Motor unit action potential duration and muscle length.
Motor unit action potential (MUAP) components are investigated by means of single fiber computer simulations and clinical measurements. The single fiber simulations have essentially full bandwidth without noise, whereas the clinical measurements were made with a 3-10,000-Hz bandwidth utilizing approximately 1000 averages to reduce noise optimally. These parameters allow the recording of a MUAP's complete "physiologic" duration including its very slow onset and termination. The simulation results demonstrate a constant waveform onset regardless of the electrode's recording location along the fiber. A far-field potential is initiated when the action potential encounters the muscle fiber's termination. The simulated waveform's and clinically recorded MUAP's near-field component extends between the potential's onset and its corresponding far-field potential's onset. This near-field component's duration should vary with fiber length, and this prediction is clinically confirmed by measuring three different muscle lengths. The far-field potential reveals a constant duration, independent of fiber length, and appears to be associated with the muscle fiber's intracellular action potential duration. A more complete understanding of the components contributing to MUAP duration should provide a more fundamental basis for quantitative clinical MUAP duration measurements.


Caloric restriction ameliorates angiotensin II-induced mitochondrial remodeling and cardiac hypertrophy.
Angiotensin II-induced cardiac damage is associated with oxidative stress-dependent mitochondrial dysfunction. Caloric restriction (CR), a dietary regimen that increases mitochondrial activity and cellular stress resistance, could provide protection. We tested that hypothesis in double transgenic rats harboring human renin and angiotensinogen genes (dTGRs). CR (60% of energy intake for 4 weeks) decreased mortality in dTGRs. CR ameliorated angiotensin II-induced cardiomyocyte hypertrophy, vascular inflammation, cardiac damage and fibrosis, cardiomyocyte apoptosis, and cardiac atrial natriuretic peptide mRNA overexpression. The effects were blood pressure independent and were linked to increased endoplasmic reticulum stress, autophagy, serum adiponectin level, and 5' AMP-activated protein kinase phosphorylation. CR decreased cardiac p38 phosphorylation, nitrotyrosine expression, and serum insulin-like growth factor 1 levels. Mitochondria from dTGR hearts showed clustered mitochondrial patterns, decreased numbers, and volume fractions but increased trans-sectional areas. All of these effects were reduced in CR dTGRs. Mitochondrial proteomic profiling identified 43 dTGR proteins and 42 Sprague-Dawley proteins, of which 29 proteins were in common in response to CR. We identified 7 proteins in CR dTGRs that were not found in control dTGRs. In contrast, 6 mitochondrial proteins were identified from dTGRs that were not detected in any other group. Gene ontology annotations with the Panther protein classification system revealed downregulation of cytoskeletal proteins and enzyme modulators and upregulation of oxidoreductase activity in dTGRs. CR provides powerful, blood pressure-independent, protection against angiotensin II-induced mitochondrial remodeling and cardiac hypertrophy. The findings support the notion of modulating cardiac bioenergetics to ameliorate angiotensin II-induced cardiovascular complications.


Anti-parasitic effect of vitamin C alone and in combination with benznidazole against Trypanosoma cruzi.
Drugs currently used for the treatment of Chagas' disease, nifurtimox and benznidazole, have a limited effectiveness and toxic side effects. With the aim of finding new therapeutic approaches, in vitro and in vivo anti-Trypanosoma cruzi activity of vitamin C alone and combined with benznidazole were investigated. The trypanocidal activity on epimastigote and trypomastigote forms was evaluated by counting parasites in a Neubauer chamber after treatment with the compounds. For the amastigote stage, transgenic parasites expressing β-galactosidase were used and quantified by measuring the β-galactosidase activity. The cytotoxicity of compounds was tested on Vero cells. The redox state of the parasite was evaluated by determining the reduced thiol levels (spectrophotometric assay) and the intracellular oxidative state (by flow cytometry). The in vivo trypanocidal activity was evaluated on a murine model of Chagas' disease. The trypanocidal activity of vitamin C and benznidazole was similar for the three parasite forms. When combining both drugs, vitamin C did not induce any change in the antiparasitic activity of benznidazole on trypomastigotes; however, on mammal cells, vitamin C diminished the cytotoxicity degree of benznidazole. Two mechanisms of action may be postulated for vitamin C: a lethal pro-oxidant effect on the parasite when used alone, and an antioxidant effect, when combined with benznidazole. A similar behavior was observed on infected mice; i.e., parasite counts in infected mice treated with vitamin C were lower than that of the control group. Animals treated with benznidazole presented lower parasitemia levels, as compared with those treated with vitamin C alone. Again, vitamin C did not cause any effect on the antiparasitic profile of benznidazole. Even though a combined treatment was employed, the antioxidant effect of vitamin C on the host was evidenced; a 100% survival was observed and the weight loss occurring during the acute phase of the infection was reduced. Based on these results, the combination of vitamin C with benznidazole could be considered as an alternative treatment for Chagas' disease. These preliminary results encourage further research to improve the treatment of Chagas' disease.


T-cell exhaustion correlates with improved outcomes in kidney transplant recipients.
Continuous antigen stimulation during chronic infection or malignancy can promote functional T cell silencing, a phenomenon called T cell exhaustion. The prevalence and impact of T cell exhaustion following organ transplantation, another immune stimulus with persistently high antigen load, are unknown. Here, we characterized serially collected peripheral blood mononuclear cells from 26 kidney transplant recipients using time-of-flight mass cytometry (CyTOF) to define distinct subsets of circulating exhausted T cells and their relationship to induction therapy and allograft function. We observed an increase in specific subsets of CD4+ and CD8+ exhausted T cells from pre-transplant to 6-months post-transplant, with greater increases in participants given anti-thymocyte globulin induction than in participants who received no induction or non-depleting induction. The percentages of exhausted T cells at 6 months correlated inversely with adenosine triphosphate (ATP) production (a surrogate of T cell function) and with allograft interstitial fibrosis. Guided by the CyTOF data, we delineated a PD-1+CD57- phenotype for CD4+ and CD8+ exhausted T cells, and confirmed that these cells have limited capacity for cytokine secretion and ATP production. In an independent cohort of 50 kidney transplant recipients, we confirmed the predicted increase of PD-1+CD57- exhausted T cells after lymphocyte-depleting induction therapy and its direct correlation with better allograft function. Our findings suggest that monitoring T cell exhaustion can be useful for post-transplant risk assessment and support the need to develop and test strategies aimed at augmenting T cell exhaustion following kidney transplantation.


Fundamental Investigations of Bond Behaviour of High-Strength Micro Steel Fibres in Ultra-High Performance Concrete under Cyclic Tensile Loading.
The objective of the contribution is to understand the fatigue bond behaviour of brass-coated high-strength micro steel fibres embedded in ultra-high performance concrete (UHPC). The study contains experimental pullout tests with variating parameters like load amplitude, fibre orientation, and fibre-embedded length. The test results show that fibres are generally pulled out of the concrete under monotonic loading and rupture partly under cyclic tensile loading. The maximum tensile stress per fibre is approximately 1176 N/mm2, which is approximately one third of the fibre tensile strength (3576 N/mm2). The load-displacement curves under monotonic loading were transformed into a bond stress-slip relationship, which includes the effect of fibre orientation. The highest bond stress occurs for an orientation of 30° by approximately 10 N/mm2. Under cyclic loading, no rupture occurs for fibres with an orientation of 90° within 100,000 load changes. Established S/N-curves of 30°- and 45°-inclined fibres do not show fatigue resistance of more than 1,000,000 load cycles for each tested load amplitude. For the simulation of fibre pullout tests with three-dimensional FEM, a model was developed that describes the local debonding between micro steel fibre and the UHPC-matrix and captures the elastic and inelastic stress-deformation behaviour of the interface using plasticity theory and a damage formulation. The model for the bond zone includes transverse pressure-independent composite mechanisms, such as adhesion and micro-interlocking and transverse pressure-induced static and sliding friction. This allows one to represent the interaction of the coupled structures with the bond zone. The progressive cracking in the contact zone and associated effects on the fibre load-bearing capacity are the decisive factors concerning the failure of the bond zone. With the developed model, it is possible to make detailed statements regarding the stress-deformation state along the fibre length. The fatigue process of the fibre-matrix bond with respect to cyclic loading is presented and analysed in the paper.


RNAi-based strategies for cyclooxygenase-2 inhibition in cancer.
Cyclooxygenase-2 (COX-2) enzyme has been involved in the tumorigenesis and in the progression of colorectal cancer (CRC). The use of traditional nonsteroidal anti-inflammatory drugs (NSAIDs) or selective COX-2 inhibitors has been proposed for the prevention and the treatment of this relevant neoplastic disease. In the light of an innovative alternative to these pharmacological approaches, we review here the possible strategies to achieve a strong and selective inhibition of COX-2 enzyme by using the mechanism of RNA Interference (RNAi) targeted against its mRNA. Anti-COX-2 siRNA molecules (siCOX-2) can be generated in CRC cells from short hairpin RNA (shRNA) precursors, delivered in vitro by a retroviral expression system, and induce a significant and stable silencing of overexpressed COX-2 in human colon cancer cells. As a safer alternative to viral approach, nonpathogenic bacteria (E. coli) can be engineered to invade eukaryotic cells and to generate siCOX-2 molecules in cancer cells. Moreover, the involvement of miRNAs in COX-2 posttranscriptional regulation opens up the possibility to exploit an endogenous silencing mechanism to knockdown overexpressed COX-2. Thus, these recent strategies disclose new challenging perspectives for the development of clinically compatible siRNA or miRNA capable of selectively inhibiting COX-2 enzyme.


Percutaneous needle biopsy and synovial histology.
Percutaneous needle biopsies of synovium are successfully used for diagnosis and investigation of joint disease by an increasing number of groups around the world. This procedure can be done in the office with little morbidity; a large number of samples can minimize the potential limitation of sampling error. Clinical indications for 'imaging the joint' by looking at morphological and other features of the actual tissue include undiagnosed acute or chronic mono- or oligoarthritis, haemarthrosis, suspected deposition diseases, new developments in previous stable disease and less often unexplained polyarthritis. Research into any joint disease can be helped by study of synovium especially using newer immunohistochemical, EM and molecular techniques. This report has reviewed other methods used for obtaining synovium, described the different percutaneous biopsy needles, detailed the methods used for biopsy with the Parker-Pearson needle and described how our group handles tissue so as to obtain maximal impact. The very few side effects of needle biopsy include haemarthrosis and, rarely, needle breakage. Finally, we have provided a brief overview of normal synovium and some aspects of synovium in a variety of joint diseases.


Quantitative analysis of retinal vasculature in normal eyes using ultra-widefield fluorescein angiography.
To quantify the area and density of retinal vascularity by ultra-widefield fluorescein angiography (UWFA). In a retrospective study, UWFA images were obtained using an ultra-widefield imaging device in 42 normal eyes of 42 patients. Central and peripheral steered images were used to define the edge of retinal vasculature by a certified grader. The length from the center of the optic disc to the edge of retinal vascularity (RVL) in each quadrant and the total retinal vascular perfusion area (RVPA) were determined by the grader using OptosAdvance software. The density of retinal vascularity (RVD) was quantified in different zones of central-steered images using Image J software. Among 42 healthy eyes, the values for mean RVL in each quadrant were 19.007±0.781 mm (superior), 18.467±0.869 mm (inferior), 17.738±0.622 mm (nasal) and 24.241±1.336 mm (temporal). The mean RVPA was 1140.117±73.825 mm2. The mean RVD of the total retina was 4.850%±0.638%. RVD varied significantly between different retina zones (P<0.001), and significant differences existed in the RVD values for total retinal area in patients over 50 years old compared to those under 50 years old (P=0.033). No gender difference was found. The UWFA device can be a promising tool for analyzing the overall retinal vasculature and may provide a better understanding of retinal vascular morphology in normal eyes. Aging may be related to lower RVD.


Distribution and genotypic characterization of Campylobacter jejuni isolated from poultry in Split and Dalmatia County, Croatia.
Consumption of poultry contaminated with Campylobacter jejuni has been recognized worldwide as the leading cause of campylobacteriosis. Therefore, the aim of our study was to investigate the prevalence and genotype diversity of Campylobacter jejuni in poultry meat intended for consumption in Split and Dalmatia County, which is the second biggest County in Croatia. Furthermore, we also wanted to discover possibly stable clones of C. jejuni appearing in different samples and periods of time, which would indicate their ability to persist in or adapt to poultry. In the period from March 2008 until June 2010, 834 samples of poultry from various sources were examined using a surface swab technique. Isolation of C. jejuni was performed by Preston broth and Karmali agar. Identification of the isolates was carried out using biochemical tests. C. jejuni was found in 84 of 574 chicken samples (14.6%) and in nine of 260 samples of turkey (3.5%). Pulse-field gel electrophoresis (PFGE) was used to analyse 61 obtained isolates using SmaI and KpnI. Of 22 different macrorestriction profiles (MRP) that were found, five were detected in poultry from both different locations and periods of time. Samples from 11 locations were found to be contaminated with more than two different genotypes of C. jejuni. Interestingly, the same MRP were found both in poultry declared to be of domestic origin and in the poultry imported from abroad. The prevalence of C. jejuni in poultry samples was in accordance with previously reported results. Genotypic analysis indicated that the population of C. jejuni in Split and Dalmatia County was diverse and that multiple strains of C. jejuni could be found in the same poultry samples. Furthermore, the same genotypes were identified from the samples obtained from different locations and periods of time, which could support the theory of a global existence of certain MRP that are able to persist in or adapt to poultry.


New and Pipeline Drugs for Gout.
Gout is the most common inflammatory arthropathy in the western world. Affecting millions and accounting for lost wages, increased health care costs, and significant disability, it remains a burden for those afflicted, their families, and the health care system. Despite the availability of a number of effective therapies, gout is often inadequately treated, and its impact on the patients overall health and well-being is underestimated by physicians and patients alike. For many decades, controlling acute flares was the priority in the management of gout. More recently, however, a deeper understanding of gout pathophysiology has resulted in a new appreciation that gout impacts the patient with consequences well beyond the episodes of acute inflammatory arthritis. Reflecting the chronic nature of the disease, gout treatment needs to be chronic as well, and aimed at reducing the underlying cause of gout-hyperuricemia-as well as the symptom of acute attacks. Therapy therefore requires both urate lowering and anti-inflammatory strategies. Unfortunately, the most commonly used urate lowering and anti-inflammatory treatments may be problematic in some gout patients, who often have multiple comorbidities that establish relative contraindications. Novel urate lowering therapies, and new medications to treat and prevent acute gouty flares, can not only improve care of the individual; they can also lead to a better discourse for the edification of those who manage and are managed for this underestimated disease. In this paper, we discuss new and pipeline drugs for acute gout, prophylactic anti-inflammatory therapies as well as urate lowering therapies.


Changes over time in thresholds for electrical stimulation of the cochlea.
The purpose of this paper is to better characterize changes over time that occurred in psychophysical detection thresholds for electrical stimulation of the cochlea. Threshold changes observed in nonhuman primates implanted with cochlear electrode arrays can be divided into at least three types based on the patterns of change over time. Short-term increases and subsequent decreases in threshold were commonly observed during the first months after implantation and were often followed by periods of long-term threshold stability. Long-term slow increases in thresholds and more rapid increases after a period of threshold stability have also been observed. The threshold changes may be divided into at least two classes based on their dependence on the waveforms used for the threshold measurements. Some changes occurred primarily in thresholds for long phase-duration signals while other changes were equal in magnitude (in decibels) for all tested stimuli. This suggests that at least two mechanisms underlay these threshold changes. The observed changes in thresholds have implications for experimental studies of electrical stimulation and for clinical application of auditory prostheses.


Transjugular intrahepatic portosystemic shunts (TIPS): effects of respiratory state and patient position on the measurement of Doppler velocities.
The purpose of this prospective study was to examine the effects of patient position and respiratory state on the measurements of Doppler velocities in transjugular intrahepatic portosystemic shunts. Thirty-eight transjugular intrahepatic portosystemic shunts in 34 consecutive patients were studied using Doppler sonography. Peak velocities were measured in the mid shunt with the patient in three positions (supine, sitting upright, and left lateral decubitus) and two respiratory states (deep inspiration and quiet respiration). A mixed linear regression model was used to assess statistically significant differences among the six velocity measurements. Peak velocities in the mid stent averaged 22 cm/sec greater in quiet respiration than in deep inspiration, which was a significant difference (p < 0.00001). Differences in velocities in the three patient positions were not significant (p = 0.53). Using 90-190 cm/sec as the normal range, the peak velocity shifted from normal to abnormal levels by changing respiratory state in 17 (45%) of 38 studies. Using 60 cm/sec as the lower normal limit, the peak velocity fell below the normal range with inspiration in 10 (26%) of 38 studies. In 12 (32%) of 38 studies, a decline in peak velocity exceeding 50 cm/sec could be induced by inspiration. Peak systolic velocity in transjugular intrahepatic portosystemic shunts is substantially altered by the respiratory state of the patient at the time of the measurement, but not by the patient position. Respiratory state must be taken into account in the interpretation of peak velocity for shunt stenosis.


Effects of food quality on trade-offs among growth, immunity and survival in the greater wax moth Galleria mellonella.
The resources available to an individual in any given environment are finite, and variation in life history traits reflect differential allocation of these resources to competing life functions. Nutritional quality of food is of particular importance in these life history decisions. In this study, we tested trade-offs among growth, immunity and survival in 3 groups of greater wax moth (Galleria mellonella) larvae fed on diets of high and average nutritional quality. We found rapid growth and weak immunity (as measured by encapsulation response) in the larvae of the high-energy food group. It took longer to develop on food of average nutritional quality. However, encapsulation response was stronger in this group. The larvae grew longer in the low-energy food group, and had the strongest encapsulation response. We observed the highest survival rates in larvae of the low-energy food group, while the highest mortality rates were observed in the high-energy food group. A significant negative correlation between body mass and the strength of encapsulation response was found only in the high-energy food group revealing significant competition between growth and immunity only at the highest rates of growth. The results of this study help to establish relationships between types of food, its nutritional value and life history traits of G. mellonella larvae.


Access to safe water in rural Artibonite, Haiti 16 months after the onset of the cholera epidemic.
Haiti has the lowest improved water and sanitation coverage in the Western Hemisphere and is suffering from the largest cholera epidemic on record. In May of 2012, an assessment was conducted in rural areas of the Artibonite Department to describe the type and quality of water sources and determine knowledge, access, and use of household water treatment products to inform future programs. It was conducted after emergency response was scaled back but before longer-term water, sanitation, and hygiene activities were initiated. The household survey and source water quality analysis documented low access to safe water, with only 42.3% of households using an improved drinking water source. One-half (50.9%) of the improved water sources tested positive for Escherichia coli. Of households with water to test, 12.7% had positive chlorine residual. The assessment reinforces the identified need for major investments in safe water and sanitation infrastructure and the importance of household water treatment to improve access to safe water in the near term.


Quantitative two-dimensional gel electrophoretic analysis of clock-controlled proteins in cultured chick pineal cells: circadian regulation of tryptophan hydroxylase.
The progression of the circadian oscillator through its cycle and the circadian rhythm of melatonin production in dissociated chick pineal cultures both require daily de novo protein synthesis during defined circadian phases. To identify specific proteins involved in these two processes, we have performed a quantitative two-dimensional polyacrylamide gel electrophoretic screen of proteins that are synthesized at different times of the day in chick pineal cell cultures. Out of approximately 700 proteins analyzed, we have identified several proteins whose levels of 35S incorporation oscillate in a light/dark cycle. One protein of 56 kDa, pI 6 (p56) undergoes a diurnal oscillation that parallels the melatonin rhythm, reaching a peak early in the night and falling to minimal levels during the day. A second protein of 22 kDa, pI 4.5 (p22) also expresses a diurnal rhythm in 35S incorporation; however, it peaks at the end of the night. The oscillations of both proteins persist, with a reduced amplitude, in constant darkness. Furthermore, the phases of the p56 and p22 rhythms are regulated by the light/dark cycle. Both p56 and p22 appear to be under direct control of the chick pineal circadian oscillator, and therefore can be described as "clock-controlled proteins." We have identified p56 as tryptophan hydroxylase by microsequencing and western blotting. Chick pineal tryptophan hydroxylase also expresses a 24-h oscillation in abundance both in vitro and in vivo. The rhythm in tryptophan hydroxylase expression represents a newly discovered level of regulation of the melatonin synthesis pathway by the circadian clock in chick pineal cells.


Influence of Maternal Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin on Socioemotional Behaviors in Offspring Rats.
Effects of dioxins on cognitive functions were reported in previous studies conducted in humans and animals. In the present study, we investigated the influence of dioxin exposure during pregnancy on social interaction and on the activity of offspring, which are related to neurodevelopmental disturbances. In addition, we analyzed neurochemical alterations of the limbic system of rat brains to suggest one mechanism of dioxin effects on brain function. We believe that this manuscript is suitable for publication in "Environmental Health Insights" because it provides an interesting topic for a wide global audience. To clarify the relationships between maternal dioxin exposure and socioemotional functions of rat offspring, dams were given TCDD (1.0 μg/kg) on gestational day 15. Social interactions and forced swimming time were compared between TCDD-exposed and control offspring in each gender. Frequency and duration of locomotion were higher, and durations per one behavior of proximity and social contact were significantly lower in the exposed males, while only the duration of proximity was lower in the exposed females. Forced swimming time on the first day was significantly longer in the exposed males. In the limbic system of the rat brain, the levels and/or activity of CaMKIIα were decreased in males and were increased in females in the exposed offspring. These results suggest that prenatal TCDD exposure induces hyperactivity and socioemotional deficits, particularly in the male offspring due to alterations in CaMKIIα activity in the limbic system of the brain.


LncRNA MALAT1 signaling pathway and clinical applications in overcome on cancers metastasis.
In spite of its high mortality rate and difficulty in finding a cure, scientific advancements have contributed to a reduction in cancer-related fatalities. Aberrant gene expression during carcinogenesis emphasizes the importance of targeting the signaling networks that control gene expression in cancer treatment. Long noncoding RNAs (lncRNAs), which are transcribed RNA molecules that play a role in gene expression regulation, are a recent innovative therapeutic approach for diagnosing and treating malignancies. MALAT1, a well-known lncRNA, functions in gene expression, RNA processing, and epigenetic control. High expression levels of MALAT1 are associated with several human disorders, including metastasis, invasion, autophagy, and proliferation of cancer cells. MALAT1 affects various signaling pathways and microRNAs (miRNAs), and this study aims to outline its functional roles in cancer metastasis and its interactions with cellular signaling pathways. Moreover, MALAT1 and its interactions with signaling pathways can be promising target for cancer treatment.


[Fatty acid composition of the cervical mucus obtained during ovulation and at the term of pregnancy].
Aim of this work is to evaluate the different fatty acid composition of cervical mucus obtained during ovulation and at the term of pregnancy. The fatty acid composition in cervical mucus was determined in 14 non pregnant women during expected ovulation (cervical score > 10) and in 12 at term pregnant women. Following extraction, (acidification and transesterification), the identification and quantification of fatty acids was performed by gas-chromatographic analysis, with the aid of a specific software. In both groups of samples, palmitic acid, stearic acid and oleic acid were the prevalent acids comprising more than half of the total amounts. Compared to non pregnant samples, in pregnant cervical mucus, elevated levels of oleic acid were pointed out, while mean levels of miristic acid and stearic acid were lower. In the samples of cervical mucus drew at the term of pregnancy, arachidonic acid levels mean values were higher when the first period of labour was started. The pregnancy-induced biochemical changes in fatty acid pattern could likely be correlate to the variations of the physiochemical properties and to the physical appearance that cervical mucus undergoes during pregnancy. The elevated levels of arachidonic acid, during the first period of labour, may be correlated with prostaglandin production by intrauterine tissues (amniotic fluid, amnion, chorion, decidua, myometrium) and probably by cervical mucus.


Characterization of and male adaptation to pheromone of femaleTrichostrongylus colubriformis (Nematoda).
Males of the ruminant nematodeTrichostrongylus colubriformis were significantly attracted to an incubate from their females during in vitro assay. Exposure of males to sera from uninfected and infected animals or selected neurotransmitters had no effect on their subsequent responsiveness to the female's pheromone. In contrast, exposure to 1500 female-hours of pheromone decreased male responsiveness after moderate rinsing prior to bioassay. Rinsing of the males with larger volumes of Tyrode's solution prior to in vitro assay increased their subsequent response to the female's pheromone. High-performance liquid chromatography yielded a presumptive pheromone peak with a fivefold increase in biological activity. This peak was soluble in alcohols and tetrahydrofuran, based on elution from reverse-phase Sep-Pak cartridges. Stability of the peak was increased by EDTA or ascorbic acid (10 mM). Storage for six weeks in ascorbic acid at 4 °C allowed recovery of 47.3% of the original material.


Do Fragments and Crystallization Additives Bind Similarly to Drug-like Ligands?
The success of fragment-based drug design (FBDD) hinges upon the optimization of low-molecular-weight compounds (MW < 300 Da) with weak binding affinities to lead compounds with high affinity and selectivity. Usually, structural information from fragment-protein complexes is used to develop ideas about the binding mode of similar but drug-like molecules. In this regard, crystallization additives such as cryoprotectants or buffer components, which are highly abundant in crystal structures, are frequently ignored. Thus, the aim of this study was to investigate the information present in protein complexes with fragments as well as those with additives and how they relate to the binding modes of their drug-like counterparts. We present a thorough analysis of the binding modes of crystallographic additives, fragments, and drug-like ligands bound to four diverse targets of wide interest in drug discovery and highly represented in the Protein Data Bank: cyclin-dependent kinase 2, β-secretase 1, carbonic anhydrase 2, and trypsin. We identified a total of 630 unique molecules bound to the catalytic binding sites, among them 31 additives, 222 fragments, and 377 drug-like ligands. In general, we observed that, independent of the target, protein-fragment interaction patterns are highly similar to those of drug-like ligands and mostly cover the residues crucial for binding. Crystallographic additives are also able to show conserved binding modes and recover the residues important for binding in some of the cases. Moreover, we show evidence that the information from fragments and drug-like ligands can be applied to rescore docking poses in order to improve the prediction of binding modes.


Protein Delivery of an Artificial Transcription Factor Restores Widespread Ube3a Expression in an Angelman Syndrome Mouse Brain.
Angelman syndrome (AS) is a neurological genetic disorder caused by loss of expression of the maternal copy of UBE3A in the brain. Due to brain-specific genetic imprinting at this locus, the paternal UBE3A is silenced by a long antisense transcript. Inhibition of the antisense transcript could lead to unsilencing of paternal UBE3A, thus providing a therapeutic approach for AS. However, widespread delivery of gene regulators to the brain remains challenging. Here, we report an engineered zinc finger-based artificial transcription factor (ATF) that, when injected i.p. or s.c., crossed the blood-brain barrier and increased Ube3a expression in the brain of an adult mouse model of AS. The factor displayed widespread distribution throughout the brain. Immunohistochemistry of both the hippocampus and cerebellum revealed an increase in Ube3a upon treatment. An ATF containing an alternative DNA-binding domain did not activate Ube3a. We believe this to be the first report of an injectable engineered zinc finger protein that can cause widespread activation of an endogenous gene in the brain. These observations have important implications for the study and treatment of AS and other neurological disorders.


[Effect of the type of reinforcement on conditioned reflex activity in male and female white rats].
On 24 males and 26 females of white rats of a mixed line, a study was carried out of the speed of formation and consolidation of instrumental drinking and defensive conditioned reflexes, elaboration of differentiation, changes in conditioned activity by the use of stochastic stereotype and by changing the meanings of the conditioned signals to opposite. Conditioned reflexes were elaborated more rapidly in female rats at drinking reinforcement and in male rats--at the defensive one. Differentiations were elaborated more rapidly and were better expressed in females than in males at both types of reinforcement.


Effects of collagen gel configuration on behavior of vascular smooth muscle cells in vitro: association with vascular morphogenesis.
The growth, behavior, and contractile protein expression of rabbit aortic smooth muscle cells (SMC) grown on, between layers, or within a collagen gel was investigated by confocal laser scanning fluorescence microscopy and Western analysis. SMC grown on collagen gel behaved similarly to those on conventional culture dishes. However, when a second layer of collagen was overlaid, cells underwent an elongated quiescent phase before onset of proliferation and a more than threefold lower logarithmic growth rate was observed. These cells self-organized into a network with ring-like structures. With increasing culture time, some of the rings developed into funnel-like, incomplete or complete tubular structures. If a tubular template preexisted within the gel, the SMC established a cylinder-shaped tube with several circularly arranged muscular layers (similar to an artery wall). This behavior mimicked endothelial cells during angiogenesis in vitro. A similar phenomenon occurred in cultures in which SMC were randomly mixed in a collagen gel, but here their behavior and morphology varied with their position within the gel. Western blot analysis showed that the SMC differentiation marker, smooth muscle myosin heavy chain-2 (SM-2), rapidly decreased, disappearing by day 10 in SMC grown on collagen, but was still detectable until day 25 in cells cultured between or within the same gel. These findings indicate that like endothelial cells, vascular SMC can display blood vessel formation behavior in vitro when an appropriate three-dimensional matrix environment is provided to keep them in a relatively higher-differentiated and low-proliferative state.


Steroid hormone regulation of vasopressinergic neurotransmission in the central nervous system.
Vasopressinergic neurotransmission is intimately linked to steroid hormone signaling. Both arginine vasopressin (VP) and the extrahypothalamic VP V1a receptors are regulated by steroid hormones. Here, we present work that has been done in our laboratory, investigating mechanisms underlying steroid hormone effects on the expression of both VP and its primary receptor in the brain, the VP V1a receptor. Data on VP receptors, their coupling to second messenger pathways, their localization in brain, and their regulation by peptide exposure are discussed. We also cover the regulation of the V1a receptor by adrenal hormones, and the molecular basis of this effect. Evidence for the existence of other receptors for VP in the brain is presented. Lastly, the regulation of the VP peptide by gonadal hormones is discussed at the transcriptional level in the rodent brain. Finally, the potential significance of the 'cross-talk' between the vasopressinergic system and the steroid hormone system is addressed.


Relationship between the DAT1 gene and the effects of methylphenidate administration in adult attention deficit hyperactivity disorder: a magnetic resonance spectroscopy study.
This study investigated the relationship between DAT1 gene polymorphisms and the effects of methylphenidate (MPH) administration on N-acetyl aspartate (NAA), creatine (Cr), and choline (Cho) levels in the anterior cingulate cortex, prefrontal cortex, striatum, and cerebellum in adult patients with attention deficit hyperactivity disorder (ADHD). This was the first study to investigate the relationship between DAT gene variable number tandem repeat (VNTR) polymorphisms and the responses of brain metabolites to MPH. Samples in this study were collected from 60 patients aged between 18 and 60 years with ADHD according to DSM-IV criteria. Genetic analysis of DAT1 gene polymorphisms was carried out using blood samples obtained after a detailed clinical evaluation. Levels of NAA, Cr, and Cho were measured in the anterior cingulate cortex, prefrontal cortex, striatum, and cerebellum by magnetic resonance spectroscopy. After this evaluation, 10 mg of MPH was given orally to patients, and the levels of the same metabolites were measured 30 min later. No marked difference in NAA, Cr, or Cho levels was detected before and after MPH administration with respect to the DAT1 gene VNTR polymorphisms. A considerable increase in Cr levels in the cerebellum was identified after MPH administration in individuals with the 10/10 repeat genotype as the DAT1 VNTR polymorphism (p=0.008). An increase in the previously decreased blood flow after MPH therapy may induce an increase in creatine levels in patients with the 10/10 repeat genotype. Our results thus suggest that the 10R allele as the DAT1 gene VNTR polymorphism might be associated with MPH-related changes in brain metabolites in adults with ADHD.


Effect of cyclooxygenase-2 inhibition with rofecoxib on endothelial dysfunction and inflammatory markers in patients with coronary artery disease.
The aim of this study was to determine whether selective cyclooxygenase-2 (COX-2) inhibition with rofecoxib can modulate endothelial dysfunction and levels of circulating inflammatory markers in patients with established coronary artery disease (CAD). Expression of COX-2 is upregulated in atherosclerosis. Thus, it has been hypothesized that COX-2 may contribute to atherogenesis by producing eicosanoids, which mediate vascular inflammation and endothelial dysfunction. In a randomized, double-blind, placebo-controlled, parallel-design trial, we studied the vascular effects of rofecoxib on brachial artery vasoreactivity and inflammatory markers in 60 patients with angiographically proven CAD who were taking concomitant low-dose aspirin. Patients were randomly assigned to receive either rofecoxib (25 mg/day; n = 30) or placebo (n = 30) for eight weeks. Brachial artery endothelium-dependent flow-mediated dilation (FMD), endothelium-independent nitroglycerin-mediated dilation (NMD), and inflammatory markers (i.e., high-sensitivity C-reactive protein [CRP], soluble intercellular adhesion molecule-1 [sICAM-1], and soluble interleukin-6 receptor [sIL-6r]) were measured at baseline and after eight-week follow-up. Baseline clinical characteristics were similar in the two groups. After eight weeks of treatment, FMD did not significantly change in either the rofecoxib or placebo group (4.0 +/- 3.0% to 4.0 +/- 3.8% vs. 2.7 +/- 2.7% to 3.1 +/- 2.7%, respectively; p = 0.6 by two-way analysis of variance). Similarly, NMD remained unchanged in both groups. Levels of CRP, sICAM-1, and sIL-6r were not significantly altered in either the rofecoxib or placebo group. The addition of selective COX-2 inhibition with rofecoxib did not appear to have any favorable or adverse effects on endothelial dysfunction or vascular inflammation in patients with CAD using concomitant low-dose aspirin.


New knowledge for old credences: asymmetric information search about in-group and out-group members.
Three experiments examined how people gather information on in-group and out-group members. Previous studies have revealed that category-based expectancies bias the hypothesis-testing process towards confirmation through the use of asymmetric-confirming questions (which are queries where the replies supporting the prior expectancies are more informative than those falsifying them). However, to date there is no empirical investigation of the use of such a question-asking strategy in an intergroup context. In the present studies, participants were asked to produce (Study 1) or to choose (Studies 2 and 3) questions in order to investigate the presence of various traits in an in-group or an out-group member. Traits were manipulated by valence and typicality. The results revealed that category-based expectancies do not always lead to asymmetric-confirming testing: whereas participants tended to ask questions that confirmed positive in-group and negative out-group stereotypical attributes, they used a more symmetric strategy when testing for the presence of negative in-group or positive out-group traits. Moreover, Study 3 also revealed a moderation effect of in-group identification. The findings point to the role played by motivational factors associated with preserving a positive social identity. Possible consequences of these hypothesis-testing processes in preserving a positive social identity for intergroup relations are discussed.


miRNAs as biomarkers of neurodegenerative disorders.
Neurodegenerative diseases (NDDs) are the result of progressive deterioration of neurons, ultimately leading to disabilities. There is no effective cure for NDDs at present; ongoing therapies are mainly aimed at treating the most bothersome symptoms. Since early treatment is crucial in NDDs, there is an urgent need for specific and sensitive biomarkers that can aid in early diagnosis of these disorders. Recently, altered expression of miRNAs has been implicated in several neurological disorders, including NDDs. miRNA expression has been extensively investigated in the cells, tissues and body fluids of patients with different types of NDDs. The aim of this review is to provide a comprehensive overview of miRNAs as biomarkers and therapeutic targets for NDDs.


Screening of copper and palladium-mediated reactions compatible with DNA-encoded chemical libraries.
The construction of DNA-encoded chemical libraries (DECLs) crucially relies on the availability of chemical reactions, which are DNA-compatible and which exhibit high conversion rates for a large number of diverse substrates. In this work, we present our optimization and validation procedures for three copper and palladium-catalyzed reactions (Suzuki cross-coupling, Sonogashira cross-coupling and copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC)), which have been successfully used by our group for the construction of large encoded libraries.


Expression of long noncoding RNA Xist is induced by glucocorticoids.
Glucocorticoids (GCs) are potent anti-inflammatory and immunosuppressive agents. However, their clinical usage is limited by severe multisystemic side effects. Glucocorticoid induced osteoporosis results in significant morbidity and mortality but the cellular and molecular mechanisms underlying GC-induced bone loss are not clear. GC use results in decreased osteoblast differentiation with increased marrow adiposity through effects on bone marrow stem cells. GC effects are transduced through its receptor (GR). To identify novel GR regulated genes, we performed RNA sequencing (RNA-Seq) analysis comparing conditional GR knockout mouse made by crossing the floxed GR animal with the Col I promoter-Cre, versus normal floxed GR without Cre, and that testing was specific for Col I promoter active cells, such as bone marrow mesenchymal stem/osteoprogenitor cells (MSCs) and osteoblasts. Results showed 15 upregulated genes (3- to 10-fold) and 70 downregulated genes (-2.7- to -10-fold), with the long noncoding RNA X-inactive specific transcript (Xist) downregulated the most. The differential expression of genes measured by RNA-Seq was validated by qRT-PCR analysis of selected genes and the GC/GR signaling-dependent expression of Xist was further demonstrated by GC (dexamethasone) treatment of GR-deficient MSCs in vitro and by GC injection of C57BL/6 mice (wild-type males and females) in vivo. Our data revealed that the long noncoding RNA Xist is a GR regulated gene and its expression is induced by GC both in vitro and in vivo. To our knowledge, this is the first evidence showing that Xist is transcriptionally regulated by GC/GR signaling.


Trials of interferon in respiratory infections of man.
Human leucocyte interferon has been shown to prevent rhinovirus infection if given intranasally to human volunteers. This finding encourages further investigation, but it is too soon to say whether interferon is likely to be clinically useful in serious virus infections of the respiratory tract.


Recombinant gp120 specifically enhances tumor necrosis factor-alpha production and Ig secretion in B lymphocytes from HIV-infected individuals but not from seronegative donors.
The effect of recombinant protein from the envelope (gp120) of the HIV on B lymphocytes purified from either HIV-infected individuals or healthy seronegative controls was examined. B cells from peripheral blood and lymph nodes of HIV-infected individuals spontaneously secreted TNF-alpha; this secretion was augmented by the presence of gp120, whereas B cells from healthy seronegative donors failed to secrete significant levels of TNF-alpha in the presence or absence of gp120. In a coculture system of B cells and chronically HIV-infected T cells (ACH-2), where viral expression is largely mediated by TNF-alpha, gp120 increased virus expression only if the B cells were obtained from HIV-infected individuals. The effects of gp120 on viral expression in this system were not mediated via CD4 receptor binding or FcR binding of anti gp120-gp120 immune complexes. Besides its effect on cytokine production, gp120 also stimulated Ig secretion in B cells from HIV-infected individuals, but not from normal donors. Finally, it was demonstrated by in situ hybridization that germinal centers of lymph nodes from HIV-infected individuals contain large amounts of HIV RNA that is in close proximity to germinal center B cells. These findings suggest that the hyperplastic germinal centers of lymph nodes provide an unique environment for virus expression and accumulation where gp120 stimulates B cells to secrete HIV inductive cytokines, such as IL-6 and TNF-alpha, and thereby further enhances virus expression in infected cells in a paracrine manner.


Latency of herpesvirus of turkey and Marek's disease virus genomes in a chicken T-lymphoblastoid cell line.
The properties of latent herpesvirus of turkey (HVT) and Marek's disease virus (MDV) genomes have been studied in virus-non-producer MDCC-BO1(T) cells, a T-lymphoblastoid cell line derived from spleen cells of an HVT-vaccinated chicken. The numbers of the two virus genomes in BO1(T) cells remained stable at 1.6 to 1.8 HVT genome equivalents/cell and 3.4 to 3.8 MDV genome equivalents/cell throughout a number of passages and were not decreased by the presence of phosphonoacetic acid in the culture. When the culture temperature of the MDV-producer MDCC-MSB1 cell line was shifted from 41 to 37 degrees C, the cells cultured at 37 degrees C contained about five times as many virus genomes as those cultured at 41 degrees C. In contrast, the numbers of the two virus genomes in BO1(T) cells were not increased by culture at 37 degrees C. RNA extracted from BO1(T) whole cells and from the polyribosomal fraction hybridized to both MDV and HVT DNAs, indicating the expression of both latent virus genomes. Digestion of cell nuclei with micrococcal nuclease revealed that both latent HVT and MDV genomes possess a nucleosomal structure. Closed circular MDV DNA was demonstrated in BO1(T) by isopycnic centrifugation of DNA in ethidium-bromide-CsCl gradients.


Tatumella ptyseos gen. nov., sp. nov., a member of the family Enterobacteriaceae found in clinical specimens.
The name Tatumella ptyseos gen. nov., sp. nov., is proposed for a group of organisms (previously called group EF-9) isolated from clinical sources in the United States, Canada, and Puerto Rico. A total of 68% of these isolates were from sputum specimens. T. ptyseos strains are gram-negative, oxidase-negative, fermentative rods that grow on MacConkey agar. The distinctive biochemical characteristics of 44 T. ptyseos isolates were as follows: acid but no gas from D-glucose, sucrose, and, usually (71%), D-xylose (62% delayed); no acid from lactose, maltose, or D-mannitol; negative tests for indole, urea, methyl red, gelatin, L-lysine decarboxylase, and L-ornithine decarboxylase; L-arginine dihydrolase variable; phenylalanine deaminase positive; Voges-Proskauer positive by the Coblentz method but negative by the O'Meara method; nonmotile at 36 degrees C but 66% weakly motile (30% delayed) at 25 degrees C; Simmons citrate positive at 25 degrees C (89%) but Simmons citrate negative at 36 degrees C. Deoxyribonucleic acid-deoxyribonucleic acid relatedness studies on 26 T. ptyseos strains showed that they were 80 to 100% related at 60 degrees C, which indicated that they comprise a single species. The deoxyribonucleic acid relatedness to other species within the Enterobacteriaceae was 7 to 38%. This is evidence that this species belongs in this family, is distinct from all described species and is best placed in a new genus. The T. ptyseos isolates studied were susceptible to all of the antimicrobial agents tested by broth dilution; these antimicrobial agents were amikacin, ampicillin, cephalothin, chloramphenicol, gentamicin, kanamycin, tetracycline, and tobramycin. Three striking differences between T. ptyseos and other members of the Enterobacteriaceae were its large zone of inhibition around penicillin (mean diameter 24 mm), its tendency to die on some laboratory media (such as blood agar) within 7 days, and its small number (usually one) of flagella. Strain H36 (=ATCC 33301, =CDC D6168, =CDC 9591-78) is the type strain of this new species. T. ptyseos is the type species for the genus Tatumella.


[Anxiety and panic in emergency medicine--the right diagnosis and treatment].
Fearful patients are in emergency situation often inattentive, unable to concentrate, agitated or even aroused. They show reduced perception and restricted willingness to cooperate. In severe conditions these patients are strongly tending towards more hazardous behavior: refusal of necessary therapy, break out or even high suicidal risk. Within disaster situations (mass accidents, fires) fearful patients with their agitated and persuasive behavior can influence other victims and with that trigger a situation of mass panic that has to be avoided at any cost. Therefore these patients must be swiftly identified and separated from the event. A diligent diagnosis process including physical-neurological examination is necessary. The recommended treatment within the emergency situation consists of a close continuous personal contact through assuring and encouraging conversations. A sense of security should be created by explaining the planned therapeutic interventions in simple, easy-to-follow and understandable words. If this necessary psycho-therapeutic intervention can not be applied a short term psychopharmacological treatment is required preferably with Benzodiazepines. Still a longterm specific therapy is highly advised, since these disturbances, if left untreated, will lead to a chronic manifestation and with that to considerable psychosocial impairments.


L-buthionine sulfoximine potentiates the antitumor effect of 4-hydroperoxycyclophosphamide when administered locally in a rat glioma model.
L-buthionine sulfoximine (BSO) inhibits glutathione synthesis and may modulate tumor resistance to some alkylating agents, but it has not been proven effective in the treatment of intracranial neoplasms. To evaluate this drug for the treatment of brain tumors, we studied the use of BSO for potentiating the antineoplastic effect of 4-hydroxyperoxycyclophosphamide (4-HC) in the rat 9L glioma model. The survival of male Fischer 344 rats with intracranial 9L gliomas was measured after implantation of controlled-release polymers containing one of the following: no drug, BSO, 4-HC, or both BSO and 4-HC. The efficacy of intracranial 4-HC treatment was assessed with and without serial systemic intraperitoneal BSO injections. Tissue glutathione levels were measured in the brains, tumors, and livers of animals treated with intraperitoneal injections or local delivery of BSO. The median survival of animals treated with intracranial polymers containing 4-HC was 2.3 times greater than that of controls. This survival benefit was doubled by local delivery of BSO. In contrast, systemic BSO therapy did not improve survival time. In animals that were treated systemically, both liver and tumor glutathione levels were significantly lower than they were in control animals. In the locally treated animals, glutathione levels were reduced in the brain tumor but not in the liver. These results demonstrate that local but not systemic delivery of BSO enhances the antineoplastic effect of 4-HC in this rat 9L glioma model. In addition, because local delivery of BSO within the brain did not deplete glutathione levels systemically, this method of treatment may be safer than systemic administration of BSO.


Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice.
Induction of phase 2 enzymes, which neutralize reactive electrophiles and act as indirect antioxidants, appears to be an effective means for achieving protection against a variety of carcinogens in animals and humans. Transcriptional control of the expression of these enzymes is mediated, at least in part, through the antioxidant response element (ARE) found in the regulatory regions of their genes. The transcription factor Nrf2, which binds to the ARE, appears to be essential for the induction of prototypical phase 2 enzymes such as glutathione S-transferases (GSTs) and NAD(P)H:quinone oxidoreductase (NQO1). Constitutive hepatic and gastric activities of GST and NQO1 were reduced by 50-80% in nrf2-deficient mice compared with wild-type mice. Moreover, the 2- to 5-fold induction of these enzymes in wild-type mice by the chemoprotective agent oltipraz, which is currently in clinical trials, was almost completely abrogated in the nrf2-deficient mice. In parallel with the enzymatic changes, nrf2-deficient mice had a significantly higher burden of gastric neoplasia after treatment with benzo[a]pyrene than did wild-type mice. Oltipraz significantly reduced multiplicity of gastric neoplasia in wild-type mice by 55%, but had no effect on tumor burden in nrf2-deficient mice. Thus, Nrf2 plays a central role in the regulation of constitutive and inducible expression of phase 2 enzymes in vivo and dramatically influences susceptibility to carcinogenesis. Moreover, the total loss of anticarcinogenic efficacy of oltipraz in the nrf2-disrupted mice highlights the prime importance of elevated phase 2 gene expression in chemoprotection by this and similar enzyme inducers.


China, the United States, and competition for resources that enable emerging technologies.
Historically, resource conflicts have often centered on fuel minerals (particularly oil). Future resource conflicts may, however, focus more on competition for nonfuel minerals that enable emerging technologies. Whether it is rhenium in jet engines, indium in flat panel displays, or gallium in smart phones, obscure elements empower smarter, smaller, and faster technologies, and nations seek stable supplies of these and other nonfuel minerals for their industries. No nation has all of the resources it needs domestically. International trade may lead to international competition for these resources if supplies are deemed at risk or insufficient to satisfy growing demand, especially for minerals used in technologies important to economic development and national security. Here, we compare the net import reliance of China and the United States to inform mineral resource competition and foreign supply risk. Our analysis indicates that China relies on imports for over half of its consumption for 19 of 42 nonfuel minerals, compared with 24 for the United States-11 of which are common to both. It is for these 11 nonfuel minerals that competition between the United States and China may become the most contentious, especially for those with highly concentrated production that prove irreplaceable in pivotal emerging technologies.


Effects of gonadectomy on indomethacin-induced ulceration and peptic activity in rats.
Castration in rats caused a reduction in the degree of ulceration produced by indomethacin. Compared with the normal intact male rats, the value of the castrated rats was highly significant (18.42 +/- 0.22 compared with 9.58 +/- 0.17) (P less than 0.01). However, the normal intact male rats had a greater degree of ulceration than the female rats at the pro-oestrous (2.33 +/- 0.13) or oestrous (2.97 +/- 0.12) phases. The ovariectomized rats, however, showed no significant reduction (P greater than 0.01) in mean ulcer score when compared with the di-oestrous female rats. The mean value of peptic activity was very high in ovariectomized rats while it was reduced in intact female rats. The ovariectomized rats were more prone to ulceration than intact female rats although the susceptibility increased in rats at di-oestrus.


Passive sodium and potassium movements in sickle erythrocytes.
Deoxygenation causes an increase in passive Na and K movements across the membrane of the sickle erythrocyte. Some investigators find that these ion movements are accompanied by cell dehydration, while others find no evidence for cell water loss with sickling. Because gelation of hemoglobin S would be enhanced by cell water loss, we reinvestigated Na and K movements in sickle cells to define further the role that ion movements might play in the pathogenesis of sickling. With deoxygenation, we found that sickle cells gained Na and lost K without losing cell water. These net ion movements were not seen in control red blood cells. For sickle cells, deoxygenation also increased passive unidirectional influxes of Na and K, effects not observed when control red blood cells were deoxygenated. The deoxygenation-induced passive influxes of Na and K in sickle cells were not diminished by anion substitution or by the addition of the diuretic furosemide. We also found differences in passive Na and K fluxes between oxygenated sickle cells and normal red blood cells. The addition of furosemide or replacement of Cl with NO3 or SCN, maneuvers that largely reduced passive Na and K movements in oxygenated normal cells, had no effect on Na and K movements in oxygenated sickle cells. These findings militate against the idea that solute and water loss occur as a consequence of deoxygenation but do indicate that there are acquired membrane abnormalities in sickle red blood cells.


Artificial enzyme innovations in electrochemical devices: advancing wearable and portable sensing technologies.
With the rapid evolution of sensing technologies, the integration of nanoscale catalysts, particularly those mimicking enzymatic functions, into electrochemical devices has surfaced as a pivotal advancement. These catalysts, dubbed artificial enzymes, embody a blend of heightened sensitivity, selectivity, and durability, laying the groundwork for innovative applications in real-time health monitoring and environmental detection. This minireview penetrates into the fundamental principles of electrochemical sensing, elucidating the unique attributes that establish artificial enzymes as foundational elements in this field. We spotlight a range of innovations where these catalysts have been proficiently incorporated into wearable and portable platforms. Navigating the pathway of amalgamating these nanoscale wonders into consumer-appealing devices presents a multitude of challenges; nevertheless, the progress made thus far signals a promising trajectory. As the intersection of materials science, biochemistry, and electronics progressively intensifies, a flourishing future seems imminent for artificial enzyme-infused electrochemical devices, with the potential to redefine the landscapes of wearable health diagnostics and portable sensing solutions.


A simple self-rating assessment method of residual work capability for occupational permanent disabilities.
To explore the validity, reliability, and determinants of a simple self-rating assessment method of residual work capability (RWC) after occupational permanent disabilities. Five hundred and thirty-nine compensated permanent disability workers answered three consecutive visual analogue questionnaires wherein they self-rated their residual work capability in terms of speed (RWCS), quality (RWCQ) and a combination of speed and quality (RWCC). At two major hospitals in Taiwan, 169 of these subjects were evaluated with physical capacity assessment (PCA), cognition and sensation assessment (CSA), the work ability index (WAI), and the 12-item Chinese health questionnaire (CHQ-12). High test-retest reliability (Pearson's correlation coefficient 0.77) and satisfactory concurrent validity were shown for RWCS and RWCC. All PCA, CSA and WAI showed significant correlation with RWCs, while CHQ-12 displayed borderline correlation. Employment status after injury and status of the victim's salary as the main source of income for his/her family before injury, were the major determinants of RWCs, in addition to the scales of PCA, CSA and WAI. The RWCC resulting from the self-rating method may be used as a simple assessment of a victim's residual work capability after occupational permanent disabilities.


Greater tree cover near residence is associated with reduced allostatic load in residents of central North Carolina.
Among urban residents, increased contacts with nature are associated with reduced morbidity and mortality. The concept of allostatic load, a biomarker-based composite measure of physiological dysregulation, can be applied to study subclinical benefits of exposure, and to elucidate pathways leading to improved health. This research explored associations between residential vegetated land cover and an allostatic load index calculated using the statistical distance measure known as Mahalanobis distance. This cross-sectional population-based study involved 186 adult residents of the Durham-Chapel Hill, North Carolina metropolitan area. Measures of tree and grass cover within 500 m of residence were derived from the U.S. Environmental Protection Agency's EnviroAtlas land cover database. Fifteen biomarkers of immune, neuroendocrine, and metabolic functions were analyzed in serum samples. Regression analysis was conducted using generalized additive models with thin-plate spline functions of geographic coordinates, adjusting for modelled traffic air pollution from local sources and sociodemographic covariates. The second and third tertiles of distance-weighted tree cover were associated with 14% (95% Confidence Limits 20%; 8%) and 15% (21%; 8%) reduction in adjusted median allostatic load, respectively, compared to the first tertile. The same tertiles of tree cover were also associated with 0.16 (0.03; 0.76) and 0.04 (0.01; 0.35) adjusted odds ratios of having allostatic load index above the 90th percentile of the sample distribution. Grass cover was inversely correlated with tree cover and was not associated with reduced allostatic load. Subclinical beneficial health effects of green spaces demonstrated in this study are consistent with reduced susceptibility to acute environmental and social stressors, and reduced risks of morbidity and mortality.


Malignant solitary fibrous tumors of the left atrial endocardium.
Solitary fibrous tumors are typically benign and usually develop in the pleura. We herein report the first case of a solitary fibrous tumor that was pathologically malignant and developed in the left atrial endocardium. A 24-year-old woman underwent resection of a malignant solitary fibrous tumor in her right forearm at another hospital. Computed tomography demonstrated a mass in her right pleura 2 months after the surgery. She was referred to our hospital, and a tumor in her left atrium was subsequently found. She underwent resection of these tumors, and pathological examination showed that they were both malignant solitary fibrous tumors.


Measurement of suppressor transfer RNA activity.
Transfer RNA (tRNA) suppression of nonsense mutations in prokaryotic systems has been widely used to study the structure and function of different prokaryotic genes. Through genetic engineering techniques, it is now possible to introduce suppressor (Su+) tRNA molecules into mammalian cells. A quantitative assay of the suppressor tRNA activity in these mammalian cells is described; it is based on the amount of tRNA-mediated readthrough of a terminating codon in the influenza virus NS1 gene after the cells are infected with virus. Suppressor activity in L cells continuously expressing Su+ (tRNAtyr) was 3.5 percent and that in CV-1 cells infected with an SV40- Su+ (tRNAtyr) recombinant was 22.5 percent.


Tumor-suppressive effect of retinoid receptor-induced gene-1 (RRIG1) in esophageal cancer.
We previously showed that induction of retinoid receptor-induced gene-1 (RRIG1) expression inhibited RhoA activation and tumor cell colony formation, invasion, and proliferation, and these effects are associated with the suppression of extracellular signal-regulated protein kinases 1 and 2 phosphorylation and cyclooxygenase-2 expression. To further elucidate its role in tumor cell growth, gene expression, and tumorigenesis, we determined RRIG1 expression in breast and esophageal tissue specimens and then stably transfected RRIG1 into a TE-8 esophageal squamous cell carcinoma (SCC) cell line. We found that RRIG1 was expressed in normal mammary glands (10 of 10) but not all ductal carcinoma in situ [11 of 19 (57.9%), P = 0.018] and invasive cancer [14 of 30 (46.7%), P = 0.0023] tissues. Similarly, RRIG1 was expressed in normal esophageal epithelium (22 of 22) but not all dysplastic [6 of 43 (14%), P = 0.0001] and SCC [50 of 122 (41%), P = 0.0001] tissues. Furthermore, RRIG1 expression correlated positively with tumor differentiation but inversely with lymph node metastasis of esophageal SCC. Finally, the stable transfection of RRIG1 inhibited esophageal SCC cell growth and the expression of extracellular signal-regulated protein kinases 1 and 2 and cell cycle-related genes (e.g., cyclin D1, phosphorylated Rb, and E2F). RRIG1-transfected sublines also inhibited tumor development in nude mice. The results of this study indicate that RRIG1 plays a role in suppressing tumorigenesis.


TL1A increases expression of CD25, LFA-1, CD134 and CD154, and induces IL-22 and GM-CSF production from effector CD4 T-cells.
Elevated levels of the cytokine TL1A is associated with several autoimmune diseases e.g. rheumatoid arthritis and inflammatory bowel disease. However, the exact role of TL1A remains elusive. In this study, we investigated the function of TL1A in a pro-inflammatory setting. We show that TL1A together with IL-12, IL-15 and IL-18 increases expression of the co-stimulatory molecules CD154 (CD40 ligand) and CD134 (OX40) on previously activated CD4+ T cells. This indicates that TL1A functions as a co-stimulatory molecule, decreasing the activation threshold of T-cells. We have previously shown that TL1A co-stimulation strongly induces IL-6 in human healthy leukocytes. Interestingly, the cytokine-activated effector T-cells did not produce IL-6 in response to TL1A, indicating distinct effects of TL1A on different cell populations. We further show that this co-stimulation increases the expression of CD25 (IL-2Rα) and CD11a (α-chain of LFA-1) on CD4 T-cells, likely governing increased IL-2/IL-15 sensitivity and cell-cell contact. Along with this, TL1A co-stimulation caused a specific induction of IL-22 and GM-CSF from the activated T-cells. These results substantially contribute to the explanation of TL1A's role in inflammation. Our results suggest that TL1A should be considered as a target for immunotherapeutic treatment of rheumatoid arthritis and inflammatory bowel disease.


A Group-Based Sexual Risk Reduction Intervention for Men Who Have Sex With Men in Ho Chi Minh City, Vietnam: Feasibility, Acceptability, and Preliminary Efficacy.
An emerging HIV epidemic can be seen among men who have sex with men (MSM) in Vietnam. There are currently no evidence-based behavioral sexual risk reduction interventions for MSM in this setting. Between October 2012 and June 2013, 100 high-risk MSM from Ho Chi Minh City were enrolled in an open pilot trial to assess feasibility and acceptability of a group-based, manualized sexual risk reduction intervention, and to preliminarily examine changes in primary and secondary outcomes. Participants completed a behavioral assessment battery and HIV testing at baseline, 3, and 6 months post-baseline. Over 80.0 % of the sample was <25 years old and 77.0 % identified as Bong kin ("hidden," masculine-appearing). Feasibility and acceptability of the program was evidenced by 87.0 % retention for the intervention sessions, 78.0 % completion of the 6 month assessment, and positive responses on evaluation forms and qualitative exit interviews. There was a decline in the number of condomless anal sex acts from baseline (6.32) to 3 month (2.06) and 6 month (2.49) follow-up (p < .0001). These data support the need for further testing of this group-based, behavioral HIV prevention intervention to reduce sexual risk behavior among MSM in Vietnam in a randomized controlled efficacy trial.


The Superior Mesenteric Artery: From Syndrome in the Young to Vascular Atherosclerosis in the Old.
This review is directed at increasing awareness of two diverse rare upper gastrointestinal problems that occur at opposite ends of the age spectrum and are difficult to diagnose and treat. The Superior Mesenteric Artery Syndrome (SMAS) likely involves a young patient, especially female, and is especially associated with rapid weight loss, resulting in relative strangulation of the duodenum by a narrowing of the angle between the Superior Mesenteric Artery (SMA) and the aorta. On the other hand, atherosclerosis of the SMA is associated most likely with postprandial upper intestinal ischemia and abdominal pain occurs in the elderly at high risk for cardiovascular (CV) disease. Medical management of the SMAS in the young involves good alimentation and weight gain to overall increase the intestinal fat pad. Medical management of SMA atherosclerotic ischemia in the elderly is directed at marked lipid lowering with atherosclerotic plaque stabilization or even regression. If needed, surgery for SMAS can be attempted laparoscopically with duodenojejunoscopy which is the most popular procedure but there are also more conservative possibilities that avoid division of the duodenum. In addition, sometimes direct vision is needed to successfully operate on SMAS. If surgery is needed for SMA atherosclerotic ischemia, it is usually attempted endoscopically with angioplasty and stent placement. Most important, in the case of these two rare clinical entities, is that the clinician have a suspicion of their presence when indicated so that the young or old patient can be spared unnecessary suffering and return to good health in a timely fashion.


[Extracardiac manifestations of undifferentiated connective tissue dysplasia].
Undifferentiated connective tissue dysplasia (UDCTD) is a common type of congenital mesenchymal defect. Among the visceral manifestations of UDCTD, cardiac anomalies have been studied in detail while the nature, extent, and clinical significance of changes in other viscera are much less known. The review summaries data on currently accepted extracardiac manifestations of UDCTD, such as venous insufficiency, tracheobronchial dyskinesia, nephroptosis, genital prolapse, gastrointestinal disorders, hemostatic pathology.


Inhibin removes the inhibitory effects of activin on steroid enzyme expression and androgen production by normal ovarian thecal cells.
Activin and inhibin are important local modulators of theca cell steroidogenesis in the ovary. Using a serum-free primary theca cell culture system, this study investigated the effects of inhibin on theca cell androgen production and expression of steroidogenic enzymes. Androstenedione secretion from theca cells cultured in media containing activin, inhibin and follistatin was assessed by RIA over 144 h. Activin (1-100 ng/ml) suppressed androstenedione production. Inhibin (1-100 ng/ml) blocked the suppressive effects of added activin, but increased androstenedione production when added alone, suggesting it was blocking endogenous activin produced by theca cells. Addition of SB-431542 (activin receptor inhibitor) and follistatin (500 ng/ml) increased androstenedione production, supporting this concept. Infection of theca cells with adenoviruses expressing inhibitory Smad6 or 7 increased androstenedione secretion, confirming that the suppressive effects of activin required activation of the Smad2/3 pathway. Activin decreased the expression levels of steroidogenic acute regulatory protein (STAR), whereas STAR expression was increased by inhibin and SB-431542, alone and in combination. CYP11A was unaffected. The expression of CYP17 encoding 17α-hydroxylase was unaffected by activin but increased by inhibin and SB-431542, and when added in combination the effect was further enhanced. The expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) was significantly decreased by activin, while inhibin alone and in combination with SB-431542 both potently increased the expression of 3β-HSD. In conclusion, activin suppressed theca cell androstenedione production by decreasing the expression of STAR and 3β-HSD. Inhibin and other blockers of activin action reversed this effect, supporting the concept that endogenous thecal activin modulates androgen production in theca cells.


Enhanced Local Disorder in a Clinically Elusive von Willebrand Factor Provokes High-Affinity Platelet Clumping.
Mutation of the cysteines forming the disulfide loop of the platelet GPIbα adhesive A1 domain of von Willebrand factor (VWF) causes quantitative VWF deficiencies in the blood and von Willebrand disease. We report two cases of transient severe thrombocytopenia induced by DDAVP treatment. Cys1272Trp and Cys1458Tyr mutations identified by genetic sequencing implicate an abnormal gain-of-function phenotype, evidenced by thrombocytopenia, which quickly relapses back to normal platelet counts and deficient plasma VWF. Using surface plasmon resonance, analytical rheology, and hydrogen-deuterium exchange mass spectrometry (HXMS), we decipher mechanisms of A1-GPIbα-mediated platelet adhesion and resolve dynamic secondary structure elements that regulate the binding pathway. Constrained by the disulfide, conformational selection between weak and tight binding states of A1 takes precedence and drives normal platelet adhesion to VWF. Less restrained through mutation, loss of the disulfide preferentially diverts binding through an induced-fit disease pathway enabling high-affinity GPIbα binding and firm platelet adhesion to a partially disordered A1 domain. HXMS reveals a dynamic asymmetry of flexible and ordered regions common to both variants, indicating that the partially disordered A1 lacking the disulfide retains native-like structural dynamics. Both binding mechanisms share common structural and thermodynamic properties, but the enhanced local disorder in the disease state perpetuates high-affinity platelet agglutination, characteristic of type 2B VWD, upon DDAVP-stimulated secretion of VWF leading to transient thrombocytopenia and a subsequent deficiency of plasma VWF, characteristic of type 2A VWD.


Using ordinal logistic regression to estimate the likelihood of colorectal neoplasia.
The utility of ordinal logistic regression in the prediction of colorectal neoplasia was demonstrated in a group of 461 consecutive patients undergoing colonoscopy in a community practice. One hundred twenty-nine patients had adenomatous polyps and 34 had colorectal adenocarcinoma. An ordinal logistic regression model developed in a random subset (292 patients) identified five predictors of colorectal neoplasia. Colorectal neoplasia risk could be predicted using the patient's age, sex, hematocrit, fecal occult blood test result and indication for colonoscopy. The risk of colorectal neoplasia in the remaining subset of patients (169) could be reliably estimated from the model. Ordinal logistic regression analysis in this select group of patients can accurately estimate the likelihood of colorectal neoplasia. Because the generalizability of our findings are unknown, the model should not be applied to other patients. However, application of this technique to an unselected group of patients not already referred for colonoscopy could provide unbiased estimates of colorectal neoplasia risk in individual patients.


['The spirit has left the bottle': the medieval Arabic physician 'Abd al-Latĭf ibn Yŭsuf al-Baghdădĭ: his medical work and his bizarre affiliation with twentieth-century spiritualism].
The Arabic physician 'Abd al-Latĭf ibn Yŭsuf al-Baghdădĭ, lived at the crossroads of the twelfth and the thirteenth century. His unbridled curiosity and his unquenchable thirst for knowledge of any kind brought him to far-away countries and regions and put him in contact with all sorts and conditions of people. The great Egyptian famine of the years 1200-1202 enabled him to study and examine thousands of human cadavers and skeletons at first hand. This led to a new understanding of the anatomical structure of the human body, and rejected the more or less antiquated ideas of the Greek doctor Galen of Pergamum. However, 'Abd al-Latĭf's vision was granted only a short life. After his death, his discovery sank into oblivion and as a consequence it was never again mentioned in Arabic medical manuals. From then on the Arabic physicians once more referred to the anatomical data which were developed and taught by Galen. Relatively few specimens of his remaining medical work were preserved for posterity. However, his Book of the two advices (or: K. al-Nasĭhatain) is of the utmost importance as a source for the medical thinking and the medical treatment in the late twelfth and the early thirteenth century A.D. During the years following World War I, 'Abd al-Latĭf's name reappeared within the spiritualistic movement in England. He became known as Abduhl Latif the great Persian physician and acted as a control of mediums. Until the late sixties, he practised the art of healing as the head of a medical mission somewhere in the Spheres.


Regulation of T cell differentiation: in vitro induction of 20 alpha-hydroxysteroid dehydrogenase in splenic lymphocytes from athymic mice by a unique lymphokine.
The enzyme 20 alpha-hydroxysteroid dehydrogenase (20 alpha SDH) has previously been shown to be a specific enzyme marker of mature T cells. In vivo, the expression of 20 alpha SDH is thymus dependent, in that splenic lymphocytes from athymic mice have only low levels of activity, although the levels of enzyme activity increase gradually with age. In vitro, 20 alpha SDH can be induced in splenic lymphocytes from nu/nu mice by conditioned media from mitogen- or alloantigen-stimulated normal lymphocytes. Induction is rapid in vitro. Beginning after a lag period of approximately 6 hr, enzyme activity increases linearly for approximately 20 to 30 hr resulting in a 5- to 10-fold increase in activity. Induction is blocked by mitomycin C, suggesting a requirement for cell proliferation. The phenotype of both the precursor and the induced lymphocyte populations is Thy 1.2-, Lyt 1-, 2-, suggesting that induction of 20 alpha SDH expression is an early step in T cell differentiation. The factor responsible for 20 alpha SDH induction has been partially purified and is distinct from other known lymphokines in both its biochemical and functional properties. The term interleukin 3 is proposed for this factor.


Origin and mechanisms of Ca2+ waves in smooth muscle as revealed by localized photolysis of caged inositol 1,4,5-trisphosphate.
The cytosolic Ca(2+) concentration ([Ca(2+)](c)) controls diverse cellular events via various Ca(2+) signaling patterns; the latter are influenced by the method of cell activation. Here, in single-voltage clamped smooth muscle cells, sarcolemma depolarization generated uniform increases in [Ca(2+)](c) throughout the cell entirely by Ca(2+) influx. On the other hand, the Ca(2+) signal produced by InsP(3)-generating agonists was a propagated wave. Using localized uncaged InsP(3), the forward movement of the Ca(2+) wave arose from Ca(2+)-induced Ca(2+) release at the InsP(3) receptor (InsP(3)R) without ryanodine receptor involvement. The decline in [Ca(2+)](c) (the back of the wave) occurred from a functional compartmentalization of the store, which rendered the site of InsP(3)-mediated Ca(2+) release, and only this site, refractory to the phosphoinositide. The functional compartmentalization arose by a localized feedback deactivation of InsP(3) receptors produced by an increased [Ca(2+)](c) rather than a reduced luminal [Ca(2+)] or an increased cytoplasmic [InsP(3)]. The deactivation of the InsP(3) receptor was delayed in onset, compared with the time of the rise in [Ca(2+)](c), persisted (>30 s) even when [Ca(2+)](c) had regained resting levels, and was not prevented by kinase or phosphatase inhibitors. Thus different forms of cell activation generate distinct Ca(2+) signaling patterns in smooth muscle. Sarcolemma Ca(2+) entry increases [Ca(2+)](c) uniformly; agonists activate InsP(3)R and produce Ca(2+) waves. Waves progress by Ca(2+)-induced Ca(2+) release at InsP(3)R, and persistent Ca(2+)-dependent inhibition of InsP(3)R accounts for the decline in [Ca(2+)](c) at the back of the wave.


A proteomic approach for the elucidation of the specificity of ectodomain shedding.
Ectodomain shedding (shedding) is a posttranslational modification mechanism, which liberates extracellular domains of membrane proteins through juxtamembrane processing. Because shedding alters cell characteristics in a rapid and irreversible manner, it must be strictly regulated. However, the regulatory mechanisms of shedding in response to environmental changes remain obscure. To evaluate the regulatory mechanisms of endogenous shedding, we previously developed a proteomic screening system to identify shedding targets. This system revealed a comprehensive picture of membrane proteins shed under defined conditions. In this study, we have improved the screening system to compare the shedding patterns in a mouse macrophage cell line treated with two different shedding inducers, lipopolysaccharide (LPS) and 12-O-tetradecanoylphorbol 13-acetate (TPA). We show here that LPS simultaneously activates the shedding of multiple membrane proteins. We further show that TPA specifically activates the shedding of αM/β2 integrin (Mac-1), which was not shed upon LPS-stimulation of macrophages. These results clearly demonstrate that the regulation of endogenous membrane protein shedding is both stimulus- and substrate-specific. The shedding targets reported to date play pivotal roles in a variety of biological phenomena, including the immunological response, cell growth, cell adhesion and cell movement. In addition, several disease-related membrane proteins are shedding targets. Thus, understanding the regulation of shedding is important for the elucidation of pathogenesis and the development of therapeutic strategies. We submit that a comprehensive characterization of endogenous shedding is indispensable for understanding the regulatory mechanisms of shedding, and thus have developed a proteomic screening system to identify shedding targets. In this study, using our screening system, we demonstrate that different extracellular stimuli activate different types of shedding, even in a single cell. Our results prove that this proteomic approach is quite effective for the elucidation of the regulatory mechanisms of shedding.


Targeting ribosome biogenesis reinforces ERK-dependent senescence in pancreatic cancer.
Pancreatic adenocarcinomas (PDAC) often possess mutations in K-Ras that stimulate the ERK pathway. Aberrantly high ERK activation triggers oncogene-induced senescence, which halts tumor progression. Here we report that low-grade pancreatic intraepithelial neoplasia displays very high levels of phospho-ERK consistent with a senescence response. However, advanced lesions that have circumvented the senescence barrier exhibit lower phospho-ERK levels. Restoring ERK hyperactivation in PDAC using activated RAF leads to ERK-dependent growth arrest with senescence biomarkers. ERK-dependent senescence in PDAC was characterized by a nucleolar stress response including a selective depletion of nucleolar phosphoproteins and intranucleolar foci containing RNA polymerase I designated as senescence-associated nucleolar foci (SANF). Accordingly, combining ribosome biogenesis inhibitors with ERK hyperactivation reinforced the senescence response in PDAC cells. Notably, comparable mechanisms were observed upon treatment with the platinum-based chemotherapy regimen FOLFIRINOX, currently a first-line treatment option for PDAC. We thus suggest that drugs targeting ribosome biogenesis can improve the senescence anticancer response in pancreatic cancer.


[Social aspects in fighting against tuberculosis].
In 1884 Robert Koch postulated in his essay on "The Etiology of Tuberculosis" favouring moments for the settling of tubercle bacilli to explain the predisposition and diversified course of tuberculosis in grown-up persons. Under the conception "disposition" he also subsumised exogen factors beside the internal conditions and proclaimed that all steps undertaken against tuberculosis should take into account the social conditions. Only to effect the treatment in a sanatorium for such patients, it is also necessary to care for their families and for the patients themselves after they left the sanatorium. Complex fighting programmes developed according to the thorough analysis of the social surroundings, including the influences of occupation, income, expenses for food, housing and clothing, the legislation with regard to the health law and education to health duty as well as the level of medical science. Briefly, there was stated that the tuberculosis mortality depends on the standard of life. From the war-epidemiology of tuberculosis at least 3 parallel curves resulted, decisively influencing the mortality, the curves of infection and charging with work as well as the curve of nourishment. So the tuberculosis was characterised as a social disease. No wonder that social appearances--and war is one of them--stamp the special picture. As a model of applied knowledge, the strategy of fighting tuberculosis in the GDR and its outstanding results are discussed. After the antituberculosis chemotherapy was introduced, which shortens the duration of the disease and removes its chronic relapsing course, the significance of the social factors, however, decreased, but concerning the situation in the Third World, their knowledge is of great importance and not only of historical interest.


Characterization of the structure and interactions of P450 BM3 using hybrid mass spectrometry approaches.
The cytochrome P450 monooxygenase P450 BM3 (BM3) is a biotechnologically important and versatile enzyme capable of producing important compounds such as the medical drugs pravastatin and artemether, and the steroid hormone testosterone. BM3 is a natural fusion enzyme comprising two major domains: a cytochrome P450 (heme-binding) catalytic domain and a NADPH-cytochrome P450 reductase (CPR) domain containing FAD and FMN cofactors in distinct domains of the CPR. A crystal structure of full-length BM3 enzyme is not available in its monomeric or catalytically active dimeric state. In this study, we provide detailed insights into the protein-protein interactions that occur between domains in the BM3 enzyme and characterize molecular interactions within the BM3 dimer by using several hybrid mass spectrometry (MS) techniques, namely native ion mobility MS (IM-MS), collision-induced unfolding (CIU), and hydrogen-deuterium exchange MS (HDX-MS). These methods enable us to probe the structure, stoichiometry, and domain interactions in the ∼240 kDa BM3 dimeric complex. We obtained high-sequence coverage (88-99%) in the HDX-MS experiments for full-length BM3 and its component domains in both the ligand-free and ligand-bound states. We identified important protein interaction sites, in addition to sites corresponding to heme-CPR domain interactions at the dimeric interface. These findings bring us closer to understanding the structure and catalytic mechanism of P450 BM3.


Glucan-induced keratoderma in acquired immunodeficiency syndrome.
Six of 20 patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex receiving intravenous infusions of soluble glucan (beta-1-3 polyglucose) developed a keratoderma of the palms and soles. The eruption began during the first two weeks of therapy and resolved two to four weeks after its discontinuation. The eruption was different in appearance from our previously reported keratoderma blennorrhagica in AIDS-associated psoriasis. None of the other 735 patients with AIDS or AIDS-related complex not treated with soluble glucan developed a similar keratoderma. The correlation between receiving glucan and the hyperkeratosis is highly significant. Since glucan is a naturally occurring component of the cell walls of yeast, fungus, and some bacterial organisms, recognition of its ability to induce such a striking reaction pattern may be of general significance and interest, although the reaction itself may be limited to patients with AIDS.


Essential Oils from Zingiber striolatum Diels Attenuate Inflammatory Response and Oxidative Stress through Regulation of MAPK and NF-κB Signaling Pathways.
Zingiber striolatum Diels (Z. striolatum), a widely popular vegetable in China, is famous for its medicinal and nutritional values. However, the anti-inflammatory effects of essential oil from Z. striolatum (EOZS) remain unclear. In this study, EOZS from seven regions in China were extracted and analyzed by GC-MS. LPS-induced RAW264.7 cells and 12-O-Tetradecanoylphorbol 13-acetate (TPA)-stimulated mice were used to evaluate the anti-inflammatory effects of EOZS. Results show that 116 compounds were identified in EOZS from seven locations. Samples 2, 4 and 5 showed the best capability on DPPH radical scavenging and NO inhibition. They also significantly reduced the production of ROS, pro-inflammatory cytokines, macrophage morphological changes, migration and phagocytic capability. Transcriptomics revealed MAPK and NF-κB signaling pathways may be involved in the anti-inflammatory mechanism, and the predictions were proven by Western blotting. In TPA-induced mice, EOZS reduced the degree of ear swelling and local immune cell infiltration by blocking the activation of MAPK and NF-κB signaling pathways, which was consistent with the in vitro experimental results. Our research unveils the antioxidant capability and potential molecular mechanism of EOZS in regulating inflammatory response, and suggests the application of EOZS as a natural antioxidant and anti-inflammatory agent in the pharmaceutical and functional food industries.


Early prediction of outcome in head-injured patients.
The relationship between Glasgow Coma Scale (GSC) scores obtained during the 1st week after head injury and outcome at 1 year was analyzed in 170 patients. Seventy-two of 76 patients with initial GCS scores of 3 or 4 lived, and only one had a favorable outcome. Favorable and unfavorable outcomes were almost equally divided when the initial GCS scores were in the intermediate range of 5, 6, or 7. No patients with an initial GCS score in this intermediate range that subsequently worsened had a favorable outcome, while over 80% of those improving to a score higher than 7 had a favorable outcome. Only 12% of those persisting with a score of 5, 6, or 7 for 1 week had favorable outcome. Outcome predictions using the multiple logistic model were made for this intermediate group of patients based on GCS scores and data on midline shift derived from computerized tomography (CT). The patients with initial scores of 5, 6, or 7 with midline shifts of less than 4.1 mm on initial CT scanning had a significantly higher favorable outcome rate compared with patients with a larger shift. However, outcome prediction made by combining shift data and initial GCS scores are not significantly more accurate than predictions based solely on initial GCS scores. Combining 48-hour GCS scores and shift data significantly improves predictive accuracy based only on coma scores. The data obtained by combining GCS scores at 72 hours and 1 week and shift data is marginally significant for improving accuracy of outcome predictions. It is concluded that GCS scores and shift data are highly accurate indicators of outcome in head-injured patients.


Altering toluene 4-monooxygenase by active-site engineering for the synthesis of 3-methoxycatechol, methoxyhydroquinone, and methylhydroquinone.
Wild-type toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 oxidizes toluene to p-cresol (96%) and oxidizes benzene sequentially to phenol, to catechol, and to 1,2,3-trihydroxybenzene. In this study T4MO was found to oxidize o-cresol to 3-methylcatechol (91%) and methylhydroquinone (9%), to oxidize m-cresol and p-cresol to 4-methylcatechol (100%), and to oxidize o-methoxyphenol to 4-methoxyresorcinol (87%), 3-methoxycatechol (11%), and methoxyhydroquinone (2%). Apparent Vmax values of 6.6 +/- 0.9 to 10.7 +/- 0.1 nmol/min/ mg of protein were obtained for o-, m-, and p-cresol oxidation by wild-type T4MO, which are comparable to the toluene oxidation rate (15.1 +/- 0.8 nmol/min/mg of protein). After these new reactions were discovered, saturation mutagenesis was performed near the diiron catalytic center at positions I100, G103, and A107 of the alpha subunit of the hydroxylase (TmoA) based on directed evolution of the related toluene o-monooxygenase of Burkholderia cepacia G4 (K. A. Canada, S. Iwashita, H. Shim, and T. K. Wood, J. Bacteriol. 184:344-349, 2002) and a previously reported T4MO G103L regiospecific mutant (K. H. Mitchell, J. M. Studts, and B. G. Fox, Biochemistry 41:3176-3188, 2002). By using o-cresol and o-methoxyphenol as model substrates, regiospecific mutants of T4MO were created; for example, TmoA variant G103A/A107S produced 3-methylcatechol (98%) from o-cresol twofold faster and produced 3-methoxycatechol (82%) from 1 mM o-methoxyphenol seven times faster than the wild-type T4MO (1.5 +/- 0.2 versus 0.21 +/- 0.01 nmol/min/mg of protein). Variant I100L produced 3-methoxycatechol from o-methoxyphenol four times faster than wild-type T4MO, and G103S/A107T produced methylhydroquinone (92%) from o-cresol fourfold faster than wild-type T4MO and there was 10 times more in terms of the percentage of the product. Variant G103S produced 40-fold more methoxyhydroquinone from o-methoxyphenol than the wild-type enzyme produced (80 versus 2%) and produced methylhydroquinone (80%) from o-cresol. Hence, the regiospecific oxidation of o-methoxyphenol and o-cresol was changed for significant synthesis of 3-methoxycatechol, methoxyhydroquinone, 3-methylcatechol, and methylhydroquinone. The enzyme variants also demonstrated altered monohydroxylation regiospecificity for toluene; for example, G103S/A107G formed 82% o-cresol, so saturation mutagenesis converted T4MO into an ortho-hydroxylating enzyme. Furthermore, G103S/A107T formed 100% p-cresol from toluene; hence, a better para-hydroxylating enzyme than wild-type T4MO was formed. Structure homology modeling suggested that hydrogen bonding interactions of the hydroxyl groups of altered residues S103, S107, and T107 influence the regiospecificity of the oxygenase reaction.


Recent advances and future directions in targeting the secretory apparatus in multiple myeloma.
Multiple myeloma is a genetically heterogeneous tumour of transformed plasma cells, terminally differentiated effectors of the B cell lineage specialized in producing large amounts of immunoglobulins. The uniquely well-developed secretory apparatus that equips normal and transformed plasma cells with the capacity for high-level protein secretion constitutes a distinctive therapeutic target. In this review we discuss how fundamental cellular processes, such as the unfolded protein response (UPR), endoplasmic reticulum (ER)-associated degradation and autophagy, maintain intracellular protein homeostasis (proteostasis) and regulate plasma cell ontogeny and malignancy. We summarize our current understanding of the cellular effects of proteasome inhibitors and the molecular bases of resistance to them. Furthermore, we discuss how improvements in our understanding of the secretory apparatus and of the complex interactions between intracellular protein synthesis and degradation pathways can disclose novel drug targets for multiple myeloma, defining a paradigm of general interest for cancer biology and disorders of altered proteostasis.