When urban taps run dry: sachet water consumption and health effects in low income neighborhoods of Accra, Ghana.
Intraurban differentials in safe drinking water in developing cities have been exacerbated by rapid population growth that exceeds expansion of local water infrastructure. In Accra, Ghana, municipal water is rationed to meet demand, and the gap in water services is increasingly being filled by private water vendors selling packaged "sachet" water. Sachets extend drinking water coverage deeper into low-income areas and alleviate the need for safe water storage, potentially introducing a health benefit over stored tap water. We explore correlates of using sachets as the primary drinking water source for 2093 women in 37 census areas classified as slums by UN-Habitat, and links between sachet water and reported diarrhea episodes in a subset of 810 children under five. We find that neighborhood rationing exerts a strong effect on a household's likelihood of buying sachet water, and that sachet customers tend to be the poorest of the poor. Sachet use is also associated with higher levels of self-reported overall health in women, and lower likelihood of diarrhea in children. We conclude with implications for sachet regulation in Accra and other sub-Saharan cities facing drinking water shortages.


Pre-hospital administration of ticagrelor in diabetic patients with ST-elevation myocardial infarction undergoing primary angioplasty: A sub-analysis of the ATLANTIC trial.
We investigated, in the contemporary era of ST-elevation myocardial infarction (STEMI) treatment, the influence of diabetes mellitus (DM) on cardiovascular outcomes, and whether pre-hospital administration of ticagrelor may affect these outcomes in a subgroup of STEMI patients with DM. DM patients have high platelet reactivity and a prothrombotic condition which highlight the importance of an effective antithrombotic regimen in this high-risk population. In toal 1,630 STEMI patients enrolled in the ATLANTIC trial who underwent primary percutaneous coronary intervention (PCI) were included. Multivariate analysis was used to explore the association of DM with outcomes and potential treatment-by-diabetes interaction was tested. A total of 214/1,630 (13.1%) patients had DM. DM was an independent predictor of poor myocardial reperfusion as reflected by less frequent ST-segment elevation resolution (≥70%) after PCI (OR 0.59, 95% CI 0.43-0.82, P < 0.01) and was an independent predictor of the composite 30-day outcomes of death/new myocardial infarction (MI)/urgent revascularization/definite stent thrombosis (ST) (OR 2.80, 95% CI 1.62-4.85, P < 0.01), new MI or definite acute ST (OR 2.46, 95% CI 1.08-5.61, P = 0.03), and definite ST (OR 10.00, 95% CI 3.54-28.22, P < 0.01). No significant interaction between pre-hospital ticagrelor vs in-hospital ticagrelor administration and DM was present for the clinical, electrocardiographic and angiographic outcomes as well as for thrombolysis in myocardial infarction major bleeding. DM remains independently associated with poor myocardial reperfusion and worse 30-day clinical outcomes. No significant interaction was found between pre-hospital vs in-hospital ticagrelor administration and DM status. Further approaches for the treatment of DM patients are needed. clinicaltrials.gov identifier: NCT01347580.


Effects of resistance exercise combined with vascular occlusion on muscle function in athletes.
The effects of resistance exercise combined with vascular occlusion on muscle function were investigated in highly trained athletes. Elite rugby players (n = 17) took part in an 8 week study of exercise training of the knee extensor muscles, in which low-intensity [about 50% of one repetition maximum] exercise combined with an occlusion pressure of about 200 mmHg (LIO, n = 6), low-intensity exercise without the occlusion (LI, n = 6), and no exercise training (untrained control, n = 5) were included. The exercise in the LI group was of the same intensity and amount as in the LIO group. The LIO group showed a significantly larger increase in isokinetic knee extension torque than that in the other two groups (P < 0.05) at all the velocities studied. On the other hand, no significant difference was seen between LI and the control group. In the LIO group, the cross-sectional area of knee extensors increased significantly (P < 0.01), suggesting that the increase in knee extension strength was mainly caused by muscle hypertrophy. The dynamic endurance of knee extensors estimated from the decreases in mechanical work production and peak force after 50 repeated concentric contractions was also improved after LIO, whereas no significant change was observed in the LI and control groups. The results indicated that low-intensity resistance exercise causes, in almost fully trained athletes, increases in muscle size, strength and endurance, when combined with vascular occlusion.


Delivery of ketotifen fumarate by commercial contact lens materials.
To investigate the uptake and delivery of the anti-allergy drug ketotifen fumarate (KF) by commercially available contact lenses. A total of 14 different commercially available contact lenses were investigated, including five frequent-replacement silicone hydrogels, three conventional hydrogels, and six daily disposable lenses. Lenses were soaked in a 0.025% KF loading solution for 24 h, and the concentration of KF in solution over time was determined by ultraviolet absorbance at 297 nm. After the 24-h loading period, lenses were placed in fresh vials containing borate buffered saline, and the release of drug into solution at 34°C was monitored for 24 h. All the lenses studied demonstrated significant uptake and release of KF into the borate buffered saline (p < 0.05 compared with initial time point). Lenses with charged surfaces [balafilcon A, etafilcon A, and etafilcon A (daily disposable)] demonstrated the greatest uptake and release of KF. Etafilcon A released 284.5 ± 29.8 μg/lens, whereas balafilcon A released 227.6 ± 14.7 μg/lens, which was substantially more (p < 0.05) than the lowest releasing lenses [nelfilcon A (40.4 ± 4.1 μg/lens) and comfilcon A (110.4 ± 8.9 μg/lens)]. The majority of lenses were able to match or exceed the total amount of KF commonly administered to the eye using twice-daily dosing of commercially available (0.025%) eye drop formulations. Most of the lenses surveyed reached a plateau concentration of KF relatively quickly, and no lens was able to release KF for longer than 4 h. Commercially available lenses demonstrated the ability to release a clinically relevant amount of KF compared with conventional eye drops. The use of commercially available contact lenses as a KF delivery system in a daily wear scenario may be feasible.


Tcstv1 and Tcstv3 elongate telomeres of mouse ES cells.
Mouse embryonic stem cell (ESC) cultures exhibit a heterogeneous mixture of metastable cells sporadically entering the 2-cell (2C)-embryo-like state, critical for ESC potency. One of 2-cell genes, Zscan4, has been shown to be responsible for telomere maintenance, genomic stability and pluripotency of mouse ESCs. Functions of other 2C-genes in ESCs remain elusive. Here we show that 2C-genes Tcstv1 and Tcstv3 play a role in regulation of telomere lengths. Overexpression or knockdown Tcstv1 and Tcstv3 does not immediately affect proliferation, pluripotency and differentiation in vitro of ESCs. However, ectopic expression of Tcstv1 or Tcstv3 results in telomere elongation, whereas Tcstv1/3 knockdown shortens telomeres of ESCs. Overexpression of Tcstv1 or Tcstv3 does not alter telomere stability. Furthermore, Tcstv1 can increase Zscan4 protein levels and telomere recombination by telomere sister chromatid exchange (T-SCE). Depletion of Tcstv1/3 reduces Zscan4 protein levels. Together, Tcstv1 and Tcstv3 are involved in telomere maintenance that is required for long-term self-renewal of mouse ESCs. Our data also suggests that Tcstv1/3 may co-operate and stabilize Zscan4 protein but the molecular bases remain to be determined.


EMS agenda for the future: where we are ... where we want to be. EMS Agenda for the Future Steering Committee.
During the past 30 years, emergency medical services (EMS) in the United States have experienced explosive growth. The American health care system is now transforming, providing an opportune time to examine what we have learned over the past three decades in order to create a vision for the future of EMS. Over the course of several months, a multidisciplinary steering committee collaborated with hundreds of EMS-interested individuals, organizations, and agencies to develop the "EMS Agenda for the Future." Fourteen EMS attributes were identified as requiring continued development in order to realize the vision established within the Agenda. They are Integration of Health Services, EMS Research, Legislation and Regulation, System Finance, Human Resources, Medical Direction, Education Systems, Public Education, Prevention, Public Access, Communication Systems, Clinical Care, Information Systems, and Evaluation. Discussion of these attributes provides important guidance for achieving a vision for the future of EMS that emphasizes its critical role in American health care.


Analysis of detergent-resistant membranes associated with apical and basolateral GPI-anchored proteins in polarized epithelial cells.
Detergent-resistant membranes (DRMs) represent specialized membrane domains resistant to detergent extraction, which may serve to segregate proteins in a specific environment in order to improve their function. Segregation of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in DRMs has been shown to be involved in their sorting to the apical membrane in polarized epithelial cells. Nonetheless, we have shown that both apical and basolateral GPI-APs associate with DRMs. In this report we investigated the lipid composition of DRMs associated with an apical and a basolateral GPI-AP. We found that apical and basolateral DRMs contain the same lipid species although in different ratios. This specific lipid ratio is maintained after mixing the cells before lysis indicating that DRMs maintain their identity after Triton extraction.


Toxoplasma modifies macrophage phagosomes by secretion of a vesicular network rich in surface proteins.
Modification of macrophage phagosomes begins shortly after formation as Toxoplasma cells secrete membranous vesicles that form a reticulate network within the vacuole. The Toxoplasma-modified compartments then resist normal endocytic processing and digestion. We have used the pronounced Ca++-dependent stability of the intraphagosomal membrane (IPM) network to purify and characterize the structural proteins of this assembly. In addition to the structural matrix, Toxoplasma secretes a discrete set of soluble proteins, including a newly described 22-kD calcium-binding protein. The IPM network adheres to intact Toxoplasma cells after host cell lysis in the presence of 1 mM Ca++; however, the network readily disperses in calcium-free buffer and was purified as vesicles that sedimented at 100,000 g. Purified IPM vesicles were specifically recognized by immune sera from mice with chronic Toxoplasma infection and consisted primarily of a 30-kD protein when analyzed by SDS PAGE. IPM network proteins share a major antigenic component located on the surface of extracellular Toxoplasma cells as shown by immunoperoxidase electron microscopy using a polyclonal antibody prepared against the IPM vesicles. Moreover, in Toxoplasma-infected macrophages, anti-IMP antibody confirmed that the extensive IPM array contains proteins also found on the Toxoplasma cell surface. Our results indicate the IMP network represents a unique structural modification of the phagosome comprised in part of Toxoplasma surface proteins.


Using multilevel spatial models to understand salamander site occupancy patterns after wildfire.
Studies of the distribution of elusive forest wildlife have suffered from the confounding of true presence with the uncertainty of detection. Occupancy modeling, which incorporates probabilities of species detection conditional on presence, is an emerging approach for reducing observation bias. However, the current likelihood modeling framework is restrictive for handling unexplained sources of variation in the response that may occur when there are dependence structures such as smaller sampling units that are nested within larger sampling units. We used multilevel Bayesian occupancy modeling to handle dependence structures and to partition sources of variation in occupancy of sites by terrestrial salamanders (family Plethodontidae) within and surrounding an earlier wildfire in western Oregon, USA. Comparison of model fit favored a spatial N-mixture model that accounted for variation in salamander abundance over models that were based on binary detection/non-detection data. Though catch per unit effort was higher in burned areas than unburned, there was strong support that this pattern was due to a higher probability of capture for individuals in burned plots. Within the burn, the odds of capturing an individual given it was present were 2.06 times the odds outside the burn, reflecting reduced complexity of ground cover in the burn. Ther was weak support that true occupancy was lower within the burned area. While the odds of occupancy in the burn were 0.49 times the odds outside the burn among the five species, the magnitude of variation attributed to the burn was small in comparison to variation attributed to other landscape variables and to unexplained, spatially autocorrelated random variation. While ordinary occupancy models may separate the biological pattern of interest from variation in detection probability when all sources of variation are known, the addition of random effects structures for unexplained sources of variation in occupancy and detection probability may often more appropriately represent levels of uncertainty.


Symptomatic remission in schizophrenia: Results from a risperidone maintenance treatment study.
This study aimed to investigate remission following the treatment of schizophrenia patients with risperidone. Clinically stabilized patients with schizophrenia (n = 374) were randomly assigned to 4-week, 26-week, or no-dose-reduction groups, in which the baseline risperidone dose was continued for 4, 26, or all weeks during 1-year period. The 'Positive and Negative Syndrome Scale' (PANNS) was assessed at baseline and monthly for six months, followed by every two months until the last recruited patient completed 1-year follow-up. Symptomatic remission was defined according to criteria established by the Schizophrenia Working Group. A Generalized Linear Mixed Model indicated significant variation in remission over time, which increased after baseline in the entire group (F = 49.32, df = 1, 3114, P < 0.001). The overall length of risperidone treatment (F = 4.34, df = 1, 416, P = 0.038) and the duration of illness (F = 8.51, df = 1, 359, P = 0.004) had significantly negative effects upon remission. Baseline remission patients were associated with a significantly increased time to relapse compared with the baseline of non-remission patients over a one year follow up period (F = 5.74, df = 1, 367, P = 0.017). One-year risperidone maintenance treatment increased remission rates in schizophrenia. A shorter illness duration, risperidone treatment length, and a lower total PANSS score were clinically useful predictors of remission. Achieving remission may postpone relapse.


The level of cell surface beta1,4-galactosyltransferase I influences the invasive potential of murine melanoma cells.
Beta1,4-Galactosyltransferase I (GalT I) is localized on the leading lamellipodia of migrating cells, where it associates with the cytoskeleton and facilitates cell spreading and migration on basal lamina matrices. It has previously been reported that a variety of highly metastatic murine and human cell lines are characterized by elevated levels of cell surface GalT I, although the intracellular biosynthetic pool is similar between cells of high and low metastatic potential. In this study, we examined whether the elevated expression of surface GalT I characteristic of metastatic cells is instructive or incidental to their metastatic behavior by altering the expression of surface GalT I and by the use of GalT I-specific perturbants. Surface GalT I levels were positively and negatively altered on murine melanoma cells by either overexpressing full-length GalT I or by homologous recombination, respectively. The consequences of altered surface GalT I expression on cell invasion in vitro and lung colonization in vivo were determined. Increasing surface GalT I expression on cells of low metastatic potential to levels characteristic of highly metastatic cells recapitulated the highly invasive phenotype in vitro. Alternatively, decreasing surface GalT I expression on highly metastatic cells to levels characteristic of low metastatic cells reduced their invasive behavior in vitro and metastatic activity in vivo. Within the physiological range of surface GalT I expression, the invasive potential of each clonal cell line correlated strongly with the level of surface GalT I expressed. As an independent means to assess the involvement of surface GalT I in metastatic behavior, cells were pretreated with two different classes of surface GalT I perturbants, a competitive oligosaccharide substrate and a substrate modifier protein. Both perturbants inhibited metastatic colonization of the lung, whereas control reagents did not. Finally, as reported by others, surface GalT I on metastatic cells selectively interacted with one glycoprotein substrate, or ligand, of approximately 100 kDa, the identity of which remains obscure. These results show that the elevated expression of surface GalT I characteristic of highly metastatic cells contributes to their invasive phenotype in vitro and to their metastatic phenotype in vivo.


Concomitant chronic subdural hematomas and arachnoid cysts in young adults.
Objective: This study aimed to evaluate the correlation between arachnoid cysts and chronic subdural hematomas in young adults. Methods: This retrospective study evaluated ten patients having concomitant chronic subdural hematomas and arachnoid cysts. Patients were evaluated with the data of age and gender, location of hematoma and arachnoid cyst, trauma history, symptoms at admission, maximum hematoma diameter, contiguity between arachnoid cyst and hematoma, and treatment methods. Results: We treated 285 patients who were diagnosed with cSDH between January 2013 and December 2019. 22 patients were under the age of 40 years. Ten of them had both cSDH and arachnoid cysts. The mean age of patients was 24.8±3.9 years. Patients with only chronic subdural hematoma had higher mean age than the patients with arachnoid cyst-related chronic subdural hematoma. In four patients, the onset of chronic subdural hematoma was reported after arachnoid cyst diagnosis. Four of the patients did not have causative trauma history, and two patients suffered minor sports-related traumas. All patients had headache, and only two patients had hemiparesis. The location of arachnoid cysts were in the middle fossa in eight patients. All patients had chronic subdural hematomas on the ipsilateral side of arachnoid cyst. Four patients who had smaller than 10 mm maximal cSDH diameter underwent conservative management. They were followed by serial neuroimaging studies and it was noted that the hematoma disappered and the size of the arachnoid cysts decreased over time without any neurological complication. In six cases, craniotomy was required, and all recovered completely. cSDH did not recur during 5-60 months of follow-up period (median 12 months). Conclusions: It seems that presence of an arachnoid cyst in young adults is a predisposing factor for the formation of chronic subdural hematoma. Coincidentally diagnosed arachnoid cyst patients may be followed up with periodical clinical examinations and neuroimaging studies.


Reliable assessment of skin flap viability using orthogonal polarization imaging.
Intraoperative evaluation of skin flap viability has primarily been dependent on clinical judgment. The purpose of this study was to determine whether an orthogonal polarization spectral imaging device could be used to accurately predict viability of random-pattern skin flaps. Orthogonal polarization spectral imaging is a newly developed technique that visualizes the microcirculation using reflected light without the use of fluorescent dyes and allows for noninvasive real-time observation of functional microvascular networks. In Sprague-Dawley rats (n = 24), three types of random skin flaps were designed with unknown zones of viability (n = 8 per group). After flap elevation, the skin flaps were evaluated by both clinical examination and orthogonal polarization spectral imaging. Areas of the flap determined to be nonviable by clinical examination were measured and marked. Orthogonal polarization spectral imaging was subsequently performed, and areas of the skin flap with stasis (i.e., cessation of red blood cell movement) in the dermal microcirculation on orthogonal polarization spectral imaging were measured and marked. The skin flaps were then secured in place. Flaps were evaluated on a daily basis for clinical signs of ischemia and necrosis. On postoperative day 7, the total amount of random skin flap necrosis was measured and recorded. Clinical examination of the random skin flaps significantly underestimated the actual amount of eventual flap necrosis, and as result was a very poor predictor of flap necrosis. By contrast, assessment of microcirculatory stasis using the orthogonal polarization spectral imaging device correlated well with the subsequent development of necrosis in all groups. In the three groups, the average amount of flap necrosis predicted by clinical examination deviated from actual necrosis by approximately 2 to 4 cm. However, the amount that orthogonal polarization spectral imaging differed from actual necrosis was 0.1 to 0.3 cm. Therefore, orthogonal polarization spectral imaging was an excellent predictor of eventual flap necrosis and much more accurate than clinical observation (p < 0.001). Intraoperative evaluation of axial and random pattern flap viability has traditionally been based on clinical examination as no other reliable, convenient test currently exists. The authors demonstrated that an orthogonal polarization spectral imaging device accurately predicts zones of necrosis in random pattern flaps by directly visualizing cessation of microcirculatory flow. Intraoperative stasis in the dermal microcirculation correlated precisely with subsequent flap necrosis. Orthogonal polarization spectral imaging was significantly more accurate than clinical examination, which consistently underestimated flap necrosis. The orthogonal polarization spectral imaging technique may have value in the intraoperative assessment of skin flap perfusion such as that required after skin-sparing mastectomy.


Preclinical screening of histone deacetylase inhibitors combined with ABT-737, rhTRAIL/MD5-1 or 5-azacytidine using syngeneic Vk*MYC multiple myeloma.
Multiple myeloma (MM) is an incurable malignancy with an unmet need for innovative treatment options. Histone deacetylase inhibitors (HDACi) are a new class of anticancer agent that have demonstrated activity in hematological malignancies. Here, we investigated the efficacy and safety of HDACi (vorinostat, panobinostat, romidepsin) and novel combination therapies using in vitro human MM cell lines and in vivo preclinical screening utilizing syngeneic transplanted Vk*MYC MM. HDACi were combined with ABT-737, which targets the intrinsic apoptosis pathway, recombinant human tumour necrosis factor-related apoptosis-inducing ligand (rhTRAIL/MD5-1), that activates the extrinsic apoptosis pathway or the DNA methyl transferase inhibitor 5-azacytidine. We demonstrate that in vitro cell line-based studies provide some insight into drug activity and combination therapies that synergistically kill MM cells; however, they do not always predict in vivo preclinical efficacy or toxicity. Importantly, utilizing transplanted Vk*MYC MM, we report that panobinostat and 5-azacytidine synergize to prolong the survival of tumor-bearing mice. In contrast, combined HDACi/rhTRAIL-based strategies, while efficacious, demonstrated on-target dose-limiting toxicities that precluded prolonged treatment. Taken together, our studies provide evidence that the transplanted Vk*MYC model of MM is a useful screening tool for anti-MM drugs and should aid in the prioritization of novel drug testing in the clinic.


Molecular analysis of the SNF4 gene of Saccharomyces cerevisiae: evidence for physical association of the SNF4 protein with the SNF1 protein kinase.
The SNF4 gene is required for expression of glucose-repressible genes in response to glucose deprivation in Saccharomyces cerevisiae. Previous evidence suggested that SNF4 is functionally related to SNF1, another essential gene in this global regulatory system that encodes a protein kinase. Increased SNF1 gene dosage partially compensates for a mutation in SNF4, and the SNF4 function is required for maximal SNF1 protein kinase activity in vitro. We have cloned SNF4 and identified its 1.2-kilobase RNA, which is not regulated by glucose repression. A 36-kilodalton SNF4 protein is predicted from the nucleotide sequence. Disruption of the chromosomal SNF4 locus revealed that the requirement for SNF4 function is less stringent at low temperature (23 degrees C). A bifunctional SNF4-lacZ gene fusion that includes almost the entire SNF4 coding sequence was constructed. The fusion protein was shown by immunofluorescence microscopy to be distributed throughout the cell, with partial localization to the nucleus. The SNF4-beta-galactosidase protein coimmunoprecipitated with the SNF1 protein kinase, thus providing evidence for the physical association of the two proteins.


New rapid one-step PCR diagnostic assay for Plasmodium falciparum infective mosquitoes.
An essential component of malaria vector control programmes is the detection of Plasmodium falciparum within its mosquito vectors, particularly in the salivary glands where the infective sporozoites reside. Several protocols have been developed for this purpose; however they require dissection of mosquito specimens prior to analysis. Here, a novel one-step RT-qPCR TaqMan diagnostic assay was developed for mosquitoes with infective Plasmodium falciparum sporozoites in the salivary glands. It is based on detection of the sporozoite-specific Pfslarp and Pfplp1 gene transcripts. These transcripts were chosen based on bioinformatics analysis, and experimentally verified to be overexpressed in the salivary gland sporozoite stage of the parasite compared to other mosquito parasite stages. The proof of principle and the performance of the assay were demonstrated using RNAlater preserved mosquito samples. Tests of analytical sensitivity showed the novel TaqMan assay to be 100% accurate, although its performance in the field needs to be further demonstrated. This method has no requirement for dissection and post-PCR processing and thus is simple and rapid to perform in individual mosquitoes or mosquito pools. It can be used in single or multiplex formats also targeting additional markers expressed in different tissues, such as detoxification enzymes associated with insecticide resistance.


A Noninvasive Imaging Toolbox Indicates Limited Therapeutic Potential of Conditionally Activated Macrophages in a Mouse Model of Multiple Organ Dysfunction.
Cell-based regenerative medicine therapies require robust preclinical safety, efficacy, biodistribution, and engraftment data prior to clinical testing. To address these challenges, we have developed an imaging toolbox comprising multispectral optoacoustic tomography and ultrasonography, which allows the degree of kidney, liver, and cardiac injury and the extent of functional recovery to be assessed noninvasively in a mouse model of multiorgan dysfunction. This toolbox allowed us to determine the therapeutic effects of adoptively transferred macrophages. Using bioluminescence imaging, we could then investigate the association between amelioration and biodistribution. Macrophage therapy provided limited improvement of kidney and liver function, although not significantly so, without amelioration of histological damage. No improvement in cardiac function was observed. Biodistribution analysis showed that macrophages homed and persisted in the injured kidneys and liver but did not populate the heart. Our data suggest that the limited improvement observed in kidney and liver function could be mediated by M2 macrophages. More importantly, we demonstrate here the utility of the imaging toolbox for assessing the efficacy of potential regenerative medicine therapies in multiple organs.


Prevention of HIV-1 Transmission Through Breastfeeding: Efficacy and Safety of Maternal Antiretroviral Therapy Versus Infant Nevirapine Prophylaxis for Duration of Breastfeeding in HIV-1-Infected Women With High CD4 Cell Count (IMPAACT PROMISE): A Randomized, Open-Label, Clinical Trial.
No randomized trial has directly compared the efficacy of prolonged infant antiretroviral prophylaxis versus maternal antiretroviral therapy (mART) for prevention of mother-to-child transmission throughout the breastfeeding period. Fourteen sites in Sub-Saharan Africa and India. A randomized, open-label strategy trial was conducted in HIV-1-infected women with CD4 counts ≥350 cells/mm (or ≥country-specific ART threshold if higher) and their breastfeeding HIV-1-uninfected newborns. Randomization at 6-14 days postpartum was to mART or infant nevirapine (iNVP) prophylaxis continued until 18 months after delivery or breastfeeding cessation, infant HIV-1 infection, or toxicity, whichever occurred first. The primary efficacy outcome was confirmed infant HIV-1 infection. Efficacy analyses included all randomized mother-infant pairs except those with infant HIV-1 infection at entry. Between June 2011 and October 2014, 2431 mother-infant pairs were enrolled; 97% of women were World Health Organization Clinical Stage I, median screening CD4 count 686 cells/mm. Median infant gestational age/birth weight was 39 weeks/2.9 kilograms. Seven of 1219 (0.57%) and 7 of 1211 (0.58%) analyzed infants in the mART and iNVP arms, respectively, were HIV-infected (hazard ratio 1.0, 96% repeated confidence interval 0.3-3.1); infant HIV-free survival was high (97.1%, mART and 97.7%, iNVP, at 24 months). There were no significant differences between arms in median time to breastfeeding cessation (16 months) or incidence of severe, life-threatening, or fatal adverse events for mothers or infants (14 and 42 per 100 person-years, respectively). Both mART and iNVP prophylaxis strategies were safe and associated with very low breastfeeding HIV-1 transmission and high infant HIV-1-free survival at 24 months.


Fragmentation of the Golgi apparatus of motor neurons in amyotrophic lateral sclerosis.
The Golgi apparatus (complex) is at the center stage of important functions of processing and transport of plasma membrane, lysosomal, and secreted proteins. The involvement of the Golgi apparatus in the pathogenesis of chronic degenerative diseases of neurons is virtually unknown. In the present study, fragmentation and atrophy of the Golgi apparatus of motor neurons in amyotrophic lateral sclerosis (ALS), has been detected with organelle specific antibodies. Approximately 30% of motor neurons in five ALS patients showed a fragmented Golgi apparatus whereas only about 1% of motor neurons from seven controls with neurologic or systemic disease showed a similar change. Morphometric studies are consistent with the hypothesis that the alteration of the Golgi apparatus is an early event in the pathogenesis of the neuronal degeneration in ALS. Immunocytochemical studies with antibodies against alpha tubulin, tau, and phosphorylated subunits of neurofilament polypeptides did not disclose differences in the staining of neurons with fragmented or normal Golgi apparatus, suggesting that the alteration of the organelle is not secondary to a gross lesion of the cytoskeleton. However, these observations do not rule out the hypothesis that the fragmentation of the Golgi apparatus is secondary to subtle changes of the polypeptides involved in the attachment of membranes of the organelle to the cytoskeleton.


Phase I and II study of azacitidine in Japanese patients with myelodysplastic syndromes.
Azacitidine, an inhibitor of DNA methyltransferase, is reported to have antileukemic efficacy and is approved for the treatment of myelodysplastic syndromes in Western countries. We have conducted a Phase I/II study of azacitidine in Japanese patients with myelodysplastic syndromes to evaluate its pharmacokinetics, efficacy, and safety. In all, 53 patients received 75 mg/m(2) azacitidine subcutaneously or intravenously once daily for seven consecutive days on a 28-day cycle. The C(max) following intravenous administration was approximately 3.7-fold higher than that following subcutaneous administration, whereas the area under the plasma concentration-time curve from time zero to infinity was comparable for subcutaneous and intravenous administration. The bioavailability of azacitidine following subcutaneous administration was 91.1%, indicating that azacitidine is nearly completely absorbed after subcutaneous administration. The hematologic improvement and hematologic response rates were 54.9% (28/51) and 28.3% (15/53), respectively, and there were no differences between the two routes of administration. Azacitidine was generally well tolerated and clinically manageable in Japanese patients with myelodysplastic syndromes. Adverse events occurred in ≥ 20% of patients included hematologic toxicity, gastrointestinal events, and general disorders, such as malaise. Grade 3/4 adverse events that occurred in ≥ 50% of patients were all due to hematologic toxicity. The safety profile of azacitidine was generally similar for both routes of administration, with the exception of injection site reactions observed following subcutaneous administration. These results indicate that azacitidine can be expected to be a useful therapeutic agent in Japanese patients with myelodysplastic syndromes.


High strength domestic wastewater treatment with submerged forward osmosis membrane bioreactor.
Forward osmosis membranes are less prone to fouling with high rejection of salts, and the osmotic membrane bioreactor (OMBR) can be considered as an innovative membrane technology for wastewater treatment. In this study, a submerged OMBR having a cellulose triacetate membrane, with the active layer facing the feed solution configuration, was operated at different organic loading rates (OLRs), i.e., 0.4, 1.2 and 2.0 kg-COD/(m(3)·d) with chemical oxygen demand (COD) concentrations of 200 mg/L, 600 mg/L and 1,000 mg/L, respectively, to evaluate the performance on varying wastewater strengths. High organic content with sufficient amount of nutrients enhanced the biomass growth. High OLR caused more extrapolymeric substances production and less dewaterability. However, no significant differences in fouling trends and flux rates were observed among different OLR operational conditions.


Metal microdrive and head cap system for silicon probe recovery in freely moving rodent.
High-yield electrophysiological extracellular recording in freely moving rodents provides a unique window into the temporal dynamics of neural circuits. Recording from unrestrained animals is critical to investigate brain activity during natural behaviors. The use and implantation of high-channel-count silicon probes represent the largest cost and experimental complexity associated with such recordings making a recoverable and reusable system desirable. To address this, we have designed and tested a novel 3D printed head-gear system for freely moving mice and rats. The system consists of a recoverable microdrive printed in stainless steel and a plastic head cap system, allowing researchers to reuse the silicon probes with ease, decreasing the effective cost, and the experimental effort and complexity. The cap designs are modular and provide structural protection and electrical shielding to the implanted hardware and electronics. We provide detailed procedural instructions allowing researchers to adapt and flexibly modify the head-gear system.


The successful use of polylactide wound dressings for chronic lower leg wounds: A retrospective analysis.
Chronic wounds are a challenging medical entity for patients, medical professionals and healthcare systems. Frequently, patients present themselves to wound specialists after months or even years of unsuccessful treatment. Recent developments have resulted in a multitude of different advanced wound dressings created to treat complex, chronic wounds, one of which is the polylactide dressing Suprathel. This study aimed at investigating the healing potential of Suprathel in chronic wounds and differentiating between old and "young", diabetic and non-diabetic chronic wounds. A prospective, multicentric, non-controlled intervention study was conducted, treating patients with chronic lower leg ulcers (>3 months) with Suprathel and assessing them weekly. Afterwards, a retrospective analysis was performed analysing the wound size initially, after 4 and after 8 weeks of treatment. Furthermore, a differentiation between diabetic and non-diabetic, and chronic wounds older and younger than 12 months, was assessed. A significant reduction in wound size was observed in the study population after 8 weeks of treatment. The effect size in the diabetic wound and the old chronic wound group even reached more than one, with the other groups still showing a large effect of the intervention. This study shows that Suprathel is a valuable tool in the armamentarium of a wound specialist. Not only could we show a positive effect on chronic wounds, we could even demonstrate a significant wound size reduction in chronic wounds of old and young, as well as diabetic wounds, with the treatment of older chronic and diabetic wounds yielding an even larger effect size. Further randomised, controlled studies are necessary to show the full potential of advanced wound dressing materials in large patient cohorts.


The role of endoplasmic reticulum calcium pumps during cytosolic calcium spiking in pancreatic acinar cells.
Many cell types show repetitive short lasting cytosolic calcium spikes with long interspike periods when stimulated with submaximal concentrations of agonists linked to the phosphoinositide signaling pathway. In pancreatic acinar cells these spikes have been shown to be evoked by constant levels of inositol trisphosphate through a mechanism of calcium-induced calcium release and do not depend acutely on the presence of external calcium. However, the processes involved in the interspike period have remained unclear. Here we report that the endoplasmic reticulum Ca(2+)-ATPase play a significant role, not only in resequestering calcium after a spike, but also in regulating the long interspike period. Decreasing the activity of the endoplasmic reticulum calcium pumps leads to shorter interspike intervals and thus higher spiking frequencies, while the duration of each spike increases. The endoplasmic reticulum Ca(2+)-ATPases are able to entirely suppress a response that can subsequently be evoked by partial inhibition of the pumps. This suggests that during the interspike period there is a considerable amount of calcium released from intracellular stores, which is rapidly buffered by the endoplasmic reticulum calcium pumps and the cytosolic calcium-binding proteins. A calcium spike will be initiated by calcium-induced calcium release only when the buffering is saturated.


Functional-anatomic fractionation of the brain's default network.
One of the most consistent observations in human functional imaging is that a network of brain regions referred to as the "default network" increases its activity during passive states. Here we explored the anatomy and function of the default network across three studies to resolve divergent hypotheses about its contributions to spontaneous cognition and active forms of decision making. Analysis of intrinsic activity revealed the network comprises multiple, dissociated components. A midline core (posterior cingulate and anterior medial prefrontal cortex) is active when people make self-relevant, affective decisions. In contrast, a medial temporal lobe subsystem becomes engaged when decisions involve constructing a mental scene based on memory. During certain experimentally directed and spontaneous acts of future-oriented thought, these dissociated components are simultaneously engaged, presumably to facilitate construction of mental models of personally significant events.


Co-Infection Patterns in Individual Ixodes scapularis Ticks Reveal Associations between Viral, Eukaryotic and Bacterial Microorganisms.
Ixodes scapularis ticks harbor a variety of microorganisms, including eukaryotes, bacteria and viruses. Some of these can be transmitted to and cause disease in humans and other vertebrates. Others are not pathogenic, but may impact the ability of the tick to harbor and transmit pathogens. A growing number of studies have examined the influence of bacteria on tick vector competence but the influence of the tick virome remains less clear, despite a surge in the discovery of tick-associated viruses. In this study, we performed shotgun RNA sequencing on 112 individual adult I. scapularis collected in Wisconsin, USA. We characterized the abundance, prevalence and co-infection rates of viruses, bacteria and eukaryotic microorganisms. We identified pairs of tick-infecting microorganisms whose observed co-infection rates were higher or lower than would be expected, or whose RNA levels were positively correlated in co-infected ticks. Many of these co-occurrence and correlation relationships involved two bunyaviruses, South Bay virus and blacklegged tick phlebovirus-1. These viruses were also the most prevalent microorganisms in the ticks we sampled, and had the highest average RNA levels. Evidence of associations between microbes included a positive correlation between RNA levels of South Bay virus and Borrelia burgdorferi, the Lyme disease agent. These findings contribute to the rationale for experimental studies on the impact of viruses on tick biology and vector competence.


Sociodemographic and survival disparities for histologic variants of bladder cancer.
To investigate the impact of perioperative factors on overall survival among patients with histologic variants of bladder cancer treated with radical cystectomy. The National Cancer Data Base was utilized to identify patients diagnosed with muscle-invasive bladder cancer (cT2-4, N0, M0) from 2004-2013. Variant histology bladder cancers (non-mucinous adenocarcinoma, mucinous/signet ring adenocarcinoma, micropapillary urothelial carcinoma, small cell carcinoma, and squamous cell carcinoma) were compared to urothelial carcinoma with respect to overall survival. Adjusted hazard ratios (aHR) and 95% confidence intervals (95% CI) were calculated from a multivariable Cox regression model to examine factors affecting overall survival, T upstaging, N upstaging, and positive surgical margins. Median survival was calculated using Kaplan-Meier analysis. A total of 5,856 patients were included in this study. Significant predictors of worse overall survival included: African-American ancestry (aHR = 1.24, 95%CI: 1.03-1.48, p = 0.021), age (1.03, 1.02-1.03, p < 0.001), comorbidity (1.30, 1.20-1.40, p < 0.001), cT3 stage (1.41, 1.26-1.57, p < 0.001), and cT4 stage (1.59, 1.38-1.84, p < 0.001). Small cell carcinoma (2.10, 1.44-3.06, p < 0.001) and non-mucinous adenocarcinoma (1.59, 1.15-2.20, p = 0.005) were significant predictors of worse overall survival compared to urothelial carcinoma. Small cell carcinoma had the worst 5 year overall survival (15.5%, 95% CI: 5.2%-30.9%) compared to urothelial carcinoma (48.7%, 95% CI: 47.2%-50.2%). Micropapillary urothelial carcinoma was a significant predictor of increased progression to node positivity and positive margin status after radical cystectomy compared to urothelial carcinoma (6.01, 3.11-11.63, p < 0.001; 4.38, 2.05-9.38; p < 0.001). Among bladder cancer patients with equal treatment and staging, small cell carcinoma and non-mucinous adenocarcinoma variant histologies were predictive of worse overall survival compared to urothelial carcinoma. Patient demographics such as African-American ancestry and age were also predictive of worse overall survival among variant histology bladder cancer and urothelial carcinoma.


Rationale and methods of the Prospective Study of Biomarkers, Symptom Improvement, and Ventricular Remodeling During Sacubitril/Valsartan Therapy for Heart Failure (PROVE-HF).
Sacubitril/valsartan is an angiotensin receptor-neprilysin inhibitor indicated for the treatment of patients with chronic heart failure (HF) with reduced ejection fraction; however, its mechanism of benefit remains unclear. Biomarkers that are linked to ventricular remodeling, myocardial injury, and fibrosis may provide mechanistic insight and important clinical guidance regarding sacubitril/valsartan use. This 52-week, multicenter, open-label, single-arm study is designed to (1) correlate biomarker changes with cardiac remodeling parameters, cardiovascular outcomes, and patient-reported outcome data and (2) determine short- and long-term changes in concentrations of biomarkers related to potential mechanisms of action and effects of sacubitril/valsartan therapy. Approximately 830 patients with HF with reduced ejection fraction will be initiated and titrated on sacubitril/valsartan according to United States prescribing information. Primary efficacy end points include the changes in N-terminal pro-B-type natriuretic peptide concentrations and cardiac remodeling from baseline to 1 year. Secondary end points include changes in concentrations of N-terminal pro-B-type natriuretic peptide and remodeling to 6 months, and changes in patient-reported outcomes using the Kansas City Cardiomyopathy Questionnaire-23 from baseline to 1 year. In addition, several other relevant biomarkers will be measured. Biomarker changes relative to the number of cardiovascular events in 12 months will also be assessed as exploratory end points. Results from the Prospective Study of Biomarkers, Symptom Improvement, and Ventricular Remodeling During Sacubitril/Valsartan Therapy for Heart Failure (PROVE-HF) will help establish a mechanistic understanding of angiotensin receptor-neprilysin inhibitor therapeutic benefits and provide clinicians with clarity on how to interpret information on biomarkers during treatment (PROVE-HF ClinicalTrials.gov identifier: NCT02887183).


Change in Hemoglobin Levels due to Anesthesia in Mice: An Important Confounder in Studies on Hematopoietic Drugs.
Analgesic and anesthetic drugs may have an impact on the results achieved from animal experiments. In the study presented here, we try to enlighten whether anesthesia with fentanyl/fluniasone and midazolam (Hypnorm and Dormicum) has an influence on measurements of hemoglobin in mice. In a cross-over study, we have compared hemoglobin levels in two groups of mice: anesthetized versus non-anesthetized and found significant decrease in hemoglobin levels in the anesthetized group (p < 0.05) unrelated to which group received the anesthesia. The mean hemoglobin levels after intraperitoneal administration of Hypnorm and Dormicum was 8.7 mmol/L compared to mean hemoglobin 9.9 mmol/L before anesthesia (p < 0.001), and the decrease lasted for more than 30 min. These results show that anesthesia can be an important confounder in studies involving measurements of hemoglobin, and this should be taken into account when planning studies and analyzing data.


Evidence for Ammonium-dependent de Novo Synthesis of Glutamate Dehydrogenase in Detached Oat Leaves.
Glutamate dehydrogenase becomes density labeled through the incorporation of deuterium and (15)N when detached oat leaves (Avena sativa var. Fulghum) are incubated in the presence of ammonia. The enzyme has been isolated by means of DEAE-cellulose chromatography, ammonium sulfate precipitation, isopycnic equilibrium centrifugation, and disc electrophoresis from leaves fed l-methionine-(35)S. Radioactivity is incorporated into isozyme 1 of glutamate dehydrogenase, whereas isozyme 2, detected only in the absence of ammonia, has not been labeled. Cycloheximide, chloramphenicol, puromycin, and 6-methyl purine inhibit the elevation of glutamate dehydrogenase by ammonia. It is suggested that the increase in glutamate dehydrogenase activity is due to de novo synthesis of isozyme 1.


Helicobacter pylori eradication: A randomised comparative trial of 7-day versus 14-day triple therapy.
Background. Helicobacter pylori is associated with several upper gastrointestinal conditions including chronic gastritis, peptic ulcer disease, and gastric malignancy. Proton pump inhibitor-based triple therapies are considered the standard regimens for H. pylori eradication, but the optimal duration of therapy is controversial. To prevent infection and complications, local studies should be undertaken to evaluate H. pylori eradication rates in a country. Objectives. We compared 7-day and 14-day regimens to determine the optimum duration of triple therapy for H. pylori eradication. Methods. We undertook a prospective randomised comparative trial of 7-day and 14-day triple therapy regimen for H. pylori eradication at the Aga Khan University Hospital, Nairobi; 120 patients with dyspepsia and H. pylori infection were randomised to receive esomeprazole, amoxicillin and clarithromycin for either 7 days (EAC 7) or 14 days (EAC 14). Compliance and side-effects were assessed 2 weeks after the start of therapy and H. pylori eradication was assessed by stool antigen tests 4 weeks after treatment. Results. Both the intention-to-treat (ITT; N=120) and per protocol (PP; N=97) analyses showed no significant differences between the eradication rates of EAC 7 (ITT 76.7%; PP 92%) and EAC 14 (ITT 73.3%; PP 93.6%) (ITT p=0.67; PP p=0.76). Poor compliance was reported in one patient in the EAC 14 group. The incidence of adverse events was comparable in the two groups. Conclusion. One-week and 2-week triple treatments for H. pylori eradication are similar in terms of efficacy, safety and patient compliance.


A stochastic self-organizing map for proximity data.
We derive an efficient algorithm for topographic mapping of proximity data (TMP), which can be seen as an extension of Kohonen's self-organizing map to arbitrary distance measures. The TMP cost function is derived in a Baysian framework of folded Markov chains for the description of autoencoders. It incorporates the data by a dissimilarity matrix D and the topographic neighborhood by a matrix H of transition probabilities. From the principle of maximum entropy, a nonfactorizing Gibbs distribution is obtained, which is approximated in a mean-field fashion. This allows for maximum likelihood estimation using an expectation-maximization algorithm. In analogy to the transition from topographic vector quantization to the self-organizing map, we suggest an approximation to TMP that is computationally more efficient. In order to prevent convergence to local minima, an annealing scheme in the temperature parameter is introduced, for which the critical temperature of the first phase transition is calculated in terms of D and H. Numerical results demonstrate the working of the algorithm and confirm the analytical results. Finally, the algorithm is used to generate a connection map of areas of the cat's cerebral cortex.


Functional characterization and localization of the Aspergillus nidulans formin SEPA.
Formins are a family of multidomain scaffold proteins involved in actin-dependent morphogenetic events. In Aspergillus nidulans, the formin SEPA participates in two actin-mediated processes, septum formation and polarized growth. In this study, we use a new null mutant to demonstrate that SEPA is required for the formation of actin rings at septation sites. In addition, we find that a functional SEPA::GFP fusion protein localizes simultaneously to septation sites and hyphal tips, and that SEPA colocalizes with actin at each site. Using live imaging, we show that SEPA localization at septation sites and hyphal tips is dynamic. Notably, at septation sites, SEPA forms a ring that constricts as the septum is deposited. Moreover, we demonstrate that actin filaments are required to maintain the proper localization pattern of SEPA, and that the amino-terminal half of SEPA is sufficient for localization at septation sites and hyphal tips. In contrast, only localization at septation sites is affected by loss of the sepH gene product. We propose that specific morphological cues activate common molecular pathways to direct SEPA localization to the appropriate morphogenetic site.


Voluntary contraction enhances spinal reciprocal inhibition induced by patterned electrical stimulation in patients with stroke.
Reciprocal inhibition (RI) may be important for recovering locomotion after stroke. Patterned electrical stimulation (PES) can modulate RI in a manner that could be enhanced by voluntary muscle contraction (VC). To investigate whether VC enhances the PES-induced spinal RI in patients with stroke. Twelve patients with chronic stroke underwent three 20 min tasks, each on different days: (1) PES (10 pulses, 100 Hz every 2 s) applied to the common peroneal nerve; (2) VC consisting of isometric contraction of the affected-side tibialis anterior muscle; (3) PES combined with VC (PES + VC). RI from the tibialis anterior to the soleus muscle was assessed before, immediately after, and 10, 20, and 30 min after the task. Compared to the baseline, PES + VC significantly increased the changes in reciprocal inhibition at immediately after and 10 min after the task. PES alone significantly increased this change immediately after the task, while VC alone showed no significant increase. VC enhanced the PES-induced plastic changes in RI in patients with stroke. This effect can potentially increase the success rate of newer neurorehabilitative approaches in achieving functional recovery after stroke.


Mutational hot spot potential of a novel base pair mutation of the CSPG2 gene in a family with Wagner syndrome.
To report a 3-generation white family clinically diagnosed variably with Wagner, Stickler, and Jansen syndromes and screened for sequence variants in the COL2A1 and CSPG2 genes. Wagner syndrome is an autosomal dominant vitreoretinopathy with a predisposition to retinal detachment and cataracts. It has significant phenotypic overlap with allelic Jansen syndrome and ocular Stickler syndrome type 1. Sticker syndrome type 1 maps to chromosome 12q13.11-q13.2, with associated COL2A1 gene mutations. Wagner syndrome maps to chromosome 5q13-q14 and is associated with mutations in CSPG2 encoding versican, a proteoglycan present in human vitreous. Genomic DNA samples derived from venous blood were collected from all family members. Complete sequencing of COL2A1 was performed on a proband. Primers for polymerase chain reaction and sequencing were designed to cover all exon and intron-exon boundaries. Direct sequencing of CSPG2 was performed on all family member samples. No detectable COL2A1 mutations were noted, making the diagnosis of ocular Stickler syndrome highly unlikely for this family. A unique base pair substitution (c.9265 + 1G>T) in intron 8 of the CSPG2 gene cosegregating with disease status was identified. This mutation occurred in a highly conserved previously reported splice site with a similar base pair substitution (G>A). Direct sequencing of this splice site mutation in 107 unrelated external controls revealed no variants, supporting the rarity of this base pair change and its causation in Wagner syndrome. This novel base pair substitution is thought to cause the deletion of exon 8 and formation of a truncated protein product. Mutation screening of CSPG2 in autosomal dominant vitreoretinopathy families is important for accurate diagnosis. This study underscores the importance of obtaining extensive pedigree information and comparative ophthalmologic clinical information, as the phenotypic findings may vary greatly among independent family members. The study also affirms the paradigm shift from diagnosis assignment based on eponyms to that based on gene mutation type.


Expression of functional proliferating-cell nuclear antigen from rice (Oryza sativa) in Escherichia coli. Activity in association with human DNA polymerase delta.
Proliferating-cell nuclear antigen (PCNA), the auxiliary protein for DNA polymerase delta, is one of the key factors for both PCNA-dependent DNA synthesis and cell-cycle progression. Plant PCNA genes have previously been cloned from rice, carrot, tobacco, and soybean cells by screening the cDNA libraries using similarity to the human or rat PCNA genes. We subcloned the relevant gene from the rice PCNA cDNA into an Escherichia coli expression vector pMAL, and the PCNA protein was expressed in the bacteria in the form of a fusion protein (70 kDa) with maltose-binding protein (MBP). Monoclonal antibody against human PCNA reacted with both purified fusion protein and a 32-kDa fragment, resulting from restriction protease (factor Xa) digestion of the fusion protein. The N-terminal amino acid sequence of the 32-kDa fragment was identical to that of rice PCNA sequence. Rice PCNA fusion protein was found to stimulate DNA synthesis catalyzed by DNA polymerase delta from human cells (although much less effectively), while having no effect on DNA polymerase alpha activity. The results indicate that plant PCNA functions as one of the cofactors of DNA synthesis as is the case with other eukaryotes.


Host genetics and HIV-1: the final phase?
This is a crucial transition time for human genetics in general, and for HIV host genetics in particular. After years of equivocal results from candidate gene analyses, several genome-wide association studies have been published that looked at plasma viral load or disease progression. Results from other studies that used various large-scale approaches (siRNA screens, transcriptome or proteome analysis, comparative genomics) have also shed new light on retroviral pathogenesis. However, most of the inter-individual variability in response to HIV-1 infection remains to be explained: genome resequencing and systems biology approaches are now required to progress toward a better understanding of the complex interactions between HIV-1 and its human host.


Clinical Profiles of Chronic Hepatitis C in a Major County Medical Center Outpatient Setting in United States.
The estimated prevalence of hepatitis C virus (HCV) infection in the US is 1.8 %. Data are limited on the clinical profile of the disease at first presentation and dynamic follow-up of ALT level, especially in publicly-funded patients. This information is critical for optimal management of these patients. The present study is aimed to assess the clinical profiles of chronic hepatitis C (CHC) at first presentation and clinical implication of dynamic follow-up of ALT level in a county medical center setting. A total of 294 patients were selected from the population consecutively evaluated in the Hepatitis Clinic at Los Angeles County-USC Medical Center between Jan. 1990 and Dec. 1998. Ethnicity of the patients was Hispanics-49.0%, Caucasian-28.6%, African American-13.6%, and Asian-8.8%. Risk factors were identifiable in 84.0% of patients, and injection drug use (IDU) represented the leading risk factor for HCV acquisition (47.4%). History of alcoholism was present in 39.1%. The initial clinical diagnoses were chronic hepatitis 76.9%; compensated cirrhosis 20.4%; and decompensated cirrhosis 2.7%. Elevation of ALT, alpha fetoprotein (AFP), ferritin, and anti-nuclear antibody (ANA) titer were seen in 219/294 (74.5%), 60/194 (30.9%), 20/83 (24.1%), and 35/97 (36.1%) patients, respectively. Anti-HBc (total) test was positive in 65/129 (50.5%) patients. The presence of cirrhosis was significantly associated with age greater than 55 years at entry, female gender, non-African American ethnicity, history of transfusion, lower level of albumin and elevated level of AFP. Longitudinal observation of ALT changes in 178 patients who had neither evidence of cirrhosis at entry nor received interferon treatment showed persistently normal, intermittently or persistently elevated ALT level in 15.2%, 38.3%, and 46.6% patients, respectively. The frequency of developing clinical evidence of cirrhosis during follow-up was significantly higher in patients with persistently (16.0%) or intermittently (7.0%) elevated ALT than that in patients with persistently normal ALT (4.0%). In conclusion, the present study analyzed the clinical profiles of CHC, assessed risk factors for developing cirrhosis, and demonstrated the clinical value of dynamic follow-up of ALT level in a cohort of publicly-funded patients. These data have major implications in designing optimal strategies for disease management, antiviral therapy, and screening for hepatocellular carcinoma in patients with CHC.


Kindlin-3 is essential for integrin activation and platelet aggregation.
Integrin-mediated platelet adhesion and aggregation are essential for sealing injured blood vessels and preventing blood loss, and excessive platelet aggregation can initiate arterial thrombosis, causing heart attacks and stroke. To ensure that platelets aggregate only at injury sites, integrins on circulating platelets exist in a low-affinity state and shift to a high-affinity state (in a process known as integrin activation or priming) after contacting a wounded vessel. The shift is mediated through binding of the cytoskeletal protein Talin to the beta subunit cytoplasmic tail. Here we show that platelets lacking the adhesion plaque protein Kindlin-3 cannot activate integrins despite normal Talin expression. As a direct consequence, Kindlin-3 deficiency results in severe bleeding and resistance to arterial thrombosis. Mechanistically, Kindlin-3 can directly bind to regions of beta-integrin tails distinct from those of Talin and trigger integrin activation. We have therefore identified Kindlin-3 as a novel and essential element for platelet integrin activation in hemostasis and thrombosis.


Effect of Natriuretic Peptide-Guided Therapy on Hospitalization or Cardiovascular Mortality in High-Risk Patients With Heart Failure and Reduced Ejection Fraction: A Randomized Clinical Trial.
The natriuretic peptides are biochemical markers of heart failure (HF) severity and predictors of adverse outcomes. Smaller studies have evaluated adjusting HF therapy based on natriuretic peptide levels ("guided therapy") with inconsistent results. To determine whether an amino-terminal pro-B-type natriuretic peptide (NT-proBNP)-guided treatment strategy improves clinical outcomes vs usual care in high-risk patients with HF and reduced ejection fraction (HFrEF). The Guiding Evidence Based Therapy Using Biomarker Intensified Treatment in Heart Failure (GUIDE-IT) study was a randomized multicenter clinical trial conducted between January 16, 2013, and September 20, 2016, at 45 clinical sites in the United States and Canada. This study planned to randomize 1100 patients with HFrEF (ejection fraction ≤40%), elevated natriuretic peptide levels within the prior 30 days, and a history of a prior HF event (HF hospitalization or equivalent) to either an NT-proBNP-guided strategy or usual care. Patients were randomized to either an NT-proBNP-guided strategy or usual care. Patients randomized to the guided strategy (n = 446) had HF therapy titrated with the goal of achieving a target NT-proBNP of less than 1000 pg/mL. Patients randomized to usual care (n = 448) had HF care in accordance with published guidelines, with emphasis on titration of proven neurohormonal therapies for HF. Serial measurement of NT-proBNP testing was discouraged in the usual care group. The primary end point was the composite of time-to-first HF hospitalization or cardiovascular mortality. Prespecified secondary end points included all-cause mortality, total hospitalizations for HF, days alive and not hospitalized for cardiovascular reasons, the individual components on the primary end point, and adverse events. The data and safety monitoring board recommended stopping the study for futility when 894 (median age, 63 years; 286 [32%] women) of the planned 1100 patients had been enrolled with follow-up for a median of 15 months. The primary end point occurred in 164 patients (37%) in the biomarker-guided group and 164 patients (37%) in the usual care group (adjusted hazard ratio [HR], 0.98; 95% CI, 0.79-1.22; P = .88). Cardiovascular mortality was 12% (n = 53) in the biomarker-guided group and 13% (n = 57) in the usual care group (HR, 0.94; 95% CI; 0.65-1.37; P = .75). None of the secondary end points nor the decreases in the NT-proBNP levels achieved differed significantly between groups. In high-risk patients with HFrEF, a strategy of NT-proBNP-guided therapy was not more effective than a usual care strategy in improving outcomes. clinicaltrials.gov Identifier: NCT01685840.


Protein-peptide association kinetics beyond the seconds timescale from atomistic simulations.
Understanding and control of structures and rates involved in protein ligand binding are essential for drug design. Unfortunately, atomistic molecular dynamics (MD) simulations cannot directly sample the excessively long residence and rearrangement times of tightly binding complexes. Here we exploit the recently developed multi-ensemble Markov model framework to compute full protein-peptide kinetics of the oncoprotein fragment 25-109Mdm2 and the nano-molar inhibitor peptide PMI. Using this system, we report, for the first time, direct estimates of kinetics beyond the seconds timescale using simulations of an all-atom MD model, with high accuracy and precision. These results only require explicit simulations on the sub-milliseconds timescale and are tested against existing mutagenesis data and our own experimental measurements of the dissociation and association rates. The full kinetic model reveals an overall downhill but rugged binding funnel with multiple pathways. The overall strong binding arises from a variety of conformations with different hydrophobic contact surfaces that interconvert on the milliseconds timescale.


Genetic steps to organ laterality in zebrafish.
All internal organs are asymmetric along the left-right axis. Here we report a genetic screen to discover mutations which perturb organ laterality. Our particular focus is upon whether, and how, organs are linked to each other as they achieve their laterally asymmetric positions. We generated mutations by ENU mutagenesis and examined F3 progeny using a cocktail of probes that reveal early primordia of heart, gut, liver and pancreas. From the 750 genomes examined, we isolated seven recessive mutations which affect the earliest left-right positioning of one or all of the organs. None of these mutations caused discernable defects elsewhere in the embryo at the stages examined. This is in contrast to those mutations we reported previously (Chen et al., 1997) which, along with left-right abnormalities, cause marked perturbation in gastrulation, body form or midline structures. We find that the mutations can be classified on the basis of whether they perturb relationships among organ laterality. In Class 1 mutations, none of the organs manifest any left-right asymmetry. The heart does not jog to the left and normally leftpredominant BMP4 in the early heart tube remains symmetric. The gut tends to remain midline. There frequently is a remarkable bilateral duplication of liver and pancreas. Embryos with Class 2 mutations have organotypic asymmetry but, in any given embryo, organ positions can be normal, reversed or randomized. Class 3 reveals a hitherto unsuspected gene that selectively affects laterality of heart. We find that visceral organ positions are predicted by the direction of the preceding cardiac jog. We interpret this as suggesting that normally there is linkage between cardiac and visceral organ laterality. Class 1 mutations, we suggest, effectively remove the global laterality signals, with the consequence that organ positions are effectively symmetrical. Embryos with Class 2 mutations do manifest linkage among organs, but it may be reversed, suggesting that the global signals may be present but incorrectly orientated in some of the embryos. That laterality decisions of organs may be independently perturbed, as in the Class 3 mutation, indicates that there are distinctive pathways for reception and organotypic interpretation of the global signals.


Context-dependent consumer control in New England tidal wetlands.
Recent studies in coastal wetlands have indicated that consumers may play an important role in regulating large-scale ecosystem processes. Predator removal experiments have shown significant differences in above-ground biomass production in the presence of higher level consumers, or predators. These results indicate that predators play an important role in regulating biomass production, but the extent to which this regulation impacts additional ecosystem functions, such as nutrient cycling and organic matter accumulation, is unclear. This study evaluated the impact that consumers have on large-scale ecosystem processes within southern New England tidal wetlands and contributes to the general understanding of trophic control in these systems. I established enclosure cages within three coastal wetlands and manipulated the presence of green crab predators to assess how trophic interactions affect ecosystem functions. Findings suggest that although these consumers may exert some top-down effects, other environmental factors, such as other consumers not studied here or bottom-up interactions, may variably play a larger role in the maintenance of ecosystem processes within the region. These results indicate that the loss of top-down control as an important mechanism influencing ecosystem functions may not hold for all wetlands along the full extent of the New England coastline.


An Evaluation of the Anti-Carcinogenic Response of Major Isothiocyanates in Non-Metastatic and Metastatic Melanoma Cells.
Malignant melanoma is one of the most deadly types of solid cancers, a property mainly attributed to its highly aggressive metastatic form. On the other hand, different classes of isothiocyanates, a class of phytochemicals, present in cruciferous vegetables have been characterized by considerable anti-cancer activity in both in vitro and in vivo experimental models. In the current study, we investigated the anti-cancer response of five isothiocyanates in an in vitro model of melanoma consisting of non-metastatic (A375, B16F-10) and metastatic (VMM1, Hs294T) malignant melanoma as well as non-melanoma epidermoid carcinoma (A431) and non-tumorigenic melanocyte-neighboring keratinocyte (HaCaT) cells. Our aim was to compare different endpoints of cytotoxicity (e.g., reactive oxygen species, intracellular glutathione content, cell cycle growth arrest, apoptosis and necrosis) descriptive of an anti-cancer response between non-metastatic and metastatic melanoma as well as non-melanoma epidermoid carcinoma and non-tumorigenic cells. Our results showed that exposure to isothiocyanates induced an increase in intracellular reactive oxygen species and glutathione contents between non-metastatic and metastatic melanoma cells. The distribution of cell cycle phases followed a similar pattern in a manner where non-metastatic and metastatic melanoma cells appeared to be growth arrested at the G2/M phase while elevated levels of metastatic melanoma cells were shown to be at sub G1 phase, an indicator of necrotic cell death. Finally, metastatic melanoma cells were more sensitive apoptosis and/or necrosis as higher levels were observed compared to non-melanoma epidermoid carcinoma and non-tumorigenic cells. In general, non-melanoma epidermoid carcinoma and non-tumorigenic cells were more resistant under any experimental exposure condition. Overall, our study provides further evidence for the potential development of isothiocyanates as promising anti-cancer agents against non-metastatic and metastatic melanoma cells, a property specific for these cells and not shared by non-melanoma epidermoid carcinoma or non-tumorigenic melanocyte cells.


Clinical management of epidermal growth factor receptor mutation-positive non-small cell lung cancer patients after progression on previous epidermal growth factor receptor tyrosine kinase inhibitors: the necessity of repeated molecular analysis.
One of the most important advances in the treatment of non-small cell lung cancer (NSCLC) has been the identification of molecular alterations vulnerable to targeted inhibition, such as mutations in the epidermal growth factor receptor (EGFR) gene. EGFR tyrosine kinase inhibitors (EGFR-TKIs) are targeted agents used to treat EGFR mutation-positive advanced NSCLC showing significant improvements in terms of response rate (RR) and progression-free survival (PFS) compared to conventional chemotherapy. However, all patients eventually develop resistance to first-line EGFR-TKIs. The most common mechanism of acquired resistance is the secondary acquisition of a single missense mutation within exon 20 in the EGFR gene, known as the T790M mutation (49-60%). New agents targeting the T790M mutation have undergone clinical development, and among these, osimertinib has shown significant activity in relapsing EGFR mutation positive patients harbouring the T790M mutation. Although precision medicine is a reality for NSCLC, obtaining relevant tissue for repeated molecular analysis from these patients remains a challenge. In this article, a group of experts from the Spanish Society of Medical Oncology (SEOM) and the Spanish Lung Cancer Group (GECP) evaluated the role of rebiopsy and the potential application of plasma-testing methodologies in advanced EGFR mutation patients progressing after EGFR-TKI.


Salmonella enterica Serovar Typhimurium Exploits Cycling through Epithelial Cells To Colonize Human and Murine Enteroids.
Enterobacterial pathogens infect the gut by a multistep process, resulting in colonization of both the lumen and the mucosal epithelium. Due to experimental constraints, it remains challenging to address how luminal and epithelium-lodged pathogen populations cross-feed each other in vivo Enteroids are cultured three-dimensional miniature intestinal organs with a single layer of primary intestinal epithelial cells (IECs) surrounding a central lumen. They offer new opportunities to study enterobacterial infection under near-physiological conditions, at a temporal and spatial resolution not attainable in animal models, but remain poorly explored in this context. We employed microinjection, time-lapse microscopy, bacterial genetics, and barcoded consortium infections to describe the complete infection cycle of Salmonella enterica serovar Typhimurium in both human and murine enteroids. Flagellar motility and type III secretion system 1 (TTSS-1) promoted Salmonella Typhimurium targeting of the intraepithelial compartment and breaching of the epithelial barrier. Strikingly, however, TTSS-1 also potently boosted colonization of the enteroid lumen. By tracing the infection over time, we identified a cycle(s) of TTSS-1-driven IEC invasion, intraepithelial replication, and reemergence through infected IEC expulsion as a key mechanism for Salmonella Typhimurium luminal colonization. These findings suggest a positive feed-forward loop, through which IEC invasion by planktonic bacteria fuels further luminal population expansion, thereby ensuring efficient colonization of both the intraepithelial and luminal niches.IMPORTANCE Pathogenic gut bacteria are common causes of intestinal disease. Enteroids-cultured three-dimensional replicas of the mammalian gut-offer an emerging model system to study disease mechanisms under conditions that recapitulate key features of the intestinal tract. In this study, we describe the full life cycle of the prototype gut pathogen Salmonella enterica serovar Typhimurium within human and mouse enteroids. We map the consecutive steps and define the bacterial virulence factors that drive colonization of luminal and epithelial compartments, as well as breaching of the epithelial barrier. Strikingly, our work reveals how bacterial colonization of the epithelium potently fuels expansion also in the luminal compartment, through a mechanism involving the death and expulsion of bacterium-infected epithelial cells. These findings have repercussions for our understanding of the Salmonella infection cycle. Moreover, our work provides a comprehensive foundation for the use of microinjected enteroids to model gut bacterial diseases.


Cryogenics free production of hyperpolarized 129Xe and 83Kr for biomedical MRI applications.
As an alternative to cryogenic gas handling, hyperpolarized (hp) gas mixtures were extracted directly from the spin exchange optical pumping (SEOP) process through expansion followed by compression to ambient pressure for biomedical MRI applications. The omission of cryogenic gas separation generally requires the usage of high xenon or krypton concentrations at low SEOP gas pressures to generate hp (129)Xe or hp (83)Kr with sufficient MR signal intensity for imaging applications. Two different extraction schemes for the hp gasses were explored with focus on the preservation of the nuclear spin polarization. It was found that an extraction scheme based on an inflatable, pressure controlled balloon is sufficient for hp (129)Xe handling, while (83)Kr can efficiently be extracted through a single cycle piston pump. The extraction methods were tested for ex vivo MRI applications with excised rat lungs. Precise mixing of the hp gases with oxygen, which may be of interest for potential in vivo applications, was accomplished during the extraction process using a piston pump. The (83)Kr bulk gas phase T1 relaxation in the mixtures containing more than approximately 1% O2 was found to be slower than that of (129)Xe in corresponding mixtures. The experimental setup also facilitated (129)Xe T1 relaxation measurements as a function of O2 concentration within excised lungs.


Later school start times for supporting the education, health, and well-being of high school students.
A number of school systems worldwide have proposed and implemented later school start times as a means of avoiding the potentially negative impacts that early morning schedules can have on adolescent students. Even mild sleep deprivation has been associated with significant health and educational concerns: increased risk for accidents and injuries, impaired learning, aggression, memory loss, poor self-esteem, and changes in metabolism. Although researchers have begun to explore the effects of delayed school start time, no one has conducted a rigorous review of evidence to determine whether later school start times support adolescent health, education, and well-being. We aimed to assess the effects of a later school start time for supporting health, education, and well-being in high school students.Secondary objectives were to explore possible differential effects of later school start times in student subgroups and in different types of schools; to identify implementation practices, contextual factors, and delivery modes associated with positive and negative effects of later start times; and to assess the effects of later school start times on the broader community (high school faculty and staff, neighborhood, and families). We conducted the main search for this review on 28 October 2014 and updated it on 8 February 2016. We searched CENTRAL as well as 17 key electronic databases (including MEDLINE, Embase, ERIC, PsycINFO, and Sociological Abstracts), current editions of relevant journals and organizational websites, trial registries, and Google Scholar. We included any randomized controlled trials, controlled before-and-after studies, and interrupted time series studies with sufficient data points that pertained to students aged 13 to 19 years and that compared different school start times. Studies that reported either primary outcomes of interest (academic outcomes, amount or quality of sleep, mental health indicators, attendance, or alertness) or secondary outcomes (health behaviors, health and safety indicators, social outcomes, family outcomes, school outcomes, or community outcomes) were eligible. At least two review authors independently determined inclusion and exclusion decisions through screening titles, abstracts, and full-text reports. Two review authors independently extracted data for all eligible studies. We presented findings through a narrative synthesis across all studies. When two or more study samples provided sufficient information to permit effect size calculations, we conducted random-effects meta-analyses to synthesize effects across studies. Our search located 17 eligible records reporting on 11 unique studies with 297,994 participants; the studies examined academic outcomes, amount and quality of sleep, mental health indicators, attendance, and student alertness. Overall, the quality of the body of evidence was very low, as we rated most studies as being at high or unclear risk of bias with respect to allocation, attrition, absence of randomization, and the collection of baseline data. Therefore, we cannot be confident about the effects of later school start times.Preliminary evidence from the included studies indicated a potential association between later school start times and academic and psychosocial outcomes, but quality and comparability of these data were low and often precluded quantitative synthesis. Four studies examined the association between later school start times and academic outcomes, reporting mixed results. Six studies examined effects on total amount of sleep and reported significant, positive relationships between later school start times and amount of sleep. One study provided information concerning mental health outcomes, reporting an association between decreased depressive symptoms and later school start times. There were mixed results for the association between later school start times and absenteeism. Three studies reported mixed results concerning the association between later school start times and student alertness. There was limited indication of potential adverse effects on logistics, as the qualitative portions of one study reported less interaction between parents and children, and another reported staffing and scheduling difficulties. Because of the insufficient evidence, we cannot draw firm conclusions concerning adverse effects at this time.It is important to note the limitations of this evidence, especially as randomized controlled trials and high-quality primary studies are difficult to conduct; school systems are often unwilling or unable to allow researchers the necessary control over scheduling and data collection. Moreover, this evidence does not speak to the process of implementing later school starts, as the included studies focused on reporting the effects rather than exploring the process. This systematic review on later school start times suggests several potential benefits for this intervention and points to the need for higher quality primary studies. However, as a result of the limited evidence base, we could not determine the effects of later school start times with any confidence.


Cilia proteins are biomarkers of altered flow in the vasculature.
Cilia, microtubule-based organelles that project from the apical luminal surface of endothelial cells (ECs), are widely regarded as low-flow sensors. Previous reports suggest that upon high shear stress, cilia on the EC surface are lost, and more recent evidence suggests that deciliation-the physical removal of cilia from the cell surface-is a predominant mechanism for cilia loss in mammalian cells. Thus, we hypothesized that EC deciliation facilitated by changes in shear stress would manifest in increased abundance of cilia-related proteins in circulation. To test this hypothesis, we performed shear stress experiments that mimicked flow conditions from low to high shear stress in human primary cells and a zebrafish model system. In the primary cells, we showed that upon shear stress induction, indeed, ciliary fragments were observed in the effluent in vitro, and effluents contained ciliary proteins normally expressed in both endothelial and epithelial cells. In zebrafish, upon shear stress induction, fewer cilia-expressing ECs were observed. To test the translational relevance of these findings, we investigated our hypothesis using patient blood samples from sickle cell disease and found that plasma levels of ciliary proteins were elevated compared with healthy controls. Further, sickled red blood cells demonstrated high levels of ciliary protein (ARL13b) on their surface after adhesion to brain ECs. Brain ECs postinteraction with sickle RBCs showed high reactive oxygen species (ROS) levels. Attenuating ROS levels in brain ECs decreased cilia protein levels on RBCs and rescued ciliary protein levels in brain ECs. Collectively, these data suggest that cilia and ciliary proteins in circulation are detectable under various altered-flow conditions, which could serve as a surrogate biomarker of the damaged endothelium.


Higher Intraocular Pressure Levels Associated With Lower Hysteresis In Type 2 Diabetes.
To investigate the differences of corneal biomechanic characteristics using Ocular Response Analyzer (ORA, Reichert; USA) on type 2 diabetics and healthy subjects. One hundred eyes of 100 subjects (between the ages of 17-91) who applied to Adnan Menderes University's Ophthalmology Clinic between January-March 2015 were included in this study, 50 diabetics (Group 1) and 50 healthy controls (Group 2). The eyes included in the study were randomly chosen. Corneal Hysteresis (CH), Corneal Resistance Factor (CRF), Goldmann correlated Intraocular Pressure (IOPg) and corneal compensated Intraocular Pressure (IOPcc) of patients were measured by ORA. Detailed ophthalmological examinations were done for every subject. Kolmogorov-Smirnov test was used to analyze the distribution of quantitative variables and t test was used for the data that were normally distributed. Any p value <0.05 was considered as statistically significant. The mean ages were 63.3±9.0 and 61.7±11.6 in Group 1 and 2, respectively (p=0.459). 25 (50.0%) were female, 25 (50.0%) were male in Group 1 and 26 (52.0%) were female, 24 (48.0%) were male in Group 2 (p=1.000). Mean IOPcc values were 17.8±3.6 (12.1-29.0) and 16.0±3.1 (10.9-23.8) mmHg (p=0.006); mean IOPg values were 16.9±3.5 (10.9-25.9) and 15.4±2.9 (9.0-24.7) mmHg (p=0.032); mean CH values were 9.9±1.5 (6.1-13.3) and 10.5±1.7 (6.5-15.7) (p=0.080) and mean CRF values were 10.4±1.6 (7.5-14.0) and 10.5±1.7 (6.6-15.4) (p=0.730) in Groups 1 and 2, respectively. There was no any statistical difference between the groups in terms of CH and CRF. However, mean CH and CRF values were found less in diabetic group. Corneal biomechanical differences seen in diabetic patients may be associated with a statistically significantly higher IOP measurements.


Purificaton of a functional mouse Fc receptor through the use of a monoclonal antibody.
We recently reported the isolation of a rat monoclonal antibody designated 2.4G2 (9) that is directed against the mouse trypsin-resistant Fc receptor (FcR) for IgG2b and IgG1 immune aggregates. We have now utilized the Fab fragment of 2.4G2 as an affinity reagent to purify FcR from the macrophage cell line J774 to apparent homogeneity. The antigen isolated from J774 cells consisted of two general types of polypeptides with broad electrophoretic mobilities of approximately 60,000 and 47,000 mol wt. Similar broad bands ranging from 47,000 to 70,000 mol wt were isolated from various FcR-bearing cell lines of B, T, and null lymphocyte, as well as of macrophage origin. J774 FcR was judged to be a glycoprotein based on the sensitivity of its isoelectric point to neuraminidase digestion, its labeling with galactose oxidase/NaB[3H4], and its binding to concanavalin A-Sepharose. In phosphate-buffered saline, the isolated protein formed large aggregates that were shown to retain FcR activity, albeit with a somewhat altered IgG subclass specificity. The FcR aglutinated erythrocytes that were coated with both IgG2b and IgG2a that did not otherwise hemagglutinate. In addition, iodinated FcR bound to Sephadex beads coated with rabbit IgG, mouse IgG1, IgG2b, and IgG2a, but not to beads coated with mouse IgG3 or rabbit F(ab')2 fragments. The binding of the purified receptor to all IgG classes was inhibited by the Fab fragments of 2.4G2. In contrast, the binding of IgG2a to intact macrophages was inhibited by 2.4G2 Fab by only 15%, whereas rabbit IgG immune aggregate binding was almost completely abolished.


Evaluation of glucocorticoid treatment on different pathological types of primary nephrotic syndrome.
This study aimed to assess the distribution of pathotypes in primary nephrotic syndrome (PNS) and their relationship with glucocorticoid treatment efficacy. The study included 120 patients who were treated in the nephrology, internal medicine and pediatrics wards of The Second Affiliated Hospital of Qiqihar Medical University between March 2014 and October 2017 and who underwent renal biopsy to confirm PNS. The patients with PNS were divided into a child group (40 cases, aged 0~17 years) and an adult group (80 cases, aged over 18 years). We evaluated the correlation of the curative effect of glucocorticoid with age, pathological type, renal tubulointerstitial damage retinol binding protein and total urine protein. The main pathological types of PNS were glomerular minor lesion (GML) and mesangial proliferative nephritis (MPGN). The glucocorticoid treatment had an improved effect on children compared to adults, and also the effect decreased with age. The pathotypes of PNS were correlated with hormone resistance: tubulointerstitial lesions were associated with glucocorticoid resistance which was also associated with the degree of tubular damage. In both adults and children the retinol binding protein (RBP) urinary levels were positively associated with the degree of renal tubular injury. In conclusion, age, pathological type, renal tubulointerstitial damage, and RBP urinary level were related to the therapeutic effect of glucocorticoid treatment in adults and children with PNS.


The pedagogical value of a student-run community-based experiential learning project: The Yong Loo Lin School of Medicine Public Health Screening.
We assessed the pedagogical value of a student-led community-based experiential learning project called the Public Health Screening (PHS) run by medical and nursing students of the National University of Singapore's Yong Loo Lin School of Medicine (NUS YLLSoM). We conducted a cross-sectional study using a self-administered anonymised questionnaire on medical and nursing students who participated in PHS using the Fund for the Improvement of Postsecondary Education (FIPSE) Survey Instrument. Participants also gave an overall score for their learning experience at the PHS. The participation rate was 93.1% (576/619) for medical students and 100% (37/37) for nursing students. All participants gave the PHS learning experience a high rating (median = 8 out of maximum of 10, inter-quartile range, 7 to 9). A majority of participants felt that PHS had helped them to improve across all domains surveyed. For medical students, those in preclinical years and females were independently more likely to feel that PHS had helped them to improve in communication skills, teamwork, ability to identify social issues, taking action, and gaining and applying their knowledge than those in clinical years and males. Improved ability to interact with patients (β=1.64, 95%CI, 1.01-2.27), appreciation of challenges to healthcare faced by Singaporeans from lower income groups (β=0.93, 95%CI, 0.49-1.37), thinking of others (β=0.70, 95%CI, 0.04-1.37) and tolerance of different people (β =0.63, 95%CI, 0.17-1.10) were strongly associated with the overall rating score. PHS was a positive learning experience in a wide range of domains for all students involved. This suggests that student-organised community-based experiential learning projects have potential educational value for both medical and nursing students.


p53 down-regulates SARS coronavirus replication and is targeted by the SARS-unique domain and PLpro via E3 ubiquitin ligase RCHY1.
Highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) has developed strategies to inhibit host immune recognition. We identify cellular E3 ubiquitin ligase ring-finger and CHY zinc-finger domain-containing 1 (RCHY1) as an interacting partner of the viral SARS-unique domain (SUD) and papain-like protease (PL(pro)), and, as a consequence, the involvement of cellular p53 as antagonist of coronaviral replication. Residues 95-144 of RCHY1 and 389-652 of SUD (SUD-NM) subdomains are crucial for interaction. Association with SUD increases the stability of RCHY1 and augments RCHY1-mediated ubiquitination as well as degradation of p53. The calcium/calmodulin-dependent protein kinase II delta (CAMK2D), which normally influences RCHY1 stability by phosphorylation, also binds to SUD. In vivo phosphorylation shows that SUD does not regulate phosphorylation of RCHY1 via CAMK2D. Similarly to SUD, the PL(pro)s from SARS-CoV, MERS-CoV, and HCoV-NL63 physically interact with and stabilize RCHY1, and thus trigger degradation of endogenous p53. The SARS-CoV papain-like protease is encoded next to SUD within nonstructural protein 3. A SUD-PL(pro) fusion interacts with RCHY1 more intensively and causes stronger p53 degradation than SARS-CoV PL(pro) alone. We show that p53 inhibits replication of infectious SARS-CoV as well as of replicons and human coronavirus NL63. Hence, human coronaviruses antagonize the viral inhibitor p53 via stabilizing RCHY1 and promoting RCHY1-mediated p53 degradation. SUD functions as an enhancer to strengthen interaction between RCHY1 and nonstructural protein 3, leading to a further increase in in p53 degradation. The significance of these findings is that down-regulation of p53 as a major player in antiviral innate immunity provides a long-sought explanation for delayed activities of respective genes.


Diffusion and Relaxation Edited Proton NMR Spectroscopy of Plasma Reveals a High-Fidelity Supramolecular Biomarker Signature of SARS-CoV-2 Infection.
We have applied nuclear magnetic resonance spectroscopy based plasma phenotyping to reveal diagnostic molecular signatures of SARS-CoV-2 infection via combined diffusional and relaxation editing (DIRE). We compared plasma from healthy age-matched controls (n = 26) with SARS-CoV-2 negative non-hospitalized respiratory patients and hospitalized respiratory patients (n = 23 and 11 respectively) with SARS-CoV-2 rRT-PCR positive respiratory patients (n = 17, with longitudinal sampling time-points). DIRE data were modelled using principal component analysis and orthogonal projections to latent structures discriminant analysis (O-PLS-DA), with statistical cross-validation indices indicating excellent model generalization for the classification of SARS-CoV-2 positivity for all comparator groups (area under the receiver operator characteristic curve = 1). DIRE spectra show biomarker signal combinations conferred by differential concentrations of metabolites with selected molecular mobility properties. These comprise the following: (a) composite N-acetyl signals from α-1-acid glycoprotein and other glycoproteins (designated GlycA and GlycB) that were elevated in SARS-CoV-2 positive patients [p = 2.52 × 10-10 (GlycA) and 1.25 × 10-9 (GlycB) vs controls], (b) two diagnostic supramolecular phospholipid composite signals that were identified (SPC-A and SPC-B) from the -+N-(CH3)3 choline headgroups of lysophosphatidylcholines carried on plasma glycoproteins and from phospholipids in high-density lipoprotein subfractions (SPC-A) together with a phospholipid component of low-density lipoprotein (SPC-B). The integrals of the summed SPC signals (SPCtotal) were reduced in SARS-CoV-2 positive patients relative to both controls (p = 1.40 × 10-7) and SARS-CoV-2 negative patients (p = 4.52 × 10-8) but were not significantly different between controls and SARS-CoV-2 negative patients. The identity of the SPC signal components was determined using one and two dimensional diffusional, relaxation, and statistical spectroscopic experiments. The SPCtotal/GlycA ratios were also significantly different for control versus SARS-CoV-2 positive patients (p = 1.23 × 10-10) and for SARS-CoV-2 negatives versus positives (p = 1.60 × 10-9). Thus, plasma SPCtotal and SPCtotal/GlycA are proposed as sensitive molecular markers for SARS-CoV-2 positivity that could effectively augment current COVID-19 diagnostics and may have value in functional assessment of the disease recovery process in patients with long-term symptoms.


Childhood abuse in patients with conversion disorder.
Despite the fact that the assumption of a relationship between conversion disorder and childhood traumatization has a long history, there is little empirical evidence to support this premise. The present study examined this relation and investigated whether hypnotic susceptibility mediates the relation between trauma and conversion symptoms, as suggested by Janet's autohypnosis theory of conversion disorder. A total of 54 patients with conversion disorder and 50 matched comparison patients with an affective disorder were administered the Structured Trauma Interview as well as measures of cognitive (Dissociative Experiences Scale) and somatoform (20-item Somatoform Dissociation Questionnaire) dissociative experiences. Patients with conversion disorder reported a higher incidence of physical/sexual abuse, a larger number of different types of physical abuse, sexual abuse of longer duration, and incestuous experiences more often than comparison patients. In addition, within the group of patients with conversion disorder, parental dysfunction by the mother-not the father-was associated with higher scores on the Dissociative Experiences Scale and the Somatoform Dissociation Questionnaire. Physical abuse was associated with a larger number of conversion symptoms (Structured Clinical Interview for DSM-IV Axis I Disorders). Hypnotic susceptibility proved to partially mediate the relation between physical abuse and conversion symptoms. The present results provide evidence of a relationship between childhood traumatization and conversion disorder.