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Skeletal muscle fiber composition and capillary density influence physical performance and whole-body metabolic properties. ~45% of the variance in fiber type is heritable, which motivated us to perform a genome-wide association study of skeletal muscle histology from 656 Swedish men. Four independent variants were associated (p − 8) with proportion of type IIx fibers or capillary-to-fiber ratio (C:F). The strongest signal localized to the rs115660502 variant, where the G-allele corresponded with increased C:F and reduced skeletal muscle expression of the proximal gene, RAB3 GTPase Activating Non-Catalytic Protein Subunit 2 (RAB3GAP2). The G-allele was less frequent in elite short-track sprinters and more frequent in endurance athletes than in matched non-athlete (population) controls; RAB3GAP2 expression was reduced by high-intensity intermittent training. RAB3GAP2 protein was not uniformly expressed in muscle tissue but localized to the endothelium and capillaries. Experimental reduction of RAB3GAP2 in human endothelial cells led to increased tube formation in vitro, to regulation of secreted factors promoting angiogenesis and T-cell activation, to reduced intracellular levels of von Willebrand factor (VWF) and, post-implantation, to increased endothelial cell density in vivo in mice. The amount of RAB3GAP2 in skeletal muscle was positively associated with exercise-induced release of VWF in vivo in humans. By regulating the release of protein factors (VWF, CD70, TNC, TNXB, MCP1, IGFBP3, COL1A1, TFPI2 and tPA), RAB3GAP2 influences fitness adaptation after exercise by improving muscle healing and promotion of capillary formation.

We review the international literature on a neglected aspect of maternal mortality: maternal homicide. Reported rates range from 0.97 to 10.6 per 100,000 live births. Women murdered in the perinatal period constituted a highly vulnerable group: they were younger, more likely to be from minority ethnic groups, and unmarried. Domestic violence was a significant risk factor for attempted and completed homicide. Compared to other countries, pregnancy-associated homicide rates were highest in the US. It is unclear how much of the difference to attribute to better case identification or to actual risk. Our review demonstrates pregnancy-associated homicide is an important contributor to maternal mortality, with rates comparable to suicide. Central to any prevention strategy will be identification of those at risk. The predictions are very weak because definitions, data collection, and analysis are so variable from study to study. Our findings reinforce the importance of screening for current and previous domestic violence.

Based on input from key operators in the Middle East region, a new rotary steerable system (RSS) was launched after a compressed development schedule. This paper describes the development and introduction of the larger tool sizes needed for both onshore and offshore hole sections, including hole sizes from 12 in. and up, in the Middle East. It also outlines the deliberate design of the tool for local assembly and repair. Large diameter (9-1/2 and 11 in.) RSS designs used an existing, smaller design for Middle East applications in both offshore and onshore wells as a basis. When designing these new sizes, engineers took note of lessons learned with smaller sizes of the tool and incorporated design elements for local manufacturing, assembly, and repair. The resulting simple, modular construction enables increased levels of local content and provides for significant reductions in transportation, and therefore associated emissions. Of course, although local content and sustainability are highly desirable, performance is essential, and this paper describes case histories demonstrating how well the new tool worked in real-world Middle East applications. In one notable example, the newly introduced 9 1/2-in. diameter RSS was used to drill an offshore section in the Gulf of Arabia. The tool was mobilized after two older generation RSS had become stuck for days. Consisting primarily of argillaceous limestone, the formation had a history of stuck-pipe events. The new RSS was recommended for this application because of a slicker construction, with a fully rotational bias unit, minimal bottom hole assembly (BHA) stabilization, and an optimized junk slot area, which together help to reduce stuck-pipe risks. The tool drilled to the target depth in a single run, thereby achieving all directional requirements. Notably, after reaching the target depth, the assembly was tripped out of the hole without any requirement for backreaming. This seamless exit, in turn, indicated achieving a smooth wellbore. Other case histories demonstrate results with both new sizes of this tool. The paper also discusses in detail the ability to repair locally and engage the local supply chain. Specifically with Middle East applications in mind, a new, simple RSS design in large diameter versions has demonstrated success in offshore and onshore applications across the region. The design has also proven capabilities for manufacturing and repair local to operations, which enables maximizing in-country value, optimizing use of the tools, and energizing local supply chains.

The Sf9 cell line, originally isolated from the insect Spodoptera frugiperda, is commonly used alongside the baculovirus expression vector system (BEVS) to produce recombinant proteins and other biologics. As more BEVS-derived vaccines and therapeutics are approved by regulators and manufactured at scale, there is increasing interest in improving the Sf9 cell line to improve bioprocess robustness and increase product yields. CRISPR-Cas9 is a powerful genome-editing tool with great potential to improve cell line characteristics. Nevertheless, reports of genome-editing in Sf9 cells are scarce, and targets for engineering are elusive. To evaluate the effectiveness of CRISPR-Cas9 to improve BEVS yields, we generated Sf9 cell lines with functional knockouts in the Sf-Caspase-1 gene, which encodes an effector caspase involved in the execution of apoptosis. Deletion of Sf-Caspase-1 abolished the hallmarks of apoptotic cell death including plasma membrane blebbing and effector caspase activity. Following infection of Sf-Caspase-1 knockout Sf9 cultures with a recombinant baculovirus expressing β-galactosidase, we did not observe any differences in cell death kinetics or increases in productivity. Similar results were obtained when Sf-Caspase-1 expression was suppressed via RNA interference. We anticipate that the CRISPR-Cas9 workflow reported here will spur future efforts to rationally engineer Sf9 cells for improved baculovirus expression.

Tumor progression alters the composition and physical properties of the extracellular matrix. Particularly, increased matrix stiffness has profound effects on tumor growth and metastasis. While endothelial cells are key players in cancer progression, the influence of tumor stiffness on the endothelium and the impact on metastasis is unknown. Through quantitative mass spectrometry, we find that the matricellular protein CCN1/CYR61 is highly regulated by stiffness in endothelial cells. We show that stiffness-induced CCN1 activates β-catenin nuclear translocation and signaling and that this contributes to upregulate N-cadherin levels on the surface of the endothelium, in vitro This facilitates N-cadherin-dependent cancer cell-endothelium interaction. Using intravital imaging, we show that knockout of Ccn1 in endothelial cells inhibits melanoma cancer cell binding to the blood vessels, a critical step in cancer cell transit through the vasculature to metastasize. Targeting stiffness-induced changes in the vasculature, such as CCN1, is therefore a potential yet unappreciated mechanism to impair metastasis. ispartof: EMBO Journal vol:36 issue:16 pages:2373-2389 ispartof: location:England status: published

Baculoviruses are recognized as viral workhorses of biotechnology, being used for production of vaccines, complex recombinant proteins, gene delivery vectors' and safe biological pesticides. Improving production yields and understanding the interactions of the virus and its host cell are important aspects of ensuring baculovirus-based processes are commercially competitive. This study aims at potential optimization of host cells used in in vitro virus production by systemically investigating changes in host gene expression in response to virus replication and transcription inside host cells. The study focuses on in vitro interactions of the Helicoverpa armigera virus with Helicoverpa zea insect cells. We used 22 genome-wide microarrays to simultaneously measure both virus and host genes in infected cells in multiple batch suspension cultures, representing high- and low-producing infection conditions. Among 661 differentially expressed genes, we identified a core set of 59 host genes consistently overexpressed post infection, with strong overrepresentation of genes involved in retrotransposition, protein processing in the endoplasmic reticulum, and ubiquitin-mediated proteolysis. Applying a whole genome correlation network analysis to link gene expression to productivity, we revealed 18 key genes significantly associated to virus yield. In addition, this study is among the first to perform a genome-wide expression study for a major baculovirus group II strain, the H. armigera virus, extending current understanding of baculovirus-insect interactions, which mainly focuses on group I viruses.

This article reviews research from the 1960s to the present pertaining to suicide in people suffering with schizophrenia. An attempt has been made to examine suggested risk factors and generate recommendations for management, with particular attention to recent Department of Health guidelines regarding the care of the severely mentally ill. The literature was reviewed using Medline and PsychInfo, supplemented with a manual literature search. The studies reviewed were of variable quality. Many were limited by small numbers, lack of control groups and diagnostic heterogeneity. In combination with the low base-rate of suicide, and the relative homogeneity within groups of patients with schizophrenia, prediction remains problematic. Current initiatives suggest increased supervision of the at-risk population with little emphasis on attempting to reduce suicidal intent. There remains a need for continuing research into this difficult area of management. Specific areas for consideration include the management of hopelessness and depression in this group of patients, and the effect of the environment. The effect of the living environment, as well as the ongoing reductions in in-patient hospital beds and the current emphasis on increased turnover merit reappraisal.

BackgroundHealth anxiety is an under-recognised but frequent cause of distress that is potentially treatable, but there are few studies in secondary care.ObjectiveTo determine the clinical effectiveness and cost-effectiveness of a modified form of cognitive–behaviour therapy (CBT) for health anxiety (CBT-HA) compared with standard care in medical outpatients.DesignRandomised controlled trial.SettingFive general hospitals in London, Middlesex and Nottinghamshire.ParticipantsA total of 444 patients aged 16–75 years seen in cardiology, endocrinology, gastroenterology, neurology and respiratory medicine clinics who scored ≥ 20 points on the Health Anxiety Inventory (HAI) and satisfied diagnostic requirements for hypochondriasis. Those with current psychiatric disorders were excluded, but those with concurrent medical illnesses were not.InterventionsCognitive–behaviour therapy for health anxiety – between 4 and 10 1-hour sessions of CBT-HA from a health professional or psychologist trained in the treatment. Standard care was normal practice in primary and secondary care.Main outcome measuresPrimary – researchers masked to allocation assessed patients at baseline, 3, 6, 12, 24 months and 5 years. The primary outcome was change in the HAI score between baseline and 12 months. Main secondary outcomes – costs of care in the two groups after 24 and 60 months, change in health anxiety (HAI), generalised anxiety and depression [Hospital Anxiety and Depression Scale (HADS)] scores, social functioning using the Social Functioning Questionnaire and quality of life using the EuroQol-5 Dimensions (EQ-5D), at 6, 12, 24 and 60 months, and deaths over 5 years.ResultsOf the 28,991 patients screened over 21 months, 5769 had HAI scores of ≥ 20 points. Improvement in HAI scores at 3 months was significantly greater in the CBT-HA group (mean number of sessions = 6) than in the standard care, and this was maintained over the 5-year period (overallp p = 0.0018) and depression scores (p = 0.0065) on the HADS were similar in both groups over the 5-year period. Gastroenterology and cardiology patients showed the greatest CBT gains. The outcomes for nurses were superior to those of other therapists. Deaths (n = 24) were similar in both groups; those in standard care died earlier than those in CBT-HA. Patients with mild personality disturbance and higher dependence levels had the best outcome with CBT-HA. Total costs were similar in both groups over the 5-year period (£12,590.58 for CBT-HA; £13,334.94 for standard care). CBT-HA was not cost-effective in terms of quality-adjusted life-years, as measured using the EQ-5D, but was cost-effective in terms of HAI outcomes, and offset the cost of treatment.LimitationsMany eligible patients were not randomised and the population treated may not be representative.ConclusionsCBT-HA is a highly effective treatment for pathological health anxiety with lasting benefit over 5 years. It also improves generalised anxiety and depressive symptoms more than standard care. The presence of personality abnormality is not a bar to successful outcome. CBT-HA may also be cost-effective, but the high costs of concurrent medical illnesses obscure potential savings. This treatment deserves further research in medical settings.Trial registrationCurrent Controlled Trials ISRCTN14565822.FundingThis project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full inHealth Technology Assessment; Vol. 21, No. 50. See the NIHR Journals Library website for further project information.

Purpose– The purpose of this paper is to investigate the influence of leaders on knowledge creation and mobilization.Design/methodology/approach– This mixed methods study included three high-performing districts based on provincial assessment results and socio-economic factors. Interviews and questionnaires were used to gather data from 53 participants including: 11 principals, 11 teacher leaders, 26 teachers, and five system leaders.Findings– The findings of the study emphasized the importance of leaders supporting knowledge creation and mobilization processes through practices such as engaging school-based knowledge influencers and fostering cultures of trust and risk taking. The author defined knowledge influencers as leaders, formal or informal, who have access to knowledge creating groups at the local and system level. These leaders influenced knowledge mobilization at different levels of the district.Research limitations/implications– A research limitation of this study was present based on the sole use of high-performing districts and schools. Participation was determined via comparisons of provincial assessment results (Ontario, Canada) and socio-economic status (SES) factors. Although causal effects are cautioned, districts and schools from various SES communities (high, medium, low) were chosen to support broad generalizations and associations.Practical implications– This study provided pragmatic considerations and recommendations for system and school leaders, those charged with increasing student achievement (e.g. use of knowledge influencers and an expanded array of data use while creating knowledge).Originality/value– A knowledge creation model was developed by the author based on a synthesis of the findings. The model and study will be of interest to those wishing to further implement or study the creation and mobilization of knowledge within organizations.

A flow cytometric method (RAPID-B™) with detection sensitivity of one viable cell of Escherichia coli serotype O157:H7 in fresh spinach (Spinacia oleracea) was developed and evaluated. The major impediment to achieving this performance was mistaking autofluorescing spinach particles for tagged target cells. Following a 5 h non-selective enrichment, artificially inoculated samples were photobleached, using phloxine B as a photosensitizer. Samples were centrifuged at high speed to concentrate target cells, then gradient centrifuged to separate them from matrix debris. In external laboratory experiments, RAPID-B and the reference method both correctly detected E. coli O157:H7 at inoculations of ca. 15 cells. In a follow-up study, after 4 cell inoculations of positives and 6 h enrichment, RAPID-B correctly identified 92% of 25 samples. The RAPID-B method limit of detection (LOD) was one cell in 25 g. It proved superior to the reference method (which incorporated real time-PCR, selective enrichment, and culture plating elements) in accuracy and speed.

Baculovirus-insect cell technologies are applied in the production of complex proteins, veterinary and human vaccines, gene delivery vectors‚ and biopesticides. Better understanding of how baculoviruses and insect cells interact would facilitate baculovirus-based production. While complete genomic sequences are available for over 58 baculovirus species, little insect genomic information is known. The release of the Bombyx mori and Plutella xylostella genomes, the accumulation of EST sequences for several Lepidopteran species, and especially the availability of two genome-scale analysis tools, namely oligonucleotide microarrays and next generation sequencing (NGS), have facilitated expression studies to generate a rich picture of insect gene responses to baculovirus infections. This review presents current knowledge on the interaction dynamics of the baculovirus-insect system‚ which is relatively well studied in relation to nucleocapsid transportation, apoptosis, and heat shock responses, but is still poorly understood regarding responses involved in pro-survival pathways, DNA damage pathways, protein degradation, translation, signaling pathways, RNAi pathways, and importantly metabolic pathways for energy, nucleotide and amino acid production. We discuss how the two genome-scale transcriptomic tools can be applied for studying such pathways and suggest that proteomics and metabolomics can produce complementary findings to transcriptomic studies.

The performance of bioprocesses involving baculoviruses largely depends on an efficient infection of cells by concentrated budded virus (BV) inoculums. Baculovirus expression vector systems have been established using Autographa californica nucleopolyhedrovirus (AcMNPV), a group I NPV that displays rapid virus kinetics, whereas bioprocesses using group II baculovirus-based biopesticides such as Helicoverpa armigera nucleopolyhedrovirus (HearNPV) have the limitation of low levels of BV, as these viruses often display poor BV production kinetics. In this study, the effect of key parameters involved in the quality of progeny virions, including cell line, virus phylogenetics and medium, on viral DNA replication, virus trafficking to the extracellular environment, and the yield of recombinant protein or polyhedra were investigated in synchronous infections of HearNPV and AcMNPV. HearNPV showed higher vDNA replication in its optimum medium, SF900III, when compared to AcMNPV, but both viruses had similar specific extracellular virion content. However, the ratio of AcMNPV extracellular virions to the total number of progeny virions produced was higher, and their quality was tenfold higher than that of HearNPV extracellular virions. The results of infection of two different cell lines, High Five and Sf9, with AcMNPV, along with HearNPV infection of HzAM1 cells in three different media, suggest that the host cells and the nutritional state of the medium as well as the phylogenetics of the virus affect the BV yields produced by different baculovirus/cell line/medium combinations.

The cell density effect is a well-established constraint in the baculovirus-insect cell expression platform, in which cell-specific productivity declines with increasing cell density, hence limiting the maximum achievable volumetric yield of protein product. A deeper elucidation of this phenomenon is sought in this study, by tracking the peak production of viral DNA (vDNA), recombinant LacZ mRNA, and β-galactosidase (β-gal) protein, over a wide range of cell densities. Sf9 suspension cell cultures were propagated in Sf-900 III serum-free medium and synchronously infected with rAcMNPV at multiple infection cell densities (ICDs) of between 0.5 and 8 × 10(6) cells/mL. There was a strong negative linear correlation between the specific β-gal yield and the peak cell density (PCD) post-infection, but contrary to previous reports, the yield decline started at a lower PCD of around 1 × 10(6) cells/mL. Most interestingly, there also was a corresponding strong negative linear correlation between the specific vDNA or LacZ mRNA yield, and the PCD. Comparing the infections at the highest and lowest PCDs tested, the yield decline was most dramatic for β-gal protein (95 %) and LacZ mRNA (90 %), while it was more moderate for vDNA (50 %). These declines were significantly reduced but not completely arrested, when spent medium was replaced with fresh at the ICD. These findings suggest that protein yield deterioration with increasing cell density originated from limitations during upstream events such as virus gene replication or transcription, rather than during the translational phase. Such limitations may be largely nutritional, but a more complex mechanism may be implicated.

While large-scale culture of insect cells will need to be conducted using bioreactors up to 10,000 l scale, many of the main challenges for cell culture-based production of insecticidal viruses can be studied using small-scale (20-500 ml) shaker/spinner flasks, either in free suspension or using microcarrier-based systems. These challenges still relate to the development of appropriate cell lines, stability of virus strains in culture, enhancing virus yields per cell, and the development of serum-free media and feeds for the desired production systems. Hence this chapter presents mainly the methods required to work with and analyze effectively insect cell systems using small-scale cultures. Outlined are procedures for quantifying cells and virus and for establishing frozen cells and virus stocks. The approach for maintaining cell cultures and the multiplicity of infection (MOI) and time of infection (TOI) parameters that should be considered for conducting infections are discussed.The methods described relate, in particular, to the suspension culture of Helicoverpa zea and Spodoptera frugiperda cell lines to produce the baculoviruses Helicoverpa armigera nucleopolyhedrovirus, HearNPV, and Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, AgMNPV, respectively, and the production of the nonoccluded Oryctes nudivirus, OrNV, using an adherent coleopteran cell line.

Breast tumors of the basal-like, hormone receptor-negative, subtype remain an unmet clinical challenge, as patients exhibit a high rate of recurrence and poor survival. Co-evolution of the malignant mammary epithelium and its underlying stroma instigates cancer-associated fibroblasts (CAFs) to endorse most, if not all, hallmarks of cancer progression. Here, we delineate a previously unappreciated role for CAFs as determinants of the molecular subtype of breast cancer. We identified a paracrine cross-talk between cancer cells expressing platelet-derived growth factor (PDGF)-CC and CAFs expressing the cognate receptors in human basal-like mammary carcinomas. Genetic or pharmacological intervention with PDGF-CC activity in mouse models of cancer resulted in conversion of basal-like breast cancers into a hormone receptor-positive state that conferred sensitivity to endocrine therapy in previously impervious tumors. We conclude that specification of the basal-like subtype of breast cancer is under microenvironmental control and therapeutically actionable in order to achieve sensitivity to endocrine therapy.

Baculovirus-based Insect Cell Technology (ICT) is widely used for the expression of recombinant heterologous proteins and baculovirus bioinsecticides, and has recently gained momentum as a commercial manufacturing platform for human and veterinary vaccines. The three key components of ICT are the Lepidopteran insect cell line, the baculovirus vector, and the growth medium. Insect cell growth media have evolved significantly in the past five decades, from basal media supplemented with hemolymph or animal serum, to highly optimized serum-free media and feeds (SFM and SFF) capable of supporting very high cell densities and recombinant protein yields. The substitution of animal sera with protein hydrolysates in SFM results in greatly reduced medium costs and much improved process scalability. However, both sera and hydrolysates share the disadvantage of lot-to-lot variability, which is detrimental to process reproducibility. Hence, the industrialization of ICT would benefit greatly from chemically defined media (CDM) for insect cells, which are not yet commercially available. On the other hand, applications such as baculovirus bioinsecticides would need truly low cost serum-free media and feeds (LC-SFM and LC-SFF) for economic viability, which require the substitution of a majority of expensive added amino acids with even higher levels of hydrolysates, hence increasing the risk of a variable process. CDM developments are anticipated to benefit both conventional and low cost ICT applications, by identifying key growth factors in hydrolysates for more targeted media and feed design.

While large-scale culture of insect cells will need to be conducted using bioreactors up to 10,000 l scale, many of the main challenges for cell culture-based production of insecticidal viruses can be studied using small-scale (20-500 ml) shaker/spinner flasks, either in free suspension or using microcarrier-based systems. These challenges still relate to the development of appropriate cell lines, stability of virus strains in culture, enhancing virus yields per cell, and the development of serum-free media and feeds for the desired production systems. Hence this chapter presents mainly the methods required to work with and analyze effectively insect cell systems using small-scale cultures. Outlined are procedures for quantifying cells and virus and for establishing frozen cells and virus stocks. The approach for maintaining cell cultures and the multiplicity of infection (MOI) and time of infection (TOI) parameters that should be considered for conducting infections are discussed.The methods described relate, in particular, to the suspension culture of Helicoverpa zea and Spodoptera frugiperda cell lines to produce the baculoviruses Helicoverpa armigera nucleopolyhedrovirus, HearNPV, and Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, AgMNPV, respectively, and the production of the nonoccluded Oryctes nudivirus, OrNV, using an adherent coleopteran cell line.

Baculovirus-based Insect Cell Technology (ICT) is widely used for the expression of recombinant heterologous proteins and baculovirus bioinsecticides, and has recently gained momentum as a commercial manufacturing platform for human and veterinary vaccines. The three key components of ICT are the Lepidopteran insect cell line, the baculovirus vector, and the growth medium. Insect cell growth media have evolved significantly in the past five decades, from basal media supplemented with hemolymph or animal serum, to highly optimized serum-free media and feeds (SFM and SFF) capable of supporting very high cell densities and recombinant protein yields. The substitution of animal sera with protein hydrolysates in SFM results in greatly reduced medium costs and much improved process scalability. However, both sera and hydrolysates share the disadvantage of lot-to-lot variability, which is detrimental to process reproducibility. Hence, the industrialization of ICT would benefit greatly from chemically defined media (CDM) for insect cells, which are not yet commercially available. On the other hand, applications such as baculovirus bioinsecticides would need truly low cost serum-free media and feeds (LC-SFM and LC-SFF) for economic viability, which require the substitution of a majority of expensive added amino acids with even higher levels of hydrolysates, hence increasing the risk of a variable process. CDM developments are anticipated to benefit both conventional and low cost ICT applications, by identifying key growth factors in hydrolysates for more targeted media and feed design.

Metabolomics refer to the global analysis of small molecule metabolites in a biological system, and can be a powerful tool to elucidate and optimize cellular processes, particularly when integrated into a systems biology framework. Determining the endometabolome in cultured animal cells is especially challenging, due to the conflicting demands for rapid quenching of metabolism and retention of membrane integrity, while cells are separated from the complex medium. The challenge is magnified in virus infected cells due to increased membrane fragility. This paper describes an effective methodology for quantitative intracellular metabolite analysis of the baculovirus-insect cell expression system, an important platform for the production of heterologous proteins and baculovirus-based biopesticides. These two applications were represented by Spodoptera frugiperda (Sf9) and Helicoverpa zea (HzAM1) cells infected with recombinant Autographa californica and wild-type Helicoverpa armigera nucleopolyhedroviruses (AcMNPV and HaSNPV), respectively. Specifically, an ice-cold quenching solution comprising 1.1% w/v NaCl and 0.2% w/v Pluronic® F-68 (NaCl+P) was found to be efficacious in preserving cell viability and minimizing cell leakage during quenching and centrifugation-based washing procedures (prior to extraction using cold 50% v/v acetonitrile). Good recoveries of intracellular adenosine triphosphate, total adenosine phosphates and amino acids were obtained after just one wash step, for both uninfected and infected insect cells. The ability to implement wash steps is critical, as insect cell media are metabolites-rich, while infected insect cells are much more fragile than their uninfected counterparts. Hence, a promising methodology has been developed to facilitate endometabolomic analysis of insect cell-baculovirus systems for bioprocess optimization.

Scanning ion occlusion sensing (SIOS), a technique that uses a tunable pore to detect the passage of individual nano-scale objects, is applied here for the rapid, accurate and direct measurement of synthetic and biological nanoparticle concentrations. SIOS is able to characterize smaller particles than other direct count techniques such as flow cytometry or Coulter counters, and the direct count avoids approximations such as those necessary for turbidity measurements. Measurements in a model system of 210–710 nm diameter polystyrene particles demonstrate that the event frequency scales linearly with applied pressure and concentration, and that measured concentrations are independent of particle type and size. Both an external-calibration and a calibration-free measurement method are demonstrated. SIOS is then applied to measure concentrations of Baculovirus occlusion bodies, with a diameter of ∼1 μm, and the marine photosynthetic cyanobacterium Prochlorococcus , with a diameter of ∼600 nm. The determined concentrations agree well with results from counting with microscopy (a 17% difference between the mean concentrations) and flow cytometry (6% difference between the mean concentrations), respectively.

Baculovirus pesticides are increasingly being used as effective biological control agents against caterpillar pests worldwide. Increasing occlusion body (OB) yields per cell in culture is the main challenge to enable commercialization of in vitro production of baculovirus pesticides. Isolating clones from a heterogeneous cell population may allow development of a high virus producing cell clone. To date, the selection of insect clones has been based mainly on laborious cell serial dilution methods which create few viable clones. This work used an automated robotic clone picking system to establish over 250 insect clones of a Helicoverpa zea cell population to be screened for virus production. However, the higher producing clones only produced 10-30% higher OB yields than the original cell population. This study suggested that unless screening of thousands of clones is performed, obtaining a 2-fold increase in OB/cell yield compared to the parent population is unlikely. Nevertheless, it creates pure clones for manufacturing. In addition, two clones that were at least 2-3 times different in OB yields were isolated. Hence, this method can create a high contrast system (OB/cell yield basis), for comparative studies using a systems biology approach, which should inform a more targeted approach to engineer genetically a production cell line.

Large-scale commercialization of baculovirus biopesticides for the control of insect pests requires a cell culture production process, and knowledge of the infection kinetics is a vital prerequisite for process optimization. Well-characterized kinetic parameters have so far only been reported for the commercially established recombinant Autographa californica nucleopolyhedrovirus (AcMNPV), a Group I NPV. In this work, key infection kinetic parameters of the Group II NPV Helicoverpa armigera nucleopolyhedrovirus (HaSNPV), and its Few Polyhedra (FP) mutant, were well characterized for the first time, in suspension HzAM1 insect cell cultures, to facilitate the scale-up of an HaSNPV-based biopesticide. The FP mutant had a selective advantage over wild-type HaSNPV in cell cultures, and the kinetic analysis showed that this was due to a superior budding rate, rather than a faster binding rate (BR) or longer budding duration. Another finding was that wild-type HaSNPV had very poor infection kinetics when compared with AcMNPV, exhibiting an 18-fold lower BR, a more than 50-fold lower budding rate, and a 60-fold lower extracellular/total progeny virus ratio. Such poor infection kinetics have serious implications during scale-up of an HaSNPV biopesticide production process, including the requirement for large volumes of virus inocula and the difficulty of achieving synchronous infections. Groups I and II NPVs may have very different infection kinetics because of their different envelope fusion proteins. This study is the first to compare the two groups of NPVs in terms of well-characterized cell-specific infection kinetics, and the findings may indicate a phylogenetic basis for kinetic differences.

To compare the effects of an acute one versus three-set full body resistance training (RT) bout in eight overweight (mean ± SD, BMI = 25.6 ± 1.5 kg m−2) young (21.0 ± 1.5 years) adults on resting energy expenditure (REE) measured on four consecutive mornings following each protocol. Participants performed a single one-set or three-set whole body (10 exercises, 10 repetition maximum) RT bout following the American College of Sports Medicine (ACSM) guidelines for RT. REE and respiratory exchange ratio (RER) by indirect calorimetry were measured at baseline and at 24, 48, and 72 h after the RT bout. Participants performed each protocol in randomized, counterbalanced order separated by 7 days. There was no difference between protocols for REE or RER. However, REE was significantly (p < 0.05) elevated (~5% or ~ 400 kJ day−1) in both the protocols at 24, 48, and 72 h post RT bout compared with baseline. There was a no change in RER in both the protocols at 72 h compared to baseline. A one-set RT bout following the ACSM guidelines for RT and requiring only ~ 15 min to complete was as effective as a three-set RT bout (~ 35 min to complete) in elevating REE for up to 72 h post RT in overweight college males, a group at high risk of developing obesity. The one-set RT protocol may provide an attractive alternative to either aerobic exercise or multiple-set RT programs for weight management in young adults, due to the minimal time commitment and the elevation in REE post RT bout.

Chronic fatigue syndrome affects between 0.006% and 3% of the population depending on the criteria of definition used, with women being at higher risk than men.We conducted a systematic overview, aiming to answer the following clinical question: What are the effects of selected treatments for chronic fatigue syndrome? We searched: Medline, Embase, The Cochrane Library, and other important databases up to November 2013 (Clinical Evidence reviews are updated periodically; please check our website for the most up-to-date version of this review).At this update, searching of electronic databases retrieved 169 studies. After deduplication and removal of conference abstracts, 86 records were screened for inclusion in the overview. Appraisal of titles and abstracts led to the exclusion of 71 studies and the further review of 15 full publications. Of the 15 full articles evaluated, two systematic reviews, one RCT, and one further follow-up report of an RCT were added at this update. We performed a GRADE evaluation for 23 PICO combinations.In this systematic overview, we categorised the effectiveness of four interventions based on information relating to the effectiveness and safety of antidepressants, cognitive behavioural therapy, corticosteroids, and graded exercise therapy.

Endothelial cells (ECs) play a key role to maintain the functionality of blood vessels. Altered EC permeability causes severe impairment in vessel stability and is a hallmark of pathologies such as cancer and thrombosis. Integrating label-free quantitative proteomics data into genome-wide metabolic modeling, we built up a model that predicts the metabolic fluxes in ECs when cultured on a tridimensional matrix and organize into a vascular-like network. We discovered how fatty acid oxidation increases when ECs are assembled into a fully formed network that can be disrupted by inhibiting CPT1A, the fatty acid oxidation rate-limiting enzyme. Acute CPT1A inhibition reduces cellular ATP levels and oxygen consumption, which are restored by replenishing the tricarboxylic acid cycle. Remarkably, global phosphoproteomic changes measured upon acute CPT1A inhibition pinpointed altered calcium signaling. Indeed, CPT1A inhibition increases intracellular calcium oscillations. Finally, inhibiting CPT1A induces hyperpermeability in vitro and leakage of blood vessel in vivo, which were restored blocking calcium influx or replenishing the tricarboxylic acid cycle. Fatty acid oxidation emerges as central regulator of endothelial functions and blood vessel stability and druggable pathway to control pathological vascular permeability.

The phenomenon of the reduction in the cell-specific yield with increasing infection cell density (ICD), the cell density effect, is one of the main hurdles for improving virus yields in vitro. In the current study, the reduction in cell-specific yields (viral DNA [vDNA], polyhedrin mRNA and occlusion body [OB]) with increasing ICD for Helicoverpa armigera nucleopolyhedrovirus (HearNPV)-infected HzAM1 (Helicoverpa zea) insect cells has been investigated. HzAM1 cells were propagated in Sf900™ III serum-free medium and synchronously infected with wild-type HearNPV at various ICDs of 0.5-5 × 10(6) cells/mL at an MOI of 5 PFU/cell. Infection was conducted either in the original medium or in fresh medium. As found previously for Sf9 and High Five cells, there were negative correlations between the three key virus infection indicators (vDNA, mRNA and OB) and the peak cell density (PCD). Generally, the yield decline with increasing PCD was most pronounced for OB, followed by mRNA, and was more moderate for vDNA. The decline was significantly reduced, but not totally arrested, when fresh medium was used. There were also strong correlations between OB and mRNA, mRNA and vDNA, and OB and vDNA levels. These results suggest that the reduction in baculovirus yield (OB) at high PCDs is associated with limitations during the upstream processes of replication and transcription together with limitations during protein translation. Furthermore, the peak protein productivity per unit of cell volume in the HzAM1/HearNPV system was shown to be higher than that of the Sf9/rAcMNPV system, but lower than that of the High Five/rAcMNPV system.

A case is described in which a patient with idiopathic cervical dystonia is treated successfully with cognitive-behavioural therapy. It was hypothesised that although the dystonia was organic in origin, it was exacerbated and perpetuated by catastrophic thoughts and abnormal illness beliefs, and that modification of maintaining cognitive and behavioural responses would lead to improvement. Self-report outcome measures were consistent with this hypothesis and improvements were maintained at 6-month follow-up.

Symptoms of depression occur commonly in medical illness, yet it is not always clear at which point such symptoms merit clinical attention. There is a common misperception that all depression is an ‘understandable’ psychological reaction to a serious illness. This belief may result in patients being denied appropriate treatment. Depression in the physically ill has been shown to have a significant impact, with increased symptom burden, impaired functioning and reduced quality of life. Treatment may lead not only to an improvement in depressive symptoms but also improvement in somatic symptoms, functional abilities, and a reduction in healthcare costs.

Rapid formation and selection of FP (few polyhedra) mutants occurs during serial passaging of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) in insect cell culture. The production of HaSNPV for use as biopesticides requires the passaging of the virus over a number of passages to produce enough virus inoculum for large-scale fermentation. During serial passaging in cell culture, FP mutants were rapidly selected, resulting in declined productivity and reduced potency of virus. Budded virus (BV) is usually harvested between 72 and 96 h postinfection (hpi) in order to obtain a high titer virus stock. In this study, the effect of time of harvest (TOH) for BV on the selection rate of HaSNPV FP mutants during serial passaging was investigated. BV were harvested at different times postinfection, and each series was serially passaged for six passages. The productivity and percentage of FP mutants at each passage were determined. It was found that the selection of FP mutants can be reduced by employing an earlier TOH for BV. Serial passaging with BV harvested at 48 hpi showed a slower accumulation of FP mutants compared to that of BV harvested after 48 hpi. Higher cell specific yields were also maintained when BV were harvested at 48 hpi. When BV that were formed between 48 and 96 hpi were harvested and serially passaged, FP mutants quickly dominated the virus population. This suggests that the BV formed and released between 48 and 96 hpi are most likely from FP mutant infected cells.

Sodium dodecyl sulfate (SDS) is commonly used to extract polyhedra from infected cells and diseased dead larval tissues. It was found, however, that 80% of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) polyhedra produced via cell culture were damaged after 30 min of 0.5% SDS treatment whereas only 20% of in vivo produced polyhedra were damaged by the same treatment. Transmission and scanning electron microscopy revealed that the damaged polyhedra had lost their polyhedron envelopes and virions were dislodged from the polyhedrin matrix, leaving empty spaces that were previously occupied by the occluded virions. Up to 20% in vitro produced polyhedra were resistant to SDS and remained intact, even after a 24 h exposure to SDS. This sensitivity to SDS was observed across a range of cell culture media, including serum supplemented media. Electron microscopy also revealed that the inferior polyhedron envelope of in vitro produced polyhedra is likely due to poor interaction between the polyhedron envelope, polyhedron envelope protein (PEP), and polyhedrin matrix. The PEP gene was cloned and sequenced and mutations in this gene were ruled out as an explanation. In vitro produced polyhedra that were passed through insect larva once were resistant to SDS, indicating that a critical component is lacking in insect cell culture medium used for producing HaSNPV or the cells growing in culture are inefficient in some ways in relation to production of polyhedra. (C) 2002 Elsevier Science (USA). All rights reserved.

A perennial problem in recombinant protein expression is low yield of the product of interest. A strategy which has been shown to increase the production of baculovirus-expressed proteins is to utilise fed-batch cultures. One disadvantage of this approach is the time-consuming task of optimising the feeding strategy. Previously, a statistical optimisation routine was applied to develop a feeding strategy that increased the yield of beta-Galactosidase (beta-Gal) by 2.4-fold (Biotechnol. Bioeng. 59 (1998) 178). This involves the single addition of nutrient concentrates (amino acids, lipids, glucose and yeastolate ultrafiltrate) into Sf 9 cell cultures grown in SF 900II medium. In this study, it is demonstrated that this optimised fed-batch strategy developed for a high-yielding intracellular product beta-Gal could be applied successfully to a relatively low-yielding glycosylated and secreted product such as the dengue virus glycoprotein NS1. Optimised batch infections yielded 4 microg/ml of NS1 at a peak cell density of 4.2 x 10 (6) cells/ml. In contrast, optimised fed-batch infections exhibited a 3-fold improvement in yield, with 12 microg/ml of NS1 produced at a peak cell density of 11.3 x 10 (6) cells/ml. No further improvements in yield were recorded when the feed volumes were doubled and the peak cell density was increased to 23 x 10 (6) cells/ml, unless the cultures were stimulated by the addition of 4 microg/ml of 20-Hydroxyecdysone (an insect moulting hormone). In this case, the NS1 yield was increased to 20 microg/ml, which was nearly 5-fold higher than optimised batch cultures.

Rapid accumulation of few polyhedra (FP) mutants was detected during serial passaging of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) in cell culture. 100% FP infected cells were observed by passage 6. The specific yield decreased from 178 polyhedra per cell at passage 2 to two polyhedra per cell at passage 6. The polyhedra at passage 6 were not biologically active, with a 28-fold reduction in potency compared to passage 3. Electron microscopy studies revealed that very few polyhedra were produced in an FP infected cell (Autographa californica nucleopolyhedrovirus and Galleria mellonella nucleopolyhedrovirus. Within an HaSNPV 25K FP gene homologue, a single base-pair insertion (an adenine residue) within a region of repetitive sequences (seven adenine residues) was identified in one plaque-purified HaSNPV FP mutant. Furthermore, the sequences obtained from individual clones of the 25K FP gene PCR products of a late passage revealed point mutations or single base-pair insertions occurring throughout the gene. The mechanism of FP mutation in HaSNPV is likely similar to that seen for Lymantria dispar nucleopolyhedrovirus, involving point mutations or small insertions/deletions of the 25K FP gene.

The study of viral-based processes is hampered by (a) their complex, transient nature, (b) the instability of products, and (c) the lack of accurate diagnostic assays. Here, we describe the use of real-time quantitative polymerase chain reaction to characterize baculoviral infection. Baculovirus DNA content doubles every 1.7 h from 6 h post-infection until replication is halted at the onset of budding. No dynamic equilibrium exists between replication and release, and the kinetics are independent of the cell density at the time of infection. No more than 16% of the intracellular virus copies bud from the cell.

The increased stiffness of a tumor triggers a multitude of responses that aid cancer cell dissemination. Stiffness-induced expression of CCN1 mediates autocrine signaling in the endothelium to upregulate N-Cadherin levels. This permits more stable interactions with cancer cells and increases their ability to spread into the circulation.