Insights in the regulation of trimetylamine N-oxide production using a comparative biomimetic approach suggest a metabolic switch in hibernating bears.
Experimental studies suggest involvement of trimethylamine N-oxide (TMAO) in the aetiology of cardiometabolic diseases and chronic kidney disease (CKD), in part via metabolism of ingested food. Using a comparative biomimetic approach, we have investigated circulating levels of the gut metabolites betaine, choline, and TMAO in human CKD, across animal species as well as during hibernation in two animal species. Betaine, choline, and TMAO levels were associated with renal function in humans and differed significantly across animal species. Free-ranging brown bears showed a distinct regulation pattern with an increase in betaine (422%) and choline (18%) levels during hibernation, but exhibited undetectable levels of TMAO. Free-ranging brown bears had higher betaine, lower choline, and undetectable TMAO levels compared to captive brown bears. Endogenously produced betaine may protect bears and garden dormice during the vulnerable hibernating period. Carnivorous eating habits are linked to TMAO levels in the animal kingdom. Captivity may alter the microbiota and cause a subsequent increase of TMAO production. Since free-ranging bears seems to turn on a metabolic switch that shunts choline to generate betaine instead of TMAO, characterisation and understanding of such an adaptive switch could hold clues for novel treatment options in burden of lifestyle diseases, such as CKD.


Accessibility issues with long-term disabilities.
Home modifications for barrier-free accessibility will assist the physically challenged populations by increasing their independence. By providing an accessible environment, an individual can become more independent and require less assistance for functional activities, such as kitchen appliance access, door widening, open floor plan, elevated electric outlets, roll-under sinks, roll-in showers and MobiLife elevating wheelchair.


Antimutagenicity of hops (Humulus lupulus L.): bioassay-directed fractionation and isolation of xanthohumol.
Bioassay-directed fractionation with a Salmonella/microsomal assay against the food borne mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was used to identify antimutagenic components of hops. Hops pellets extracted with diethylether showed antimutagenic activity against mutations induced by IQ. Fractionation of the diethylether extract (DE) by column chromatography, followed by semi-preparative HPLC yielded two fractions (E4b and E4d) with strong antimutagenic activity against IQ induced mutations. Separation of fraction E4b resulted in inactive fractions, while fraction E4d has been identified to be xanthohumol. In mammalian test system with human hepatoma HepG2 cells fraction E4d at 10mug/ml completely prevented formation of IQ induced DNA damage. These results indicate that xanthohumol is a very promising potential protective agent against genotoxicity of food borne carcinogens, which warrants further investigation.


Respiration in man affected by TVR contractions elicited in inspiratory and expiratory intercostal muslces.
Vibration-induced effects on respiration in man were studied by recording the electrical activity (EMG) from the intercostal muscles and the diaphragm. The vibration was applied in an upper thoracic region where inspiratory muscle activity prevailed or in a lower thoracic region where expiratory muscle activity prevailed. The effects were also studied by recording the movements of the thorax and the respiratory air flow. Sustained vibration in the upper region enhanced the activity of the underlying inspiratory muscles and caused an expansion of the rib cage whereas it had little or weak effects on diaphragm-activity or on expiratory intercostal muscles. Sustained vibration in the lower region enhanced the activity of the underlying expiratory muscles, often inhibited the inspiratory activity and caused a depression of the rib cage. It also tended to inhibit the diaphragm activity. It was also found that bilateral vibration, timed by the respiratory movements and alternating between upper and lower regions could aid or counteract the ventilation if it was applied respectively 'in phase' or 'out of phase' with the rhythmical contractions in the underlying muscles. The motor responses described are largely explicable in terms of tonic vibration reflexes (TVR) arising in the inspiratory and expiratory intercostal muscles underlying the vibrators.


Sensorimotor integration to cutaneous afferents in humans: the effect of the size of the receptive field.
Transcranial magnetic stimulation (TMS) can be used to study sensorimotor integration in humans non-invasively. Motor excitability has been found to be inhibited when afferent stimuli are given to a peripheral nerve and precede TMS at interstimulus intervals (ISIs) of 20-50 ms. This phenomenon has been referred to as short-latency afferent inhibition (SAI). To better understand the functional meaning of these phenomena, we examined the effect of the size of the receptive field on SAI to cutaneous afferents in upper-limb sensorimotor areas in humans. We examined the effect of the stimulation of the isolated right first (D1), second (D2) and third finger (D3), the right second and third finger together (D23) and the right first three fingers together (D123) on the amplitude of motor evoked potentials (MEPs) to TMS in hand and forearm muscles. We examined the right abductor pollicis brevis (APB), first dorsal interosseous (FDI), extensor carpi radialis (ECR) and flexor carpi radialis (FCR) muscles. Digital stimulation preceded TMS at ISIs of 20-50 ms. The effect of D2 stimulation was MEP inhibition (SAI), which was more marked and consistent in APB and FDI muscles than in ECR and FCR muscles. Similarly, D1 and D3 stimulation caused MEP reduction, while no MEP enhancement could be found to single finger stimulation. In contrast, D123 stimulation induced less effective SAI in upper-limb muscles. MEP potentiation was recorded in some muscles to D123 stimulation. A significant difference between D2 and D123 stimulation was found in APB (ISIs = 30-50 ms) and FDI (ISIs = 40-50 ms) muscles, but not in forearm muscles. The effect to D23stimulation on MEP amplitude was intermediate between those to D2 and D123 stimulation. Our data suggest that motor excitability to cutaneous afferents may be influenced by the size of the receptive fields, this effect being the result of increasing convergence between hand afferents in the somatosensory system. These phenomena appear to be topographically arranged across the representation of upper-limb muscles. These findings may help to understand the functional significance of SAI in normal physiology and pathophysiology.


Relative finger lengths, sex differences, and psychological traits.
Various finger length and personality and ability measures were obtained for a sample of Australian adolescent twins (306 boys and 397 girls). A new measure of relative finger length (the length of a given finger relative to the sum of all four fingers) was investigated, and shown to be superior to the traditional 2D:4D for discriminating between the sexes. It also had the advantage of permitting a more analytic approach: for example, the 2nd finger-length contributed much more than the 4th finger length to the sex difference in 2D:4D, and a smooth gradient of sex differences across the hand was evident. Sex differences on right hands were greater than those for left hands. Within-sex correlations were obtained between the various finger-length measures and a personality and an ability scale that showed relatively large sex differences (Eysenck's Psychoticism scale and the spatial subscale from Jackson's Multidimensional Aptitude Battery). The correlations were low, but on the whole consistent with the between-sex differences for the girls. For the boys, this was so for Psychoticism, but spatial ability was, if anything, correlated in the opposite direction.


Immunohistochemical detection of uPA, tPA, and PAI-1 in a stasis-induced deep vein thrombosis model and its application to thrombus age estimation.
We immunohistochemically examined the expression of urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), and plasminogen-activator inhibitor type-1 (PAI-1) using venous thrombi developed by ligation of the inferior vena cava (IVC) in mice. The uPA-, tPA- and PAI-1-positive cells could be firstly detected 5, 7, and 3 days, respectively, after IVC ligation. Morphometrically, the number of PAI-1-positive cells was significantly higher than those of uPA- and tPA-positive cells at later than 7 days. In all of the thrombus samples aged 10-21 days, the uPA/PAI-1 and tPA/PAI-1 ratios were >0.1 and >0.2, respectively. In contrast, all of the thrombus samples aged 1-7 days had uPA/PAI-1 of <0.1 and tPA/PAI-1 ratios of <0.2. These findings implied that uPA/PAI-1 of >0.1 and tPA/PAI-1 of >0.2 indicated an age of 10 days or more. Moreover, in four of five samples aged 10 days, uPA/PAI-1 ratios were <0.3, and the remaining one had uPA/PAI-1 of 0.32. All thrombi aged 14-21 days showed values greater than 0.3. Thus, uPA/PAI-1 ratios, markedly exceeding 0.3, strongly indicated an age of more than 14 days. The present study demonstrated that the immunohistochemical detection of uPA, tPA, and PAI-1 was suitable to estimate the age of venous thrombi.


T Cell Development and Function.
T cells play critical roles in the immune system, including in responses to cancer, autoimmunity, and tissue regeneration. T cells arise from common lymphoid progenitors (CLPs) that differentiate from hematopoietic stem cells in the bone marrow. CLPs then traffic to the thymus, where they undergo thymopoiesis through a number of selection steps, resulting in mature single positive naive CD4 helper or CD8 cytotoxic T cells. Naive T cells are home to secondary lymphoid organs like lymph nodes and are primed by antigen-presenting cells, which scavenge for both foreign and self-antigens. Effector T cell function is multifaceted, including direct target cell lysis and secretion of cytokines, which regulate the functions of other immune cells (refer to "Graphical Abstract"). This review will discuss T cell development and function, from the development of lymphoid progenitors in the bone marrow to principles that govern T cell effector function and dysfunction, specifically within the context of cancer.


Functional Monomerization of a ClC-Type Fluoride Transporter.
Anion channels and antiporters of the ClC superfamily have been found to be exclusively dimeric in nature, even though each individual monomer contains the complete transport pathway. Here, we describe the destabilization through mutagenesis of the dimer interface of a bacterial F(-)/H(+) antiporter, ClC(F)-eca. Several mutations that produce monomer/dimer equilibrium of the normally dimeric transporter were found, simply by shortening a hydrophobic side chain in some cases. One mutation, L376W, leads to a wholly monomeric variant that shows full activity. Furthermore, we discovered a naturally destabilized homologue, ClC(F)-rla, which undergoes partial monomerization in detergent without additional mutations. These results, in combination with the previous functional monomerization of the distant relative ClC-ec1, demonstrate that the monomer alone is the functional unit for several clades of the ClC superfamily.


Relative efficacy and safety of early lactate clearance-guided therapy resuscitation in patients with sepsis: A meta-analysis.
Compelling evidence has shown that aggressive resuscitation bundles are one of the cornerstones of the successful treatment of patients with sepsis. Recent studies suggest that lactate normalization during resuscitation is a more powerful indicator of resuscitative adequacy; however, early lactate clearance-guided therapy is still not recommended. We performed this meta-analysis to evaluate the effect of early lactate clearance-directed therapy as a potentially more effective resuscitation target. Studies were identified using PubMed, Embase, and the Cochrane Library without region, publication type, or language restrictions. Randomized trials were included when they compared the efficacy and safety of lactate clearance-guided resuscitation versus central venous oxygen saturation (ScvO2)-guided therapy. The primary outcome was mortality, and the secondary outcomes were intensive care unit (ICU) stay, length of hospital stay, mechanical ventilation time, Acute Physiology and Chronic Health Evaluation-II (APACHE-II) score, and Sepsis-related Organ Failure Assessment (SOFA) score. Seven randomized controlled trials encompassing 1301 cases were reviewed. Compared with guided ScvO2 therapy, early lactate clearance-directed therapy was associated with decreased in-hospital mortality (relative ratio [RR] 0.68, 95% confidence interval [CI] 0.56 to 0.82), shorter ICU stay (mean difference [MD] -1.64 days, 95% CI -3.23 to -0.05), shorter mechanical ventilation time (MD -10.22 hours, 95% CI -15.94 to -4.5), and lower APACHE-II scores (MD -4.47, 95% CI -7.25 to -1.69). However, patients undergoing early lactate clearance-guided therapy had similar lengths of hospital stay and similar SOFA scores. As a specific indicator of resuscitation outcome, lactate clearance alone is superior to ScvO2 alone during a standard resuscitation paradigm. The optimal or desired rate of lactate clearance is still a contentious area. To guide resuscitation and normalize lactate levels in patients, repeating lactate measurements every 2 hours until the patient has met a lactate clearance of 10% or greater may be helpful. PROSPERO CRD42018100515.


Evaluating U.S. gamers' metacognitions about digital entertainment: Validation of Metacognition about Online Gaming Scale in the U.S. context.
Online gaming has become an important practice for several audiences in the last decade. Video games like Fortnite and League of Legends are played by millions of people daily, raising concerns about both addiction and disruptive attitudes. Several analytical instruments have been deployed for exploring this phenomenon, including the Metacognitions about Online Gaming Scale (MOGS). MOGS measures the role of metacognitions toward online gaming, echoing the increasing importance of such a construct in media consumption and perception. This article presents the highlights of a study involving 690 American gamers aimed at two purposes. First, the MOGS was validated for with U.S. gamers, who represent a leading niche in the current digital entertainment and were not involved with the original MOGS development. Second, the three subscales of MOGS were analyzed as mediators of the relationship between hours spent playing online gaming and Internet Gaming Disorder (IGD). The results from Confirmatory Factor Analysis suggested the MOGS has promising reliability and validity for use with U.S. gamers. The results also indicated that three subscales of the MOGS partially mediated the relationship between hours spent on playing online gaming and Internet Gaming Disorder.


Clinicians' guide to antibiotics. Chloramphenicol.
Chloramphenicol, despite its well-recognized toxic side effects, is a cheap and useful antibiotic in the management of meningitis, typhoid, brain abscess and severe Haemophilus influenza infections. Where possible, drug levels should be monitored.


Nurses as practice change facilitators for healthy behaviors.
This study used nurses as practice change consultants to help primary care medical practices improve their delivery of health behavior services to patients. Nurse consultants worked with 20 practices from 2 healthcare systems. In each practice, the nurses helped clinicians and staff to develop a practice-specific protocol so that they could identify and intervene with the health behavior of their patients. As a result of the nurse consultant intervention, health behavior delivery was improved. This article describes the specific methods and the lessons learned through this study. We encourage practices to use nurse consultants as one way of improving quality of care.


High precision, simulated cutting efficiency measurement of endodontic root canal instruments: influence of file configuration and lubrication.
A high precision method is presented for measurement of root canal instrument cutting efficiency in pure, linear movements. Operator-independent instrumentation is achieved by means of an automatic, mechanical test rig mounted as an accessory in an Instron tensile testing machine. It is now possible to do reliable cutting simulation that closely represents clinical motions. Cutting efficiency is assessed via "wear" volume measurement by means of three-dimensional surface mapping on bovine bone serving as a dentin substitute. A volume precision of 3 x 10(-3) mm3 is achieved which is equivalent to a weight resolution better than 10 micrograms. In particular, cutting efficiency data for K-type and H-type files is presented showing an enhanced performance (by roughly a factor of 3) in the latter case. Lubrication is also shown to increase the cutting efficiency (+200% for the K file and +30% for the H file). Water and 2.5% sodium hypochlorite solutions have equivalent effects.


Differential chemical diagnosis of primary hyperoxaluria type II. Highly sensitive analysis of optical isomers of glyceric acid by GC/MS as diastereoisomeric derivatives.
We established a separation method for the optical isomers of glyceric acid in urine by modifying the derivatization steps of the procedure used for the screening and diagnosis. The trimethylsilyl derivatization step in the mass screening procedure was replaced by O-acetyl-(+)-2-butylation, and the samples were analyzed under equivalent GC/MS conditions by capillary gas chromatography on a DB-5MS column. This method can be applied to cases that show a high urinary concentration of glyceric acid to obtain a differential diagnosis of primary hyperoxaluria type II and d-glyceric aciduria easily. l-Glyceric acid was also isolated from the urine of healthy controls as one of the main peaks.


Twinfilin bypasses assembly conditions and actin filament aging to drive barbed end depolymerization.
Cellular actin networks grow by ATP-actin addition at filament barbed ends and have long been presumed to depolymerize at their pointed ends, primarily after filaments undergo "aging" (ATP hydrolysis and Pi release). The cytosol contains high levels of actin monomers, which favors assembly over disassembly, and barbed ends are enriched in ADP-Pi actin. For these reasons, the potential for a barbed end depolymerization mechanism in cells has received little attention. Here, using microfluidics-assisted TIRF microscopy, we show that mouse twinfilin, a member of the ADF-homology family, induces depolymerization of ADP-Pi barbed ends even under assembly-promoting conditions. Indeed, we observe in single reactions containing micromolar concentrations of actin monomers the simultaneous rapid elongation of formin-bound barbed ends and twinfilin-induced depolymerization of free barbed ends. The data show that twinfilin catalyzes dissociation of subunits from ADP-Pi barbed ends and thereby bypasses filament aging prerequisites to disassemble newly polymerized actin filaments.


Impact of COVID-19 pandemic on sickness absence for mental ill health in National Health Service staff.
To explore the patterns of sickness absence in National Health Service (NHS) staff attributable to mental ill health during the first wave of the COVID-19 epidemic in March-July 2020. Case-referent analysis of a secondary dataset. NHS Trusts in England. Pseudonymised data on 959 356 employees who were continuously employed by NHS trusts during 1 January 2019 to 31 July 2020. Trends in the burden of sickness absence due to mental ill health from 2019 to 2020 according to demographic, regional and occupational characteristics. Over the study period, 164 202 new sickness absence episodes for mental ill health were recorded in 12.5% (119 525) of the study sample. There was a spike of sickness absence for mental ill health in March-April 2020 (899 730 days lost) compared with 519 807 days in March-April 2019; the surge was driven by an increase in new episodes of long-term absence and had diminished by May/June 2020. The increase was greatest in those aged >60 years (227%) and among employees of Asian and Black ethnic origin (109%-136%). Among doctors and dentists, the number of days absent declined by 12.7%. The biggest increase was in London (122%) and the smallest in the East Midlands (43.7%); the variation between regions reflected the rates of COVID-19 sickness absence during the same period. Although the COVID-19 epidemic led to an increase in sickness absence attributed to mental ill health in NHS staff, this had substantially declined by May/June 2020, corresponding with the decrease in pressures at work as the first wave of the epidemic subsided.


Docosahexaenoic Acid Modulates Invasion and Metastasis of Human Ovarian Cancer via Multiple Molecular Pathways.
We investigated the effect of docosahexaenoic acid (DHA) on the invasion and metastasis of ovarian cancer cells (A2780, HO8910, and SKOV-3). Cytotoxicity assay was performed to determine the optimal doses of DHA in this experiment. The effects of DHA on invasion ability were assessed by invasion assay. The expressions of messenger RNA and/or proteins associated with invasion or metastasis were detected by quantitative Real Time-Polymerase Chain Reaction or Western blot. The effect of DHA on cell metastasis was assessed in xenograft model of zebrafish. Docosahexaenoic acid and α-linolenic acid could reduce the cell vitalities in dose-dependent manner. However, DHA inhibited the invasion and metastasis of ovarian cancer cells, but α-linolenic acid did not (**P < 0.01). Docosahexaenoic acid could downregulate the expressions of WAVE3, vascular endothelial cell growth factor, and MMP-9, and upregulate KISS-1, TIMP-1, and PPAR-γ, which negatively correlated with cell invasion and metastasis (*P < 0.05). Docosahexaenoic acid restrained the development of subintestinal vessels and cancer cell metastasis in xenograft model of zebrafish (**P < 0.01). Docosahexaenoic acid inhibited the invasion and metastasis of ovarian cancer cells in vitro and in vivo through the modulation of NF-κB signaling pathway, suggesting that DHA is a promising candidate for ovarian cancer therapy.


Interaction between variants in CLU and MS4A4E modulates Alzheimer's disease risk.
Ebbert et al. reported gene-gene interactions between rs11136000-rs670139 (CLU-MS4A4E) and rs3865444-rs670139 (CD33-MS4A4E). We evaluate these interactions in the largest data set for an epistasis study. We tested interactions using 3837 cases and 4145 controls from Alzheimer's Disease Genetics Consortium using meta-analyses and permutation analyses. We repeated meta-analyses stratified by apolipoprotein E (APOE) ε4 status, estimated combined odds ratio (OR) and population attributable fraction (cPAF), and explored causal variants. Results support the CLU-MS4A4E interaction and a dominant effect. An association between CLU-MS4A4E and APOE ε4 negative status exists. The estimated synergy factor, OR, and cPAF for rs11136000-rs670139 are 2.23, 2.45, and 8.0, respectively. We identified potential causal variants. We replicated the CLU-MS4A4E interaction in a large case-control series and observed APOE ε4 and possible dominant effect. The CLU-MS4A4E OR is higher than any Alzheimer's disease locus except APOE ε4, APP, and TREM2. We estimated an 8% decrease in Alzheimer's disease incidence without CLU-MS4A4E risk alleles and identified potential causal variants.


Evidence for a late chondritic veneer in the Earth's mantle from high-pressure partitioning of palladium and platinum
The high-pressure solubility in silicate liquids of moderately siderophile 'iron-loving' elements (such as nickel and cobalt) has been used to suggest that, in the early Earth, an equilibrium between core-forming metals and the silicate mantle was established at the bottom of a magma ocean. But observed concentrations of the highly siderophile elements--such as the platinum-group elements platinum, palladium, rhenium, iridium, ruthenium and osmium--in the Earth's upper mantle can be explained by such a model only if their metal-silicate partition coefficients at high pressure are orders of magnitude lower than those determined experimentally at one atmosphere (refs 3-8). Here we present an experimental determination of the solubility of palladium and platinum in silicate melts as a function of pressure to 16 GPa (corresponding to about 500 km depth in the Earth). We find that both the palladium and platinum metal-silicate partition coefficients, derived from solubility, do not decrease with pressure--that is, palladium and platinum retain a strong preference for the metal phase even at high pressures. Consequently the observed abundances of palladium and platinum in the upper mantle seem to be best explained by a 'late veneer' addition of chondritic material to the upper mantle following the cessation of core formation.


Focal gastric mucosal blood flow in aspirin-induced ulceration.
Focal gastric mucosal blood flow was studied during aspirin injury by hydrogen gas clearance in a chambered segment model of canine gastric corpus. Measurements were made simultaneously every 15 minutes at ulcerated and nonulcerated areas 1.5 hours before, during (20 mM of aspirin in 150 mM of HCl for 1 hour), and 2 hours after exposure of the mucosa to topical aspirin. There was a highly significant decrease (p less than 0.001) in flow at the ulcerated areas 30 minutes after exposure to aspirin, coinciding with the appearance of focal mucosal pallor followed by subsequent hemorrhagic foci and ulceration. This was not followed by recovery to basal flow values. Blood flow to the non-ulcerated areas was significantly but less severely reduced than in the ulcerated areas (p less than 0.05) 90 minutes after exposure to aspirin. This was followed by recovery to basal levels. It is proposed that aspirin induces reduction of focal mucosal blood flow of varying degrees and that mucosal areas with flow reduced to below a "critical value" develop gross damage.


A novel nanoemulsion vaccine induces mucosal Interleukin-17 responses and confers protection upon Mycobacterium tuberculosis challenge in mice.
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) is contracted via aerosol infection, typically affecting the lungs. Mycobacterium bovis bacillus Calmette-Guerin (BCG) is the only licensed vaccine and has variable efficacy in protecting against pulmonary TB. Additionally, chemotherapy is associated with low compliance contributing to development of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mtb. Thus, there is an urgent need for the design of more effective vaccines against TB. Experimental vaccines delivered through the mucosal route induce robust T helper type 17 (Th17)/ Interleukin (IL) -17 responses and provide superior protection against Mtb infection. Thus, the development of safe mucosal adjuvants for human use is critical. In this study, we demonstrate that nanoemulsion (NE)-based adjuvants when delivered intranasally along with Mtb specific immunodominant antigens (NE-TB vaccine) induce potent mucosal IL-17T-cell responses. Additionally, the NE-TB vaccine confers significant protection against Mtb infection, and when delivered along with BCG, is associated with decreased disease severity. These findings strongly support the development of a NE-TB vaccine as a novel, safe and effective, first-of-kind IL-17 inducing mucosal vaccine for potential use in humans.


Fine-tuning the oxidative ability of persistent radicals: electrochemical and computational studies of substituted 2-pyridylhydroxylamines.
N-tert-butyl-N-2-pyridylhydroxylamines were synthesized from 2-halopyridines and 2-methyl-2-nitrosopropane using magnesium-halogen exchange. The use of Turbo Grignard generated the metallo-2-pyridyl intermediate more reliably than alkyllithium reagents. The hydroxylamines were characterized using NMR, electrochemistry, and density functional theory. Substitution of the pyridyl ring in the 3-, 4-, and 5-positions was used to vary the potential of the nitroxyl/oxoammonium redox couple by 0.95 V. DFT computations of the electrochemical properties agree with experiment and provide a toolset for the predictive design of pyridyl nitroxides.


Enteric controlled-release pantoprazole-loaded microparticles prepared by using Eudragit S100 and poly(epsilon-caprolactone) blend.
Microparticles of poly(epsilon-caprolactone) and of its blend with Eudragit S100 were prepared by emulsion/solvent evaporation technique to provide controlled release and gastro-resistance for an acid labile drug. This drug was sodium pantoprazole, a proton pump inhibitor. Both formulations were successfully prepared, but only the microparticles prepared with the blend were capable of stabilizing the drug in the acid medium. Furthermore, this formulation showed in vivo protection of stomachs against ulceration caused by ethanol in rats. These microparticles were tabletted, and the tablets demonstrated slower drug release and higher acid protection than the microparticles before tabletting.


Immunomodulation of T and B cell functions in multiple myeloma patients treated with combined erythropoietin and alpha-interferon therapy.
T and B lymphocyte functions were investigated in the course of a long-term trial of recombinant human erythropoietin in patients with progressing multiple myeloma. Peripheral mononuclear cell as well as T and B lymphocyte cultures were established at the 1st, 13th, and last week of the 24-week protocol from 16 treated and 15 untreated patients. Control cultures from healthy individuals were also obtained. A suppression of phytohemagglutinin-induced proliferation of T cells was noted in all 1st-week cultures, whereas a variable increase of 3H-thymidine uptake was noted at the end of the trial in the cultures from erythropoietin-treated patients. A significant increase was observed, however, in cultures from 5 erythropoietin-treated patients who also received alpha-interferon when their cells were grown in the presence of the hormone. In contrast, the pokeweed mitogen-driven in vitro synthesis of immunoglobulins was not significantly influenced by the duration of erythropoietin treatment, nor by addition of the hormone. IgG secretion by Epstein-Barr virus-transformed B cells in cultures from 9 erythropoietin-treated and 6 untreated patients was enhanced in the presence of both recombinant human erythropoietin and alpha-interferon. These data suggest that synergy between the two cytokines may variably modulate certain immune functions in vitro. This effect might account for the increase of serum IgM levels noted in some patients who received alpha-interferon.


Methotrexate and 5-aminoimidazole-4-carboxamide riboside exert synergistic anticancer action against human breast cancer and hepatocellular carcinoma.
To investigate the influences of methotrexate (MTX) on the anticancer actions and pharmacokinetics of 5-aminoimidazole-4-carboxamide riboside (AICA riboside) in human breast cancer and hepatocellular carcinoma. Human breast cancer cell line MCF-7 and human hepatocellular carcinoma cell line HepG2 were examined. The cell proliferation was assessed using a sulforhodamine B assay. Western blotting and radioactivity assays were used to analyze the phosphorylation of AMPK. The DNA synthesis was analyzed with BrdU incorporation. Nude mice bearing MCF-7 cell xenografts were used to for in vivo study. MTX (50 mg/kg, ip, per week) and AICA riboside (200 mg/kg, ip, every other day) were administered the animals for 2 weeks. The concentrations of AICA riboside and its active metabolite AICA ribotide in the plasma and tumors were measured with HPLC. Synergistic cytotoxicity in vitro was observed with MTX (0.1, 0.5, and 1 μmol/L) combined with AICA riboside (0.25-1 mmol/L) in MCF-7 cells, and with MTX (0.5 and 1 μmol/L) combined with AICA riboside (0.5 and 1 mmol/L) in HepG2 cells. MTX (1 μmol/L) significantly enhanced the AICA riboside-induced AMPK activation and BrdU incorporation in both MCF-7 and HepG2 cells. Co-treatment with MTX and AICA riboside exerted more potent inhibition on the tumor growth in nude mice than either drug alone. After injection of AICA riboside (200 mg/kg, iv) in nude mice bearing MCF-7 xenografts, MTX (50 mg/kg, iv) significantly increased the concentrations of AICA riboside and its active metabolite AICA ribotide in tumors. MTX and AICA riboside exert synergistic anticancer action against MCF-7 and HepG2 cells in vitro and in vivo. MTX increases the concentration of AICA riboside and its active metabolite AICA ribotide in tumors in vivo.


Retrograde pyelography via ureteral stents.
Retrograde pyelography to establish ureteral patency prior to removal of ureteral stent can be done via the stent. A simple technique is described.


Event-related potential measures of consciousness: two equations with three unknowns.
This is a brief review of event-related brain potentials (ERPs) as indices of cortical information processing in conditions in which conscious perception of stimuli is supposed to be absent: sleep, coma, vegetative state, general anesthesia, neglect as well as presentation of subliminal or masked stimuli. Exogenous ERP components such as N1 and P2 are much more likely to remain in all these conditions than endogenous components. Further, all varieties of the late posterior positive ERP waves (e.g., P3b, P600, late positive complex) are most difficult to be elicited in such conditions, indicating that the cortical activity underlying the late posterior positivity may have a particularly close relationship to brain mechanisms of conscious perception. Contrary to what might be expected, reliable ERP effects indicating complex analysis of semantic stimulus features (i.e., meaning) can be recorded without conscious awareness, generally, as easy as (in some conditions, even easier than) ERP components related to rather simple physical stimulus features. It should be emphasized, however, that we never should overestimate our confidence about the degree of subjects' unawareness. Particularly in the conditions in which no behavioral response can be obtained (e.g., sleep, coma, anesthesia), residual conscious processing, at least in some subjects and on some trials, cannot be ruled out.


Tissue breakdown and exposure associated with orbital hydroxyapatite implants.
Tissue breakdown and exposure of a hydroxyapatite implant were observed in eight patients: in four of six patients after evisceration and in four of 31 after enucleation. The reasons for evisceration were a blind, painful eye and endophthalmitis in two patients each. The reasons for enucleation were a choroidal melanoma in two patients and endophthalmitis and irreparable traumatic damage in one patient each. The patients with endophthalmitis received the implant in a second surgical procedure after intensive antibiotic treatment. Small tissue defects healed spontaneously, whereas large defects showed little tendency to heal by secondary intention. Tissue breakdown over a hydroxyapatite implant may be related to delayed ingrowth of fibrovascular tissue, and possibly related to an inflammatory reaction incited by the hydroxyapatite. Careful case selection, facilitation of tissue penetration by drilling holes into the hydroxyapatite sphere, delayed fitting of the prosthesis, and vaulting of the posterior surface of the initial prosthesis to reduce pressure on the tissues covering the anterior pole of the implant may alleviate the problems of tissue breakdown and exposure.


Oxygen delivery at sea level and altitude (after slow ascent to 5000 meters), at rest and in mild exercise.
Oxygen delivery (DO2) calculated from cardiac output, haematocrit (Hct) and arterial oxygen saturation (SaO2), has been obtained on six subjects at sea level (London) and after slow ascent to 5000 meters (Chamlang base camp) at rest and during mild exercise (25 watts and 50 watts). Haematocrit was increased in all six subjects at 5000 m and expressed as haemoglobin (Hb) rose from a mean (+/- standard error; SEM) of 13.8 +/- 0.1 g (100 ml)(-1) to 15.8 +/-0.3 g (100 ml)(-1) (t = 6.3, p = 0.0014). SaO2 was almost constant with exercise at sea level (rest 98.5%, 25 w 98.3% and 50 w 98.3%) but declined more steeply with exercise at 5000 m (rest 88.8 +/-0.6%, 25 w 85.4 +/-0.4% and 50 w 84.4 +/- 0.5%). Arterial oxygen content (CaO2) was very similar for 25 watts exercise at altitude (5000 m, 18.1 ml per decilitre--dl) as at sea level (London, CaO2 18.2 ml dl(-1)). At rest CaO2 was higher at altitude (18.8 +/-0.2 ml dl(-1)) than at sea level (18.3 +/- 0.4 ml dl(-1)) and at 50 w CaO2 was lower at altitude (17.9 +/- 0.4 ml dl(-1)) than at sea level (18.2 +/- 0.2 ml dl(-1)). Hence, similar cardiac output values at rest (sea level, 5.0 +/- 0.4 litres min(-1) l min(-1); altitude, 5.6 +/- 0.31 min(-1)-) and at 25 w exercise (sea level, 8.2 +/-0.7 1 min(-1); altitude, 8.3 +/-0 .9 1 min'(-1) resulted in similar values for DO2 at rest (sea level, 0.9 +/-0.1 l min(-1) altitude, 1.0 +/-0.1 l min(-1) and 25 w exercise (sea level, 1.5 +/-0.1 l min(-1) altitude, 1.5 +/- 0.2 l min(-1). For 50 w exercise cardiac output and oxygen delivery were greater at altitude in one subject but were significantly reduced for the remaining five (cardiac output mean difference 3.0 +/- 0.91 min(-1), p = 0.015; DO2 mean difference, 0.56 +/- 0.21 l min(-1) p = 0.028). Acclimatization was therefore adequate to sustain a normal value for oxygen delivery for rest and 25 watts exercise (via compensatory erythropoiesis) but insufficient for 50-watt exercise in five of the six subjects.


The relationship between lower extremity injury and the hip abductor to extensor strength ratio in collegiate athletes.
To determine the relationship of previous lower extremity injury and the measured ratio of hip abduction to extension strength in collegiate athletes. Cohort study of college athletes at time of pre-participation screening physical. An NCAA Division I college. Two hundred and thirty-six college athletes from a NCAA Division I school (162) males and (74) females. The ratio of maximal hip abduction to extension strength was calculated, following raw data collection with a specially designed dynamometer anchoring station. Injury to the lower extremities, in the past year, was recorded via personal interview at the time of screening and verified by review of previous injury records. A significant difference in the ratio of hip abduction to extension strength was noted on the left lower extremity of athletes with reported lower extremity (LE) injury compared to those without injury. Upon further review of data, hip extension weakness appears to be the likely cause of this difference. Athletes with reported lower extremity injury demonstrated a significant residual difference in the ratio of hip abduction to extension strength. This may be the result of injury related muscle weakness, altered muscle firing patterns, central inhibition or unknown compensatory strategies which all may be risk factors for recurrent injury. Further research is underway to identify the cause/effect relationship of this finding. This study may advance our understanding of potential compensatory strategies about the hip which theoretically may result from previous lower extremity injury or injuries which are incompletely rehabilitated. Additionally, this study provides some reasoning to support the screening of hip strength during the pre-participation physical, as it may be an important factor to prevent recurrent injury.


Drinking water microbiology--new directions toward water quality enhancement.
Drinking water microbiology has emerged from decades of relative complacency to recognize there can be major concerns with potable water quality. Many of these issues are a result of an explosion of information on new waterborne agents, treatment problems with raw-source water qualities, biofilm development in some distribution systems and specialized requirements in water quality unique to hospitals and industries. Protozoan cyst survival after some disinfection practices involving surface water impoundments and virus occurrence in poorly protected groundwaters have provided reasons for expanding minimum treatment of surface waters and for requiring disinfection of all groundwaters unless there is a demonstrative data base to support exceptions in treatment requirements. Official monitoring of small water supplies must be increased on a monthly basis and a rapid alert established to inform water plant operators of unsatisfactory water qualities. As an option, application of operational tests to analyse water quality in terms of chlorine residual, turbidity, total coliforms and heterotrophic bacterial counts in small water plant operations should be encouraged. This would provide the operator at remote locations with the opportunity to utilize the information to make necessary treatment adjustments or corrections in water distribution deficiencies promptly and be a supplement to the official regional monitoring program. Application of drinking water alternative sources (bottled water and water from point-of-use treatment devices) should be viewed by the health authorities as only a temporary solution, not as a permanent fix for a public water supply known to present some established health risk to consumers. The public must also recognize that bottled water is not frequently monitored by health laboratories for acceptable quality and the use of home treatment devices places the responsibility of proper maintenance on the user. Microbial quality improvements in drinking water to hospitals and food industries can frequently be achieved through a routine, systematic flushing program for building plumbing networks and associated attachment devices. In other situations, use of booster disinfection or point-of-use devices may provide the important special water quality requirements for certain industrial applications. In any event, these supplemental treatment measures will require careful in-plant monitoring and maintenance to prevent reversals in water quality enhancement.


[Spreading and mechanisms of antimicrobial resistance of microorganisms, producing beta-lactamases. Phenotypical screening for MBL producers (carbapenemases B1) among strains of Pseudomonas genus, isolated in cases of nosocomial infections].
Intrahospital strains (215) of the bacterial genus Pseudomonas isolated from patients of 30 Medical centers of 15 Russian regions have been investigated for antibiotic resistance. The bacterial cultures resistant to imipenem and/or meropenem were considered as metallo-beta-lactamase (MBL) producers. Production of subclass B1 MBL (carbapenemases) was evaluated by means of the double-disk approximation test using MBL inhibitor, EDTA. There were 55 P. aeroginosa strains (25.6%) resistant to imipenem and meropenem simultaneously; 19 isolates (8.8%) of P. aeroginosa were characterized by synergism between carbapenem and EDTA. The subclass B1 MBL producers are widely distributed in the intrahospital strain obtained from Moscow, Yaroslavl, Ekaterinburg, Omsk, and Tomsk hospitals.


[Profilament actin in muscle cells of starfish ambulacra].
In gelatinated extracts of starfish ambulacral podia the actin polymerized during gel formation exists as oligomers incapable of binding to rabbit myosin. Actin is also the major protein extractable from the cells by glycerol- and Triton-containing solutions, which do not destroy the contractile mechanism of the cells. The weakly bound gel proteins washed from gel by low ionic strength solutions comprise a factor hampering the polymerization of rabbit actin. The data obtained suggest that the ambulacral sarcoplasmic proteins contain actin (profilament form) coupled to the factor preventing its polymerization.


Differential expression of interleukin-32 in chronic rhinosinusitis with and without nasal polyps.
Chronic rhinosinusitis (CRS) is a heterogeneous disease characterized by local inflammation of the upper airways and sinuses and is frequently divided into polypoid CRS (CRSwNP) and nonpolypoid CRS (CRSsNP). However, the mechanism of inflammation in CRS has still not been fully elucidated. The aim of the study was to investigate the role of interleukin-32 (IL-32), a recently discovered proinflammatory cytokine, in CRS. We collected nasal epithelial cells and nasal tissue from patients with CRS and control subjects. We assayed mRNA for IL-32 by real-time PCR and measured IL-32 protein using ELISA, Western blot, and immunohistochemistry. The expression of mRNA for IL-32 was elevated in epithelial cells from uncinate tissue from patients with CRSsNP compared with patients with CRSwNP (P < 0.05), control subjects (P=0.06), and epithelial cells from nasal polyp (NP) tissue (P < 0.05). Production of IL-32 was induced by IFN-γ, TNF, and dsRNA in primary airway epithelial cells. In whole-tissue extracts, the expression of IL-32 protein was significantly elevated in patients with CRSwNP compared with patients with CRSsNP and control subjects. Immunohistochemistry data showed that IL-32 was detected in mucosal epithelial cells and inflammatory cells in the lamina propria. Levels of IL-32 were correlated with the levels of CD3 and macrophage mannose receptor in NP tissue. Immunofluorescence data showed IL-32 co-localization with CD3-positive T cells and CD68-positive macrophages in NPs. Overproduction of IL-32 may be involved in the pathogenesis of CRS, although the role of IL-32 in the inflammation in CRSsNP and CRSwNP may be different.


The effect of organics transformation and migration on pore structure of bituminous coal and lignite during biomethane production.
Biomethane generation by coal degradation not only can increase coalbed methane (CBM) reserves, namely, microbially enhanced coalbed methane (MECBM), but also has a significant effect on the pore structure of coal which is the key factor in CBM extraction. The transformation and migration of organics in coal are essential to pore development under the action of microorganisms. Here, the biodegradation of bituminous coal and lignite to produce methane and the cultivation with inhibition of methanogenic activity by 2-bromoethanesulfonate (BES) were performed to analyze the effect of biodegradation on coal pore development by determining the changes of the pore structure and the organics in culture solution and coal. The results showed that the maximum methane productions from bituminous coal and lignite were 117.69 μmol/g and 166.55 μmol/g, respectively. Biodegradation mainly affected the development of micropore whose specific surface area (SSA) and pore volume (PV) decreased while the fractal dimension increased. After biodegradation, various organics were generated which were partly released into culture solution while a large number of them remained in residual coal. The content of newly generated heterocyclic organics and oxygen-containing aromatics in bituminous coal was 11.21% and 20.21%. And the content of heterocyclic organics in bituminous coal was negatively correlated with SSA and PV but positively correlated with the fractal dimension which suggested that the retention of organics contributed greatly to the decrease of pore development. But the retention effect on pore structure was relatively poor in lignite. Besides, microorganisms were observed around fissures in both coal samples after biodegradation which would not be conducive to the porosity of coal on the micron scale. These results revealed that the effect of biodegradation on pore development of coal was governed by the combined action of organics degradation to produce methane and organics retention in coal whose contributions were antagonistic and determined by coal rank and pore aperture. The better development of MECBM needs to enhance organics biodegradation and reduce organics retention in coal.


Atrial High-Rate Event Incidence and Predictors in Patients With Permanent Pacemaker Implantation.
Objective: The present study aims to investigate the incidence and predictors of atrial high-rate events (AHREs) in patients with permanent pacemaker implants. Methods: A total of 289 patients who were implanted with a dual-chamber pacemaker due to complete atrioventricular block or symptomatic sick sinus syndrome (SSS) and had no previous history of atrial fibrillation were included in the present study. AHREs are defined as events with an atrial frequency of ≥175 bpm and a duration of ≥5 min. The patients were divided into two groups according to whether or not AHREs were detected during the follow-up: group A (AHRE+, n = 91) and group N (AHRE-, n = 198). Results: During the 12-month follow-up period, AHREs were detected in 91 patients (31.5%). The multivariate Cox regression analysis revealed that patient age [odds ratio [OR] = 1.041; 95% confidence interval [CI], 1.018-1.064; and P < 0.001], pacemaker implantation due to symptomatic SSS (OR = 2.225; 95% CI, 1.227-4.036; and P = 0.008), and the percentage of atrial pacing after pacemaker implantation (OR = 1.010; 95% CI, 1.002-1.017; and P = 0.016) were independent AHRE predictors. Conclusion: The AHRE detection rate in patients with pacemaker implants was 31.5%. Patient age, pacemaker implantation due to symptomatic SSS, and the percentage of atrial pacing after pacemaker implantation were independent AHRE predictors.


Efficacy and safety of low dosage amantadine hydrochloride as prophylaxis for influenza A.
The efficacy and safety of prophylactic low dose amantadine hydrochloride was assessed in two double-blind, placebo-controlled, randomized studies. In a study of 476 subjects aged 18 to 55 years, adverse reactions were not significantly different between the group receiving 100 mg/day amantadine and the placebo group but significantly greater in the group given 200 mg/day (P less than 0.009). The influenza attack rate in this study was too low to assess efficacy. In an experimental challenge study of influenza A/Beth/1/85 in 78 subjects of similar age the prophylactic administration of 50 mg, 100 mg or 200 mg/day doses of amantadine were more effective than placebo in preventing influenza illness (P less than 0.02, 66, 74 and 82% protection, respectively), and in suppressing viral replication (P = 0.02). There was no significant difference between amantadine groups in influenza illness or viral shedding. Compared with the placebo group the 100 and 200 mg amantadine groups showed a significant decrease in infection rate (100 mg: 40% protection: P = 0.012; 200 mg: 32% protection: P = 0.045) whereas the 50 mg group did not (20% protection: P = 0.187). These results suggest that 100 mg/day of amantadine will reduce toxicity but maintain the prophylactic efficacy seen with 200 mg/day.


Highly sensitive hydrazine chemical sensor based on mono-dispersed rapidly synthesized PEG-coated ZnS nanoparticles.
Monodispersed PEG-coated ZnS (P-ZnS) nanoparticles (NPs) were synthesized by facile microwave process and utilized as efficient electron mediators for the fabrication of highly sensitive hydrazine chemical sensor. The detailed morphological and structural properties revealed the monodispersity and good crystallinity for synthesized P-ZnS NPs. A high-sensitivity of ∼ 89.3 μA cm(-2) μM and low limit of detection of 1.07 μM, based on S/N ratio, were obtained for the fabrication of hydrazine chemical sensor based on P-ZnS NPs. To the best of our knowledge, this is the first report which demonstrates the utilization of P-ZnS NPs for the fabrication of efficient hydrazine chemical sensor. By this work, it could be concluded that simply synthesized ZnS NPs can be used as efficient electron mediators for the fabrication of effective hydrazine chemical sensors.


Down-regulation of death-associated protein kinase-2 is required for beta-catenin-induced anoikis resistance of malignant epithelial cells.
Resistance of solid tumor cells to anoikis, apoptosis induced by cell detachment from the extracellular matrix, is thought to be critical for the ability of these cells to grow anchorage independently within three-dimensional tumor masses and from metastases. Beta-catenin, a major oncoprotein, can inhibit anoikis of cancer cells via unknown mechanisms. In an effort to identify these mechanisms we found that beta-catenin blocks anoikis of malignant kidney and intestinal epithelial cells and promotes their anchorage-independent growth by down-regulating death-associated protein kinase-2 (DAPk-2), a pro-apoptotic protein whose cellular functions have so far remained unexplored. We found that beta-catenin-induced down-regulation of DAPk-2 requires the presence of the transcription factor Tcf-4, a known mediator of beta-catenin signaling. We also observed that DAPk-2 contributes to the execution of anoikis of the non-malignant epithelial cells. Thus, beta-catenin-induced down-regulation of DAPk-2 represents a novel signaling mechanism by which beta-catenin promotes the survival of malignant epithelial cells following their detachment from the ECM and enables these cells to grow in an anchorage-independent manner.


Cloning of double stranded DNAs derived from polysomal mRNA of maize endosperm: isolation and characterisation of zein clones.
Starting from polysomal RNA of developing maize endosperm and applying the cloning procedure of integrating dsDNA into the Pst I site of plasmid pBR 322, clones containing sequences complementary to endosperm mRNAs were obtained. 25 per cent of these clones were identified as containing zein specific DNA sequences which hybridized either with the zein mRNA coding for the 22 000 Mr protein or with the zein mRNA coding for the 19 000 Mr protein. The zein-specific DNA inserts of the recombinant plasmids were further characterized by restriction enzyme analysis.


Delayed rupture of traumatic anterior cerebral artery A4 segment aneurysm: A case report.
Delayed rupture of traumatic intracranial aneurysms is relatively rare, and traumatic anterior A4 segment aneurysms may be associated with trauma to the cerebral falx. The mortality rate in patients with delayed traumatic rupture of intracranial aneurysms is>50%. Therefore, early diagnosis and treatment are critical. Herein, we present a case of a patient who did not have an intracranial aneurysm on computed tomographic angiography (CTA) after admission. Subsequently, the patient consciousness deteriorated, and CTA revealed aneurysm and bleeding. A 55-year-old man fell from a 3-meter-high truck and was unconscious. During the following few hours, the gradually regained consciousness. No intracranial aneurysms were found on CTA of the patient head immediately after admission. The final diagnosis was delayed rupture of traumatic intracranial aneurysms. The patient underwent endovascular and symptomatic treatments. The patient gradually recovered and was referred to the rehabilitation department for further treatment. Considering the catastrophic consequences of the disease, we should review CTA or digital subtraction angiography many times after admission, and take appropriate surgical procedures in time.


Allergic rhinitis--a total genome-scan for susceptibility genes suggests a locus on chromosome 4q24-q27.
Allergic rhinitis is a common disease of complex inheritance and is characterised by mucosal inflammation caused by allergen exposure. The genetics of closely related phenotypes such as asthma, atopy and to some extend atopic dermatitis has attracted attention in recent years. Genetic reports of allergic rhinitis on the contrary have as yet been most sparse. To identify candidate regions holding genes for allergic rhinitis we performed a total genome-scan on affected sib-pair families. From 100 Danish sib-pair families selected for allergy, families containing sib-pairs matching a phenotype definition of both clinical allergic rhinitis and confirmed specific allergy were chosen. Thirty-three affected sib-pair families qualified for the scan that was undertaken using 446 microsatellite markers. Non-parametric linkage results were obtained from MAPMAKER/SIBS computer program. The study revealed one major candidate region on chromosome 4q24-q27 (LOD=2.83) and eight minor candidate regions 2q12-q33, 3q13, 4p15-q12, 5q13-q15, 6p24-p23, 12p13, 22q13, and Xp21 (LOD=1.04-1.63) likely to contain susceptibility genes for allergic rhinitis. Our findings did not support a previous report of linkage of allergic rhinitis to chromosome 12q14-q24 but they added positive evidence to the asthma and atopy candidate regions 2q33 and 6p23. Further identification of the specific genes involved in allergic rhinitis will give opportunities for improved diagnosis and treatment.


Does preoperative 3D CT planning helps in predicting the component size determination and alignment in automatic robotic total knee arthroplasty (RA-TKA).
Image-based Robotic Total knee Arthroplasty (RA-TKA)was developed with the purpose of enhancing the accuracy in determining the component sizes preoperatively and helping surgeons in minimizing errors and improve patient outcomes. The research aims to find the reliability of robotic-assisted TKR based on images in determining the correct component sizes using preoperative three-dimensional (3D) computer tomography. After ethical approval, we conducted a prospective study from March 2022 to December 2022. A total of 100 knees underwent image-based RA-TKA having grade 4 Osteoarthritis knee (Kellegren Lawrence classification). A single senior surgeon performed on all patients. Postoperative implant sizes and fit were assessed by five radiographic markers by an independent observer. In our study, we found the mean age was (64.96 ± 7.3) years, with female to male ratio of 43:22. The preoperative 3D CT accuracy is 100% for femoral component sizing and 97% for the tibial component. There was a statistically significant improvement in varus deformity from preoperative 7.370 ± 3.70° to 1.24 0 ± 0.910° after surgery., p = 0.001. Improvement in flexion deformity correction was from preoperative 6.50 ± 6.30 to postoperative 1.640 ± 1.770, p = 0.001. Our study concludes that the use of pre-operative 3D CT helps in predicting the component sizes, minimizes surgical time, and enhances implant position accuracy, as well as improves postoperative limb alignment in the coronal and sagittal planes.


In vivo detection of free radicals in mouse septic encephalopathy using molecular MRI and immuno-spin trapping.
Free radicals are known to play a major role in sepsis. Combined immuno-spin trapping and molecular magnetic resonance imaging (MRI) was used to detect in vivo and in situ levels of free radicals in murine septic encephalopathy after cecal ligation and puncture (CLP). DMPO (5,5-dimethyl pyrroline N-oxide) was injected over 6h after CLP, before administration of an anti-DMPO probe (anti-DMPO antibody bound to albumin-gadolinium-diethylene triamine pentaacetic acid-biotin MRI targeting contrast agent). In vitro assessment of the anti-DMPO probe in oxidatively stressed mouse astrocytes significantly decreased T1 relaxation (p < 0.0001) compared to controls. MRI detected the presence of anti-DMPO adducts via a substantial decrease in %T1 change within the hippocampus, striatum, occipital, and medial cortex brain regions (p < 0.01 for all) in septic animals compared to shams, which was sustained for over 60 min (p < 0.05 for all). Fluorescently labeled streptavidin was used to target the anti-DMPO probe biotin, which was elevated in septic brain, liver, and lungs compared to sham. Ex vivo DMPO adducts (qualitative) and oxidative products, including 4-hydroxynonenal and 3-nitrotyrosine (quantitative, p < 0.05 for both), were elevated in septic brains compared to shams. This is the first study that has reported on the detection of in vivo and in situ levels of free radicals in murine septic encephalopathy.


Current developments and challenges in the search for a naturally selected Diels-Alderase.
Only a very few examples of enzymes known to catalyze pericyclic reactions have been reported, and presently no enzyme has been demonstrated unequivocally to catalyze a Diels-Alder reaction. Nevertheless, research into secondary metabolism has led to the discovery of numerous natural products exhibiting the structural hallmarks of [4+2] cycloadditions, prompting efforts to characterize the responsible enzymatic processes. These efforts have resulted in a growing collection of enzymes believed to catalyze pericyclic [4+2] cycloaddition reactions; however, in each case the complexity of the substrates and catalytic properties of these enzymes poses significant challenges in substantiating these hypotheses. Herein we consider the principles motivating these efforts and the enzymological systems currently under investigation.


Superior fluorescent probe for detection of potassium ion.
Here, a simple, and highly sensitive fluorescent assay is designed to monitor K(+). The versatile, robust biosensing strategy is based on the specific recognition utility of label-free aptamers with their targets and PicoGreen dye as the signal probe. The aptamers undergo a conformational change to a secondary structure such as G-quadruplex in the presence of targets. In addition to a conformational change with its targets, the remaining single-stranded DNA (ssDNA) aptamer form a duplex structure with its complete complementary sequence. Conformational changes of aptamers as well as fluorescence amplification produce clear signal-off in the presence of targets. Fluorescent assay employing this mechanism for the detection of K(+) is highly sensitive, and selective. The detection limit of the K(+) assay is determined to be 2.37 pM. The sensing strategy is low-cost and simple in its operation without requirement for complex labeling of probe DNA or sophisticated synthesis of the fluorescent compound. Also, the method has less structural requirement of complexes of aptamers with their targets, thus rending its wilder applications for various targets.


Vascular chymase: pathophysiological role and therapeutic potential of inhibition.
Chymase is a chymotrypsin-like serine protease secreted from mast cells. Mammalian chymases are classified into two subgroups (alpha and beta) according to structure and substrate specificity; human chymase is an alpha-chymase. An important action of chymase is the ACE-independent conversion of Ang I to Ang II, but chymase also degrades the extracellular matrix, activates TGF-beta1 and IL-1beta, forms 31-amino acid endothelins and is involved in lipid metabolism. Under physiological conditions, the role of chymase in blood vessels is uncertain. In pathological situations, however, chymase may be important. In animal models of hypertension and atherosclerosis, chymase may be involved in lipid deposition and intimal and smooth muscle hyperplasia, at least in some vessels. In addition, chymase has pro-angiogenic properties. In human diseased blood vessels (e.g. atherosclerotic and aneurysmal aorta; remodeled pulmonary blood vessels), there are increases in chymase-containing mast cells and/or in chymase-dependent conversion of Ang I to Ang II. These findings have raised the possibility that inhibition of chymase may have a role in the therapy of vascular disease. The effects of chymase can theoretically be attenuated either by reducing availability of the enzyme, with a mast cell stabiliser, or alternatively with specific chymase inhibitors. The mast cell stabiliser, tranilast, was shown to be beneficial in animal models of atherosclerosis, where a prevention protocol was used, but was not effective in clinical trials where it was administered after angioplasty. Chymase inhibitors could have the advantage of being effective even if used after injury. Several orally active inhibitors, including SUN-C8257, BCEAB, NK3201 and TEI-E548, are now available. These have yet to be tested in humans, but promising results have been obtained in animal models of atherosclerosis and angiogenesis. It is concluded that orally active inhibitors of chymase may have a place in the treatment of vascular diseases where injury-induced mast cell degranulation contributes to the pathology.


Phthalate ester effects on rat Sertoli cell function in vitro: effects of phthalate side chain and age of animal.
Mono(2-ethylhexyl) phthalate (MEHP), the active metabolite of the testicular toxicant di(2-ethylhexyl) phthalate, inhibits FSH-stimulated rat Sertoli cell cAMP accumulation, stimulates basal lactate production, and decreases intracellular ATP levels in vitro. Dibutyl phthalate and dipentyl phthalate but not diethyldimethyl or dipropyl are also age-dependent testicular toxicants in vivo. We therefore examined the effect of animal age and phthalate monoester on the Sertoli cell FSH-stimulated cAMP accumulation, lactate secretion, and ATP levels in order to determine if these effects are part of the mechanism of action of phthalate esters in vivo. MEHP, monobutyl and monopentyl phthalates but not the monoethyl, monomethyl, or monopropyl phthalates inhibited FSH-stimulated cAMP accumulation, a segregation which matches the in vivo toxicity potential of these agents. MEHP and monopentyl, but not monobutyl phthalates, also stimulated Sertoli cell lactate secretion. The effect of the active phthalates on FSH-stimulated cAMP accumulation and lactate secretion is not dependent on age of animal over a range of 13-80 days, suggesting that the age-related toxicity in vivo may be related to differences in metabolism and disposition rather than tissue sensitivity. Since the ED50 of MEHP inhibition of cAMP accumulation and lactate secretion is similar, these two effects may be related to a common initial effect of the active phthalates. Inhibition of intracellular ATP levels is specific for MEHP and is lost with age (greater than 28 days of age) and thus is not likely to be an essential part of the in vivo mechanism of action of phthalate diesters.


Prospective observational study of treatment pattern, effectiveness and safety of zoledronic acid therapy beyond 24 months in patients with multiple myeloma or bone metastases from solid tumors.
To study the treatment patterns, effectiveness and safety of zoledronic acid (ZOL) beyond 2 years of therapy, given the paucity of data on long-term treatment in daily clinical practice. Patients with multiple myeloma (MM) or solid tumor bone metastases (STM) and at least 24 months of regular q3-4w ZOL therapy were followed prospectively for an additional 18 months beyond the 24 months required for study entry. End-points included ZOL exposure, incidence of skeletal related events (SRE), and safety. In all, 298 evaluable patients were enrolled. The mean continuation rate of ZOL was 90.6%. Exposure to ZOL decreased with time in all patients, but was lower (50.0% vs. 67.6%; p<0.001) and with higher discontinuation rates (incidence rate ratio [IRR]=1.95; p=0.002) in MM compared to the STM group. ZOL suppressed the rate of SREs similarly during the study as compared to before inclusion (0.12 vs. 0.13 events per person-year; p=0.7). At 18 months, 84.5% remained SRE-free. In STM patients, persistent ZOL therapy was associated with lower SRE risk (hazard ratio [HR]=0.42; p=0.01), but not in MM. Renal deterioration occurred in 3.7% and osteonecrosis of the jaw (ONJ) developed in 6.0%, with dental trauma increasing ONJ risk (HR=4.67; p=0.002). Beyond 2 years of therapy, treatment patterns of ZOL were heterogeneous and SRE rates were low. The safety profile of ZOL was acceptable, and interrupting ZOL in patients with solid tumors was associated with a higher risk of SREs.


Evaluation of auditory function in a population of clinically healthy cats using evoked otoacoustic emissions.
Cats may demonstrate deafness due to a variety of aetiologies and the current preferred method for assessing auditory function is the brainstem auditory evoked response (BAER). The BAER has largely been replaced by otoacoustic emission (OAE) testing in human neonatal deafness screening as the equipment is more readily available, is cheaper and the test is less invasive and simpler. This is the first study to demonstrate that transient evoked OAEs (TEOAE) and distortion product OAEs (DPOAE) can be recorded in cats using commercially available equipment. Protocols for recording the emissions and analysing the results are given. DPOAE testing is suggested to be quicker in this population of healthy cats and shows promise in rapidly providing detailed information about auditory function at a variety of different frequencies.


Study of mitoxantrone for the treatment of recurrent neuromyelitis optica (Devic disease).
Neuromyelitis optica is a severe demyelinating disease that selectively involves the optic nerves and the spinal cord but usually spares the brain. It is considered to have a B-cell-induced pathogenesis. Mitoxantrone hydrochloride, a synthetic anthracenedione approved for worsening relapsing-remitting multiple sclerosis and secondary progressive multiple sclerosis, has been shown to primarily suppress the humoral response. To evaluate the benefit of mitoxantrone treatment in patients with relapsing neuromyelitis optica. Prospective 2-year study. Academic multiple sclerosis center. Five patients (3 women and 2 men) with an age range of 20 to 51 years and an Expanded Disability Status Scale score of 2.5 to 6.5 (mean +/- SD, 4.40 +/- 1.88). Monthly intravenous infusions of mitoxantrone hydrochloride, 12 mg/m2, for 6 months followed by 3 additional treatments every 3 months. Expanded Disability Status Scale score measured every 3 months and during relapses; findings on orbital, brain, and spinal cord magnetic resonance images performed at baseline and at 3, 6, 12, 18, and 24 months; and visual evoked potentials and results of ophthalmologic evaluations performed at baseline and annually. During the 2 years of treatment, 2 patients each had a relapse once within the initial 5 months of treatment (1 severe and 1 moderate). Improvement was seen clinically and on magnetic resonance images in 4 patients. Patients generally tolerated the treatment well, although 1 patient had a reversible decrease in cardiac ejection fraction. Our results suggest a beneficial effect of mitoxantrone treatment for relapsing neuromyelitis optica.


Comparison of Long-Term Oncological Results in Young Women with Breast Cancer between BRCA-Mutation Carriers Versus Non-Carriers: How Tumor and Genetic Risk Factors Influence the Clinical Prognosis.
Breast cancer (BC) is very uncommon in young women (YW) and it is unclear whether a BRCA mutation has prognostic implications. Our aim was to evaluate the characteristics of YW with BC by comparing the long-term oncological results between BRCA-mutation carriers and non-carriers. We retrospectively reviewed all the consecutive YW (aged 18-40 years) diagnosed with BC. Endpoints were disease-free survival (DFS), distant disease-free survival (DDFS), and overall survival (OS). 63 YW with a BRCA mutation were compared with 339 YW without BRCA mutation. BRCA-mutation carriers were younger (60.3% versus 34.8% if age ≤ 35 years, p = 0.001) and presented with more aggressive tumors (66.7% versus 40.7% if G3, p = 0.001; 57.2% versus 12.4% if biological subtype triple-negative, p = 0.001; 73.0% versus 39.2% if Ki67 ≥ 25%, p = 0.001). Non-carriers presented significantly better DFS, DDFS, and OS compared with BRCA-mutation carriers. Neoadjuvant chemotherapy was found to be an independent protective factor for OS in BRCA-mutation carriers. BC is more likely to present at a younger age (≤ 35 years) and with more aggressive characteristics (G3, triple-negative, Ki67 ≥ 25%) in YW with BRCA mutation compared with their non-mutated counterparts. Young BRCA-mutation carriers showed a poorer prognosis in terms of recurrence and survival compared with non-carriers. The implementation of neoadjuvant chemotherapy may improve survival in YW with BC and BRCA mutation.


Search for dual function inhibitors for Alzheimer's disease: synthesis and biological activity of acetylcholinesterase inhibitors of pyridinium-type and their Abeta fibril formation inhibition capacity.
Alzheimer's disease (AD) represents the most common neurodegenerative disorder, which is expressed through decline of mental function. Current treatment approaches include acetylcholinesterase inhibitors and NMDA-receptor partial antagonists. The most explored recent approaches that are closely related to the pathogenesis of this disease based on formally articulated amyloid hypothesis are: Abeta fibril formation inhibitors, amyloid precursor protein, and secretase inhibitors. [Scarpini, E.; Scheltens, P.; Feldman, H. Lancet Neurol.2003, 2, 539] In view of the development of new AChE inhibitors as drugs capable of reducing the symptoms of AD, the capacity of newly synthesized AChE inhibitors of pyridinium-type to inhibit the AChE was examined and compared to those of other inhibitors of this type presented earlier. [Kapková, P.; Stiefl, N.; Sürig, U.; Engels, B.; Baumann, K.; Holzgrabe, U. Arch. Pharm. Pharm. Med. Chem.2003, 336, 523; Alptüzun, V.; Kapková, P.; Baumann, K.; Erciyas, E.; Holzgrabe, U. J. Pharm. Pharmacol.2003, 55, 1397] Furthermore, the anti-Abeta fibril formation property of AChE inhibitors of pyridinium- and bispyridinium-type was evaluated to expand their activity profile and to reveal potential additive pharmacological effects which may reinforce their therapeutic application besides their capacity of increasing acetylcholine levels. Abeta fibril formation studies were performed by means of thioflavin T fluorescence assay.


Anterior resection of rectal cancer without bowel preparation and diverting stoma.
Since the introduction of total mesorectal excision (TME) as the standard operation technique for rectal cancer, anastomotic leakage percentages of up to 18% have been reported. To prevent such leakage, the use of mechanical bowel preparation and also the construction of a diverting ileostoma or colostomy have been standard procedures for years. In our institute, however, all patients undergoing colorectal surgery are operated upon without these measures. The present study was undertaken to investigate the results of this strategy in terms of the occurrence of postoperative anastomotic leakage. All patients who underwent an elective (low) anterior resection between January 1996 and December 2001 (n = 144) entered the study. The clinical and pathological records of these patients were reviewed retrospectively. The exclusion criteria were patients with fixed rectal carcinoma who received preoperative radiotherapy and/or a stoma only at operation, emergency operations, abdominoperoneal resections, and Hartmann's procedures. Anastomotic leakage occurred in 7 out of 144 patients (4.9%). There was a trend toward a higher leakage frequency in men, in patients with a distal anastomosis, in patients with a stapled anastomosis, and in patients with a T3-T4 tumor or with positive lymph nodes. None of these factors, however, had a significant prognostic value based on a univariate or multivariate analysis. Those who died after leakage tended to be older than those who did not (P < 0.05). A (low) anterior resection can be performed safely without mechanical bowel preparation or a diverting stoma, and results in an anastomotic leakage percentage of less than 5%. Appropriate selection of patients may be important, but none of the investigated patient- or tumor-related factors could be identified as decisive.


Social-life cycle assessment of oil palm plantation smallholders in Bengkulu province, Indonesia.
Smallholders are often seen as a weak point in the development of oil palm plantation production. The quality and yield of their production are considered low according to world market standards; the continuity of their production is irregular; and finally, improving the welfare of farmers is difficult to achieve. However, smallholder plantations have an important and strategic role in achieving sustainable development. This study aims to assess the social life cycle assessment of smallholder oil palm plantations in Bengkulu Province by involving stakeholders consisting of workers, local communities, farm owners, and value chain actors. A total of 600 respondents were selected using quota sampling and interviewed using a structured questionnaire. Data analysis was conducted using social-life cycle assessment, involving various social issues and relevant social indicators for each stakeholder. The findings in our research show that smallholder oil palm plantations still do not meet the minimum wage for workers, and the equipment used by workers is still very minimal in terms of security and safety guarantees. The price of palm oil continues to fluctuate, and plantation policies are constantly changing, making it difficult for oil palm owners to meet the standards and various rules that are set for managing oil palm plantations. In addition, oil palm plantations are no longer a guarantee for the fulfillment of food security conditions for household owners, workers, communities, and actors involved in the oil palm institutional chain. Our finding is that, surprisingly, there is a low level of commitment by smallholders and the government to sustainability in oil palm plantations. The Indonesian Sustainable Palm Oil and Roundtable on Sustainable Palm Oil as standards for sustainable oil palm plantations, as well as global standards for oil palm plantations to demonstrate environmentally friendly production processes, have not been able to compel smallholders to implement sustainable development in oil palm. Various policies formulated by the Indonesian government are perceived to have not addressed the core issues faced by small-scale farmers. Policies supporting small-scale farmers, particularly those related to increasing productivity, fostering and monitoring the environmental management of palm oil plantation activities, and ensuring ownership of plantation, are necessary for the achievement of sustainable smallholder palm oil plantation development.


The Sigma-2 receptor / transmembrane protein 97 (σ2R/TMEM97) modulator JVW-1034 reduces heavy alcohol drinking and associated pain states in male mice.
Alcohol Use Disorder (AUD) is a chronic relapsing disorder characterized by compulsive alcohol intake, loss of control over alcohol intake, and a negative emotional state when access to alcohol is prevented. AUD is also closely tied to pain, as repeated alcohol drinking leads to increased pain sensitivity during withdrawal. The sigma-2 receptor, recently identified as transmembrane protein 97 (σ2R/TMEM97), is an integral membrane protein involved in cholesterol homeostasis and lipid metabolism. Selective σ2R/Tmem97 modulators have been recently shown to relieve mechanical hypersensitivity in animal models of neuropathic pain as well as to attenuate alcohol withdrawal signs in C. elegans and to reduce alcohol drinking in rats, suggesting a potential key role for this protein in alcohol-related behaviors. In this study, we tested the effects of a potent and selective σ2R/TMEM97 ligand, JVW-1034, on heavy alcohol drinking and alcohol-induced heightened pain states in mice using an intermittent access model. Administration of JVW-1034 decreased both ethanol intake and preference for ethanol, without affecting water intake, total fluid intake, or food intake. Notably, this effect was specific for alcohol, as JVW-1034 had no effect on sucrose intake. Furthermore, JVW-1034 reduced both thermal hyperalgesia and mechanical hypersensitivity in ethanol withdrawn mice. Our data provide important evidence that modulation of σ2R/TMEM97 with small molecules can mediate heavy alcohol drinking as well as chronic alcohol-induced heightened pain sensitivity, thereby identifying a promising novel pharmacological target for AUD and associated pain states.


Under-prescribing and non-adherence to medications after coronary bypass surgery in older adults: strategies to improve adherence.
The focus for this clinical review is under-prescribing and non-adherence to medication guidelines in older adults after coronary artery bypass grafting (CABG) surgery. Non-adherence occurs in all age groups, but older adults have a unique set of challenges including difficulty hearing, comprehending, and remembering instructions; acquiring and managing multiple medications; and tolerating drug-drug and drug-disease interactions. Still, non-adherence leads to increased morbidity, mortality, and costs to the healthcare system. Factors contributing to non-adherence include failure to initiate therapy before hospital discharge; poor education about the importance of each medication by hospital staff; poor education about medication side effects; polypharmacy; multiple daily dosing; excessive cost; and the physician's lack of knowledge of clinical indicators for use of medications. To improve adherence, healthcare systems must ensure that (i) all patients are prescribed the appropriate medications at discharge; (ii) patients fill and take these medications post-operatively; and (iii) patients continue long-term use of these medications. Interventions must target central administrative policies within healthcare institutions, the difficulties facing providers, as well as the concerns of patients. Corrective efforts need to be started early during the hospitalization and involve practitioners who can follow patients after the date on which surgical care is no longer needed. A solid, ongoing relationship between patients and their primary-care physicians and cardiologists is essential. This review summarizes the post-operative medication guidelines for CABG surgery, describes barriers that limit the adherence to these guidelines, and suggests possible avenues to improve medication adherence in older cardiac surgery patients.


Binding of iodomercurates to sulfhydryl-blocked beta-lactoglobulin-A, -B, and -C.
Sulfhydryl-blocked beta-lactoglobulins (beta-LG-S-SCH2CH2OH)-A, -B, and -C bind only one iodomercurate species, HgI3-, at only one site, with a dissociation constant of 4.0 X 10(-5) M at 25 degrees, pH 5.0, 0.10 ionic strength. (Binding to native beta-LG-SH-A, -B, and -C is more complex, involving the sulfhydryl and two other sites and several iodomercurates.) The red shift of the HgI3- spectrum on binding would ordinarily suggest a hydrophobic site, but the HgI3- site is distinct from, and independent of, the alkane-binding site of native and blocked beta-LG; HgI3- may bind a group that shifts its trigonal planar structure toward the tetrahedron of HgI4(2-). Binding of HgI3- to blocked beta-LG interferes with the well-known association of beta-LG-A to octamers at pH 4.6 and low temperature. The relation of the HgI3- site to the crystallographic iodomercurate-binding sites of beta-LG-SH is examined. To facilitate these and future studies of iodomercurate binding, the 200-400 nm spectra of HgI2, HgI3-, and HgI4(2-) in aqueous solutions and the thermodynamic formation constants at 25 degrees for the equilibria HgI2 + I- = HgI3- (4.9 X 10(3) M-1) and HgI3- + I- = HgI4(2-) (0.118 X 10(3) M-1) were obtained.


Mouse Crumbs3 sustains epithelial tissue morphogenesis in vivo.
The human apical protein CRB3 (Crb3 in mouse) organizes epithelial cell polarity. Loss of CRB3 expression increases the tumorogenic potential of cultured epithelial cells and favors metastasis formation in nude mice. These data emphasize the need of in vivo models to study CRB3 functions. Here, we report the phenotypic analysis of a novel Crb3 knockout mouse model. Crb3-deficient newborn mice show improper clearance of airways, suffer from respiratory distress and display perinatal lethality. Crb3 is also essential to maintain apical membrane identity in kidney epithelial cells. Numerous kidney cysts accompany these polarity defects. Impaired differentiation of the apical membrane is also observed in a subset of cells of the intestinal epithelium. This results in improper remodeling of adhesive contacts in the developing intestinal epithelium, thereby leading to villus fusion. We also noted a strong increase in cytoplasmic β-catenin levels in intestinal epithelial cells. β-catenin is a mediator of the Wnt signaling pathway, which is overactivated in the majority of colon cancers. In addition to clarifying the physiologic roles of Crb3, our study highlights that further functional analysis of this protein is likely to provide insights into the etiology of diverse pathologies, including respiratory distress syndrome, polycystic kidney disease and cancer.


Percutaneous revascularization of coronary chronic total occlusion: Toward a reappraisal of the available evidence.
Chronic total occlusion (CTO) is a challenging subset of coronary artery disease that is commonly encountered in real-world practice; it is associated with worse long-term prognosis. Observational studies suggest that percutaneous coronary intervention (PCI) for CTO is associated with reduction in myocardial ischemia and improvement in quality of life and left ventricular function. Some observational studies suggested that CTO-PCI is associated with improvement of the 'hard' clinical endpoints; others did not. Nearly all these studies compared the clinical outcome of successful versus failed PCI, rather than comparing the outcome of a whole CTO-PCI cohort versus a 'true' control group. Interestingly, in observational studies that compared the outcome of CTO-PCI versus optimal medical treatment, long-term mortality was comparable between the two strategies. In patients with multi-vessel disease and CTO, complete revascularization is more often achieved by coronary artery bypass grafting than by PCI; the SYNTAX score of these patients often favors surgical revascularization according to the current guidelines. The current guidelines reflect the divergence of opinion on the usefulness/benefit of CTO-PCI, mainly due to the lack of randomized trials. Evidence is awaited from three ongoing randomized controlled trials comparing PCI versus optimal medical treatment in the setting of CTO.


Fast modulation of μ-opioid receptor (MOR) recycling is mediated by receptor agonists.
The μ-opioid receptor (MOR) is a member of the G protein-coupled receptor family and the main target of endogenous opioid neuropeptides and morphine. Upon activation by ligands, MORs are rapidly internalized via clathrin-coated pits in heterologous cells and dissociated striatal neurons. After initial endocytosis, resensitized receptors recycle back to the cell surface by vesicular delivery for subsequent cycles of activation. MOR trafficking has been linked to opioid tolerance after acute exposure to agonist, but it is also involved in the resensitization process. Several studies describe the regulation and mechanism of MOR endocytosis, but little is known about the recycling of resensitized receptors to the cell surface. To study this process, we induced internalization of MOR with [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO) and morphine and imaged in real time single vesicles recycling receptors to the cell surface. We determined single vesicle recycling kinetics and the number of receptors contained in them. Then we demonstrated that rapid vesicular delivery of recycling MORs to the cell surface was mediated by the actin-microtubule cytoskeleton. Recycling was also dependent on Rab4, Rab11, and the Ca(2+)-sensitive motor protein myosin Vb. Finally, we showed that recycling is acutely modulated by the presence of agonists and the levels of cAMP. Our work identifies a novel trafficking mechanism that increases the number of cell surface MORs during acute agonist exposure, effectively reducing the development of opioid tolerance.


A miniaturized method for multiple and inexpensive electrophoreses on polyacrylamide slab gels.
The design, construction, and use of a simple inexpensive system for multiple slab gel electrophoreses are described. Each gel slab is cooled from both sides by an electrode buffer. The electrophoresis time of each gel can be easily controlled independently from the others in the set. Because this set is very simple, it was completely handmade by the author. Depending on the purpose for which the set is to be used, its size, thickness, and shape can be changed.


Anandamide deficiency and heightened neuropathic pain in aged mice.
Damaging of peripheral nerves may result in chronic neuropathic pain for which the likelihood is increased in the elderly. We assessed in mice if age-dependent alterations of endocannabinoids contributed to the heightened vulnerability to neuropathic pain at old age. We assessed nociception, endocannabinoids and the therapeutic efficacy of R-flurbiprofen in young and aged mice in the spared nerve injury model of neuropathic pain. R-flurbiprofen was used because it is able to reduce neuropathic pain in young mice in part by increasing anandamide. Aged mice developed stronger nociceptive hypersensitivity after sciatic nerve injury than young mice. This was associated with low anandamide levels in the dorsal root ganglia, spinal cord, thalamus and cortex, which further decreased after nerve injury. In aged mice, R-flurbiprofen had only weak antinociceptive efficacy and it failed to restore normal anandamide levels after nerve injury. In terms of the mechanisms, we found that fatty acid amide hydrolase (FAAH) which degrades anandamide, was upregulated after nerve injury at both ages, so that this upregulation likely did not account for the age-dependent differences. However, enzymes contributing to oxidative metabolism of anandamide, namely cyclooxygenase-1 and Cyp2D6, were increased in the brain of aged mice, possibly enhancing the oxidative breakdown of anandamide. This may overwhelm the capacity of R-flurbiprofen to restore anandamide homeostasis and may contribute to the heightened risk for neuropathic pain at old age.


Cross-tissue, single-cell stromal atlas identifies shared pathological fibroblast phenotypes in four chronic inflammatory diseases.
Pro-inflammatory fibroblasts are critical for pathogenesis in rheumatoid arthritis, inflammatory bowel disease, interstitial lung disease, and Sjögren's syndrome and represent a novel therapeutic target for chronic inflammatory disease. However, the heterogeneity of fibroblast phenotypes, exacerbated by the lack of a common cross-tissue taxonomy, has limited our understanding of which pathways are shared by multiple diseases. We profiled fibroblasts derived from inflamed and non-inflamed synovium, intestine, lungs, and salivary glands from affected individuals with single-cell RNA sequencing. We integrated all fibroblasts into a multi-tissue atlas to characterize shared and tissue-specific phenotypes. Two shared clusters, CXCL10+CCL19+ immune-interacting and SPARC+COL3A1+ vascular-interacting fibroblasts, were expanded in all inflamed tissues and mapped to dermal analogs in a public atopic dermatitis atlas. We confirmed these human pro-inflammatory fibroblasts in animal models of lung, joint, and intestinal inflammation. This work represents a thorough investigation into fibroblasts across organ systems, individual donors, and disease states that reveals shared pathogenic activation states across four chronic inflammatory diseases. Grant from F. Hoffmann-La Roche (Roche) AG.


A conserved subcomplex within the bacterial cytokinetic ring activates cell wall synthesis by the FtsW-FtsI synthase.
Cell division in bacteria is mediated by a multiprotein assembly called the divisome. A major function of this machinery is the synthesis of the peptidoglycan (PG) cell wall that caps the daughter poles and prevents osmotic lysis of the newborn cells. Recent studies have implicated a complex of FtsW and FtsI (FtsWI) as the essential PG synthase within the divisome; however, how PG polymerization by this synthase is regulated and coordinated with other activities within the machinery is not well understood. Previous results have implicated a conserved subcomplex of division proteins composed of FtsQ, FtsL, and FtsB (FtsQLB) in the regulation of FtsWI, but whether these proteins act directly as positive or negative regulators of the synthase has been unclear. To address this question, we purified a five-member Pseudomonas aeruginosa division complex consisting of FtsQLB-FtsWI. The PG polymerase activity of this complex was found to be greatly stimulated relative to FtsWI alone. Purification of complexes lacking individual components indicated that FtsL and FtsB are sufficient for FtsW activation. Furthermore, support for this activity being important for the cellular function of FtsQLB was provided by the identification of two division-defective variants of FtsL that still form normal FtsQLB-FtsWI complexes but fail to activate PG synthesis. Thus, our results indicate that the conserved FtsQLB complex is a direct activator of PG polymerization by the FtsWI synthase and thereby define an essential regulatory step in the process of bacterial cell division.


Recovery of functional human lymphocytes from Leukotrap filters.
We report here a method by which functional human PBL may be recovered from a blood product preparation unit (Leukotrap) which is normally employed by blood banks to prepare a leukocyte-poor blood product and which is commonly discarded after use. Blood filters which were back-flushed with cold, phosphate-buffered saline (PBS) and the resulting buffy coats collected and separated on Histopaque gradients yielded an average of 2.67 x 10(8) (+/- 1.17 x 10(8] cells per filter processed (n = 38), with viability greater than 95%. The percentages of cell types recovered before or after Histopaque separation closely approximated those reported for freshly drawn blood when examined using differential staining and morphological criteria. Fluorescent antibody analysis revealed a normal distribution of Leu-4 (T), Leu-12 (B), Leu-3a (T helper), Leu-2a (T suppressor) and Leu-M3 (macrophage) positive cell subsets. Recovered lymphocytes were fully responsive to phytohemagglutinin (PHA), concanavalin A (ConA) and pokeweed (PWM) mitogens. Random donor lymphocytes which were co-cultured in a two-way mixed lymphocyte response (MLR) were fully capable of proliferative responses compared to the same populations cultured alone. The results from our study indicate that Leukotrap filters provide a readily available, easily processed and convenient source of functional human PBL for use in the in vitro study of the human immune system.


Genetic diversity and recombination analysis of grapevine leafroll-associated virus 1 from China.
Grapevine leafroll-associated virus 1 (GLRaV-1) is one of the causal agents of grapevine leafroll disease (GLD). To investigate the prevalence and genetic variation of GLRaV-1 in China, 132 grapevine samples from 14 Chinese provinces and regions were tested using reverse transcription PCR (RT-PCR) and reverse transcription nested PCR (RT-nPCR). The samples included symptomatic and asymptomatic cultivars, and 36.4% of them tested positive for GLRaV-1. 'Beida' samples, previously identified as virus-free rootstocks, were also found to be infected with GLRaV-1 with an incidence of 40 . GLRaV-1 coat protein (CP) genes and heat-shock protein 70 (HSP70) genes from 43 GLRaV-1 isolates were selected and sequenced. Phylogenetic analysis of global CP and HSP70 gene sequences showed that all variants belonged to eight and seven groups, respectively. For CP gene sequence variants, group 4 was a new group that included only Chinese isolates. The results also showed that natural selection, rather than random processes, led to the evolution of variants belonging to CP gene sequence variants in group 2 and group 8. Furthermore, three new recombination events were identified in the GLRaV-1 CP gene population. This is the first report on the genetic variation of GLRaV-1 isolates in China, and this study will benefit grape clean-plant programs in China.


Dual cross-linked starch hydrogel for eugenol encapsulation and the formation of hydrogen bonds on textural hydrogel.
Physical and chemical cross-linked hydrogels combining N, N'-Methylenebisacrylamide (MBA)-grafted starch (MBAS) and sorbitol were successfully prepared and encapsulated with eugenol in this work. The dense porous structure with diameter of 10-15 μm and strong skeleton after restructuring inside the hydrogel was confirmed by SEM. The band shifts between 3258 cm-1 and 3264 cm-1 clarified the presence of a large number of hydrogen bonds in physical and chemical cross-linked hydrogels. The robust structure of the hydrogel was confirmed by mechanical and thermal property measurements. Molecular docking techniques were used to help understand the bridging pattern between three raw materials and to assess the advantageous conformation, which demonstrate sorbitol is beneficial to improve the characteristics of textural hydrogel by the formation of hydrogen bonds, creating a denser network, structural recombination and new intermolecular hydrogen bonds between starch and sorbitol afforded considerably junction zones. Compared to ordinary starch-based hydrogels, eugenol-loaded starch-sorbitol hydrogels (ESSG) exhibited a more attractive internal structure, swelling properties, viscoelasticity. Moreover, the ESSG showed excellent antimicrobial activity for typical undesired microorganisms in foods.