Circadian stress tolerance in adult Caenorhabditis elegans

Circadian rhythms control several behaviors through neural networks, hormones and gene expression. One of these outputs in invertebrates, vertebrates and plants is the stress resistance behavior. In this work, we studied the circadian variation in abiotic stress resistance of adult C. elegans as well as the genetic mechanisms that underlie such behavior. Measuring the stress resistance by tap response behavior we found a rhythm in response to osmotic (NaCl LC(50) = 340 mM) and oxidative (H(2)O(2) LC(50) = 50 mM) shocks, with a minimum at ZT0 (i.e., lights off) and ZT12 (lights on), respectively. In addition, the expression of glutathione peroxidase (C11E4.1) and glycerol-3-phosphate dehydrogenase (gpdh-1) (genes related to the control of stress responses) also showed a circadian fluctuation in basal levels with a peak at night. Moreover, in the mutant osr-1 (AM1 strain), a negative regulator of the gpdh-1 pathway, the osmotic resistance rhythms were masked at 350 mM but reappeared when the strain was treated with a higher NaCl concentration. This work demonstrates for the first time that in the adult nematode, C. elegans stress responses vary daily, and provides evidence of an underlying rhythmic gene expression that governs these behaviors.     

Effect of CMV-immunoglobulins (cytotect biotest) prophylaxis on CMV pneumonia after lung transplantation

Lung transplant (LT) recipients among solid organ transplant recipients are at high risk for cytomegalovirus (CMV) infections. We evaluated the effect of CMV-Immunoglobulins (CMV-IG) (Cytotect Biotest) on CMV pneumonia diagnosed in 303 follow-up transbronchial biopsies (TBB) of lung transplant recipients. 24 patients (control group, 155 TBB from 1999 to 2002) received acyclovir for 24 months and 33 recipients (study group, 148 TBB from 2003 to 2008) received a combined CMV prophylaxis consisting of CMV-IG (Cytotect Biotest) for 12 months and a short Ganciclovir or Valganciclovir therapy from 21th to 42th postoperative day followed by acyclovir up to 24 months. In our study the percentage of pneumonia at first month TBB was similar in the study group vs the control group, 9.1% (3/33) vs 8.3% (2/24), p=0.9 ns, but after the first month the percentage was significantly lower in the study group in the first year at follow-up TBB, 1% (1/99) vs 6.4% (5/78), p=0.048, and in first two years follow-up TBB, 0.8% (1/122) vs 6.5% 8/124), p=0.018 (Statistical analysis: Chi-square test for proportion differences). Our data suggest a strong efficacy of CMV-IG prophylaxis in reducing CMV pneumonia after first month in lung transplant recipients.     

Structure-function relationship in cyclodextrin glycosyltransferase from Bacillus circulans DF 9R

Cyclodextrin glycosyltransferases (CGTases E.C.2.4.1.19) catalyze cyclomaltooligosaccharides (cyclodextrins) production, an important industrial process. We herein report structural features of Bacillus circulans DF 9R cyclodextrin glycosyltransferase including its sequence and several aspects of enzyme structure-function relationship. Protein ethoxyformylation, under our experimental conditions, indicated that only one out of the 13 enzyme histidines was modified leading to a drastic drop in cyclizing and hydrolytic activity. Besides, tryptic digestion of the ¹⁴C ethoxyformylated protein and studies of the peptide mixture showed that histidine 233 is the most reactive histidine residue. This is the first cyclodextrin glycosyltransferase with a known primary structure and a glutamine instead of glycine residue at position 179 in the highly conserved −6 subsite, shown to be involved in substrate binding. The presence of glycine at that position was considered as a requirement for such binding following the induced-fit mechanism already proposed. Moreover, the enzyme has all the features previously described for an α- or α/β-cyclodextrin producer.     

Differential proliferative response of the ventral prostate and seminal vesicle to testosterone replacement

We have investigated epithelial cell proliferation and the rate of glandular recovery of the ventral prostate (VP) and seminal vesicle (SV) promoted by testosterone replacement (TR) in castration-induced regressed glands. Adult male Wistar rats were castrated and, after 21 days, they were treated with testosterone propionate (4 mg/kg/day). Intact (CT) and castrated rats without TR (CS) were also analysed. VP and SV were processed for histochemistry, morphometric-stereological analysis and immunocytochemistry to determine the PCNA index (PI). After 10 days of TR, the VP weight reached approximately 72% of the CT values, while the SV weight exceeded approximately 17% of the CT values. By the third day of TR, VP and SV presented a mean PI of 34% and 94% for distal region and 14% and 22% for proximal region, respectively. SV also had more luminal cells PCNA-positive than VP, mainly in the distal region. The PI values fell on days 5, 7 and 10, but were still higher than CT. These findings indicate that epithelial cells from involuted SV are more responsive to TR than those from VP when stimulated to proliferate and replace the luminal cell population, suggesting a different mechanism regulating cell proliferation in response to androgenic stimuli.     

IL-12, but not IL-18, is critical to neutrophil activation and resistance to polymicrobial sepsis induced by cecal ligation and puncture

Sepsis is a systemic inflammatory response resulting from local infection due, at least in part, to impaired neutrophil migration. IL-12 and IL-18 play an important role in neutrophil migration. We have investigated the mechanism and relative role of IL-12 and IL-18 in polymicrobial sepsis induced by cecal ligation and puncture (CLP) in mice. Wild-type (WT) and IL-18(-/-) mice were resistant to sublethal CLP (SL-CLP) sepsis. In contrast, IL-12(-/-) mice were susceptible to SL-CLP sepsis with high bacteria load in peritoneal cavity and systemic inflammation (serum TNF-alpha and lung neutrophil infiltration). The magnitude of these events was similar to those observed in WT mice with lethal CLP sepsis. The inability of IL-12(-/-) mice to restrict the infection was not due to impairment of neutrophil migration, but correlated with decrease of phagocytosis, NO production, and microbicidal activities of their neutrophils, and with reduction of systemic IFN-gamma synthesis. Consistent with this observation, IFN-gamma(-/-) mice were as susceptible to SL-CLP as IL-12(-/-) mice. Moreover, addition of IFN-gamma to cultures of neutrophils from IL-12(-/-) mice restored their phagocytic, microbicidal activities and NO production. Mortality of IL-12(-/-) mice to SL-CLP was prevented by treatment with IFN-gamma. Thus we show that IL-12, but not IL-18, is critical to an efficient host defense in polymicrobial sepsis. IL-12 acts through induction of IFN-gamma and stimulation of phagocytic and microbicidal activities of neutrophils, rather than neutrophil migration per se. Our data therefore provide further insight into the defense mechanism against this critical area of infectious disease.     

Attachment and detachment of bacteria on surfaces with tunable and switchable wettability

Controlling accumulations of unwanted biofilms requires an understanding of the mechanisms that organisms use to interact with submerged substrata. While the substratum properties influencing biofilm formation are well studied, those that may lead to cellular or biofilm detachment are not. Surface-grafted stimuli-responsive polymers, such as poly (N-isopropylacrylamide) (PNIPAAm) release attached cells upon induction of environmentally-triggered phase changes. Altering the physicochemical characteristics of such polymeric systems for systematically studying release, however, can alter the phase transition. The physico-chemical changes of thin films of PNIPAAm grafted from initiator-modified self-assembled monolayers (SAMs) of ω-substituted alkanethiolates on gold can be altered by changing the composition of the underlying SAM, without affecting the overlying polymer. This work demonstrates that the ability to tune such changes in substratum physico-chemistry allows systematic study of attachment and release of bacteria over a large range of water contact angles. Such surfaces show great promise for studying a variety of interactions at the biointerface. Understanding of the source of this tunability will require further studies into the heterogeneity of such films and further investigation of interactions beyond those of water wettability.     

Homogeneous Canine Chest Phantom Construction: A Tool for Image Quality Optimization

Digital radiographic imaging is increasing in veterinary practice. The use of radiation demands responsibility to maintain high image quality. Low doses are necessary because workers are requested to restrain the animal. Optimizing digital systems is necessary to avoid unnecessary exposure, causing the phenomenon known as dose creep. Homogeneous phantoms are widely used to optimize image quality and dose. We developed an automatic computational methodology to classify and quantify tissues (i.e., lung tissue, adipose tissue, muscle tissue, and bone) in canine chest computed tomography exams. The thickness of each tissue was converted to simulator materials (i.e., Lucite, aluminum, and air). Dogs were separated into groups of 20 animals each according to weight. Mean weights were 6.5 ± 2.0 kg, 15.0 ± 5.0 kg, 32.0 ± 5.5 kg, and 50.0 ± 12.0 kg, for the small, medium, large, and giant groups, respectively. The one-way analysis of variance revealed significant differences in all simulator material thicknesses (p < 0.05) quantified between groups. As a result, four phantoms were constructed for dorsoventral and lateral views. In conclusion, the present methodology allows the development of phantoms of the canine chest and possibly other body regions and/or animals. The proposed phantom is a practical tool that may be employed in future work to optimize veterinary X-ray procedures.     

HMGB1 Mediates Cognitive Impairment in Sepsis Survivors

Severe sepsis, a syndrome that complicates infection and injury, affects 750,000 annually in the United States. The acute mortality rate is approximately 30%, but, strikingly, sepsis survivors have a significant disability burden: up to 25% of survivors are cognitively and physically impaired. To investigate the mechanisms underlying persistent cognitive impairment in sepsis survivors, here we developed a murine model of severe sepsis survivors following cecal ligation and puncture (CLP) to study cognitive impairments. We observed that serum levels of high mobility group box 1 (HMGB1), a critical mediator of acute sepsis pathophysiology, are increased in sepsis survivors. Significantly, these levels remain elevated for at least 4 wks after CLP. Sepsis survivors develop significant, persistent impairments in learning and memory, and anatomic changes in the hippocampus associated with a loss of synaptic plasticity. Administration of neutralizing anti-HMGB1 antibody to survivors, beginning 1 wk after onset of peritonitis, significantly improved memory impairments and brain pathology. Administration of recombinant HMGB1 to na?ve mice recapitulated the memory impairments. Together, these findings indicate that elevated HMGB1 levels mediate cognitive decline in sepsis survivors, and suggest that it may be possible to prevent or reverse cognitive impairments in sepsis survivors by administration of anti-HMGB1 antibodies.