Formulation approaches to improving the delivery of an antiviral drug with activity against seasonal flu.
The main objective of the present study was to develop formulations of noscapine hydrochloride hydrate with enhanced solubility and bioavailability using co-solvent- and cyclodextrin-based approaches. Different combinations of co-solvents, which were selected on the basis of high-throughput solubility screening, were subjected to in vitro intestinal drug permeability studies conducted with Ussing chambers. Vitamin E tocopherol polyethylene glycol succinate and propylene glycol based co-solvent formulations provided the maximum permeability coefficient for the drug. Inclusion complexes of the drug were prepared using hydroxypropyl-β-cyclodextrin and sulphobutylether cyclodextrins. Pharmacokinetic studies were carried out in male Sprague-Dawley rats for the selected formulations. The relative bioavailabilities of the drug with the co-solvent- and cyclodextrin-based formulations were found to be similar.


Acute mitral valve obstruction during infective endocarditis.
Five patients were found during surgery or at necropsy to have the mitral valve orifice obstructed by vegetations. They had had unexplained severe and recurrent episodes of acute febrile pulmonary oedema, and four had few cardiac ausculatory findings. Three patients died suddenly and unexpectedly; the other two were operated on and survived. In view of its ominous prognosis, acute mitral valve obstruction should be considered in patients whose pulmonary symptoms are compatible with endocarditis and are not adequately explained by the findings on examination of the heart. The condition, which should be confirmed by echocardiography, requires emergency surgery.


Steroid injection for hip osteoarthritis: efficacy under ultrasound guidance.
To determine the efficacy of IA corticosteroid (CS) injection in pain reduction for hip OA under ultrasound (US) guidance. Forty patients [mean age 62.78 (8.16) years] fulfilling ACR criteria for hip OA, with synovitis detected at US, gave their consent for IA US-guided CS injection because of pain refractory to conventional therapy. At baseline, at 1 and 3 months, patients filled up a visual analogue scale (VAS) pain on walking, performed the Lequesne index and were checked by US for synovitis. Results were compared with age-matched controls. The occurrence of side effects both short and long term was monitored. IA steroid deposition was performed under US guidance. After 1 and 3 months, walking pain VAS was significantly reduced vs baseline (P < 0.001) and had high correlation with Lequesne index. Synovial hypertrophy was reduced in 75% of the hips after 1 and 3 months vs baseline (P < 0.001). In the group of controls, hip walking pain VAS, Lequesne index and synovial hypertrophy were not changed at 3 months vs baseline (P > 0.05). Transient facial rash was present in 16 patients during the first 24-48 h after injection. No side effects were reported. US-guided steroid injections in hip OA is an efficacious and safe therapeutic approach to achieve pain control and reduction of synovial hypertrophy avoiding the use of X-ray-guided procedure.


Curcuma comosa improves learning and memory function on ovariectomized rats in a long-term Morris water maze test.
Curcuma comosa extract and some purified compounds from this plant have been reported to have estrogenic-like effects, and estrogen improves learning in some animals and potentially in postmenopausal women; therefore, this study tested the hypothesis that Curcuma comosa and estrogen have similar beneficial effects on spatial learning and memory. Curcuma comosa hexane extract, containing 0.165 mg of (4E,6E)-1,7-diphenylhepta-4,6-dien-3-one per mg of the crude extract, was orally administered to ovariectomized Wistar rats at the doses of 250 or 500 mg/kg body weight. 17beta-estradiol (10 microg/kg body weight, subcutaneously) was used as a positive control. Thirty days after the initiation of treatment, animals were tested in a Morris water maze for spatial learning and memory. They were re-tested every 30 days and a final probe trial was run on day 119. Compared to control rats, OVX rats displayed significant memory impairment for locating the platform in the water maze from day 67 after the surgery, onward. In contrast, OVX rats treated with either Curcuma comosa or estrogen were significantly protected from this decline in cognitive function. Further, the protection of cognitive effects by Curcuma comosa was larger at higher dose. These results suggest that long-term treatment with Curcuma comosa has beneficial effects on learning and memory function in rats.


Practices and determinants of delivery by skilled birth attendants in Bangladesh.
Utilization of Skilled Birth Attendants (SBAs) at birth is low (20%) in Bangladesh. Birth attendance by SBAs is considered as the "single most important factor in preventing maternal deaths". This paper examined the practices and determinants of delivery by SBAs in rural Bangladesh. The data come from the post-intervention survey of a cluster-randomized community controlled trial conducted to evaluate the impact of limited post-natal care (PNC) services on healthcare seeking behavior of women with a recent live birth in rural Bangladesh (n = 702). Multivariable logistic regression model was used to identify the potential determinants of delivery by SBAs. The respondents were aged between 16 and 45, with the mean age of 24.41 (± 5.03) years. Approximately one-third (30.06%) of the women had their last delivery by SBAs. Maternal occupation, parity, complications during pregnancy and antenatal checkup (ANC) by SBAs were the significant determinants of delivery by SBAs. Women who took antenatal care by SBAs were 2.62 times as likely (95% CI: 1.66, 4.14; p < 0.001) to have their delivery conducted by SBAs compared to those who did not, after adjusting for other covariates. Our findings suggest that ANC by SBAs and complications during pregnancies are significant determinants of delivery by SBAs. Measure should be in place to promote antenatal checkup by SBAs to increase utilization of SBAs at birth in line with achieving the Millennium Development Goal-5. Future research should focus in exploring the unmet need for, and potential barriers in, the utilization of delivery by SBAs.


Anti-Inflammation Property of Syzygium cumini (L.) Skeels on Indomethacin-Induced Acute Gastric Ulceration.
Indomethacin, nonsteroidal anti-inflammatory drug (NSAIDs), induced gastric damage and perforation through the excess generation of reactive oxygen species (ROS). Syzygium cumini (L.) Skeels is commonly used as a medicinal plant and is claimed to have antioxidant activities. The effects of Syzygium cumini (L.) Skeels aqueous extract (SCC) on antifree radical, anti-inflammation, and antiulcer of SCC on indomethacin induced acute gastric ulceration were determined in our study. Scavenging activity at 50% of SCC is higher than ascorbic acid in in vitro study. Mice treated with indomethacin revealed mucosal hemorrhagic lesion and inhibited mucus content. Pretreatment with SCC caused discernible decrease in indomethacin induced gastric lesion and lipid peroxide content. In addition, oxidized glutathione (GSSG), glutathione peroxidase (GPx), nitric oxide (NO) levels, and gastric wall mucus were restored on acute treated mice model. Indomethacin induced inflammation by activated inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-α) proinflammatory cytokines to release large amount of ROS/RNS which were ameliorated in mice pretreatment with SCC. SCC showed restoration of the imbalance of oxidative damage leading to amelioration of cyclooxygenase enzyme (COX). In conclusion, SCC acts as an antioxidant, anti-inflammation, and antiulcer against indomethacin.


Germinal Center T Follicular Helper Cells Are Highly Permissive to HIV-1 and Alter Their Phenotype during Virus Replication.
HIV-1 replication is concentrated within CD4(+) T cells in B cell follicles of secondary lymphoid tissues during asymptomatic disease. Limited data suggest that a subset of T follicular helper cells (TFH) within germinal centers (GC) is highly permissive to HIV-1. Whether GC TFH are the major HIV-1 virus-producing cells in vivo has not been established. In this study, we investigated TFH permissivity to HIV-1 ex vivo by spinoculating and culturing tonsil cells with HIV-1 GFP reporter viruses. Using flow cytometry, higher percentages of GC TFH (CXCR5(high)PD-1(high)) and CXCR5(+)programmed cell death-1 (PD-1)(low) cells were GFP(+) than non-GC TFH (CXCR5(+)PD-1(intermediate)) or extrafollicular (EF) (CXCR5(-)) cells. When sorted prior to spinoculation, however, GC TFH were substantially more permissive than CXCR5(+)PD-1(low) or EF cells, suggesting that many GC TFH transition to a CXCR5(+)PD-1(low) phenotype during productive infection. In situ hybridization on inguinal lymph node sections from untreated HIV-1-infected individuals without AIDS revealed higher frequencies of HIV-1 RNA(+) cells in GC than non-GC regions of follicle or EF regions. Superinfection of HIV-1-infected individuals' lymph node cells with GFP reporter virus confirmed the permissivity of follicular cells ex vivo. Lymph node immunostaining revealed 96% of CXCR5(+)CD4(+) cells were located in follicles. Within sorted lymph node cells from four HIV-infected individuals, CXCR5(+) subsets harbored 11-66-fold more HIV-1 RNA than CXCR5(-) subsets, as determined by RT PCR. Thus, GC TFH are highly permissive to HIV-1, but downregulate PD-1 and, to a lesser extent, CXCR5 during HIV-1 replication. These data further implicate GC TFH as the major HIV-1-producing cells in chronic asymptomatic HIV-1 infection.


Preliminary model assessing the cost-effectiveness of preoperative chlorhexidine mouthwash at reducing postoperative pneumonia among abdominal surgery patients in South Africa.
Pneumonia is a common and severe complication of abdominal surgery, it is associated with increased length of hospital stay, healthcare costs, and mortality. Further, pulmonary complication rates have risen during the SARS-CoV-2 pandemic. This study explored the potential cost-effectiveness of administering preoperative chlorhexidine mouthwash versus no-mouthwash at reducing postoperative pneumonia among abdominal surgery patients. A decision analytic model taking the South African healthcare provider perspective was constructed to compare costs and benefits of mouthwash versus no-mouthwash-surgery at 30 days after abdominal surgery. We assumed two scenarios: (i) the absence of COVID-19; (ii) the presence of COVID-19. Input parameters were collected from published literature including prospective cohort studies and expert opinion. Effectiveness was measured as proportion of pneumonia patients. Deterministic and probabilistic sensitivity analyses were performed to assess the impact of parameter uncertainties. The results of the probabilistic sensitivity analysis were presented using cost-effectiveness planes and cost-effectiveness acceptability curves. In the absence of COVID-19, mouthwash had lower average costs compared to no-mouthwash-surgery, $3,675 (R 63,770) versus $3,958 (R 68,683), and lower proportion of pneumonia patients, 0.029 versus 0.042 (dominance of mouthwash intervention). In the presence of COVID-19, the increase in pneumonia rate due to COVID-19, made mouthwash more dominant as it was more beneficial to reduce pneumonia patients through administering mouthwash. The cost-effectiveness acceptability curves shown that mouthwash surgery is likely to be cost-effective between $0 (R0) and $15,000 (R 260,220) willingness to pay thresholds. Both the absence and presence of SARS-CoV-2, mouthwash is likely to be cost saving intervention for reducing pneumonia after abdominal surgery. However, the available evidence for the effectiveness of mouthwash was extrapolated from cardiac surgery; there is now an urgent need for a robust clinical trial on the intervention on non-cardiac surgery.


A comparison of the effectiveness of swabbing and flossing as a means of recovering spermatozoa from the oral cavity.
This study examined whether flossing the teeth is a more effective collection method in recovering spermatozoa than conventional swabbing techniques. It was hypothesized that inclusion of flossing as a collection method would extend the recovery of spermatozoa to longer postcoital intervals (PCIs). Eighteen individuals provided 174 oral cavity samples. Successful recovery of spermatozoa was assessed with respect to the collection method and reported activity in the oral cavity during the PCI. Samples were subjected to a differential extraction procedure prior to microscopic evaluation of the extracted pellet. The results indicate that swabbing is more effective than flossing when the PCI falls within 1.5-12 h. However, spermatozoa were recovered from seven floss samples where the corresponding swabs gave negative results. When combining the results from the two collection methods, the percentage of subjects from whom spermatozoa are recovered increases for each PCI beyond the 0-h interval.


Relationship characteristics and sexual risk-taking in young men who have sex with men.
Young men who have sex with men (MSM), particularly young men of color, are experiencing the largest increase in HIV incidence of any risk group in the United States Epidemiological research suggests that the majority of transmissions among MSM are occurring in the context of primary partnerships, but little research has been done on the processes within these dyads that increase HIV risk behaviors. The aim of this study was to use longitudinal partnership-level data to explore the effects of partner and relationship characteristics on the frequency of unprotected sex within young MSM relationships. One hundred twenty-two young MSM (age 16-20 at baseline) were assessed at three time-points six months apart, with 91% retention at the 12-month follow-up wave. Over 80% were racial/ethnic minorities. At each wave, participants reported on characteristics of the relationships and partners for up to three sexual partners. Hierarchical linear modeling was used for analyses. The largest effect was for considering the relationship to be serious, which was associated with nearly an eightfold increase in the rate of unprotected sex. Other factors that increased risk behaviors included older partners, drug use prior to sex, physical violence, forced sex, and partnership lasting more than six months. Partners met online were not associated with significantly more sexual risk. These data provide insight into the relationship processes that should be addressed in prevention programs targeted at young MSM. Relationships may serve as a promising unit for HIV prevention interventions, although more formative research will be required to address potential logistical obstacles to implementing such interventions. The partner-by-partner analytic approach (i.e., evaluating situational variables associated with several partners for a given participant) holds promise for future HIV behavioral research.


Non-acid-fastness in Mycobacterium tuberculosis ΔkasB mutant correlates with the cell envelope electron density.
The acid-fastness is the most important and the most specific characteristics in mycobacteria, the mechanism of which is not clear but may be attributed to the lipid rich cell wall of this bacterium. While the exact component(s) responsible for this staining method remained unidentified, a Mycobacterium tuberculosis mutant, attenuated strain that produced shorter mycolic acids with defects in trans-cyclopropanation was shown to be acid fast negative. In this study, we examined the ultrastructure of the cell envelope (CE) of the mutant strain ΔkasB (missing a beta-ketoacyl-ACP synthase involved in mycolic acid biosynthesis), the parental CDC1551 (wild type strain) and kasB complemented strain, and compared ultrastructural differences among them with conventional transmission electron microscopy (TEM) and cryo-transmission electron microscopy (CEM). Conventional TEM revealed that there were no detectable differences in the thickness of the cell envelope among three strains (wild-type: 43.35 ± 6.13 nm; ΔkasB: 45.98 ± 11.32 nm; complement: 40.71 ± 6.3 nm). However, CEM data demonstrated that the region between the inner and outer membranes of the mutant strain, which is composed mainly of cell wall anchored mycolic acids (MA), showed a significant decrease in electron density as compared to the wild type and kasB complement strain (567.1 ± 372.7 vs. 301.4 ± 262.1, or vs. 235.2 ± 174.9, p < 0.02 or p < 0.001, respectively). These results suggested that altered MA patterns in the kasB mutant may have affected the packing of the lipid rich layer of the M. tuberculosis cell envelope, resulting in a reduced electron density of this layer as seen by CEM and loss of acid-fastness in light microscopical observation, and we propose a novel model of the cell envelope structure in tubercle bacilli.


Implementing a Standardized Communication Tool in an Intensive Care Unit.
Effective communication is essential in critical care settings. Use of the SBAR (Situation, Background, Assessment, Recommendation) tool has been shown to standardize and improve communication among health care providers. This quality improvement project was designed to improve communication in an intensive care unit that lacked a standardized communication protocol. Communication practices differed greatly between nurses and advanced practice providers. As a result, patient safety was put at risk owing to incomplete, inaccurate, or delayed information when clinical concerns were reported or escalated. This project used a pre-post design in which surveys were used to gather information on staff perceptions of communication and collaboration between nurses and advanced practice providers before and after an educational intervention. The 2 groups received identical education on SBAR guidelines adapted for use in the intensive care unit setting and patient safety. Results showed improvement in all areas of communication. Significant improvements were found on the General Perceptions subscale among advanced practice providers (P = .04) and among nurses (P = .007). In the combined study population, improvements were observed on all subscales, with significant results for the Open Communication (P = .03) and General Perceptions (P = .002) subscales. A significant increase was found in the percentage of nurses using the SBAR tool after the intervention (95%) compared with before the intervention (66%; P < .001). Implementation of the SBAR communication tool significantly improved general perceptions of communication in this intensive care unit.


Prenatal diagnosis of 18p deletion and isochromosome 18q mosaicism in a fetus with a cystic hygroma.
Although, deletion of short arm of chromosome 18 is one of the most frequent autosomal terminal deletions, mosaic form of 18p deletion is infrequently observed. Furthermore, prenatally detected cases of 18p deletion and isochromosome 18q mosaicism are extremely rare. Herein, we present a case of del(18p)/i(18q) mosaicism, prenatally detected after chori- onic villus sampling. A 37-year-old woman was referred for prenatal diagnosis because of fetal septated cystic hygroma measuring 4.3 mm. Cytogenetic analysis showed a mosaic 46,XX,del(18)(p11.2)/46,XX,i(18)(q10) karyotype in both, short- and long-term culture. Parents elected to terminate the pregnancy. Fetal mosaic karyotype was confirmed by chromosomal analysis of cultured skin fibroblasts. Molecular characterization of chromosome 18 structural aberrations was performed by fluorescence in situ hybridization (FISH). Considering variable ultrasound findings among cases with del(18p)/i(18q) mosaicism, we emphasized that first and second trimester ultrasound screening examinations for fetal malformations, followed by cytogenetic and molecular evaluations, are very important in the management of prenatally detected cases.


Rational design of indoleamine 2,3-dioxygenase inhibitors.
Indoleamine 2,3-dioxygenase (IDO) is an important therapeutic target for the treatment of diseases such as cancer that involve pathological immune escape. We have used the evolutionary docking algorithm EADock to design new inhibitors of this enzyme. First, we investigated the modes of binding of all known IDO inhibitors. On the basis of the observed docked conformations, we developed a pharmacophore model, which was then used to devise new compounds to be tested for IDO inhibition. We also used a fragment-based approach to design and to optimize small organic molecule inhibitors. Both approaches yielded several new low-molecular weight inhibitor scaffolds, the most active being of nanomolar potency in an enzymatic assay. Cellular assays confirmed the potential biological relevance of four different scaffolds.


Genetic structure of West Greenland populations of lumpfish Cyclopterus lumpus.
In this study, 11 microsatellite markers were used to determine the structure of West Greenlandic lumpfish Cyclopterus lumpus populations across six spawning locations spanning >1500 km and compared with neighbouring populations in Canada and Iceland. To evaluate whether data allow for identification of origin of C. lumpus in Greenlandic waters, genetic assignment analysis was performed for 86 C. lumpus sampled on a feeding migration. Significant structuring with isolation by distance was observed in the West Greenland samples and two major subpopulations, north and south, were suggested. Based on FST values, closer relationships were observed between Greenland and Canada, than Greenland and Iceland. Surprisingly, the North Greenland population showed more similarities with Canadian samples, than did the geographically closer south-west Greenland population. Origin could be assigned for a high proportion of non-spawning fish and demonstrated a marked east-west spatial separation of fish of Greenlandic and Icelandic genotypes.


Novel uromodulin mutation in familial juvenile hyperuricemic nephropathy.
Familial juvenile hyperuricemic nephropathy (FJHN) is an autosomal dominant disorder characterized by early onset of hyperuricemia, decreased fractional renal urate excretion and progressive interstitial nephropathy. Mutations in the uromodulin (UMOD) gene encoding uromodulin/Tamm-Horsfall, a glycosylphosphatidylinositol (GPI)-anchored protein, cause this disease. One Chinese family with 13 FJHN-affected individuals is described. Clinical data, blood and urine samples of 7 affected members (all alive patients in this family) and 15 unaffected members were collected. Mutation analysis of the UMOD gene was performed by polymerase chain reaction and direct sequencing. Urinary uromodulin from affected or unaffected members of this family and healthy controls was examined by enzyme-linked immunosorbent assay kit. Expression of uromodulin in renal tissue was shown with immunofluorescence. A novel mutation (p.T605G) within the uromodulin GPI anchor signal segment was identified in the affected individuals of this FJHN family. There was a markedly increased expression of uromodulin in renal tissue and significantly decreased urinary excretion of uromodulin in affected patients with an estimated glomerular filtration rate <60 ml/min/1.73 m(2). The present study reported a novel mutation in exon 9 of UMOD in the Chinese Han population, within the GPI anchor signal segment of uromodulin. Since the GPI anchor is linked with the release or secretion of proteins, our finding may provide further evidence for the underlying mechanism of decreased urinary excretion of uromodulin in FJHN.


Upper extremity access for fenestrated endovascular aortic aneurysm repair is not associated with increased morbidity.
Fenestrated endovascular aortic aneurysm repair (FEVAR) is an alternative to open repair in patients with complex abdominal aortic aneurysms who are neither fit nor suitable for standard open or endovascular repair. Chimney and snorkel grafts are other endovascular alternatives but frequently require bilateral upper extremity access that has been associated with a 3% to 10% risk of stroke. However, upper extremity access is also frequently required for FEVAR because of the caudal orientation of the visceral vessels. The purpose of this study was to assess the use of upper extremity access for FEVAR and the associated morbidity. During a 5-year period, 148 patients underwent FEVAR, and upper extremity access for FEVAR was used in 98 (66%). Outcomes were compared between those who underwent upper extremity access and those who underwent femoral access alone. The primary end point was a cerebrovascular accident or transient ischemic attack, and the secondary end point was local access site complications. The mean number of fenestrated vessels was 3.07 ± 0.81 (median, 3) for a total of 457 vessels stented. Percutaneous upper extremity access was used in 12 patients (12%) and open access in 86 (88%). All patients who required a sheath size >7F underwent high brachial open access, with the exception of one patient who underwent percutaneous axillary access with a 12F sheath. The mean sheath size was 10.59F ± 2.51F (median, 12F), which was advanced into the descending thoracic aorta, allowing multiple wire and catheter exchanges. One hemorrhagic stroke (one of 98 [1%]) occurred in the upper extremity access group, and one ischemic stroke (one of 54 [2%]) occurred in the femoral-only access group (P = .67). The stroke in the upper extremity access group occurred 5 days after FEVAR and was related to uncontrolled hypertension, whereas the stroke in the femoral group occurred on postoperative day 3. Neither patient had signs or symptoms of a stroke immediately after FEVAR. The right upper extremity was accessed six times without a stroke (0%) compared with the left being accessed 92 times with one stroke (1%; P = .8). Four patients (4%) had local complications related to upper extremity access. One (1%) required exploration for an expanding hematoma after manual compression for a 7F sheath, one (1%) required exploration for hematoma and neurologic symptoms after open access for a 12F sheath, and two patients (2%) with small hematomas did not require intervention. Two (two of 12 [17%]) of these complications were in the percutaneous access group, which were significantly more frequent than in the open group (two of 86 [2%]; P = .02). Upper extremity access appears to be a safe and feasible approach for patients undergoing FEVAR. Open exposure in the upper extremity may be safer than percutaneous access during FEVAR. Unlike chimney and snorkel grafts, upper extremity access during FEVAR is not associated with an increased risk of stroke, despite the need for multiple visceral vessel stenting.


Diet and pharyngeal cancer.
A population-based case-control study was conducted to examine the effect of ingestion of vitamin C, carotenoids and retinol on risk of pharyngeal cancer. Data were available from 166 cases of pharyngeal cancer or their next-of-kin, and from 547 controls similar in age and sex to the cases. Odds ratios (ORs) relating consumption of vitamin C, carotenoids and retinol from foods and vitamins C and A from supplements to risk of pharyngeal cancer were calculated using multiple logistic regression analysis. After adjustment for smoking and alcohol consumption, a significant increase in risk associated with low intake of vitamin C from foods was observed. Compared to the highest quartile, the OR for the lowest quartile of intake was 2.5 (95% confidence interval, 1.5-4.2). No overall effect of dietary carotenoid or retinol consumption was noted after adjustment for smoking and alcohol. Decreasing use of either vitamin C or vitamin A supplements was associated with increasing cancer risk among the case group as a whole. However, the effect of vitamin C supplement use was substantially lower when next-of-kin respondents were excluded from analysis. Also, the OR for use of vitamin A supplements is based on a very small number of cases reporting use, and must be viewed with caution. The results of our study suggest that intake of vitamin C may be protective against pharyngeal cancer, and are consistent with the results of previous studies which reported a decreased risk of pharyngeal cancer associated with intake of fruits and vegetables.


Effects of a web-based educational module on pediatric emergency medicine physicians' knowledge, attitudes, and behaviors regarding youth violence.
Youth seen in the emergency department (ED) with injuries from youth violence (YV) have increased risk for future violent injury and death. Pediatric emergency medicine (PEM) physicians rarely receive training in, or perform, YV screening and intervention. Our objective was to examine effects of a web-based educational module on PEM physicians' knowledge, attitudes, and behaviors regarding YV screening and interventions in the ED. We invited all PEM fellows and attendings at an urban Level I pediatric trauma center to complete an interactive web-based education module (and 1-month booster) with information on YV's public health impact and how to screen, counsel and refer YV-involved patients. Consenting subjects completed electronic assessments of YV prevention knowledge and attitudes (using validated measures when possible) before and after the initial module and after the booster. To measure behavior change, chart review identified use of YV-specific discharge instructions in visits by YV-injured PEM patients (age 12-17; identified by E codes) 6 months before and after the intervention. We analyzed survey data were analyzed with Fisher's exact for binary outcomes and Kruskal-Wallis for Likert responses. Proportion of patients given YV discharge instructions before and after the intervention was compared using chi-square. Eighteen (67%) of 27 PEM physicians participated; 1 was lost at post-module assessment and 5 at 1 month. Module completion time ranged from 15-30 minutes. At baseline, 50% of subjects could identify victims' re-injury rate; 28% were aware of ED YV discharge instructions. After the initial module and at 1 month, there were significant increases in knowledge (p<0.001) and level of confidence speaking with patients about avoiding YV (p=0.01, df=2). Almost all (94%) said the module would change future management. In pre-intervention visits, 1.6% of patients with YV injuries were discharged with YV instructions, versus 15.7% in the post-intervention period (p=0.006, 95%CI for difference 3.6%-24.5%). A brief web-based module influenced PEM physicians' knowledge and attitudes about YV prevention and may have affected behavior changes related to caring for YV victims in the ED. Further research should investigate web-based educational strategies to improve care of YV victims in a larger population of PEM physicians.


Non-disclosure of tuberculosis diagnosis by patients to their household members in south western Uganda.
Tuberculosis (TB) non-disclosure by adult patients to all household members is a setback to TB control efforts. It reduces the likelihood that household contacts will seek early TB screening, initiation on preventive or curative treatment, but also hinders the implementation of infection controls and home-based directly observed treatment. Therefore, the purpose of this study was to determine the level of TB non-disclosure, its predictors and the effects of disclosure among adult TB patients in Uganda. We conducted a cross-sectional study at a large regional referral hospital in Mbarara, south-western Uganda. Questionnaires were administered to collect patients' sociodemographic and their TB disclosure data. Non-disclosure was considered if a patient did not reveal their TB diagnosis to all household members within 2 weeks post-treatment initiation. Univariate and multivariate logistic regression models were fitted for predictors of non-disclosure. We enrolled 62 patients, 74% males, mean age of 32 years, and median of five people per household. Non-disclosure rate was 30.6%. Post-disclosure experiences were positive in 98.3% of patients, while negative experiences suggestive of severe stigma occurred in 12.3% of patients. Being female (OR 6.5, 95% CI: 1.4-29.3) and belonging to Muslim faith (OR 12.4, 95% CI: 1.42-109.1) were significantly associated with TB non-disclosure to household members. There is a high rate of TB non-disclosure to all household members by adult patients in rural Uganda, particularly among women and muslim patients. Interventions enhancing TB disclosure at household level while minimizing negative effects of stigma should be developed and prioritized.


Atmospheric hydrogen scavenging: from enzymes to ecosystems.
We have known for 40 years that soils can consume the trace amounts of molecular hydrogen (H2) found in the Earth's atmosphere.This process is predicted to be the most significant term in the global hydrogen cycle. However, the organisms and enzymes responsible for this process were only recently identified. Pure culture experiments demonstrated that several species of Actinobacteria, including streptomycetes and mycobacteria, can couple the oxidation of atmospheric H2 to the reduction of ambient O2. A combination of genetic, biochemical, and phenotypic studies suggest that these organisms primarily use this fuel source to sustain electron input into the respiratory chain during energy starvation. This process is mediated by a specialized enzyme, the group 5 [NiFe]-hydrogenase, which is unusual for its high affinity, oxygen insensitivity, and thermostability. Atmospheric hydrogen scavenging is a particularly dependable mode of energy generation, given both the ubiquity of the substrate and the stress tolerance of its catalyst. This minireview summarizes the recent progress in understanding how and why certain organisms scavenge atmospheric H2. In addition, it provides insight into the wider significance of hydrogen scavenging in global H2 cycling and soil microbial ecology.


Improving psychotherapy research: The example of mindfulness based interventions.
The increasing number and sophistication of available psychotherapies suggests that a critical appraisal of the methodological issues of psychotherapy studies is highly needed. Several key questions regarding the efficacy of a given intervention, the understanding of whether positive effects observed following the delivery of a psychotherapeutic intervention are specifically attributable to the intervention itself or to other "non specific" factors, such as benefit expectations, therapist attention and support, and the possibility of improving psychotherapy research need an answer. This, in turn, could provide clinicians with more rigorous information about psychotherapy outcomes and could properly address several shortcomings that are frequently observed in current psychotherapy studies. Accordingly, in this editorial I will highlight some of the most important critical issues that a well designed psychotherapy study should take into account, including the need for appropriate control groups, appropriate randomization and blinding procedures, and the importance of performing appropriately powered studies that include a sufficiently long follow-up period. Finally, I will build on my expertise in the field of mindfulness based interventions, in particular mindfulness based stress reduction and mindfulness based cognitive therapy, to show how such issues have been and can be successfully implemented in the design of future psychotherapy studies.


Molecular Detection of Theileria ovis and Theleiria equi in Livestock from Palestine.
Theileria and Babesia are intracellular protozoan parasites infecting a wide range of animals. In Palestine, there is limited information on the prevalence of Theileria and Babesia spp. in livestock. We used PCR of the 18S ribosomal RNA gene followed by DNA sequencing to detect and identify parasite DNA in blood samples from sheep (n = 49), goats (n = 48), horses (n = 40), camels (n = 34), donkeys (n = 28) and mules (n = 2) from four districts of Palestine. DNA of T. ovis and T. equi was detected in 19 and 2 ovine blood samples, respectively. None of the camels, donkeys, and goats were positive for T. ovis. Sheep had a significantly higher rate of infection than other animals (P < 0.05). Theileria ovis is highly prevalent in sheep, while T. equi DNA was detected in a small proportion of the equids in Palestine.


Effects of reduced free fatty acid availability on hormone-sensitive lipase activity in human skeletal muscle during aerobic exercise.
Hormone-sensitive lipase (HSL) catalyzes the hydrolysis of intramuscular triacylglycerol (IMTG); however, its regulation in skeletal muscle is poorly understood. To examine the effects of reduced free fatty acid (FFA) availability on HSL activity in skeletal muscle during aerobic exercise, 11 trained men exercised at 55% maximal O2 uptake for 40 min after the ingestion of nicotinic acid (NA) or nothing (control). Muscle biopsies were taken at rest and 5, 20, and 40 min of exercise. Plasma FFA were suppressed (P < 0.05) in NA during exercise ( approximately 0.40 +/- 0.04 vs. approximately 0.07 +/- 0.01 mM). The respiratory exchange ratio (RER) was increased throughout exercise (0.020 + 0.008) after NA ingestion. However, the provision of energy from fat oxidation only decreased from 33% of the total in the control trial to 26% in the NA trial, suggesting increased IMTG oxidation in the NA trial. Mean HSL activity was 2.25 + 0.15 mmol x kg dry mass(-1) x min(-1) at rest and increased (P < 0.05) to 2.94 +/- 0.20 mmol x kg dry mass(-1) x min(-1) at 5 min in control. Contrary to the hypothesis, mean HSL was not activated to a greater extent in the NA trial during exercise (2.20 + 0.28 at rest to 2.88 + 0.21 mmol x kg dry mass(-1) x min(-1) at 5 min). No further HSL increases were observed at 20 or 40 min in both trials. There was variability in the response to NA ingestion, as some subjects experienced a large increase in RER and decrease in fat oxidation, whereas other subjects experienced no shift in RER and maintained fat oxidation despite the reduced FFA availability in the NA trial. However, even in these subjects, HSL activity was not further increased during the NA trial. In conclusion, reduced plasma FFA availability accompanied by increased epinephrine concentration did not further activate HSL beyond exercise alone.


Exome sequencing followed by genotyping suggests SYPL2 as a susceptibility gene for morbid obesity.
Recently developed high-throughput sequencing technology shows power to detect low-frequency disease-causing variants by deep sequencing of all known exons. We used exome sequencing to identify variants associated with morbid obesity. DNA from 100 morbidly obese adult subjects and 100 controls were pooled (n=10/pool), subjected to exome capture, and subsequent sequencing. At least 100 million sequencing reads were obtained from each pool. After several filtering steps and comparisons of observed frequencies of variants between obese and non-obese control pools, we systematically selected 144 obesity-enriched non-synonymous, splicing site or 5' upstream single-nucleotide variants for validation. We first genotyped 494 adult subjects with morbid obesity and 496 controls. Five obesity-associated variants (nominal P-value<0.05) were subsequently genotyped in 1425 morbidly obese and 782 controls. Out of the five variants, only rs62623713:A>G (NM_001040709:c.A296G:p.E99G) was confirmed. rs62623713 showed strong association with body mass index (beta=2.13 (1.09, 3.18), P=6.28 × 10(-5)) in a joint analysis of all 3197 genotyped subjects and had an odds ratio of 1.32 for obesity association. rs62623713 is a low-frequency (2.9% minor allele frequency) non-synonymous variant (E99G) in exon 4 of the synaptophysin-like 2 (SYPL2) gene. rs62623713 was not covered by Illumina or Affymetrix genotyping arrays used in previous genome-wide association studies. Mice lacking Sypl2 has been reported to display reduced body weight. In conclusion, using exome sequencing we identified a low-frequency coding variant in the SYPL2 gene that was associated with morbid obesity. This gene may be involved in the development of excess body fat.


Natural selection shaped regional mtDNA variation in humans.
Human mtDNA shows striking regional variation, traditionally attributed to genetic drift. However, it is not easy to account for the fact that only two mtDNA lineages (M and N) left Africa to colonize Eurasia and that lineages A, C, D, and G show a 5-fold enrichment from central Asia to Siberia. As an alternative to drift, natural selection might have enriched for certain mtDNA lineages as people migrated north into colder climates. To test this hypothesis we analyzed 104 complete mtDNA sequences from all global regions and lineages. African mtDNA variation did not significantly deviate from the standard neutral model, but European, Asian, and Siberian plus Native American variations did. Analysis of amino acid substitution mutations (nonsynonymous, Ka) versus neutral mutations (synonymous, Ks) (kaks) for all 13 mtDNA protein-coding genes revealed that the ATP6 gene had the highest amino acid sequence variation of any human mtDNA gene, even though ATP6 is one of the more conserved mtDNA proteins. Comparison of the kaks ratios for each mtDNA gene from the tropical, temperate, and arctic zones revealed that ATP6 was highly variable in the mtDNAs from the arctic zone, cytochrome b was particularly variable in the temperate zone, and cytochrome oxidase I was notably more variable in the tropics. Moreover, multiple amino acid changes found in ATP6, cytochrome b, and cytochrome oxidase I appeared to be functionally significant. From these analyses we conclude that selection may have played a role in shaping human regional mtDNA variation and that one of the selective influences was climate.


Identification of proctolin in the central nervous system of the horseshoe crab, Limulus polyphemus.
A proctolin-like peptide was isolated from the prosomal CNS of the chelicerate arthropod, Limulus, and purified using size exclusion, ion exchange and high performance liquid chromatography. Coincident bioassay (cockroach hindgut) and radioimmunoassay were employed to identify fractions which contained proctolin-like material. Proctolin-like activity coeluted with synthetic proctolin with all three chromatographic techniques employed. When applied to either the Limulus heart or hindgut preparations, purified Limulus proctolin produced excitatory responses which were indistinguishable from those produced by the synthetic peptide. Purified samples of the Limulus proctolin-like peptide were subjected to Edman degradation and tandem mass spectrometry and the amino acid sequence of the Limulus peptide was determined to be identical to that of cockroach proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH). The presence of proctolin in the Limulus CNS and its biological action on the isolated heart and hindgut suggest a physiological role for this peptide in the regulation of cardiac output and hindgut motility.


In-depth sequence analysis of the tomato chromosome 12 centromeric region: identification of a large CAA block and characterization of pericentromere retrotranposons.
We sequenced a continuous 326-kb DNA stretch of a microscopically defined centromeric region of tomato chromosome 12. A total of 84% of the sequence (270 kb) was composed of a nested complex of repeat sequences including 27 retrotransposons, two transposable elements, three MITEs, two terminal repeat retrotransposons in miniature (TRIMs), ten unclassified repeats and three chloroplast DNA insertions. The retrotransposons were grouped into three families of Ty3-Gypsy type long terminal repeat (LTR) retrotransposons (PCRT1-PCRT3) and one LINE-like retrotransposon (PCRT4). High-resolution fluorescence in situ hybridization analyses on pachytene complements revealed that PCRT1a occurs on the pericentromere heterochromatin blocks. PCRT1 was the prevalent retrotransposon family occupying more than 60% of the 326-kb sequence with 19 members grouped into eight subfamilies (PCRT1a-PCRT1h) based on LTR sequence. The PCRT1a subfamily is a rapidly amplified element occupying tens of megabases. The other PCRT1 subfamilies (PCRT1b-PCRT1h) were highly degenerated and interrupted by insertions of other elements. The PCRT1 family shows identity with a previously identified tomato-specific repeat TGR2 and a CENP-B like sequence. A second previously described genomic repeat, TGR3, was identified as a part of the LTR sequence of an Athila-like PCRT2 element of which four copies were found in the 326-kb stretch. A large block of trinucleotide microsatellite (CAA)n occupies the centromere and large portions of the flanking pericentromere heterochromatin blocks of chromosome 12 and most of the other chromosomes. Five putative genes in the remaining 14% of the centromere region were identified, of which one is similar to a transcription regulator (ToCPL1) and a candidate jointless-2 gene.


Missed opportunities in the referral of high-risk infants to early intervention.
Using a statewide population-based data source, we describe current neonatal follow-up referral practices for high-risk infants with developmental delays throughout California. From a cohort analysis of quality improvement data from 66 neonatal follow-up programs in the California Children's Services and California Perinatal Quality Care Collaborative High-Risk Infant Follow-Up Quality of Care Initiative, 5129 high-risk infants were evaluated at the first visit between 4 and 8 months of age in neonatal follow-up. A total of 1737 high-risk infants were evaluated at the second visit between 12 and 16 months of age. We calculated referral rates in relation to developmental status (high versus low concern) based on standardized developmental testing or screening. Among infants with low concerns (standard score >70 or passed screen) at the first visit, 6% were referred to early intervention; among infants with high concerns, 28% of infants were referred to early intervention. Even after including referrals to other (private) therapies, 34% infants with high concerns did not receive any referrals. These rates were similar for the second visit. In spite of the specialization of neonatal follow-up programs to identify high-risk infants with developmental delays, a large proportion of potentially eligible infants were not referred to early intervention.


Synthetic virus-like particles prepared via protein corona formation enable effective vaccination in an avian model of coronavirus infection.
The ongoing battle against current and rising viral infectious threats has prompted increasing effort in the development of vaccine technology. A major thrust in vaccine research focuses on developing formulations with virus-like features towards enhancing antigen presentation and immune processing. Herein, a facile approach to formulate synthetic virus-like particles (sVLPs) is demonstrated by exploiting the phenomenon of protein corona formation induced by the high-energy surfaces of synthetic nanoparticles. Using an avian coronavirus spike protein as a model antigen, sVLPs were prepared by incubating 100 nm gold nanoparticles in a solution containing an optimized concentration of viral proteins. Following removal of free proteins, antigen-laden particles were recovered and showed morphological semblance to natural viral particles under nanoparticle tracking analysis and transmission electron microscopy. As compared to inoculation with free proteins, vaccination with the sVLPs showed enhanced lymphatic antigen delivery, stronger antibody titers, increased splenic T-cell response, and reduced infection-associated symptoms in an avian model of coronavirus infection. Comparison to a commercial whole inactivated virus vaccine also showed evidence of superior antiviral protection by the sVLPs. The study demonstrates a simple yet robust method in bridging viral antigens with synthetic nanoparticles for improved vaccine application; it has practical implications in the management of human viral infections as well as in animal agriculture.


Global characterization of the Dicer-like protein DrnB roles in miRNA biogenesis in the social amoeba Dictyostelium discoideum.
Micro (mi)RNAs regulate gene expression in many eukaryotic organisms where they control diverse biological processes. Their biogenesis, from primary transcripts to mature miRNAs, have been extensively characterized in animals and plants, showing distinct differences between these phylogenetically distant groups of organisms. However, comparably little is known about miRNA biogenesis in organisms whose evolutionary position is placed in between plants and animals and/or in unicellular organisms. Here, we investigate miRNA maturation in the unicellular amoeba Dictyostelium discoideum, belonging to Amoebozoa, which branched out after plants but before animals. High-throughput sequencing of small RNAs and poly(A)-selected RNAs demonstrated that the Dicer-like protein DrnB is required, and essentially specific, for global miRNA maturation in D. discoideum. Our RNA-seq data also showed that longer miRNA transcripts, generally preceded by a T-rich putative promoter motif, accumulate in a drnB knock-out strain. For two model miRNAs we defined the transcriptional start sites (TSSs) of primary (pri)-miRNAs and showed that they carry the RNA polymerase II specific m7G-cap. The generation of the 3'-ends of these pri-miRNAs differs, with pri-mir-1177 reading into the downstream gene, and pri-mir-1176 displaying a distinct end. This 3´-end is processed to shorter intermediates, stabilized in DrnB-depleted cells, of which some carry a short oligo(A)-tail. Furthermore, we identified 10 new miRNAs, all DrnB dependent and developmentally regulated. Thus, the miRNA machinery in D. discoideum shares features with both plants and animals, which is in agreement with its evolutionary position and perhaps also an adaptation to its complex lifestyle: unicellular growth and multicellular development.


Cost-effective fluorescent amplified fragment length polymorphism (AFLP) analyses using a three primer system.
The amplified fragment length polymorphism (AFLP) technique is a widely used multi-purpose DNA fingerprinting tool. The ability to size-separate fluorescently labelled AFLP fragments on a capillary electrophoresis instrument has provided a means for high-throughput genome screening, an approach particularly useful in studying the molecular ecology of nonmodel organisms. While the 'per-marker-generated' costs for AFLP are low, fluorescently labelled oligonucleotides remain costly. We present a cost-effective method for fluorescently end-labelling AFLPs that should make this tool more readily accessible for laboratories with limited budgets. Both standard fluorescent AFLPs and the end-labelled alternatives presented here are repeatable and produce similar numbers of fragments when scored using both manual and automated scoring methods. While it is not recommended to combine data using the two approaches, the results of the methods are qualitatively comparable, indicating that AFLP end-labelling is a robust alternative to standard methods of AFLP genotyping. For researchers commencing a new AFLP project, the AFLP end-labelling method outlined here is easily implemented, as it does not require major changes to PCR protocols and can significantly reduce the costs of AFLP studies.


Mitral annular dilatation and papillary muscle dislocation without mitral regurgitation in sheep.
Asymmetrical mitral annular (MA) dilatation and papillary muscle dislocation are implicated in the pathogenesis of functional mitral regurgitation (MR). To determine the mechanism by which annular and papillary muscle geometric alterations result in MR, we implanted radiopaque markers in the left ventricle, mitral annulus, anterior and posterior mitral leaflets, and papillary muscle tips and bases in 2 groups of sheep. One group served as controls (CTL, n=7); an experimental group (EXP, n=9) underwent topical phenol application to obliterate anterior annular and leaflet muscle (confirmed histologically ex vivo). After 1 week of recovery, markers were imaged with biplane videofluoroscopy, and hemodynamic data were recorded. MA area (computed from 3-dimensional marker coordinates) was 11% to 13% larger in the EXP group than in the CTL group (P<0.05 by ANOVA). This area increase resulted exclusively from intercommissural axis increase except in 1 heart with large (>1 cm) increases in both the intercommissural and septolateral annular axes. The anterior papillary muscle tip in EXP was displaced from CTL by 2.9+/-0.23 mm toward the anterolateral left ventricle and 2.5+/-0.12 mm toward the mitral annulus at end systole; the posterior papillary muscle geometry was unchanged. Transthoracic echocardiography revealed MR only in the heart exhibiting biaxial annular enlargement. MA dilatation in the intercommissural dimension with anterior papillary muscle tip displacement toward the annulus is insufficient to produce MR in sheep. Functional MR may require MA dilatation in the septolateral axis, as observed with proximal circumflex coronary occlusion.


Tuberculous radiculomyelitis presenting as urinary retention in a child with Down's syndrome.
Tuberculous radiculomyelitis (TBRM) is an uncommon complication of TB meningitis. The authors report the case of a 10-year-old Asian girl with trisomy 21, who presented with acute urinary retention and fever. She was initially treated for a urinary tract infection. After an acute neurological deterioration she was found to have evidence of TB meningitis with TBRM. She developed acute hydrocephalus requiring ventriculo-peritoneal shunt. She was treated with quadruple antituberculous therapy and high dose intravenous dexamethasone. She needed tracheostomy with continuous positive airway pressure (CPAP) support. Although she showed gradual neurological improvement in her cognitive functions, she persisted to have quadriparesis with the need for tracheostomy and CPAP support overnight and gastrostomy feeding. Acute urinary retention in children is uncommon, and should serve as a 'red flag' to consideration of further underlying neurological problems. This presentation and subsequent events should serve as a learning point to clinicians.


Update on multi-gene panel testing and communication of genetic test results.
With technological advances, multi-gene panel testing has become increasingly used to identify patients at risk for hereditary breast cancer (HBC). There are currently evidence-based interventions and breast cancer screening strategies that exist for cancer prevention and early detection among patients with HBC. Moreover, in addition to the personal impact of identifying HBC, this information may be shared with at-risk family members to amplify the benefits of testing and subsequent care among those at high risk. Opportunities and challenges with the utilization of updated multi-gene panel testing for HBC, including: (a) tumor sequencing with germline consequences; (b) genetic counseling implications; and (c) strategies to improve the communication of genetic test results to family members will be reviewed. With the advances and expansion of genetic testing, all health care providers need to be updated on both the importance and complexities of HBC counseling and testing, in order to optimize patient care.


Hydration and packing are crucial to amyloidogenesis as revealed by pressure studies on transthyretin variants that either protect or worsen amyloid disease.
The formation of amyloid aggregates is the hallmark of the amyloidogenic diseases. Transthyretin (TTR) is involved in senile systemic amyloidosis (wild-type protein) and familial amyloidotic polyneuropathy (point mutants). Through the use of high hydrostatic pressure (HHP), we compare the stability among wild-type (wt) TTR, two disease-associated mutations (V30M and L55P) and a trans-suppressor mutation (T119M). Our data show that the amyloidogenic conformation, easily populated in the disease-associated mutant L55P, can be induced by a cycle of compression-decompression with the wt protein rendering the latter highly amyloidogenic. After decompression, the recovered wt structure has weaker subunit interactions (loosened tetramer, T(4)(*)) and presents a stability similar to L55P, suggesting that HHP induces a defective fold in the wt protein, converting it to an altered conformation already present in the aggressive mutant, L55P. On the other hand, glucose, a chemical chaperone, can mimic the trans-suppression mutation by stabilizing the native state and by decreasing the amyloidogenic potential of the wt TTR at pH 5.0. The sequence of pressure stability observed was: L55P<V30M<wt<<T119M. The pressure dissociation of L55P at 1 degrees C exhibited dependence on protein concentration, allowing us to assess the volume change of association and the free-energy change. After a cycle of compression-decompression at 37 degrees C and pH 5.6 or lower, all amyloidogenic variants underwent aggregation. Binding of bis-(8-anilinonaphthalene-1-sulfonate) (bis-ANS) revealed that the species formed under pressure retained part of its tertiary contacts (except T119M). However, at neutral pH, where aggregation did not take place after decompression, bis-ANS binding was absent. Thus, TTR has to experience this partially folded conformation to undergo aggregation after decompression. Overall, our studies provide evidence that amyloidogenesis correlates with less packed structures (larger volume changes) and high susceptibility to water infiltration. The hydration effects can be counteracted by osmolytes or by a specific mutation.


Constitutive expression of a grapevine polygalacturonase-inhibiting protein affects gene expression and cell wall properties in uninfected tobacco.
Polygalacturonase-inhibiting proteins (PGIPs) directly limit the effective ingress of fungal pathogens by inhibiting cell wall-degrading endopolygalacturonases (ePGs). Transgenic tobacco plants over-expressing grapevine (Vitis vinifera) Vvpgip1 have previously been shown to be resistant to Botrytis infection. In this study we characterized two of these PGIP over-expressing lines with known resistance phenotypes by gene expression and hormone profiling in the absence of pathogen infection. Global gene expression was performed by a cross-species microarray approach using a potato cDNA microarray. The degree of potential cross-hybridization between probes was modeled by a novel computational workflow designed in-house. Probe annotations were updated by predicting probe-to-transcript hybridizations and combining information derived from other plant species. Comparing uninfected Vvpgip1-overexpressing lines to wild-type (WT), 318 probes showed significant change in expression. Functional groups of genes involved in metabolism and associated to the cell wall were identified and consequent cell wall analysis revealed increased lignin-levels in the transgenic lines, but no major differences in cell wall-derived polysaccharides. GO enrichment analysis also identified genes responsive to auxin, which was supported by elevated indole-acetic acid (IAA) levels in the transgenic lines. Finally, a down-regulation of xyloglucan endotransglycosylase/hydrolases (XTHs), which are important in cell wall remodeling, was linked to a decrease in total XTH activity. This evaluation of PGIP over-expressing plants performed under pathogen-free conditions to exclude the classical PGIP-ePG inhibition interaction indicates additional roles for PGIPs beyond the inhibition of ePGs.


Significance and therapeutic potential of the natriuretic peptides/cGMP/cGMP-dependent protein kinase pathway in vascular regeneration.
Natriuretic peptides (NPs), which consist of atrial, brain, and C-type natriuretic peptides (ANP, BNP, and CNP, respectively), are characterized as cardiac or vascular hormones that elicit their biological effects by activation of the cGMPcGMP-dependent protein kinase (cGK) pathway. We recently reported that adenoviral gene transfer of CNP into rabbit blood vessels not only suppressed neointimal formation but also accelerated reendothelialization, a required step for endothelium-dependent vasorelaxation and antithrombogenicity. Accordingly, we investigated the therapeutic potential of the NPscGMPcGK pathway for vascular regeneration. In transgenic (Tg) mice that overexpress BNP in response to hindlimb ischemia, neovascularization with appropriate mural cell coating was accelerated without edema or bleeding, and impaired angiogenesis by the suppression of nitric oxide production was effectively rescued. Furthermore, in BNP-Tg mice, inflammatory cell infiltration in ischemic tissue and vascular superoxide production were suppressed compared with control mice. Ischemia-induced angiogenesis was also significantly potentiated in cGK type I Tg mice, but attenuated in cGK type I knockout mice. NPs significantly stimulated capillary network formation of cultured endothelial cells by cGK stimulation and subsequent Erk12 activation. Furthermore, gene transfer of CNP into ischemic muscles effectively accelerated angiogenesis. These findings reveal an action of the NPscGMPcGK pathway to exert multiple vasculoprotective and regenerative actions in the absence of apparent adverse effects, and therefore suggest that NPs as the endogenous cardiovascular hormone can be used as a strategy of therapeutic angiogenesis in patients with tissue ischemia.


A simple approach for synthesis, characterization and bioactivity of bovine bones to fabricate the polyurethane nanofiber containing hydroxyapatite nanoparticles.
In the present study, we had introduced polyurethane (PU) nanofibers that contain hydroxyapatite (HAp) nanoparticles (NPs) as a result of an electrospinning process. A simple method that does not depend on additional foreign chemicals had been employed to synthesize HAp NPs through the calcination of bovine bones. Typically, a colloidal gel consisting of HAp/PU had been electrospun to form nanofibers. In this communication, physiochemical aspects of prepared nanofibers were characterized by FE-SEM, TEM and TEM-EDS, which confirmed that nanofibers were well-oriented and good dispersion of HAp NPs, over the prepared nanofibers. Parameters, affecting the utilization of the prepared nanofibers in various nano-biotechnological fields have been studied; for instance, the bioactivity of the produced nanofiber mats was investigated while incubating in simulated body fluid (SBF). The results from incubation of nanofibers, indicated that incorporation of HAp strongly activates the precipitation of the apatite-like particles, because of the HAp NPs act as seed, that accelerate crystallization of the biological HAp from the utilized SBF.


[Construction of a Japanese version of the "Regret and Maximization Scale" in decision making].
This study developed a Japanese version of the "Regret and Maximization Scale" in decision making, which was originally constructed by Schwartz, Ward, Monterosso, Lyubomirsky, White, and Lehman (2002). This scale measures assess the tendency to experience regret, and individual differences in the desire to maximize or to satisfy. In Study 1, the original version of the "Regret and Maximization Scale" was translated into Japanese and administered to 307 Japanese university students responded the scale. Factor analysis did not replicate the finding of Schwartz et al. (2002). In Study 2, we developed new items, and constructed a "Japanese Version of the Regret and Maximization Scale", based on the interpretation of the factor analysis in Study 1. This new version of the scale was administered to 163 Japanese university students. The result of factor analysis and reliability analysis indicated that this "Japanese Version of the Regret and Maximization Scale" had a considerably high Cronbach's alpha and conceptual validity.


New evidence in the treatment of overactive bladder.
Overactive bladder (OAB) in women is highly prevalent. It can be very bothersome and affect quality of life. Treatment differs significantly depending on the clinical presentation, and includes behavioural modification, medicinal therapies and surgical procedures. New treatment options have become available in recent years, and can offer great benefit to patients with persistent, bothersome OAB. First-line management for OAB consists of lifestyle and behavioural therapy, followed by antimuscarinic therapy or oral beta-3 agonists. Sacral neuromodulation, onabotulinumtoxinA and posterior tibial nerve stimulation are additional well tolerated and efficacious treatment options that should be considered in carefully selected, refractory OAB patients. These recent advances in OAB therapy are discussed in this review. The treatment of OAB is often complex, and can require trials of multiple different treatment modalities. Although the treatment advances discussed in this review have augmented treatment of OAB, the condition continues to negatively impact quality of life for our ageing female population. Clinical trials and development of novel therapies continue to be paramount.


Xylitol for the prevention of acute otitis media episodes in children aged 2-4 years: protocol for a pragmatic randomised controlled trial.
Xylitol (or 'birch sugar') is a naturally occurring sugar with antibacterial properties that has been used as a natural non-sugar sweetener in chewing gums, confectionery, toothpaste and medicines. In this preventative randomised trial, xylitol will be tested for the prevention of acute otitis media (AOM), a common and costly condition in young children. The primary outcome will be the incidence of AOM. Secondary outcomes will include upper respiratory tract infections (URTIs) and dental caries. This study will be a pragmatic, blinded (participant and parents, practitioners and analyst), two-armed superiority, placebo-controlled randomised trial with 1:1 allocation, stratified by clinical site. The trial will be conducted in the 11 primary care group practices participating in the TARGet Kids! research network in Canada. Eligible participants between the ages of 2-4 years will be randomly assigned to the intervention arm of regular xylitol syrup use or the control arm of regular sorbitol use for 6 months. We expect to recruit 236 participants, per treatment arm, to detect a 20% relative risk reduction in AOM episodes. AOM will be identified through chart review. The secondary outcomes of URTIs and dental caries will be identified through monthly phone calls with specified questions. Ethics approval from the Research Ethics Boards at the Hospital for Sick Children and St. Michael's Hospital has been obtained for this study and also for the TARGet Kids! research network. Results will be submitted for publication to a peer-reviewed journal and will be discussed with decision makers. NCT03055091; Pre-results.


Large-scale identification and evolution indexing of tyrosine phosphorylation sites from murine brain.
Metazoans employ reversible tyrosine phosphorylation to regulate innumerable biological processes. Thus, the large-scale identification of tyrosine phosphorylation sites from primary tissues is an essential step toward a molecular systems understanding of dynamic regulation in vivo. The relative paucity of phosphotyrosine has greatly limited its identification in large-scale phosphoproteomic experiments. However, using antiphosphotyrosine peptide immunoprecipitations, we report the largest study to date of tyrosine phosphorylation sites from primary tissue, identifying 414 unique tyrosine phosphorylation sites from murine brain. To measure the conservation of phosphorylated tyrosines and their surrounding residues, we constructed a computational pipeline and identified patterns of conservation within the signature of phosphotyrosine.


Connexin 43 Plays a Role in Pulmonary Vascular Reactivity in Mice.
Pulmonary arterial hypertension (PAH) is a chronic condition characterized by vascular remodeling and increased vaso-reactivity. PAH is more common in females than in males (~3:1). Connexin (Cx)43 has been shown to be involved in cellular communication within the pulmonary vasculature. Therefore, we investigated the role of Cx43 in pulmonary vascular reactivity using Cx43 heterozygous (Cx43+/−) mice and 37,43Gap27, which is a pharmacological inhibitor of Cx37 and Cx43. Contraction and relaxation responses were studied in intra-lobar pulmonary arteries (IPAs) derived from normoxic mice and hypoxic mice using wire myography. IPAs from male Cx43+/− mice displayed a small but significant increase in the contractile response to endothelin-1 (but not 5-hydroxytryptamine) under both normoxic and hypoxic conditions. There was no difference in the contractile response to endothelin-1 (ET-1) or 5-hydroxytryptamine (5-HT) in IPAs derived from female Cx43+/−mice compared to wildtype mice. Relaxation responses to methacholine (MCh) were attenuated in IPAs from male and female Cx43+/− mice or by pre-incubation of IPAs with 37,43Gap27. Nω-Nitro-L-arginine methyl ester (l-NAME) fully inhibited MCh-induced relaxation. In conclusion, Cx43 is involved in nitric oxide (NO)-induced pulmonary vascular relaxation and plays a gender-specific and agonist-specific role in pulmonary vascular contractility. Therefore, reduced Cx43 signaling may contribute to pulmonary vascular dysfunction.


[The place of living-donor liver transplants in the Netherlands].
There is a shortage of donor livers from deceased persons, which means there is a relatively high mortality rate among patients on the national liver-transplant waiting list in the Netherlands. This justifies expanding the current programme for liver transplants from living donors. The new law on donor organs law came into effect on 1 July 2020. The expectation is that this will lead to a greater supply of post-mortem donor livers. New methods for organ preservation could also lead to an increase in the number of available donor livers. An increase in the supply of donor livers could lead to changes in donor policy. These changes should take into consideration that a liver transplant from a living donor can take place earlier in the course of the disease and that the quality of the donor liver is better, which means that outcomes for the recipient are also better.


Conjugative plasmids of Neisseria gonorrhoeae.
Many clinical isolates of the human pathogen Neisseria gonorrhoeae contain conjugative plasmids. The host range of these plasmids is limited to Neisseria species, but presence of a tetracycline (tetM) determinant inserted in several of these plasmids is an important cause of the rapid spread of tetracycline resistance. Previously plasmids with different backbones (Dutch and American type backbones) and with and without different tetM determinants (Dutch and American type tetM determinants) have been identified. Within the isolates tested, all plasmids with American or Dutch type tetM determinants contained a Dutch type plasmid backbone. This demonstrated that tetM determinants should not be used to differentiate between conjugal plasmid backbones. The nucleotide sequences of conjugative plasmids with Dutch type plasmid backbones either not containing the tetM determinant (pEP5233) or containing Dutch (pEP5289) or American (pEP5050) type tetM determinants were determined. Analysis of the backbone sequences showed that they belong to a novel IncP1 subfamily divergent from the IncP1alpha, beta, gamma, delta and epsilon subfamilies. The tetM determinants were inserted in a genetic load region found in all these plasmids. Insertion was accompanied by the insertion of a gene with an unknown function, and rearrangement of a toxin/antitoxin gene cluster. The genetic load region contains two toxin/antitoxins of the Zeta/Epsilon toxin/antitoxin family previously only found in Gram positive organisms and the virulence associated protein D of the VapD/VapX toxin/antitoxin family. Remarkably, presence of VapX of pJD1, a small cryptic neisserial plasmid, in the acceptor strain strongly increased the conjugation efficiency, suggesting that it functions as an antitoxin for the conjugative plasmid. The presence of the toxin and antitoxin on different plasmids might explain why the host range of this IncP1 plasmid is limited to Neisseria species. The isolated plasmids conjugated efficiently between N. gonorrhoeae strains, but did not enhance transfer of a genetic marker.


The testicular soma of Tsc22d3 knockout mice supports spermatogenesis and germline transmission from spermatogonial stem cell lines upon transplantation.
Spermatogonial stem cells (SSCs) are adult stem cells that are slowly cycling and self-renewing. The pool of SSCs generates very large numbers of male gametes throughout the life of the individual. SSCs can be cultured in vitro for long periods of time, and established SSC lines can be manipulated genetically. Upon transplantation into the testes of infertile mice, long-term cultured mouse SSCs can differentiate into fertile spermatozoa, which can give rise to live offspring. Here, we show that the testicular soma of mice with a conditional knockout (conKO) in the X-linked gene Tsc22d3 supports spermatogenesis and germline transmission from cultured mouse SSCs upon transplantation. Infertile males were produced by crossing homozygous Tsc22d3 floxed females with homozygous ROSA26-Cre males. We obtained 96 live offspring from six long-term cultured SSC lines with the aid of intracytoplasmic sperm injection. We advocate the further optimization of Tsc22d3-conKO males as recipients for testis transplantation of SSC lines.


Mental representation of verb meaning: behavioral and electrophysiological evidence.
Previous psycholinguistic research has debated the nature of the mental representation of verbs and the access of relevant verb information in sentence processing. In this study, we used behavioral and electrophysiological methods to examine the representation of verbs in and out of sentence contexts. In five experiments, word naming and event-related potential (ERP) components were used to measure the speed and the amplitude, respectively, associated with different verb-object combinations that result in different degrees of fit between the verb and its object. Both naming speed and ERP amplitudes (N400) are proven to be sensitive indices of the degree of fit, varying as a function of how well the object fits the verb in terms of selectional restrictions. The results suggest that the semantic features of the verb's arguments are an integral part of the mental representation of verbs, and such information of the verb is accessed and used on-line during sentence processing. Implications of these results are discussed in light of recent computational semantic models that view the lexicon through high-order lexical co-occurrences in language use.


Upregulating mTOR/ERK signaling with leonurine for promoting angiogenesis and tissue regeneration in a full-thickness cutaneous wound model.
Wound therapy remains a clinical challenge due to the poor vascularization during the healing process and the high demand to achieve functional and aesthetically satisfactory scars. Newly-formed blood vessels are necessary for wound healing since they can deliver nutrients and oxygen to the wound area. In this study, the role of leonurine (LN), a traditional Chinese medicine isolated from Herba leonuri, in promoting angiogenesis and its function in wound healing have been investigated. The results of co-culture with human umbilical vein endothelial cells (HUVECs) demonstrated that LN treatment (5-20 μM) could promote the proliferation and migration and enhance the ability of in vitro angiogenesis through up-regulating the mTOR/ERK signaling pathway. Furthermore, a full-thickness cutaneous wound model was used to investigate the healing effect of LN in vivo. Intragastric administration of 20 mg per kg per day LN stimulated the regeneration of more blood vessels at the wound sites, which confirmed the in vitro results of promoting angiogenesis. Due to fast vascularization, the collagen matrix deposition and remodeling processes were also accelerated in LN treated wounds, resulting in efficient wound healing. In summary, LN promoted angiogenesis of endothelial cells in vitro by activating the mTOR/ERK pathway, and could efficiently enhance the angiogenesis and collagen deposition of the regenerated tissue, together with facilitating the wound healing process in vivo. This study provides evidence for LN-stimulated angiogenesis and tissue regeneration in skin wounds, especially in ischemic wounds.


Nonequilibrium Response of Nanosystems Coupled to Driven Quantum Baths.
Commonly, nanosystems are characterized by their response to time-dependent external fields in the presence of inevitable environmental fluctuations. The direct impact of the external driving on the environment is generally neglected. While this approach is satisfactory for macroscopic systems, on the nanoscale, an interaction of external fields with the environment is often unavoidable on principle. We extend the standard linear response theory of quantum dissipative systems to strongly driven baths. Significant modifications are found for two paradigm examples. First, we evaluate the polarizability of a molecule immersed in a strongly polarizable medium that responds to terahertz radiation. We find an increase of the molecular polarizability by about 30%. Second, we determine the response of a semiconductor quantum dot in close proximity to a metallic nanoparticle. Both are placed in a polarizable medium and exposed to electromagnetic irradiation. We show that the response of the quantum dot is qualitatively modified by the driven nanoparticle, including the generation of an additional channel of stimulated emission.


First clinical and myopathological description of a myofibrillar myopathy with congenital onset and homozygous mutation in FLNC.
Filamin C (encoded by the FLNC gene) is a large actin-cross-linking protein involved in shaping the actin cytoskeleton in response to signaling events both at the sarcolemma and at myofibrillar Z-discs of cross-striated muscle cells. Multiple mutations in FLNC are associated with myofibrillar myopathies of autosomal-dominant inheritance. Here, we describe for the first time a boy with congenital onset of generalized muscular hypotonia and muscular weakness, delayed motor development but no cardiac involvement associated with a homozygous FLNC mutation c.1325C>G (p.Pro442Arg). We performed ultramorphological, proteomic, and functional investigations as well as immunological studies of known marker proteins for dominant filaminopathies. We show that the mutant protein is expressed in similar quantities as the wild-type variant in control skeletal muscle fibers. The proteomic signature of quadriceps muscle is altered and ultrastructural perturbations are evident. Moreover, filaminopathy marker proteins are comparable both in our homozygous and a dominant control case (c.5161delG). Biochemical investigations demonstrate that the recombinant mutant protein is less stable and more prone to degradation by proteolytic enzymes than the wild-type variant. The unusual congenital presentation of the disease clearly demonstrates that homozygosity for mutations in FLNC severely aggravates the phenotype.


Spatial distribution of damage affects the healing, growth, and morphology of coral.
Many predators and herbivores do not kill their prey, but rather remove or damage tissue. Prey are often able to heal or regenerate this lost tissue. If the prey are modular organisms (e.g., some plants and cnidarians), regeneration is frequently influenced by other modules interconnected to damaged ones. For example, many coral predators remove tissue from colonies consisting of many polyps, and these polyps often share resources with their neighbors. Thus, the distribution of tissue loss on a coral colony could affect the coral's response. I hypothesized that spatially aggregated damage might be slow to heal due to competing demands on nearby polyps. To explore the spatial patterns of corallivory and their implications, I conducted: (1) field surveys documenting the spatial distribution of lesions on corals; (2) field experiments testing the effect of the distance between lesions on coral tissue healing, skeletal growth, and morphology; and (3) field surveys relating corallivore presence to coral growth and morphology. In the field surveys, lesions were aggregated at multiple spatial scales, and most lesions had other lesions within 2 cm. When lesions were near one another, coral tissue regeneration was depressed, although there was no effect on whole colony growth. After a year, however, linear extension was lower in the neighborhood of the lesions. Additionally, gastropod corallivores (Coralliophila violacea) with low movement decreased coral growth and increased coral topographical complexity. These results suggest that corallivores that create clusters of coral damage have a greater effect on coral growth and recovery from damage than corallivores that spread damage throughout the colony.


Platelet-rich plasma for managing pain and inflammation in osteoarthritis.
Osteoarthritis (OA) is a common disease involving joint damage, an inadequate healing response and progressive deterioration of the joint architecture. Autologous blood-derived products, such as platelet-rich plasma (PRP), are key sources of molecules involved in tissue repair and regeneration. These products can deliver a collection of bioactive molecules that have important roles in fundamental processes, including inflammation, angiogenesis, cell migration and metabolism in pathological conditions, such as OA. PRP has anti-inflammatory properties through its effects on the canonical nuclear factor κB signalling pathway in multiple cell types including synoviocytes, macrophages and chondrocytes. PRP contains hundreds of different molecules; cells within the joint add to this milieu by secreting additional biologically active molecules in response to PRP. The net results of PRP therapy are varied and can include angiogenesis, the production of local conditions that favour anabolism in the articular cartilage, or the recruitment of repair cells. However, the molecules found in PRP that contribute to angiogenesis and the protection of joint integrity need further clarification. Understanding PRP in molecular terms could help us to exploit its therapeutic potential, and aid the development of novel treatments and tissue-engineering approaches, for the different stages of joint degeneration.


Good clinical outcome after ischemic stroke with successful revascularization is time-dependent.
Trials of IV recombinant tissue plasminogen activator (rt-PA) have demonstrated that longer times from ischemic stroke symptom onset to initiation of treatment are associated with progressively lower likelihoods of clinical benefit, and likely no benefit beyond 4.5 hours. How the timing of IV rt-PA initiation relates to timing of restoration of blood flow has been unclear. An understanding of the relationship between timing of angiographic reperfusion and clinical outcome is needed to establish time parameters for intraarterial (IA) therapies. The Interventional Management of Stroke pilot trials tested combined IV/IA therapy for moderate-to-severe ischemic strokes within 3 hours from symptom onset. To isolate the effect of time to angiographic reperfusion on clinical outcome, we analyzed only middle cerebral artery and distal internal carotid artery occlusions with successful reperfusion (Thrombolysis in Cerebral Infarction 2-3) during the interventional procedure (<7 hours). Time to angiographic reperfusion was defined as time from stroke onset to procedure termination. Good clinical outcome was defined as modified Rankin Score 0-2 at 3 months. Among the 54 cases, only time to angiographic reperfusion and age independently predicted good clinical outcome after angiographic reperfusion. The probability of good clinical outcome decreased as time to angiographic reperfusion increased (unadjusted p = 0.02, adjusted p = 0.01) and approached that of cases without angiographic reperfusion within 7 hours. We provide evidence that good clinical outcome following angiographically successful reperfusion is significantly time-dependent. At later times, angiographic reperfusion may be associated with a poor risk-benefit ratio in unselected patients.


Non-Viral Generation of Neural Precursor-like Cells from Adult Human Fibroblasts.
Recent studies have reported direct reprogramming of human fibroblasts to mature neurons by the introduction of defined neural genes. This technology has potential use in the areas of neurological disease modeling and drug development. However, use of induced neurons for large-scale drug screening and cell-based replacement strategies is limited due to their inability to expand once reprogrammed. We propose it would be more desirable to induce expandable neural precursor cells directly from human fibroblasts. To date several pluripotent and neural transcription factors have been shown to be capable of converting mouse fibroblasts to neural stem/precursor-like cells when delivered by viral vectors. Here we extend these findings and demonstrate that transient ectopic insertion of the transcription factors SOX2 and PAX6 to adult human fibroblasts through use of non-viral plasmid transfection or protein transduction allows the generation of induced neural precursor (iNP) colonies expressing a range of neural stem and pro-neural genes. Upon differentiation, iNP cells give rise to neurons exhibiting typical neuronal morphologies and expressing multiple neuronal markers including tyrosine hydroxylase and GAD65/67. Importantly, iNP-derived neurons demonstrate electrophysiological properties of functionally mature neurons with the capacity to generate action potentials. In addition, iNP cells are capable of differentiating into glial fibrillary acidic protein (GFAP)-expressing astrocytes. This study represents a novel virusfree approach for direct reprogramming of human fibroblasts to a neural precursor fate.


Graphene oxide layers modified by light energetic ions.
In this paper, the effect of light ion irradiation on graphene oxide foil structure and composition was studied. Due to the excellent properties of graphene based materials suitable for application in electronics, optoelectronics, micro-mechanics and space technologies, the interaction of energetic ions with graphene based structures is worth studying. From the fundamental point of view, it is also interesting to get information about graphene oxide structure modification and the possible functional properties after irradiation by energetic ions. The light ion irradiation of graphene oxide (GO) foil was performed using 2.5 MeV H+ and 5.1 MeV He2+ ions. The change in the elemental composition of the GO foils after ion irradiation was investigated using Rutherford Backscattering Spectrometry and Elastic Recoil Detection Analysis. The influence of ion irradiation was further studied by microscopy methods. The chemical composition and structural changes of the GO foil surface were characterized by spectroscopy techniques including XPS, FTIR and Raman spectroscopy. Although the results of ion beam analysis indicated no significant compositional changes in the bulk of GO foils connected to ion irradiation, XPS, ATR-FTIR and Raman spectroscopy revealed reduction and removal of oxygen functionalities on the surface of graphene oxide. This reduction leads to a surface resistivity decrease after ion irradiation dependent on the ion species, fluence and energy.


Conclusion: applying South East Asia Rainforest Research Programme science to land-use management policy and practice in a changing landscape and climate.
The context and challenges relating to the remaining tropical rainforest are briefly reviewed and the roles which science can play in addressing questions are outlined. Key messages which articles in the special issue, mainly based on projects of the Royal Society South East Asia Rainforest Research Programme (SEARRP), have raised of relevance to policies on land use, land management and REDD+ are then considered. Results from the atmospheric science and hydrology papers, and some of the ecological ones, demonstrate the very high ecosystem service values of rainforest (compared with oil palm) in maintaining high biodiversity, good local air quality, reducing greenhouse emissions, and reducing landslide, flooding and sedimentation consequences of climate change-and hence provide science to underpin the protection of remaining forest, even if degraded and fragmented. Another group of articles test ways of restoring forest quality (in terms of biodiversity and carbon value) or maintaining as high biodiversity and ecological functioning levels as possible via intelligent design of forest zones and fragments within oil palm landscapes. Finally, factors that have helped to enhance the policy relevance of SEARRP projects and dissemination of their results to decision-makers are outlined.


A novel staging system for caries severity in the primary dentition.
Caries in the primary dentition (CIPD) has a high prevalence in U.S. children compared to other diseases, with substantial disparities among different population groups. Few reports correlate CIPD prevalence with clinical impairment of children's quality of life, such as tooth pain, speech delay or trauma to the child from operative restorations, which we collectively term morbidity. Likewise, current case definitions (ECC, S-ECC) and disease metrics (mean dmfs/dmft) are not helpful in assessing morbidity for individual or groups of children. We describe a construct to stage caries severity for children ages 0 -5, called "CIPD Levels." This metric is based on small interval age-group dmft scores, and has a direct link to current and predicted morbidity for the child. It is modeled after staging systems for medical diseases in which the various stages or levels are correlated with the probability of morbidity or mortality. We created a matrix in which CIPD Levels 0-4 are assigned for dmft scores 0-7 depending on a child's age. CIPD Level-4 is the highest level, and frequently results in clinical adverse outcomes, including pain and extensive restorations. We next tested this matrix with data from a high-risk population. Among children with any cavitated caries at age <24 months, 82.8% reached the adverse outcomes threshold (CIPD Level-4) at age 36 months. For children with dmft = 0 at 24 months, 71.4% did not reach CIPD Level-4 at age 36 months. Our new metric is useful for quantifying disease burden from caries for high-risk children.


Involvement of the Fas/FasL pathway in the pathogenesis of germ cell tumours of the adult testis.
Induction of apoptosis by Fas ligand (FasL) of Fas-containing cells is a known mechanism involved in the eradication of inappropriate cells during normal development. Alterations of the Fas/FasL pathway have been found in various types of cancer, leading to circumvention of attack of the tumour by the immune system. An alternative way to circumvent eradication by induction of apoptosis is through changes in the downstream inhibitors. For example, Fas-associating phosphatase-1 (Fap-1) binds directly to the Fas receptor and results in a block of the downstream signalling. To shed more light on the role of the Fas/FasL pathway in the development of human testicular germ cell tumours of the adult testis, this study investigated the presence of Fas, FasL, Fap-1, HLA class I and II molecules, CD45 (lymphocyte marker), and CD57 [natural killer (NK) cell marker] by immunohistochemistry on frozen sections of 41 cases of seminomas, non-seminomas, and spermatocytic seminomas. Every germ cell tumour was positive for Fap-1 and negative for HLA classes I and II, like their non-malignant cells of origin. The infiltrating lymphocytes, predominantly present in seminomas, showed consistently positive staining for Fas and CD45, but not for Fap-1. No Fas was found on NK cells. All seminomas and non-seminomas (except teratomas), including their precursor stages, carcinoma in situ, intratubular seminoma and intratubular non-seminoma, showed positive staining for FasL, but not for Fas. Teratoma showed no staining for FasL and was positive for Fas. In contrast, both Fas and FasL were detectable on spermatocytic seminoma. These data indicate a different regulation of the Fas/FasL system in seminoma and spermatocytic seminoma, supporting a separate pathogenesis for these germ cell-derived tumours. The presence of Fap-1 in all histological variants of germ cell tumours might be related to the consistently positive staining in cells of the germ lineage. This study indicates that production of FasL by the germ cell tumour cells might be involved in the early development of these types of adult testicular cancer by inducting apoptosis of Fas-positive, Fap-1-negative tumour-infiltrating lymphocytes.


Distinguishing Subgroups Based on Psychomotor Functioning among Patients with Major Depressive Disorder.
Retardation and agitation are symptoms of major depressive disorder (MDD), and their presence could play a role in determining clinically meaningful depressive subtypes such as nonmelancholic depression (NMD) and melancholic depression (MD). In this project, we explored whether three depression subgroups (NMD, MD with psychotic symptoms, and MD without psychotic symptoms) could be distinguished based on objective measures of psychomotor functioning. Sixty-nine patients with MDD underwent extensive clinical and psychomotor testing prior to treatment with electroconvulsive therapy. Psychomotor functioning was assessed subjectively using the Core Assessment of Psychomotor Change (CORE) and objectively by means of both 24-h actigraphy and performance on a fine motor drawing task. The daytime activity levels measured by actigraphy were significantly lower (F = 7.1, p = 0.0004) in MD patients both with and without psychotic symptoms than in those with NMD. No objective psychomotor variable was able to distinguish between melancholic patients with and those without psychotic symptoms. The depression subtypes NMD, MD with psychotic symptoms, and MD without psychotic symptoms are not marked by increasing psychomotor retardation, possibly because psychomotor disturbance in MD with psychotic symptoms often consists of agitation rather than retardation, or a mixture of the two. However, psychomotor functioning as measured by actigraphy can be used to distinguish between NMD patients and MD patients.


Decidual small extracellular vesicles induce trophoblast invasion by upregulating N-cadherin.
Small extracellular vesicles (sEVs) are important mediators of cell-to-cell communication involved in the successful establishment of a pregnancy. Human decidual stromal cells play a key role in regulating trophoblast invasion. Nevertheless, the regulatory functions of decidual stromal cells-derived sEVs in human trophoblast cells are still unclear. In this study, primary human decidual stromal cells were isolated, and immortalized human endometrial stromal cell line (HESCs) were decidualized into human decidual stromal cells (HDSCs) using hormonal cocktail containing medroxy progesterone 17-acetate (MPA), estrogen and cAMP analog. HDSC-sEVs were isolated from both primary human decidual stromal cells and immortal HDSCs, respectively, and identified by transmission electron microscopy and western blotting. EV uptake assay indicated that HDSC-sEVs could be uptaken by trophoblast cells. HDSC-sEVs could increase the invasiveness and the expression level of N-cadherin of trophoblast cells with elevated phosphorylation of SMAD2 and SMAD3 in the cells. Silencing of N-cadherin could block cell invasion induced by HDSC-sEVs, while knockdown of SMAD2 and SMAD3 could inhibit the upregulation of N-cadherin in trophoblast cells. Taken together, our results suggested a regulatory effect of HDSC-sEVs in the invasion of trophoblast cells, and HDSC-sEVs may be important mediators of trophoblasts during embryo implantation and placentation.


Functional analysis of the two-gene lysis system of the pneumococcal phage Cp-1 in homologous and heterologous host cells.
The two lysis genes cph1 and cpl1 of the Streptococcus pneumoniae bacteriophage Cp-1 coding for holin and lysozyme, respectively, have been cloned and expressed in Escherichia coli. Synthesis of the Cph1 holin resulted in bacterial cell death but not lysis. The cph1 gene was able to complement a lambda Sam mutation in the nonsuppressing E. coli HB101 strain to produce phage progeny, suggesting that the holins encoded by both phage genes have analogous functions and that the pneumococcal holin induces a nonspecific lesion in the cytoplasmic membrane. Concomitant expression of both holin and lysin of Cp-1 in E. coli resulted in cell lysis, apparently due to the ability of the Cpl1 lysozyme to hydrolyze the peptidoglycan layer of this bacterium. The functional analysis of the cph1 and cpl1 genes cloned in a pneumococcal mutant with a complete deletion of the lytA gene, which codes for the S. pneumoniae main autolysin, provided the first direct evidence that, in this gram-positive-bacterium system, the Cpl1 endolysin is released to its murein substrate through the activity of the Cph1 holin. Demonstration of holin function was achieved by proving the release of pneumolysin to the periplasmic fraction, which strongly suggested that the holin produces a lesion in the pneumococcal membrane.