Corneal Changes and Strategies to Improve Survival of Hypomorphic Collagen VII-Deficient Mice for the Study of Ocular Dystrophic Epidermolysis Bullosa

Ophthalmic study of collagen CVII hypomorphic mice is uniquely challenging due to the strain’s published survival rate to weaning of 24%. Because chronic ocular fibrosis requires time to develop, optimizing the survival rate is of critical importance. In this study, standard husbandry practices were enhanced by the addition of sterilized diet and drug delivery gels, acidified water, irradiated food pellets, cellulose fiber bedding, minimal handling, removal of siblings within 2-3 wk from birth, and a preferred housing location. Survival rates per breeding cycle, sex, weight, and cause of early euthanasia were recorded and analyzed over 43 mo. Overall, 49% of mice survived to weaning and 76% of weaned mice survived to 20 wk of age. Corneal opacities were seen in 65% of mice by 20 wk, but only 10% of eyes showed the sustained opacification that was indicative of fibrosis. Corneal opacities occurred at the same rate as in humans with epidermolysis bullosa. 66% of the mice showed weight loss at 11 wk. Males required early euthanasia 4 times more often than did females. Euthanasia was required for urinary obstruction due to penile prolapse in 88% of males. With our enhanced care protocol, hypomorphic mice in our colony survived at twice the published rate. With this revised husbandry standard, experiments planned with termination endpoints of 14 wk for males and 17 wk for females are more likely to reach completion.     

A long-term porcine model for measurement of gastrointestinal motility

Animal models have become an essential tool in the investigations of gut motility under experimental conditions. To determine the influence of various anaesthetic drugs on the motility pattern of the gastroduodenal tract, a new long-term model has had to be developed for allowing measurements in conscious and unrestrained as well as in sedated and analgosedated pigs. Since mechanical ventilation influences gut motility, it was necessary that this animal model enabled the investigation of the effect of drugs causing sedation and analgosedation during spontaneous breathing. Seven male, castrated pigs, German landrace, 32-40 kg bodyweight (BW) were investigated in this study. After habituation of the pigs to local housing conditions over 5 days, the animals were trained over 4 days to prepare for experimental situations and investigators. Pigs were inserted with a central venous catheter and with percutaneous enterogastrostomy (PEG) under general anaesthesia. Intestinal motility was measured by intraluminal impedancometry. The catheter was introduced over the PEG into the stomach and positioned into the duodenum by duodenoscopy. Measurements were done in conscious, unrestrained pigs and with sedated, and analgosedated animals on subsequent days. The habituation and training of the pigs to the investigators and for the laboratory conditions took between 7 and 9 days. The initial anaesthesia protocol for the instrumentation using remifentanil/propofol led to pyloric spasm and was thus unsuitable for duodenal intubation with an endoscope. In contrast, a combination of ketamine/propofol enabled this procedure. It was practicable to measure gut motility in conscious, unrestrained pigs. Spontaneous breathing was sufficient under propofol sedation and analgosedation using fentanyl-propofol. Systematically local application of polividon iodine in the area of the subcutaneous catheters avoided the necessity of using systemic prophylactic antibiotics. In conclusion, the habituation and training for 9 days enabled the measurement of gut motility by intraluminal impedancometry in conscious pigs. The insertion of the catheter was done during general anaesthesia using a combination of propofol and ketamine. For the future determination of gut motility performed under general anaesthesia, each sedation and analgosedation concept has to be evaluated to see whether it allows spontaneous breathing or whether mechanical ventilation is necessary.     

The UCS domain protein She4p binds to myosin motor domains and is essential for class I and class V myosin function

BACKGROUND: Myosins are motor proteins involved in processes like cell motility, vesicle transport, or cytokinesis. In a variety of organisms, a novel group of proteins forming the UCS (UNC-45/CRO1/SHE4) domain-containing family are essential for proper myosin function. The Saccharomyces cerevisae UCS domain protein She4p is involved in two myosin-requiring events, endocytosis and mRNA localization. RESULTS: In contrast to UCS domain proteins from other organisms that interact with class II myosins, we demonstrate that She4p associates with yeast class I and class V myosins. She4p binds to motor domains of class V myosin Myo4p and class I myosin Myo5p, and this binding depends on She4p's UCS domain. In vivo, She4p is essential for the function and localization of Myo3p, Myo4p, and Myo5p (but not of Myo2p) and for colocalization of class I myosins with cortical actin patches. In vitro, She4p stimulates binding of Myo5p to filamentous actin. Wild-type She4p, but not a mutant lacking the UCS domain, accumulates in a cap-like structure at the bud tip. This localization requires Myo2p and actin, suggesting a Myo2-dependent mechanism by which She4p is targeted to the bud cap. Localization of She4p is essential for proper positioning and myosin-actin association of cortical Myo5p. CONCLUSIONS: Our results suggest that She4p is a novel myosin motor domain binding protein and operates as a localized regulator of myosin function of class I and likely class V myosins.     

Impaired insulin secretion and glucose tolerance in beta cell-selective Ca(v)1.2 Ca2+ channel null mice

Insulin is secreted from pancreatic beta cells in response to an elevation of cytoplasmic Ca(2+) resulting from enhanced Ca(2+) influx through voltage-gated Ca(2+) channels. Mouse beta cells express several types of Ca(2+) channel (L-, R- and possibly P/Q-type). beta cell-selective ablation of the gene encoding the L-type Ca(2+) channel subtype Ca(v)1.2 (betaCa(v)1.2(-/-) mouse) decreased the whole-cell Ca(2+) current by only approximately 45%, but almost abolished first-phase insulin secretion and resulted in systemic glucose intolerance. These effects did not correlate with any major effects on intracellular Ca(2+) handling and glucose-induced electrical activity. However, high-resolution capacitance measurements of exocytosis in single beta cells revealed that the loss of first-phase insulin secretion in the betaCa(v)1.2(-/-) mouse was associated with the disappearance of a rapid component of exocytosis reflecting fusion of secretory granules physically attached to the Ca(v)1.2 channel. Thus, the conduit of Ca(2+) entry determines the ability of the cation to elicit secretion.     

Conquered from the Deep Sea? A New Deep-Sea Isopod Species from the Antarctic Shelf Shows Pattern of Recent Colonization

The Amundsen Sea, Antarctica, is amongst the most rapidly changing environments of the world. Its benthic inhabitants are barely known and the BIOPEARL 2 project was one of the first to biologically explore this region. Collected during this expedition, Macrostylis roaldi sp. nov. is described as the first isopod discovered on the Amundsen-Sea shelf. Amongst many characteristic features, the most obvious characters unique for M. roaldi are the rather short pleotelson and short operculum as well as the trapezoid shape of the pleotelson in adult males. We used DNA barcodes (COI) and additional mitochondrial markers (12S, 16S) to reciprocally illuminate morphological results and nucleotide variability. In contrast to many other deep-sea isopods, this species is common and shows a wide distribution. Its range spreads from Pine Island Bay at inner shelf right to the shelf break and across 1,000 m bathymetrically. Its gene pool is homogenized across space and depth. This is indicative for a genetic bottleneck or a recent colonization history. Our results suggest further that migratory or dispersal capabilities of some species of brooding macrobenthos have been underestimated. This might be relevant for the species’ potential to cope with effects of climate change. To determine where this species could have survived the last glacial period, alternative refuge possibilities are discussed.     

Uptake and biotransformation of pure commercial microcystin-LR versus microcystin-LR from a natural cyanobacterial bloom extract in the aquatic fungus <Emphasis Type="Italic">Mucor hiemalis</Emphasis>

Objectives

To evaluate the remediation efficiency of Mucor hiemalis by comparing media elimination, uptake, and biotransformation of microcystin-LR with exposure to pure toxin versus a crude bloom extract.

Results

With exposure to the extract, the elimination rate of microcystin-LR from the media, which was 0.28 ng MC-LR l^?1 h^?1, was significantly higher compared to that achieved with exposure to the pure toxin (0.16 ng MC-LR l^?1 h^?1) after 24 h. However, intracellular breakdown of microcystin-LR was significantly lower in the extract exposed pellets compared to the pure toxin treated fungal pellets over time. This coincided with reduced intracellular glutathione S-transferase activity with crude extract exposure which could be responsible for the detection of only the glutathione conjugate of microcystin-LR.

Conclusion

This paper signifies the importance of using laboratory exposure scenarios which resemble conditions in nature to fully understand and evaluate remediation efficiency. There is merit in using M. hiemalis for mycoremediation of cyanotoxins in surface waters.

     

The "ins" and "outs" of the high-affinity choline transporter CHT1

Maintenance of acetylcholine (ACh) synthesis depends on the activity of the high-affinity choline transporter (CHT1), which is responsible for the reuptake of choline from the synaptic cleft into presynaptic neurons. In this review, we discuss the current understanding of mechanisms involved in the cellular trafficking of CHT1. CHT1 protein is mainly found in intracellular organelles, such as endosomal compartments and synaptic vesicles. The presence of CHT1 at the plasma membrane is limited by rapid endocytosis of the transporter in clathrin-coated pits in a mechanism dependent on a dileucine-like motif present in the carboxyl-terminal region of the transporter. The intracellular pool of CHT1 appears to constitute a reserve pool of transporters, important for maintenance of cholinergic neurotransmission. However, the physiological basis of the presence of CHT1 in intracellular organelles is not fully understood. Current knowledge about CHT1 indicates that stimulated and constitutive exocytosis, in addition to endocytosis, will have major consequences for regulating choline uptake. Future investigations of CHT1 trafficking should elucidate such regulatory mechanisms, which may aid in understanding the pathophysiology of diseases that affect cholinergic neurons, such as Alzheimer's disease.     

Soil-transmitted helminth infection,anemia, and malnutrition among preschool-age children in Nangapanda subdistrict,Indonesia

BackgroundSoil-transmitted helminth (STH) infections are still prevalent in Indonesia, with roughly one-third of infected population being preschool-age children (PSC), which are generally at higher risk of morbidity such as malnutrition and anemia. This study aimed to investigate the association of STH infections with nutritional status and anemia among PSC in Nangapanda subdistrict, Ende, East Nusa Tenggara.MethodsA cross-sectional survey involving PSC ranging from 12 to 59 months old from Nangapanda subdistrict, Ende district, East Nusa Tenggara was performed. Socio-demographic, breastfeeding, and complementary feeding information was obtained from structured questionnaires, while nutritional and anemia status was determined from anthropometry and hemoglobin measurements, respectively. Anthropometric z-scores were calculated based on the World Health Organization 2006 standards and stool samples were examined using Kato-Katz method.ResultsA total of 393 PSC randomly selected from 22 villages were examined. The prevalence of underweight, stunting, wasting, and anemia were 33.1%, 40.2%, 17.1%, and 60.3%, respectively. STH infection, predominated by Ascaris lumbricoides, was found in 160 (58.8%) PSC. Single STH infection, but not multiple infection, was independently associated with a lower risk of anemia (odds ratio [OR] 0.320, 95% confidence interval [CI]: 0.126–0.809, p = 0.016). Similar association with anemia was also found on mild STH infection (OR 0.318 [95% CI: 0.114–0.887], p = 0.029). On the other hand, younger children were found to have a higher risk of anemia and stunting. None of the examined variables were independently associated with underweight and wasting.ConclusionSTH infection as well as anemia and malnutrition were prevalent in this region. However in this study, current STH infections seemed to have minimal negative impact on children’s nutritional status.     

Immunohistochemical analysis of muscle cytochromec oxidase deficiency in children

Despite the demonstration of a clear biochemical defect, the genetic alterations causing childhood forms of cytochromec oxidase (COX) deficiency remain unknown. The double genetic origin (nuclear and mitochondrial DNA), and the complexity of COX enzyme structure and regulation, indicate the need for genetic iinvestigations of the molecular structure of individual COX subunits. In the present study a new monoclonal antibody, which reacts exclusively with heart-type human COX subunit VIIa (VIIa-H), and other monoclonal antibodies against human COX subunits, were used in the immunohistochemical analysis of skeletal muscle from children with different forms of mitochondrial myopathy with COX deficiency. By immunohistochemical investigation a normal reaction was seenn with antibodies to COX subunits IV, Va+Vb, and VIa+VIc in all four cases, and in two cases with antibodies to COX VIIa-H and VIIa+VIIb. In muscle from a fatal infantile case with cardiac and skeletal muscle involvement, no immunohistochemical reaction was seen with the monoclonal antibody against the tissue-specific subunit VIIa-H. In muscle from an 11-year-old boy with exclusive muscular symptoms and signs, immunohistological reactions were absent with COX subunit VIIa-H and COX subunits VIIa+VIIb, and slightly decreased with COX subunit II, thus demonstrating a different molecular mechanism in each case. It is concluded that the molecular basis of COX deficiency in childhood may vary greatly between patients.     

Food perception with age and its relationship to pleasantness

Differences between elderly subjects (n = 46, 61-86 years) and young subjects (n = 36, 18-25 years) in food perception and food liking were investigated. Intensity and liking ratings were assessed for custard dessert, in which flavor enrichment, textural change, and irritant addition were incorporated as strategies to compensate for sensory losses with increasing age. The sensory acuity (taste, olfaction, irritation, chewing efficiency) of both young and elderly subjects was measured with the help of different sensitivity tests. The elderly perceived the custards differently from the young, mainly as less intense in flavor (cherry/vanilla) and less intense in creaminess/swallowing effort. Several of the observed interaction effects were different for the elderly and the young. The majority of these differences manifested as lower intensity slopes for the elderly. Losses in sensitivity to taste and to olfactory and trigeminal stimuli as well as a reduced chewing efficiency were observed on average for the elderly compared with the young. Furthermore, subgroups of the elderly were observed in which the compensatory strategies flavor enrichment, textural change, and irritant addition led to an increase in food liking. However, these subgroups did not differ in their sensory acuity. The present study does not support the assumption that age-associated changes in food perception-caused by losses in sensory acuity-inevitably reduce the food liking of the elderly.