronectin and grade were identified as important markers. High levels of fibronectin and cyclin D1 were associated with poor survival (p = 0.018), and with markers of tumour aggressiveness. Upregulation of both FN1 and CCND1 messenger RNA was related to cancer invasion and mesenchymal phenotype.
We demonstrate that data-driven prediction models, adding protein markers to clinical information, have potential to significantly improve preoperative identification of patients with LNM in EEC.
Protein levels and function are poorly predicted by genomic and transcriptomic analysis of patient tumours. Therefore, direct study of the functional proteome has the potential to provide a wealth of ...information that complements and extends genomic, epigenomic and transcriptomic analysis in The Cancer Genome Atlas (TCGA) projects. Here we use reverse-phase protein arrays to analyse 3,467 patient samples from 11 TCGA 'Pan-Cancer' diseases, using 181 high-quality antibodies that target 128 total proteins and 53 post-translationally modified proteins. The resultant proteomic data are integrated with genomic and transcriptomic analyses of the same samples to identify commonalities, differences, emergent pathways and network biology within and across tumour lineages. In addition, tissue-specific signals are reduced computationally to enhance biomarker and target discovery spanning multiple tumour lineages. This integrative analysis, with an emphasis on pathways and potentially actionable proteins, provides a framework for determining the prognostic, predictive and therapeutic relevance of the functional proteome.
Cancer cell lines are major model systems for mechanistic investigation and drug development. However, protein expression data linked to high-quality DNA, RNA, and drug-screening data have not been ...available across a large number of cancer cell lines. Using reverse-phase protein arrays, we measured expression levels of ∼230 key cancer-related proteins in >650 independent cell lines, many of which have publically available genomic, transcriptomic, and drug-screening data. Our dataset recapitulates the effects of mutated pathways on protein expression observed in patient samples, and demonstrates that proteins and particularly phosphoproteins provide information for predicting drug sensitivity that is not available from the corresponding mRNAs. We also developed a user-friendly bioinformatic resource, MCLP, to help serve the biomedical research community.
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•Targeted protein expression of >650 cancer cell lines were characterized•Cell line data recapitulate mutation effects on protein expression in patients•RPPA-based protein markers are powerful predictors of drug sensitivity•A data portal enables exploring proteomics and related molecular/drug data
Li et al. analyze the levels of cancer-related total and phosphorylated proteins using RPPA in a large set of human cancer cell lines, many also with DNA, RNA, and drug-screening data available. These proteins can recapitulate mutational patterns seen in patients and show ability to predict drug sensitivity.
There is a growing body of evidence demonstrating that tree survival is influenced by negative density-dependence, but it is still controversial how the effect may vary with life-stage, and to what ...extent it plays a role in regulating tree survival in heterogeneous subtropical forests. In this study, we investigated density-dependent effects on tree survival of six tree species in a 5-ha subtropical forest in eastern China. The roughly 45 000 individuals in the forest were fully censused in 2003 and 2008. For each of these species, we used an inhomogeneous pair-correlation function to quantify the change in spatial distribution for different size classes, and a case-control design to study seedling—adult associations in 2003. Autologistic regression was used to determine the influence of neighborhood factors on individual survival from 2003 to 2008. We found that seedlings of five species were repulsed by distance to nearest conspecific adults in terms of their survival, consistent with predictions of the Janzen—Connell mechanism. By contrast, only the least shade-tolerant Schima superba had a negative relationship with individual survival and conspecific distance-weighted basal area. This suggests that the Janzen—Connell effect is only prevalent at the early seedling stage or seed-to-seedling phase. The strength of clustering significantly declined at sapling—pole and pole—adult transitions for Sycopsis sinensis and at seedling—sapling transition for Cleyera pachyphylla. Correlations between individual survival and conspecific abundance for these species were consistent with trends in the strength of clustering. These results suggest that density dependence plays a limited role in individual survival and species spatial structure beyond the early seedling stage (i.e. after true leaves growing) in this forest. In addition, this study indicates that including individuals from early life-stages and factoring out potential confounding factors such as habitat preference are important in studies that seek evidence for density dependence in forest trees.
Schematic diagram of Database-assisted workflow to prescreen scFv from hybridomas and the development of one-step CLEIA based on scFv-AP.
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•Database-assisted workflow to prescreen scFv ...from hybridomas was presented.•The rearrangement between functional and the aberrant light chain was identified.•Characterization of scFv candidates was conducted by one-step ELISA.•A sensitive competitive CLEIA was developed based on scFv-AP fusion protein.•The developed CLEIA was applied for determination of total MG in tilapia fish.
To determine malachite green (MG) and its major metabolite, leucomalachite green (LMG) residual levels in tilapia fish, chemiluminescent enzyme immunoassay (CLEIA) was developed based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein. At first, VH and VL gene sequences were cloned from hybridoma cell lines secreting monoclonal antibody against LMG, and then thoroughly by database-assisted sequence analysis. Finally, the productive VH and VL were assembled to an intact scFv sequence and engineered to produce scFv-AP fusion protein. The fusion protein was further identified as a bifunctional reagent for immunoassay, then a sensitive one-step CLEIA against LMG was developed with a half-maximal inhibitory concentration (IC50) and limit of detection (LOD) of 1.3 and 0.04 ng/mL, respectively. The validation results of this novel competitive CLEIA was in line with those obtained by classical HPLC method for determination of total MG in spiked and field incurred samples.
Neuropeptide Y (NPY) receptors belong to the G-protein-coupled receptor superfamily and have important roles in food intake, anxiety and cancer biology
. The NPY-Y receptor system has emerged as one ...of the most complex networks with three peptide ligands (NPY, peptide YY and pancreatic polypeptide) binding to four receptors in most mammals, namely the Y
, Y
, Y
and Y
receptors, with different affinity and selectivity
. NPY is the most powerful stimulant of food intake and this effect is primarily mediated by the Y
receptor (Y
R)
. A number of peptides and small-molecule compounds have been characterized as Y
R antagonists and have shown clinical potential in the treatment of obesity
, tumour
and bone loss
. However, their clinical usage has been hampered by low potency and selectivity, poor brain penetration ability or lack of oral bioavailability
. Here we report crystal structures of the human Y
R bound to the two selective antagonists UR-MK299 and BMS-193885 at 2.7 and 3.0 Å resolution, respectively. The structures combined with mutagenesis studies reveal the binding modes of Y
R to several structurally diverse antagonists and the determinants of ligand selectivity. The Y
R structure and molecular docking of the endogenous agonist NPY, together with nuclear magnetic resonance, photo-crosslinking and functional studies, provide insights into the binding behaviour of the agonist and for the first time, to our knowledge, determine the interaction of its N terminus with the receptor. These insights into Y
R can enable structure-based drug discovery that targets NPY receptors.
Small-cell lung cancer (SCLC) is the most aggressive and lethal subtype of lung cancer, for which, better understandings of its biology are urgently needed. Single-cell sequencing technologies ...provide an opportunity to profile individual cells within the tumor microenvironment (TME) and investigate their roles in tumorigenic processes. Here, we performed high-precision single-cell transcriptomic analysis of ~5000 individual cells from primary tumors (PTs) and matched normal adjacent tissues (NATs) from 11 SCLC patients, including one patient with both PT and relapsed tumor (RT). The comparison revealed an immunosuppressive landscape of human SCLC. Malignant cells in SCLC tumors exhibited diverse states mainly related to the cell cycle, immune, and hypoxic properties. Our data also revealed the intratumor heterogeneity (ITH) of key transcription factors (TFs) in SCLC and related gene expression patterns and functions. The non-neuroendocrine (non-NE) tumors were correlated with increased inflammatory gene signatures and immune cell infiltrates in SCLC, which contributed to better responses to immune checkpoint inhibitors. These findings indicate a significant heterogeneity of human SCLC, and intensive crosstalk between cancer cells and the TME at single-cell resolution, and thus, set the stage for a better understanding of the biology of SCLC as well as for developing new therapeutics for SCLC.
Methane is a major contributor to anthropogenic greenhouse gas emissions. Identifying large sources of methane, particularly from the oil and gas sectors, will be essential for mitigating climate ...change. Aircraft-based methane sensing platforms can rapidly detect and quantify methane point-source emissions across large geographic regions, and play an increasingly important role in industrial methane management and greenhouse gas inventory. We independently evaluate the performance of five major methane-sensing aircraft platforms: Carbon Mapper, GHGSat-AV, Insight M, MethaneAIR, and Scientific Aviation. Over a 6 week period, we released metered gas for over 700 single-blind measurements across all five platforms to evaluate their ability to detect and quantify emissions that range from 1 to over 1,500 kg(CH4)/h. Aircraft consistently quantified releases above 10 kg(CH4)/h, and GHGSat-AV and Insight M detected emissions below 5 kg(CH4)/h. Fully blinded quantification estimates for platforms using downward-facing imaging spectrometers have parity slopes ranging from 0.76 to 1.13, with R 2 values of 0.61 to 0.93; the platform using continuous air sampling has a parity slope of 0.5 (R 2 = 0.93). Results demonstrate that aircraft-based methane sensing has matured since previous studies and is ready for an increasingly important role in environmental policy and regulation.
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