Localization of [gamma]-Glutamylcysteine Synthetase and Glutathione Synthetase Activity in Maize Seedlings

Fresh weight, protein, cysteine, [gamma]-glutamylcysteine, glutathione, and the extractable activity of the enzymes of glutathione biosynthesis, [gamma]-glutamylcysteine synthetase (EC 6.3.2.2) and glutathione synthetase (EC 6.3.2.3), were measured in roots, scutella, endosperms, and shoots of 3-, 7-, and 11-d-old maize (Zea mays L. cv LG 9) seedlings. In 3-d-old seedlings, the scutella represented 14% of the seedling fresh weight, containing 43% of total protein and 63 and 55% of the activity of [gamma]-glutamylcysteine synthetase and glutathione synthetase, respectively; in 11-d-old seedlings, the corresponding values were 4.5% for fresh weight, 8.0% for protein content, and 14 and 20% for the enzyme activities. The highest concentrations of thiols were found for cysteine (0.27 mM) in the roots, for glutathione (4.4 mM) in the shoots, and for [gamma]-glutamylcysteine (13 [mu]M) in the scutella of 3-d-old seedlings. The enzyme activities of roots were localized in subcellular fractions after sucrose density gradient centrifugation. Nearly half of the [gamma]-glutamylcysteine synthetase activity was detected in the root proplastids of 4-d-old seedlings, whereas <10% of the glutathione synthetase activity was localized in this organelle. Our results demonstrate the importance of scutella in glutathione synthesis in the early stage of seedling development. Unlike chloroplasts, root plastids show only a small proportion of glutathione synthetase activity

Rats Selectively Bred for High Voluntary Physical Activity Behavior are Not Protected from the Deleterious Metabolic Effects of a Western Diet When Sedentary

Background: Physical activity and diet are well-established modifiable factors that influence chronic disease risk. We developed a selectively bred, polygenic model for high and low voluntary running (HVR and LVR, respectively) distances. After 8 generations, large differences in running distance were noted. Despite these inherent behavioral differences in physical activity levels, it is unknown whether HVR rats would be inherently protected from diet-induced metabolic dysfunction. Objectives: The aim of this study was to determine whether HVR rats without voluntary running wheels would be inherently protected from diet-induced metabolic dysfunction. Methods: Young HVR, LVR, and a wild-type (WT) control group were housed with no running wheel access and fed either a normal diet (ND) or a high-sugar/fat Western diet (WD) for 8 wk. Body weight, percentage body fat (by dual-energy X-ray absorptiometry scan), blood lipids [total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides (TGs), nonesterified fatty acids], and hepatic TG content were measured, and indices of insulin sensitivity were determined via an intravenous glucose tolerance test. Additionally, weekly energy intake and feed efficiency were calculated. Results: After 8 wk, significant differences in body weight and body fat percentage were noted in all WD animals compared with ND animals, with the LVR-WD exhibiting the greatest increase due, in part, to their enhanced feed efficiency. Lipid dysregulation was present in all WD rat lines compared with ND counterparts. Furthermore, LVR-WD rats had higher total cholesterol, HDL cholesterol, and TG concentrations, and higher areas under the curve (AUC) for insulin than HVR-WD and WT-WD, although HVR-WD animals had higher AUCglucose than both LVR-WD and WT-WD and higher LDL than WT-WD. Conclusions: In the absence of high voluntary running behavior, the genetic predisposition for high running in HVR did not largely protect them from the deleterious effects of a WD compared with LVR, suggesting genetic factors influencing physical activity levels may, in part, be independent from genes influencing metabolism

Nondestructive analysis of urinary calculi using micro computed tomography

BACKGROUND: Micro computed tomography (micro CT) has been shown to provide exceptionally high quality imaging of the fine structural detail within urinary calculi. We tested the idea that micro CT might also be used to identify the mineral composition of urinary stones non-destructively. METHODS: Micro CT x-ray attenuation values were measured for mineral that was positively identified by infrared microspectroscopy (FT-IR). To do this, human urinary stones were sectioned with a diamond wire saw. The cut surface was explored by FT-IR and regions of pure mineral were evaluated by micro CT to correlate x-ray attenuation values with mineral content. Additionally, intact stones were imaged with micro CT to visualize internal morphology and map the distribution of specific mineral components in 3-D. RESULTS: Micro CT images taken just beneath the cut surface of urinary stones showed excellent resolution of structural detail that could be correlated with structure visible in the optical image mode of FT-IR. Regions of pure mineral were not difficult to find by FT-IR for most stones and such regions could be localized on micro CT images of the cut surface. This was not true, however, for two brushite stones tested; in these, brushite was closely intermixed with calcium oxalate. Micro CT x-ray attenuation values were collected for six minerals that could be found in regions that appeared to be pure, including uric acid (3515 – 4995 micro CT attenuation units, AU), struvite (7242 – 7969 AU), cystine (8619 – 9921 AU), calcium oxalate dihydrate (13815 – 15797 AU), calcium oxalate monohydrate (16297 – 18449 AU), and hydroxyapatite (21144 – 23121 AU). These AU values did not overlap. Analysis of intact stones showed excellent resolution of structural detail and could discriminate multiple mineral types within heterogeneous stones. CONCLUSIONS: Micro CT gives excellent structural detail of urinary stones, and these results demonstrate the feasibility of identifying and localizing most of the common mineral types found in urinary calculi using laboratory CT

Locally contextualizing understandings of depression, the EPDS, and PHQ-9 among a sample of postpartum women living with HIV in Malawi

Background: The Edinburgh Postnatal Depression Scale (EPDS) and Patient Health Questionnaire-9 (PHQ-9) are widely used depression screening tools, yet perceptions and understandings of their questions and of depression are not well defined in cross-cultural research. Methods: 30 postpartum women living with HIV in Malawi were recruited from a cohort study and participated in in-depth cognitive interviews. Transcripts were evaluated following an inductive approach to identify common themes. Results: Participants most frequently described looking sad or different than usual, self-isolation, ‘thinking too much,’ and anger as key symptoms of being depressed. HIV-associated stigma was commonly identified as a cause of depression. The EPDS and PHQ-9 were generally well understood but did not capture all the important symptoms of depression that women described. Participants sometimes requested clarification or rephrasing of certain EPDS and PHQ-9 questions when asked to explain the questions’ meanings in their own words, and requested rephrasing more often for EPDS questions than PHQ-9 questions. Few women believed either tool was sufficient to detect depression. Limitations: Our results may not be generalizable, but are locally contextualized. Women suffering with depression may have been more or less likely to agree to the qualitative interview depending on their comfort level discussing any current depressive symptoms. Conclusions: Researchers and practitioners who use the EPDS and PHQ-9 should be aware of the tools’ limitations in their context and population. New instruments may need to be developed or adaptations to existing tools made to improve accuracy of depression screening and diagnosis in different cultural contexts

Micro-morphologic changes around biophysically-stimulated titanium implants in ovariectomized rats

<p>Abstract</p> <p>Background</p> <p>Osteoporosis may present a risk factor in achievement of osseointegration because of its impact on bone remodeling properties of skeletal phsiology. The purpose of this study was to evaluate micro-morphological changes in bone around titanium implants exposed to mechanical and electrical-energy in osteoporotic rats.</p> <p>Methods</p> <p>Fifteen 12-week old sprague-dowley rats were ovariectomized to develop osteoporosis. After 8 weeks of healing period, two titanium implants were bilaterally placed in the proximal metaphyses of tibia. The animals were randomly divided into a control group and biophysically-stimulated two test groups with five animals in each group. In the first test group, a pulsed electromagnetic field (PEMF) stimulation was administrated at a 0.2 mT 4 h/day, whereas the second group received low-magnitude high-frequency mechanical vibration (MECHVIB) at 50 Hz 14 min/day. Following completion of two week treatment period, all animals were sacrificed. Bone sites including implants were sectioned, removed <it>en bloc </it>and analyzed using a microCT unit. Relative bone volume and bone micro-structural parameters were evaluated for 144 μm wide peri-implant volume of interest (VOI).</p> <p>Results</p> <p>Mean relative bone volume in the peri-implant VOI around implants PEMF and MECHVIB was significantly higher than of those in control (<it>P </it>< .05). Differences in trabecular-thickness and -separation around implants in all groups were similar (<it>P </it>> .05) while the difference in trabecular-number among test and control groups was significant in all VOIs (<it>P </it>< .05).</p> <p>Conclusion</p> <p>Biophysical stimulation remarkably enhances bone volume around titanium implants placed in osteoporotic rats. Low-magnitude high-frequency MECHVIB is more effective than PEMF on bone healing in terms of relative bone volume.</p

Multilayer regulatory mechanisms control cleavage factor I proteins in filamentous fungi

Cleavage factor I (CFI) proteins are core components of the polyadenylation machinery that can regulate several steps of mRNA life cycle, including alternative polyadenylation, splicing, export and decay. Here, we describe the regulatory mechanisms that control two fungal CFI protein classes in Magnaporthe oryzae: Rbp35/CfI25 complex and Hrp1. Using mutational, genetic and biochemical studies we demonstrate that cellular concentration of CFI mRNAs is a limited indicator of their protein abundance. Our results suggest that several post-transcriptional mechanisms regulate Rbp35/CfI25 complex and Hrp1 in the rice blast fungus, some of which are also conserved in other ascomycetes. With respect to Rbp35, these include C-terminal processing, RGG-dependent localization and cleavage, C-terminal autoregulatory domain and regulation by an upstream open reading frame of Rbp35-dependent TOR signalling pathway. Our proteomic analyses suggest that Rbp35 regulates the levels of proteins involved in melanin and phenylpropanoids synthesis, among others. The drastic reduction of fungal CFI proteins in carbon-starved cells suggests that the pre-mRNA processing pathway is altered. Our findings uncover broad and multilayer regulatory mechanisms controlling fungal polyadenylation factors, which have profound implications in pre-mRNA maturation. This area of research offers new avenues for fungicide design by targeting fungal-specific proteins that globally affect thousands of mRNAs

Multidrug-Resistant Gram-Negative Bacteria in Burn Patients

Patients with burn injuries are at high risk for infectious complications, and infections are the most common cause of death after the first 72 h of hospitalization. Hospital-acquired infections caused by multidrug resistant (MDR) Gram-negative bacteria (GNB) in this population are concerning. Here, we evaluated carriage with MDR GNB in patients in a large tertiary-care burn intensive care unit. Twenty-nine patients in the burn unit were screened for intestinal carriage. Samples were cultured on selective media. Median time from admission to the burn unit to first sample collection was 9 days (IQR 5 – 17 days). In 21 (72%) patients, MDR GNB were recovered; the most common bacterial species isolated was Pseudomonas aeruginosa, which was found in 11/29 (38%) of patients. Two of these patients later developed bloodstream infections with P. aeruginosa. Transmission of KPC-31-producing ST22 Citrobacter freundii was detected. Samples from two patients grew genetically similar C. freundii isolates that were resistant to ceftazidime-avibactam. On analysis of whole-genome sequencing, blaKPC-31 was part of a Tn4401b transposon that was present on two different plasmids in each C. freundii isolate. Plasmid curing experiments showed that removal of both copies of blaKPC-31 was required to restore susceptibility to ceftazidime-avibactam. In summary, MDR GNB colonization is common in burn patients and patient-to-patient transmission of highly resistant GNB occurs. These results emphasize the ongoing need for infection prevention and antimicrobial stewardship efforts in this highly vulnerable population

Reproducibility of a peripheral quantitative computed tomography scan protocol to measure the material properties of the second metatarsal

Background: Peripheral quantitative computed tomography (pQCT) is an established technology that allows for the measurement of the material properties of bone. Alterations to bone architecture are associated with an increased risk of fracture. Further pQCT research is necessary to identify regions of interest that are prone to fracture risk in people with chronic diseases. The second metatarsal is a common site for the development of insufficiency fractures, and as such the aim of this study was to assess the reproducibility of a novel scanning protocol of the second metatarsal using pQCT. Methods. Eleven embalmed cadaveric leg specimens were scanned six times; three times with and without repositioning. Each foot was positioned on a custom-designed acrylic foot plate to permit unimpeded scans of the region of interest. Sixty-six scans were obtained at 15% (distal) and 50% (mid shaft) of the second metatarsal. Voxel size and scan speed were reduced to 0.40 mm and 25 mm.sec-1. The reference line was positioned at the most distal portion of the 2nd metatarsal. Repeated measurements of six key variables related to bone properties were subject to reproducibility testing. Data were log transformed and reproducibility of scans were assessed using intraclass correlation coefficients (ICC) and coefficients of variation (CV%). Results: Reproducibility of the measurements without repositioning were estimated as: trabecular area (ICC 0.95; CV% 2.4), trabecular density (ICC 0.98; CV% 3.0), Strength Strain Index (SSI) - distal (ICC 0.99; CV% 5.6), cortical area (ICC 1.0; CV% 1.5), cortical density (ICC 0.99; CV% 0.1), SSI - mid shaft (ICC 1.0; CV% 2.4). Reproducibility of the measurements after repositioning were estimated as: trabecular area (ICC 0.96; CV% 2.4), trabecular density (ICC 0.98; CV% 2.8), SSI - distal (ICC 1.0; CV% 3.5), cortical area (ICC 0.99; CV%2.4), cortical density (ICC 0.98; CV% 0.8), SSI - mid shaft (ICC 0.99; CV% 3.2). Conclusions: The scanning protocol generated excellent reproducibility for key bone properties measured at the distal and mid-shaft regions of the 2 nd metatarsal. This protocol extends the capabilities of pQCT to evaluate bone quality in people who may be at an increased risk of metatarsal insufficiency fractures

Correlates of physical activity among community-dwelling adults aged 50 or over in six low- and middle-income countries

Background: Considering that physical activity is associated with healthy ageing and helps to delay, prevent, or manage a plethora of non-communicable diseases in older adults, there is a need to investigate the factors that influence physical activity participation in this population. Thus, we investigated physical activity correlates among community-dwelling older adults (aged ≥50 years) in six low- and middle-income countries. Methods: Cross-sectional data were analyzed from the World Health Organization’s Study on Global Ageing and Adult Health. Physical activity was assessed by the Global Physical Activity Questionnaire. Participants were dichotomized into low (i.e., not meeting 150 minutes of moderate physical activity per week) and moderate-to-high physically active groups. Associations between physical activity and a range of correlates were examined using multivariable logistic regressions. Results: The overall prevalence (95%CI) of people not meeting recommended physical activity levels in 34,129 participants (mean age 62.4 years, 52.1% female) was 23.5% (22.3%-24.8%). In the multivariable analysis, older age and unemployment were significant sociodemographic correlates of low physical activity. Individuals with low body mass index (<18.5kg/m2), bodily pain, asthma, chronic back pain, chronic obstructive pulmonary disease, hearing problems, stroke, visual impairment, slow gait, and weak grip strength were less likely to meet physical activity targets in the overall sample (P<0.05). The associations varied widely between countries. Conclusion: Our data illustrates that a multitude of factors influence physical activity target achievement in older adults, which can inform future interventions across low- and middle-income countries to assist people of this age group to engage in regular physical activity. Future prospective cohort studies are also required to investigate the directionality and mediators of the relationships observed