The Sac1 phosphoinositide phosphatase regulates Golgi membrane morphology and mitotic spindle organization in mammals

Phosphoinositides (PIPs) are ubiquitous regulators of signal transduction events in eukaryotic cells. PIPs are degraded by various enzymes, including PIP phosphatases. The integral membrane Sac1 phosphatases represent a major class of such enzymes. The central role of lipid phosphatases in regulating PIP homeostasis notwithstanding, the biological functions of Sac1-phosphatases remain poorly characterized. Herein, we demonstrate that functional ablation of the single murine Sac1 results in preimplantation lethality in the mouse and that Sac1 insufficiencies result in disorganization of mammalian Golgi membranes and mitotic defects characterized by multiple mechanically active spindles. Complementation experiments demonstrate mutant mammalian Sac1 proteins individually defective in either phosphoinositide phosphatase activity, or in recycling of the enzyme from the Golgi system back to the endoplasmic reticulum, are nonfunctional proteins in vivo. The data indicate Sac1 executes an essential household function in mammals that involves organization of both Golgi membranes and mitotic spindles and that both enzymatic activity and endoplasmic reticulum localization are important Sac1 functional properties.     

Identification and localization of a putative ATP-binding cassette transporter in sea lice (Lepeophtheirus salmonis) and host Atlantic salmon (Salmo salar)

Some members of the ABC-transporter superfamily, such as P-glycoprotein and the multidrug resistance associated protein, may confer resistance to the avermectin subclass of macrocyclic lactones. The aim of this study was to examine the presence of ABC transporters in both sea lice (Lepeophtheirus salmonis) and its Atlantic salmon host (Salmo salar) using monoclonal antibodies (C219 and JSB-1, with high selectivity for P-gp) and a new polyclonal antibody (SL0525) generated against a putative sea louse ABC transporter. The antibody raised to SL0525 did not react with rat P-gp, suggesting that an ABC transporter, not necessarily P-gp, was isolated. C219 was the only antibody to localize P-gp in all 3 salmon tissues (intestine, kidney and liver). American lobster (Homarus americanus) was used as a reference crustacean for L. salmonis immunostaining reactions and showed positive staining in the hepatopancreatic and intestinal tissues with all 3 antibodies. The L. salmonis showed positive staining in the intestinal epithelial lining with all antibodies. This report represents the first documented evidence for the expression of ABC transporters in L. salmonis, its Atlantic salmon host, and the American lobster.     

An Evidenced-Based Scale of Disease Severity following Human Challenge with Enteroxigenic Escherichia coli

Background

Experimental human challenge models have played a major role in enhancing our understanding of infectious diseases. Primary outcomes have typically utilized overly simplistic outcomes that fail to entirely account for complex illness syndromes. We sought to characterize clinical outcomes associated with experimental infection with enterotoxigenic Escherichia coli (ETEC) and to develop a disease score.

Methods

Data were obtained from prior controlled human ETEC infection studies. Correlation and univariate regression across sign and symptom severity was performed. A multiple correspondence analysis was conducted. A 3-parameter disease score with construct validity was developed in an iterative fashion, compared to standard outcome definitions and applied to prior vaccine challenge trials.

Results

Data on 264 subjects receiving seven ETEC strains at doses from 1x10⁵ to 1x10¹⁰ cfu were used to construct a standardized dataset. The strongest observed correlation was between vomiting and nausea (r = 0.65); however, stool output was poorly correlated with subjective activity-impacting outcomes. Multiple correspondence analyses showed covariability in multiple signs and symptoms, with severity being the strongest factor corresponding across outcomes. The developed disease score performed well compared to standard outcome definitions and differentiated disease in vaccinated and unvaccinated subjects.

Conclusion

Frequency and volumetric definitions of diarrhea severity poorly characterize ETEC disease. These data support a disease severity score accounting for stool output and other clinical signs and symptoms. Such a score could serve as the basis for better field trial outcomes and gives an additional outcome measure to help select future vaccines that warrant expanded testing in pivotal pre-licensure trials.     

Inactivation of Venezuelan Equine Encephalomyelitis Virus by gamma-Radiation

Exposure of Venezuelan equine encephalomyelitis (VEE) virus (at -70 C) to 6 × 10⁶ r γ-radiation (⁶⁰Co) resulted in loss of lethality for young adult mice and guinea pigs, and loss of capacity to produce plaques or cytopathic effects in tissue culture. The suckling mouse was more sensitive for detecting live virus in radiated suspensions than was the adult mouse or guinea pig. Live virus was demonstrable in preparations exposed to 6 × 10⁶ r but not in suspensions exposed to 8 × 10⁶ r and more. The rate of inactivation of VEE virus by γ-radiation was an exponential function of the dosage.     

Time of ovarian follicle selection during the porcine estrous cycle

Two experiments were conducted to: 1) determine the time during the procine estrous cycle when compensation in ovulation rate after unilateral ovariectomy (ULO) ceases to be complete, 2) compare the follicle selection process in gilts selected for high ovulation rate with unselected control gilts and 3) determine the number of follicles on the right ovary at various stages of the estrous cycle. Experiment I included 25 crossbred gilts, while Experiment II included 17 gilts selected for high ovulation rate and 16 unselected control gilts. The right ovary was removed via a mid-ventral laparotomy on either day 13, 15, 17 or 19 of the cycle. In Experiment I, compensation in ovulation rate ceased between days 13 and 15; whereas, in Experiment II, cessation occurred between days 15 and 17. Selected and control gilts responded alike to ULO, indicating similarity in the follicle selection process. Follicle numbers in the right ovary showed a general decline, especially between days 17 and 19, indicating that atresia was occurring during the follicular phase. The results indicate that the selection of ovarian follicles for ovulation at the ensuing estrus occurs before day 17 of the porcine estrous cyle.     

Lipid Composition of Whole Roots and of Ca2+, Mg2+-activated Adenosine Triphosphatases from Wheat and Oat as Related to Mineral Nutrition

Lipid composition of whole roots of wheat (Triticum vulgare Vill. cv. Svenno Spring Wheat) and oat (Avena sativa L. cv. Brighton) and of cell wall fractions, mitochondrial fractions and microsomal fractions of these roots were studied. Lipid composition depended upon the level of mineral nutrition. In wheat total phospholipids, phosphatidyl choline and sulfolipid content was highest in the roots grown at the higher salt concentration, while the reverse was true for oat roots. In both species glycolipid and sterol content was lower in the high salt roots, at the same time as higher proportions of them were built into the microsomal fraction. Phosphatidyl choline content of the wheat root membrane fractions increased with the salt level, while the opposite occurred in the oat roots. The phosphatidyl choline content may be correlated with the (Ca²⁺, Mg²⁺)-stimulated ATPase activity.     

Self Reported Incidence and Morbidity of Acute Respiratory Illness among Deployed U.S. Military in Iraq and Afghanistan

Background

Historically, respiratory infections have had a significant impact on U.S. military missions. Deployed troops are particularly at high risk due to close living conditions, stressful work environments and increased exposure to pathogens. To date, there are limited data available on acute respiratory illness (ARI) among troops deployed in support of ongoing military operations, specifically Operation Enduring Freedom (OEF) and Operation Iraqi Freedom (OIF).

Methods

Using self-report data from two sources collected from troops deployed to Iraq, Afghanistan and the surrounding region, we analyzed incidence and risk factors for ARI. Military personnel on mid-deployment Rest & Recuperation (R&R) or during redeployment were eligible to participate in the voluntary self-report survey.

Results

Overall, 39.5% reported having at least one ARI. Of these, 18.5% sought medical care and 33.8% reported having decreased job performance. The rate of self-reported ARI was 15 episodes per 100 person-months among those taking the voluntary survey, and 24.7 episodes per 100 person-months among those taking the clinic health questionnaire. Negative binomial regression analysis found female sex, Navy branch of service and lack of flush toilets to be independently associated with increased rates of ARI. Deployment to OIF, increasing age and higher rank were also positively associated with ARI risk.

Conclusions

The overall percentage of deployed military personnel reporting at least one acute respiratory illness decreased since earlier parts of OIF/OEF. However, the reported effect on job performance increased tremendously. The most important factors associated with increased respiratory infection are female sex, Navy branch of service, lack of improved latrine facilities, deployment to OIF, increasing age and higher rank.     

Alveolar macrophage activation is a key initiation signal for acute lung ischemia-reperfusion injury

Lung ischemia-reperfusion (I/R) injury is a biphasic inflammatory process. Previous studies indicate that the later phase is neutrophil-dependent and that alveolar macrophages (AMs) likely contribute to the acute phase of lung I/R injury. However, the mechanism is unclear. AMs become activated and produce various cytokines and chemokines in many inflammatory responses, including transplantation. We hypothesize that AMs respond to I/R by producing key cytokines and chemokines and that depletion of AMs would reduce cytokine/chemokine expression and lung injury after I/R. To test this, using a buffer-perfused, isolated mouse lung model, we studied the impact of AM depletion by liposome-clodronate on I/R-induced lung dysfunction/injury and expression of cytokines/chemokines. I/R caused a significant increase in pulmonary artery pressure, wet-to-dry weight ratio, vascular permeability, tumor necrosis factor (TNF)-alpha, monocyte chemoattractant protein (MCP)-1, and macrophage inflammatory protein (MIP)-2 expression, as well as decreased pulmonary compliance, when compared with sham lungs. After AM depletion, the changes in each of these parameters between I/R and sham groups were significantly attenuated. Thus AM depletion protects the lungs from I/R-induced dysfunction and injury and significantly reduces cytokine/chemokine production. Protein expression of TNF-alpha and MCP-1 are positively correlated to I/R-induced lung injury, and AMs are a major producer/initiator of TNF-alpha, MCP-1, and MIP-2. We conclude that AMs are an essential player in the initiation of acute lung I/R injury.