Cancer-associated fibroblasts rewire the estrogen receptor response in luminal breast cancer, enabling estrogen independence.

Advanced breast cancers represent a major therapeutic challenge due to their refractoriness to treatment. Cancer-associated fibroblasts (CAFs) are the most abundant constituents of the tumor microenvironment and have been linked to most hallmarks of cancer. However, the influence of CAFs on therapeutic outcome remains largely unchartered. Here, we reveal that spatial coincidence of abundant CAF infiltration with malignant cells was associated with reduced estrogen receptor (ER)-α expression and activity in luminal breast tumors. Notably, CAFs mediated estrogen-independent tumor growth by selectively regulating ER-α signaling. Whereas most prototypical estrogen-responsive genes were suppressed, CAFs maintained gene expression related to therapeutic resistance, basal-like differentiation, and invasion. A functional drug screen in co-cultures identified effector pathways involved in the CAF-induced regulation of ER-α signaling. Among these, the Transforming Growth Factor-ß and the Janus kinase signaling cascades were validated as actionable targets to counteract the CAF-induced modulation of ER-α activity. Finally, genes that were downregulated in cancer cells by CAFs were predictive of poor response to endocrine treatment. In conclusion, our work reveals that CAFs directly control the luminal breast cancer phenotype by selectively modulating ER-α expression and transcriptional function, and further proposes novel targets to disrupt the crosstalk between CAFs and tumor cells to reinstate treatment response to endocrine therapy in patients.

Sushi domain-containing protein 4 binds to epithelial growth factor receptor and initiates autophagy in an EGFR phosphorylation independent manner.

BACKGROUND: Sushi domain-containing protein 4 (SUSD4) is a recently discovered protein with unknown cellular functions. We previously revealed that SUSD4 can act as complement inhibitor and as a potential tumor suppressor. METHODS: In a syngeneic mouse model of breast cancer, tumors expressing SUSD4 had a smaller volume compared with the corresponding mock control tumors. Additionally, data from three different expression databases and online analysis tools confirm that for breast cancer patients, high mRNA expression of SUSD4 in the tumor tissue correlates with a better prognosis. In vitro experiments utilized triple-negative breast cancer cell lines (BT-20 and MDA-MB-468) stably expressing SUSD4. Moreover, we established a cell line based on BT-20 in which the gene for EGFR was knocked out with the CRISPR-Cas9 method. RESULTS: We discovered that the Epithelial Growth Factor Receptor (EGFR) interacts with SUSD4. Furthermore, triple-negative breast cancer cell lines stably expressing SUSD4 had higher autophagic flux. The initiation of autophagy required the expression of EGFR but not phosphorylation of the receptor. Expression of SUSD4 in the breast cancer cells led to activation of the tumor suppressor LKB1 and consequently to the activation of AMPKα1. Finally, autophagy was initiated after stimulation of the ULK1, Atg14 and Beclin-1 axis in SUSD4 expressing cells. CONCLUSIONS: In this study we provide novel insight into the molecular mechanism of action whereby SUSD4 acts as an EGFR inhibitor without affecting the phosphorylation of the receptor and may potentially influence the recycling of EGFR to the plasma membrane.

Knockout of Sf-Caspase-1 generates apoptosis-resistant Sf9 cell lines: Implications for baculovirus expression.

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 ß-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.

Canonical and non-canonical roles for ATG8 proteins in autophagy and beyond.

During autophagy, the ATG8 family proteins have several well-characterized roles in facilitating early, mid, and late steps of autophagy, including autophagosome expansion, cargo recruitment and autophagosome-lysosome fusion. Their discovery has importantly allowed for precise experimental monitoring of the pathway, bringing about a huge expansion of research in the field over the last decades. In this review, we discuss both canonical and non-canonical roles of the autophagic lipidation machinery, with particular focus on the ATG8 proteins, their post-translational modifications and their increasingly uncovered alternative roles mediated through their anchoring at different membranes. These include endosomes, macropinosomes, phagosomes and the plasma membrane, to which ATG8 proteins can bind through canonical or alternative lipidation. Beyond new ATG8 binding partners and cargo types, we also explore several open questions related to alternative outcomes of autophagic machinery engagement beyond degradation. These include their roles in plasma membrane repair and secretion of selected substrates as well as the physiological implications hereof in health and disease.

Restarting Neglected Tropical Diseases Programs in West Africa during the COVID-19 Pandemic: Lessons Learned and Best Practices.

Countries across West Africa began reporting COVID-19 cases in February 2020. By March, the pandemic began disrupting activities to control and eliminate neglected tropical diseases (NTDs) as health ministries ramped up COVID-19-related policies and prevention measures. This was followed by interim guidance from the WHO in April 2020 to temporarily pause mass drug administration (MDA) and community-based surveys for NTDs. While the pandemic was quickly evolving worldwide, in most of West Africa, governments and health ministries took quick action to implement mitigation measures to slow the spread. The U.S. Agency for International Development's (USAID) Act to End NTDs | West program (Act | West) began liaising with national NTD programs in April 2020 to pave a path toward the eventual resumption of activities. This process consisted of first collecting and analyzing COVID-19 epidemiological data, policies, and standard operating procedures across the program's 11 countries. The program then developed an NTD activity restart matrix that compiled essential considerations to restart activities. By December 2020, all 11 countries in Act | West safely restarted MDA and certain surveys to monitor NTD prevalence or intervention impact. Preliminary results show satisfactory MDA program coverage, meaning that enough people are taking the medicine to keep countries on track toward achieving their NTD disease control and elimination goals, and community perceptions have remained positive. The purpose of this article is to share the lessons and best practices that have emerged from the adoption of strategies to limit the spread of the novel coronavirus during MDA and other program activities.

Implementation of mass drug administration for neglected tropical diseases in Guinea during the COVID-19 pandemic.

BACKGROUND: Guinea reported its first case of COVID-19 on March 12, 2020. Soon thereafter, a national state of emergency was declared, all land borders were closed, schools were shut down, and public gatherings were limited. Many health activities, including field-based activities targeting neglected tropical diseases (NTDs), were paused. The World Health Organization (WHO) issued updated guidance on the resumption of NTD field-based activities on July 27, 2020. In response, the Guinea Ministry of Health (MoH) and its partners planned and resumed mass drug administration (MDA) in mid-August to September 2020 in 19 health districts. METHODOLOGY/PRINCIPAL FINDINGS: A risk-benefit assessment was conducted to identify potential risks associated with the MDA in the COVID-19 context. Following this assessment, a risk mitigation plan with barrier measures was developed to guide MDA implementation. These measures included COVID-19 testing for all national staff leaving Conakry, mask wearing, social distancing of two meters, and hand washing/sanitizing. A checklist was developed and used to monitor compliance to risk mitigation measures. Data on adherence to risk mitigation measures were collected electronically during the MDA. A total of 120 checklists, representing 120 community drug distributor (CDD) teams (two CDDs per team) and 120 households, were completed. Results indicated that washing or disinfecting hands was practiced by 68.3% of CDD teams, compared to 45.0% among households. Face masks to cover the mouth and nose were worn by 79.2% of CDD teams, while this was low among households (23.3%). In 87.5% of households, participants did not touch the dose pole and in 88.3% of CDD teams, CDDs did not touch the hands of the participants while giving the drugs. A large majority of CDD teams (94.2%) and household members (94.2%) were willing to participate in the MDA despite the pandemic. The epidemiological coverage was ≥65% for lymphatic filariasis, onchocerciasis and soil-transmitted helminths in 10 out of 19 HDs and ≥75% for schistosomiasis for school-aged children in 7 out of 11 HDs. CONCLUSIONS/SIGNIFICANCE: Guinea was one of the first countries in Africa to resume MDA activities during the COVID-19 pandemic without causing an observed increase of transmission. The development of a risk mitigation plan and a method to monitor adherence to barrier measures was critical to this unprecedented effort. The rapid incorporation of COVID-19 barrier measures and their acceptance by CDDs and household members demonstrated both the adaptability of the National NTD Program to respond to emerging issues and the commitment of the MoH to implement NTD programs.

Mapping lymphatic filariasis in Loa loa endemic health districts naïve for ivermectin mass administration and situated in the forested zone of Cameroon.

BACKGROUND: The control of lymphatic filariasis (LF) caused by Wuchereria bancrofti in the Central African Region has been hampered by the presence of Loa loa due to severe adverse events that arise in the treatment with ivermectin. The immunochromatographic test (ICT) cards used for mapping LF demonstrated cross-reactivity with L. loa and posed the problem of delineating the LF map. To verify LF endemicity in forest areas of Cameroon where mass drug administration (MDA) has not been ongoing, we used the recently developed strategy that combined serology, microscopy and molecular techniques. METHODS: This study was carried out in 124 communities in 31 health districts (HDs) where L. loa is present. At least 125 persons per site were screened. Diurnal blood samples were investigated for circulating filarial antigen (CFA) by FTS and for L. loa microfilariae (mf) using TBF. FTS positive individuals were further subjected to night blood collection for detecting W. bancrofti. qPCR was used to detect DNA of the parasites. RESULTS: Overall, 14,446 individuals took part in this study, 233 participants tested positive with FTS in 29 HDs, with positivity rates ranging from 0.0 to 8.2%. No W. bancrofti mf was found in the night blood of any individuals but L. loa mf were found in both day and night blood of participants who were FTS positive. Also, qPCR revealed that no W. bancrofti but L.loa DNA was found with dry bloodspot. Positive FTS results were strongly associated with high L. loa mf load. Similarly, a strong positive association was observed between FTS positivity and L loa prevalence. CONCLUSIONS: Using a combination of parasitological and molecular tools, we were unable to find evidence of W. bancrofti presence in the 31 HDs, but L. loa instead. Therefore, LF is not endemic and LF MDA is not required in these districts.

Homicide in pregnant and postpartum women worldwide: a review of the literature.

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.

Baseline trachoma prevalence in Guinea: Results of national trachoma mapping in 31 health districts.

BACKGROUND: Based on previous studies, historical records and risk factors, trachoma was suspected to be endemic in 31 health districts (HDs) in Guinea. To facilitate planning for the elimination of trachoma as a public health problem, national trachoma surveys were conducted between 2011 and 2016 to determine the prevalence of trachomatous inflammation-follicular (TF) and trachomatous trichiasis (TT) in all 31 endemic HDs. METHODOLOGY/PRINCIPAL FINDINGS: A total of 27 cross-sectional surveys were conducted, each using two-stage cluster sampling (one survey in 2011 covered five HDs). Children aged 1-9 years and adults aged ≥15 years were examined for TF and TT, respectively, using the World Health Organization (WHO) simplified grading system. Indicators of household access to water, sanitation and hygiene (WASH) were also collected. A total of 100,051 people from 13,725 households of 556 clusters were examined, of whom 44,899 were male and 55,152 were female. 44,209 children aged 1-9-years and 48,745 adults aged ≥15 years were examined. The adjusted prevalence of TF varied between 1.0% (95%CI: 0.6-1.5%) to 41.8% (95%CI: 39.4-44.2%), while the adjusted prevalence of TT ranged from 0.0% (95%CI: 0.0-0.2%) to 2.8% (95%CI: 2.3-3.5%) in the 27 surveys. In all, 18 HDs had a TF prevalence ≥5% in children aged 1-9 years and 21 HDs had a TT prevalence ≥0.2% in adults aged ≥15 years. There were an estimated 32,737 (95% CI: 19,986-57,811) individuals with TT living in surveyed HDs at the time of surveys. CONCLUSIONS/SIGNIFICANCE: Trachoma is a public health problem in Guinea. 18 HDs required intervention with at least one round of mass drug administration and an estimated 32,737 persons required TT surgery in the country. The results provided clear evidence for Guinea to plan for national trachoma elimination.

Microenvironmental control of breast cancer subtype elicited through paracrine platelet-derived growth factor-CC signaling.

Breast tumors of the basal-like, hormone receptor-negative subtype remain an unmet clinical challenge, as there is high rate of recurrence and poor survival in patients following treatment. Coevolution of the malignant mammary epithelium and its underlying stroma instigates cancer-associated fibroblasts (CAFs) to support 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 paracrine crosstalk 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 of PDGF-CC activity in mouse models of cancer resulted in conversion of basal-like breast cancers into a hormone receptor-positive state that enhanced sensitivity to endocrine therapy in previously resistant tumors. We conclude that specification of breast cancer to the basal-like subtype is under microenvironmental control and is therapeutically actionable.

Prevalence of trachoma in the Kayes region of Mali eight years after stopping mass drug administration.

BACKGROUND: In 2009, three years after stopping mass treatment with azithromycin, a trachoma impact survey in four health districts in the Kayes region of Mali found a prevalence of trachomatous inflammation-follicular (TF) among children aged 1 to 9 years of >5% and a trachomatous trichiasis (TT) prevalence within the general population (≥1-year-old) of <1%. As a result, the government's national trachoma program expanded trichiasis surgery and related activities required to achieve trachoma elimination. METHODOLOGY/PRINCIPAL FINDINGS: In 2015, to assess progress towards elimination, a follow-up impact survey was conducted in the Kayes, Kéniéba, Nioro and Yélimané health districts. The survey used district level two-stage cluster random sampling methodology with 20 clusters of 30 households in each evaluation unit. Subjects were eligible for examination if they were ≥1 year. TF and TT cases were identified and confirmed by experienced ophthalmologists. In total 14,159 people were enumerated and 11,620 (82%) were examined. TF prevalence (95% confidence interval (CI)) was 0.5% (0.3-1%) in Kayes, 0.8% (0.4-1.7%) in Kéniéba, 0.2% (0-0.9%) in Nioro and 0.3% (0.1-1%) in Yélimané. TT prevalence (95% CI) was 0.04% (0-0.25%) in Kayes, 0.29% (0.11-0.6%) in Kéniéba, 0.04% (0-0.25%) in Nioro and 0.07% (0-0.27%) in Yélimané. CONCLUSIONS/SIGNIFICANCE: Eight years after stopping MDA and intensifying trichiasis surgery outreach campaigns, all four districts reached the TF elimination threshold of <5% and three of four districts reached the TT elimination threshold of <0.1%.

Tumor stiffness extends its grip on the metastatic microenvironment.

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.

Cognitive-behaviour therapy for health anxiety in medical patients (CHAMP): a randomised controlled trial with outcomes to 5 years.

BACKGROUND: Health anxiety is an under-recognised but frequent cause of distress that is potentially treatable, but there are few studies in secondary care. OBJECTIVE: To 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. DESIGN: Randomised controlled trial. SETTING: Five general hospitals in London, Middlesex and Nottinghamshire. PARTICIPANTS: A 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. INTERVENTIONS: Cognitive-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 MEASURES: Primary - 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. RESULTS: Of 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 (overall p < 0.0001), with no loss of efficacy between 2 and 5 years. Differences in the generalised anxiety (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. LIMITATIONS: Many eligible patients were not randomised and the population treated may not be representative. CONCLUSIONS: CBT-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 REGISTRATION: Current Controlled Trials ISRCTN14565822. FUNDING: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 21, No. 50. See the NIHR Journals Library website for further project information.

Tumor matrix stiffness promotes metastatic cancer cell interaction with the endothelium.

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 ß-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.

In vitro production of baculoviruses: identifying host and virus genes associated with high productivity.

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.

Cell Culture for Production of Insecticidal Viruses.

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.

Development of Serum-Free Media for Lepidopteran Insect Cell Lines.

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.

Chronic fatigue syndrome.

INTRODUCTION: 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. METHODS AND OUTCOMES: 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). RESULTS: 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. CONCLUSIONS: 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.

Decline in Helicoverpa armigera nucleopolyhedrovirus occlusion body yields with increasing infection cell density in vitro is strongly correlated with viral DNA levels.

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.

Proteomics-based metabolic modeling reveals that fatty acid oxidation (FAO) controls endothelial cell (EC) permeability.

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.