Changes in the distribution of LFA-1, catenins, and F-actin during transendothelial migration of monocytes in culture.
To determine changes in the spatial and temporal distribution of cell-cell adhesion molecules during transendothelial migration of monocytes, we examined an in vitro model system of diapedesis using high resolution laser scanning confocal microscopy. Human arterial endothelial cells were cultured to confluence on coverslips coated with Matrigel and activated with IL-1beta before the addition of monocytic THP-1 cells. Seventy per cent of monocytes transmigrated through the endothelium within one hour. Diapedesis, but not adhesion and spreading, was inhibited 8-fold in co-cultures that contained endothelial cell conditioned medium, suggesting the release of an endothelial derived inhibitor. Double immunofluorescence labeling with antibodies to LFA-1, alpha- and beta-catenin, VE-cadherin and with Texas Red phalloidin, identified a circular transmigration passage in endothelial cell-cell contact regions. This passage was formed by an LFA-1-containing pseudopodium that penetrated between endothelial cells. Apical to the transmigration passage, monocytes remained round in shape, while underneath the endothelium, they spread along the Matrigel. The margins of the transmigration passage contained high levels of LFA-1 and F-actin, suggesting a major role of these molecules during the transmigration process itself. Endothelial adherens junctions, as judged by the presence of VE-cadherin and alpha-catenin adjacent to the passage, remained intact during diapedesis. The presence of catenins at heterotypic contact regions between monocytes and endothelial cells during diapedesis suggested cadherin-mediated interactions between the two cell types. These results reveal dynamic changes in the distribution of adhesion molecules and the actin cytoskeleton during monocyte transendothelial migration in culture.


Assessment of pharmacokinetics and tolerability of intranasal diazepam relative to rectal gel in healthy adults.
Diazepam rectal gel (RG) is currently the only approved rescue therapy for outpatient management of seizure clusters in the United States. There is an unmet medical need for an alternative rescue therapy for seizure clusters that is effective, and more convenient to administer with a socially acceptable method of delivery. An intranasal diazepam formulation has been developed, and this study evaluates the tolerability and bioavailability of diazepam nasal spray (NS) relative to an equivalent dose of diazepam-RG in healthy adults. Twenty-four healthy adults were enrolled in a phase 1, open-label, 3-period crossover study. Plasma diazepam and metabolite concentrations were measured by serial sampling. Dose proportionality for 5- and 20-mg intranasal doses and the bioavailability of 20mg diazepam-NS relative to 20mg diazepam-RG were assessed by maximum plasma concentration (Cmax) and systemic exposure parameters (AUC0-∞ and AUC0-24). The mean Cmax values for 20mg diazepam-NS and 20mg diazepam-RG were 378 ± 106 and 328 ± 152 ng/mL, achieved at 1.0 and 1.5h, respectively. Subjects administered intranasal and rectal gel formulations experienced nasal and rectal leakage, respectively. Diazepam absorption following intranasal administration was consistent but 3 subjects with diazepam-RG had low plasma drug levels at the earliest assessment of 5 min, due to poor retention, and were excluded from analysis. Excluding them, the treatment ratios (20mg diazepam-NS:20mg diazepam-RG) and 90% confidence intervals for diazepam Cmax and AUC0-24 were 0.98 (0.85-1.14) and 0.89 (0.80-0.98), respectively, suggesting that the bioavailability was comparable between the two formulations. Dose proportionality was observed between the lowest and highest dose-strengths of intranasal formulation. Both intranasal and rectal treatments were well tolerated with mild to moderate adverse events. Results suggest that a single-dose of 20mg diazepam-NS is tolerable and comparable in bioavailability to that of diazepam-RG. The intranasal formulation may provide caregivers and patients with a more socially acceptable and convenient alternative rescue therapy in the acute treatment of seizure clusters.


Human liver stem cells improve liver injury in a model of fulminant liver failure.
Liver transplantation is currently the only effective therapy for fulminant liver failure, but its use is limited by the scarcity of organs for transplantation, high costs, and lifelong immunosuppression. Here we investigated whether human liver stem cells (HLSCs) protect from death in a lethal model of fulminant liver failure induced by intraperitoneal injection of D-galactosamine and lipopolysaccharide in SCID mice. We show that injection of HLSCs and of HLSC-conditioned medium (CM) significantly attenuates mouse mortality in this model. Histopathological analysis of liver tissue showed reduction of liver apoptosis and enhancement of liver regeneration. By optical imaging we observed a preferential localization of labeled HLSCs within the liver. HLSCs were detected by immunohistochemistry in large liver vessels (at 24 hours) and in the liver parenchyma (after day 3). Fluorescence in situ hybridization analysis with the human pan-centromeric probe showed that positive cells were cytokeratin-negative at 24 hours. Coexpression of cytokeratin and human chromosome was observed at 7 and, to a lesser extent, at 21 days. HLSC-derived CM mimicked the effect of HLSCs in vivo. Composition analysis of the HLSC-CM revealed the presence of growth factors and cytokines with liver regenerative properties. In vitro experiments showed that HLSC-CM protected human hepatocytes from apoptosis and enhanced their proliferation. These data suggest that fulminant liver failure may potentially benefit from treatment with HLSCs or HLSC-CM.


Optimization of Optoelectronic Properties of Patterned Single-Walled Carbon Nanotube Films.
We propose a novel strategy to enhance optoelectrical properties of single-walled carbon nanotube (SWCNT) films for transparent electrode applications by film patterning. First, we theoretically considered the effect of the conducting pattern geometry on the film quality factor and then experimentally examined the calculated structures. We extend these results to show that the best characteristics of patterned SWCNT films can be achieved using the combination of initial film properties: low transmittance and high conductivity. The proposed strategy allows the patterned layers of SWCNTs to outperform the widely used indium-tin-oxide electrodes on both flexible and rigid substrates.


Requirement of the zinc-binding domain of ClpX for Spx proteolysis in Bacillus subtilis and effects of disulfide stress on ClpXP activity.
Spx, a transcriptional regulator of the disulfide stress response in Bacillus subtilis, is under the proteolytic control of the ATP-dependent protease ClpXP. Previous studies suggested that ClpXP activity is down-regulated in response to disulfide stress, resulting in elevated concentrations of Spx. The effect of disulfide stress on ClpXP activity was examined using the thiol-specific oxidant diamide. ClpXP-catalyzed degradation of either Spx or a green fluorescent protein derivative bearing an SsrA tag recognized by ClpXP was inhibited by diamide treatment in vitro. Spx is also a substrate for MecA/ClpCP-catalyzed proteolysis in vitro, but diamide used at the concentrations that inhibited ClpXP had little observable effect on MecA/ClpCP activity. ClpX bears a Cys4 Zn-binding domain (ZBD), which in other Zn-binding proteins is vulnerable to thiol-reactive electrophiles. Diamide treatment caused partial release of Zn from ClpX and the formation of high-molecular-weight species, as observed by electrophoresis through nonreducing gels. Reduced Spx proteolysis in vitro and elevated Spx concentration in vivo resulted when two of the Zn-coordinating Cys residues of the ClpX ZBD were changed to Ser. This was reflected in enhanced Spx activity in both transcription activation and repression in cells expressing the Cys-to-Ser mutants. ClpXP activity in vivo is reduced when cells are exposed to diamide, as shown by the enhanced stability of an SsrA-tagged protein after treatment with the oxidant. The results are consistent with the hypothesis that inhibition of ClpXP by disulfide stress is due to structural changes to the N-terminal ZBD of ClpX.


Ibrutinib for the treatment of chronic lymphocytic leukemia and mantle cell lymphoma.
Ibrutinib is a novel oral tyrosine kinase inhibitor that irreversibly binds and inhibits tyrosine-protein kinase BTK (Bruton tyrosine kinase). BTK has been found to be important in the function of B-cell receptor signaling and therefore in the maintenance and expansion of various B-cell malignancies including chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). Targeting BTK with ibrutinib has been found to be an effective strategy in treating these malignancies. Phase I clinical testing in non-Hodgkin's lymphomas and CLL showed that the drug was extremely well tolerated with no major dose-limiting toxicities and a 54% overall response rate. Subsequently, two phase Ib/II studies were performed on patients with CLL, one in relapsed/refractory CLL and one in previously untreated elderly patients with CLL. Both of these studies continued to show good tolerability of the drug and an overall response rate of about 71% with extended duration of response. Another phase II study using ibrutinib in relapsed/refractory MCL was conducted and also showed that it was well tolerated with an overall response rate of 68% and extended duration of response. Due to these results, the U.S. Food and Drug Administration granted accelerated approval for ibrutinib in November 2013 for patients with MCL who had received at least one prior therapy and in February 2014 for patients with CLL who had received at least one prior therapy. This review will discuss the preclinical pharmacology, pharmacokinetics and clinical efficacy to date of ibrutinib in the treatment of CLL and MCL.


Bone marrow chimeras-a vital tool in basic and translational research.
Bone marrow chimeras are used routinely in immunology research as well as in other fields of biology. Here, we provide a concise state-of-the-art review about the types of chimerisms that can be achieved and the type of information that each model generates. We include separate sections for caveats and future developments. We provide examples from the literature in which different types of chimerism were employed to answer specific questions. While simple bone marrow chimeras allow to dissect the role of genes in distinct cell populations such as the hematopoietic cells versus non-hematopoietic cells, mixed bone marrow chimeras can provide detailed information about hematopoietic cell types and the intrinsic and extrinsic roles of individual genes. The advantages and caveats of bone marrow chimerism for the study of microglia are addressed, as well as alternatives to irradiation that minimize blood-brain-barrier disruption. Elementary principles are introduced and their potential is exemplified through summarizing recent studies.


The design and conduct of Keep It Off: An online randomized trial of financial incentives for weight-loss maintenance.
Background Obesity continues to be a serious public health challenge. Rates are increasing worldwide, with nearly 70% of the US adults overweight or obese, leading to increased clinical and economic burden. While successful approaches for achieving weight loss have been identified, techniques for long-term maintenance of initial weight loss have largely been unsuccessful. Financial incentive interventions have been shown in several settings to be successful in motivating participants to adopt healthy behaviors. Purpose Keep It Off is a three-arm randomized controlled trial that compares the efficacy of a lottery-based incentive, traditional direct payment incentive, and control of daily feedback without any incentive for weight-loss maintenance. This design allows comparison of a traditional direct payment incentive with one based on behavioral economic principles that consider the underlying psychology of decision-making. Methods Participants were randomized in a 2:1 ratio for each active arm relative to control, with a targeted 188 participants in total. Eligible participants were those aged 30-80 who lost at least 11 lb (5 kg) during the first 4 months of participation in Weight Watchers, a national weight-loss program, with whom we partnered. The interventions lasted 6 months (Phase I); participants were followed for an additional 6 months without intervention (Phase II). The primary outcome is weight change from baseline to the end of Phase I, with the change at the end of Phase II a key secondary endpoint. Keep It Off is a pragmatic trial that recruited, consented, enrolled, and followed patients electronically. Participants were provided a wireless weight scale that electronically transmitted daily self-monitored weights. Weights were verified every 3 months at a Weight Watchers center local to the participant and electronically transmitted. Results Using the study web-based platform, we integrated recruitment, enrollment, and follow-up procedures into a digital platform that required little staff effort to implement and manage. We randomized 191 participants in less than 1 year. We describe the design of Keep It Off and implementation of enrollment. Lessons Learned We demonstrated that our pragmatic design was successful in rapid accrual of participants in a trial of interventions to maintain weight loss. Limitations Despite the nationwide reach of Weight Watchers, the generalizability of study findings may be limited by the characteristics of its members. The interventions under study are appropriate for settings where an entity, such as an employer or health insurance company, could offer them as a benefit. Conclusions Keep It Off was implemented and conducted with minimal staff effort. This study has the potential to identify a practical and effective weight-loss maintenance strategy.


Pathways to renal biopsy and diagnosis among patients with ANCA small-vessel vasculitis.
Antineutrophil cytoplasmic antibody small-vessel vasculitis (ANCA-SVV) is an autoimmune systemic process increasingly recogniSed since the advent of antibody testing for the disease. Prompt diagnosis and institution of immunosuppressive therapy has been shown to improve patient outcome. The goal of this study was to better understand how patients navigate the health care system from symptom presentation to biopsy diagnosis, and to study the effects of prompt versus delayed diagnosis. Disease symptoms and number of physicians seen prior to renal biopsy were assessed for 127 ANCA-SVV patients. Direct, delayed, and quest pathways to diagnosis and treatment of vasculitis were defined for both patients and providers. Kruskal-Wallis and Fisher exact tests were used to evaluate continual measures and compare categorical variables across pathways. Among patients who sought direct care, physician delay in referral to a nephrologist was common (49/127, 71%, p=0.0023). Patients who delayed seeking care also experienced a delayed diagnosis 57% of the time (p=0.0023). Patients presenting with prodromal flu or upper respiratory involvement were more likely to have a delay/quest patient pathway (56% and 55%, respectively) than a direct patient pathway (44%, p=0.033 and 45%, p=0.019, respectively). There was a trend for patients with more severe loss of renal function to have a more direct referral to a nephrologist. Delay in diagnosis of ANCA SVV may be due to lack of or non-specific symptoms, especially in patients who present with non-renal manifestations of disease. Better algorithms are needed to identify extra-renal manifestations, expedite diagnosis and improve patient outcomes.


Cardiovascular adaptations of pregnancy in T and B cell-deficient mice.
The pathophysiology of gestational hypertensive disorders is incompletely defined. T lymphocytes are implicated. Both T and natural killer (NK) cells express RAS and, in implantation sites, NK cells are highly enriched. We hypothesized that T cells and/or NK cells contribute to circulatory control during pregnancy. Using radiotelemetry of arterial pressure, heart rate, and activity, mice without T and B cells (genotypes BALB/c-Rag2(-/-) and NOD.scid) were examined at baseline and across pregnancy. These strains differ in NK cell competency, with Rag2(-/-) being normal and NOD.scid impaired. Circulatory features differed between these inbred strains. Rag2(-/-); had blood pressure responses to pregnancy that did not differ from congenic normal mice. NOD.scid had higher midgestational blood pressure compared with normoglycemic NOD mice (3-5 mm Hg greater than NOD; P < 0.004). In comparison to controls, both T and B strains had much higher heart rates after first trimester that did not remit until parturition (>30 bpm greater than control; P < 0.0001). NOD.scid had additional anomalies, including 90% depletion of circulating NK cells and elevated (57%) proliferation of uterine NK cells within implantation sites. These data demonstrate immune control of midgestational heart rate and suggest NK cells contribute to midpregnancy regulation of mean arterial pressure.


Ruptured hepatic hydatid cyst and an unusual case of pseudomonas septicaemia.
Pseudomonas aeruginosa is a very rare cause of septicaemia in adults without significant underlying disease. A case of pseudomonas septicaemia occurring as a complication of a ruptured hydatid cyst in an apparently healthy young soldier is presented. This patient also had unusual pulmonary sequelae resembling pulmonary metastases together with massive haemoptysis.


Molecular analysis of the 21-kb bacteriocin-encoding plasmid pEF1 from Enterococcus faecium 6T1a.
The complete 21,344-bp DNA sequence of the bacteriocin-encoding plasmid pEF1 from Enterococcus faecium 6T1a was determined. Thirty-four putative open reading frames which could code for proteins longer than 42 amino acids were found. Those included the structural genes encoding for the previously described bacteriocins enterocin I and J (also named as enterocins L50A and L50B). After comparison to sequences in public databases, analysis of the gene organization of pEF1 suggests a modular structure with three different functional domains: the replication region, the bacteriocin region and the mobilization plus UV-resistance region. This genetic mosaic structure most probably evolved through recombination events promoted by transposable elements. The hypothesis that the bacteriocin cluster on pEF1 could act as a functional plasmid stabilization module in E. faecium 6T1a is discussed.


The Bystander Effect of Ultraviolet Radiation and Mediators.
A bystander effect is biological changes in non-irradiated cells by transmitted signals from irradiated bystander cells, which causes the radiation toxic effects on the adjacent non-irradiated tissues. This phenomenon occurs by agents such as ionizing radiation, ultraviolet radiation (UVR) and chemotherapy. The bystander effect includes biological processes such as damage to DNA, cell death, chromosomal abnormalities, delay and premature mutations and micronuclei production. The most involved genes in creating this phenomenon are cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), the nuclear factor of kappa B (NFkB) and Mitogen-Activated Protein Kinases (MAPKs). Radiation generated reactive oxygen species (ROS) can damage DNA, membranes and protein buildings. Studies have shown that Vitamin C, Hesperidin, and melatonin can reduce the number of ROS and have a protective role. Silver nanoparticles (Ag NPs) are the most abundant nanoparticles produced and when they enter cells, they can create DNA damage. Studies have shown that combined treatment with UVR and silver nanoparticles could form γ-H2AX and 8-hydroxy-2'-deoxyguanosine (8-OHdG) synergistically. This article reviews the direct and the bystander effects of UVR on the nuclear DNA, the effect of radioprotectors and Ag NPs on these effects.


Capacity to increase walking speed is limited by impaired hip and ankle power generation in lower functioning persons post-stroke.
It is well known that stroke patients walk with reduced speed, but their potential to increase walking speed can also be impaired and has not been thoroughly investigated. We hypothesized that failure to effectively recruit both hip flexor and ankle plantarflexor muscles of the paretic side limits the potential to increase walking speed in lower functioning hemiparetic subjects. To test this hypothesis, we measured gait kinematics and kinetics of 12 persons with hemiparesis following stroke at self-selected and fast walking conditions. Two groups were identified: (1) lower functioning subjects (n=6) who increased normalized walking speed from 0.52 leg lengths/s (ll/s, SEM: 0.04) to 0.72 ll/s (SEM: 0.03) and (2) higher functioning subjects (n=6) who increased walking speed from 0.88 ll/s (SEM: 0.04) to 1.4 ll/s (SEM 0.03). Changes in spatiotemporal parameters, joint kinematics and kinetics between self-selected and fast walking were compared to control subjects examined at matched walking speeds (0.35 ll/s (SEM: 0.03), 0.63 ll/s (SEM: 0.03), 0.92 ll/s (SEM: 0.04) and 1.4 ll/s (SEM: 0.04)). Similar to speed-matched controls, the higher functioning hemiparetic subjects increased paretic limb hip flexion power and ankle plantarflexion power to increase walking speed. The lower functioning hemiparetic subjects did not increase power generation at the hip or ankle to increase walking speed. This observation suggests that impaired ankle power generation combined with saturation of hip power generation limits the potential to increase walking speed in lower functioning hemiparetic subjects.


Atypical tetanus in a completely immunized 14-year-old boy.
We report the uncommon clinical course of tetanus in a completely immunized 14-year-old boy. His initial symptoms, which included a flaccid paralysis, supported a diagnosis of botulism. Preliminary mouse-test results with combined botulinum antitoxins A, B, and E, obtained from tetanus-immunized horses, backed this diagnosis. The change in his clinical course from paralysis to rigor and the negative, more specific, botulinum mouse test with isolated botulinum antitoxins A, B, and E, obtained from nonvaccinated rabbits, disproved the diagnosis of botulism. Tetanus was suspected despite complete vaccination. The final results of a positive mouse test performed with isolated tetanus antitoxin confirmed the diagnosis. Adequate treatment was begun, and the boy recovered completely.


A generalized method for controlling end-tidal respiratory gases during nonsteady physiological conditions.
While forcing of end-tidal gases by regulating inspired gas concentrations is a common technique for studying cardiorespiratory physiology, independently controlling end-tidal gases is technically challenging. Feedforward control methods are challenging because end-tidal values vary as a dynamic function of both inspired gases and other nonregulated physiological parameters. Conventional feedback control is limited by delays within the lungs and body tissues and within the end-tidal forcing system itself. Consequently, modern end-tidal forcing studies have generally restricted their analysis to simple time courses of end-tidal gases and to resting steady-state conditions. To overcome these limitations, we have designed and validated a more generalized end-tidal forcing system that removes the need for manual tuning and rule-of-thumb based control heuristics, while allowing for accurate control of gases along spontaneous and complicated time courses and under nonsteady physiological conditions. On average during resting, steady walking, and walking with time varying speed, our system achieved step changes in PetCO2 within 3.0 ± 0.9 (mean ± SD) breaths and PetO2 within 4.4 ± 0.9 breaths, while also maintaining small steady-state errors of 0.1 ± 0.2 mmHg for PetCO2 and 0.3 ± 0.8 mmHg for PetO2 The system also accurately tracked more complicated changes in end-tidal values through a bandwidth of 1/10 the respiratory (sampling) frequency, a practical limit of feedback control systems. The primary mechanism enabling this controller performance is a generic mathematical model of the cardiopulmonary system that captures the breath-by-breath relationship between inspired and end-tidal gas concentrations, with secondary contributions from reduced delays in controlled air delivery.


Non-invasive means to study the functional status of sinusoidal liver endothelial cells.
Circulating connective tissue macromolecules are efficiently eliminated by receptor-mediated endocytosis in liver endothelial cells (LEC). In patients with a seriously diseased liver, for example in liver cirrhosis or when a transplanted liver is about to be rejected, dysfunctional LEC or altered blood perfusion through the liver results in significantly increased serum levels of connective tissue macromolecules and other substances that are normally taken up by LEC. With the aid of high affinity antibodies or other kinds of binding proteins it has been possible to measure a number of connective tissue macromolecules in the blood, and some of these substances have been used to diagnose serious liver disease. However, the metabolic patterns of these molecules are such that it is difficult to extrapolate directly on the basis of increased serum levels to conclude that the main underlying cause of increased serum levels is dysfunctional LEC. Elevated serum levels of many connective tissue proteins may reflect either increased synthesis, increased release from cell or tissue depots, and/or decreased removal by LEC. Therefore, to use measurement of serum connective tissue molecule levels as indicators of LEC function, it is imperative to know the catabolic routes of these substances in health and disease. Due to the fact that we presently know more about the total metabolic pattern of hyaluronan (HYA) than of other connective tissue macromolecules that are cleared solely by LEC, serum HYA should be preferentially used to monitor the function of LEC, in particular in liver transplantation and cirrhosis.


How and why weight stigma drives the obesity 'epidemic' and harms health.
In an era when obesity prevalence is high throughout much of the world, there is a correspondingly pervasive and strong culture of weight stigma. For example, representative studies show that some forms of weight discrimination are more prevalent even than discrimination based on race or ethnicity. In this Opinion article, we review compelling evidence that weight stigma is harmful to health, over and above objective body mass index. Weight stigma is prospectively related to heightened mortality and other chronic diseases and conditions. Most ironically, it actually begets heightened risk of obesity through multiple obesogenic pathways. Weight stigma is particularly prevalent and detrimental in healthcare settings, with documented high levels of 'anti-fat' bias in healthcare providers, patients with obesity receiving poorer care and having worse outcomes, and medical students with obesity reporting high levels of alcohol and substance use to cope with internalized weight stigma. In terms of solutions, the most effective and ethical approaches should be aimed at changing the behaviors and attitudes of those who stigmatize, rather than towards the targets of weight stigma. Medical training must address weight bias, training healthcare professionals about how it is perpetuated and on its potentially harmful effects on their patients. Weight stigma is likely to drive weight gain and poor health and thus should be eradicated. This effort can begin by training compassionate and knowledgeable healthcare providers who will deliver better care and ultimately lessen the negative effects of weight stigma.


A collagen-binding protein on the surface of ejaculated rabbit spermatozoa.
A rabbit antibody to mouse 3T3 cell fibronectin was used in conjunction with a fluorescein-tagged second antibody to detect fibronectin-like activity on the surface of rabbit spermatozoa. Only ejaculated sperm displayed an intense and highly localized fluorescence over the acrosomal region. Cauda epididymal sperm of the rabbit as well as several other species did not exhibit any reaction. The fluorescent activity could be eliminated by trypsin treatment but was re-established by incubation in cell-free seminal fluid. Sperm recovered from females 10-12 h after mating showed a reduction or absence of antifibronectin fluorescence, suggesting that this component's loss could be a factor in sperm capacitation. Because fibronectins show strong binding to collagen, mixtures of ejaculated sperm and collagen were examined in the light and electron microscope. Living sperm appear to have a strong affinity for collagen and quickly adhere to the filaments by their heads, while continuing vigorous flagellations. Surface labeling of sperm with the galactose-oxidase-NaB[3H]4 technique, extraction with urea-detergent mixtures and affinity chromatography of extracts on gelatin-Sepharose revealed a single radioactive band of mot wt approximately 40,000 after SDS polyacrylamide gel electrophoresis and fluorography.


NMR studies of lysozyme surface accessibility by using different paramagnetic relaxation probes.
Paramagnetic probes, whose approach to proteins can be monitored by nuclear magnetic resonance (NMR) studies, have been found to be of primary relevance for investigating protein surfaces' accessibility. Here, a Gd(III) neutral complex which contains two metal ions, [Gd2(L7)(H2O)2], is suggested as a paramagnetic probe particularly suited for systematic NMR investigation of protein surface accessibility, due to an expected high relaxivity and to the lack of electric charge which could favor specific interactions. Hen egg white lysozyme has been used as a model system to verify the absence of preferential approaches of this paramagnetic probe to specific protein moieties by comparing paramagnetic perturbation profiles of 1H-13C HSQC signals obtained in the presence of TEMPOL and [Gd2(L7)(H2O)2]. From the similarity of the measured paramagnetic perturbation profiles induced by the two different probes, specific interactions of [Gd2(L7)(H2O)2] with the enzyme could be ruled out. The large size of the latter probe is suggested to be responsible for the strong paramagnetic perturbations observed for CalphaH groups which are located in convex surface-exposed regions. The combined use of the two probes reveals fine details of the dynamics controlling their approach toward the protein surface.


Heparin-induced thrombocytopenia: an estimate of the average cost in the hospital setting in France.
Heparin-induced thrombocytopenia is a severe drug adverse effect with possible dramatic consequences. The risk is 0.1% to 5%. The costs of heparin-induced thrombocytopenia in France were estimated using the Programme Médicalisé des Systèmes d'Information (PMSI) national discharge database. Hospitalizations with heparin-induced thrombocytopenia were identified using diagnostic codes. Costs were assessed from the perspective of the French Sickness Fund or hospitals. Heparin-induced thrombocytopenia could be the reason of admission or could occur during the stay and lead to a different tariff or to additional costs associated with extra length of stay. Direct costs were also estimated from experts' opinions. A sensitivity analysis was performed from data collected in 1 center. During 2005, 445 hospitalizations with heparin-induced thrombocytopenia codes were identified. For 45 patients, the main diagnosis was heparin-induced thrombocytopenia; for the remaining 400 patients, heparin-induced thrombocytopenia occurred during the hospital stay. Tariffs and extra costs were used to estimate an overall average cost of 3230 for heparin-induced thrombocytopenia. For patients with heparin-induced thrombocytopenia as main diagnosis, the average cost was 3400; for the patients with heparin-induced thrombocytopenia that occurred during the stay, 1910 was due to an increased of the tariff and 3348 to an increased length of stay. Estimated direct costs of an episode were 3350 to 3700. Different methods were used to arrive at an estimated cost of 3500 for a heparin-induced thrombocytopenia episode for inpatients. One limitation of the study is that heparin-induced thrombocytopenia tends to be underreported by physicians during hospitalization.


Transformation of Solanum tuberosum plastids allows high expression levels of β-glucuronidase both in leaves and microtubers developed in vitro.
Plastid genome transformation offers an attractive methodology for transgene expression in plants, but for potato, only expression of gfp transgene (besides the selective gene aadA) has been published. We report here successful expression of β-glucuronidase in transplastomic Solanum tuberosum (var. Desiree) plants, with accumulation levels for the recombinant protein of up to 41% of total soluble protein in mature leaves. To our knowledge, this is the highest expression level reported for a heterologous protein in S. tuberosum. Accumulation of the recombinant protein in soil-grown minitubers was very low, as described in previous reports. Interestingly, microtubers developed in vitro showed higher accumulation of β-glucuronidase. As light exposure during their development could be the trigger for this high accumulation, we analyzed the effect of light on β-glucuronidase accumulation in transplastomic tubers. Exposure to light for 8 days increased β-glucuronidase accumulation in soil-grown tubers, acting as a light-inducible expression system for recombinant protein accumulation in tuber plastids. In this paper we show that plastid transformation in potato allows the highest recombinant protein accumulation in foliar tissue described so far for this food crop. We also demonstrate that in tubers high accumulation is possible and depends on light exposure. Because tubers have many advantages as protein storage organs, these results could lead to new recombinant protein production schemes based on potato.


Correlated electron dynamics in nonsequential double ionization by orthogonal two-color laser pulses.
We have investigated the correlated electron dynamics in nonsequential double ionization (NSDI) of helium by the orthogonally polarized two-color pulses that consisted of an 800-nm and a 400-nm laser fields using the classical ensemble model. Depending on the relative phase of the two-color field, the electron momentum distributions along the polarization direction of the 800-nm field exhibit a surprisingly strong anticorrelated or correlated behavior. Back analysis reveals that recollisions eventually leading to NSDI are concentrated in a time window as short as several hundreds attoseconds with this scheme. By changing the relative phase of the two-color field, the revisit time of recolliding electron wave packet has been controlled with attosecond precision, which is responsible for the various correlated behaviors of the two electrons. Our results reveal that the orthogonally polarized two-color field can serve as a powerful tool to control the correlated electron dynamics in NSDI.


Streptozotocin is equally diabetogenic whether administered to fed or fasted mice.
Streptozotocin (STZ) is a selective pancreatic β cell toxin used to generate experimental hyperglycemia in rodent models. Several laboratory animal protocols suggest that STZ be administered to fasted rodents to minimize competition between STZ and glucose for low affinity GLUT2 transporters on β cells. However, whether the diabetogenic effects of multiple low dose (MLD)-STZ administration are enhanced by fasting has not been addressed. Given that repeated bouts of fasting can cause undue metabolic stress in mice, we compared the efficacy of MLD-STZ injections (50 mg/kg body weight daily for 5 days) to induce experimental hyperglycemia in both NOD/SCID/γchain(null) and C57BL/6J mice that were either ad libitum fed (STZ-Fed) or that had been fasted for 6 h (STZ-Fasted) prior to the time of STZ administration. Both STZ-Fed and STZ-Fasted mice had significantly worse glucose tolerance than vehicle-treated control mice 10 days after initiation of the MLD-STZ regimen. In C57BL/6J mice, fasting glucose levels, serum insulin levels, β cell mass, and glucose disposal during intraperitoneal glucose tolerance tests (IPGTTs) were indistinguishable between STZ-Fed and STZ-Fasted mice 20 days after MLD-STZ. The glucose intolerant phenotypes persisted for 20 weeks thereafter, irrespective of whether C57BL/6J mice were fed or fasted at the time of STZ injections. However, STZ-Fasted C57BL/6J mice experienced significant weight loss during the repeated bouts of fasting/re-feeding that were required to complete the MLD-STZ protocol. In summary, induction of experimental hyperglycemia can be achieved using the MLD-STZ protocol without repeated bouts of fasting, which have the potential to cause metabolic stress in laboratory mice.


Mercury content of common foods determined by neutron activation analysis.
The mercury contents in samples of flour, sugar, nonfat dry milk, potatoes, hamburger, chicken breast, shrimp, liver, eggs, and whole milk were determined by neutron activation analysis. The mercury was separated by anion exchange chromatography and precipitated as the sulfide. The mercury concentrations for all these foods were below 50 parts per billion.


Diagnostic radiation exposure in pediatric trauma patients.
The amount of imaging studies performed for disease diagnosis has been rapidly increasing. We examined the amount of radiation exposure that pediatric trauma patients receive because they are an at-risk population. Our hypothesis was that pediatric trauma patients are exposed to high levels of radiation during a single hospital visit. Retrospective review of children who presented to Johns Hopkins Pediatric Trauma Center from July 1, 2004, to June 30, 2005. Radiographic studies were recorded for each patient and doses were calculated to give a total effective dose of radiation. All radiographic studies that each child received during evaluation, including any associated hospital admission, were included. A total of 945 children were evaluated during the study year. A total of 719 children were included in the analysis. Mean age was 7.8 (±4.6) years. Four thousand six hundred three radiographic studies were performed; 1,457 were computed tomography (CT) studies (31.7%). Average radiation dose was 12.8 (±12) mSv. We found that while CT accounted for only 31.7% of the radiologic studies performed, it accounted for 91% of the total radiation dose. Mean dose for admitted children was 17.9 (±13.8) mSv. Mean dose for discharged children was 8.4 (±7.8) mSv (p<0.0001). Burn injuries had the lowest radiation dose [1.2 (±2.6) mSv], whereas motor vehicle collision victims had the highest dose [18.8 (±14.7) mSv]. When the use of radiologic imaging is considered essential, cumulative radiation exposure can be high. In young children with relatively long life spans, the benefit of each imaging study and the cumulative radiation dose should be weighed against the long-term risks of increased exposure.


A glycan-phosphatidylinositol-specific phospholipase D in human serum.
A group of proteins anchored to the cell by phosphatidylinositol (PI) has recently been identified. The significance of this new class of membrane anchor is unknown; one possibility is that it facilitates release of the molecule by phospholipases. In fact, phospholipase C enzymes specific for the complex carboxyl-terminal glycolipids of these proteins have been isolated from African trypanosomes and from hepatocyte plasma membranes. This study reports the discovery of a glycan-PI-specific phospholipase D in human serum that cleaves both the membrane form of the variant surface glycoprotein of African trypanosomes and its glycolipid precursor, but not phosphatidylethanolamine, phosphatidylcholine, or phosphatidylinositol. Decay-accelerating factor, another PI-anchored molecule, is also cleaved by the enzyme and converted from a hydrophobic to a soluble protein. The enzyme is Ca2+-dependent, heat labile, and not affected by the inhibitor of serine proteases, phenylmethylsulfonylfluoride. Its function is not known, but the present findings indicate that it participates in the metabolism of glycolipid-anchored membrane proteins.


Transgenerational interactions between pesticide exposure and warming in a vector mosquito.
While transgenerational plasticity may buffer ectotherms to warming and pesticides separately, it remains unknown how combined exposure to warming and pesticides in the parental generation shapes the vulnerability to these stressors in the offspring. We studied the transgenerational effects of single and combined exposure to warming (4°C increase) and the pesticide chlorpyrifos on life-history traits of the vector mosquito Culex pipiens. Parental exposure to a single stressor, either warming or the pesticide, had negative effects on the offspring: parental exposure to both warming and the pesticide resulted in an overall lower offspring survival, and a delayed offspring metamorphosis. Parental exposure to a single stressor did, however, not alter the vulnerability of the offspring to the same stressor in terms of survival. Parental pesticide exposure resulted in larger offspring when the offspring experienced the same stressor as the parents. Within both the parental and offspring generations, warming made the pesticide more toxic in terms of survival. Yet, this synergism disappeared in the offspring of parents exposed to both stressors simultaneously because in this condition, the pesticide was already more lethal at the lower temperature. Our results indicate that transgenerational effects will not increase the ability of this vector species to deal with pesticides in a warming world. Bifactorial transgenerational experiments are crucial to understand the combined impact of warming and pesticides across generations, hence to assess the efficacy of vector control in a warming world.


Multiscale brain research on a microfluidic chip.
One major challenge in current brain research is generating an integrative understanding of the brain's functions and disorders from its multiscale neuronal architectures and connectivity. Thus, innovative neurotechnology tools are urgently required for deciphering the multiscale functional and structural organizations of the brain at hierarchical scales from the molecular to the organismal level by multiple brain research initiatives launched by the European Union, United States, Australia, Canada, China, Korea, and Japan. To meet this demand, microfluidic chips (μFCs) have rapidly evolved as a trans-scale neurotechnological toolset to enable multiscale studies of the brain due to their unique advantages in flexible microstructure design, multifunctional integration, accurate microenvironment control, and capacity for automatic sample processing. Here, we review the recent progress in applying innovative μFC-based neuro-technologies to promote multiscale brain research and uniquely focus on representative applications of μFCs to address challenges in brain research at each hierarchical level. We discuss the current trend of combinational applications of μFCs with other neuro- and biotechnologies, including optogenetics, brain organoids, and 3D bioprinting, for better multiscale brain research. In addition, we offer our insights into the existing outstanding questions at each hierarchical level of brain research that could potentially be addressed by advancing microfluidic techniques. This review will serve as a timely guide for bioengineers and neuroscientists to develop and apply μFC-based neuro-technologies for promoting basic and translational brain research.


Findings of metabolic bone disease in infants with unexplained fractures in contested child abuse investigations: a case series of 75 infants.
Background Infants who present with multiple unexplained fractures (MUF) are often diagnosed as victims of child abuse when parents deny wrongdoing and cannot provide a plausible alternative explanation. Herein we describe evidence of specific and commonly overlooked radiographic abnormalities and risk factors that suggest a medical explanation in such cases. Methods We evaluated such infants in which we reviewed the radiographs for signs of poor bone mineralization. We reviewed medical, pregnancy and family histories. Results Seventy-five of 78 cases showed poor bone mineralization with findings of healing rickets indicating susceptibility to fragility fractures that could result from a wide variety of causes other than child abuse. We found risk factors that could explain the poor bone mineralization: maternal and infant vitamin D deficiency (VDD), decreased fetal bone loading, prematurity and others. Most infants had more than one risk factor indicating that this bone disorder is a multifactorial disorder that we term metabolic bone disease of infancy (MBDI). Maternal and infant VDD were common. When tested, 1,25-dihydroxyvitamin D levels were often elevated, indicating metabolic bone disease. Conclusions Child abuse is sometimes incorrectly diagnosed in infants with MUF. Appreciation of the radiographic signs of MBDI (healing rickets), risk factors for MBDI and appropriate laboratory testing will improve diagnostic accuracy in these cases.


Structural and functional events on the porcine zona pellucida during maturation, fertilization and embryonic development: a scanning electron microscopy analysis.
Porcine oocytes and pre-implantation embryos from the same, as well as from different animals, have an extremely heterogeneous morphology of the zona pellucida (ZP) surface, as shown by scanning electron microscopy. For years, it has been believed that this heterogeneous morphology plays an important role in the sperm-oocyte interaction. The aim of this study was to analyse the zona morphology and sperm-binding patterns on the porcine ZP. Oocytes were divided into four categories: immature, matured in vivo, or matured in vitro over a time period of 24 or 48 h. The zona morphology of early embryos grown in vivo or in vitro was also investigated. Four different types of zona morphology were detectable. They ranged from a porous, net-like structure to a nearly smooth and compact surface. No correlation could be established between the different kinds of maturation in terms of these zona types. All oocytes exhibited extremely heterogeneous zona morphologies, with no clear trend. During subsequent in vivo embryo development, the zona surface changes from a porous structure to one with a compact surface, while the morphology of in vitro embryos remained compact at all stages of development. The analysis of the number and distribution patterns of spermatozoa trapped in the ZP revealed extremely variable patterns, regardless of the zona morphology. Differences were only present if sorted or unsorted spermatozoa were used for insemination. Regardless of the number of inseminated spermatozoa after sorting, only a few (1-2) could be detected on the ZP. Whether oocytes were matured in vivo or in vitro was not a relevant factor. Unsorted spermatozoa bound in higher numbers than sorted ones. The number was directly dependent on the number of spermatozoa used for insemination.


Developing a measure to assess attitudes toward epilepsy in the US population.
The aim of this study was to develop an instrument to measure the US public's attitudes toward people with epilepsy and to assess the initial reliability and validity of the instrument. A 46-item attitudinal instrument was developed and tested using a proportional, stratified, national, random-digit dial household telephone survey of adults aged > or = 18 (n=758). Exploratory factor analyses revealed four underlying constructs that accounted for 34.4% of the variance in the factor analysis: negative stereotypes (alpha=0.73); risk and safety concerns (alpha=0.85); work and role expectations (alpha=0.76); and personal fear and social avoidance (alpha=0.79). Knowledge was also assessed; participants with less knowledge about epilepsy had more negative attitudes. The results of these analyses provided evidence for reliability and construct validity of the instrument. Additional tests of the reliability, validity, and factor structure of the scales are necessary to refine the instrument.


A comparison of the fully repositionable and retrievable Boston Lotus and direct flow medical valves for the treatment of severe aortic stenosis: A single center experience.
Second generation transcatheter aortic valve implantation (TAVI) devices have been designed to reduce the rate of paravalvular leak (PVL) and other complications. An important technological advancement has been the ability to fully reposition devices to facilitate optimal implantation depth and position to reduce the likelihood of PVL. To compare procedural and 30-day outcomes according to the Valve Academic Research Consortium (VARC)-2 criteria following TAVI with the fully repositionable and retrievable Lotus and DFM devices. 175 patients with severe aortic stenosis underwent transfemoral TAVI with the Lotus (n = 60) and DFM (n = 115) valve. Baseline clinical characteristics did not differ between the two groups. All devices were successfully implanted, with one case of valve embolization in the Lotus group. Device success (95 vs. 98.2%, P = 0.89), VARC-defined combined safety (90 vs. 93%, P = 0.48), and clinical efficacy (86.7 vs. 90.4%, P = 0.65) rates at 30-days were similar between Lotus and DFM groups. There was no severe PVL; one patient in both Lotus and DFM group developed moderate PVL after the procedure. The Lotus valve was associated with a higher rate of new pacemaker implantation (37.3 vs. 11.2%, P < 0.001) and a lower mean aortic gradient (9.4 ± 5 vs. 12.3 ± 5, P < 0.001) at 30-days as compared with the DFM valve. In this single-center, retrospective analysis, both Lotus and DFM devices demonstrated excellent device success, safety and efficacy at 30-day follow-up. The DFM valve was associated with minimally higher transvalvular gradients but lower new pacemaker implantation rates when compared to the Lotus valve.


Prediction of treatment response at 5-year follow-up in a randomized clinical trial of behaviorally based couple therapies.
Building on earlier work examining predictors of short- and moderate-term treatment response, demographic, intrapersonal, communication, and interpersonal variables were examined as predictors of clinically significant outcomes 5 years after couples completed 1 of 2 behaviorally based couple therapies. One hundred and thirty-four couples were randomly assigned to Integrative Behavioral Couple Therapy (IBCT; Jacobson & Christensen, 1998) or Traditional Behavioral Couple Therapy (TBCT; Jacobson & Margolin, 1979) and followed for 5 years after treatment. Outcomes include clinically significant change categories of relationship satisfaction and marital status at 5-year follow-up. Optimal subsets of predictors were selected using an automated, bootstrapped selection procedure based on Bayesian information criterion. Higher levels of commitment and being married for a longer period of time were associated with decreased likelihood of divorce or separation (odds ratio [OR] = 1.39, p = .004; OR = 0.91, p = .015). Being married for a longer period of time was also associated with increased likelihood of positive, clinically significant change (OR = 1.12, p = .029). Finally, higher levels of wife-desired closeness were associated with increased odds of positive, clinically significant change and decreased odds of divorce for moderately distressed, IBCT couples (OR = 1.16, p = .002; OR = 0.85, p = .007, respectively), whereas the opposite was true for moderately distressed, TBCT couples (OR = 0.77, p < .001; OR = 1.17, p = .002, respectively). Commitment-related variables are associated with clinically significant outcomes at 5-year follow-up as well as at termination and moderate-term follow-up.


Enucleation of blind, painful eyes.
To determine the demographics and results of treatment in patients undergoing enucleation of blind, painful eyes. Medical records were reviewed to determine the precipitating cause, preoperative symptoms, prior therapy, and results of treatment in 58 patients in whom enucleation was performed to relieve ocular discomfort. Patient perception of symptoms and treatment was assessed by a follow-up mail survey. Forty-five percent of patients had sustained prior trauma. Ocular discomfort was attributed to phthisis (43%), elevated intraocular pressure (40%), or corneal decompensation (10%). Preoperative symptoms included ocular pain (91%), periocular headache (17%), tearing (26%), and ipsilateral photophobia (26%). Eight patients experienced contralateral photophobia. Enucleation provided complete symptomatic relief in 54 patients (93%). Two of the four patients with persistent discomfort experienced painful facial paresthesias, attributed to prior retrobulbar alcohol injections. Ninety-one percent of the 32 patients who completed the follow-up survey reported pain relief after surgery, whereas 94% would recommend enucleation to others with blind, painful eyes. Trauma is the most common precipitating condition in patients undergoing enucleation of blind, painful eyes. These patients experience a variety of symptoms, including ocular pain, facial pain or paresthesias, tearing, and photophobia. Contralateral photophobia is not uncommon. Enucleation is highly effective in relieving these symptoms and should be considered in patients unresponsive to topical therapy. Facial paresthesias in patients who have received prior retrobulbar alcohol injections are not relieved by enucleation.


Ultrasound-Guided Motor-Sparing Knee Blocks for Postoperative Analgesia Following Total Knee Arthroplasty: A Randomized Blinded Study.
Pain following total knee arthroplasty (TKA) is often severe and can inhibit rehabilitation. Motor-sparing analgesic techniques such as periarticular infiltrations and adductor canal blocks have been popularized for knee analgesia since they preserve motor strength and permit early mobilization. Our primary objective was to compare the duration of analgesia from motor-sparing blocks with that of a standard periarticular infiltration. We used the time to first rescue analgesia as the end point. We randomized 82 patients scheduled for elective TKA to receive either the preoperative motor-sparing block (0.5% ropivacaine, 2.5 μg/mL of epinephrine, 10 mg of morphine, and 30 mg of ketorolac) or intraoperative periarticular infiltration (0.3% ropivacaine, 2.5 μg/mL of epinephrine, 10 mg of morphine, and 30 mg of ketorolac). For the motor-sparing block, we modified the ultrasound-guided adductor canal block by combining it with a lateral femoral cutaneous nerve block and posterior knee infiltration. The patients, surgeons, anesthetists administering the blocks, and outcome assessors all remained blinded to group allocation. Our primary outcome was duration of analgesia (time to first rescue analgesia). Secondary outcomes included quadriceps strength, function, side effects, satisfaction, and length of hospital stay. The duration of analgesia was significantly longer (mean difference, 8.8 hours [95% confidence interval = 3.98 to 13.62], p < 0.01) for the motor-sparing-block group (mean [and standard error], 18.1 ± 1.7 hours) compared with the periarticular infiltration group (mean, 9.25 ± 1.7 hours). The infiltration group had significantly higher scores for pain at rest for the first 2 postoperative hours and for pain with knee movement at 2 and 4 hours. There were no significant differences between groups with regard to any other secondary outcomes. In patients undergoing a TKA, a motor-sparing block provides longer analgesia than periarticular infiltration with retention of quadriceps muscle strength, function, patient satisfaction, and a short hospital stay. Therapeutic Level I. See Instructions for Authors for a complete description of levels of evidence.


Sunlight intensity based global positioning system for near-surface underwater sensors.
Water monitoring is important in domains including documenting climate change, weather prediction and fishing. This paper presents a simple and energy efficient localization strategy for near surface buoy based sensors. Sensors can be dropped randomly in the ocean and thus self-calibrate in terms of geographic location such that geo-tagged observations of water quality can be made without the need for costly and energy consuming GPS-hardware. The strategy is based on nodes with an accurate clock and light sensors that can regularly sample the level of light intensity. The measurements are fitted into a celestial model of the earth motion around the sun. By identifying the trajectory of the sun across the skies one can accurately determine sunrise and sunset times, and thus extract the longitude and latitude of the sensor. Unlike previous localization techniques for underwater sensors, the current approach does not rely on stationary or mobile reference points.


Heritability of attention problems in children: longitudinal results from a study of twins, age 3 to 12.
Twin studies of childhood behavior problems support the conclusion that individual differences in impulsivity, hyperactivity, and inattention are largely due to genetic influences. Non-genetic variation is due to environmental influences that are unique to the individual, and possibly to rater contrast effects. In the present longitudinal twin study, we report on the size of genetic and environmental effects on individual differences in attention problems at ages 3, 7, 10 and 12 years. Mothers were asked to complete the CBCL for their twin offspring when the children were 3 (n = 11,938), 7 (n = 10,657), 10 (n = 6,192), and 12 years old (n = 3,124). We focus on the Overactivity (OA) scale in the Child Behavior Checklist (CBCL/2-3), and on the Attention Problem (AP) scale of the CBCL/4-18. The data were analyzed using longitudinal structural equation modeling. Broad heritability of OA and AP is estimated at nearly 75%, at each age. A contrast effect was observed at age 3 only. The results revealed less stability of OA at age 3 to AP at age 7 (r = .40), compared to the stability from AP at age 7 and beyond (r = .70). Genetic effects explained between 76% and 92% of the covariance between OA and AP. OA and AP are highly heritable at all ages in both genders. The same set of genes appears to be expressed in boys and girls. The size of genetic and environmental contributions remains the same across the ages studied. Stability in OA and AP is accounted for by genetic influences. Children who do not display OA or AP at a given age are unlikely to develop these problems at a subsequent age.


Sugar metabolism by fusobacteria: regulation of transport, phosphorylation, and polymer formation by Fusobacterium mortiferum ATCC 25557.
Strains of eight Fusobacterium species differed in the ability to use sugars as energy sources for growth. For Fusobacterium russii ATCC 25533, F. gonidiaformans ATCC 25563, and F. nucleatum ATCC 10953 (except for fructose), growth was marginal to poor on all of the sugars tested. Other species displayed reasonable growth on glucose, fructose, mannose, and galactose, and two strains of F. mortiferum (ATCC 25557 and ATCC 9817) grew well on six of the sugars tested, including sucrose and maltose. Glucose transport by resting cells of most of the species was dependent upon (or markedly stimulated by) the presence of a fermentable amino acid. By contrast, F. mortiferum cells rapidly accumulated glucose and other sugars in the absence of amino acids. Although these cells were constitutive for glucose uptake, accumulation of other sugars was specifically induced by growth of F. mortiferum on the appropriate sugar. Spectrophotometric analyses and in situ staining of anionic polyacrylamide gels showed that glucose and fructose (mannose) are phosphorylated by separate ATP-dependent kinases. Fructokinase was stable in air at 4 degrees C, but under these conditions, greater than 70% of the glucokinase activity was lost. After overnight dialysis of the extract, no glucokinase activity was detectable; however, 65% of the initial enzyme activity was retained by inclusion of 1 mM dithiothreitol in the dialysis buffer. Thin-section electron microscopy showed that cells of F. mortiferum produced various amounts of intracellular glycogen during growth on the following sugars (in decreasing order of formation): galactose greater than sucrose greater than glucose greater than mannose greater than fructose. Mechanisms for sugar transport regulation, phosphorylation, and polymer synthesis by F. mortiferum cells are proposed.


Optical detection of polycations via polymer film-modified microtiter plates: response mechanism and bioanalytical applications.
Microtiter plate wells modified with thin (approximately 20 microm) polymeric films capable of optically sensing macromolecular protamine and other polycationic species are described. The plates are prepared by coating the bottom of each well of a conventional 96-well polypropylene plate with an adherent polymer film (a mixture of poly(vinyl chloride) and polyurethane) containing a lipophilic 2',7'-dichlorofluorescein derivative. Surprisingly, optical response toward polycations is shown to result from the extraction of the fluorescein derivative from the polymer film into a lyophobic colloidal phase at the sample/film interface. This new phase is likely composed of a micellular-type ion pair complex between the analyte polycation from aqueous sample phase and the deprotonated form of the fluorescein derivative. Accumulation of the deprotonated fluorescein species in this interfacial region induces an absorbance change measured at 540 nm. Optimized plates can be used to sense protamine concentrations in the range of 0-100 microg/mL in 10 min with little or no response to physiological levels of common cationic species (Na+, K+, Ca2+, etc.). The modified plates are shown to be useful as simple optical detectors for measuring heparin levels in plasma via titrations with protamine and for monitoring protease activities (trypsin and plasmin) that cleave polycationic peptides/proteins such as protamine into smaller peptide fragments that are not detected by the sensing films. Assays for "clot busting" plasminogen activators (streptokinase, urokinase, and tissue plasminogen activator) are also demonstrated using this relatively simple microtiter plate-based polycation detection system.


Hepatitis B and C in the liver transplant recipient.
Liver disease related to chronic viral hepatitis is the leading indication for orthotopic liver transplantation (OLT) worldwide. The evolution of our understanding of hepatitis B and C infection in the OLT recipient has been rapid in the last decade. The spontaneous risk for viral recurrence after transplantation is high but has been effectively decreased in hepatitis B infected recipients with the use of HBIG and lamivudine with dramatic improvements in patient and graft survivals. HCV recurrence as defined by histologic injury is almost universal although graft or patient outcomes for the first decade after OLT do not appear to be limited by HCV infection for most patients. However, individual patients do suffer severe graft injury and even graft loss due to recurrent HCV. With longer follow up into the second decade, the prevalence of HCV-related graft failure is likely to increase. In addition, the role of different immunosuppressive protocols on disease recurrence requires further study. Thus, although hepatitis B recurrence has been effectively contained by use of HBIG with or without lamivudine, the more intractable problem of managing recurrent HCV has as yet no obvious solutions. Optimal antiviral strategies for hepatitis C post-OLT have yet to be identified.


[Resistance of the slow potential and inhibition of afferent input of the frog olfactory bulb to hypoxia and blockade of synaptic transmission by manganese, cobalt and magnesium ions].
The effects of hypoxia and Mn++, Co++ and Mg++ on the electrical responses of the olfactory bulb to natural stimulation and single direct electric stimulation of olfactory nerve were studied in a frog. The slow potential evoked by natural stimulation and related inhibition of afferent olfactory input proved to be much more resistant to depression of synaptic transmission in the olfactory bulb with any methods used than postsynaptic components of the olfactory bulb orthodromic potential and related postsynaptic inhibition. Slow potentials were recorded after total blockade of synaptic transmission by Mg++ ions. It is concluded that the slow potential and related inhibition of afferent olfactory input are mainly of nonsynaptic nature. It is suggested that the slow potential reflects the depolarization of glial cells in a glomerular layer evoked by accumulation of K+ ions. Possible mechanisms for inhibition of olfactory bulb afferent input are discussed.


A retrospective analysis of the trends in modern contraceptive use among adolescents and young women (15-24 years) in the Democratic Republic of the Congo in the last two decades.
The increasing rate of unsafe abortion among adolescents and young women (AYW) in Sub-Saharan Africa may be attributed to a high rate of undesirable pregnancies and limited access to contraception services. The study looked at the prevalence of current contraception use among young women (15-24 years old) in the Democratic Republic of the Congo (DRC). The study analyzed secondary data from the demographic health survey (DHS) and UNICEF concerning AYW aged 15-24 years using IBM Corp. Released 2021. IBM SPSS Statistics for Windows, Version 28.0. (Armonk, NY: IBM Corp.). The study population included 15,319 female adolescents and young adults in DRC. DHS datasets for 2001, 2010, 2017/2018, and UNICEF 2007 and 2013-2014 were downloaded after permission to use the data. The AYW aged 20-24 accounted for 67% of the study population. The use of modern contraceptives has followed a roller coaster course. It dropped significantly from 2.6% in 2001 to 0.1% in 2007; it climbed to 2.7% in 2010 and 3.3% in 2013/14 but dropped to 2% in 2017/18. The most used method was male condoms (1.76%). A higher proportion of unmarried (2.3%) used any methods than married (1.8%). Those who attained secondary/higher education (2.6%) used modern contraceptives more than those with no formal education (1.5%), P < 0.0001. Those residing in the urban areas (4.5%) used modern contraceptives compared to those who lived in rural areas (1.7%) P < 0.0001. Modern contraceptive use among those with birth histories (2.6%) was comparatively higher than those who have not (2.3%), P < 0.0001. Those who lived in urban areas were adjusted odds ratio (AOR) = 2.504 times (95% confidence interval (CI): 1.793-3.497) more likely to use birth control than those in rural areas (P < 0.001). As reported in this study, the proportion of AYW's awareness of modern contraceptives is deficient in DRC, which might be attributed not only due to a lack of knowledge or enlightenment but also due to healthcare facilities' limited capacity to provide reproductive and sexual health services. Educating the AYW about modern contraceptive methods and interventions, including parents, the school system, and youth organizations, and strengthening the capacity of health facilities to provide modern contraception should be considered.


Targeting p35/Cdk5 signalling via CIP-peptide promotes angiogenesis in hypoxia.
Cyclin-dependent kinase-5 (Cdk5) is over-expressed in both neurons and microvessels in hypoxic regions of stroke tissue and has a significant pathological role following hyper-phosphorylation leading to calpain-induced cell death. Here, we have identified a critical role of Cdk5 in cytoskeleton/focal dynamics, wherein its activator, p35, redistributes along actin microfilaments of spreading cells co-localising with p(Tyr15)Cdk5, talin/integrin beta-1 at the lamellipodia in polarising cells. Cdk5 inhibition (roscovitine) resulted in actin-cytoskeleton disorganisation, prevention of protein co-localization and inhibition of movement. Cells expressing Cdk5 (D144N) kinase mutant, were unable to spread, migrate and form tube-like structures or sprouts, while Cdk5 wild-type over-expression showed enhanced motility and angiogenesis in vitro, which was maintained during hypoxia. Gene microarray studies demonstrated myocyte enhancer factor (MEF2C) as a substrate for Cdk5-mediated angiogenesis in vitro. MEF2C showed nuclear co-immunoprecipitation with Cdk5 and almost complete inhibition of differentiation and sprout formation following siRNA knock-down. In hypoxia, insertion of Cdk5/p25-inhibitory peptide (CIP) vector preserved and enhanced in vitro angiogenesis. These results demonstrate the existence of critical and complementary signalling pathways through Cdk5 and p35, and through which coordination is a required factor for successful angiogenesis in sustained hypoxic condition.


Addressing a Mental Health Intervention Gap in Juvenile Detention: A Pilot Study.
Research suggests that 60-70% of adolescents detained in the juvenile justice system meet criteria for a mental health disorder compared to 20% of the general adolescent population; however, the vast majority do not receive services. Unfortunately, mental health symptoms often worsen during detainment, and detainment is linked to lower levels of educational attainment and increased risk of adult recidivism. Thus, not only are these adolescents unlikely to receive needed mental health care, but also the lack of interventions in detention may exacerbate inequities of contact with the criminal justice system in adulthood. In addition to these youth being an underserved population broadly, youth of color are also disproportionately incarcerated compared to their white counterparts. The current paper describes results of a pilot study of an Acceptance and Commitment Therapy (ACT)-based behavioral skills intervention, aimed at providing evidence-based mental health treatment for an adolescent population at risk of long-term adverse mental health outcomes. The study included 128 males aged 14-17 who resided in juvenile detention. Results demonstrated that the intervention was acceptable to participants, feasible to provide in detention, and could be implemented with fidelity and competency. Intervention participants demonstrated declines in symptoms of mental health, and ACT-specific constructs of experiential avoidance, cognitive fusion, and perceived barriers to moving toward their values. These results have important implications for the possibility of an effective intervention that could disrupt systemic inequity in youth mental health, and thus support further testing of this intervention in a randomized controlled trial.


The consequences of 3,4-methylenedioxymethamphetamine induced CYP2D6 inhibition in humans.
3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is a widely abused substituted amphetamine. MDMA is predominantly O-demethylenated in humans by cytochrome P450 isoforms 2D6 and 1A2 (CYP2D6 and CYP CYP1A2, respectively). MDMA is also a mechanism-based inhibitor of CYP2D6. A controlled clinical trial was conducted in 15 healthy male subjects whereby a probe drug, dextromethorphan (DEX), was administered after an oral dose of 1.5 mg/kg MDMA. The pharmacokinetics of DEX and its metabolites were used to evaluate changes in CYP2D6 activity. The urinary metabolic ratio of DEX and dextrorphan was used to calculate a recovery half-life of CYP2D6. After MDMA, DEX Cmax and area under the curve increased approximately 10-fold with corresponding decreases in dextrorphan pharmacokinetic parameters. The metabolic ratio increased almost 100-fold from 0.0061 +/- 0.0056 to 0.4322 +/- 0.2848 after MDMA administration, with 67% of the subjects having a value greater than the antimode of 0.3 for assigning the poor metabolizer phenotype. CYP2D6 activity recovered after 10 days with a recovery half-life of 46.6 hours. In addition to the possible long-term serotonergic effects of MDMA, users must be warned of the consequences of such an inhibition.


The GPCR-Gαs-PKA signaling axis promotes T cell dysfunction and cancer immunotherapy failure.
Immune checkpoint blockade (ICB) targeting PD-1 and CTLA-4 has revolutionized cancer treatment. However, many cancers do not respond to ICB, prompting the search for additional strategies to achieve durable responses. G-protein-coupled receptors (GPCRs) are the most intensively studied drug targets but are underexplored in immuno-oncology. Here, we cross-integrated large singe-cell RNA-sequencing datasets from CD8+ T cells covering 19 distinct cancer types and identified an enrichment of Gαs-coupled GPCRs on exhausted CD8+ T cells. These include EP2, EP4, A2AR, β1AR and β2AR, all of which promote T cell dysfunction. We also developed transgenic mice expressing a chemogenetic CD8-restricted Gαs-DREADD to activate CD8-restricted Gαs signaling and show that a Gαs-PKA signaling axis promotes CD8+ T cell dysfunction and immunotherapy failure. These data indicate that Gαs-GPCRs are druggable immune checkpoints that might be targeted to enhance the response to ICB immunotherapies.


Toward Community-Driven Big Open Brain Science: Open Big Data and Tools for Structure, Function, and Genetics.
As acquiring bigger data becomes easier in experimental brain science, computational and statistical brain science must achieve similar advances to fully capitalize on these data. Tackling these problems will benefit from a more explicit and concerted effort to work together. Specifically, brain science can be further democratized by harnessing the power of community-driven tools, which both are built by and benefit from many different people with different backgrounds and expertise. This perspective can be applied across modalities and scales and enables collaborations across previously siloed communities.


The Homburg Lung: Efficacy and Safety of a Minimal-Invasive Pump-Driven Device for Veno-Venous Extracorporeal Carbon Dioxide Removal.
Extracorporeal carbon dioxide removal (ECCO2R) is increasingly considered a viable therapeutic approach in the management of hypercapnic lung failure to avoid intubation or to allow lung-protective ventilator settings. This study aimed to analyze efficacy and safety of a minimal-invasive ECCO2R device, the Homburg lung. The Homburg lung is a pump-driven system for veno-venous ECCO2R with ¼″ tubing and a 0.8 m surface oxygenator. Vascular access is usually established via a 19F/21 cm bilumen cannula in the right internal jugular vein. For this work, we screened patient registries from two German centers for patients who underwent ECCO2R with the Homburg lung because of hypercapnic lung failure since 2013. Patients who underwent extracorporeal membrane oxygenation before ECCO2R were excluded. Patients who underwent ECCO2R more than one time were only included once. In total, 24 patients (aged 53.86 ± 12.49 years; 62.5% male) were included in the retrospective data analysis. Ventilatory failure occurred because of chronic obstructive pulmonary disease (50%), cystic fibrosis (16.7%), acute respiratory distress syndrome (12.5%), and other origins (20.8%). The system generated a blood flow of 1.18 ± 0.23 liters per minute (lpm). Sweep gas flow was 3.87 ± 2.97 lpm. Within 4 hours, paCO2 could be reduced significantly from 82.05 ± 15.57 mm Hg to 59.68 ± 12.27 mm Hg, thereby, increasing pH from 7.23 ± 0.10 to 7.36 ± 0.09. Cannulation-associated complications were transient arrhythmia (1/24 patients) and air embolism (1/24). Fatal complications did not occur. In conclusion, the Homburg lung provides effective carbon dioxide removal in hypercapnic lung failure. The cannulation is a safe procedure, with complication rates comparable to those in central venous catheter implantation.


LATTICE FORMATION IN COMPLEMENT FIXATION: STUDIES WITH UNIVALENT RABBIT ANTIBODY.
Hybrid univalent 6.5S antibody molecules, formed by recombination of half-molecules of rabbit antibody to ovalbumin with those of normal rabbit gamma(G)-globulin, fail to fix complement in reactions with homologous antigen. Such hybrid molecules, however, block complement fixation by intact antibody to ovalbumin. Molecules of antibody reconstituted in the absence of other protein retain the capacity to fix complement. The data suggest that small complexes containing excess univalent antibody do not fix complement and that lattice formation is required for fixation.


Systemic anticoagulation in the setting of vascular extremity trauma.
There is conflicting data regarding if patients with vascular extremity trauma who undergo surgical treatment need to be systematically anticoagulated. We hypothesized that intraoperative systemic anticoagulation (ISA) decreased the risk of repair thrombosis or limb amputation after traumatic vascular injury of the extremities. We analyzed a composite risk of repair thrombosis and/or limb amputation (RTLA) between patients who did and did not undergo ISA during arterial injury repair. Patient data was collected in the American Association for the Surgery of Trauma PROspective Vascular Injury Treatment (PROOVIT) registry. This registry contains demographic, diagnostic, treatment, and outcome data. Between February 2013 and August 2015, 193 patients with upper or lower extremity arterial injuries who underwent open operative repair were entered into the PROOVIT registry. The majority were male (87%) with a mean age of 32.6 years (range 4-91) and 74% injured by penetrating mechanism. 63% of the injuries were described as arterial transection and 37% had concomitant venous injury. 62% of patients underwent ISA. RTLA occurred in 22 patients (11%) overall, with no significant difference in these outcomes between patients who received ISA and those that did not (10% vs. 14%, p=0.6). There was, however, significantly higher total blood product use noted among patients treated with ISA versus those that did not receive ISA (median 3 units vs. 1 unit, p=0.002). Patients treated with ISA also stayed longer in the ICU (median 3days vs. 1day, p=0.001) and hospital (median 9.5days vs. 6days, p=0.01). In this multicenter prospective cohort, intraoperative systemic anticoagulation was not associated with a difference in rate of repair thrombosis or limb loss; but was associated with an increase in blood product requirements and prolonged hospital stay. Our data suggest there is no significant difference in outcome to support use of ISA for repair of traumatic arterial injuries.


Synthesis and antifungal activities of miltefosine analogs.
Miltefosine is an alkylphosphocholine that shows broad-spectrum in vitro antifungal activities and limited in vivo efficacy in mouse models of cryptococcosis. To further explore the potential of this class of compounds for the treatment of systemic mycoses, nine analogs (3a-3i) were synthesized by modifying the choline structural moiety and the alkyl chain length of miltefosine. In vitro testing of these compounds against the opportunistic fungal pathogens Candida albicans, Candida glabrata, Candida krusei, Aspergillus fumigatus, and Cryptococcus neoformans revealed that N-benzyl-N,N-dimethyl-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3a), N,N-dimethyl-N-(4-nitrobenzyl)-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3d), and N-(4-methoxybenzyl)-N,N-dimethyl-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3e) exhibited minimum inhibitory concentrations (MIC) of 2.5-5.0 μg/mL against all tested pathogens, when compared to miltefosine with MICs of 2.5-3.3 μg/mL. Compound 3a showed low in vitro cytotoxicity against three mammalian cell lines similar to miltefosine. In vivo testing of 3a and miltefosine against C. albicans in a mouse model of systemic infection did not demonstrate efficacy. The results of this study indicate that further investigation will be required to determine the potential usefulness of the alkylphosphocholines in the treatment of invasive fungal infections.


Neuropeptide S reduces duodenal bicarbonate secretion and ethanol-induced increases in duodenal motility in rats.
Alcohol disrupts the intestinal mucosal barrier by inducing metabolic and functional changes in epithelial cells. Recently, we showed that neuropeptide S (NPS) decreases duodenal motility and increases mucosal paracellular permeability, suggesting a role of NPS in the pathogenesis of disorders and dysfunctions in the small intestine. The aim of the present study was to investigate the effects of NPS on ethanol- and HCl-induced changes of duodenal mucosal barrier function and motility. Rats were anaesthetized with thiobarbiturate, and a 30-mm segment of the proximal duodenum with an intact blood supply was perfused in situ. The effects on duodenal bicarbonate secretion, the blood-to-lumen clearance of 51Cr-EDTA, motility and transepithelial net fluid flux were investigated. Intravenous (i.v.) administration of NPS significantly reduced duodenal mucosal bicarbonate secretion and stimulated mucosal transepithelial fluid absorption, mechanisms dependent on nitrergic signaling. NPS dose-dependently reduced ethanol-induced increases in duodenal motility. NPS (83 pmol·kg-1·min-1, i.v.) reduced the bicarbonate and fluid secretory response to luminal ethanol, whereas a 10-fold higher dose stimulated fluid secretion but did not influence bicarbonate secretion. In NPS-treated animals, duodenal perfusion of acid (pH 3) induced greater bicarbonate secretory rates than in controls. Pre-treating animals with Nω-nitro-L-arginine methyl ester (L-NAME) inhibited the effect of NPS on bicarbonate secretion. In response to luminal acid, NPS-treated animals had significantly higher paracellular permeability compared to controls, an effect that was abolished by L-NAME. Our findings demonstrate that NPS reduces basal and ethanol-induced increases in duodenal motility. In addition, NPS increases luminal alkalinization and mucosal permeability in response to luminal acid via mechanisms that are dependent on nitric oxide signaling. The data support a role for NPS in neurohumoral regulation of duodenal mucosal barrier function and motility.


Is Exposure Necessary? A Randomized Clinical Trial of Interpersonal Psychotherapy for PTSD.
Exposure to trauma reminders has been considered imperative in psychotherapy for posttraumatic stress disorder (PTSD). The authors tested interpersonal psychotherapy (IPT), which has demonstrated antidepressant efficacy and shown promise in pilot PTSD research as a non-exposure-based non-cognitive-behavioral PTSD treatment. The authors conducted a randomized 14-week trial comparing IPT, prolonged exposure (an exposure-based exemplar), and relaxation therapy (an active control psychotherapy) in 110 unmedicated patients who had chronic PTSD and a score >50 on the Clinician-Administered PTSD Scale (CAPS). Randomization stratified for comorbid major depression. The authors hypothesized that IPT would be no more than minimally inferior (a difference <12.5 points in CAPS score) to prolonged exposure. All therapies had large within-group effect sizes (d values, 1.32-1.88). Rates of response, defined as an improvement of >30% in CAPS score, were 63% for IPT, 47% for prolonged exposure, and 38% for relaxation therapy (not significantly different between groups). CAPS outcomes for IPT and prolonged exposure differed by 5.5 points (not significant), and the null hypothesis of more than minimal IPT inferiority was rejected (p=0.035). Patients with comorbid major depression were nine times more likely than nondepressed patients to drop out of prolonged exposure therapy. IPT and prolonged exposure improved quality of life and social functioning more than relaxation therapy. This study demonstrated noninferiority of individual IPT for PTSD compared with the gold-standard treatment. IPT had (nonsignificantly) lower attrition and higher response rates than prolonged exposure. Contrary to widespread clinical belief, PTSD treatment may not require cognitive-behavioral exposure to trauma reminders. Moreover, patients with comorbid major depression may fare better with IPT than with prolonged exposure.


Interferon inducible GBPs restrict Burkholderia thailandensis motility induced cell-cell fusion.
Innate immunity responds to pathogens by producing alarm signals and activating pathways that make host cells inhospitable for pathogen replication. The intracellular bacterium Burkholderia thailandensis invades the cytosol, hijacks host actin, and induces cell fusion to spread to adjacent cells, forming multinucleated giant cells (MNGCs) which promote bacterial replication. We show that type I interferon (IFN) restricts macrophage MNGC formation during B. thailandensis infection. Guanylate-binding proteins (GBPs) expressed downstream of type I IFN were required to restrict MNGC formation through inhibition of bacterial Arp2/3-dependent actin motility during infection. GTPase activity and the CAAX prenylation domain were required for GBP2 recruitment to B. thailandensis, which restricted bacterial actin polymerization required for MNGC formation. Consistent with the effects in in vitro macrophages, Gbp2-/-, Gbp5-/-, GbpChr3-KO mice were more susceptible to intranasal infection with B. thailandensis than wildtype mice. Our findings reveal that IFN and GBPs play a critical role in restricting cell-cell fusion and bacteria-induced pathology during infection.


Shikonin suppresses proliferation and induces apoptosis in human leukemia NB4 cells through modulation of MAPKs and c‑Myc.
Acute promyelocytic leukemia (APL) is a special subtype of acute myeloid leukemia that responds to treatment with all‑trans retinoic acid and arsenic trioxide. However, severe side effects and drug resistance limit the effectiveness of these treatments. Hence, new drugs for APL are required urgently. Shikonin, an active naphthoquinone derived from the Chinese medical herb Zi Cao exerts antitumor activity in several cancers. In the present study, the effects of shikonin on proliferation and apoptosis in NB4 cells, as well as related mechanisms were assessed. Treatment of NB4 cells with shikonin inhibited proliferation in a concentration‑ and time‑dependent manner. The cell cycle was arrested in the G1 phase. NB4 cells treated with shikonin exhibited more apoptosis and higher levels of cleaved caspase‑3 and poly ADP‑ribose polymerase than control cells. Western blotting results demonstrated that the expression of p‑p38 mitogen‑activated protein kinase (p‑p38MAPK) and p‑c‑Jun N‑terminal kinase (p‑JNK) was increased significantly by shikonin treatment, while the expression of p‑ERK and c‑Myc was decreased. In summary, these findings indicated that shikonin inhibited cell proliferation and induced apoptosis partly through modulation of the MAPKs and downregulation of c‑Myc.


Expression status of p16 protein is associated with human papillomavirus oncogenic potential in cervical and genital lesions.
The p16 protein (p16) is a cyclin-dependent kinase (CDK) inhibitor that decelerates the cell cycle by inactivating the CDKs that phosphorylate retinoblastoma (Rb) protein. Recent biological studies have revealed that p16 expression is markedly influenced by the status of Rb expression, and p16 overexpression has been demonstrated in cervical cancers because of functional inactivation of Rb by human papillomavirus (HPV) E7 protein. To clarify the relationship between p16 overexpression and HPV infection in cervical carcinogenesis, immunohistochemical analysis of p16 and detection of HPV by in situ hybridization and polymerase chain reaction were performed on 139 formalin-fixed and paraffin-embedded samples of cervical and genital condylomatous and neoplastic lesions. Marked overexpression of p16 protein, ie, diffuse and strong immunostaining, was observed in all cervical cancers and preneoplastic lesions with infection by high- and intermediate-risk HPVs, ie, subtypes 16, 18, 31, 33, 52, and 58. Condylomata acuminata and low-grade squamous intraepithelial lesions with infection by low-risk HPV such as HPV-6/11 showed focal and weak immunohistochemical staining for p16. Our results clearly showed that the mode of p16 expression in lesions with high- and intermediate-risk HPVs differed from its expression in lesions with low-risk HPVs and thus might be attributable to differences in functional inactivation of Rb protein by different HPVs.


Breast-feeding practices among Thai women in Australia.
to examine the perceptions and experience of breast-feeding practices among Thai women who are now living in Australia. ethnographic interviews and participant observation among Thai women in Australia. Melbourne Metropolitan Area,Victoria, Australia. Thai women see breast milk as the most valuable food for newborn babies and young children. Breast milk is seen as 'human milk', while infant formula is referred to as 'animal milk'. Breast feeding is seen as beneficial not only to babies' health, but also to the mother's health. Women consume certain foods such as drinking hot water and consuming hot soup during the confinement period as a way to ensure breast milk production. Women appear to know that 'breast is best', but some lack accurate knowledge about breast feeding. They also receive inaccurate information regarding feeding practices from health professionals. there is no doubt that Thai women see that 'breast is best', therefore, the main message for those involved in giving breast-feeding advice is to 'make it easy for women to do it'. Only then can health services and care be made more meaningful to the many women who have decided to breast feed their babies in their new country.


Continuous subcutaneous insulin infusion: an approach to achieving normoglycaemia.
A study was performed to examine the feasibility of achieving long periods of near-normoglycaemia in patients with diabetes mellitus by giving a continuous subcutaneous infusion of insulin solution from a miniature, battery-driven, syringe pump. Twelve insulin-dependent diabetics had their insulin pumped through a subcutaneously implanted, fine nylon cannula; the basal infusion rate was electronically stepped up eightfold before meals. The blood glucose profile of these patients was closely monitored during the 24 hours of the subcutaneous infusion and compared with the profile on a control day, when the patients were managed with their usual subcutaneous insulin. Diet and exercise were standardised on both days. In five out of 14 studies the subcutaneous insulin infusion significantly lowered the mean blood glucose concentration without producing hypoglycaemic symptoms; in another six patients the mean blood glucose concentration was maintained. As assessed by the M value the level of control was statistically improved in six out of 14 studies by the infusion method and maintained in six other patients. To assess the effects of blood glucose control on diabetic microvascular disease it will be necessary to achieve long-term normoglycaemia in selected diabetics. The results of this preliminary study suggest that a continuous subcutaneous insulin infusion may be a means of maining physiological glucose concentrations in diabetics. Though several problems remain--for example, in determining the rate of infusion--longer-term studies with the miniature infusion pumps are now needed.


The phase image: its relationship to patterns of contraction and conduction.
To determine the relationship of phase changes and abnormalities of ventricular contraction and conduction, we performed phase image analysis of blood pool scintigrams in 29 patients. Eleven patients had no evidence of blood pool contraction or ECG conduction abnormalities, four had contraction abnormalities, seven had abnormal conduction and seven had abnormalities of both variables. The phase delay generally related to the degree of contraction abnormality. The mean phase delay in hypokinetic segments differed from that in normokinetic segments in the same patient (p less than 0.025), the phase delay of akinetic and dyskinetic segments differed from that in normokinetic segments (p less than 0.001) and the phase delay in dyskinetic segments differed from that in akinetic segments (p less than 0.005), but there was a significant overlap in the phase delay in normal and hypokinetic segments. Also, in patients with conduction abnormalities, the minimal associated regional phase delay presented a phase dispersion and a pattern of contraction consistent with the pattern of conduction and different from normal. A single study performed both at rest and with stress demonstrated the effect of heart rate on phase assessment and confirmed the independent effects of contraction and conduction on phase delay. Acquisition and analytic methods should add significantly to the resolution of the phase method.


Nanoparticle surface electromagnetic fields studied by single-particle nonlinear optical spectroscopy.
Polarization-resolved second harmonic generation (SHG) measurements were performed on solid gold nanosphere (SGN) dimers at the single-particle level. The results indicated that single-particle SHG measurements could be used to quantify the localized electromagnetic surface fields that result from excitation of inter-particle localized surface plasmon modes. For several dimers, the polarization-resolved measurements revealed that the surface fields localized between SGNs in a dimer were chiral. Quantitative analysis of SHG line shapes obtained from single-particle continuous polarization variation (CPV-SHG) experiments confirmed that the chirality originated from non-zero magnetic-dipolar contributions to the SGN dimer nonlinear optical response. Correlation of SEM images and single-particle SHG measurements obtained for several SGN dimers, as well as lithographically generated nanostructures, were used to demonstrate the structure sensitivity of the CPV-SHG method.


Analysis of an abscisic acid (ABA)-responsive gene promoter belonging to the Asr gene family from tomato in homologous and heterologous systems.
Asr is a family of genes that maps to chromosome 4 of tomato. Asr2, a recently reported member of this family, is believed to be regulated by abscisic acid (ABA), stress and ripening. A genomic Asr2 clone has been fully sequenced, and candidate upstream regulatory elements have been identified. To prove that the promoter region is functional in vivo, we fused it upstream of the beta-glucuronidase (GUS) reporter gene. The resulting chimeric gene fusion was used for transient expression assays in papaya embryogenic calli and leaves. In addition, the same construct was used to produce transgenic tomato, papaya, tobacco, and potato plants. Asr2 upstream sequences showed promoter function in all of these systems. Under the experimental conditions tested, ABA stimulated GUS expression in papaya and tobacco, but not in tomato and potato systems.


Molecular phylogenetic relationships among members of the family Phytolaccaceae sensu lato inferred from internal transcribed spacer sequences of nuclear ribosomal DNA.
The phylogeny of a phylogenetically poorly known family, Phytolaccaceae sensu lato (s.l.), was constructed for resolving conflicts concerning taxonomic delimitations. Cladistic analyses were made based on 44 sequences of the internal transcribed spacer of nuclear ribosomal DNA from 11 families (Aizoaceae, Basellaceae, Didiereaceae, Molluginaceae, Nyctaginaceae, Phytolaccaceae s.l., Polygonaceae, Portulacaceae, Sarcobataceae, Tamaricaceae, and Nepenthaceae) of the order Caryophyllales. The maximum parsimony tree from the analysis resolved a monophyletic group of the order Caryophyllales; however, the members, Agdestis, Anisomeria, Gallesia, Gisekia, Hilleria, Ledenbergia, Microtea, Monococcus, Petiveria, Phytolacca, Rivinia, Schindleria, Seguieria, Stegnosperma, and Trichostigma, which belong to the family Phytolaccaceae s.l., did not cluster under a single clade, demonstrating that Phytolaccaceae is polyphyletic.


Percutaneous transluminal angioplasty in the lower extremities: a 5-year experience.
From January 1979 to March 1984, percutaneous transluminal angioplasty (PTA) was used to treat 148 limbs of 135 Mayo Clinic patients with occlusive arterial disease of the lower extremities. The procedure was technically successful in more than 95% of the attempts. The outcome was clinical improvement in 89 limbs and no improvement in 40 limbs; in 19 limbs, PTA was technically successful but the patient was dismissed from the hospital and lost to follow-up before the extent of improvement could be determined. Mean ankle/brachial pressure indices increased after PTA in those with clinical improvement but not in those without improvement. Clinical improvement was less likely to follow PTA in patients with advanced age, diabetes, severe initial symptoms, low ankle/brachial indices, or distal occlusive disease. In patients with improvement after PTA, the mean follow-up period was 33 months; during that time, failure (defined as recurrence of the original symptoms or the need for repeat PTA or operation) occurred at a rate of 6.4% per year. Serious complications occurred after three procedures (2.0%). We conclude that PTA is technically feasible and generally safe for many patients with occlusive arterial disease of the lower limbs.