Fine-tuned coupling between human parahippocampal ripples and sleep spindles.
Sleep-associated memory consolidation is thought to rely on coordinated information transfer between the hippocampus and neocortex brought about during slow wave sleep (SWS) by distinct local field potential oscillations. Specifically, findings in animals have led to the concept that ripples originating from hippocampus combine with spindles to provide a fine-tuned temporal frame for a persistent transfer of memory-related information to the neocortex. The present study focused on characterizing the temporal relationship between parahippocampal ripple activity (80-140 Hz) and spindles recorded from frontal, parietal and parahippocampal cortices in 12 epilepsy patients implanted with parahippocampal foramen ovale electrodes. Overall, parietal and parahippocampal spindles showed closer relationships to parahippocampal ripple activity than frontal spindles, with the latter following parietal and parahippocampal spindles at a variable delay of up to 0.5 s. On a timescale of seconds, ripple activity showed a continuous increase before the peak of parietal and parahippocampal spindles, and decreased thereafter. At a fine timescale of milliseconds, parahippocampal ripple activity was tightly phase-locked to the troughs of these spindles. The demonstration of spindle phase-locked ripple activity in humans is consistent with the idea of a temporally fine-tuned hippocampus-to-neocortex transfer of information taking place during SWS.


Effect of pinitol treatment on insulin action in subjects with insulin resistance.
Endogenous low-molecular-weight glycans containing pinitol (3-O-methyl-D-chiro-inositol) and D-chiro-inositol are thought to mediate certain actions of insulin. We tested the hypothesis that oral administration of soybean-derived pinitol would improve insulin sensitivity in obese subjects (BMI = 36.6 kg/m2) with diet-treated type 2 diabetes or glucose intolerance (HbA1c = 6.8%). There were 22 subjects randomized to receive either pinitol 20 mg x kg(-1) x day(-1) (n = 12) or placebo (n = 10) in a 28-day double-blinded trial. No toxicity due to the pinitol was observed during the study The sensitivity of glucose and lipid metabolism to insulin were assessed by measurement of whole-body glucose, palmitate, and glycerol kinetics during basal conditions and a hyperinsulinemic-euglycemic clamp. Metabolic measurements were made at baseline and again at the end of the study Final plasma levels of pinitol were 48-fold (1.06 +/- 0.15 vs. 0.02 +/- 0.01 micromol/l, P < 0.0001) and D-chiro-inositol levels 14-fold (0.56 +/- 0.08 vs. 0.04 +/- 0.02 micromol/l, P < 0.0001) greater in the pinitol group compared with placebo. Four weeks of pinitol treatment did not alter baseline glucose production, insulin-mediated glucose disposal, or rates of appearance of free fatty acids and glycerol in plasma. We conclude that plasma levels of both pinitol and D-chiro-inositol are very responsive to pinitol ingestion, but insulin sensitivity does not increase after pinitol treatment in individuals with obesity and mild type 2 diabetes.


Tree-structured risk stratification of in-hospital mortality after percutaneous coronary intervention for acute myocardial infarction: a report from the New York State percutaneous coronary intervention database.
Previous risk scores have shown excellent performance. However, the need for real-time risk score computation makes their implementation in an emergent situation challenging. A more simplified approach can provide practitioners with a practical bedside risk stratification tool. We developed an easy-to-use tree-structured risk stratification model for patients undergoing early percutaneous coronary intervention (PCI) for acute myocardial infarction. The model was developed on the New York State PCI database for 1999 to 2000 (consisting of 5385 procedures) and was validated using the subsequent 2001 to 2002 database (consisting of 7414 procedures). Tree-structured modeling identified 3 key presenting features: cardiogenic shock, congestive heart failure, and age. In the validation data set, this risk stratification model identified patient groups with in-hospital mortality ranging from 0.5% to 20.6%, more than a 20-fold increased risk. The performance of this model was similar to the Mayo Clinic Risk Score with a discriminative capacity of 82% (95% confidence interval, 79%-84%) versus 80% (95% confidence interval, 77%-82%), respectively. Patients undergoing PCI for acute myocardial infarction can be readily stratified into risk categories using the tree-structured model. This provides practicing cardiologists with an internally validated and easy-to-use scheme for in-hospital mortality risk stratification.


Screening of remote Rhododendron species and hybrids for interspecific hybridization with R. simsii hybrids.
To introduce yellow, blue or orange pigmentation in the Rhododendron subgenus Tsutsusi (including R. simsii hybrids) by classical breeding, intersubgeneric pollination experiments involving Hymenanthes, Pentanthera and Rhododendron (including Vireya) species and hybrids were started. Prefertilization research by pollen tube staining revealed a different behaviour between bilateral crosses. Ovule culture was applied as a means to save a substantially larger amount of hybrid embryos from abortion. Though flower/seed capsule abortion eliminated many crosses in an early developmental stage, ovules of many crosses could be initiated in vitro on various time intervals after pollination. Germination was achieved on basal Rhododendron medium + 50 mg/l GA3. Green seedlings were obtained after Tsutsusi x Hymenanthes, Hymenanthes x Tsutsusi, Tsutsusi x Rhododendron, Rhododendron x Tsutsusi and Vireya x Tsutsusi pollination. Many seedlings have little growth vigour and next to green seedlings albino seedlings occur frequently, depending on the pollination type. The first true leaves of many assumed hybrids obviously lack chlorophyll. The ploidy level of Vireya x Tsutsusi and Tsutsusi x Pentanthera seedlings is intermediate, which strongly suggests their hybrid origin. Molecular analysis through SSR confirmed the interspecific charachter of seedlings in half of the crosses tested.


Resonant States of H3+ and D2H+.
Vibrational resonances for H(3) (+) and D(2)H(+), as well as H(3) (+) at J=3, are calculated using a complex absorbing potential (CAP) method with an automated procedure to find stability points in the complex plane. Two different CAP functional forms and different CAP extents are used to analyze the consistency of the results. Calculations are performed using discrete variable representation continuum basis elements calculated to high levels of accuracy by diagonalizing large, dense, Hamiltonian matrices. For D(2)H(+), two energy regions are analyzed: the one where D(2)+H(+) is the only dissociation product and the one where HD+D(+) can also be formed. Branching ratios are obtained in the latter case by using different CAPs. It is shown that H(3) (+) and D(2)H(+) support some narrow Feshbach-type resonances but that higher angular momentum states must be studied to model the pre-dissociation spectrum recorded by Carrington and co-workers [J. Chem. Phys. 98, 1073 (1993)].


Olfactory impairment in familial ataxias.
The main clinical manifestations of the spinocerebellar ataxias (SCAs) result from the involvement of the cerebellum and its connections. Cerebellar activity has been consistently observed in functional imaging studies of olfaction, but the anatomical pathways responsible for this connection have not yet been elucidated. Previous studies have demonstrated olfactory deficit in SCA2, Friedreich's ataxia and in small groups of ataxia of diverse aetiology. The authors used a validated version of the 16-item smell identification test from Sniffin' Sticks (SS-16) was used to evaluate 37 patients with genetically determined autosomal dominant ataxia, and 31 with familial ataxia of unknown genetic basis. This data was also compared with results in 106 Parkinson's disease patients and 218 healthy controls. The SS-16 score was significantly lower in ataxia than in the control group (p<0.001, 95% CI for β=0.55 to 1.90) and significantly higher in ataxia than in Parkinson's disease (p<0.001, 95% CI for β=-4.58 to -3.00) when adjusted for age (p=0.001, 95% CI for β=-0.05 to -0.01), gender (p=0.19) and history of tobacco use (p=0.41). When adjusted for general cognitive function, no significant difference was found between the ataxia and control groups. This study confirms previous findings of mild hyposmia in ataxia, and further suggests this may be due to general cognitive deficits rather than specific olfactory problems.


Assembling and rotating erythrocyte aggregates by acoustofluidic pressure enabling full phase-contrast tomography.
The combined use of ultrasound radiation and microfluidics is a promising tool for aiding the development of lab-on-a-chip devices. In this study, we show that the rotation of linear aggregates of micro-particles can be achieved under the action of acoustic field pressure. This novel manipulation is investigated by tracking polystyrene beads of different sizes through the 3D imaging features of digital holography (DH). From our analysis it is understood that the positioning of the micro-particles and their aggregations are associated with the effect of bulk acoustic radiation forces. The observed rotation is instead found to be compatible with the presence of acoustic streaming patterns as evidenced by our modelling and the resulting numerical simulation. Furthermore, the rotation frequency is shown to depend on the input voltage applied on the acoustic device. Finally, we demonstrate that we can take full advantage of such rotation by combining it with quantitative phase imaging of DH for a significant lab-on-a-chip biomedical application. In fact, we demonstrate that it is possible to put in rotation a linear aggregate of erythrocytes and rely on holographic imaging to achieve a full phase-contrast tomography of the aforementioned aggregate.


[The Munster Prognosis Score (MPS)--prognostic validity of a practical instrument for prediction of progress in schizophrenic psychosis].
The prognostic validity of a short and easy to handle prognostic instrument (Münster prognosis score, MPS) for schizophrenic patients is proved in this study. The MPS consists of 4 items: number of hospitalisations, stability of course of illness in the last year, premorbid development, and stability of psychosocial environment. Each item has to be rated as favourable or unfavourable. With three or four favourable ratings the prognosis is good, otherwise bad. The interrater reliability proved with 50 DSM-III schizophrenic patients had a kappa of 0.84, which is sufficient. In an 8-years follow-up with 66 DMS-III schizophrenic outpatients, the MPS had a comparably good prognostic validity according to the Strauss-Carpenter scale and the Philips scale in respect of the total symptom score (AMDP) and psychosocial functioning (GAS). According to prognosis of rehospitalisations, the MPS was as good as the SCS, and both were better than the Phillips scale.


Is microevolution the only emergency exit in a warming world? Temperature influences egg laying but not its underlying mechanisms in great tits.
Many bird species have advanced their seasonal timing in response to global warming, but we still know little about the causal effect of temperature. We carried out experiments in climate-controlled aviaries to investigate how temperature affects luteinizing hormone, prolactin, gonadal development, timing of egg laying and onset of moult in male and female great tits. We used both natural and artificial temperature patterns to identify the temperature characteristics that matter for birds. Our results show that temperature has a direct, causal effect on onset of egg-laying, and in particular, that it is the pattern of increase rather than the absolute temperature that birds use. Surprisingly, the pre-breeding increases in plasma LH, prolactin and in gonadal size are not affected by increasing temperature, nor do they correlate with the onset of laying. This suggests that the decision to start breeding and its regulatory mechanisms are fine-tuned by different factors. We also found similarities between siblings in the timing of both the onset of reproduction and associated changes in plasma LH, prolactin and gonadal development. In conclusion, while temperature affects the timing of egg laying, the neuroendocrine system does not seem to be regulated by moderate temperature changes. This lack of responsiveness may restrain the advance in the timing of breeding in response to climate change. But as there is heritable genetic variation on which natural selection can act, microevolution can take place, and may represent the only way to adapt to a warming world.


The phenomenon of the late recovered Bell's palsy: treatment options to improve facial symmetry.
Bell's palsy is an idiopathic neuropathy of cranial nerve VII, and the incidence ranges from 15 to 40 per 100,000. The majority of patients recover, but up to 16 percent of patients have significant sequelae. The phenomenon of the "late recovered" Bell's palsy has the following specific features and has not formerly been described: (1) tightening of the facial muscles, with a deepening nasolabial fold and reduced palpebral fissure; (2) blepharospasm; and (3) incomplete recovery of peripheral VIIth nerve branches, with ipsilateral forehead paralysis, reduced depressor anguli oris function, and poor excursion of the angle of the mouth on smiling. Nonsurgical treatment involved four monthly botulinum toxin injections. Patients had injections to paralyze the ipsilateral orbicularis oculi, contralateral forehead rhytides, and depressor anguli oris and to treat blepharospasm and muscle tightness. The effectiveness of the botulinum toxin injections on facial symmetry and patient appreciation of this were assessed by measuring brow height and teeth exposure before and 3 weeks after injection. Twenty-three patients were followed up for a mean period of 37 months. The difference in brow height and teeth exposure after injection was less than preinjection measurements, but this did not reach statistical significance. Patient self-assessments showed improvements in their appreciation of the facial symmetry, ability to go out in public, and feelings of self-worth (visual analogue scale). Surgical treatment options include ipsilateral brow lift, division of the contralateral frontal branch, contralateral tarsorrhaphy to equalize the palpebral fissures, and bilateral upper blepharoplasty. The true benefit of botulinum toxin injections was more apparent during facial animation and not when the face was static. The patients greatly appreciated the improvement in facial symmetry. Various treatment options are available to improve the quality of life for patients with late recovered Bell's palsy.


Navigation bronchoscopy for diagnosis and small nodule location.
Lung cancer continues to be the most common cause of cancer death. Screening programs for high risk patients with the use of low-dose computed tomography (CT) has led to the identification of small lung lesions that were difficult to identify using previous imaging modalities. Electromagnetic navigational bronchoscopy (ENB) is a novel technique that has shown to be of great utility during the evaluation of small, peripheral lesions, that would otherwise be challenging to evaluate with conventional bronchoscopy. The diagnostic yield of navigational bronchoscopy however is highly variable, with reports ranging from 59% to 94%. This variability suggests that well-defined selection criteria and standardized protocols for the use of ENB are lacking. Despite this variability, we believe that this technique is a useful tool evaluating small peripheral lung lesions when patients are properly selected.


Developmental origin of the neuronal subtypes that comprise the amygdalar fear circuit in the mouse.
We have taken a genetic-based fate-mapping approach to determine the specific contributions of telencephalic progenitors to the structures that comprise the amygdalar fear circuit including the central (CA), lateral (LA), and basolateral (BLA) amygdala. Our data indicate that progenitors in the ventral pallium (VP) contribute projection neurons to the LA and BLA but not the CA. Rather, the CA appears to derive, at least in part, from progenitors located in the ventral lateral ganglionic eminence (vLGE). Diverse groups of interneurons populate these amygdalar nuclei, and as predicted our data support the notion that they originate from subpallial progenitors. A rather specific population of amygdalar interneurons, the intercalated cells (ITCs), is known to play a fundamental role in fear-related behaviors. However, no information on their specific origin has, as yet, been provided. Our findings suggest that the ITCs arise from the dorsal lateral ganglionic eminence (dLGE) and migrate in the lateral migratory stream to populate the paracapsular regions as well as the main intercalated mass of the amygdala (IA). Germ-line Gsx2 mutants are known to exhibit an expansion of the VP into the LGE and a concomitant reduction in the dLGE and vLGE. Accordingly, Gsx2 conditional mutants display a significantly enlarged LA and a significant reduction in ITCs both within the paracapsular regions and the IA. Additional support for a dLGE origin of the ITCs was obtained in conditional mutants of the dLGE gene Sp8. Thus, our findings indicate diverse origins for the neuronal components that comprise the amygdalar fear circuit.


Increased microenvironment stiffness in damaged myofibers promotes myogenic progenitor cell proliferation.
The stiffness of the myogenic stem cell microenvironment markedly influences the ability to regenerate tissue. We studied the effect of damaged myofibers on myogenic progenitor cell (MPC) proliferation and determined whether the structural integrity of the microenvironment contributes to phenotypic changes. Individual myofibers were isolated and cultured for 6 days. During this period, the cytoskeleton of myofibers and transcription factors regulating MPC differentiation were characterized by immunostaining. Atomic Force Microscopy (AFM) was performed to measure stiffness of cultured myofibers. Healthy and damaged myofibers, and their associated MPCs, were studied in skeletal muscle from dystrophic and tenotomy mouse models. MPCs were cultured on stiffness-tunable substrates, and their phenotypes were assessed by immunostaining of myogenic transcription factors. We showed that individual myofibers tend to shrink or collapse when cultured ex vivo starting from day 1 and that this is associated with a marked increase in the number of proliferative MPCs (Pax7(+)MyoD(+)). The myofibers collapsed due to a loss of viability as shown by Evans blue dye uptake and the disorganization of their cytoskeletons. Interestingly, collapsed myofibers in mdx skeletal muscles were similar to damaged myofibers in that they lose their viability, have a disorganized cytoskeleton (actin and α-actinin), and display local MPC (MyoD(+)) proliferation at their periphery. In a tenotomy model that causes loss of muscle tension, the cytoskeletal disorganization of myofibers also correlated with the activation/proliferation of MPCs. A deeper analysis of collapsed myofibers revealed that they produce trophic factors that influence MPC proliferation. In addition, collapsed myofibers expressed several genes related to the basal lamina. Immunostaining revealed the presence of fibronectin in the basal lamina and the cytoplasm of damaged myofibers. Lastly, using atomic force microscopy (AFM), we showed that collapsed myofibers exhibit greater stiffness than intact myofibers. Growing MPCs on a 2-kPa polyacrylamide-based substrate, exempt of additional microenvironmental cues, recapitulated proliferation and reduced spontaneous differentiation compared to growth on a 0.5-kPa substrate. Our results support the notion that collapsed or damaged myofibers increase the structural stiffness of the satellite cell microenvironment, which in addition to other cues such as trophic factors and changes in extracellular matrix composition, promotes the proliferation and maintenance of MPCs, required for myofiber repair.


[The criteria for selecting the measurement methods in drinking water certification].
The drinking water certification system has been put into use since January 1, 1998. The paper analyzes this system-associated terminological problems and proposes a new wording of a criterion for requirements for water certification.


Indwelling pleural catheters.
Indwelling pleural catheters (IPC) are now established as one of the major tools for the management of recurrent pleural effusions. Their traditional role, which saw them only as second line treatment for malignant effusions, has now expanded. Recent evidence has not only suggested that they may be effectively employed as first-line therapy in some malignant cases, but that there is a wider spectrum of diseases which may be managed by their use. The majority of patients are likely to experience symptomatic benefit and some may also go on to achieve pleurodesis. IPCs are relatively simple to insert and maintain, and theoretically allow patients to be managed entirely as an outpatient, meaning that they are likely to be cost-effective in the longer term. They can also dramatically improve the quality of life in patients who have typically needed lengthy hospital admissions or who have terminal malignant disease.


Does competition for transplantable hearts encourage 'gaming' of the waiting list?
Transplant centers may "game" the severity of listed patients to increase their patients' likelihood of receiving transplantable organs. Recent lawsuits allege gaming at some centers, and listing policies were modified in 1999 to clarify listing criteria. We tested for gaming and its relationship to heart transplant center competition. We found that increased competition resulted in more patients listed in the most severe illness category (p < .01), consistent with the gaming hypothesis. Gaming was mitigated after the 1999 policy change (p > .05), which suggests that the new rules were effective. Continued monitoring is warranted, given prior gaming and recent accusations.


[Neurologic manifestations of subarachnoid and parenchymatous hemorrhages caused by arterial aneurysm].
Overall 155 patients with subarachnoidal and parenchymatous hemorrhages from arterial aneurysms, mainly of the anterior part of the circle of Willis, were examined. The intensity of subarachnoidal and parenchymatous hemorrhages varied, with the ++diencephalo-hypothalamic area being largely involved. The volume of intraparenchymatous hemorrhages ranged from 10 to 90 ml. Massive basal SAH was accompanied, in a number of cases, by blood congestion in the fourth ventricle. Correlations were established between the gravity of the health status, clinical cerebral decompensation, and the intensity of hemorrhage to the basal subarachnoidal space and cerebral parenchyma. Three variants of clinical decompensation of the brain were revealed: it ran a torpid course in the majority of cases (64%), it increased dramatically in every fourth patient, and gradually regressed in every 10th patient.


Identification of the functional PD-L1 interface region responsible for PD-1 binding and initiation of PD-1 signaling.
The PD-1/PD-L1 checkpoint pathway is important for regulating immune responses and can be targeted by immunomodulatory drugs to treat a variety of immune disorders. However, the precise protein-protein interactions required for the initiation of PD-1/PD-L1 signaling are currently unknown. Previously, we designed a series of first-generation PD-1 targeting peptides based on the native interface region of programmed death ligand 1 (PD-L1) that effectively reduced PD-1/PD-L1 binding. In this work, we further characterized the previously identified lead peptide, MN1.1, to identify key PD-1 binding residues and design an optimized peptide, MN1.4. We show MN1.4 is significantly more stable than MN1.1 in serum and retains the ability to block PD-1/PD-L1 complex formation. We further characterized the immunomodulatory effects of MN1.4 treatment by measuring markers of T cell activation in a co-culture model with ovarian cancer cells and peripheral blood mononuclear cells. We found MN1.4 treatment reduced cytokine secretion and suppressed T cell responses in a similar manner as recombinant PD-L1. Therefore, the PD-L1 interface region used to design MN1.4 appeared sufficient to initiate PD-1 signaling and likely represents the minimum necessary region of PD-L1 required for PD-1 recognition. We propose a peptide agonist for PD-1, such as MN1.4, could have several applications for treating autoimmune disorders caused by PD-1 deficiencies such as type 1 diabetes, inflammatory arthritis, or autoimmune side effects arising from monoclonal antibody-based cancer immunotherapies.


Antioxidant and anti-inflammatory role of zingerone in ethanol-induced hepatotoxicity.
Alcoholic liver disease is a direct result of alcohol-induced hepatotoxicity coupled with impaired hepatic regenerative activity. Our aim of the study was to investigate the beneficial effect of zingerone on hepatic oxidative stress and inflammation induced by ethanol in experimental rats. Male albino Wistar rats were divided into four groups. Rats of groups 1 and 2 received isocaloric glucose and dimethyl sulfoxide (2 % DMSO). Hepatotoxicity was induced in groups 3 and 4 by supplementing 30 % ethanol post orally for 60 days. Rats of groups 2 and 4 received zingerone (20 mg/kg body weight in 2 % DMSO p.o) daily during the final 30 days of the experimental period. Ethanol alone administered rats showed significant increase in the plasma and tissue lipid peroxidation markers such as thiobarbituric acid reactive substances, lipid hydroperoxides, conjugated dienes, and a significant decrease in the activities of plasma and tissue enzymic and non-enzymic antioxidants such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, reduced glutathione, vitamin C, and vitamin E. Moreover, the presence of mast cells and increase in the expressions of inflammatory markers such as NF-κB, COX-2, TNF-α, and IL-6 and decrease in the expression of Nrf2 in the liver was observed in ethanol-fed rats. Supplementation with zingerone to ethanol-fed rats reversed the changes induced by ethanol in the experimental rats. Thus, zingerone, through its antioxidant and anti-inflammatory effects, may represent a therapeutic option to protect against ethanol-induced hepatotoxicity.


Identification of risk loci for necrotizing meningoencephalitis in Pug dogs.
Due to their unique population structure, purebred dogs have emerged as a key model for the study of complex genetic disorders. To evaluate the utility of a newly available high-density canine whole-genome array with >170,000 single nucleotide polymorphisms (SNPs), genome-wide association was performed on a small number of case and control dogs to determine disease susceptibility loci in canine necrotizing meningoencephalitis (NME), a disorder with known non-Mendelian inheritance that shares clinical similarities with atypical variants of multiple sclerosis in humans. Genotyping of 30 NME-affected Pug dogs and 68 healthy control Pugs identified 2 loci associated with NME, including a region within dog leukocyte antigen class II on chromosome 12 that remained significant after Bonferroni correction. Our results support the utility of this high-density SNP array, confirm that dogs are a powerful model for mapping complex genetic disorders and provide important preliminary data to support in depth genetic analysis of NME in numerous affected breeds.


Two phase I studies of low dose recombinant human IL-12 with Melan-A and influenza peptides in subjects with advanced malignant melanoma.
Preclinical studies have shown that low dose IL-12 can potentiate cytotoxic lymphocyte responses. Since previous trials have demonstrated significant toxicity from high dose recombinant human IL-12 (rhIL-12), we sought to determine an optimal biological dose for rhIL-12 at lower doses when combined with peptide antigens. Two studies were undertaken. The rhIL-12 was administered at doses of 0 (placebo), 10, 30 and 100 ng/kg, subcutaneously in one study and intravenously in the other. Apart from IL-12 dosing, the studies were identical. Subjects had evaluable stage III or IV melanoma which expressed Melan-A by RT-PCR or immunohistochemistry. Melan-A (26-35) (EAAGIGILTV) and influenza matrix (58-66) (GILGFVFTL) peptides were administered intradermally on weeks 1, 2, 3, 4 and 9. Twenty-eight subjects were enrolled, of whom 24 were evaluable for clinical and immunological responses. Therapy was well tolerated, the main adverse event being influenza-like symptoms. Immunological monitoring included the evaluation of cutaneous reactions and assays for antigen-specific T-cells. Clinical responses included a complete response in a subject with small volume subcutaneous disease, a partial response in a subject with hepatic metastases, and mixed responses in pulmonary, pleural and nodal disease. Biopsies of accessible tumors showed infiltration with CD4+ and CD8+ lymphocytes capable of lysing Melan-A peptide-pulsed targets in vitro. No clear dose-dependent effect of rhIL-12 could be determined. The rhIL-12 given either s.c. or i.v. was well tolerated at doses of 10-100 ng/kg. Clinical and immunological activity has been observed in this study where peptides were administered either with or without low dose rhIL-12.


Effect of low frequency electromagnetic field on the behavior and bioenergetics of the polychaete Hediste diversicolor.
The aim of the present study was to determine the effect of an electromagnetic field (EMF) of value typically recorded in the vicinity of submarine cables (50 Hz, 1 mT) on the behavior and bioenergetics of the polychaete Hediste diversicolor. No avoidance or attraction behavior to EMF was shown, but the burrowing activity of H. diversicolor was enhanced in EMF treatment, indicating a stimulating effect on bioturbation potential. The polychaete maintained a positive energy balance and high amount (85% of assimilated energy) of energy available for individual production (scope for growth) after exposure to EMF. Food consumption and respiration rates were not affected but ammonia excretion rate was significantly reduced in EMF-exposed animals compared to the control conditions (geomagnetic field). The mechanisms behind this effect remain, however, unclear. This is the first study demonstrating the effects of environmentally realistic EMF value on the behavior and physiology of marine invertebrates, thus there is a need for more research.


Haemophilus influenzae ophthalmia neonatorum.
Haemophilus influenzae is an occasional cause of ophthalmia neonatorum. Recently, this organism has exhibited a change in its epidemiology and has been identified as a cause of severe neonatal infections. Ophthalmologists should be aware of the changing susceptibility of neonates to severe infections with H influenzae and the possibility that H influenzae ophthalmia neonatorum can be associated with these infections.


Transient ischemic injury in the rat retina caused by thrombotic occlusion-thrombolytic reperfusion.
To establish a clinically relevant model of transient retinal ischemia by thrombotic occlusion-thrombolytic reperfusion of the central retinal artery of the rat. Thrombus was photochemically induced in the central retinal artery by the combination of intravenous injection of photo-sensitive dye, rose bengal, and green laser irradiation focused on the artery. Transient retinal ischemia for 60 minutes was achieved by a subsequent systemic administration of tissue-type plasminogen activator to reperfuse the occluded vessel. Samples of retinas were excised from the animals killed 3, 9, 12, 24, 48, and 78 hours after the reperfusion. The experimental data were processed using the TdT-dUTP terminal nick-end labeling (TUNEL) method to detect apoptotic cells. The transient retinal ischemia caused time-sequential apoptotic changes in the retinal cells as evaluated by counting the number of TUNEL-positive cells. The most remarkable changes occurred in the central area of retina, and further on the sections taken 24 hours after reperfusion. The peripheral area was less affected, and the outer nuclear cell layer was almost unaffected throughout the observation period. The proposed method to cause retinal transient ischemia is highly reproducible, and it is easy to simulate the progress and topographical distribution of retinal changes observed in the clinical cases of central retinal arterial occlusion and its subsequent thrombolytic reperfusion. This may provide a useful tool for constructing the effective thrombolytic strategies against the central retinal arterial occlusion and for evaluating the effects of neuroprotective agents.


Biliopancreatic Diversion with a New Type of Gastrectomy.
In an attempt to improve the results of biliopancreatic diversion in the treatment of morbid obesity, two aspects of the procedure performed at Laval Hospital were modified to reduce adverse physiological consequences. The distal gastrectomy was replaced by a parietal gastrectomy which preserves vagal continuity along with the lesser curvature, and leaves intact the antro-pyloroduodenal pump. The duodenum was stapled shut and nutrients were diverted through a duodeno-ileal anastomosis. The biliopancreatic diverting intestinal limb was anastomosed to the nutrient ileal limb 100 cm proximal to the ileocaecal valve instead of 50 cm proximal to it, thus doubling the length of the common ileal absorptive segment. Weight loss after either operation was greater than 70% of initial excess weight. Following the new operation, there was a lesser prevalence of side-effects, especially loose stools and malodorous gas, a lesser degree of hypocalcemia and no hypoalbuminemia. The duodenum recanalized at the staple line in 20% of the patients who had the new operation. When data from these patients were excluded, weight loss following the new operation was greater than that seen after the old one. The prevalence of side-effects and the degree of calcium and protein malabsorption remained significantly lower. Weight loss remained satisfactory with a common limb measuring 100 cm. The parietal gastrectomy was not restrictive as shown by the failure to lose further weight when the duodenal stapled diversion failed. Weight loss was thus mainly a function of biliopancreatic diversion, but increased weight loss in the new procedure despite a doubling of the common ileal limb suggests that parietal gastrectomy contributed to weight loss. Because duodenal recanalization can be corrected surgically and now prevented, the modified biliopancreatic bypass is preferred.


Suppression of TLR2-induced IL-12, reactive oxygen species, and inducible nitric oxide synthase expression by Mycobacterium tuberculosis antigens expressed inside macrophages during the course of infection.
We report the enrichment of and immune responses mediated by genes expressed by Mycobacterium tuberculosis inside macrophages as a function of time. Results indicate that M. tuberculosis expresses different genes at different times postinfection. Genes expressed early (day 1) following infection enhance M. tuberculosis-mediated activation of dendritic cells (DCs), whereas genes expressed later (day 5) in the infection prevent DC activation. However, all genes downmodulated MHC class I and II expression on infected macrophages, thus compromising their ability to interact with Ag-specific T cells. Day-1 and -5 genes downmodulated proinflammatory cytokine production from DCs, thus impairing signal 3 during DC-T cell cognate interactions. Consequently, T cells activated by Ag-experienced DCs secreted low levels of IFN-gamma and IL-17 but maintained high IL-10 secretion, thus inducing suppressor responses. Further characterization revealed that day-1 and -5 genes increased TLR2-induced expression of suppressors of cytokine signaling 1 from DCs and downmodulated IL-12 expression. In addition, day-1 and -5 genes prevented the generation of reactive oxygen species in DCs. In contrast, although day-5 genes increased TLR2-mediated suppressors of cytokine signaling 1 expression in macrophages, day-1 genes downmodulated the expression of inducible NO synthase 2. Similar downregulation of immune responses was observed upon exogenous stimulation with day-1 or -5 Ags. Finally, day-1 and -5 genes promoted enhanced survival of M. tuberculosis inside DCs and macrophages. These results indicate that M. tuberculosis genes, expressed inside infected macrophages as a function of time, collectively suppress protective immune responses by using multiple and complementary mechanisms.


The Streptomyces reticuli alpha-chitin-binding protein CHB2 and its gene.
When co-cultivated with chitin-containing fungi, Streptomyces reticuli secretes the chitin-binding protein CHB2. Microscopical and immunological investigations revealed that CHB2 acts like a glue to mediate the contact between the fungal and the Streptomyces hyphae. CHB2 was purified to homogeneity, and the sequence of its N-terminal amino acids was determined and used to deduce an oligonucleotide, which was then used to probe a subgenomic library. The chb2 gene was cloned, sequenced and overexpressed. The deduced mature protein has a molecular mass of 18.6 kDa, and a large number of its amino acids are identical to those of CHB1 from Streptomyces olivaceoviridis. CHB2 effectively targets different types of alpha-chitin, but no other polysaccharide. The dissociation constant (Kd) for binding to purified crab shell chitin is 0.27 microM. Immunological studies suggest that homologues of CHB1 and CHB2 are secreted by streptomycetes while growing in the presence of alpha-chitin-containing substrates.


Comparison on phenolic compounds and antioxidant properties of cabernet sauvignon and merlot wines from four wine grape-growing regions in China.
The antioxidant activities in the Cabernet Sauvignon and Merlot wines from four wine grape-growing regions in China were measured by different analytical assays: 2,2-diphenyl-1-picrylhydrazyl (DPPH·), cupric reducing antioxidant capacity (CUPRAC), superoxide radical-scavenging activity (SRSA) and the contents of total phenols, total flavonoids, total flavanols and total anthocyanins were determined. The results showed that the contents of phenolic compounds and the levels of antioxidant activity in the wine samples greatly varied with cultivar and environmental factors of vine growth. The contents of phenolic compounds and antioxidant activities in Cabernet Sauvignon and Merlot wines from the Yuquanying region of Ningxia were significantly higher than other three regions, followed by the wines from Shacheng region of Hebei, and these parameters were the lowest in Cabernet Sauvignon and Merlot wines from the Changli regions of Hebei and Xiangning region of Shanxi. Taken together, a close relationship between phenolic subclasses and antioxidant activity was observed for the wine samples. Moreover, there were significant discrepancies in the individual phenolic composition and content of four regional Cabernet Sauvignon and Merlot wines, among which the individual phenolic compounds (catechin, epicatechin, cinnamic acid, quercetin-3-O-glucuronide, quercetin-3-O-glucoside, laricitrin-3-O-glucoside and isorhamnetin-3-O-glucoside) revealed a significant correlation (p < 0.05) with the antioxidant capacity in present study, especially for catechin and epicatechin.


Synthesis, structure, and photophysical and electrochemical properties of a pi-stacked polymer.
Dibenzofulvene (DBF) was polymerized using anionic initiators to afford a vinyl polymer. Oligo(DBF)s having from two to eight side-chain fluorene moieties bearing different chain-terminal groups were isolated by preparative size-exclusion chromatography. The structures of the isolated oligomers were revealed by single-crystal X-ray and (1)H NMR analyses. Both in solution and in crystal, the in-chain fluorene moieties stacked on top of each other, while the terminal conformation varied depending on the terminal group. These conformational characteristics were supported by molecular mechanics and dynamics calculations. The oligomers and polymers indicated hypochromism and red shift in UV absorption spectra and exclusive excimer emission in fluorescence spectra. In addition, reduced oxidation potentials were observed for the oligomers in electrochemical analyses, which suggests charge delocalization over the pi-stacked electron systems. The photophysical and electrochemical effects increased with the chain length of the oligomers and leveled off around the chain length of an oligomer consisting of five fluorene units.


Neuropathology of HAND With Suppressive Antiretroviral Therapy: Encephalitis and Neurodegeneration Reconsidered.
HIV-1 infiltrates the central nervous system (CNS) during the initial infection and thereafter plays a persistent role in producing CNS dysfunction as the disease progresses. HIV-associated neurocognitive disorders (HAND) are highly prevalent in HIV-infected patient populations, including currently infected patients with good access to suppressive antiretroviral therapy (cART). cART decreased the severity of CNS dysfunction dramatically and, in doing so, upended the neuropathological foundation of HAND pathophysiology. It is clear that the working concept of pathophysiology prior to cART, which was driven by inflammation, encephalitis, and neurodegeneration, needs to be replaced. The NeuroAIDS field is reluctant to take that important step. This review explores the fact that the neuropathological concept that drove the field before the era of cART no longer seems to fit with what is commonly observed in patients treated successfully with cART. The field clings to the pre-cART idea that HAND is sequentially driven by virus replication in CNS, brain inflammation (encephalitis), and neurodegeneration. Neurovirological, clinicopathological, and gene expression correlations in cART-treated patients, however, provide little strong support for it. Introducing cART into clinical practice decreased HIVE, inflammation, and degeneration but did not cure HAND. Brain gene array data suggest that the neurovascular unit is a critical target in virally suppressed patients with HAND. The NeuroAIDS field needs an infusion of new ideas to steer research toward issues of the highest relevance to virally suppressed patients. With no suitable replacement immediately within reach, devaluating formative ideas is understandably difficult to accept. The cliniconeuropathological correlation in virally suppressed patients needs to be better defined.


A point mutation within a distinct conserved region of the herpes simplex virus DNA polymerase gene confers drug resistance.
We have shown that a drug-resistant mutant from a clinical isolate of herpes simplex virus contains a single point mutation in the DNA polymerase gene that confers resistance to both acyclovir and foscarnet. The mutated amino acid is located within a distinct conserved region shared among alpha-like DNA polymerases which we designate region VII. We infer that these conserved sequences are directly or indirectly involved in the recognition and binding of nucleotide and PPi substrates.


Direct measurement of change in muscle glycogen concentration after a mixed meal in normal subjects.
Postprandial storage of carbohydrate as glycogen in muscle was quantitated in normal subjects (n = 8) by natural abundance 13C-nuclear magnetic resonance spectroscopy with proton decoupling in a 4.7-tesla magnet. After an overnight fast three basal measurements of gastrocnemius muscle glycogen were made and a mixed meal was given. Muscle glycogen concentration rose from 83.3 +/- 5.2 to a maximum of 100.2 +/- 6.7 mmol/l muscle at 4.9 h (P < 0.01) and fell thereafter to 90.6 +/- 5.9 mmol/l muscle at 7 h postprandially (P < 0.006). The meal brought about an increase in plasma glucose from 5.4 +/- 0.2 to 7.3 +/- 0.4 mmol/l at 30 min but this was followed by a rapid fall to 6.2 +/- 0.4 mmol/l at 75 min. Plasma insulin rose from 62.4 +/- 11.4 to 900 +/- 216 pmol/l at 30 min and declined steadily thereafter. It was calculated from total muscle mass measurements and estimation of carbohydrate absorption rates that at peak muscle glycogen concentrations between 26 and 35% of the absorbed carbohydrate was stored as muscle glycogen. These data quantitate the role of skeletal muscle glycogen synthesis in postprandial carbohydrate storage and demonstrate that this tissue acts as a dynamic buffer to maintain glucose homeostasis during postprandial substrate storage.


Chemoprevention of colorectal cancer through inhibition of cyclooxygenase-2.
Two events in the last decade have set the stage for the large-scale clinical testing of chemopreventive agents for colorectal cancer in people at low to moderate risk for this disease. One of these is the discovery of a cause-effect relationship between the activities of cyclooxygenases (COX) and carcinogenesis in the colon, which can be interdicted by inhibitors of the enzymes. The other is the development of selective inhibitors of COX-2. These agents, when used in animals, also inhibit carcinogenesis in the colon. Additionally, they appear to be safe enough in humans to allow large-scale testing in healthy people. We review the key data implicating a causal relationship between the activity of COX and carcinogenesis and its possible mechanisms of action. We also emphasize work that points to other molecular targets for chemoprevention of colorectal cancer, which is emerging from studies of the link between COX and carcinogenesis.


Immunologic memory to phosphocholine. VII. Lack of T15 V1 gene utilization in Xid anti-PC hybridomas.
CBA/N mice carrying the Xid defect fail to make antibodies expressing the T15 idiotype in response to immunization with PC-KLH. Antibodies predominating in the Xid response have binding properties characteristic of group II antibodies that emerge in the memory response in BALB/c; the prototype group II antibody utilizes a VH gene product distinct from the V1 gene product expressed by T15 idiotype-positive antibodies. To examine VH gene usage in the anti-PC response of Xid B cells, hybridomas were produced from Xid mice immune to PC-KLH. Four hybridomas possessing properties typical of the predominant group II antibody response in Xid mice and two representing minor components of the response were studied. Analysis of DNA by Southern blot hybridization revealed that none of the hybridomas utilized the T15 V1 gene segment, nor did they share use of a common VDJ gene product. These results indicate that Xid group II antibodies either make use of different VH gene segments or use the same VH in combination with various D and JH segments.


Opposite effect of intracellular Ca2+ and protein kinase C on the expression of inwardly rectifying K+ channel 1 in mouse skeletal muscle.
The level of inwardly rectifying K+ channel 1 (IRK1) mRNA decreased upon denervation and increased during muscle differentiation in mouse skeletal muscle. To identify the mechanism(s) underlying the regulation of IRK1 mRNA expression, we examined its expression using the well differentiated C2C12 mouse skeletal muscle cell line as a model system. Since nerve-induced muscle activity results in contraction, it was questioned whether the changes in IRK1 expression might be relevant to the increased intracellular calcium that functions as a cytoplasmic messenger in excitation-contraction coupling. Indeed, activation of either L-type calcium channels or ryanodine receptors increased the level of IRK1 mRNA. More directly, ionomycin activated the IRK1 expression in time- and dose-dependent manners, which was abolished by treatment with EGTA. Genistein, a tyrosine kinase inhibitor, also abolished the stimulating effect of ionomycin. Meanwhile, activation of protein kinase C by 12-O-tetradecanoylphorbol acetate (TPA) markedly decreased the level of IRK1 mRNA, which required ongoing protein synthesis. Actinomycin D experiments revealed that ionomycin increased the half-life of IRK1 mRNA from 0.86 to 1.97 h, but TPA decreased it to 0.38 h. However, neither ionomycin nor TPA appreciably altered the rate of IRK1 gene transcription. Based on these observations, we conclude that intracellular calcium and protein kinase C are oppositely involved in the muscle activity-dependent regulation of IRK1 gene expression and that both act at the level of mRNA stability.


[Patient liaison service and social work unit].
The present article describes the activity of the Patient Liaison Service and Social Work Unit on March 11 and subsequent days. The service centered its activity on providing support, advice, and information to relatives, as well as on resource management and identification and localization of victims. The organization of activities and the response of the various professionals that comprise the team, as well as the results of their interventions are discussed.


Targeted regulation of transcription in primary cells using CRISPRa and CRISPRi.
Targeted transcriptional activation or interference can be induced with the CRISPR-Cas9 system (CRISPRa/CRISPRi) using nuclease-deactivated Cas9 fused to transcriptional effector molecules. These technologies have been used in cancer cell lines, particularly for genome-wide functional genetic screens using lentiviral vectors. However, CRISPRa and CRISPRi have not yet been widely applied to ex vivo cultured primary cells with therapeutic relevance owing to a lack of effective and nontoxic delivery modalities. Here we develop CRISPRa and CRISPRi platforms based on RNA or ribonucleoprotein (RNP) delivery by electroporation and show transient, programmable gene regulation in primary cells, including human CD34+ hematopoietic stem and progenitor cells (HSPCs) and human CD3+ T cells. We show multiplex and orthogonal gene modulation using multiple sgRNAs and CRISPR systems from different bacterial species, and we show that CRISPRa can be applied to manipulate differentiation trajectories of HSPCs. These platforms constitute simple and effective means to transiently control transcription and are easily adopted and reprogrammed to new target genes by synthetic sgRNAs. We believe these technologies will find wide use in engineering the transcriptome for studies of stem cell biology and gene function, and we foresee that they will be implemented to develop and enhance cellular therapeutics.


Delusion proneness in nonclinical individuals and cognitive insight: the contributions of rumination and reflection.
Although previous research demonstrates that clinical individuals with delusions score low on one of the facets of cognitive insight, self-reflection, and high on the other facet, self-certainty, analogous studies of delusion proneness in nonclinical individuals have found that delusion proneness in nonclinical individuals associates with higher levels of both self-certainty and self-reflection. The present study sought to reconcile these inconsistent results by examining the contributions of different facets of self-reflection, rumination and reflection, to delusion proneness. One hundred fifty-two individuals completed three questionnaires: the Beck Cognitive Insight Scale (BCIS), the Peters et al. Delusions Inventory (PDI), and the Rumination-Reflection Questionnaire (RRQ). The results showed that the individuals scoring higher on delusion proneness demonstrated higher levels of both self-certainty and self-reflection on the BCIS as well as higher levels of rumination and reflection on the RRQ. As predicted, the strength of the relationship between BCIS self-reflection and delusion proneness was diminished when rumination was controlled for. These findings suggest that the previously observed positive relation between BCIS self-reflection and nonclinical delusion proneness might be driven, in part, by the ruminative aspect of self-reflection.


Identification and characterization of alpha 3 beta 1 integrin on primary and transformed rat islet cells.
Dispersed rat islet cells embedded in a matrix of collagen I are known to form aggregates in vitro reminiscent of native islets. Furthermore, it appears that islet function and survival are better maintained in vitro when cells are grown in the presence of extracellular matrix. These studies suggest an important role of cell--matrix interactions in the formation and maintenance of islet structure and function. The molecular basis of these interactions is mostly unknown. In the present study, we confirm the presence of beta 1 integrins on primary and transformed (RIN-2A line) rat islet cells. Perturbation studies in vitro show that beta 1 integrins play a role in islet cell attachment and spreading on bovine extracellular matrix and on the matrix produced by A-431 cells. The alpha 3 integrin subunit is coimmunoprecipitated with beta 1 from extracts of both primary and transformed islet cells, and immunodepletion studies suggest that alpha 3 beta 1 represents nearly half of the total beta 1 integrins expressed on primary islet cells. In situ, alpha 3 and beta 1 are expressed on the surface of all islet cell types, as shown by indirect immunocytochemistry on paraformaldehyde-fixed sections of rat pancreas. In conclusion, the study demonstrates the presence of alpha 3 beta 1 on primary and transformed rat islet cells, and an important role of beta 1 integrins in islet cell attachment and spreading in vitro.


Machine Learning to Classify Cardiotocography for Fetal Hypoxia Detection.
Fetal hypoxia can cause damaging consequences on babies' such as stillbirth and cerebral palsy. Cardiotocography (CTG) has been used to detect intrapartum fetal hypoxia during labor. It is a non-invasive machine that measures the fetal heart rate and uterine contractions. Visual CTG suffers inconsistencies in interpretations among clinicians that can delay interventions. Machine learning (ML) showed potential in classifying abnormal CTG, allowing automatic interpretation. In the absence of a gold standard, researchers used various surrogate biomarkers to classify CTG, where some were clinically irrelevant. We proposed using Apgar scores as the surrogate benchmark of babies' ability to recover from birth. Apgar scores measure newborns' ability to recover from active uterine contraction, which measures appearance, pulse, grimace, activity and respiration. The higher the Apgar score, the healthier the baby is.We employ signal processing methods to pre-process and extract validated features of 552 raw CTG. We also included CTG-specific characteristics as outlined in the NICE guidelines. We employed ML techniques using 22 features and measured performances between ML classifiers. While we found that ML can distinguish CTG with low Apgar scores, results for the lowest Apgar scores, which are rare in the dataset we used, would benefit from more CTG data for better performance. We need an external dataset to validate our model for generalizability to ensure that it does not overfit a specific population.Clinical Relevance- This study demonstrated the potential of using a clinically relevant benchmark for classifying CTG to allow automatic early detection of hypoxia to reduce decision-making time in maternity units.


Prevention of breast cancer-induced osteolytic bone resorption by benzyl isothiocyanate.
Osteolytic bone resorption is the primary cause of pain and suffering (e.g. pathological bone fracture) in women with metastatic breast cancer. The current standard of care for patients with bone metastasis for reducing the incidence of skeletal complications includes bisphosphonates and a humanized antibody (denosumab). However, a subset of patients on these therapies still develops new bone metastasis or experiences adverse effects. Moreover, some bisphosphonates have poor oral bioavailability. Therefore, orally-bioavailable and non-toxic inhibitors of breast cancer-induced osteolytic bone resorption are still clinically desirable. We have shown previously that benzyl isothiocyanate (BITC) decreases the incidence of breast cancer in a transgenic mouse model without any side effects. The present study provides in vivo evidence for inhibition of breast cancer-induced osteolytic bone resorption by BITC. Plasma achievable doses of BITC (0.5 and 1 μM) inhibited in vitro osteoclast differentiation induced by co-culture of osteoclast precursor cells (RAW264.7) and breast cancer cells representative of different subtypes. This effect was accompanied by downregulation of key mediators of osteoclast differentiation, including receptor activator of nuclear factor-κB ligand and runt-related transcription factor 2 (RUNX2), in BITC-treated breast cancer cells. Doxycycline-inducible knockdown of RUNX2 augmented BITC-mediated inhibition of osteoclast differentiation. Oral administration of 10 mg BITC/kg body weight, 5 times per week, inhibited MDA-MB-231-induced skeletal metastasis multiplicity by ~81% when compared with control (P = 0.04). The present study indicates that BITC has the ability to inhibit breast cancer-induced osteolytic bone resorption in vivo.


Acetabular reconstruction with impacted morsellised cancellous bone graft and cement. A 10- to 15-year follow-up of 60 revision arthroplasties.
We report a long-term review of 60 acetabular components revised using impacted, morsellised bone allografts and a cemented polyethylene cup. The acetabular defects were cavitary (37) or combined (23). Follow-up was for a mean 11.8 years (10 to 15). Further revision was needed in five hips, two for septic and three for aseptic loosening. The overall survival rate at 11.8 years was 90%; excluding the septic cases it was 94%. Acetabular reconstruction with impacted morsellised cancellous grafts and cement gives satisfactory long-term results.


On the sure criticality of tasks in activity networks with imprecise durations.
The notion of the necessary criticality (both with respect to path and to activity) of a network with imprecisely defined (by means of intervals or fuzzy intervals) activity duration times is introduced and analyzed. It is shown, in the interval case, that both the problem of asserting whether a given path is necessarily critical and the problem of determining an arbitrary necessarily critical path (more exactly, a subnetwork covering all the necessarily critical paths) are easy. The corresponding solution algorithms are proposed. However, the problem of evaluating whether a given activity is necessarily critical does not seem to be so easy. Certain conditions are formulated which, in some situations (but not in all possible situations), allow the necessary criticality of activities to be evaluated. The results obtained for networks with interval activity duration times are generalized to the case of networks with fuzzy activity duration times. Two effective algorithms for calculating the degree of necessary criticality of a fixed path, as well as an algorithm for determining the paths that are necessarily critical to the maximum degree, are proposed.


Partial purification and characterization of a novel neutral proteinase from human uterine cervix.
1. Human uterine cervical stroma was found to contain a Ca(2+)-independent neutral proteinase against casein and N-benzoyl-dl-arginine p-nitroanilide (Bz-dl-Arg-Nan). This enzyme was tightly bound to an insoluble material (20000g pellet) and was solubilized by high concentrations of NaCl or KCl. High concentrations of them in the reaction system, however, inhibited reversibly the activity of this enzyme. 2. The neutral proteinase was partially purified by extraction with NaCl, gel filtration on Sephadex G-200 and affinity chromatography on casein-Sepharose. 3. The optimal pH of this partially purified enzyme was 7.4-8.0 against casein and Bz-dl-Arg-Nan. The molecular weight of the enzyme was found to be about 1.4x10(5) by gel filtration on Sephadex G-200. 4. The enzyme was significantly inhibited by di-isopropyl phosphorofluoridate (0.1mm). High concentration of phenylmethanesulphonyl fluoride (5mm), 7-amino-1-chloro-3-l-tosylamidoheptan-2-one (0.5mm), antipain (10mum) or leupeptin (10mum) was also found to be inhibitory, but chymostatin (40mug/ml), soya-bean trypsin inhibitor (2.5mg/ml), human plasma (10%, v/v), p-chloromercuribenzoate (1mm), EDTA (10mm) and 1-chloro-4-phenyl-3-l-tosylamidobutan-2-one (1mm) had no effect on the enzyme. 5. The neutral proteinase hydrolysed casein, Bz-dl-Arg-Nan and heat-denatured collagen, but was inactive towards native collagen and several synthetic substrates, such as 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg, 3-carboxypropionyl-Ala-Ala-Ala p-nitroanilide and 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-d-Arg, and also proteoglycan. The enzyme did not act as a plasminogen activator. 6. These properties suggested that a neutral proteinase in the human uterine cervix was different from enzymes previously reported.


Secretin: specific binding to rat brain membranes.
The binding of [125I]secretin to rat brain membranes was investigated. Radiolabeled secretin bound with high affinity (KD = 0.2 nM) to a single class of noninteracting sites. Binding was specific, saturable, and reversible. Regional distribution studies indicated that the specific binding was greatest in the cerebellum, intermediate in the cortex, thalamus, striatum, hippocampus, and hypothalamus, and lowest in the midbrain and medulla/pons. Pharmacological studies indicated that only secretin, but not other peptides, inhibits binding of [125I]secretin with high affinity. Also, certain guanine nucleotides inhibited high affinity binding. These data indicate that rat brain membranes possess high affinity binding sites specific for secretin and that with the use of [125I] secretin the kinetics, stoichiometry, specificity, and distribution of secretin receptors can be directly investigated.


Comparative analysis of the CD19+ and CD138+ cell antibody repertoires in the cerebrospinal fluid of patients with multiple sclerosis.
Increased amounts of intrathecally synthesized IgG and oligoclonal bands have long been recognized as a hallmark of multiple sclerosis (MS). B cells and plasma cells are components of the inflammatory infiltrates in both active and chronic MS lesions, and increased numbers of these cells are present in MS cerebrospinal fluid (CSF). Single-cell RT-PCR was used to analyze both the CD19+ B cell and CD138+ plasma cell populations in CSF of two patients with clinically definite MS and of one MS patient whose CSF was obtained after a clinically isolated syndrome, but before the second episode. Sequence analysis of amplified IgG V region sequences identified the rearranged germline segments, extent of somatic mutation, and clonal relationships within and between the two cell populations in the three MS patients. Expanded B cell and plasma cell clones were detected in each MS CSF and in all three patients the CD138+ IgG repertoire was more restricted. However, little if any significant sequence overlap was observed between the CD19+ and CD138+ repertoires of each donor. Detection of plasma cell clones by single-cell PCR will facilitate the in vitro production of recombinant Abs useful in identifying disease-relevant Ags.


Effect of antimicrobial therapy with amoxicillin and cefprozil on bacterial interference and beta-lactamase production in the adenoids.
To compare the effects on the bacterial flora of the adenoids of antimicrobial therapy with amoxicillin or cefprozil, we enrolled in a prospective randomized study 60 children scheduled for elective adenoidectomy because of recurrent otitis media. They were randomized before surgery into 3 groups of 20 patients each: a no-therapy group, and groups with 10 days of either amoxicillin or cefprozil therapy. Core adenoid materials were quantitatively cultured for aerobic and facultative bacteria. The in vitro ability of alpha-hemolytic streptococci (AHS) to inhibit the growth of non-type B Haemophilus influenzae and Streptococcus pneumoniae was determined. The number of organisms in adenoids obtained from patients treated with either antibiotic was reduced as compared to that in adenoids from controls. However, in patients treated with amoxicillin, a significant decline in the number of AHS, and an increase (in Staphylococcus aureus) or no change in the number of other beta-lactamase-producing bacteria (BLPB) was noted. In contrast, in those treated with cefprozil, no change was noted in the frequency of recovery of AHS, and the number of BLPB decreased. Interfering AHS were more often recovered in patients treated with cefprozil. We conclude that the adenoids contain more interfering AHS after therapy with a second-generation oral cephalosporin (cefprozil) than after amoxicillin therapy. This study suggests a potential beneficial effect of using an antimicrobial that selectively spares interfering AHS while inhibiting BLPB.


Smoothened signaling in vertebrates is facilitated by a G protein-coupled receptor kinase.
Smoothened, a heptahelical membrane protein, functions as the transducer of Hedgehog signaling. The kinases that modulate Smoothened have been thoroughly analyzed in flies. However, little is known about how phosphorylation affects Smoothened in vertebrates, mainly, because the residues, where Smoothened is phosphorylated are not conserved from Drosophila to vertebrates. Given its molecular architecture, Smoothened signaling is likely to be regulated in a manner analogous to G protein-coupled receptors (GPCRs). Previously, it has been shown, that arrestins and GPCR kinases, (GRKs) not only desensitize G protein-dependent receptor signaling but also function as triggers for GPCR trafficking and formation of signaling complexes. Here we describe that a GRK contributes to Smoothened-mediated signaling in vertebrates. Knockdown of the zebrafish homolog of mammalian GRK2/3 results in lowered Hedgehog transcriptional responses, impaired muscle development, and neural patterning. Results obtained in zebrafish are corroborated both in cell culture, where zGRK2/3 phosphorylates Smoothened and promotes Smoothened signal transduction and in mice where deletion of GRK2 interferes with neural tube patterning. Together, these data suggest that a GRK functions as a vertebrate kinase for Smoothened, promoting Hedgehog signal transduction during early development.


The use of a new chemical device based on silver and cationic surfactants as a new approach for daily oral hygiene: A preliminary study on a group of periodontal patients.
The aim of this study was to evaluate the abatement power of oral microbial loading of a new gel formulation based on the complex silver-2-mercaptobenzoate, chlorhexidine digluconate and didecyldimethylammonium chloride (named ADC) through polymerase chain reaction (PCR). The study sample consists of a group of 20 patients with chronic periodontal disease. Patients were over 25 years of age and did not undergo surgical or non-surgical periodontal treatment in the previous 6 months. The study sample was allotted into two groups of 10 patients each, homogeneous by age and sex. The test group received a bottle containing ADC gel, while the control group received an identical one containing placebo, similar to ADC in consistence, colour, taste and odour. Sub-gingival samples of four sites, one in each quadrant, of greatest probing depth in each patient were used. Microbiological analyses were performed at baseline and at day 15. Paired t test was performed to detect statistical significant reduction in total bacterial loading and oral pathogens in the study groups. The analysis showed a statistically significant reduction in the total bacterial loading evaluated pre- and post-treatment (P = 0.029) in the study groups. In the control group, the decrease in total bacterial loading was not significant (P = 0.279). Clinically, ADC gel does not have any side effects and discomfort such as pain, burning, tingling sensation or numbness and produces no adverse reactions in time. Our study aimed to evaluate the efficacy of a new chemical formulation with antibacterial properties to use for daily oral hygiene with a preliminary study. Our results showed a statistically significant reduction in total bacterial loading after treatment, but the limitations of our study do not allow us to demonstrate the clinical efficacy of the ADC gel.


Impact of weight loss on markers of systemic inflammation in obese Saudi children with asthma.
Weight loss studies were conducted in children without asthma have demonstrated a reduction in systemic inflammation. However, the impact of weight loss in the obese paediatric population with asthma has not been investigated. To measure the effects of weight loss on markers of systemic inflammation in obese children with bronchial asthma. Eighty obese children with bronchial asthma (42 boys, and 38 girls) with mean age 13.86 ± 3.21 years were divided into two equal groups. The training group received diet regimen, exercise training in addition to the medical treatment for two months, where the control group received the medical treatment only. There was a 17.5%, 15.5%, 22.4%, 14.1% and 15.9% reduction in mean values of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), interleukin-8 (IL-8), Leptin and body mass index (BMI) respectively and 38.7 % increase in mean values of adiponectin in the training group. While, there was a 0.7%, 9.0%, 2.8%, 1.6% and 1.2% increase in mean values of TNF-alpha, IL-6, IL-8, Leptin and BMI respectively and 3.9% decrease in mean values of adiponectin in the control group. The mean values of TNF-alpha, IL-6, IL-8, Leptin and BMI was decreased and adiponectin was increased significantly in the training group, however the results of the control group were not significant. Also, there were significant differences between both groups at the end of the study. Weight loss improves markers of systemic inflammation in obese Saudi children with bronchial asthma.


Inhibition of LRIG3 gene expression via RNA interference modulates the proliferation, cell cycle, cell apoptosis, adhesion and invasion of glioblastoma cell (GL15).
LRIG3 (leucine-rich repeats and immunoglobulin-like domains, LRIG) gene is both under-and over-expressed in human cancers and its role on tumor growth is not fully clarified. Here, we used a human U6 promoter-driven DNA template approach to induce short hairpin RNA (shRNA)-triggered RNA interference (RNAi) to block LRIG3 gene expression in the human glioma cell line GL15. Specific knockdown of LRIG3 by shRNA resulted in significantly increase of the GL15 invasion and adhesion activity in vitro and markedly promoted cell growth. LRIG3 repression also induced increment of the proportion of G0/G1 cells and inhibited apoptosis in GL15 cells. Our results demonstrated that RNAi against LRIG3 could effectively down regulate LRIG3 gene expression. LRIG3 might be involved in the regulation of EGFR signaling, and serve as a tumor suppressor gene in the pathogenesis of glioma.


A systematic review of blunt abdominal aortic injury and analysis of predictors of death.
At present, research on blunt abdominal aortic injury (BAAI) is limited, with the majority being case reports. Consequently, there is a significant knowledge gap concerning this condition. To address this, we conducted a systematic review by extensively searching major databases. We included all literature that provided individual (non-identifiable) data on BAAI patients, irrespective of the study design. Furthermore, we undertook regression analyses to identify predictors of death after BAAI. The search yielded 2,099 results, leading to the inclusion of 102 case reports and one conference abstract. Using the Joanna Briggs Institute (JBI) checklist for assessment, all studies were deemed of medium to high quality. In total, 133 patients were included, with a median age of 34 years, and 73.7% being male. The predominant clinical manifestation was pain, reported in 65.6% of patients. The most frequently observed aortic lesion severity was grade A (intimal tear or intramural hematoma) at 46.9%, and the most common lesion location was zone III (infrarenal aorta) in 88.3% of cases. The overall mortality after BAAI was 15.3%. Multivariate regression analyses revealed the following predictors of death after BAAI: lower limb ischemia (relative risk [RR] = 7.137, 95% confidence interval [CI] 1.154 - 44.161), cardiopulmonary arrest (RR = 10.250, 95% CI 1.452 - 72.344), and injuries to body parts other than the abdomen and lumbar spine (RR = 2.593, 95% CI 1.189 - 5.655). In conclusion, this review provides a detailed quantitative summary of BAAI's clinical manifestations, diagnosis, treatment, and prognosis, emphasizing its high mortality rate and identifying three critical variables as predictors of death.


Pediatric obesity pharmacotherapy: current state of the field, review of the literature and clinical trial considerations.
Despite the increasing number of medications recently approved to treat obesity among adults, few agents have been formally evaluated in children or adolescents for this indication. Moreover, there is a paucity of guidance in the literature addressing best practices with regard to pediatric obesity pharmacotherapy clinical trial design, and only general recommendations have been offered by regulatory agencies on this topic. The purposes of this article are to (1) offer a background of the current state of the field of pediatric obesity medicine, (2) provide a brief review of the literature summarizing pediatric obesity pharmacotherapy clinical trials, and (3) highlight and discuss some of the unique aspects that should be considered when designing and conducting high-quality clinical trials evaluating the safety and efficacy of obesity medications in children and adolescents. Suggestions are offered in the areas of target population and eligibility criteria, clinical trial end-point selection, trial duration, implementation of lifestyle modification therapy and recruitment and retention of participants. Efforts should be made to design and conduct trials appropriately to ensure that high-quality evidence is generated on the safety and efficacy of various medications used to treat pediatric obesity.


Efficacy of influenza vaccine in the elderly in welfare nursing homes: reduction in risks of mortality and morbidity during an influenza A (H3N2) epidemic.
The effect of influenza vaccination on the occurrence and severity of influenza virus infection in a population residing in nursing homes for the elderly was studied during an influenza A (H3N2) epidemic in Japan. Of 22,462 individuals living in 301 welfare nursing homes, 10,739 received either one dose (2027 subjects) or two doses (8712 subjects) of inactivated, subunit trivalent influenza vaccine. During the period Nov. 1998 to March 1999, there were 950 cases of influenza infection diagnosed clinically, with virus isolation or serology. There were statistically significantly fewer cases of influenza, hospital admissions due to severe infection and deaths due to influenza in the vaccinated cohort (256 cases, 32 hospital admissions, 1 death) than in the unvaccinated controls (694 cases, 150 hospital admissions, 5 deaths; reduction rates 59.8%, 76.9% and 79.1% respectively). Vaccination was almost equally effective in those who received one dose of vaccine and those who received two doses. No serious adverse reactions to vaccination were recorded. Thus influenza vaccination is safe and effective in this population, and should be an integral part of the routine care of persons aged > or =65 years residing in nursing homes.


Determination of diffusion coefficients in live cells using fluorescence recovery after photobleaching with wide-field fluorescence microscopy.
Fluorescence recovery after photobleaching (FRAP) enables characterization of quantitative dynamic properties such as diffusion coefficients of fluorescent molecules in living cells by analyzing the recovery of fluorescence intensity after photobleaching in a specific cellular compartment or area. To quantitatively determine high intracellular diffusion coefficients, a suitable optical system as well as an appropriate model for fast diffusion analysis is necessary. Here, we propose a procedure to quantify the diffusion coefficient of rapidly-diffusing fluorescent molecules that makes use of an epi-fluorescence microscope with a photobleaching laser in combination with established models for diffusion analysis. Analysis for the diffusion coefficients of tandemly oligomerized green flurescent proteins (GFPs) in living cells when changing the photobleaching times showed that photobleaching with shorter times than the diffusion speed indicated not the only way to obtain appropriate diffusion coefficients of fast-moving molecules. Our results also showed that the apparent spreading of the effective radius of the photobleached area works as a correction factor for determining appropriate diffusion coefficients of fast-moving molecules like monomeric GFPs. Our procedure provides a useful approach for quantitative measurement of diffusion coefficients in living cells. This procedure is relevant for characterizing dynamic molecular interactions, especially of fast-moving molecules, and is relevant for studies in many biological fields.


Melt analysis of mismatch amplification mutation assays (Melt-MAMA): a functional study of a cost-effective SNP genotyping assay in bacterial models.
Single nucleotide polymorphisms (SNPs) are abundant in genomes of all species and biologically informative markers extensively used across broad scientific disciplines. Newly identified SNP markers are publicly available at an ever-increasing rate due to advancements in sequencing technologies. Efficient, cost-effective SNP genotyping methods to screen sample populations are in great demand in well-equipped laboratories, but also in developing world situations. Dual Probe TaqMan assays are robust but can be cost-prohibitive and require specialized equipment. The Mismatch Amplification Mutation Assay, coupled with melt analysis (Melt-MAMA), is flexible, efficient and cost-effective. However, Melt-MAMA traditionally suffers from high rates of assay design failures and knowledge gaps on assay robustness and sensitivity. In this study, we identified strategies that improved the success of Melt-MAMA. We examined the performance of 185 Melt-MAMAs across eight different pathogens using various optimization parameters. We evaluated the effects of genome size and %GC content on assay development. When used collectively, specific strategies markedly improved the rate of successful assays at the first design attempt from ~50% to ~80%. We observed that Melt-MAMA accurately genotypes across a broad DNA range (~100 ng to ~0.1 pg). Genomic size and %GC content influence the rate of successful assay design in an independent manner. Finally, we demonstrated the versatility of these assays by the creation of a duplex Melt-MAMA real-time PCR (two SNPs) and conversion to a size-based genotyping system, which uses agarose gel electrophoresis. Melt-MAMA is comparable to Dual Probe TaqMan assays in terms of design success rate and accuracy. Although sensitivity is less robust than Dual Probe TaqMan assays, Melt-MAMA is superior in terms of cost-effectiveness, speed of development and versatility. We detail the parameters most important for the successful application of Melt-MAMA, which should prove useful to the wider scientific community.


Longitudinal relationships between financial difficulties and eating attitudes in undergraduate students.
Previous research has shown a relationship between financial difficulties and poor mental health in students, but there has been no research examining such a relationship for eating attitudes. A group of 444 British undergraduate students completed the Index of Financial Stress and the Eating Attitudes Test (26-item version) at up to four time points across a year at university. Higher baseline financial difficulties significantly predicted higher eating attitudes scores at Times 3 and 4 (up to a year), after adjusting for demographic variables and baseline eating attitudes score. Lower family affluence also predicted higher eating attitudes scores at Time 4 (up to a year). A higher eating attitudes score at baseline also significantly predicted greater financial difficulties at Time 2 (3-4 months). When considering these relationships by gender, they were significant for women only. Greater financial difficulties and lower family affluence predict a worsening in eating attitudes over time in female students. The relationship appears to be partially bi-directional, with financial difficulties driving poorer eating attitudes in the shorter term.


Long-term Results of Living Donors in Simultaneous Kidney and Liver Transplantations.
Because of the shortage of organs available for transplantation, living related sequential transplantation with the use of liver and a kidney from the same donor has emerged as a reasonable therapeutic alternative. However, there is insufficient literature about the complications that living donors experience after simultaneous kidney and liver transplantations. From December 2001 to October 2009, 5 living donors provided simultaneous donation of livers and kidneys and 1 living donor donated first her kidney and then her liver. Demographic data of the donors and information concerning the surgery and postoperative observation were collected prospectively. All of the donors were female. The median age was 27.5 (range, 19-36) years. Indications requiring the simultaneous transplantation of livers and kidneys were primary hyperoxaluria type 1 (PH1) in 5 potential recipients and cirrhosis due to chronic hepatitis B infection and idiopathic chronic renal insufficiency in 1 potential recipient. Four recipients underwent right hepatectomy (segments 5-8) and right nephrectomy; 1 recipient underwent left hepatectomy (segments 2-4) and right nephrectomy; and 1 recipient underwent left lobectomy (segments 2-3) and right nephrectomy. There were no complications except in 1 donor (postoperative ileus). No donor developed hypertension or microalbuminuria. With the right indications, appropriate preoperative evaluation, meticulous surgical technique, proper postoperative care, and long-term close monitoring to minimize morbidity and mortality risks, liver and kidney donation from the same donor can be considered for simultaneous kidney and liver transplantation.


A multidisciplinary approach to digital mapping of dinosaurian tracksites in the Lower Cretaceous (Valanginian-Barremian) Broome Sandstone of the Dampier Peninsula, Western Australia.
The abundant dinosaurian tracksites of the Lower Cretaceous (Valanginian-Barremian) Broome Sandstone of the Dampier Peninsula, Western Australia, form an important part of the West Kimberley National Heritage Place. Previous attempts to document these tracksites using traditional mapping techniques (e.g., surface overlays, transects and gridlines combined with conventional photography) have been hindered by the non-trivial challenges associated with working in this area, including, but not limited to: (1) the remoteness of many of the tracksites; (2) the occurrence of the majority of the tracksites in the intertidal zone; (3) the size and complexity of many of the tracksites, with some extending over several square kilometres. Using the historically significant and well-known dinosaurian tracksites at Minyirr (Gantheaume Point), we show how these issues can be overcome through the use of an integrated array of remote sensing tools. A combination of high-resolution aerial photography with both manned and unmanned aircraft, airborne and handheld high-resolution lidar imaging and handheld photography enabled the collection of large amounts of digital data from which 3D models of the tracksites at varying resolutions were constructed. The acquired data encompasses a very broad scale, from the sub-millimetre level that details individual tracks, to the multiple-kilometre level, which encompasses discontinuous tracksite exposures and large swathes of coastline. The former are useful for detailed ichnological work, while the latter are being employed to better understand the stratigraphic and temporal relationship between tracksites in a broader geological and palaeoecological context. These approaches and the data they can generate now provide a means through which digital conservation and temporal monitoring of the Dampier Peninsula's dinosaurian tracksites can occur. As plans for the on-going management of the tracks in this area progress, analysis of the 3D data and 3D visualization will also likely provide an important means through which the broader public can experience these spectacular National Heritage listed landscapes.


Treasure of the Past: IV: THE STANDARD-CELL COMPARATOR, A SPECIALIZED POTENTIOMETER.
The standard cell plays a very important role in the maintenance of the electrical units and in correlating the units of the various national laboratories. Modern standard cells have attained such a high degree of reproducibility and permanence as to warrant the use of apparatus of the utmost precision and reliability in their intercomparison. The paper describes a new potentiometer developed especially for this purpose. Although it actually measures the small difference between the known emf of a reference cell and that of the cell under test, it contains a simple mechanical computing feature which automatically adds this small difference algebraically to the emf of the reference cell and thereby indicates directly the value of the emf under measurement. The design of the instrument is such that no readjustment of its coils will be required when the impending changes in the ohm and the volt are accomplished. The new instrument has been given the distinctive name, "standard-cell comparator".


Correlating agricultural use of organophosphates with outdoor air concentrations: a particular concern for children.
For the organophosphate pesticide chlorpyrifos, median inhalation noncancer, acute children's exposures in agricultural communities are elevated above reference doses; for diazinon, similar exposures are nearly elevated. We used multivariate linear regression analysis to examine the temporal and spatial associations between agricultural use and measured air concentrations of chlorpyrifos, chlorpyrifos oxon, diazinon, and malathion. Agricultural use within a 3-mile radius on the monitoring day and use on the 2-4 prior days were significantly associated with air concentrations (p < 0.01) for all analytes except malathion; chlorpyrifos oxon showed the strongest association (p < 0.0001). In the final models, which included weather parameters, the proportion of variance (r2, adjusted for the number of model variables) for all analytes ranged from 0.28 (p < 0.01) for malathion to 0.65 (p < 0.0001) for diazinon. Recent cellular, animal, and human evidence of toxicity, particularly in newborns, supports the public health concern indicated by initial risk estimates. Agricultural applications of organophosphates and their oxon products may have substantial volatization and off-field movement and are a probable source of exposures of public health concern.


A multiple microwave ablation strategy in patients with initially unresectable colorectal cancer liver metastases - A safety and feasibility study of a new concept.
Resection for colorectal cancer liver metastases is indicated when an R0 resection with preservation of a sufficient future liver remnant (FLR) is achievable. Multimodality conversion of initially unresectable patients to resectable is possible in some patients. We present results of a downstaging strategy using microwave ablation (MWA). In patients where resection was precluded by absence of a tumour-free FLR due to the extent of segmental tumour engagement, but with the potential to clear the whole liver with multiple local ablations, MWA was performed at laparotomy using ultrasound guidance or computer-assisted navigation. Mortality and morbidity was recorded and the overall and disease-free survival of the ablated patients was compared to data of two historic cohorts. Ten of twenty treated patients were alive at median follow-up 25 months. There was no perioperative mortality, with MWA-associated complications being mild to moderate. The MWA group showed a 4-year overall survival of 41%, compared to 70% for a historic cohort of primarily resected patients and 4% for patients with palliative treatment. Results of the multiple ablation strategy in the defined population suggest a survival benefit, compared to palliative chemotherapy alone with acceptable associated morbidity and no perioperative mortality.


The effects of ageing on the biomechanical properties of root dentine and fracture.
Knowledge of the mechanical behaviour of root dentine can facilitate better understanding of spontaneous vertical root fracture (VRF), an age-related disease initiated mainly at the root apex. We tested the hypothesis that the biomechanical properties of root dentine change with ageing. Sixteen human premolars were divided into "old" (17-30 years) and "young" (50-80 years) groups. The elastic modulus, nano-hardness, micro-hardness, elemental contents, tubular density/area of root dentine in cervical, middle and apical root regions were evaluated using atomic force microscopy-based nano-indentation, Knoop indentation, scanning electron microscopy and energy dispersive X-ray spectroscopy, respectively. The apical dentine showed a lower nano-hardness, a lower elastic modulus, a lower calcium content, a lower calcium-to-phosphorus ratio and a smaller tubular density/area than the cervical dentine in both age groups, whereas spatial differences in micro-hardness were observed only in old roots. Compared with young dentine, old dentine showed a greater hardness, a higher elastic modulus, a greater mineral content and a smaller tubular size in the cervical portion, whereas the age-induced changes in tubular density were insignificant. Finite element analysis revealed that due to its higher elastic modulus, old apical dentine has a higher stress level than young dentine. The intrinsic material properties of root dentine have spatial variations, and they are altered by ageing. The higher stress level in old apical dentine may be one reason, if not the most important one, why spontaneous VRFs are more likely to occur in the elderly population.


Phosphorylation of cyclophilin D at serine 191 regulates mitochondrial permeability transition pore opening and cell death after ischemia-reperfusion.
The mitochondrial permeability transition pore (mPTP) plays a critical role in the pathogenesis of cardiovascular diseases, including ischemia/reperfusion injury. Although the pore structure is still unresolved, the mechanism through which cyclophilin D (CypD) regulates mPTP opening is the subject of intensive studies. While post-translational modifications of CypD have been shown to modulate pore opening, specific phosphorylation sites of CypD have not yet been identified. We hypothesized here that phosphorylation of CypD on a serine residue controls mPTP opening and subsequent cell death at reperfusion. We combined in silico analysis with in vitro and genetic manipulations to determine potential CypD phosphorylation sites and their effect on mitochondrial function and cell death. Importantly, we developed an in vivo intramyocardial adenoviral strategy to assess the effect of the CypD phosphorylation event on infarct size. Our results show that although CypD can potentially be phosphorylated at multiple serine residues, only the phosphorylation status at S191 directly impacts the ability of CypD to regulate the mPTP. Protein-protein interaction strategies showed that the interaction between CypD and oligomycin sensitivity-conferring protein (OSCP) was reduced by 45% in the phosphoresistant S191A mutant, whereas it was increased by 48% in the phosphomimetic S191E mutant cells. As a result, the phosphoresistant CypD S191A mutant was protected against 18 h starvation whereas cell death was significantly increased in phosphomimetic S191E group, associated with mitochondrial respiration alteration and ROS production. As in vivo proof of concept, in S191A phosphoresistant rescued CypD-KO mice developed significantly smaller infarct as compared to WT whereas infarct size was drastically increased in S191E phosphomimetic rescued mice. We conclude that CypD phosphorylation at S191 residue leads to its binding to OSCP and thus sensitizes mPTP opening for the subsequent cell death.


Induction of nitric oxide synthase in rat intestine by interleukin-1alpha may explain diarrhea associated with zinc deficiency.
Synthesis of inducible nitric oxide synthase (iNOS) in the intestine may result in local tissue damage. We investigated whether a challenge with interleukin-1alpha could give rise to intestinal iNOS expression and diarrhea in rats of differing zinc status. Weaning male rats were fed a zinc-deficient (ZD) diet (2 mg zinc/kg) for 4 wk to induce zinc deficiency or a zinc-supplemented diet [50.8 mg zinc/kg; controls, including pair-fed (PF ) and ad libitum (AL) consumption groups], and then subcutaneously injected with interleukin-1alpha (2 x 10(7) units/kg body wt). Without the interleukin-1alpha challenge, ZD rats had significantly lower plasma zinc concentration than the other groups. Intestinal metallothionein-1 mRNA abundance was lower in ZD rats than in AL rats. iNOS was expressed in the intestine of ZD rats but not in the others. None of the rats experienced diarrhea during the feeding period. Interleukin-1alpha led to a reduction in plasma zinc concentration, enhancement in intestinal metallothionein-1 mRNA levels, and expression of the intestinal iNOS gene in all groups. However, the abundance of iNOS mRNA was significantly higher in ZD rats than in the other groups. The presence of iNOS protein was demonstrated by immunohistochemical staining in the intestine of ZD rats that had been treated with interleukin-1alpha 12 h earlier. In addition, diarrhea occurred in most of the ZD rats and some of the PF rats but not in AL rats after interleukin-1alpha treatment. We conclude that ZD rats respond to interleukin-1alpha challenge more severely than controls, reflected by a more marked and prolonged iNOS expression and a greater incidence of diarrhea.


Reconstitution of the energy-linked transhydrogenase activity in membranes from a mutant strain of Escherichia coli K12 lacking magnesium ion- or calcium ion-stimulated adenosine triphosphatase.
1. We have isolated a mutant of Escherichia coli K12 (strain AN295) that forms de-repressed amounts of Mg(2+),Ca(2+)-stimulated adenosine triphosphatase. 2. The Mg(2+),Ca(2+)-stimulated triphosphatase activity was separated from membrane preparations from strain AN295 by extraction with 5mm-Tris-HCl buffer containing EDTA and dithiothreitol, resulting in a loss of the ATP-dependent transhydrogenase activity. The non-energy-linked transhydrogenase activity remained in the membrane residue. 3. The solubilized Mg(2+),Ca(2+)-stimulated adenosine triphosphatase activity from strain AN295 was partially purified by repeated gel filtration. The addition of the purified Mg(2+),Ca(2+)-stimulated adenosine triphosphatase to the membrane residue from strain AN295 reactivated the ATP-dependent transhydrogenase activity. 4. Strain AN296, lacking Mg(2+),Ca(2+)-stimulated adenosine triphosphatase activity, was derived by transducing the mutant allele, uncA401, into strain AN295. The ATP-dependent transhydrogenase activity was lost but the non-energy linked transhydrogenase was retained. 5. The ATP-dependent transhydrogenase activity in membrane preparations from strain AN296 (uncA(-)) could not be re-activated by the purified Mg(2+),Ca(2+)-stimulated adenosine triphosphatase from strain AN295. However, after extraction by 5mm-Tris-HCl buffer containing EDTA and dithiothreitol, the ATP-dependent transhydrogenase activity could be re-activated by the addition of the purified Mg(2+),Ca(2+)-stimulated adenosine triphosphatase from strain AN295 to the membrane residue from strain AN296 (uncA(-)).


miR-21 and its target gene CCL20 are both highly overexpressed in the microenvironment of colorectal tumors: significance of their regulation.
Recently, we reported a functional interaction between miR-21 and its identified chemokine target CCL20 in colorectal cancer (CRC) cell lines. Here, we investigated whether such functional interactions are permitted at the cellular level which would require an inverse correlation of expression and also co-expression of miR-21 and CCL20 in the same cell. Expression profiling was performed using qPCR, and ELISA, in situ hybridization and immunohistochemistry were applied for the presentation of their cellular localization. We demonstrated that miR-21 as well as CCL20 were both significantly upregulated in CRC tissues; thus, showing no antidromic expression pattern. This provided an initial clue that miR-21 and CCL20 may not be expressed in the same cell. In addition, we located miR-21 expression at the cellular level predominantly in stromal cells such as tumor-associated fibroblasts and to a minor degree in immune cells such as macrophages and lymphocytes. Likewise, CCL20 expression was primarily detected in tumor-infiltrating immune cells. Thus, investigating the cellular localization of miR-21 and its target CCL20 revealed that both molecules are expressed predominantly in the microenvironment of CRC tumors.