Dynamics of HIV-1 assembly and release.
Assembly and release of human immunodeficiency virus (HIV) occur at the plasma membrane of infected cells and are driven by the Gag polyprotein. Previous studies analyzed viral morphogenesis using biochemical methods and static images, while dynamic and kinetic information has been lacking until very recently. Using a combination of wide-field and total internal reflection fluorescence microscopy, we have investigated the assembly and release of fluorescently labeled HIV-1 at the plasma membrane of living cells with high time resolution. Gag assembled into discrete clusters corresponding to single virions. Formation of multiple particles from the same site was rarely observed. Using a photoconvertible fluorescent protein fused to Gag, we determined that assembly was nucleated preferentially by Gag molecules that had recently attached to the plasma membrane or arrived directly from the cytosol. Both membrane-bound and cytosol derived Gag polyproteins contributed to the growing bud. After their initial appearance, assembly sites accumulated at the plasma membrane of individual cells over 1-2 hours. Assembly kinetics were rapid: the number of Gag molecules at a budding site increased, following a saturating exponential with a rate constant of approximately 5 x 10(-3) s(-1), corresponding to 8-9 min for 90% completion of assembly for a single virion. Release of extracellular particles was observed at approximately 1,500+/-700 s after the onset of assembly. The ability of the virus to recruit components of the cellular ESCRT machinery or to undergo proteolytic maturation, or the absence of Vpu did not significantly alter the assembly kinetics.


Core research teams: A strategy to research critically ill pregnant and postnatal women.
There has been little research conducted on critically ill pregnant and postnatal women. When developing a research protocol to conduct a prospective multi-centre survey on this study population, we found there were vital concerns that needed addressing prior to the research proceeding. Prompt identification of the study population and valid, reliable data collection were two aspects that needed particular attention with study recruitment potentially occurring in a total of eleven clinical areas from seven hospitals. In this paper we outline the particular issues faced by us when conducting multi-centre research on a study population that occurs infrequently and unpredictably, and when there is a necessary urgency to identify eligible study participants. The key strategy to overcome these difficulties, was the creation of 'Core Research Teams' in each clinical area. Our experience of using Core Research Teams in our pilot study is described in this paper. We found that the Core Research Team model is a very positive strategy to overcome the methodological challenges when operating a multi-centre study.


Transport of the harmful bloom alga Aureococcus anophagefferens by oceangoing ships and coastal boats.
It is well established that cyst-forming phytoplankton species are transported in ships' ballast tanks. However, there is increasing evidence that other phytoplankton species which do not encyst are also capable of surviving ballast transit. These species have alternative modes of nutrition (hetero- or mixotrophy) and/or are able to survive long-term darkness. In our studies of no-ballast-on-board vessels arriving in the Great Lakes, we tested for the presence of the harmful algal bloom species Aureococcus anophagefferens (brown tide) in residual (i.e., unpumpable) ballast water using methods based on the PCR. During 2001, the brown tide organism was detected in 7 of 18 ballast water tanks in commercial ships following transit from foreign ports. Furthermore, it was detected after 10 days of ballast tank confinement during a vessel transit in the Great Lakes, a significant result given the large disparity between the salinity tolerance for active growth of Aureococcus (>22 ppt) and the low salinity of the residual ballast water (approximately 2 ppt). We also investigated the potential for smaller, recreational vessels to transport and distribute Aureococcus. During the summer of 2002, 11 trailered boats from the inland bays of Delaware and coastal bays of Maryland were sampled. Brown tide was detected in the bilge water in the bottoms of eight boats, as well as in one live-well sample. Commercial ships and small recreational boats are therefore implicated as potential vectors for long-distance transport and local-scale dispersal of Aureococcus.


Syllable duration changes during babbling: a longitudinal study of French infant productions.
At the babbling stage, the syllable does not have the temporal characteristics of adult syllables because of the infant's limited oro-motor skills. This research aims to further our knowledge of syllable duration and temporal variability and their evolution with age as an indicator of the development of articulatory skills. The possible impact of syllable position, as well as that of type of intrasyllabic associations and intersyllabic articulatory changes on these parameters has also been tested. Oral productions of 22 French infants were recorded monthly from 8 to 14 months. 11 261 Consonant-Vowel (CV) syllables were annotated and temporally analyzed. The mean duration varied according to syllable position, but not to the intrasyllabic or intersyllabic articulatory changes. Moreover, the syllable duration decreased significantly from the age of 10 months onwards, whereas the temporal variability remained the same.


[Use of urography by magnetic resonance for the study of the urinary apparatus versus conventional urography].
Conventional urography (IVU) is an essential examination for the assessment of urinary tract but it is not free of complications, such as adverse reactions to contrast agents used (vasovagal and anaphylactic reactions), neurotoxicity, nephrotoxicity, as well as the damage due to the ionizing irradiation applied to the patient. For this reason, alternative imaging techniques such as magnetic resonance (MR) urography or uro-resonance have been developed. We present a case study assessing the diagnostic accuracy, specificity and sensitivity of uro-resonance and IVU as a morphological and functional examination of the urinary tract: and a quality study of the urographic images obtained with MR versus IVU. 150 patients have submited to a MR study, 63 of them with an IVU study already performed, acquiring high-intensity signals at T2 corresponding to abdominal and retroperitoneal fluid, initially using furosemide at low doses and, in a final study, administering gadolinium at a rate of 0.1 mg/Kg. The test was indicated in patients with antecedents of adverse reactions to iodine contrast, acute or chronic kidney failure, functional cancellation of the kidneys, pregnant patients and those in paediatric age. The capacity of diagnosis of urinary obstruction and the aetiology of this obstruction of both tests was studied, as well as the quality of the images obtained by the urographic study using MR. High resolution images were obtained of all the upper urinary tracts using MR, especially in the renal pelvis, without artefacts caused by peristalsis or intestinal fluid interposition. In 83.3% of cases, examinations revealed urological pathology. The diagnostic accuracy of the involvement cause of the urinary tract was 83.3%, with a sensitivity of 89.6%, a specificity of 69.2%. a positive predictive value of 86.6% and a negative predictive value of 75%. MR urography is a high sensitive technique for the study of urinary tract, used as an alternative to conventional urography particularly in cases of the contraindication of ionizing radiation or allergy to the contrast agent, as well as in patients with renal failure, and offers a wider morphological and functional study, with a high image quality, able to displace conventional examinations in the short or medium term.


Prevalence of Trichomonas vaginalis infection in women attending a major gynaecological hospital in Greece: a cross-sectional study.
The prevalence of Trichomonas vaginalis is not accurately estimated, since it is not a reportable disease. To assess the prevalence of T vaginalis infection in women attending a Greek gynaecological hospital and to evaluate four diagnostic methods for T vaginalis infection. 255 symptomatic and 247 asymptomatic women were included in the study during 2006-07; 372 were Greek and 130 were immigrants. T vaginalis was detected in vaginal samples, using wet mount, culture in modified Diamond's medium, antigen detection and two PCR assays, targeting different regions of T vaginalis genome. Specimens were considered positive for T vaginalis, when tested positive either by culture or by both PCRs. 23 women (4.6%) were positive for T vaginalis. Seven of the 23 positive samples (30.4%) were only PCR-positive. Infection was more prevalent in symptomatic women (6.7%) than in asymptomatic ones (2.4%). T vaginalis was more frequently detected in immigrants (7.9%) than in Greek women (3.3%). Gardnerella vaginalis infection was significantly more frequent in women infected with T vaginalis. PCR was the most sensitive method (100%), followed by culture (69.6%), wet mount (69.6%) and latex agglutination (54.6%). Agreement between PCR and culture as well as wet mount examination was very good (kappa=0.79). The study shows a relatively low percentage of trichomoniasis in the female population living in Athens. The infection was more prevalent among immigrants, and a proportion of the infected women was asymptomatic. The tested methods had good agreement and PCR was found to improve the diagnostic yield considerably.


Should Technical and Anatomical Difficulties Discourage Operators From Embarking on Transradial Access for Percutaneous Coronary Intervention?
Use of the radial approach for PCI procedures is increasing due to lower rates of access-site complications/bleeding, and patient preference. However, femoral operators switching may be discouraged by the learning curve and by anatomical issues that may complicate the procedure. We aimed to define the frequency of anatomic variants and success rates during transradial access for PCI. We retrospectively analyzed 2588 cases of PCI attempted by the radial route; radial/brachial and subclavian angiography was performed when obstructions were encountered. Presence of anatomical variants, spasm, and ability to complete the procedure were noted. Radial procedures were successfully completed in 2741/2588 cases (98.2%); in the remainder, switching to femoral approach was necessary. Local arteriography was performed in 221/2588 cases (8.5%) due to difficulties encountered; of these, 131/221 difficulties (59%) were due to problems at the radial arterial level, 58/221 (26%) were due to problems at the subclavian level, and 32/221 (15%) were due to problems at brachial arterial sites. Extreme radial tortuosity (18%) and radial loop (20%) had relatively lower rates of success followed by subclavian tortuosity (73%). Females had significantly higher incidences of radial spasm (6% vs 1.9% in men; P<.001), radial tortuosity (3.4% vs 1.7% in men; P=.01), and subclavian tortuosity (3.8% vs 1.8% in men; P<.01). Inability to successfully complete invasive procedures via the radial approach is uncommon. Even when encountered, most difficulties can be overcome with the use of vasodilators and hydrophilic wires. These data provide reassurance for would-be radial converts that the learning curve may not be as steep as envisaged.


Reducing patient barriers to pain and fatigue management.
Pain and fatigue are recognized as critical symptoms that impact the quality of life of cancer patients. The barriers to pain and fatigue relief have been classified into three categories: patient, professional, and system barriers. The overall objective of this trial as to test the effects of the "Passport to Comfort" intervention on reducing barriers to pain and fatigue management for ambulatory care cancer patients. This quasi-experimental, comparative study uses a Phase 1 control group of usual care followed sequentially by a Phase 2 intervention group in which educational and system-change efforts were directed toward improved pain and fatigue management. A sample of 187 cancer patients with breast, lung, colon, or prostate cancers, and a pain and/or fatigue rating of 4 or more (moderate to severe), were recruited. Patients in the intervention group received four educational sessions on pain/fatigue assessment and management, whereas patients in the control group received usual care. Pain and fatigue barriers and patient knowledge were measured at baseline, one month, and three months post-accrual. Patients in the intervention group experienced significant improvements in pain and fatigue measures immediately postintervention, and these improvements were sustained over time. The "Passport to Comfort" intervention was effective in reducing patient barriers to pain and fatigue management as well as in increasing patient knowledge regarding pain and fatigue. This intervention demonstrates innovation by translating the evidence-based guidelines for pain and fatigue as developed by the National Comprehensive Cancer Network into practice.


Two-photon luminescence properties of gold nanorods.
Gold nanorods can be internalized by macrophages (an important early cellular marker in atherosclerosis and cancer) and used as an imaging contrast agent for macrophage targeting. Objective of this study is to compare two-photon luminescence (TPL) properties of four aspect ratios of gold nanorods with surface plasmon resonance at 700, 756, 844 and 1060 nm respectively. TPL from single nanorods and Rhodamine 6G particles was measured using a laser-scanning TPL microscope. Nanorod TPL emission spectrum was recorded by a spectrometer. Quadratic dependence of luminescence intensity on excitation power (confirming a TPL process) was observed below a threshold (e.g., <1.6 mW), followed by photobleaching at higher power levels. Dependence of nanorod TPL intensity on excitation wavelength indicated that the two-photon action cross section (TPACS) is plasmon-enhanced. Largest TPACS of a single nanorod (12271 GM) was substantially larger than a single Rhodamine 6G particle (25 GM) at 760 nm excitation. Characteristics of nanorod TPL emission spectrum can be explained by plasmon-enhanced interband transition of gold. Comparison results of TPL brightness, TPACS and emission spectrum of nanorods can guide selection of optimal contrast agent for selected imaging applications.


Spotlight on Tdap₅ vaccine (Covaxis®) as a single-booster immunization for the prevention of tetanus, diphtheria, and pertussis: in children (aged ≥4 years), adolescents, and adults.
Covaxis® (also licensed as Triaxis® or Adacel® in individual countries) is a Tdap₅ (i.e. combined tetanus toxoid, reduced diphtheria toxoid, five component acellular pertussis [namely detoxified pertussis toxin, filamentous hemagglutinin, pertactin, and fimbriae types 2 and 3]) vaccine for the prevention of diphtheria, tetanus, and pertussis. It is approved for use in Europe as a single intramuscular booster dose in children (aged ≥4 years), adolescents, and adults, and in the US it is approved for use in individuals aged 11-64 years. In large, randomized, controlled clinical trials conducted in the UK and North America, a single intramuscular booster dose of Covaxis® induced robust immune responses for all of its component antigens when given to children (aged ≥4 years), adolescents, and adults. In addition, Covaxis® vaccine was safe and generally well tolerated in terms of solicited and unsolicited local injection-site and systemic adverse events, most of which were of mild intensity and resolved without sequelae. Furthermore, the immunogenicity of each individual component and the reactogenicity of Covaxis® vaccine in children, adolescents, and adults was generally similar to that of comparator vaccines. Despite being a vaccine-preventable disease and having >90% primary vaccination coverage worldwide, pertussis remains uncontrolled, particularly amongst adolescents and adults. Given the changing epidemiology of pertussis and the requirement to reduce infection in adolescents and adults (including healthcare workers) and thereby prevent transmission of the disease from these individuals to very young infants, the new 'cocoon strategy' recommended in current vaccination guidelines has become a key strategy in the management of morbidity and mortality associated with pertussis. This strategy focuses on the immunization of healthcare workers, and the parents and family members of infants who are too young to have undergone primary immunization, so as to prevent the transmission of pertussis to these young at-risk infants. The implementation of the 'cocoon strategy' may finally give countries the ability to control pertussis infections in these at-risk infants and ultimately provide the desired herd immunity against pertussis. In line with this strategy, a booster dose of Covaxis® vaccine provides a valuable option to reduce pertussis morbidity and mortality, and to maintain seroprotection against diphtheria and tetanus in children (aged ≥4 years), adolescents, and adults.


Speech therapy after thyroidectomy.
Common complaints of patients who have received thyroidectomy include dysphonia (voice dysfunction) and dysphagia (difficulty swallowing). One cause of these surgical outcomes is recurrent laryngeal nerve paralysis. Many studies have discussed the effectiveness of speech therapy (e.g., voice therapy and dysphagia therapy) for improving dysphonia and dysphagia, but not specifically in patients who have received thyroidectomy. Therefore, the aim of this paper was to discuss issues regarding speech therapy such as voice therapy and dysphagia for patients after thyroidectomy. Another aim was to review the literature on speech therapy for patients with recurrent laryngeal nerve paralysis after thyroidectomy. Databases used for the literature review in this study included, PubMed, MEDLINE, Academic Search Primer, ERIC, CINAHL Plus, and EBSCO. The articles retrieved by database searches were classified and screened for relevance by using EndNote. Of the 936 articles retrieved, 18 discussed "voice assessment and thyroidectomy", 3 discussed "voice therapy and thyroidectomy", and 11 discussed "surgical interventions for voice restoration after thyroidectomy". Only 3 studies discussed topics related to "swallowing function assessment/treatment and thyroidectomy". Although many studies have investigated voice changes and assessment methods in thyroidectomy patients, few recent studies have investigated speech therapy after thyroidectomy. Additionally, some studies have addressed dysphagia after thyroidectomy, but few have discussed assessment and treatment of dysphagia after thyroidectomy.


Accuracy of an HRP-2/panLDH rapid diagnostic test to detect peripheral and placental Plasmodium falciparum infection in Papua New Guinean women with anaemia or suspected malaria.
The diagnosis of malaria during pregnancy is complicated by placental sequestration, asymptomatic infection, and low-density peripheral parasitaemia. Where intermittent preventive treatment (IPT) with sulfadoxine-pyrimethamine is threatened by drug resistance, or is inappropriate due to low transmission, intermittent screening and treatment (ISTp) with rapid diagnostic tests for malaria (RDT) could be a valuable alternative. Therefore, the accuracy of RDTs to detect peripheral and placental infection was assessed in a declining transmission setting in Papua New Guinea (PNG). The performance of a combination RDT detecting histidine-rich protein-2 (HRP-2) and Plasmodium lactate dehydrogenase (pLDH), and light microscopy (LM), to diagnose peripheral Plasmodium falciparum and Plasmodium vivax infections during pregnancy, were assessed using quantitative real-time PCR (qPCR) as the reference standard. Participants in a malaria prevention trial in PNG with a haemoglobin ≤90 g/L, or symptoms suggestive of malaria, were tested. Ability of RDT and LM to detect active placental infection on histology was evaluated in some participants. Among 876 women, 1162 RDTs were undertaken (anaemia: 854 [73.5 %], suspected malaria: 308 [26.5 %]). qPCR detected peripheral infection during 190 RDT episodes (165 P. falciparum, 19 P. vivax, 6 mixed infections). Overall, RDT detected peripheral P. falciparum infection with 45.6 % sensitivity (95 % CI 38.0-53.4), a specificity of 96.4 % (95.0-97.4), a positive predictive value of 68.4 % (59.1-76.8), and a negative predictive value of 91.1 % (89.2-92.8). RDT performance to detect P. falciparum was inferior to LM, more so amongst anaemic women (18.6 vs 45.3 % sensitivity, Liddell's exact test, P < 0.001) compared to symptomatic women (72.9 vs 82.4 % sensitivity, P = 0.077). RDT and LM missed 88.0 % (22/25) and 76.0 % (19/25) of P. vivax infections, respectively. In a subset of women tested at delivery and who had placental histology (n = 158) active placental infection was present in 19.6 %: all three peripheral blood infection detection methods (RDT, LM, qPCR) missed >50 % of these infections. In PNG, HRP-2/pLDH RDTs may be useful to diagnose peripheral P. falciparum infections in symptomatic pregnant women. However, they are not sufficiently sensitive for use in intermittent screening amongst asymptomatic (anaemic) women. These findings have implications for the management of malaria in pregnancy. The adverse impact of infections undetected by RDT or LM on pregnancy outcomes needs further evaluation.


Bolstering Components of the Immune Response Compromised by Prior Exposure to Adenovirus: Guided Formulation Development for a Nasal Ebola Vaccine.
The severity and longevity of the current Ebola outbreak highlight the need for a fast-acting yet long-lasting vaccine for at-risk populations (medical personnel and rural villagers) where repeated prime-boost regimens are not feasible. While recombinant adenovirus (rAd)-based vaccines have conferred full protection against multiple strains of Ebola after a single immunization, their efficacy is impaired by pre-existing immunity (PEI) to adenovirus. To address this important issue, a panel of formulations was evaluated by an in vitro assay for their ability to protect rAd from neutralization. An amphiphilic polymer (F16, FW ∼39,000) significantly improved transgene expression in the presence of anti-Ad neutralizing antibodies (NAB) at concentrations of 5 times the 50% neutralizing dose (ND50). In vivo performance of rAd in F16 was compared with unformulated virus, virus modified with poly(ethylene) glycol (PEG), and virus incorporated into poly(lactic-co-glycolic) acid (PLGA) polymeric beads. Histochemical analysis of lung tissue revealed that F16 promoted strong levels of transgene expression in naive mice and those that were exposed to adenovirus in the nasal cavity 28 days prior to immunization. Multiparameter flow cytometry revealed that F16 induced significantly more polyfunctional antigen-specific CD8+ T cells simultaneously producing IFN-γ, IL-2, and TNF-α than other test formulations. These effects were not compromised by PEI. Data from formulations that provided partial protection from challenge consistently identified specific immunological requirements necessary for protection. This approach may be useful for development of formulations for other vaccine platforms that also employ ubiquitous pathogens as carriers like the influenza virus.


Obesity and Insulin Resistance Are Inversely Associated with Serum and Adipose Tissue Carotenoid Concentrations in Adults.
Low tissue concentrations of carotenoids have been suggested to contribute to insulin resistance in obesity. The objectives of the study were to 1) evaluate the relations of adipose tissue and serum carotenoids with body fat, abdominal fat distribution, muscle, adipose tissue and liver insulin resistance, and dietary intake; 2) evaluate the relations and distributions of carotenoids detected in adipose tissue and serum; and 3) compare serum carotenoids and retinol concentrations in subjects with and without obesity. Post hoc analysis of serum and adipose tissue carotenoids in individuals [n = 80; 31 men, 49 women; age (mean ± SEM): 51.4 ± 1.1 y] who participated in 2 separate studies conducted at the Clinical Research Facility at the Garvan Institute of Medical Research (Sydney) between 2008 and 2013. Retinol, α-carotene, β-carotene, ζ-carotene, lutein, lycopene, phytoene, and phytofluene were measured using HPLC. Body composition was measured by dual-energy X-ray absorptiometry. Insulin resistance was measured by 2-step hyperinsulinemic-euglycemic clamps with deuterated glucose (n = 64), and subcutaneous and visceral abdominal volume and liver and pancreatic fat by MRI (n = 60). Periumbilical subcutaneous fat biopsy was performed and carotenoids and retinol measured in the tissue (n = 16). We found that ζ-carotene, phytoene, and phytofluene were stored in considerable amounts in adipose tissue (25% of adipose tissue carotenoids). Carotenoid concentrations in adipose tissue and serum correlated significantly, but they followed different distributions: ζ-carotene was 3-fold higher in adipose tissue compared with serum, while lutein and lycopene made up 20% and 21% of serum carotenoids compared with 2% and 12% of adipose tissue carotenoids, respectively. Liver (P ≤ 0.028) and adipose tissue (P = 0.023), but not muscle (P ≥ 0.16), insulin resistance correlated inversely with many of the serum carotenoids. Multiple serum and adipose tissue carotenoids are associated with favorable metabolic traits, including insulin sensitivity in liver and adipose tissue in humans.


Methodological considerations for measuring spontaneous physical activity in rodents.
When exploring biological determinants of spontaneous physical activity (SPA), it is critical to consider whether methodological factors differentially affect rodents and the measured SPA. We determined whether acclimation time, sensory stimulation, vendor, or chamber size affected measures in rodents with varying propensity for SPA. We used principal component analysis to determine which SPA components (ambulatory and vertical counts, time in SPA, and distance traveled) best described the variability in SPA measurements. We compared radiotelemetry and infrared photobeams used to measure SPA and exploratory activity. Acclimation time, sensory stimulation, vendor, and chamber size independently influenced SPA, and the effect was moderated by the propensity for SPA. A 24-h acclimation period prior to SPA measurement was sufficient for habituation. Principal component analysis showed that ambulatory and vertical measurements of SPA describe different dimensions of the rodent's SPA behavior. Smaller testing chambers and a sensory attenuation cubicle around the chamber reduced SPA. SPA varies between rodents purchased from different vendors. Radiotelemetry and infrared photobeams differ in their sensitivity to detect phenotypic differences in SPA and exploratory activity. These data highlight methodological considerations in rodent SPA measurement and a need to standardize SPA methodology.


Continental data on cave-dwelling spider communities across Europe (Arachnida: Araneae).
Spiders (Arachnida: Araneae) are widespread in subterranean ecosystems worldwide and represent an important component of subterranean trophic webs. Yet, global-scale diversity patterns of subterranean spiders are still mostly unknown. In the frame of the CAWEB project, a European joint network of cave arachnologists, we collected data on cave-dwelling spider communities across Europe in order to explore their continental diversity patterns. Two main datasets were compiled: one listing all subterranean spider species recorded in numerous subterranean localities across Europe and another with high resolution data about the subterranean habitat in which they were collected. From these two datasets, we further generated a third dataset with individual geo-referenced occurrence records for all these species. Data from 475 geo-referenced subterranean localities (caves, mines and other artificial subterranean sites, interstitial habitats) are herein made available. For each subterranean locality, information about the composition of the spider community is provided, along with local geomorphological and habitat features. Altogether, these communities account for > 300 unique taxonomic entities and 2,091 unique geo-referenced occurrence records, that are made available via the Global Biodiversity Information Facility (GBIF) (Mammola and Cardoso 2019). This dataset is unique in that it covers both a large geographic extent (from 35° south to 67° north) and contains high-resolution local data on geomorphological and habitat features. Given that this kind of high-resolution data are rarely associated with broad-scale datasets used in macroecology, this dataset has high potential for helping researchers in tackling a range of biogeographical and macroecological questions, not necessarily uniquely related to arachnology or subterranean biology.


"Can't you initiate me here?": Challenges to timely initiation on antiretroviral therapy among methadone clients in Dar es Salaam, Tanzania.
Despite dramatic improvement in antiretroviral therapy (ART) access globally, people living with HIV who inject drugs continue to face barriers that limit their access to treatment. This paper explores barriers and facilitators to ART initiation among clients attending a methadone clinic in Dar es Salaam, Tanzania. We interviewed 12 providers and 20 clients living with HIV at the Muhimbili National Hospital methadone clinic between January and February 2015. We purposively sampled clients based on sex and ART status and providers based on job function. To analyze interview transcripts, we adopted a content analysis approach. Participants identified several factors that hindered timely ART initiation for clients at the methadone clinic. These included delays in CD4 testing and receiving CD4 test results; off-site HIV clinics; stigma operating at the individual, social and institutional levels; insufficient knowledge of the benefits of early ART initiation among clients; treatment breakdown at the clinic level possibly due to limited staff; and initiating ART only once one feels physically ill. Participants perceived social support as a buffer against stigma and facilitator of HIV treatment. Some clients also reported that persistent monitoring and follow-up on their HIV care and treatment by methadone clinic providers led them to initiate ART. Health system factors, stigma and limited social support pose challenges for methadone clients living with HIV to initiate ART. Our findings suggest that on-site point-of-care CD4 testing, a peer support system, and trained HIV treatment specialists who are able to counsel HIV-positive clients and initiate them on ART at the methadone clinic could help reduce barriers to timely ART initiation for methadone clients.


A histopathologic study of salivary gland tumors in Kano, northern Nigeria.
Significant differences in the global distribution of salivary gland tumors have been reported, but no formal study has been carried out here in Kano, the largest city in northern Nigeria. We therefore undertook this eight-year retrospective study of all histologically diagnosed salivary neoplasms at the histopathology laboratory of our referral teaching hospital in Kano. Seventy-eight salivary gland tumors were diagnosed during the eight-year study period accounting for 0.4% of all neoplasms. Benign tumors were more prevalent, comprising 56.4%, while malignancies were 43.6%. Pleomorphic adenoma and mucoepidermoid carcinoma were the commonest histological types constituting 48.7% and 23.1% respectively, while the most frequent sites were parotid, submandibular and minor salivary glands accounting for 49%, 26% and 24% respectively. As in most studies of black populations there were no adenolymphomas. Age distribution was bimodal with a benign peak in the third decade and a malignant peak in the sixth. Our findings were broadly similar to most other African reports but somewhat at variance with Western literature. Improved hospital attendance with more comprehensive reporting would yield more representative data.


A comparison of indirect methods for continuous estimation of arterial PCO2 in men.
Four different measures (PETCO2, PACO2, PADCO2, and PJCO2) for indirectly estimating arterial PCO2 (PaCO2) from respired gas at the mouth have been investigated. PETCO2 was the end-tidal PCO2. PACO2 was calculated using a reconstruction of the alveolar oscillation of PCO2 obtained from the end-tidal "plateau" in PCO2. PADCO2 was calculated as for PACO2 except that the effects of dead space were incorporated. PJCO2 was calculated from an empirical relationship involving PETCO2 and tidal volume. Six subjects were studied at rest and during cycle ergometry at 50 and 100 W while breathing a variety of gas mixtures. Arterial samples were drawn for determination of true PaCO2. The differences for each method between estimated and true PaCO2 at rest and at 50 and 100 W were as follows: PETCO2, -1.35 +/- 2.64, 1.67 +/- 2.31, and 2.67 +/- 2.02 (SD) Torr; PaCO2, -2.15 +/- 2.73, -0.80 +/- 2.18, and -0.35 +/- 2.31 (SD) Torr; PADCO2, -1.55 +/- 2.54, 0.25 +/- 2.16, and 0.63 +/- 2.26 (SD) Torr; and PJCO2, -1.41 +/- 2.30, 0.12 +/- 1.79, and 0.08 +/- 1.96 (SD) Torr. It is concluded that, at rest, all methods significantly underestimate true PaCO2 and during exercise PETCO2 significantly overestimates PaCO2, but no bias was detected for any of the other methods.


[Holy Trinity monument in the city of Osijek].
Plague was one of the most deadly epidemic diseases of the Baroque period. Responses to it were not only medical, but religious as well. A good example of the latter is the Most Holy Trinity monument in the city of Osijek, which was in the 18th century the biggest town of the Kingdom of Slavonia and today is the regional centre in the Republic of Croatia. The monument was erected between 1729 and 1730 on the main square of the Osijek military fortress Tvrđa by the widow of the General Maksimilijan Petraš who died during the 1728 plague epidemic. Inscription on it implores the mercy of God as a protection against plague. Its foundation could be also interpreted as a part of the Catholic Revival, which was implemented by the Habsburgs in Osijek and Slavonia after their liberation from the Ottomans. But although, on the one hand, it could be interpreted as a symbol of the successful implementation of the Habsburg unifying religious policies due to its strong resemblance with the similar columns throughout the Habsburg Monarchy, on the other hand, it represented a continuation of the theurgic understanding of medicine, which could be interpreted as the failure of the Habsburg enlightened medical policies. Thus the archival documents from the Osijek State Archive together with the Osijek plague column itself were analysed with the aim of explaining the above mentioned ambiguities.


Nucleophosmin interacts directly with c-Myc and controls c-Myc-induced hyperproliferation and transformation.
The transcription factor c-Myc is essential for cellular proliferation and is one of the most frequently activated oncogenes, but the molecular mechanism mediating its critical role in transformation is unclear. Like c-Myc, multifunctional nucleophosmin (NPM) is tightly regulated during proliferation and is overexpressed in several different types of cancer. Overexpression of NPM enhances proliferation and oncogene-mediated transformation, but the mechanism mediating these effects is unknown. We examined whether NPM stimulates proliferation and transformation by affecting c-Myc. Here, we show that NPM is essential for the activities of oncogenic c-Myc and that overexpressed NPM dramatically stimulates c-Myc-induced hyperproliferation and transformation. Endogenous and exogenous NPM directly interact with c-Myc and regulate the expression of endogenous c-Myc target genes at the promoter. Therefore, NPM is a key cofactor for the transforming activity of c-Myc and the interaction with c-Myc may mediate the enhancement of proliferation and transformation induced by NPM overexppression.


Evaluation of the minimum volume of salvage blood required for the successful use of two different autotransfusion devices.
Autotransfusion in pediatric anesthesia is beneficial in several clinical settings; however, more frequent usage is deterred by process-related costs and the fact that the absolute volume of blood returned may be minimal. Two autotransfusion devices (CATS, Fresenius Kabi AG, and Sorin Xtra, Sorin GmbH) with different technologies were evaluated to determine the minimum volume necessary to efficiently process salvage blood. Banked blood was diluted to simulate different clinical scenarios. Volume and red cell mass in returned blood were analyzed as a function of initial volume and red cell mass in the collection reservoir using incremental increases in the volume of blood added to the reservoir. The volume of the returned processed blood for infusion was dependent on degree of dilution and blood volume in the reservoir. The CATS required a smaller initial minimum volume to process and successfully return; however, the Xtra provided a more efficient recovery at low initial collected volumes. Using test blood of various degrees of dilution, the CATS and Xtra were able to return blood for retransfusion after a mean initial red cell mass of 48.7 ml (±4.9 ml) and 53.7 ml (±5.0 ml) were processed, respectively. Red cell mass rather than salvaged blood volume represents a reliable predictor of the successful use of an autotransfusion device. Measuring the hematocrit of the salvaged blood could improve the use of the devices. The investigated devices are likely to be roughly equivalent in effectiveness.


Characteristics of, and insurance payments for, injuries to cyclists in Tasmania, 1990-2010.
To describe the characteristics and costs of injuries to cyclists resulting in a 3rd party insurance claim in Tasmania. Data on injuries to cyclists were obtained from the Motor Accident Insurance Board (MAIB) for the period 1990-2010. Frequency and insurance costs of injuries to cyclists were compared to injuries incurred by other road users. Descriptive analyses of cycling injuries and insurance costs by year, age and sex of claimant, and type and location of injury are presented. Annual costs of insurance claims by cyclists averaged AUD 3.9 million. There was a significant decrease in the frequency of claims made by all road users combined over the study period, but not for cyclists. Cycling injuries made up 2.0% of claims but accounted for 3.4% of the total costs and were among the road user groups with the highest mean costs per claim. Fractures (20.7%) were the most common cycling injury. Brain injuries led to the highest mean claim costs (AUD 1,559,032), and accounted for 66.8% of claim costs made by cyclists. Mean costs per claim for cycling injuries are high compared to those made by most other road users. The costs of these injuries impose a substantial burden on insurance payers. The high costs and severity of claims by cyclists compared to other road users demonstrates the high vulnerability of cyclists, and lends support to increasing separation of cyclists from motor vehicles.


Haptoglobin: a natural bacteriostat.
The combination of bacteria and blood in a wound can have lethal consequences, probably because hemoglobin iron supports prolific bacterial growth. Rats inoculated intraperitoneally with pathogenic Escherichia coli and small amounts of hemoglobin die. Simultaneous administration of haptoglobin, a naturally occurring hemoglobin-binding protein, fully protects against lethality. Therefore, haptoglobin may not only accelerate the clearance of free hemoglobin, but also limit its utilization by adventitious bacteria. Haptoglobin may have therapeutic potential in the treatment of life-threatening, hemoglobin-driven bacterial infections.


First application of a transcutaneous optical single-port glucose monitoring device in patients with type 1 diabetes mellitus.
The combination of continuous glucose monitoring (CGM) and continuous subcutaneous insulin infusion can be used to improve the treatment of patients with diabetes. The aim of this study was to advance an existing preclinical single-port system for clinical application by integrating the sensors of a phosphorescence based CGM system into a standard insulin infusion set. The extracorporeal optical phase fluorimeter was miniaturised and is now comparable with commercial CGM systems regarding size, weight and wear comfort. Sensor chemistry was adapted to improve the adhesion of the sensor elements on the insulin infusion set. In-vitro tests showed a linear correlation of R2=0.998 between sensor values and reference glucose values in the range of 0-300mg/dl. Electrical and cytotoxicity tests showed no negative impact on human health. Two single-port devices were tested in each of 12 patients with type 1 diabetes mellitus in a clinical set-up for 12h. Without additional data processing, the overall median absolute relative difference (median ARD) was 22.5%. For some of the used devices the median ARD was even well below 10%. The present results show that individual glucose sensors performance of the single-port system is comparable with commercial CGM systems but further improvements are needed. The new system offers a high extent of safety and usability by combining insulin infusion and continuous glucose measurement in a single-port system which could become a central element in an artificial pancreas for an improved treatment of patients with type 1 diabetes mellitus.


Adults with probable developmental coordination disorder selectively process early visual, but not tactile information during action preparation. An electrophysiological study.
Developmental coordination disorder (DCD) is a neurodevelopmental condition affecting motor coordination in children and adults. Here, EEG signals elicited by visual and tactile stimuli were recorded while adult participants with and without probable DCD (pDCD) performed a motor task. The task cued reaching movements towards a location in visible peripersonal space as well as an area of unseen personal space. Event-related potentials elicited by visual and tactile stimuli revealed that visual processing was strongly affected by movement preparation in the pDCD group, even more than in controls. However, in contrast to the controls, tactile processing in unseen space was unaffected by movement preparation in the pDCD group. The selective use of sensory information from vision and proprioception is fundamental for the adaptive control of movements, and these findings suggest that this is impaired in DCD. Additionally, the pDCD group showed attenuated motor rhythms (beta: 13-30 Hz) over sensorimotor regions following cues to prepare movements towards unseen personal space. The results reveal that individuals with pDCD exhibit differences in the neural mechanisms of spatial selection and action preparation compared to controls, which may underpin the sustained difficulties they experience. These findings provide new insights into the neural mechanisms potentially disrupted in this highly prevalent disorder.


Isoform distribution and tissue contents of contractile and cytoskeletal proteins in hypertrophied smooth muscle from rat portal vein.
Growth of the smooth muscle in the rat portal vein was initiated by an increased transmural pressure. After 7 days, the cross-sectional area of the vessel wall and the maximal active force of the longitudinal muscle layer had increased twofold. Electron microscopy showed that the cell cross-sectional area was increased, suggesting cellular hypertrophy. Increased amounts of intermediate (10 nm) filaments were observed in the hypertrophied cells. The hypertrophied vessels had decreased DNA content per unit wet weight compared with the control vessels (hypertrophied, 1.5 +/- 0.1; control, 1.9 +/- 0.1 micrograms/mg; p less than 0.01). Protein composition was studied with electrophoretic methods. Compared with control preparations the hypertrophied veins had similar myosin and actin contents per unit wet weight (myosin: hypertrophied, 4.4 +/- 0.8; control, 5.9 +/- 0.9; actin: hypertrophied 12.2 +/- 0.6; control, 11.8 +/- 1.0 mg/g). Two different forms of the myosin heavy chain were detected with 5% sodium dodecyl sulfate-polyacrylamide gels. The proportion of the lower molecular weight heavy chain relative to total heavy chain content was about 30% and similar in both preparations. The relation filamin/myosin was increased in the hypertrophied vessels. Pyrophosphate gel electrophoresis revealed two protein bands, with an increase in the slower migrating band in the hypertrophied vessels possibly reflecting an increase in filamin content in the extracts. In the control portal vein alpha-actin is the dominating isoform constituting about 55% of total actin. In hypertrophied vessels, alpha-actin decreased (by 15%) and gamma-actin increased (by 20%). The portal vein contained desmin and vimentin in a ratio of about 6:1. The hypertrophied vessels showed a marked increase in the amount of these proteins (desmin/actin: hypertrophied, 0.32; control, 0.14). In conclusion, during pressure-induced growth of the portal vein, contractile protein contents increase in proportion to the increase in weight. A change in isoforms of actin occurs but no evidence for a change in myosin isoforms was found. The structural proteins increase relative to tissue weight, possibly associated with the increased number of intermediate filaments demonstrated with electron microscopy.


Association of silicofluoride treated water with elevated blood lead.
Previous epidemiological studies have associated silicofluoride-treated community water with enhanced child blood lead parameters. Chronic, low-level dosage of silicofluoride (SiF) has never been adequately tested for health effects in humans. We report here on a statistical study of 151,225 venous blood lead (VBL) tests taken from children ages 0-6 inclusive, living in 105 communities of populations from 15,000 to 75,000. The tests are part of a sample collected by the New York State Department of Children's Health, mostly from 1994-1998. Community fluoridation status was determined from the CDC 1992 Fluoridation Census. Covariates were assigned to each community using the 1990 U.S. Census. Blood lead measures were divided into groups based on race and age. Logistic regressions were carried out for each race/age group, as well as above and below the median of 7 covariates to test the relationship between known risk factors for lead uptake, exposure to SiF-treated water, and VBL >10 microg/dL. For every age/race group, there was a consistently significant association of SiF treated community water and elevated blood lead. Logistic regressions above and below the median value of seven covariates show an effect of silicofluoride on blood lead independent of those covariates. The highest likelihood of children having VBL> 10 microg/dL occurs when they are both exposed to SiF treated water and likely to be subject to another risk factor known to be associated with high blood lead (e.g., old housing). Results are consistent with prior analyses of surveys of children's blood lead in Massachusetts and NHANES III. These data contradict the null hypothesis that there is no difference between the toxic effects of SiF and sodium fluoride, pointing to the need for chemical studies and comprehensive animal testing of water treated with commercial grade silicofluorides.


KCa1.1, a calcium-activated potassium channel subunit alpha 1, is targeted by miR-17-5p and modulates cell migration in malignant pleural mesothelioma.
Malignant pleural mesothelioma (MPM) is an aggressive, locally invasive, cancer elicited by asbestos exposure and almost invariably a fatal diagnosis. To date, we are one of the leading laboratory that compared microRNA expression profiles in MPM and normal mesothelium samples in order to identify dysregulated microRNAs with functional roles in mesothelioma. We interrogated a significant collection of MPM tumors and normal pleural samples in our biobank in search for novel therapeutic targets. Utilizing mRNA-microRNA correlations based on differential gene expression using Gene Set Enrichment Analysis (GSEA), we systematically combined publicly available gene expression datasets with our own MPM data in order to identify candidate targets for MPM therapy. We identified enrichment of target binding sites for the miR-17 and miR-30 families in both MPM tumors and cell lines. RT-qPCR revealed that members of both families were significantly downregulated in MPM tumors and cell lines. Interestingly, lower expression of miR-17-5p (P = 0.022) and miR-20a-5p (P = 0.026) was clearly associated with epithelioid histology. We interrogated the predicted targets of these differentially expressed microRNA families in MPM cell lines, and identified KCa1.1, a calcium-activated potassium channel subunit alpha 1 encoded by the KCNMA1 gene, as a target of miR-17-5p. KCa1.1 was overexpressed in MPM cells compared to the (normal) mesothelial line MeT-5A, and was also upregulated in patient tumor samples compared to normal mesothelium. Transfection of MPM cells with a miR-17-5p mimic or KCNMA1-specific siRNAs reduced mRNA expression of KCa1.1 and inhibited MPM cell migration. Similarly, treatment with paxilline, a small molecule inhibitor of KCa1.1, resulted in suppression of MPM cell migration. These functional data implicating KCa1.1 in MPM cell migration support our integrative approach using MPM gene expression datasets to identify novel and potentially druggable targets.


Population pharmacokinetics and pharmacodynamics of piperaquine in children with uncomplicated falciparum malaria.
Dihydroartemisinin-piperaquine is being increasingly used as a first-line artemisinin combination treatment for malaria. The aim of this study was to describe the pharmacokinetic and pharmacodynamic properties of piperaquine in 236 children with uncomplicated falciparum malaria in Burkina Faso. They received a standard body weight-based oral 3-day fixed-dose dihydroartemisinin-piperaquine regimen. Capillary plasma concentration-time profiles were characterized using nonlinear mixed-effects modeling. The population pharmacokinetics of piperaquine were described accurately by a two-transit-compartment absorption model and a three-compartment distribution model. Body weight was a significant covariate affecting clearance and volume parameters. The individually predicted day 7 capillary plasma concentration of piperaquine was an important predictor (P < 0.0001) of recurrent malaria infection after treatment. Young children (2-5 years of age) received a significantly higher body weight-normalized dose than older children (P = 0.025) but had significantly lower day 7 piperaquine concentrations (P = 0.024) and total piperaquine exposures (P = 0.021), suggesting that an increased dose regimen for young children should be evaluated.


Metronomic scheduling of imatinib abrogates clonogenicity of neuroblastoma cells and enhances their susceptibility to selected chemotherapeutic drugs in vitro and in vivo.
Imatinib is currently in early clinical trials as targeted therapy for relapsed neuroblastomas and other childhood solid tumors expressing platelet-derived growth factor receptors (PDGFR) or c-Kit. Short-term treatment with imatinib in clinically achievable concentrations is ineffective in neuroblastoma in vitro. However, clinically, imatinib is administered daily over long time periods. The effects of combining imatinib with chemotherapy in neuroblastoma are unknown. Here, a panel of neuroblastoma cell lines (n = 5) were studied, representing tumors with different biological (MYCN-amplification +/-) and clinical (drug resistance) features. Using a protracted low-dose treatment schedule (1-3 weeks; 0.5-5microM) imatinib dose-dependently inhibited proliferation and clonogenic survival for all tested cell lines with IC50 <2.5microM. In contrast, short-term treatment (<96 hrs) was ineffective. Low-dose imatinib was synergistic in combination with doxorubicin and caused increased G2/M- and S-phase arrest and apoptosis as evidenced by enhanced caspase-3 activation and sub-G1 DNA accumulation. A significant but less pronounced effect was observed when imatinib was combined with etoposide or vincristine, as opposed to cisplatin, melphalan, or irinotecan. All cell lines expressed PDGFRbeta, whereas no protein expression of PDGFRalpha was detected in MYCN amplified cell lines. PDGF-BB caused PDGFRbeta phosphorylation and partially rescued neuroblastoma cells from doxorubicin-induced apoptosis, in an imatinib-sensitive manner. In vivo, treatment with imatinib in combination with doxorubicin induced a significant growth inhibition of established neuroblastoma xenografts. These findings suggest clinical testing of imatinib in combination with selected chemotherapeutic drugs, in particular doxorubicin, in children with high-risk neuroblastoma.


Neuroinflammation and its modulation by flavonoids.
There is increasing evidence to suggest that neuroinflammatory processes contribute to the cascade of events that lead to the progressive neuronal damage observed in neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. Therefore, treatment regimes aimed at modulating neuroinflammatory processes may act to slow the progression of these debilitating brain disorders. Recently, a group of dietary polyphenols known as flavonoids have been shown to exert neuroprotective effects in vivo and in neuronal cell models. In this review we discuss the evidence relating to the modulation of neuroinflammation by flavonoids. We highlight the evidence which suggests their mechanism of action involves: 1) attenuation of the release of cytokines, such as interleukin-1beta (IL-1beta and tumor necrosis factor-alpha (TNF-alpha); 2) an inhibitory action against inducible nitric oxide synthase (iNOS) induction and subsequent nitric oxide (NO(*)) production; 3) inhibition of the activation of NADPH oxidase and subsequent reactive oxygen species generation; 4) a capacity to down-regulate the activity of pro-inflammatory transcription factors such as nuclear factor-kappaB (NF-kappaB); and 5) the potential to modulate signalling pathways such as mitogen-activated protein kinase (MAPK) cascade. We also consider the potential of these dietary compounds to represent novel therapeutic agents by considering their metabolism in the body and their ability to access the brain via the blood brain barrier. Finally, we discuss future areas of study which are necessary before dietary flavonoids can be established as therapeutic agents against neuroinflammation.


EVALUATION OF MULTIPLEX LIGATION DEPENDENT PROBE AMPLIFICATION AS A TOOL FOR DIAGNOSIS AND CARRIER DETECTION IN FAMILIES WITH A DYSTROPHINOPATHY.
We set out to evaluate multiplex ligation dependent probe amplification (MLPA) as a tool for diagnosis and carrier detection in families with a dystrophinopathy. Fifty three Indian families with provisional diagnosis of Duchene muscular dystrophy or Becker muscular dystrophy were evaluated by MLPA and multiplex polymerase chain reaction (PCR). Sanger sequencing was used to analyze the entire gene in one patient. Mothers were tested for carrier status whenever possible. Molecular analysis of DMD gene by combining MLPA and multiplex PCR yielded a mutation detection rate of 62% (33/53). Deletions were detected in 27/53 (51%) cases, duplications in 5/53 (9%) cases, a small deletion one case and Sanger sequencing detected a nonsense mutation in one case. Mutation was not detected in 36% (19/53) cases. Fifty six percent of mothers (9/16) were found to be carriers. MLPA helped to refine the results of multiplex PCR testing in 22 patients (5 duplications, 16 deletions and one small deletion). We also describe a situation where a deletion of single exon on MLPA (but not detected by multiplex PCR) was actually due to a deletion of two nucleotides in the probe ligation site. MLPA appears to score over multiplex PCR in diagnosis and carrier detection, specifically by detecting deletions and duplications that are not detected by traditional multiplex PCR.


Single-molecule studies of high-mobility group B architectural DNA bending proteins.
Protein-DNA interactions can be characterized and quantified using single molecule methods such as optical tweezers, magnetic tweezers, atomic force microscopy, and fluorescence imaging. In this review, we discuss studies that characterize the binding of high-mobility group B (HMGB) architectural proteins to single DNA molecules. We show how these studies are able to extract quantitative information regarding equilibrium binding as well as non-equilibrium binding kinetics. HMGB proteins play critical but poorly understood roles in cellular function. These roles vary from the maintenance of chromatin structure and facilitation of ribosomal RNA transcription (yeast high-mobility group 1 protein) to regulatory and packaging roles (human mitochondrial transcription factor A). We describe how these HMGB proteins bind, bend, bridge, loop and compact DNA to perform these functions. We also describe how single molecule experiments observe multiple rates for dissociation of HMGB proteins from DNA, while only one rate is observed in bulk experiments. The measured single-molecule kinetics reveals a local, microscopic mechanism by which HMGB proteins alter DNA flexibility, along with a second, much slower macroscopic rate that describes the complete dissociation of the protein from DNA.


Sexual Activity and Cognitive Decline in Older Adults.
This prospective study tested whether sexual activity and emotional closeness during partnered sexual activity relate to cognitive decline (episodic memory performance) in older adulthood. In total, 6016 adults aged 50 and over (2672 men, 3344 women; M age = 66.0 ± 8.8 years) completed an episodic memory task and self-report questions related to health, sexual activity, and emotional closeness. Two years later, participants again completed the episodic memory task. After controlling for demographic and health-related lifestyle factors, more frequent sexual activity and greater emotional closeness during partnered sexual activity were associated with better memory performance. The association between sexual activity and memory performance was stronger among older participants in the sample. Memory performance worsened over 2 years, but change in memory performance was unrelated to sexual activity or emotional closeness during partnered sexual activity. These findings build on experimental research that has found sexual activity enhances episodic memory in non-human animals. Further research using longer timeframes and alternative measures of cognitive decline is recommended.


Photomixing at 1.55 microm in ion-irradiated In(0.53)Ga(0.47)As on InP.
We report the first demonstration of a terahertz photomixer made of ion-irradiated In(0.53)Ga(0.47)As lattice-matched to InP and fiber-optic coupled with the drive lasers. A continuous-wave radiation is generated at frequencies up to 0.8 THz by photomixing two continuous-wave laser diodes around 1.55 microm. The measured 3dB-down bandwidth of 300 GHz yields a carrier lifetime of 0.53 ps, in agreement with the value of 0.41 ps measured in pump probe experiments. The detected signal is at the most 15 dB lower than the one obtained from similar photomixers fabricated from low-temperature-grown GaAs.


Cellular distribution of anionic antimicrobial peptide in normal lung and during acute pulmonary inflammation.
Anionic peptides (APs) are small antimicrobial peptides present in human and ovine lung. In this study APs were also detected in bovine lung, and production of APs in lungs with acute inflammation induced by various stimuli was determined. The distribution and intensity of APs were determined by immunohistochemistry in lungs of 1) neonatal calves (1-3 days of age) inoculated with Mannheimia (Pasteurella) haemolytica, a known inducer of the bovine beta-defensin lingual antimicrobial peptide (LAP) or pyrogen-free saline (PFS), and 2) growing calves (3 months of age) similarly inoculated with M. haemolytica, a lipopolysaccharide (LPS) from M. haemolytica, an LPS-associated protein from M. haemolytica, or PFS. APs were also detected by western blots with the same antibody in lungs of the calves above, as well as in calves inoculated with Pseudomonas aeruginosa, and an adult cow. Anionic peptide (AP) immunoreactivity was detected in bands (approximate weights) in the western blots of lung at 28-30 (strongest signal), 31, 45, and 52-60 kd regardless of inoculum. The adult cow lacked bands at 45 kd, but it had additional bands at 64 (inconsistently) and 35-38 kd. All these band sizes are consistent with those of the western blots of human and ovine lung. The cellular distribution of APs in lung of neonatal and growing cattle was similar to that in lung of human and sheep. In lungs with acute inflammation induced by live bacteria, LPS, or protein, AP distribution and intensity were similar to those in control (PFS-inoculated) lungs and slightly decreased in bronchioles. This work demonstrates that AP is present in lung of cattle and is thereby conserved among two ruminant species and man. Distribution and intensity of AP production are not enhanced by infection or acute inflammation and are decreased in bronchioles, which suggests that AP is not induced like beta-defensins such as LAP, but, instead, is produced constitutively.


C-terminal truncations in human 3'-5' DNA exonuclease TREX1 cause autosomal dominant retinal vasculopathy with cerebral leukodystrophy.
Autosomal dominant retinal vasculopathy with cerebral leukodystrophy is a microvascular endotheliopathy with middle-age onset. In nine families, we identified heterozygous C-terminal frameshift mutations in TREX1, which encodes a 3'-5' exonuclease. These truncated proteins retain exonuclease activity but lose normal perinuclear localization. These data have implications for the maintenance of vascular integrity in the degenerative cerebral microangiopathies leading to stroke and dementias.


Geological gas-storage shapes deep life.
Around the world, several dozen deep sedimentary aquifers are being used for storage of natural gas. Ad hoc studies of the microbial ecology of some of them have suggested that sulfate reducing and methanogenic microorganisms play a key role in how these aquifers' communities function. Here, we investigate the influence of gas storage on these two metabolic groups by using high-throughput sequencing and show the importance of sulfate-reducing Desulfotomaculum and a new monophyletic methanogenic group. Aquifer microbial diversity was significantly related to the geological level. The distance to the stored natural gas affects the ratio of sulfate-reducing Firmicutes to deltaproteobacteria. In only one aquifer, the methanogenic archaea dominate the sulfate-reducers. This aquifer was used to store town gas (containing at least 50% H2 ) around 50 years ago. The observed decrease of sulfates in this aquifer could be related to stimulation of subsurface sulfate-reducers. These results suggest that the composition of the microbial communities is impacted by decades old transient gas storage activity. The tremendous stability of these gas-impacted deep subsurface microbial ecosystems suggests that in situ biotic methanation projects in geological reservoirs may be sustainable over time.


Potent human broadly SARS-CoV-2-neutralizing IgA and IgG antibodies effective against Omicron BA.1 and BA.2.
Memory B-cell and antibody responses to the SARS-CoV-2 spike protein contribute to long-term immune protection against severe COVID-19, which can also be prevented by antibody-based interventions. Here, wide SARS-CoV-2 immunoprofiling in Wuhan COVID-19 convalescents combining serological, cellular, and monoclonal antibody explorations revealed humoral immunity coordination. Detailed characterization of a hundred SARS-CoV-2 spike memory B-cell monoclonal antibodies uncovered diversity in their repertoire and antiviral functions. The latter were influenced by the targeted spike region with strong Fc-dependent effectors to the S2 subunit and potent neutralizers to the receptor-binding domain. Amongst those, Cv2.1169 and Cv2.3194 antibodies cross-neutralized SARS-CoV-2 variants of concern, including Omicron BA.1 and BA.2. Cv2.1169, isolated from a mucosa-derived IgA memory B cell demonstrated potency boost as IgA dimers and therapeutic efficacy as IgG antibodies in animal models. Structural data provided mechanistic clues to Cv2.1169 potency and breadth. Thus, potent broadly neutralizing IgA antibodies elicited in mucosal tissues can stem SARS-CoV-2 infection, and Cv2.1169 and Cv2.3194 are prime candidates for COVID-19 prevention and treatment.


Contractile properties of knee-extensors in one single family with nemaline myopathy: central and peripheral aspects of muscle activation.
Patients with nemaline myopathy, a muscle disorder primarily affecting the thin filaments, suffer from weakness which is poorly understood. As disturbed excitation-contraction coupling has been suggested as a possible mechanism, the present study was designed to investigate whether the contractile properties of the knee-extensor muscles in patients from a single family with nemaline myopathy were different from able-bodied individuals. To assess central neural as well as more peripheral intrinsic aspects of muscle activation, isometric voluntary and electrically elicited quadriceps contractions were evoked at different knee angles. Interestingly, across the range of 30-70 degrees of knee flexion, the capacity to achieve maximal voluntary activation of the muscles, assessed by a super-imposed stimulation technique, was significantly higher in patients compared with controls. Furthermore, the torque-frequency relation differed between groups, with the muscles of patients producing higher torques at low (twitch and 10 Hz) stimulation frequencies relative to maximal (150 Hz) stimulation than controls at both 30 degrees and 60 degrees of knee flexion. These results suggest that no impairment was present at relatively low activation frequencies. It may, however, be indicative for a reduced cross-bridge attachment as part of the excitation-contraction coupling specifically at high activation frequencies. In conclusion, the quadriceps weakness observed in this specific patient group cannot be explained by an impaired capacity to maximally activate these muscles. However, the data of relatively high torques produced at submaximal activation frequencies are compatible with the hypothesis that patients with nemaline myopathy may have an impaired acto-myosin interaction specifically at high levels of activation.


Milk Options Observation (MOO): A Mixed-Methods Study of Chocolate Milk Removal on Beverage Consumption and Student/Staff Behaviors in a Rural Elementary School.
Providing flavored milk in school lunches is controversial, with conflicting evidence on its impact on nutritional intake versus added sugar consumption and excess weight gain. Nonindustry-sponsored studies using individual-level analyses are needed. Therefore, we conducted this mixed-methods study of flavored milk removal at a rural primary school between May and June 2012. We measured beverage selection/consumption pre- and post-chocolate milk removal and collected observation field notes. We used linear and logistic mixed models to assess beverage waste and identified themes in staff and student reactions. Our analysis of data from 315 unique students and 1,820 beverages choices indicated that average added sugar intake decreased by 2.8 g postremoval, while average reductions in calcium and protein consumption were negligible (12.2 mg and 0.3 g, respectively). Five thematic findings emerged, including concerns expressed by adult staff about student rebellion following removal, which did not come to fruition. Removing flavored milk from school-provided lunches may lower students' daily added sugar consumption without considerably decreasing calcium and protein intake and may promote healthy weight.


Gβγ subunit activation promotes dopamine efflux through the dopamine transporter.
The dopamine transporter (DAT) is an important regulator of brain dopamine (DA) homeostasis, controlling the intensity and duration of DA signaling. DAT is the target for psychostimulants-like cocaine and amphetamine-and plays an important role in neuropsychiatric disorders, including attention-deficit hyperactivity disorder and drug addiction. Thus, a thorough understanding of the mechanisms that regulate DAT function is necessary for the development of clinical interventions to treat DA-related brain disorders. Previous studies have revealed a plethora of protein-protein interactions influencing DAT cellular localization and activity, suggesting that the fine-tuning of DA homeostasis involves multiple mechanisms. We recently reported that G-protein beta-gamma (Gβγ) subunits bind directly to DAT and decrease DA clearance. Here we show that Gβγ induces the release of DA through DAT. Specifically, a Gβγ-binding/activating peptide, mSIRK, increases DA efflux through DAT in heterologous cells and primary dopaminergic neurons in culture. Addition of the Gβγ inhibitor gallein or DAT inhibitors prevents this effect. Residues 582 to 596 in the DAT carboxy terminus were identified as the primary binding site of Gβγ. A TAT peptide containing the Gβγ-interacting domain of DAT blocked the ability of mSIRK to induce DA efflux, consistent with a direct interaction of Gβγ with the transporter. Finally, activation of a G-protein-coupled receptor, the muscarinic M5R, results in DAT-mediated DA efflux through a Gβγ-dependent mechanism. Collectively, our data show that Gβγ interacts with DAT to promote DA efflux. This novel mechanism may have important implications in the regulation of brain DA homeostasis.


Absent pericardium during coronary bypass.
The importance of recognizing congenitally absent left pericardium in the setting of acquired cardiovascular disease was recently appreciated during a case of coronary artery bypass grafting. An associated defect in the left hemidiaphragm and an abnormal location of the left phrenic nerve were found. Careful dissection of the phrenic nerve from the surface of the heart and closure of the diaphragmatic defect were necessary to avoid injury to the former and prevent hernia through the latter structure. Absent left pericardium discovered during cardiac surgery requires attention to the location of the left phrenic nerve and recognition of associated defects in the diaphragm.


Inhibition of de novo Methyltransferase 3B is a Potential Therapy for Hepatocellular Carcinoma.
Aberrant epigenetic patterns, including inactivation of tumor suppressor genes due to DNA methylation, have been described in many human cancers. Epigenetic therapeutic is a new and rapidly developing area of tumor treatment because DNA methyltransferase (DNMT) inhibitors can reverse its changes. We attempted to identify potential approach for epigenetic therapy of hepatocellular carcinoma. We knocked down the expression of DNMT 1 or DNMT 3B by siRNA, and inhibited DNA methyltranferases by 5-Aza-2'-deoxycytidine. We used high-density oligonucleotide gene expression microarrays to examine the induced genes in human hepatocellular carcinoma cell line SMMC-7721 after suppressing DNA methyltranferases. The 5' ends of up-regulated genes were analyzed by BLAST database to determine whether they have promoter CpG islands, and then the identical induced genes were compared among different inhibition of DNA methyltranferases. Our results show that 9 genes were found to be over expressed by more than two-fold induced by DNMT1 siRNA and 5-Aza-CdR, and 30 genes were found to be over expressed by more than two-fold induced by DNMT3B siRNA and 5-Aza-CdR in SMMC-7721. Among them, 76.6% up-regulated genes conjectural contained 5' CpG islands. The DNMT3B siRNA could induce more genes identical to demethylation agent in SMMC-7721. DNMT3B might be a new potential target for therapy of hepatocellular carcinoma.


Betacoronavirus Adaptation to Humans Involved Progressive Loss of Hemagglutinin-Esterase Lectin Activity.
Human beta1-coronavirus (β1CoV) OC43 emerged relatively recently through a single zoonotic introduction. Like related animal β1CoVs, OC43 uses 9-O-acetylated sialic acid as receptor determinant. β1CoV receptor binding is typically controlled by attachment/fusion spike protein S and receptor-binding/receptor-destroying hemagglutinin-esterase protein HE. We show that following OC43's introduction into humans, HE-mediated receptor binding was selected against and ultimately lost through progressive accumulation of mutations in the HE lectin domain. Consequently, virion-associated receptor-destroying activity toward multivalent glycoconjugates was reduced and altered such that some clustered receptor populations are no longer cleaved. Loss of HE lectin function was also observed for another respiratory human coronavirus, HKU1. This thus appears to be an adaptation to the sialoglycome of the human respiratory tract and for replication in human airways. The findings suggest that the dynamics of virion-glycan interactions contribute to host tropism. Our observations are relevant also to other human respiratory viruses of zoonotic origin, particularly influenza A virus.


Circulating concentrations of 1,25-dihydroxyvitamin D3 in patients with primary hyperparathyroidism.
Vitamin D metabolism was studied in 65 patients with surgically proven primary hyperparathyroidism. The mean concentration of 1,25-dihydroxyvitamin D3 was 51.7 +/- 34 pg/ml (mean +/- SD) and was not significantly different from normal. Renal function was normal in 60 of these patients and in this group circulating 1,25-dihydroxyvitamin D3 was below the lower limit of normal in three and elevated in 17; it was related to the serum concentrations of amino-terminal parathyroid hormone, but was independent of serum calcium and the urinary excretion of calcium. The incidence of nephrolithiasis or hyperparathyroid bone disease or combined nephrolithiasis and bone disease in these patients was not related to the circulating concentration of 1,25-dihydroxyvitamin D3. In the remaining five patients, in whom renal impairment was present, circulating 1,25-dihydroxyvitamin D3 was below the lower limit of normal in four. Thus, in primary hyperparathyroidism the circulating concentration of 1,25-dihydroxyvitamin D3 is elevated in only a minority of patients and appears to be unrelated to the occurrence of nephrolithiasis or bone disease.


Neurotrophin gene augmentation by electrotransfer to improve cochlear implant hearing outcomes.
This Review outlines the development of DNA-based therapeutics for treatment of hearing loss, and in particular, considers the potential to utilize the properties of recombinant neurotrophins to improve cochlear auditory (spiral ganglion) neuron survival and repair. This potential to reduce spiral ganglion neuron death and indeed re-grow the auditory nerve fibres has been the subject of considerable pre-clinical evaluation over decades with the view of improving the neural interface with cochlear implants. This provides the context for discussion about the development of a novel means of using cochlear implant electrode arrays for gene electrotransfer. Mesenchymal cells which line the cochlear perilymphatic compartment can be selectively transfected with (naked) plasmid DNA using array - based gene electrotransfer, termed 'close-field electroporation'. This technology is able to drive expression of brain derived neurotrophic factor (BDNF) in the deafened guinea pig model, causing re-growth of the spiral ganglion peripheral neurites towards the mesenchymla cells, and hence into close proximity with cochlear implant electrodes within scala tympani. This was associated with functional enhancement of the cochlear implant neural interface (lower neural recruitment thresholds and expanded dynamic range, measured using electrically - evoked auditory brainstem responses). The basis for the efficiency of close-field electroporation arises from the compression of the electric field in proximity to the ganged cochlear implant electrodes. The regions close to the array with highest field strength corresponded closely to the distribution of bioreporter cells (adherent human embryonic kidney (HEK293)) expressing green fluorescent reporter protein (GFP) following gene electrotransfer. The optimization of the gene electrotransfer parameters using this cell-based model correlated closely with in vitro and in vivo cochlear gene delivery outcomes. The migration of the cochlear implant electrode array-based gene electrotransfer platform towards a clinical trial for neurotrophin-based enhancement of cochlear implants is supported by availability of a novel regulatory compliant mini-plasmid DNA backbone (pFAR4; plasmid Free of Antibiotic Resistance v.4) which could be used to package a 'humanized' neurotrophin expression cassette. A reporter cassette packaged into pFAR4 produced prominent GFP expression in the guinea pig basal turn perilymphatic scalae. More broadly, close-field gene electrotransfer may lend itself to a spectrum of potential DNA therapeutics applications benefitting from titratable, localised, delivery of naked DNA, for gene augmentation, targeted gene regulation, or gene substitution strategies.


Do older patients and their family caregivers agree about the quality of chronic illness care?
Family caregivers often accompany patients to medical visits; however, it is unclear whether caregivers rate the quality of patients' care similarly to patients. This study aimed to (1) quantify the level of agreement between patients' and caregivers' reports on the quality of patients' care and (2) determine how the level of agreement varies by caregiver and patient characteristics. Cross-sectional analysis. Multimorbid older (aged 65 and above) adults and their family caregivers (n = 247). Quality of care was rated separately by patients and their caregivers using the Patient Assessment of Chronic Illness Care (PACIC) instrument. The level of agreement was examined using a weighted kappa statistic (Kw). Agreement of caregivers' and patients' PACIC scores was low (Kw = 0.15). Patients taking ten or more medications per day showed less agreement with their caregivers about the quality of care than patients taking five or fewer medications (Kw = 0.03 and 0.34, respectively, P < 0.05). Caregivers who reported greater difficulty assisting patients with health care tasks had less agreement with patients about the quality of care being provided when compared with caregivers who reported no difficulty (Kw = -0.05 and 0.31, respectively, P < .05). Patient-caregiver dyads had greater agreement on objective questions than on subjective questions (Kw = 0.25 and 0.15, respectively, P > 0.05). Patient-caregiver dyads following a more complex treatment plan (i.e. taking many medications) or having more difficulty following a treatment plan (i.e. having difficulty with health care tasks) had less agreement. Future qualitative research is needed to elucidate the underlying reasons patients and caregivers rate the quality of care differently.


HCF-CRS: A Hybrid Content based Fuzzy Conformal Recommender System for providing recommendations with confidence.
A Recommender System (RS) is an intelligent system that assists users in finding the items of their interest (e.g. books, movies, music) by preventing them to go through huge piles of data available online. In an effort to overcome the data sparsity issue in recommender systems, this research incorporates a content based filtering technique with fuzzy inference system and a conformal prediction approach introducing a new framework called Hybrid Content based Fuzzy Conformal Recommender System (HCF-CRS). The proposed framework is implemented to be used in the domain of movies and it provides quality recommendations to users with a confidence level and an improved accuracy. In our proposed framework, first, a Content Based Filtering (CBF) technique is applied to create a user profile by considering the history of each user. CBF is useful in the situations like: lack of demographic information and the data sparsity problems. Second, a Fuzzy based technique is incorporated to find the similarities and differences between the user profile and the movies in the dataset using a set of fuzzy rules to get a predicted rating for each movie. Third, a Conformal prediction algorithm is implemented to calculate the non-conformity measure between the predicted ratings produced by fuzzy system and the actual ratings from the dataset. A p-value (confidence measure) is computed to give a level of confidence to each recommended item and a bound is set on the confidence level called a significance level ε, according to which the movies only above the specified significance level are recommended to user. By building a confidence centric hybrid conformal recommender system using the content based filtering approach with fuzzy logic and conformal prediction algorithm, the reliability and the accuracy of the system is considerably enhanced. The experiments are evaluated on MovieLens and Movie Tweetings datasets for recommending movies to the users and they are compared with other state-of-the-art recommender systems. Finally, the results confirm that the proposed algorithms perform better than the traditional ones.


Host specific diversity in Lactobacillus johnsonii as evidenced by a major chromosomal inversion and phage resistance mechanisms.
Genetic diversity and genomic rearrangements are a driving force in bacterial evolution and niche adaptation. We sequenced and annotated the genome of Lactobacillus johnsonii DPC6026, a strain isolated from the porcine intestinal tract. Although the genome of DPC6026 is similar in size (1.97 mbp) and GC content (34.8%) to the sequenced human isolate L. johnsonii NCC 533, a large symmetrical inversion of approximately 750 kb differentiated the two strains. Comparative analysis among 12 other strains of L. johnsonii including 8 porcine, 3 human and 1 poultry isolate indicated that the genome architecture found in DPC6026 is more common within the species than that of NCC 533. Furthermore a number of unique features were annotated in DPC6026, some of which are likely to have been acquired by horizontal gene transfer (HGT) and contribute to protection against phage infection. A putative type III restriction-modification system was identified, as were novel Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) elements. Interestingly, these particular elements are not widely distributed among L. johnsonii strains. Taken together these data suggest intra-species genomic rearrangements and significant genetic diversity within the L. johnsonii species and indicate towards a host-specific divergence of L. johnsonii strains with respect to genome inversion and phage exposure.


Assignment of the structural gene for the third component of human complement to chromosome 19.
The third component of complement (C3) is synthesized and secreted by cultured human primary fibroblasts. A monoclonal antibody having specificity for an antigenic determinant carried by human but not mouse C3 was used to study the continued expression of human C3 in three panels of independently derived human-mouse somatic cell hybrids. Expression of the human product was shown to segregate with human chromosome 19 and with no other chromosome or group of chromosomes. A unique-sequence human genomic C3 DNA clone was isolated and used as a probe in DNA hybridization experiments with DNA prepared from appropriate human-mouse somatic cell hybrids to confirm assignment of the human C3 gene to chromosome 19.


Antibacterial activity and chemical characteristics of several Western Australian honeys compared to manuka honey and pasture honey.
The physicochemical parameters and antibacterial activity of 10 Western Australian (WA) and two comparator honeys were determined. Honeys showed a pH range of 4.0-4.7, colour range of 41.3-470.7 mAU, methylglyoxal levels ranging from 82.2 to 325.9 mg kg-1 and hydrogen peroxide levels after 2 h of 22.7-295.5 µM. Antibacterial activity was assessed by the disc diffusion assay, phenol equivalence assay, determination of minimum inhibitory and bactericidal concentrations and a time-kill assay. Activity was shown for all honeys by one or more method, however, activity varied according to which assay was used. Minimum inhibitory concentrations for WA honeys against 10 organisms ranged from 4.0 to >32.0% (w/v). Removal of hydrogen peroxide activity by catalase resulted in decreased activity for several honeys. Overall, the data showed that honeys in addition to those derived from Leptospermum spp. have antimicrobial activity and should not be overlooked as potential sources of clinically useful honey.


Identification of histidine 105 in the beta1 subunit of soluble guanylate cyclase as the heme proximal ligand.
Soluble guanylate cyclase isolated from bovine and rat lung is a heterodimeric hemoprotein composed of alpha1 and beta1 subunits. The heme binding region has been localized to residues 1-385 of the beta1 subunit [beta1(1-385)], while the catalytic site(s) have been localized to the C-terminal region of sGC. There are four conserved histidine residues in the heme binding region of sGC. H220 and H346 are conserved among all known sGC subunits (alpha and beta), while H105 and H134 are conserved only in the beta subunits (beta1 and beta2). Site-directed mutagenesis was used to individually change each of the conserved histidines in sGC beta1(1-385) to alanine or glycine, and the resulting mutants were expressed in E. coli. All of the mutants except for H105A and H105G had heme bound as isolated. Imidazole (Im) was able to rescue heme binding to H105G when added to the growth medium and purification buffers. The heme in H105G isolated in the presence of imidazole [H105G(Im)] was ferric and a mixture of 5-coordinate, high-spin and 6-coordinate, low-spin complexes. After reduction, the ferrous heme in H105G(Im) was 5-coordinate, high-spin as indicated by resonance Raman spectroscopy. When imidazole in H105G(Im) was exchanged with N-methylimidazole (MeIm), the Fe-N(Im/MeIm) stretching frequency was shifted from 221 to 212 cm-1. A shift of this magnitude is expected when the ligand is directly coordinated to the heme iron. All of the data are consistent with the conclusion that H105 in the beta1 subunit is the heme proximal ligand.


Transient down-regulation of androgen receptor messenger ribonucleic acid (mRNA) expression in Sertoli cells by follicle-stimulating hormone is followed by up-regulation of androgen receptor mRNA and protein.
Cooperative actions of FSH and androgens on initiation, maintenance, and restoration of spermatogenesis have been described. In the present experiments the regulatory effects of FSH on androgen receptor (AR) gene expression in Sertoli cells were studied. In immature rats injection of FSH (1 microgram/g BW, ip) resulted in a rapid down-regulation of testicular AR mRNA expression (4 h), followed by recovery to the control level (10 h). Using cultured immature Sertoli cells, a similar transient effect on AR mRNA expression was observed after the addition of FSH (500 ng/ml) or (Bu)2cAMP (0.5 mM). Cycloheximide treatment of the cells did not prevent the rapid FSH-induced down-regulation of AR mRNA expression, indicating that de novo protein synthesis is not required for this effect. Furthermore, using a transcriptional run-on assay, no marked decrease in the rate of AR gene transcription was found upon treatment of the cultured Sertoli cells with FSH for 2 or 4 h. This demonstrates that the short term effect of FSH or AR mRNA expression reflects a change in mRNA stability. The AR protein level was not markedly affected by the transient decrease in AR mRNA expression. When immature Sertoli cells were incubated with FSH for longer time periods (24-72 h), both AR mRNA and protein expression were increased. In Sertoli cells isolated from 15-day-old rats, this increase was higher (mRNA, 2- to 3-fold; protein, 2-fold) than in Sertoli cells isolated from 25-day-old animals. The results indicate that FSH plays a complex role in the regulation of AR expression in immature rat Sertoli cells.


In vivo MR acoustic radiation force imaging in the porcine liver.
High intensity focused ultrasound (HIFU) in the abdomen can be sensitive to acoustic aberrations that can exist in the beam path of a single sonication. Having an accurate method to quickly visualize the transducer focus without damaging tissue could assist with executing the treatment plan accurately and predicting these changes and obstacles. By identifying these obstacles, MR acoustic radiation force imaging (MR-ARFI) provides a reliable method for visualizing the transducer focus quickly without damaging tissue and allows accurate execution of the treatment plan. MR-ARFI was used to view the HIFU focus, using a gated spin echo flyback readout-segmented echo-planar imaging sequence. HIFU spots in a phantom and in the livers of five live pigs under general anesthesia were created with a 550 kHz extracorporeal phased array transducer initially localized with a phase-dithered MR-tracking sequence to locate microcoils embedded in the transducer. MR-ARFI spots were visualized, observing the change of focal displacement and ease of steering. Finally, MR-ARFI was implemented as the principle liver HIFU calibration system, and MR-ARFI measurements of the focal location relative to the thermal ablation location in breath-hold and breathing experiments were performed. Measuring focal displacement with MR-ARFI was achieved in the phantom and in vivo liver. In one in vivo experiment, where MR-ARFI images were acquired repeatedly at the same location with different powers, the displacement had a linear relationship with power [y = 0.04x + 0.83 μm (R(2) = 0.96)]. In another experiment, the displacement images depicted the electronic steering of the focus inside the liver. With the new calibration system, the target focal location before thermal ablation was successfully verified. The entire calibration protocol delivered 20.2 J of energy to the animal (compared to greater than 800 J for a test thermal ablation). ARFI displacement maps were compared with thermal ablations during seven breath-hold ablations. The error was 0.83 ± 0.38 mm in the S/I direction and 0.99 ± 0.45 mm in the L/R direction. For six spots in breathing ablations, the mean error in the nonrespiration direction was 1.02 ± 0.89 mm. MR-ARFI has the potential to improve free-breathing plan execution accuracy compared to current calibration and acoustic beam adjustment practices. Gating the acquisition allows for visualization of the focal spot over the course of respiratory motion, while also being insensitive to motion effects that can complicate a thermal test spot. That MR-ARFI measures a mechanical property at the focus also makes it insensitive to high perfusion, of particular importance to highly perfused organs such as the liver.


Factor XII promotes blood coagulation independent of factor XI in the presence of long-chain polyphosphates.
Inorganic polyphosphates (polyP), which are secreted by activated platelets (short-chain polyP) and accumulate in some bacteria (long-chain polyP), support the contact activation of factor XII (FXII) and accelerate the activation of FXI. The aim of the present study was to evaluate the role of FXI in polyP-mediated coagulation activation and experimental thrombus formation. Pretreatment of plasma with antibodies that selectively inhibit FXI activation by activated FXII (FXIIa) or FIX) activation by activated FXI (FXIa) were not able to inhibit the procoagulant effect of long or short-chain polyP in plasma. In contrast, the FXIIa inhibitor, corn trypsin inhibitor, blocked the procoagulant effect of long and short polyP in plasma. In a purified system, long polyP significantly enhanced the rate of FXII and prekallikrein activation and the activation of FXI by thrombin but not by FXIIa. In FXI-deficient plasma, long polyP promoted clotting of plasma in an FIX-dependent manner. In a purified system, the activation of FXII and prekallikrein by long polyP promoted FIX activation and prothombin activation. In an ex vivo model of occlusive thrombus formation, inhibition of FXIIa with corn trypsin inhibitor but not of FXI with a neutralizing antibodies abolished the prothrombotic effect of long polyP. We propose that long polyP promotes FXII-mediated blood coagulation bypassing FXI. Accordingly, some polyp-containing pathogens may have evolved strategies to exploit polyP-initiated FXII activation for virulence, and selective inhibition of FXII may improve the host response to pathogens.


Systematic review of outcome of downstaging hepatocellular cancer before liver transplantation in patients outside the Milan criteria.
The aim of this systematic review was to assess the evidence on tumour downstaging before liver transplantation in patients with hepatocellular carcinoma (HCC) initially staged beyond the Milan criteria. MEDLINE (from 1952), Embase (from 1980) and the Cochrane Library were searched. The review included cohort studies that reported the outcomes of patients with HCC outside the Milan criteria who underwent downstaging before transplantation. Eight studies met the inclusion criteria and included a total of 720 patients who underwent transplantation following downstaging after initial presentation with disease outside the Milan criteria. The rate of successful downstaging varied from 24 to 69 per cent of patients. Reported survival rates ranged from 82 to 100 per cent, 79 to 100 per cent and 54·6 to 94 per cent at 1, 3 and 5 years respectively. These were comparable with results for patients presenting within the Milan criteria. Successful downstaging of HCC to within the Milan criteria is feasible in a proportion of patients. Absolute and disease-free survival rates in patients transplanted following downstaging are comparable to those in patients within the Milan criteria.


Effect of lime juice on Vibrio parahaemolyticus and Salmonella enterica inactivation during the preparation of the raw fish dish ceviche.
Ceviche is a raw fish dish common in Peru and other Latin American counties. The most characteristic feature of ceviche is the use of lime juice for marinating or "cooking" the raw fish. Confirmed cases of cholera in Peru, New Jersey, and Florida have been associated with ceviche. Although the effect of organic acids on pathogenic bacteria has been well characterized, few data exist on the effect of these acids in seafood systems. The objective of the study was to evaluate the effects of lime juice marination on pathogens likely to be present in ceviche. Tilapia (Oreochromis niloticus) fillet pieces were inoculated with Vibrio parahaemolyticus and Salmonella enterica (>7 log CFU/g) and incubated at 25 and 4°C for 30 or 120 min in the presence of fresh lime juice at concentrations typical for the preparation of ceviche. Similar levels of cells were also inoculated into fresh lime juice without tilapia. Surviving cells were enumerated on selective (xylose lysine Tergitol 4 and thiosulfate-bile-citrate-sucrose) and nonselective (tryptic soy agar) media. V. parahaemolyticus levels were reduced to below detection limits (∼5-log reduction) under all conditions studied. Salmonella strains on tilapia were much more resistant to inactivation and were only slightly reduced (∼1- to 2-log reduction). Salmonella and V. parahaemolyticus inoculated directly into lime juice without tilapia were all reduced to below detection limits (∼5-log reduction). A typical ceviche recipe reduces V. parahaemolyticus risk significantly but is less effective for control of S. enterica.


The cohesin subunit Rad21 is a negative regulator of hematopoietic self-renewal through epigenetic repression of Hoxa7 and Hoxa9.
Acute myelogenous leukemia (AML) is a high-risk hematopoietic malignancy caused by a variety of mutations, including genes encoding the cohesin complex. Recent studies have demonstrated that reduction in cohesin complex levels leads to enhanced self-renewal in hematopoietic stem and progenitors (HSPCs). We sought to delineate the molecular mechanisms by which cohesin mutations promote enhanced HSPC self-renewal as this represents a critical initial step during leukemic transformation. We verified that RNAi against the cohesin subunit Rad21 causes enhanced self-renewal of HSPCs in vitro through derepression of polycomb repressive complex 2 (PRC2) target genes, including Hoxa7 and Hoxa9. Importantly, knockdown of either Hoxa7 or Hoxa9 suppressed self-renewal, implying that both are critical downstream effectors of reduced cohesin levels. We further demonstrate that the cohesin and PRC2 complexes interact and are bound in close proximity to Hoxa7 and Hoxa9. Rad21 depletion resulted in decreased levels of H3K27me3 at the Hoxa7 and Hoxa9 promoters, consistent with Rad21 being critical to proper gene silencing by recruiting the PRC2 complex. Our data demonstrates that the cohesin complex regulates PRC2 targeting to silence Hoxa7 and Hoxa9 and negatively regulate self-renewal. Our studies identify a novel epigenetic mechanism underlying leukemogenesis in AML patients with cohesin mutations.