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Background Recent efforts have described the evolution of glioblastoma from initial diagnosis to post-treatment recurrence on a genomic and transcriptomic level. However, the evolution of the proteomic landscape is largely unknown. Methods Sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) was used to characterize the quantitative proteomes of two independent cohorts of paired newly diagnosed and recurrent glioblastomas. Recurrence-associated proteins were validated using immunohistochemistry and further studied in human glioma cell lines, orthotopic xenograft models, and human organotypic brain slice cultures. External spatial transcriptomic, single-cell, and bulk RNA sequencing data were analyzed to gain mechanistic insights. Results Although overall proteomic changes were heterogeneous across patients, we identified BCAS1, INF2, and FBXO2 as consistently upregulated proteins at recurrence and validated these using immunohistochemistry. Knockout of FBXO2 in human glioma cells conferred a strong survival benefit in orthotopic xenograft mouse models and reduced invasive growth in organotypic brain slice cultures. In glioblastoma patient samples, FBXO2 expression was enriched in the tumor infiltration zone and FBXO2-positive cancer cells were associated with synaptic signaling processes. Conclusions These findings demonstrate a potential role of FBXO2-dependent glioma-microenvironment interactions to promote tumor growth. Furthermore, the published datasets provide a valuable resource for further studies.

Complex traits are characterized by multiple genes and variants acting simultaneously on a phenotype. However, studying the contribution of individual pairs of genes to complex traits has been challenging since human genetics necessitates very large population sizes, while findings from model systems do not always translate to humans. Here, we combine genetics with combinatorial RNAi (coRNAi) to systematically test for pairwise additive effects (AEs) and genetic interactions (GIs) between 30 lipid genome-wide association studies (GWAS) genes. Gene-based burden tests from 240,970 exomes show that in carriers with truncating mutations in both, APOB and either PCSK9 or LPL (“human double knock-outs”) plasma lipid levels change additively. Genetics and coRNAi identify overlapping AEs for 12 additional gene pairs. Overlapping GIs are observed for TOMM40/APOE with SORT1 and NCAN. Our study identifies distinct gene pairs that modulate plasma and cellular lipid levels primarily via AEs and nominates putative drug target pairs for improved lipid-lowering combination therapies., Studying the contribution of pairs of genes to complex traits has been challenging. Here, the authors combine exome and genotype data with RNAi to screen for genetic interactions between 30 genes identified in lipid GWAS to hint at pairs whose joint modulation may improve lipid-lowering therapies.

Formalin-fixed, paraffin-embedded (FFPE), biobanked tissue samples offer an invaluable resource for clinical and biomarker research. Here we developed a pressure cycling technology (PCT)-SWATH mass spectrometry workflow to analyze FFPE tissue proteomes and applied it to the stratification of prostate cancer (PCa) and diffuse large B-cell lymphoma (DLBCL) samples. We show that the proteome patterns of FFPE PCa tissue samples and their analogous fresh frozen (FF) counterparts have a high degree of similarity and we confirmed multiple proteins consistently regulated in PCa tissues in an independent sample cohort. We further demonstrate temporal stability of proteome patterns from FFPE samples that were stored between one to 15 years in a biobank and show a high degree of the proteome pattern similarity between two types histological region of small FFPE samples, i.e. punched tissue biopsies and thin tissue sections of micrometer thickness, despite the existence of certain degree of biological variations. Applying the method to two independent DLBCL cohorts we identified myeloperoxidase (MPO), a peroxidase enzyme, as a novel prognostic marker. In summary, this study presents a robust proteomic method to analyze bulk and biopsy FFPE tissues and reports the first systematic comparison of proteome maps generated from FFPE and FF samples. Our data demonstrate the practicality and superiority of FFPE over FF samples for proteome in biomarker discovery. Promising biomarker candidates for PCa and DLBCL have been discovered.

Summary Deciphering how TCR signals are modulated by coinhibitory receptors is of fundamental and clinical interest. Using quantitative interactomics, we define the composition and dynamics of the PD-1 and BTLA coinhibitory signalosomes in primary effector T cells and at the T cell-antigen-presenting cell interface. We also solve the existing controversy regarding the role of the SHP-1 and SHP-2 protein-tyrosine phosphatases in mediating PD-1 coinhibition. PD-1 predominantly recruits SHP-2, but when absent, it recruits SHP-1 and remains functional. In contrast, BTLA predominantly recruits SHP-1 and to a lesser extent SHP-2. By separately analyzing the PD-1-SHP-1 and PD-1-SHP-2 complexes, we show that both dampen the TCR and CD28 signaling pathways equally. Therefore, our study illustrates how comparison of coinhibitory receptor signaling via quantitative interactomics in primary T cells unveils their extent of redundancy and provides a rationale for designing combinations of blocking antibodies in cancer immunotherapy on the basis of undisputed modes of action., Graphical Abstract, Highlights • Proteomic profiling of PD-1 and BTLA signalosome assembly in primary T cells • PD-1 and BTLA show marked quantitative difference in SHP-1 and SHP-2 use • PD-1 predominantly recruits SHP-2, which can be replaced by SHP-1 when absent • PD-1 bound to SHP-2 or SHP-1 inhibits both the TCR and CD28 signaling pathways, The respective contributions of the SHP-1 and SHP-2 protein-tyrosine phosphatases to PD-1 coinhibition remain debated. Using quantitative interactomics, Celis-Gutierrez et al. define the composition of the PD-1 signalosomes in primary T cells. Deconstructing it into PD-1-SHP-2 and PD-1-SHP-1 complexes showed that they target the TCR and CD28 pathways equally.

SUMMARYGenetic interactions (GIs), the joint impact of different genes or variants on a phenotype, are foundational to the genetic architecture of complex traits. However, identifying GIs through human genetics is challenging since it necessitates very large population sizes, while findings from model systems not always translate to humans. Here, we combined exome-sequencing and genotyping in the UK Biobank with combinatorial RNA-interference (coRNAi) screening to systematically test for pairwise GIs between 30 lipid GWAS genes. Gene-based protein-truncating variant (PTV) burden analyses from 240,970 exomes revealed additive GIs for APOB with PCSK9 and LPL, respectively. Both, genetics and coRNAi identified additive GIs for 12 additional gene pairs. Overlapping non-additive GIs were detected only for TOMM40 at the APOE locus with SORT1 and NCAN. Our study identifies distinct gene pairs that modulate both, plasma and cellular lipid levels via additive and non-additive effects and nominates drug target pairs for improved lipid-lowering combination therapies.

Summary Cancer is mostly incurable when diagnosed at a metastatic stage, making its early detection via blood proteins of immense clinical interest. Proteomic changes in tumor tissue may lead to changes detectable in the protein composition of circulating blood plasma. Using a proteomic workflow combining N-glycosite enrichment and SWATH mass spectrometry, we generate a data resource of 284 blood samples derived from patients with different types of localized-stage carcinomas and from matched controls. We observe whether the changes in the patient's plasma are specific to a particular carcinoma or represent a generic signature of proteins modified uniformly in a common, systemic response to many cancers. A quantitative comparison of the resulting N-glycosite profiles discovers that proteins related to blood platelets are common to several cancers (e.g., THBS1), whereas others are highly cancer-type specific. Available proteomics data, including a SWATH library to study N-glycoproteins, will facilitate follow-up biomarker research into early cancer detection.

Predicting how a system behaves under changing conditions is an essential component of science and engineering. The ability to make accurate predictions about the system indicates that it is well understood and provides the opportunity to simulate the response to conditions that would be empirically difficult or impossible to test. In the life sciences, the term systems biology was introduced to articulate the notion that the molecular and phenotypic response of a cell or organism to perturbations is the result of interplay of a multitude of molecules. The ability to predict the behavior of such complex molecular systems remains challenging and inevitably requires the involvement of different types of models and data that support them. In this article, we discuss a range of data-driven models that have proven particularly useful for predicting the behavior of biological systems at different levels of complexity and the matching data generation methods that support them. We specifically focus on predictions based on protein or proteome data generated by mass spectrometry. We describe three case studies that represent frequently encountered situations in systems biology.

Sequential window acquisition of all theoretical mass spectra (SWATH-MS) requires a spectral library to extract quantitative measurements from the mass spectrometry data acquired in data-independent acquisition mode (DIA). Large combined spectral libraries containing SWATH assays have been generated for humans and several other organisms, but so far no publicly available library exists for measuring the proteome of zebrafish, a rapidly emerging model system in biomedical research. Here, we present a large zebrafish SWATH spectral library to measure the abundance of 104,185 proteotypic peptides from 10,405 proteins. The library includes proteins expressed in 9 different zebrafish tissues (brain, eye, heart, intestine, liver, muscle, ovary, spleen, and testis) and provides an important new resource to quantify 40% of the protein-coding zebrafish genes. We employ this resource to quantify the proteome across brain, muscle, and liver and characterize divergent expression levels of paralogous proteins in different tissues. Data are available via ProteomeXchange (PXD010876, PXD010869) and SWATHAtlas (PASS01237)., Scientific Data, 6, ISSN:2052-4463

Fish species display huge differences in physical activity ranging from lethargy to migration of thousands of miles, making them an interesting model to identify determinants of physical fitness. Here, we show a remarkable plasticity of zebrafish in response to exercise and induction of PGC1α (encoded byPPARGC1A), a dominant regulator of mitochondrial biogenesis. Forced expression of humanPPARGC1Ainduces mitochondrial biogenesis, an exercise-like gene expression signature, and physical fitness comparable to wild-type animals trained in counter-current swim tunnels. Quantifying transcriptional and proteomic changes in response to exercise or PGC1α, we identify conserved ‘exercise’ adaptations, including a stoichiometric induction of the electron transport chain (ETC) that re-organizes into respiratory supercomplexes in both conditions. We further show that ndufa4/ndufa4l, previously assigned to complex I, associates to free and supramolecular complex IVin vivo. Thus, zebrafish is a useful and experimentally tractable vertebrate model to study exercise biology, including ETC expression and assembly.HIGHLIGHTSPGC1α reprograms zebrafish skeletal muscle to a ‘red fiber’ phenotype and increases exercise performanceZebrafish show a high molecular plasticity in response to PGC1α and exerciseSWATH-MS proteomics show a stoichiometric induction of the electron transport chain that organizes as supercomplexes in response to PGC1α and exercisendufa4/ndufa4l associate to free and supramolecular complex IVin vivo

The ability to reliably and reproducibly measure any protein of the human proteome in any tissue or cell type would be transformative for understanding systems-level properties as well as specific pathways in physiology and disease. Here, we describe the generation and verification of a compendium of highly specific assays that enable quantification of 99.7% of the 20,277 annotated human proteins by the widely accessible, sensitive, and robust targeted mass spectrometric method selected reaction monitoring, SRM. This human SRMAtlas provides definitive coordinates that conclusively identify the respective peptide in biological samples. We report data on 166,174 proteotypic peptides providing multiple, independent assays to quantify any human protein and numerous spliced variants, non-synonymous mutations, and post-translational modifications. The data are freely accessible as a resource at http://www.srmatlas.org/, and we demonstrate its utility by examining the network response to inhibition of cholesterol synthesis in liver cells and to docetaxel in prostate cancer lines.

Cancer genome sequencing has shown that driver genes can often be distinguished not only by the elevated mutation frequency but also by specific nucleotide positions that accumulate changes at a high rate. However, properties associated with a residue9s potential to drive tumorigenesis when mutated have not yet been systematically investigated. Here, using a novel methodological approach, we identify and characterize a compendium of 180 hotspot residues within 160 human proteins which occur with a significant frequency and are likely to have functionally relevant impact. We find that such mutations (i) are more prominent in proteins that can exist in the on and off state, (ii) reflect the identity of a tumor of origin, and (iii) often localize within interfaces which mediate interactions with other proteins or ligands. Following, we further examine structural data for human protein complexes and identify a number of additional protein interfaces that accumulate cancer mutations at a high rate. Jointly, these analyses suggest that disruption and dysregulation of protein interactions can be instrumental in switching functions of cancer proteins and activating downstream changes.

Summary In individuals, heterogeneous drug-response phenotypes result from a complex interplay of dose, drug specificity, genetic background, and environmental factors, thus challenging our understanding of the underlying processes and optimal use of drugs in the clinical setting. Here, we use mass-spectrometry-based quantification of molecular response phenotypes and logic modeling to explain drug-response differences in a panel of cell lines. We apply this approach to cellular cholesterol regulation, a biological process with high clinical relevance. From the quantified molecular phenotypes elicited by various targeted pharmacologic or genetic treatments, we generated cell-line-specific models that quantified the processes beneath the idiotypic intracellular drug responses. The models revealed that, in addition to drug uptake and metabolism, further cellular processes displayed significant pharmacodynamic response variability between the cell lines, resulting in cell-line-specific drug-response phenotypes. This study demonstrates the importance of integrating different types of quantitative systems-level molecular measurements with modeling to understand the effect of pharmacological perturbations on complex biological processes., Graphical Abstract, Highlights • Mass spectrometry-based quantification of multi-level cellular response to drugs • Generation of a large multi-omics dataset from different perturbed cell lines • Capturing the cell-type-specific responses to drugs with mathematical models • Identification of pharmacodynamic variability as major determinant of drug response, Blattmann et al. combine proteomic and metabolomic profiling with mechanistic modeling to characterize the complex processes underlying the heterogeneous drug response in cellular cholesterol regulation across a panel of different human cell lines.

Mass spectrometry-based proteomics has been contributing greatly to systems biology studies and its role in systems biology research is likely to grow in the next years. Its unique capability of quantifying proteins and protein modifications at large scale and throughput make it a prime technology in systems biology studies, specifically those focused on complex biological processes and diseases. In this article we first introduce the motivation and challenges of using proteomics in systems biology studies. Second, we give an overview of the most common methods to measure and quantify proteins using liquid-chromatography coupled tandem mass spectrometry. In the third section we highlight how mass spectrometry-based proteomics contributes to conceptually different approaches in studying the proteome (i.e., mapping the proteome, mapping the interactions, and relating the proteome to the genome), and then we focus on three exemplary biological processes to show how mass spectrometry based studies could increase our understanding of them.

Expanded proteomic analysis of metabolism Combined analysis of large data sets characterizing genes, transcripts, and proteins can elucidate biological functions and disease processes. Williams et al. report an exceptionally detailed characterization of mitochondrial function in a genetic reference panel of recombinant inbred mice. They measured the metabolic function of nearly 400 mice under various environmental conditions and collected detailed quantitative information from livers of the animals on over 25,000 transcripts. These data were integrated with quantitation of over 2500 proteins and nearly 1000 metabolites. Such analysis showed a frequent lack of correlation of transcript and protein abundance, enabled the identification of genomic variants of mitochondrial enzymes that caused inborn errors in metabolism, and revealed two genes that appear to function in cholesterol metabolism. Science , this issue p. 10.1126/science.aad0189

Indirect evidence suggests that water supply to fleshy fruits during the final stages of development occurs through the phloem, with the xylem providing little water, or acting as a pathway for water loss back to the plant. This inference was tested by examining the water balance and vascular functioning of ripening kiwifruit berries (Actinidia chinensis var. chinensis 'Hort16A') exhibiting a pre-harvest 'shrivel' disorder in California, and normal development in New Zealand. Dye labelling and mass balance experiments indicated that the xylem and phloem were both functional and contributed approximately equally to the fruit water supply during this stage of development. The modelled fruit water balance was dominated by transpiration, with net water loss under high vapour pressure deficit (D(a)) conditions in California, but a net gain under cooler New Zealand conditions. Direct measurement of pedicel sap flow under controlled conditions confirmed inward flows in both the phloem and xylem under conditions of both low and high D(a). Phloem flows were required for growth, with gradual recovery after a step increase in D(a). Xylem flows alone were unable to support growth, but did supply transpiration and were responsive to D(a)-induced pressure fluctuations. The results suggest that the shrivel disorder was a consequence of a high fruit transpiration rate, and that the perception of complete loss or reversal of inward xylem flows in ripening fruits should be re-examined.

Gold kiwifruit (Actinidia chinensis Planch. var. chinensis ‘Hort16A’) is an important crop for New Zealand. In this trial, all nutrients, except nitrogen (N), were applied at levels comparable to commercial practice. The control treatment received approximately 145 kg N/ha/y, a conservative rate of N application for kiwifruit. The zero-N treatment vines received all nutrients except N, and the high-N treatment received double the control levels of N. Carbohydrate concentrations in the leaves, fruit and canes of zero-N and high-N vines were determined periodically through the season for two years. There were lower total sugar concentrations in the leaves of the zero-N vines however total carbohydrate concentration content was higher in the zero-N fruit. Vegetative vigor was reduced in the zero-N vines when compared to the high-N vines. By reducing N, the partitioning of carbon to fruit appears favored. This effect may be due to modified sink strength under reduced N.

Gold kiwifruit (Actinidia chinensis Planch. var. chinensis ‘HORT16A’, marketed as ZESPRI™' ‘HORT16A’) is an important export crop for New Zealand. Nutrient management, including the application of nitrogen (N), for ‘HORT16A’ kiwifruit has mimicked strategies for the green (A. deliciosa ‘Hayward’) cultivar, however, physiological differences between Actinidia species necessitate the development of specific nutrient regimes for ‘HORT16A’ vines. In this trial, all nutrients, except N, were applied at levels comparable to commercial practice. The Control treatment received approximately 145 kg N ha/y, and was based on estimates of total N removed each year in the harvested fruit. The Zero-N treatment vines received all nutrients except N, and the High-N treatment received double the Control, approximately 295 kg N ha/y, for two consecutive seasons. The High-N treatment represents the upper limit of N application practices in commercial ‘HORT16A’ kiwifruit production. Zero-N fruit had higher calcium (...

Root pressure was measured continuously over spring in eight clonal kiwifruit rootstocks selected from seven Actinidia species (A. chrysantha, A. deliciosa, A. eriantha, A. hemsleyana, A. kolomikta, A. macrosperma, A. polygama), using pressure transducers and miniature compression fittings. Rootstocks that promoted scion vigour developed root pressures up to 0.15 MPa before or during scion budburst, whereas those that reduced scion vigour developed root pressure up to 0.05 MPa only after scion shoot expansion. When several seasons were compared, the date of onset of root pressure and the magnitude of pressure achieved were consistent for each rootstock. Root pressure was first recorded between late July and early September in vigour-promoting rootstocks, while scion budburst and initial shoot growth were in late August and early September. Vigour-reducing rootstocks did not develop significant root pressure until October. The date of onset was similar for the grafted rootstock and ungrafted plant of the same clone, but was not clearly related to the timing of shoot growth by the ungrafted plant. In the grafted plants the leaf and xylem water potentials of the scion were more negative, midday turgor was 0.3-0.5 MPa lower, and wilting was sometimes observed in developing shoots growing on low-vigour rootstocks, indicating that water stress was contributing to reductions in growth. Leaf turgor was correlated with average root pressure but not pressure measured during the day, suggesting that root pressure was not supporting transpiration during peak flows and was, instead, indicative of higher root hydraulic conductance. The rapid temporal rise in root pressure observed each spring in the various rootstocks was not accompanied by changes in xylem sap solute potential, but when rootstock clones were compared those that developed higher root pressures had higher sap solute potentials. Xylem sap solute potential varied between rootstocks from -0.07 MPa to -0.15 MPa, while root pressures measured at the same time varied between 0.0 MPa and 0.09 MPa, suggesting that an osmotic mechanism could account for the observed root pressure. Differences in phenology between the rootstocks and scion appeared to account for the rootstock effects on shoot growth, and changes in root pressure provided a useful indication of seasonal changes in root hydraulic properties and solute transport behaviour.

Genome-wide association studies (GWAS) are powerful tools to unravel genomic loci associated with common traits and complex human disease. However, GWAS only rarely reveal information on the exact genetic elements and pathogenic events underlying an association. In order to extract functional information from genomic data, strategies for systematic follow-up studies on a phenotypic level are required. Here we address these limitations by applying RNA interference (RNAi) to analyze 133 candidate genes within 56 loci identified by GWAS as associated with blood lipid levels, coronary artery disease, and/or myocardial infarction for a function in regulating cholesterol levels in cells. Knockdown of a surprisingly high number (41%) of trait-associated genes affected low-density lipoprotein (LDL) internalization and/or cellular levels of free cholesterol. Our data further show that individual GWAS loci may contain more than one gene with cholesterol-regulatory functions. Using a set of secondary assays we demonstrate for a number of genes without previously known lipid-regulatory roles (e.g. CXCL12, FAM174A, PAFAH1B1, SEZ6L, TBL2, WDR12) that knockdown correlates with altered LDL–receptor levels and/or that overexpression as GFP–tagged fusion proteins inversely modifies cellular cholesterol levels. By providing strong evidence for disease-relevant functions of lipid trait-associated genes, our study demonstrates that quantitative, cell-based RNAi is a scalable strategy for a systematic, unbiased detection of functional effectors within GWAS loci., Author Summary Complex traits and diseases are assumed to result from interactions between multiple genes in relevant biological processes. Recent genome-wide association studies have uncovered many novel genomic loci where genes with functional significance are expected. However, functional validation of such genes has thus far remained confined to single gene approaches. Here, we use RNA interference and high-content screening microscopy to profile 133 genes at 56 loci associated with blood lipid traits, cardiovascular disease, and/or myocardial infarction for a function in regulating cellular free cholesterol levels and the efficiency of low-density lipoprotein uptake. Our results suggest that a high number of trait-associated genes have conserved cholesterol-regulatory functions in cells, with several GWAS loci harboring more than one gene of likely functional significance. For a number of genes without previously known lipid-regulatory functions, consequences upon siRNA knockdown positively correlated with cellular levels of LDL receptor, a major determinant of blood LDL levels. Moreover, GFP–tagged fusion proteins of several candidates shifted cellular cholesterol levels to inverse directions than knockdown, and subcellular localization of some candidates was sterol-dependent. Our study generates a valuable resource for prioritization of lipid-trait/CAD/MI-associated genes for future in-depth mechanistic analyses and introduces cell-based RNAi as a scalable and unbiased tool for functional follow-up of GWAS loci.

Drug-induced transcriptional modules (biclusters) were identified and annotated in three human cell lines and rat liver. These were used to assess conservation across systems and to infer and experimentally validate novel drug effects and gene functions., Biclustering of drug-induced gene expression profiles resulted in modules of drugs and genes, which were enriched in both drug and gene annotations. Identifying drug-induced transcriptional modules separately in three human cell lines and rat liver allows assessment of their conservation across model systems. About 70% of modules are conserved across cell lines, a lower bound of 15% was estimated for their conservation across organisms, and between the in vitro and in vivo systems. Drug-induced transcriptional modules can predict novel gene functions. A conserved module associated with (chole)sterol metabolism revealed novel regulators of cellular cholesterol homeostasis; 10 of them were validated in functional imaging assays. Analysis of drugs clustered into modules can give new insights into their mechanisms of action and provide leads for drug repositioning. We predicted and experimentally validated novel cell cycle inhibitors and modulators of PPARγ, estrogen and adrenergic receptors, with potential for developing new therapies against diabetes and cancer., In pharmacology, it is crucial to understand the complex biological responses that drugs elicit in the human organism and how well they can be inferred from model organisms. We therefore identified a large set of drug-induced transcriptional modules from genome-wide microarray data of drug-treated human cell lines and rat liver, and first characterized their conservation. Over 70% of these modules were common for multiple cell lines and 15% were conserved between the human in vitro and the rat in vivo system. We then illustrate the utility of conserved and cell-type-specific drug-induced modules by predicting and experimentally validating (i) gene functions, e.g., 10 novel regulators of cellular cholesterol homeostasis and (ii) new mechanisms of action for existing drugs, thereby providing a starting point for drug repositioning, e.g., novel cell cycle inhibitors and new modulators of α-adrenergic receptor, peroxisome proliferator-activated receptor and estrogen receptor. Taken together, the identified modules reveal the conservation of transcriptional responses towards drugs across cell types and organisms, and improve our understanding of both the molecular basis of drug action and human biology.

The responses of fruit and shoot growth of ‘Hayward’ kiwifruit vines to changes of temperature were determined during spring, summer or autumn. Mature vines were warmed 2–5°C above ambient temperatures by enclosing them in temperature-controlled tunnel houses for 34–89 d. Increasing temperature during spring advanced the date of flowering by 17 d and increased the rate of shoot elongation by 6 mm d–1 °C–1. The fruit on these early-flowering vines were larger and had a higher dry matter concentration than control fruit during the first part of the season. Increasing temperature during summer increased the rate of shoot elongation but reduced fruit growth, accumulation of dry matter in fruit and fruit firmness. In contrast, increasing temperature during late autumn increased fruit growth but reduced the soluble solids concentration (SSC) of fruit and thus, delayed commercial maturity. When fruit growth data for summer and autumn were combined the variation in fruit growth with temperature could be described by a single quadratic curve. Maximum fruit growth occurred at 17°C and temperatures above or below this optimum reduced fruit growth. Consequently, during summer when ambient temperatures averaged 17°C, warming vines decreased fruit growth, while during late autumn, when ambient temperatures had fallen to 13°C, warming vines increased fruit growth. Warming vines during summer reduced both the SSC of ripe fruit and the vitamin C concentration. Warming vines during autumn increased SSC but reduced the vitamin C concentration.

Whole-plant hydraulic conductance, shoot growth, and leaf photosynthetic properties were measured on kiwifruit vines with four clonal rootstocks to examine the relationship between plant hydraulic conductance and leaf stomatal conductance (gs) and to test the hypothesis that reduced hydraulic conductance can provide an explanation for reductions in plant vigour caused by rootstocks. The rootstocks were selected from four species of Actinidia and grafted with Actinidia chinensis var. chinensis 'Hort16A' (yellow kiwifruit) as the scion. Total leaf area of the scion on the least vigorous Actinidia rootstock, A. kolomikta, was 25% of the most vigorous, A. hemsleyana. Based on shoot growth and leaf area, the selections of A. kolomikta and A. polygama are low-vigour rootstocks, and A. macrosperma and A. hemsleyana are high-vigour rootstocks for A. chinensis. Whole-plant hydraulic conductance, the ratio of xylem sap flux to xylem water potential, was lower in the low-vigour rootstocks, reflecting their smaller size. However, leaf-area-specific conductance (Kl) and gs were both higher in the low-vigour rootstocks, the opposite of the expected pattern. Differences in Kl were found in the compartment from the roots to the scion stem, with no difference between rootstocks in the conductance of stems or leaves of the scion. There was no evidence that the graft union caused a significant reduction in hydraulic conductance of vines with low-vigour rootstocks. Leaf photosynthetic capacity did not vary between rootstocks, but photosynthesis and carbon isotope discrimination (Delta13C) under ambient conditions were higher in the low-vigour rootstocks because gs was higher. gs and Delta13C were positively correlated with Kl, although the mechanism for this relationship was not based on stomatal regulation of a similar xylem water potential because water potential varied between rootstocks. For Actinidia rootstocks, changes in Kl do not provide a direct explanation for changes in vigour of the scion. However, depending on the rootstock in question, changes in hydraulic conductance, biomass partitioning, and crown structure are involved in the response.

Infectious endocytosis of incoming human papillomavirus type 16 (HPV-16), the main etiological agent of cervical cancer, is poorly characterized in terms of cellular requirements and pathways. Conflicting reports attribute HPV-16 entry to clathrin-dependent and -independent mechanisms. To comprehensively describe the cell biological features of HPV-16 entry into human epithelial cells, we compared HPV-16 pseudovirion (PsV) infection in the context of cell perturbations (drug inhibition, siRNA silencing, overexpression of dominant mutants) to five other viruses (influenza A virus, Semliki Forest virus, simian virus 40, vesicular stomatitis virus, and vaccinia virus) with defined endocytic requirements. Our analysis included infection data, i.e. GFP expression after plasmid delivery by HPV-16 PsV, and endocytosis assays in combination with electron, immunofluorescence, and video microscopy. The results indicated that HPV-16 entry into HeLa and HaCaT cells was clathrin-, caveolin-, cholesterol- and dynamin-independent. The virus made use of a potentially novel ligand-induced endocytic pathway related to macropinocytosis. This pathway was distinct from classical macropinocytosis in regards to vesicle size, cholesterol-sensitivity, and GTPase requirements, but similar in respect to the need for tyrosine kinase signaling, actin dynamics, Na+/H+ exchangers, PAK-1 and PKC. After internalization the virus was transported to late endosomes and/or endolysosomes, and activated through exposure to low pH., Author Summary Human papillomavirus type 16 is the main etiological agent of cervical cancer. Despite advances in our understanding of transformation and cancer progression, as well as preventative vaccination strategies, the early events in papillomavirus infections are incompletely understood. Here, we investigated which strategies and cellular mechanisms the virus uses to enter epithelial cells. Entry was slow and asynchronous likely due to several structural alterations, which needed to occur on the cell exterior. Interestingly, the virus hijacked a potentially novel pathway of endocytosis for entry, which was distinct from classical macropinocytosis in regards to vesicle size, cholesterol-sensitivity, and GTPase requirements, but similar in respect to tyrosine kinase signaling, actin dynamics, Na+/H+ exchangers, PAK-1 and PKC requirements. This cellular mechanism may also be used by other viruses such as influenza A virus, echo virus 1, and choriomeningitis virus.

Influenza A virus (IAV) infection is normally controlled by adaptive immune responses initiated by dendritic cells (DCs). We investigated the consequences of IAV infection of human primary DCs on their ability to function as antigen-presenting cells. IAV was internalized by both myeloid DCs (mDCs) and plasmacytoid DCs but only mDCs supported viral replication. Although infected mDCs efficiently presented endogenous IAV antigens on MHC class II, this was not the case for presentation on MHC class I. Indeed, cross-presentation by uninfected cells of minute amounts of endocytosed, exogenous IAV was ∼300-fold more efficient than presentation of IAV antigens synthesized by infected cells and resulted in a statistically significant increase in expansion of IAV-specific CD8 T cells. Furthermore, IAV infection also impaired cross-presentation of other exogenous antigens, indicating that IAV infection broadly attenuates presentation on MHC class I molecules. Our results suggest that cross-presentation by uninfected mDCs is a preferred mechanism of antigen-presentation for the activation and expansion of CD8 T cells during IAV infection., Author Summary Although the interactions between viruses and dendritic cells (DCs) have been studied for many years, surprisingly little is known on the functional relationship between infection and antigen presentation in primary human DCs. Here, we asked specifically whether Influenza A virus (IAV) infection of human primary plasmacytoid DCs and myeloid DCs (pDCs and mDCs, respectively) affected their ability to function as antigen-presenting cells and activate T cells specific to IAV or other antigens. Our data confirm that pDCs are poorly infected and also present IAV antigens poorly. mDCs, on the other hand, are readily susceptible to IAV infection and present IAV antigen to T cells. However, we found that MHC class I presentation by mDCs infected with IAV are ∼300-fold less efficient relative to what mDCs are capable of achieving by cross-presentation following the endocytosis of only a very few non-infectious virions. Importantly, IAV infection of mDCs not only reduces the efficiency of IAV presentation but also reduces their ability to cross-present antigens from other viruses encountered subsequently. The reduced overall antigen processing capacity of mDCs describes a mechanism that may contribute to the suppression of immunity to secondary pathogens that appear during the course of IAV infection.

Summary Patterns of shoot development and the production of different types of shoots were compared with scion leaf area index (LAI) to identify how eight clonal Actinidia rootstocks influence scion development. Rootstocks selected from seven Actinidiaspecies (A. chrysanthaMerri., A. deliciosa(A. Chev.) C. F. Liang et A.R. Ferguson, A. eriantha Benth., A. hemsleyana Dunn, A. kolomikta(Maxim. et Rupr.) Maxim., A. macrosperma C.F. Liang and A. polygama (Sieb. et Zucc.) Maxim.) were grafted with the scion Actinidia chinensis Planch. var. chinensis‘Hort16A’(yellow kiwifruit). Based on an earlier architectural analysis of A. chinensis, axillary shoot types produced by the scion were classified as short, medium or long. Short and medium shoots produced a restricted number of preformed leaves before the shoot apex ceased growth and aborted, resulting in a ‘terminated’ shoot. The apex of long shoots continued growth and produced more nodes throughout the growing seasons. Mid-season LAI of the scion was related to the proportion of shoots that ceased growth early in the season. Scions on low-vigor rootstocks had 50% or less leaf area than scions on the most vigorous rootstocks and had a higher proportion of short and medium shoots. On low-vigor rootstocks, a higher proportion of short shoots was retained during pruning to form the parent structure of the following year. Short parent shoots produced a higher proportion of short daughter shoots than long parent shoots, thus reinforcing the effect of the low-vigor rootstocks. However, overall effects of rootstock on shoot development were consistent regardless of parent shoot type and nodal position within the parent shoot. Slower-growing shoots were more likely to terminate and scions on lowvigor rootstocks produced a higher proportion of slow-growing shoots. Shoot termination also occurred earlier on lowvigor rootstocks. The slower growth of terminating shoots was detectable from about 20 days after bud burst. Removal of a proportion of shoots at the end of bud burst increased the growth rate and decreased the frequency of termination of the remaining shoots on all rootstocks, indicating that the fate of a shoot was linked to competitive interactions among shoots during initial growth immediately after bud burst. Rootstock influ enced the process of shoot termination independently of its effect on final leaf size. Scions on low-vigor rootstocks had a higher proportion of short shoots and short shoots on all rootstocks had smaller final leaf sizes at equivalent nodes than medium or long shoots. Only later in the development of long shoots was final leaf size directly related to rootstock, with smaller leaves on low-vigor rootstocks. Thus, the most important effect of these Actinidia rootstocks on scion development occurred during the initial period of shoot growth immediately after bud burst.