Isolation and Culture of Larval Cells from C. elegans

Cell culture is an essential tool to study cell function. In C. elegans the ability to isolate and culture cells has been limited to embryonically derived cells. However, cells or blastomeres isolated from mixed stage embryos terminally differentiate within 24 hours of culture, thus precluding post-embryonic stage cell culture. We have developed an efficient and technically simple method for large-scale isolation and primary culture of larval-stage cells. We have optimized the treatment to maximize cell number and minimize cell death for each of the four larval stages. We obtained up to 7.8×104 cells per microliter of packed larvae, and up to 97% of adherent cells isolated by this method were viable for at least 16 hours. Cultured larval cells showed stage-specific increases in both cell size and multinuclearity and expressed lineage- and cell type-specific reporters. The majority (81%) of larval cells isolated by our method were muscle cells that exhibited stage-specific phenotypes. L1 muscle cells developed 1 to 2 wide cytoplasmic processes, while L4 muscle cells developed 4 to 14 processes of various thicknesses. L4 muscle cells developed bands of myosin heavy chain A thick filaments at the cell center and spontaneously contracted ex vivo. Neurons constituted less than 10% of the isolated cells and the majority of neurons developed one or more long, microtubule-rich protrusions that terminated in actin-rich growth cones. In addition to cells such as muscle and neuron that are high abundance in vivo, we were also able to isolate M-lineage cells that constitute less than 0.2% of cells in vivo. Our novel method of cell isolation extends C. elegans cell culture to larval developmental stages, and allows use of the wealth of cell culture tools, such as cell sorting, electrophysiology, co-culture, and high-resolution imaging of subcellular dynamics, in investigation of post-embryonic development and physiology

Family involvement and firms’ establishment mode choice in foreign markets

Extant literature on foreign entry increasingly recognizes firms’ heterogeneity as a potential reason for inconsistency in results on the establishment mode choice, i.e. whether and under which conditions firms should choose to enter a new country through a greenfield investment or an acquisition. Our study contributes to this debate by identifying family ownership and family involvement in management as potential powerful sources of such heterogeneity. Integrating international business studies with both corporate finance literature on family firms and recent contributions from the Socio Emotional Wealth perspective on family ownership, we claim that, due to greater risk aversion and lower access to information, the family involvement either in the firm ownership and management leads to a higher propensity towards greenfield initiatives (vs. acquisitions). However, we also find that such a propensity decreases with international experience especially in family-owned firms given the greater ability of professionalized management to overcome family-related concerns on making acquisitions. Our analysis on 1,045 foreign initiatives undertaken by 311 Italian family and non-family firms between 2003 and 2013 confirms our expectations – indicating family ownership as a significant driver of international business choices

Glutathione S-transferase genotypes modify lung function decline in the general population: SAPALDIA cohort study

BACKGROUND: Understanding the environmental and genetic risk factors of accelerated lung function decline in the general population is a first step in a prevention strategy against the worldwide increasing respiratory pathology of chronic obstructive pulmonary disease (COPD). Deficiency in antioxidative and detoxifying Glutathione S-transferase (GST) gene has been associated with poorer lung function in children, smokers and patients with respiratory diseases. In the present study, we assessed whether low activity variants in GST genes are also associated with accelerated lung function decline in the general adult population. METHODS: We examined with multiple regression analysis the association of polymorphisms in GSTM1, GSTT1 and GSTP1 genes with annual decline in FEV1, FVC, and FEF(25–75 )during 11 years of follow-up in 4686 subjects of the prospective SAPALDIA cohort representative of the Swiss general population. Effect modification by smoking, gender, bronchial hyperresponisveness and age was studied. RESULTS: The associations of GST genotypes with FEV1, FVC, and FEF(25–75 )were comparable in direction, but most consistent for FEV1. GSTT1 homozygous gene deletion alone or in combination with GSTM1 homozygous gene deletion was associated with excess decline in FEV1 in men, but not women, irrespective of smoking status. The additional mean annual decline in FEV1 in men with GSTT1 and concurrent GSTM1 gene deletion was -8.3 ml/yr (95% confidence interval: -12.6 to -3.9) relative to men without these gene deletions. The GSTT1 effect on the FEV1 decline comparable to the observed difference in FEV1 decline between never and persistent smoking men. Effect modification by gender was statistically significant. CONCLUSION: Our results suggest that genetic GSTT1 deficiency is a prevalent and strong determinant of accelerated lung function decline in the male general population

Biochemical warfare on the reef : the role of glutathione transferases in consumer tolerance of dietary prostaglandins

© 2010 The Authors. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS ONE 5 (2010): e8537, doi:10.1371/journal.pone.0008537.Despite the profound variation among marine consumers in tolerance for allelochemically-rich foods, few studies have examined the biochemical adaptations underlying diet choice. Here we examine the role of glutathione S-transferases (GSTs) in the detoxification of dietary allelochemicals in the digestive gland of the predatory gastropod Cyphoma gibbosum, a generalist consumer of gorgonian corals. Controlled laboratory feeding experiments were used to investigate the influence of gorgonian diet on Cyphoma GST activity and isoform expression. Gorgonian extracts and semi-purified fractions were also screened to identify inhibitors and possible substrates of Cyphoma GSTs. In addition, we investigated the inhibitory properties of prostaglandins (PGs) structurally similar to antipredatory PGs found in high concentrations in the Caribbean gorgonian Plexaura homomalla. Cyphoma GST subunit composition was invariant and activity was constitutively high regardless of gorgonian diet. Bioassay-guided fractionation of gorgonian extracts revealed that moderately hydrophobic fractions from all eight gorgonian species examined contained putative GST substrates/inhibitors. LC-MS and NMR spectral analysis of the most inhibitory fraction from P. homomalla subsequently identified prostaglandin A2 (PGA2) as the dominant component. A similar screening of commercially available prostaglandins in series A, E, and F revealed that those prostaglandins most abundant in gorgonian tissues (e.g., PGA2) were also the most potent inhibitors. In vivo estimates of PGA2 concentration in digestive gland tissues calculated from snail grazing rates revealed that Cyphoma GSTs would be saturated with respect to PGA2 and operating at or near physiological capacity. The high, constitutive activity of Cyphoma GSTs is likely necessitated by the ubiquitous presence of GST substrates and/or inhibitors in this consumer's gorgonian diet. This generalist's GSTs may operate as ‘all-purpose’ detoxification enzymes, capable of conjugating or sequestering a broad range of lipophilic gorgonian compounds, thereby allowing this predator to exploit a range of chemically-defended prey, resulting in a competitive dietary advantage for this species.Financial support for this work was provided by the Ocean Life Institute Tropical Research Initiative Grant (WHOI) to KEW and MEH; the Robert H. Cole Endowed Ocean Ventures Fund (WHOI) to KEW; the National Undersea Research Center - Program Development Proposal (CMRC-03PRMN0103A) to KEW; Walter A. and Hope Noyes Smith, and a National Science Foundation Graduate Research Fellowship to KEW

Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock, 2012

OBJECTIVE: To provide an update to the "Surviving Sepsis Campaign Guidelines for Management of Severe Sepsis and Septic Shock," last published in 2008. DESIGN: A consensus committee of 68 international experts representing 30 international organizations was convened. Nominal groups were assembled at key international meetings (for those committee members attending the conference). A formal conflict of interest policy was developed at the onset of the process and enforced throughout. The entire guidelines process was conducted independent of any industry funding. A stand-alone meeting was held for all subgroup heads, co- and vice-chairs, and selected individuals. Teleconferences and electronic-based discussion among subgroups and among the entire committee served as an integral part of the development. METHODS: The authors were advised to follow the principles of the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system to guide assessment of quality of evidence from high (A) to very low (D) and to determine the strength of recommendations as strong (1) or weak (2). The potential drawbacks of making strong recommendations in the presence of low-quality evidence were emphasized. Recommendations were classified into three groups: (1) those directly targeting severe sepsis; (2) those targeting general care of the critically ill patient and considered high priority in severe sepsis; and (3) pediatric considerations. RESULTS: Key recommendations and suggestions, listed by category, include: early quantitative resuscitation of the septic patient during the first 6 h after recognition (1C); blood cultures before antibiotic therapy (1C); imaging studies performed promptly to confirm a potential source of infection (UG); administration of broad-spectrum antimicrobials therapy within 1 h of the recognition of septic shock (1B) and severe sepsis without septic shock (1C) as the goal of therapy; reassessment of antimicrobial therapy daily for de-escalation, when appropriate (1B); infection source control with attention to the balance of risks and benefits of the chosen method within 12 h of diagnosis (1C); initial fluid resuscitation with crystalloid (1B) and consideration of the addition of albumin in patients who continue to require substantial amounts of crystalloid to maintain adequate mean arterial pressure (2C) and the avoidance of hetastarch formulations (1B); initial fluid challenge in patients with sepsis-induced tissue hypoperfusion and suspicion of hypovolemia to achieve a minimum of 30 mL/kg of crystalloids (more rapid administration and greater amounts of fluid may be needed in some patients (1C); fluid challenge technique continued as long as hemodynamic improvement is based on either dynamic or static variables (UG); norepinephrine as the first-choice vasopressor to maintain mean arterial pressure ≥65 mmHg (1B); epinephrine when an additional agent is needed to maintain adequate blood pressure (2B); vasopressin (0.03 U/min) can be added to norepinephrine to either raise mean arterial pressure to target or to decrease norepinephrine dose but should not be used as the initial vasopressor (UG); dopamine is not recommended except in highly selected circumstances (2C); dobutamine infusion administered or added to vasopressor in the presence of (a) myocardial dysfunction as suggested by elevated cardiac filling pressures and low cardiac output, or (b) ongoing signs of hypoperfusion despite achieving adequate intravascular volume and adequate mean arterial pressure (1C); avoiding use of intravenous hydrocortisone in adult septic shock patients if adequate fluid resuscitation and vasopressor therapy are able to restore hemodynamic stability (2C); hemoglobin target of 7-9 g/dL in the absence of tissue hypoperfusion, ischemic coronary artery disease, or acute hemorrhage (1B); low tidal volume (1A) and limitation of inspiratory plateau pressure (1B) for acute respiratory distress syndrome (ARDS); application of at least a minimal amount of positive end-expiratory pressure (PEEP) in ARDS (1B); higher rather than lower level of PEEP for patients with sepsis-induced moderate or severe ARDS (2C); recruitment maneuvers in sepsis patients with severe refractory hypoxemia due to ARDS (2C); prone positioning in sepsis-induced ARDS patients with a PaO (2)/FiO (2) ratio of ≤100 mm Hg in facilities that have experience with such practices (2C); head-of-bed elevation in mechanically ventilated patients unless contraindicated (1B); a conservative fluid strategy for patients with established ARDS who do not have evidence of tissue hypoperfusion (1C); protocols for weaning and sedation (1A); minimizing use of either intermittent bolus sedation or continuous infusion sedation targeting specific titration endpoints (1B); avoidance of neuromuscular blockers if possible in the septic patient without ARDS (1C); a short course of neuromuscular blocker (no longer than 48 h) for patients with early ARDS and a PaO (2)/FI O (2) 180 mg/dL, targeting an upper blood glucose ≤180 mg/dL (1A); equivalency of continuous veno-venous hemofiltration or intermittent hemodialysis (2B); prophylaxis for deep vein thrombosis (1B); use of stress ulcer prophylaxis to prevent upper gastrointestinal bleeding in patients with bleeding risk factors (1B); oral or enteral (if necessary) feedings, as tolerated, rather than either complete fasting or provision of only intravenous glucose within the first 48 h after a diagnosis of severe sepsis/septic shock (2C); and addressing goals of care, including treatment plans and end-of-life planning (as appropriate) (1B), as early as feasible, but within 72 h of intensive care unit admission (2C). Recommendations specific to pediatric severe sepsis include: therapy with face mask oxygen, high flow nasal cannula oxygen, or nasopharyngeal continuous PEEP in the presence of respiratory distress and hypoxemia (2C), use of physical examination therapeutic endpoints such as capillary refill (2C); for septic shock associated with hypovolemia, the use of crystalloids or albumin to deliver a bolus of 20 mL/kg of crystalloids (or albumin equivalent) over 5-10 min (2C); more common use of inotropes and vasodilators for low cardiac output septic shock associated with elevated systemic vascular resistance (2C); and use of hydrocortisone only in children with suspected or proven "absolute"' adrenal insufficiency (2C). CONCLUSIONS: Strong agreement existed among a large cohort of international experts regarding many level 1 recommendations for the best care of patients with severe sepsis. Although a significant number of aspects of care have relatively weak support, evidence-based recommendations regarding the acute management of sepsis and septic shock are the foundation of improved outcomes for this important group of critically ill patients

Evaluation of appendicitis risk prediction models in adults with suspected appendicitis

Background Appendicitis is the most common general surgical emergency worldwide, but its diagnosis remains challenging. The aim of this study was to determine whether existing risk prediction models can reliably identify patients presenting to hospital in the UK with acute right iliac fossa (RIF) pain who are at low risk of appendicitis. Methods A systematic search was completed to identify all existing appendicitis risk prediction models. Models were validated using UK data from an international prospective cohort study that captured consecutive patients aged 16–45 years presenting to hospital with acute RIF in March to June 2017. The main outcome was best achievable model specificity (proportion of patients who did not have appendicitis correctly classified as low risk) whilst maintaining a failure rate below 5 per cent (proportion of patients identified as low risk who actually had appendicitis). Results Some 5345 patients across 154 UK hospitals were identified, of which two‐thirds (3613 of 5345, 67·6 per cent) were women. Women were more than twice as likely to undergo surgery with removal of a histologically normal appendix (272 of 964, 28·2 per cent) than men (120 of 993, 12·1 per cent) (relative risk 2·33, 95 per cent c.i. 1·92 to 2·84; P < 0·001). Of 15 validated risk prediction models, the Adult Appendicitis Score performed best (cut‐off score 8 or less, specificity 63·1 per cent, failure rate 3·7 per cent). The Appendicitis Inflammatory Response Score performed best for men (cut‐off score 2 or less, specificity 24·7 per cent, failure rate 2·4 per cent). Conclusion Women in the UK had a disproportionate risk of admission without surgical intervention and had high rates of normal appendicectomy. Risk prediction models to support shared decision‐making by identifying adults in the UK at low risk of appendicitis were identified