[Our Experience with the Total Replacement of the First Metatarsophalangeal Joint by Medin PH-flex].
The first metatarsophalangeal (MTP) joint replacement ranks among the treatment methods of patients with hallux rigidus. The paper aims to evaluate the short-term to mid-term outcomes and to present clinical experience with our Medin PH-flex implant. MATERIAL AND METHODS In the period from January 2011 to 2016 we performed total replacement of the first MTP joint in 31 patients, in 4 cases bilaterally. In total, 35 implants were evaluated. The mean age of the patient at the time of surgery was 57.7 years (39-72 years). The surgery was conducted in 29 women and 2 men. The patients were evaluated using the AOFAS score (American Orthopaedic Foot and Ankle Society score), the radiographs were assessed as to the potential occurrence of radiolucent lines, with major stress put on the assessment of the mobility in MTP joint and its position. The pain was assessed based on the VAS score. RESULTS Prior to the joint replacement surgery, the mean AOFAS score in patients was 55.6 (35-65). Postoperatively, the mean AOFAS score was 80.8 (65-95). The pain suffered by patients was evaluated with the use of the Pain Visual Analogue Scale (VAS score). The preoperative mean VAS score was 5 (2-8), whereas the postoperative score improved to mean VAS 2 (0-4). The range of motion was clinically assessed with a goniometer. The mean range of motion of plantar flexion and dorsiflexion was 16.00° (5-35°) and 28.60° (10-55°), respectively. The mean range of motion was 36° (15-60°). No intraoperative complications were observed. In all the patients, the surgical wound healed per primam. In 2 female - (5.7 %) of the whole group of patients who underwent surgery a deep infection occurred, namely 10 and 21 months following the implantation. In both the female patients their condition was managed by joint revision operation and by a simple removal of the implant. DISCUSSION Joint replacement related matters were repeatedly discussed in professional literature. There are many papers published in the literature on this topic. A whole range of the first MTP joint implants of different shapes have been developed, with extremely different clinical results. CONCLUSIONS An appropriately chosen type of the implant, a fitting indication and a correctly applied implantation technique can lead to the desired good outcome. The first MTP joint replacement should be indicated after careful consideration since the management of a potential joint replacement failure can often be very technically challenging and quite mutilating for the patient. The mid-term outcomes of the Medin a.s. first MTP implant seem to be promising. It will, however, be necessary to wait for long-term outcomes in order to evaluate the final benefits of this type of implant in patients with hallux rigidus. Key words: hallux rigidus, arthroplasty of the MTP joint, hemiarthroplasty, silicone implant.


Diagnostic electron microscopy on reembedded ("popped off") areas of large Spurr epoxy sections.
A technique is described in which areas of large Spurr epoxy thick sections are directly popped off of glass slides by inverted Beem capsules filled with polymerized plastic. This technique allows the survey of large regions of tissue with the maintenance of tissue relationships and the selection of specific areas for electron microscopic studies. Within the context of diagnostic electron microscopy well-preserved and representative portions of tissues can be selected as well as individual diagnostic cells or other difficult to find structures. Many small pieces of minced, optimally fixed and processed tissue can also be surveyed by this technique. Thus, one can screen the equivalent of 20 or more standard thick sections in one step. This "pop-off" technique does not appear to alter the ultrastructure of the tissue in any way.


Sexual and vegetative compatibility genes in the aspergilli.
Gene flow within populations can occur by sexual and/or parasexual means. Analyses of experimental and in silico work are presented relevant to possible gene flow within the aspergilli. First, the discovery of mating-type (MAT) genes within certain species of Aspergillus is described. The implications for self-fertility, sexuality in supposedly asexual species and possible uses as phylogenetic markers are discussed. Second, the results of data mining for heterokaryon incompatibility (het) and programmed cell death (PCD) related genes in the genomes of two heterokaryon incompatible isolates of the asexual species Aspergillus niger are reported. Het-genes regulate the formation of anastomoses and heterokaryons, may protect resources and prevent the spread of infectious genetic elements. Depending on the het locus involved, hetero-allelism is not tolerated and fusion of genetically different individuals leads to growth inhibition or cell death. The high natural level of heterokaryon incompatibility in A. niger blocks parasexual analysis of the het-genes involved, but in silico experiments in the sequenced genomes allow us to identify putative het-genes. Homologous sequences to known het- and PCD-genes were compared between different sexual and asexual species including different Aspergillus species, Sordariales and the yeast Saccharomyces cerevisiae. Both het- and PCD-genes were well conserved in A. niger. However some point mutations and other small differences between the het-genes in the two A. niger isolates examined may hint to functions in heterokaryon incompatibility reactions.


Dramatic increase of the RNA editing for glutamate receptor subunits during terminal differentiation of clonal human neurons.
RNA editing plays an important role in determining physiological characteristics of certain glutamate-gated receptor (GluR) channels such as Ca2+ permeability and desensitization kinetics. In one case, the editing changes a gene-encoded glutamine (Q) to an arginine (R) codon located in the channel-forming domain of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit GluR-B and also the kainate receptor subunits GluR5 and GluR6. Another case of RNA editing alters an arginine (R) to a glycine (G) codon at a position termed the "R/G" site of AMPA subunits GluR-B, C, and D. Double-stranded RNA-specific adenosine deaminases (DRADA) have been implicated as agents involved in the editing. By using a human teratocarcinoma cell line, NT2, we investigated the change of the RNA editing of GluR subunits in conjunction with the expression of two DRADA members, DRADA1 and DRADA2 genes, during neuronal differentiation. Whereas Q/R and R/G site RNA editing both become progressively activated in differentiating NT2 cells, the expression of the two DRADA genes can already be detected even in the undifferentiated NT2 cells. Development of the editing machinery appears to require, in addition to DRADA enzymes, a currently unidentified mechanism(s) that may become activated during neuronal differentiation.


Exercise increases NPY/AgRP and TH neuron activity in the hypothalamus of female mice.
Recent evidence identifies a potent role for aerobic exercise to modulate the activity of hypothalamic neurons related to appetite; however, these studies have been primarily performed in male rodents. Since females have markedly different neuronal mechanisms regulating food intake, the current study aimed to determine the effects of acute treadmill exercise on hypothalamic neuron populations involved in regulating appetite in female mice. Mature, untrained female mice were exposed to acute sedentary, low- (10 m/min), moderate- (14 m/min), and high (18 m/min)-intensity treadmill exercise in a randomized crossover design. Mice were fasted 10 h before exercise, and food intake was monitored for 48 h after bouts. Immunohistochemical detection of cFOS was performed 3 h post-exercise to determine the changes in hypothalamic neuropeptide Y (NPY)/agouti-related peptide (AgRP), pro-opiomelanocortin (POMC), tyrosine hydroxylase (TH), and SIM1-expressing neuron activity concurrent with the changes in food intake. Additionally, stains for pSTAT3tyr705 and pERKthr202/tyr204 were performed to detect exercise-mediated changes in intracellular signaling. Briefly, moderate- and high-intensity exercises increased 24-h food intake by 5.9 and 19%, respectively, while low-intensity exercise had no effects. Furthermore, increases in NPY/AgRPARC, SIM1PVN, and TH neuron activity were observed 3 h after high-intensity exercise, with no effects on POMCARC neurons. While no effects of exercise on pERKthr202/tyr204 were observed, pSTAT3tyr705 was elevated specifically in NPY/AgRP neurons 3 h post-exercise. Overall, aerobic exercise increased the activity of several appetite-stimulating neuron populations in the hypothalamus of female mice, which may provide insight into previously reported sexual dimorphisms in post-exercise feeding.


The concept of the natriuretic hormone and its relation to hypertension.
Acute volume expansion increases the intrathoracic blood volume thus endowing the plasma with an increased capacity to cause a natriuresis, to inhibit Na-K-ATPase and stimulate vascular reactivity. It is not known whether these changes, which stem from a common stimulus are due to a change in the concentration of one substance or several. It is proposed that in essential hypertension a genetic abnormality of the kidney causes a difficulty in excreting sodium. This leads to an initial blood volume expansion which causes the observed rise in the plasma's capacity to inhibit sodium transport, and to the increased vascular tone. Eventually the increase in tone of the arterial smooth muscle causes the blood pressure to rise, while the increase in tone of the smooth muscle of the veins diminishes venous compliance thus causing a shift of blood from the periphery to the chest thus providing the stimulus for the persistent rise in the plasma's capacity to increase vascular reactivity, even though total blood volume may have returned to normal.


What Is the Link between Stringent Response, Endoribonuclease Encoding Type II Toxin-Antitoxin Systems and Persistence?
Persistence is a transient and non-inheritable tolerance to antibiotics by a small fraction of a bacterial population. One of the proposed determinants of bacterial persistence is toxin-antitoxin systems (TASs) which are also implicated in a wide range of stress-related phenomena. Maisonneuve E, Castro-Camargo M, Gerdes K. 2013. Cell 154:1140-1150 reported an interesting link between ppGpp mediated stringent response, TAS, and persistence. It is proposed that accumulation of ppGpp enhances the accumulation of inorganic polyphosphate which modulates Lon protease to degrade antitoxins. The decrease in the concentration of antitoxins supposedly activated the toxin to increase in the number of persisters during antibiotic treatment. In this study, we show that inorganic polyphosphate is not required for transcriptional activation of yefM/yoeB TAS, which is an indirect indication of Lon-dependent degradation of YefM antitoxin. The Δ10 strain, an Escherichia coli MG1655 derivative in which the 10 TAS are deleted, is more sensitive to ciprofloxacin compared to wild type MG1655. Furthermore, we show that the Δ10 strain has relatively lower fitness compared to the wild type and hence, we argue that the persistence related implications based on Δ10 strain are void. We conclude that the transcriptional regulation and endoribonuclease activity of YefM/YoeB TAS is independent of ppGpp and inorganic polyphosphate. Therefore, we urge for thorough inspection and debate on the link between chromosomal endoribonuclease TAS and persistence.


Growth Inhibition of Streptomyces Species by l-Serine and Its Effect on Tetracycline Biosynthesis.
The addition of serine to minimal medium inhibited the growth of Streptomyces aureofaciens and Streptomyces rimosus. Both the outgrowth of spores and the growth of vegetative cells were inhibited by l-serine. This effect was independent of the carbon source used. In rich nutrient medium, however, the serine effect was not observed. The presence of glycine and methionine in minimal medium reversed the growth inhibition imposed by serine, suggesting that a metabolic block related to the synthesis of these two amino acids was involved. A serine-tolerant mutant of S. aureofaciens isolated after ultraviolet irradiation showed a level of serine deaminase comparable to that of the wild-type strain, which indicated that tolerance to serine was not due to the presence of a more active deaminating enzyme in the mutant. Serine markedly reduced tetracycline and oxytetracycline biosynthesis with the parental strains of Streptomyces spp. The serine-tolerant mutant, however, produced almost the same amount of tetracycline in the presence or absence of serine. The final cell population in fermentation broth was not significantly reduced by l-serine, and the addition of glycine and methionine did not increase the tetracycline yields, which suggested that l-serine inhibition of antibiotic biosynthesis was by a mechanism different from that related to growth inhibition.


Morphogenesis of Bullet-Shaped Rabies Virus Particles Regulated by TSG101.
Viral protein assembly and virion budding are tightly regulated to enable the proper formation of progeny virions. At this late stage in the virus life cycle, some enveloped viruses take advantage of the host endosomal sorting complex required for transport (ESCRT) machinery, which contributes to the physiological functions of membrane modulation and abscission. Bullet-shaped viral particles are unique morphological characteristics of rhabdoviruses; however, the involvement of host factors in rhabdovirus infection and, specifically, the molecular mechanisms underlying virion formation are not fully understood. In the present study, we used a small interfering RNA (siRNA) screening approach and found that the ESCRT-I component TSG101 contributes to the propagation of rabies virus (RABV). We demonstrated that the matrix protein (M) of RABV interacts with TSG101 via the late domain containing the PY and YL motifs, which are conserved in various viral proteins. Loss of the YL motif in the RABV M or the downregulation of host TSG101 expression resulted in the intracellular aggregation of viral proteins and abnormal virus particle formation, indicating a defect in the RABV assembly and budding processes. These results indicate that the interaction of the RABV M and TSG101 is pivotal for not only the efficient budding of progeny RABV from infected cells but also for the bullet-shaped virion morphology. IMPORTANCE Enveloped viruses bud from cells with the host lipid bilayer. Generally, the membrane modulation and abscission are mediated by host ESCRT complexes. Some enveloped viruses utilize their late (L-) domain to interact with ESCRTs, which promotes viral budding. Rhabdoviruses form characteristic bullet-shaped enveloped virions, but the underlying molecular mechanisms involved remain elusive. Here, we showed that TSG101, one of the ESCRT components, supports rabies virus (RABV) budding and proliferation. TSG101 interacted with RABV matrix protein via the L-domain, and the absence of this interaction resulted in intracellular virion accumulation and distortion of the morphology of progeny virions. Our study reveals that virion formation of RABV is highly regulated by TSG101 and the virus matrix protein.


Dark Rims: Novel Sequence Enhances Diagnostic Specificity in Multiple Sclerosis.
The 2010 McDonald criteria are designed to sensitively detect MS; however, the low specificity of these criteria can occasionally lead to the misdiagnosis of MS. The purpose of this study was to determine whether a novel double inversion recovery MR imaging technique has the potential to increase the specificity of diagnostic criteria distinguishing MS from non-MS white matter lesions. This was a cross-sectional observational study. MR imaging data were acquired between 2011 and 2016. A novel double inversion recovery sequence that suppresses CSF and GM signal was used (GM-double inversion recovery). We compared WM lesions in a group of patients with multiple sclerosis and in a second group of positive controls with white matter lesions who did not have a diagnosis of MS. The presence of a rim on the GM-double inversion recovery MR imaging sequence was combined with the 2001 and 2010 McDonald disseminated-in-space criteria. Multiple MR imaging markers, including lesion location, size, and the presence of a rim, were compared between groups as well as a quantitative measure of lesion T1 hypointensity. MR images from 107 patients with relapsing-remitting MS (median age, 32 years) and 36 positive control (median age, 39 years) subjects were analyzed. No significant differences were found in age and sex. In patients with MS, 1120/3211 lesions (35%) had a rim on GM-double inversion recovery; the positive control group had only 9/893 rim lesions (1%). Rims were associated with a decrease in the lesion T1 ratio. Using the 2010 MR imaging criteria plus the presence of rims on GM-double inversion recovery, we achieved 78% and 97% specificity in subjects with ≥1 and ≥2 rim lesions, respectively. The addition of a novel GM-double inversion recovery technique enhanced specificity for diagnosing MS compared with established MR imaging criteria.


Gut glutathione metabolism and changes with 7,12-DMBA and glutamine.
The mechanism by which oral glutamine (GLN) prevents 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast cancer is unknown. While GLN triples the negative extraction of gut glutathione (GSH) in rats, DMBA significantly disrupts it. Actual gut GSH flux has not been reported. We hypothesized that the gut is a producer of GSH; DMBA blocks gut GSH production and supplemental oral GLN antagonizes this effect. Eighty Sprague-Dawley rats were randomized to four groups (n = 20/group): DMBA + GLN, DMBA + FA, OIL + GLN, OIL + FA. Rats (age 50 days) were gavaged with a one-time dose of 100 mg/kg DMBA or oil. Rats were gavaged with AES-14 as GLN (1 gm/kg/day) or an isonitrogenous amount of Freamine (FA) from 1 week before till sacrifice at 1 week after DMBA (greatest effect on gut GSH extraction). Arterial and portal blood was taken for GLN and GSH levels, and blood flow was measured using (14)C-PAH. Gut GLN and GSH fluxes (uptake or production) were calculated. DMBA abrogated the normal GSH production (negative flux) in OIL + FA while not affecting GLN metabolism. GLN maintained GSH production in DMBA + GLN. Oral GLN restores to normal GSH production in DMBA-treated animals suggesting one of the mechanism(s) by which GLN prevents breast cancer in this model. Unchanged uptake of GLN in the DMBA-treated animals may indicate a block in GSH transport rather than actual intracellular production.


Management of atrial fibrillation in the Emergency Department: current approach and future expectations.
Atrial fibrillation (AF) is the most common cardiac dysrhythmia and occurs in 3.3%-10% of emergency admissions. It is frequently quoted for people over the age of 75, but the cases of AF in young subjects without structural heart disease are also increasing, therefore, leading to the evaluation of "lonely atrial fibrillation" as a new challenge for the clinician. The first diagnosis and treatment often occur in the emergency room and the emergency physician has therefore to evaluate the initial step towards the therapeutic decisions. Although international standard guidelines are available, AF treatment in the Emergency Department (ED) is still heterogeneous in terms of the management strategy chosen. There are two main strategies for the management of AF: rate and rhythm control. Moreover, antithrombotic treatment is pivotal in AF to prevent cardioembolic stroke and it is considered a primary objective after an accurate assessment of antithrombotic treatment risks and benefits. The introduction of innovative echocardiographic approach, directly in ED, seems to improve the management and risk stratification of patients with AF. This review aims to provide an overview about the current approach and the future expectations in the management of AF in ED. This manuscript represents a synopsis of the lectures on AF management in the ED of the Third Italian GREAT Network Congress, that was hold in Rome, 15-19 October 2012. We decided to use only the most relevant references for each contribution as suggested by each participant at this review.


You Think Failure Is Hard? So Is Learning From It.
Society celebrates failure as a teachable moment. But do people actually learn from failure? Although lay wisdom suggests people should, a review of the research suggests that this is hard. We present a unifying framework that points to emotional and cognitive barriers that make learning from failure difficult. Emotions undermine learning because people find failure ego-threatening. People tend to look away from failure and not pay attention to it to protect their egos. Cognitively, people also struggle because the information in failure is less direct than the information in success and thus harder to extract. Beyond identifying barriers, this framework suggests inroads by which barriers might be addressed. Finally, we explore implications. We outline what, exactly, people miss out on when they overlook the information in failure. We find that the information in failure is often high-quality information that can be used to predict success.


Two Cases of Vasculitis with Renal Vein Thrombosis.
We present a series of two cases of ANCA associated Vasculitis (AAV) presenting as Pulmonary Renal syndrome with associated renal vein thrombosis. Although there are enough evidences suggesting association of venous thrombosis with AAVs, the incidence of renal vein thrombosis is rare. Renal vein thrombosis should be ruled out in cases where there is delay in recovery of renal function in patients with AAV. Positive laboratory values for anti-Proteinase-3 (PR3) and anti-Myeloperoxidase (MPO) ANCA in the cases that presented as Rapidly Progressive Glomerulonephritis, helped in early initiation of treatment with complete recovery of Renal function.


Migration of cultured vascular cells in response to plasma and platelet-derived factors.
Phagokinetic migration of cultured vascular cells was tested in response to human platelet-rich serum ('serum') and human platelet-poor plasma serum ('plasma'). The cell types tested included bovine aortic endothelial cells, human umbilical vein endothelial cells, human haemangiomal capillary endothelial cells, bovine adrenal microvascular pericytes, and bovine aortic smooth muscle cells. Human serum stimulated a significant increase in the rate of migration for all five cell types. Human plasma stimulated the endothelial cells to migrate but had no effect on the migration of pericytes or smooth muscle cells. Highly purified platelet-derived growth factor (PDGF) stimulated dose-dependent migration of smooth muscle cells causing a 50% increase in phagokinetic track area relative to controls. Neither pericyte nor endothelial cell migration was stimulated by PDGF. Rabbit antiserum to human PDGF completely blocked the smooth muscle cell migration induced by either 10% serum or 1 ng/ml pure PDGF. Purified platelet factor IV (PF4) stimulated migration of pericytes but not of smooth muscle cells nor endothelial cells. Sheep antiserum to human PF4 completely blocked the pericyte migration induced by either 10% serum or 1 microgram/ml pure PF4. These results indicate that PDGF is the primary factor in serum responsible for the migration of cultured aortic smooth muscle cells and that PF4 is a critical factor required to induce the migration of pericytes. Other factors present in both plasma and serum control the migration of vascular endothelial cells.


Insulin resistance in divergent strains of Holstein-Friesian dairy cows offered fresh pasture and increasing amounts of concentrate in early lactation.
The objective of this study was to determine whether the physiological response to an intravenous glucose challenge would be affected by genetic strain or concentrate supplementation in grazing Holstein-Friesian cows in early lactation. North American (NA; n = 30) or New Zealand (NZ; n = 30) cows were randomly allocated to 1 of 3 feeding treatments. All cows were offered a generous pasture allowance, and 4 of the 6 groups received either 3 or 6 kg of dry matter (DM)/cow per day of concentrates. During wk 5 of lactation, all cows underwent an intravenous glucose challenge. Cows of NA origin produced more milk than NZ cows, but there was no significant strain effect on milk fat or protein yield. Milk yield and the yield of individual components increased with increasing level of concentrate eaten, but there were no significant strain x diet interactions. During wk 1 to 6, mean body weight and body condition score decreased in all treatments. Average body weight was greater in NA cows, but body condition score was greater for NZ cows. There was no strain or diet effect on the length of the postpartum anovulatory interval, with cows ovulating before 40 d postpartum on average. Glucose fractional turnover rate was greater in NZ cows compared with those of NA origin and in all cows receiving 6 kg of DM concentrates, indicating a less severe insulin resistance in those treatments. Consistent with this, the time taken to dispose of half the peak glucose concentration was less when 6 kg of DM concentrate was fed, and tended to be less in NZ than in NA cows. There was no effect of genetic strain on glucose area under the curve (AUC) at 60 or 120 min, but AUC at both time points was less in cows receiving 6 kg of DM concentrates per day. Neither genetic strain nor nutrition affected basal or peak insulin concentrations, insulin increment, or insulin AUC, and there were no strain x diet interactions for any of the glucose challenge response variables measured. In conclusion, differences in milk production between NA and NZ cows in early lactation can, at least in part, be explained by the greater degree of insulin resistance in the NA cows, and this insulin resistance can be overcome by supplementing grazing cows with 6 kg of DM concentrates.


Central serous chorioretinopathy: A review.
Central serous chorioretinopathy (CSC) is the fourth most common non-surgical retinopathy associated with fluid leakage. The pathogenesis is not yet completely understood, but changes in the choroid, sclera and RPE have been described associated with venous congestion of choroidal outflow. CSC can be categorised into acute, chronic, and recurrent subtypes with recent classifications of simple and complex based on the area of RPE change seen on fundus autofluorescence. A multimodal imaging approach is helpful in the diagnosis and management of CSC and secondary complications such as type 1 neovascularisation. Although spontaneous resolution with relatively good visual outcomes is common, treatment should be considered in patients with persistent or recurrent SRF. Treatment options include laser, systemic medications, intravitreal therapy, and surgery. Of these, argon laser for focal extramacular fluid leaks and photodynamic therapy of leakage identified by indocyanine-green angiography currently have the greatest supportive evidence.


Validation of black blood late gadolinium enhancement (LGE) for evaluation of myocardial infarction in patients with or without pathological Q-wave on electrocardiogram (ECG).
The pathological Q-wave (QW) is an important indicator of infarcted myocardial volume indicating a worse prognosis compared to non-Q-wave (NQW) infarctions. Traditional classification divides infarcts into transmural and non-transmural based on QW and NQW. This view has been challenged by the advent of late gadolinium enhancement (LGE) MR imaging. Conventional LGE (Conv-LGE) detection of subendocardial MI is limited by bright blood pool. Dark Blood LGE imaging (DB-LGE) nulls the blood pool improving the conspicuity and accuracy of detection of subendocardial infarcts. We hypothesize that improved detection of subendocardial enhancement with DB-LGE will result in improved correlation of electrocardiogram (ECG) and extent of infarction. Sixty-four clinically confirmed infarction patients were enrolled in this prospective study. All the participants underwent cardiac MR imaging including conv-LGE and DB-LGE. Twelve-lead ECG were performed on the same day. The patients were divided into QW and NQW groups by one experienced cardiologist. MI quantitation was by MI% (the ratio of MI volume to whole myocardial volume) and transmural grading, compared using paired t-test and Wilcoxon-test, respectively. The image quality obtained by Conv-LGE and DB-LGE were evaluated according to the signal intensity ratio (SIR) and contrast-to-noise ratio (CNR). Fifty-six subjects were enrolled in the final analysis [23 (41%) QW and 33 (59%) NQW infarcts]. For the QW cohort, both sequences classified infarcts as transmural in 21/23 (91%) subjects and subendocardial in 2/23 (9%). For the NQW cohort, both sequences classified infarcts as transmural in 16/33 (48%) subjects and subendocardial in 17/33 (52%). Using BB-LGE there were significant differences in detecting subendocardial infarcts in QW and NQW cohorts (Z=-5.85, P<0.001). The MI% of QW group was greater than in NQW group (24.2±10.3 vs.15.9±9.8, P=0.003). Compared to Conv-LGE, BB-LGE provided higher CNR and SIR between infarcted myocardium and blood pool (6.3±2.6 vs. 2.1±1.3, P<0.001; 5.4±1.9 vs. 1.3±0.2, P<0.001). BB-LGE detected more subendocardial infarcted segments in the QW group and NQW group (Z=-4.24, P<0.001; Z=-5.57, P<0.001). The larger MI% was displayed in BB-LGE than in Conv-LGE in both QW group and NQW group (24.2±10.3 vs. 22.6±10.3, P<0.001; 15.9±9.8 vs.14.6±9.6, P=0.001). Compared to conventional LGE, DB-LGE can provide more accurate detection and characterization of infarction in terms of transmurality and subendocardial extent. This is important for evaluating QW and NQW MIs. Due to nulling the high signal of blood pool, DB-LGE can effectively improve the identification of subendocardial MI which may be missed on conventional LGE. Therefore, in both QW and NQW MIs, DB-LGE detects more subendocardial MIs and larger MI% is found. This may facilitate more accurate quantitative MR assessment of both QW and NQW MIs and further empower LGE volume as a predictive biomarker.


Perioperative management of the hypothalamic-pituitary-adrenal axis in patients with pituitary adenomas: an Australasian survey.
There is limited consensus regarding optimal glucocorticoid administration for pituitary surgery to prevent a potential adrenal crisis. To assess the investigation and management of the hypothalamic-pituitary-adrenal (HPA) axis in patients undergoing trans-sphenoidal hypophysectomy in Australasia. A questionnaire was sent to one endocrinologist at each of 18 centres performing pituitary surgery in Australasia. Using hypothetical case vignettes, respondents were asked to describe their investigation and management of the HPA axis for a patient with a: non-functioning macroadenoma and intact HPA axis, non-functioning macroadenoma and HPA deficiency and growth hormone secreting microadenoma undergoing trans-sphenoidal hypophysectomy. Responses were received from all 18 centres. Seventeen centres assess the HPA axis preoperatively by measuring early morning cortisol or a short synacthen test. Preoperative evaluation of the HPA status influenced glucocorticoid prescription by 10 centres, including 2/18 who would not prescribe perioperative glucocorticoids for a patient with a macroadenoma and an intact HPA axis. Tumour size influenced glucocorticoid prescribing patterns at 7/18 centres who prescribe a lower dose or no glucocorticoids for a patient with a microadenoma. Choice of investigations for definitive postoperative assessment of the HPA axis varied with eight centres requesting an insulin tolerance test, four centres a 250 µg short synacthen test and six centres requesting other tests. There is wide variability in the investigation and management of perioperative glucocorticoid requirements for patients undergoing pituitary surgery in Australasia. This may reflect limited evidence to define optimal management and that further well-designed studies are needed.


The protective effect exerted by ascorbic acid on DNA fragmentation of human leukocytes induced by Lachesis muta muta venom.
The objective of this study was to evaluate the genotoxic and mutagenic effects of the toxins present in Lachesis muta muta's venom on human peripheral blood leukocytes and the protective potential of ascorbic acid on DNA fragmentation. The venom of L. muta muta was incubated in different concentrations (1, 2.5, 5, 7.5, 10, 15, 20, 30, 40, 50, 60, and 120 µg/mL) with human blood to evaluate DNA fragmentation using the comet, agarose gel electrophoresis, and micronucleus assays. In these concentrations evaluated, the venom of L. muta muta induced genotoxicity (comet assay and agarose gel electrophoresis) and mutagenicity (micronucleus test), but they were not cytotoxic, as they did not change the rate of cell proliferation after cytokinesis blockade with cytochalasin B. The ascorbic acid significantly inhibited the genotoxicity induced by L. muta muta venom in the proportions evaluated (1:0.1 and 1:0.5, venom/ascorbic acid - w/w). Thus, future studies are needed to elucidate the protective mechanisms of ascorbic acid on the genotoxic effects induced by toxins present in snake venoms.


EUROPEAN SCIENCE POLICY: France Rebels Against Gene-Patenting Law.
France is on a collision course with the European Union over an E.U. directive that many researchers believe would allow raw DNA sequences of human genes to be patented. On 7 June, French justice minister Elisabeth Guigou told the National Assembly that the directive--which must be enacted by each of the 15 E.U. member nations by 30 July--contradicts French bioethics laws, which forbid the patenting of any part of the human body. If France maintains its defiance, it could be fined up to $600,000 daily for each day it refuses to adopt the directive.


Multi-potent Natural Scaffolds Targeting Amyloid Cascade: In Search of Alzheimer's Disease Therapeutics.
Alzheimer's Disease (AD) once considered a rare disorder emerges as a major health concern in recent times. The disease pathogenesis is very complex and yet to be understood completely. However, "Amyloid Cascade" is the central event in disease pathogenesis. Several proteins of the amyloid cascade are currently being considered as potential targets for AD therapeutics discovery. Many potential compounds are in clinical trials, but till now there is no known cure for the disease. Recent years have witnessed remarkable research interest in the search of novel concepts in drug designing for AD. Multi-targeted ligand design is a paradigm shift in conventional drug discovery. In this process rather than designing ligands targeting a single receptor, novel ligands have been designed/ synthesized that can simultaneously target many pathways involved in disease pathogenesis. Here, recent developments in computational drug designing protocols to identify multi-targeted ligand for AD have been discussed. Therapeutic potential of different multi-potent compounds also has been discussed briefly. Prime emphasis has been given to multi-potent ligand from natural resources. Polyphenols are an interesting group of compounds which show efficacy against a wide range of disease and have the property to exhibit multi-potency. Several groups attempted to identify novel multi-potent phytochemicals for AD therapy. Multi-potency of several polyphenols or compounds synthesized using the poly-phenolic scaffolds have been briefly discussed here. However, the multi-targeted drug designing for AD is still in early stages, more advancement in drug designing method/algorithm developments is urgently required to discover more efficient compounds for AD therapeutics.


Seasonal and temperature-induced changes in myosin heavy chain composition of crucian carp hearts.
Myosin heavy chain isoforms of the ventricular myocardium from crucian carp (Carassius carassius L.) hearts were analyzed in different times of the year by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis [K. A. Esser, M. O. Boluyt, and T. P. White, Am. J. Physiol. 255 (Heart Circ. Physiol. 24): H659-H663, 1988]. In winter only one myosin heavy chain type was present, but in summer about one-half of the winter myosin was replaced by more slowly moving summer myosin. The occurrence of summer myosin correlated with seasonal changes in water temperature of the pond, where the fish were caught. Furthermore, the heavy chain composition of the heart was altered by temperature acclimation in the laboratory: cold-acclimated (2 degrees C) fish had only winter myosin, but warm-acclimated (22 degrees C) fish had both summer and winter myosin in about equal amounts. Myosin adenosinetriphosphatase activity of the hearts containing both summer and winter myosin was higher than that of hearts containing only winter myosin. Functionally, changes in myosin heavy chain composition were associated with inverse thermal acclimation in the heart rate. Warm-acclimated fish had higher in vitro heart rate and shorter contraction duration than cold-acclimated animals. Present findings suggest that changes in myosin heavy chain composition together with concomitant changes in Ca2+ activation of contraction make possible large seasonal alterations in the activity of crucian carp hearts. These adjustments are needed to adapt the cardiovascular system to winter hibernation and summer activity, which are dictated by seasonally bound changes in environmental conditions.


Linezolid- and Vancomycin-resistant Enterococcus faecium in Solid Organ Transplant Recipients: Infection Control and Antimicrobial Stewardship Using Whole Genome Sequencing.
Vancomycin-resistant enterococci are an important cause of healthcare-associated infections and are inherently resistant to many commonly used antibiotics. Linezolid is the only drug currently approved by the US Food and Drug Administration to treat vancomycin-resistant enterococci; however, resistance to this antibiotic appears to be increasing. Although outbreaks of linezolid- and vancomycin-resistant Enterococcus faecium (LR-VRE) in solid organ transplant recipients remain uncommon, they represent a major challenge for infection control and hospital epidemiology. We describe a cluster of 4 LR-VRE infections among a group of liver and multivisceral transplant recipients in a single intensive care unit. Failure of treatment with linezolid in 2 cases led to a review of standard clinical laboratory methods for susceptibility determination. Testing by alternative methods including whole genome sequencing (WGS) and a comprehensive outbreak investigation including sampling of staff members and surfaces was performed. Review of laboratory testing methods revealed a limitation in the VITEK 2 system with regard to reporting resistance to linezolid. Linezolid resistance in all cases was confirmed by E-test method. The use of WGS identified a resistant subpopulation with the G2376C mutation in the 23S ribosomal RNA. Sampling of staff members' dominant hands as well as sampling of surfaces in the unit identified no contaminated sources for transmission. This cluster of LR-VRE in transplant recipients highlights the possible shortcomings of standard microbiology laboratory methods and underscores the importance of WGS to identify resistance mechanisms that can inform patient care, as well as infection control and antibiotic stewardship measures.


Spironolactone prevents chlorthalidone-induced sympathetic activation and insulin resistance in hypertensive patients.
Recent studies from our laboratory indicate that chlorthalidone triggers persistent activation of the sympathetic nervous system and promotes insulin resistance in hypertensive patients, independent of serum potassium. Mechanisms underlying these adverse effects of chlorthalidone remain unknown, but increasing evidence in rodents suggests the role of angiotensin and aldosterone excess in inducing both sympathetic overactivity and insulin resistance. Accordingly, we conducted studies in 17 subjects with untreated stage 1 hypertension, measuring sympathetic nerve activity at baseline and after 12 weeks of chlorthalidone alone (25 mg/d), chlorthalidone plus spironolactone, and chlorthalidone plus irbesartan, using randomized crossover design. We found that chlorthalidone alone decreased 24-hour ambulatory blood pressure from 135±3/84±2 to 124±2/78±2 mm Hg and significantly increased sympathetic nerve activity from baseline (from 41±3 versus 49±4 bursts per minute; P<0.01). The addition of spironolactone to chlorthalidone returned sympathetic nerve activity value to baseline (42±3 bursts per minute; P>0.05), whereas the addition of irbesartan failed to alter the sympathetic nerve activity response to chlorthalidone in the same subjects (52±2 bursts per minute; P<0.01) despite a similar reduction in ambulatory blood pressure (121±2/75±2 and 121±2/75±2 mm Hg, respectively). Chlorthalidone alone also increased indices of insulin resistance, which was not observed when used in combination with spironolactone. In conclusion, our study demonstrates beneficial effects of spironolactone in attenuating both chlorthalidone-induced sympathetic activation and insulin resistance in humans, independent of blood pressure reduction. Because sympathetic overactivity and insulin resistance contribute to the poor prognosis in patients with cardiovascular disease, combination therapy of chlorthalidone with mineralocorticoid receptor antagonists may constitute a preferable regimen than chlorthalidone alone in hypertensive patients.


Caregiver assistance among Medicare beneficiaries with atrial fibrillation and factors associated with anticoagulant treatment.
Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and disproportionately affects the elderly. This study describes patient characteristics and caregiver assistance among Medicare beneficiaries with AF and examines factors associated with receiving anticoagulant treatment. Patients with AF and age/gender-matched controls were identified from Medicare Current Beneficiary Survey data from 2001 to 2006. A logistic regression model was used to assess factors associated with receiving anticoagulants in a subgroup of patients with AF whose treatment pattern was established for 2 consecutive years. Sample weights were applied to obtain nationally representative estimates. A total of 2990 patients with AF and 5980 control patients were included in the burden of disease analysis, and 1481 patients with AF were included in the anticoagulant predictor analysis. Patients with AF had a higher level of comorbidity (Charlson Comorbidity Index: 3.3 vs 1.5; P < 0.05), worse self-perceived health status (P < 0.001), and greater level of disability (P < 0.001) than their matched counterparts. A greater proportion of patients with AF required caregiver assistance (62.8% vs 51.5%; P < 0.001). Logistic regression found that higher Charlson Comorbidity Index scores, difficulty in obtaining necessary health care, older age, being widowed, a history of psychiatric disorders, and being underweight decreased the likelihood of receiving anticoagulant therapy. In a Medicare population, a greater need for caregiver assistance was observed in patients with AF. Subgroups characterized by frailty or inability for self-care were identified as being less likely to receive anticoagulant therapy. The need for caregiver assistance among patients with AF, as well as the patient subgroups identified as less likely to receive anticoagulant therapy, should be considered when making treatment decisions.


Cranial Neuralgias.
Pain arising from cranial neuralgias represents a significant health burden. Successful treatment depends on accurate diagnosis, which requires knowledge of neuroanatomy and pathophysiology as well as familiarity with the varied clinical presentations encountered in neurologic practice. This article delineates the relevant anatomy, clinical features, and management of the most common primary and secondary cranial neuralgias. Trigeminal neuralgia, which can result from neurovascular compression or demyelination, is a particularly severe form of facial pain. Herpes zoster virus is a common cause of neuralgia that causes herpes zoster ophthalmicus acutely and postherpetic neuralgia chronically. Rarer facial pain syndromes arising from a single nerve include glossopharyngeal neuralgia, nervus intermedius neuralgia, and paratrigeminal oculosympathetic syndrome. In patients presenting with a cranial neuralgia, unless the etiology is apparent (eg, herpes zoster), cranial imaging studies should be undertaken to look for structural abnormalities such as neoplasm, granulomatous disease, demyelinating disease, or vascular malformations. Management of both common and rare cranial neuralgias is often challenging and is best guided by the most recent available evidence.


Globin synthesis in heterokaryons formed between chick erythrocytes and human K562 cells or rat L6 myoblasts.
Chick globin synthesis was studied in heterokaryons formed between chick erythrocytes and human K562 erythroleukemia cells or rat L6J1 myoblasts. It was found that chick globin synthesis was activated after fusion of definitive (17 to 19-day) chick erythrocytes obtained from 17 to 19-day embryos with K562 cells. Chick globins appeared to be of the adult alpha A, alpha D and beta types, whereas no embryonic globin synthesis could be detected. The pattern and time-course of globin synthesis was investigated after fusion of 4 to 5-day embryonic erythrocytes with rat L6J1 myoblasts. The level of globin synthesis was high at early time points but then decreased. Globin synthesis, however, was still detectable at 9 days in these heterokaryons. Chick alpha A, alpha D and epsilon-globin chain synthesis was observed both at early and late time points after fusion.


The health and well-being of Australia's future medical doctors: protocol for a 5-year observational cohort study of medical trainees.
Clinical training in the undergraduate medical course places multiple stressors on trainees, which have been held to lead to heightened distress, depression, suicide, substance misuse/abuse and poor mental health outcomes. To date, evidence for morbidity in trainees is largely derived from cross-sectional survey-based research. This limits the accuracy of estimates and the extent to which predispositional vulnerabilities (biological and/or psychological), contextual triggers and longer-term consequences can be validly identified. Longitudinal clinical assessments embedded within a biopsychosocial framework are needed before effective preventative and treatment strategies can be put in place. This study is an observational longitudinal cohort study of 330 students enrolled in the undergraduate medicine course at the University of New South Wales (UNSW) Sydney, Australia. Students will be recruited in their fourth year of study and undergo annual assessments for 4 consecutive years as they progress through increasingly demanding clinical training, including internship. Assessments will include clinical interviews for psychiatric morbidity, and self-report questionnaires to obtain health, psychosocial, performance and functioning information. Objective measures of cognitive performance, sleep/activity patterns as well as autonomic and immune function (via peripheral blood samples) will be obtained. These data will be used to determine the prevalence, incidence and severity of mental disorder, elucidate contextual and biological triggers and mechanisms underpinning psychopathology and examine the impact of psychopathology on performance and professional functioning. Ethics approval has been granted by the UNSW human research ethics committee (reference HC16340). The findings will be disseminated through peer-reviewed publications and conference presentations, and distributed to key stakeholders within the medical education sector. The outcomes will also inform targeted preventative and treatment strategies to enhance stress resilience in trainee doctors.


Cytogenetic features of infants less than 12 months of age at diagnosis of acute lymphoblastic leukemia: impact of the 11q23 breakpoint on outcome: a report of the Childrens Cancer Group.
Cytogenetic analyses of pretreatment bone marrows were performed at local institutions as part of Childrens Cancer Group (CCG) protocol CCG-107 for infants less than 1 year of age with previously untreated acute lymphoblastic leukemia (ALL). Cytogenetic analyses from 39 patients (17 males and 22 females) were accepted after review. Several unique cytogenetic features were observed. Twelve patients (31%) had a t(4;11)(q21;q23) and had a significantly shorter event-free survival (EFS) than did the other patients with adequate cytogenetic analyses (P = .009). Five additional patients had an 11q23 breakpoint, not associated with 4q21. When EFS for these 5 patients was compared with that of the t(4;11) patients, even with these small numbers there was a strong, although not significant, suggestion that the t(4;11) patients have a reduced EFS (P = .09), indicating that the specific translocation, t(4;11)(q21;q23), and not an 11q23 breakpoint per se, may be associated with the poor prognosis of these infants. Structural abnormalities were present in 27 of 28 patients with abnormal karyotypes. A new recurring abnormality, t(5;15)(p15:1;q11) or t(5;15)(p15.3;q13), was identified in 3 patients (Arthur et al, Blood 70:274a, 1987 [abstr, suppl 1]). Two females had structural abnormalities involving Xp11, a breakpoint rarely seen in ALL. Fourteen (36%) patients had a single structural abnormality, and 13 (33%) had complex karyotypes. No patients had hyperdiploidy with more than 50 chromosomes. Only normal chromosomes were observed in 11 patients (28%), and their outcome did not differ from patients with abnormal karyotypes. These cytogenetic abnormalities found in the leukemic cells of infants are clearly different from those in older children and adults, and may explain, in part, the unique biologic characteristics of infant ALL.


[MiR-24 improves beta-like globin gene expression through targeting Sp1].
We studied the function and mechanism of miR-24 in regulating beta-like globin gene expression. We first detected the expression of miR-24 during erythroid differentiation and also detected the globin gene expression in miR-24 overexpressing K562 cells through q-PCR. Dual-luciferase reporter assay and Western blotting were used to identify target genes of miR-24. "Rescue experiment" was further used to investigate the regulation of miR-24 on globin gene expression whether depending on targeting Sp1 or not. We found that miR-24 increased during hemin-induced K562 cells and EPO-induced HPCs (hematopoietic progenitor cells) erythroid differentiation. Overexpression of miR-24 in K562 cells promoted the epsilon- and gamma-globin gene expression during hemin-induced erythroid differentiation through targeting the negative globin regulator Sp1. These results suggested that miR-24 can improve the expression of beta-like globin gene through targeting Sp1.


Analgesics promote welfare and sustain tumour growth in orthotopic 4T1 and B16 mouse cancer models.
Murine orthotopic cancer models often require surgery, potentially causing pain or distress. However, analgesics are often withheld because they may alter tumour development. Two orthotopically implanted cancers were investigated in mice pre-treated with meloxicam (10 mg/kg), buprenorphine (0.2 mg/kg) or saline (1 ml/kg). Tumours were imaged and welfare was assessed using body weight, behaviour and nociceptive responses. In study 1, BALB/c mice were inoculated with 4T1 mammary carcinoma or saline during surgery or anaesthesia. As pre-treatment with a single buprenorphine dose appeared beneficial to cancer growth consistency, a second cohort of mice additionally received saline or buprenorphine at 12 and 24 h. Surgery resulted in increased mammary tumour growth and lung metastases. These unwanted effects were lessened by buprenorphine pre-treatment, especially when given repeatedly. Mammary tumour-bearing mice became less active and nociceptive thresholds declined over time, indicating some discomfort as tumours grew. In study 2, C57BL/6 mice received B16 melanoma. This non-surgical model was used to determine whether meloxicam or buprenorphine affected cancer seeding of the lungs. While meloxicam reduced B16 lung seeding, buprenorphine did not. Mechanical thresholds decreased as cancer developed in mice bearing melanoma, but the magnitude of this was insufficient to conclude that there were any significant welfare concerns. This study highlights the scientific value in utilising non-surgical models, where possible. When surgery must be performed at the time of tumour inoculation, the effects of this should be controlled with appropriate analgesics to enhance the value and possibly translation of the research.


ADAM17 in tumor associated leukocytes regulates inflammatory mediators and promotes mammary tumor formation.
The presence of inflammatory cells within the tumor microenvironment has been tightly linked to mammary tumor formation and progression. Specifically, interactions between tumor cells and infiltrating macrophages can contribute to the generation of a pro-tumorigenic microenvironment. Understanding the complex mechanisms that drive tumor cell-macrophage cross-talk will ultimately lead to the development of approaches to prevent or treat early stage breast cancers. As described here, we demonstrate that the cell surface protease a disintegrin and metalloproteinase 17 (ADAM17) is expressed by macrophages in mammary tumors and contributes to regulating the expression of pro-inflammatory mediators, including inflammatory cytokines and the inflammatory mediator cyclooxygenase-2 (Cox-2). Furthermore, we demonstrate that ADAM17 is expressed on leukocytes, including macrophages, within polyoma middle T (PyMT)-derived mammary tumors. Genetic deletion of ADAM17 in leukocytes resulted in decreased onset of mammary tumor growth, which was associated with reduced expression of the Cox-2 within the tumor. These findings demonstrate that ADAM17 regulates key inflammatory mediators in macrophages and that leukocyte-specific ADAM17 is an important promoter of mammary tumor initiation. Understanding the mechanisms associated with early stage tumorigenesis has implications for the development of preventive and/or treatment strategies for early stage breast cancers.


Nucleotide polymorphism in colicin E1 and Ia plasmids from natural isolates of Escherichia coli.
We examined DNA sequence polymorphism for the colicin gene clusters of seven ColE1 and six ColIa plasmids obtained from natural isolates of Escherichia coli. These gene clusters harbor levels of nucleotide diversity ranging from 0.006 (ColIa) to 0.054 (ColE1). This level of diversity is similar to that observed for chromosomally encoded E. coli genes. However, the variance associated with these estimates is severalfold higher for the plasmid-encoded genes. This increased variance may be due to the differing plasmid population sizes. The pattern of colicin gene cluster polymorphism suggests that the two colicins are evolving in different fashions. ColE1 accumulates polymorphism at an elevated rate in the central domain of the colicin protein, while ColIa polymorphism is distributed evenly along the gene cluster. Comparison of the patterns of divergence between colicin and related proteins of ColIa and Ib and patterns of polymorphism within ColIa suggest that this gene cluster is not evolving in a neutral fashion. These data lend support to the hypothesis that colicin gene clusters may evolve under the influence of diversifying selection.


Regional gastrointestinal contractility parameters using the wireless motility capsule: inter-observer reproducibility and influence of age, gender and study country.
The wireless motility capsule concurrently measures temperature, pH and pressure as it traverses the gastrointestinal tract. To describe normative values for motility/contractility parameters across age, gender and testing centres. Healthy participants underwent a standardised wireless motility capsule assessment following an overnight fast and consumption of a meal of known nutritional content. Traces were divided into regions of interest and analysed using 2 software packages (MotiliGI and GIMS Data Viewer). Inter-observer agreement was independently assessed by 2 investigators. Normative data for motility/contractility parameters (maximum amplitude, mean peak amplitude, contraction frequency and motility index) are presented for 107 individuals (62 male, median age 40 years, range 18-78). MotiliGI-Gastric, small bowel and colonic maximal contraction amplitude correlated with age (r = .24, P = .01; r = .22, P = .02; and r = .2, P = .04 respectively). Small bowel motility index was higher in females than males (150.4 ± 12 vs 122 ± 7.6, P = .04). Inter-observer agreement was excellent for transit times, pH and contractility/motility parameters. GIMS Data viewer-Gastric, small bowel and colonic loge motility index correlated with the respective area under the contraction curve, total contractions, sum of amplitudes and contraction frequency (all r>.35, P < .0003) but not with transit times. Our analysis provides normative data for motility/contractility parameters. Log motility index summarises a number of measures. In future, the measurement of contractile activity with the wireless motility capsule may potentially aid in the diagnosis of disease states such as visceral myopathic disorders.


Interplay between DNA methylation, histone modification and chromatin remodeling in stem cells and during development.
Genes constitute only a small proportion of the mammalian genome, the majority of which is composed of non-genic repetitive elements including interspersed repeats and satellites. A unique feature of the mammalian genome is that there are numerous tissue-dependent, differentially methylated regions (T-DMRs) in the non-repetitive sequences, which include genes and their regulatory elements. The epigenetic status of T-DMRs varies from that of repetitive elements and constitutes the DNA methylation profile genome-wide. Since the DNA methylation profile is specific to each cell and tissue type, much like a fingerprint, it can be used as a means of identification. The formation of DNA methylation profiles is the basis for cell differentiation and development in mammals. The epigenetic status of each T-DMR is regulated by the interplay between DNA methyltransferases, histone modification enzymes, histone subtypes, non-histone nuclear proteins and non-coding RNAs. In this review, we will discuss how these epigenetic factors cooperate to establish cell- and tissue-specific DNA methylation profiles.


Pre- and postjunctional effects of inflammatory mediators in horse airways.
In addition to their direct contractile effects, histamine (Hist), serotonin [5-hydroxytryptamine (5-HT)], and leukotriene (LT) D(4), in low concentrations, dramatically augment electrical field stimulation (EFS)-induced smooth muscle contractions in equine airways. To determine the mechanism of their action, we studied, in trachealis strips, the effect of these mediators on both cholinergically induced tension and the release of ACh from cholinergic nerves. All three mediators synergistically augmented the contraction of the trachealis that was due to release of endogenous ACh, i.e., EFS-induced contraction. These same mediators caused only a small but parallel shift of the ACh concentration-response curve. Comparison of the mediator effects on the responses to endogenous and exogenous ACh suggested a prejunctional effect. However, release of ACh was augmented only by Hist and 5-HT but not by LTD(4). Hist-induced contraction of trachealis was abolished by pyrilamine (H(1)-receptor antagonist) but not by ranitidine (H(2)-receptor antagonist), whereas thioperamide (H(3)-receptor antagonist) shifted the Hist response curve to the left. The augmenting effect of Hist on EFS-induced contraction was abolished by pyrilamine and unaffected by ranitidine or thioperamide. We conclude that inflammatory mediators can increase endogenous cholinergic responses of equine airways via both prejunctional and postjunctional mechanisms. LTD(4) acts solely on smooth muscle, whereas 5-HT and Hist additionally act on neuronal receptors to facilitate release of ACh. Excitatory effects of Hist, i.e., direct contractile effect, and augmentation of endogenous cholinergic response are both mediated via H(1) receptors, whereas the inhibitory H(3) receptors partially oppose the direct contractile effect of this mediator.


Phase II Clinical Trial of Eribulin-Gemcitabine Combination Therapy in Previously Treated Patients With Advanced Liposarcoma or Leiomyosarcoma.
Monotherapy with eribulin or gemcitabine has been found to be moderately effective in treating soft-tissue sarcomas (STS). In this study, we evaluated the efficacy and safety of eribulin-gemcitabine combination therapy for the two most common histologic types of STS, liposarcoma and leiomyosarcoma. In this nonrandomized, multicenter, phase II study, we included patients with progressive disease who had received one or two courses of chemotherapy that included doxorubicin. Patients were administered 1.4 mg/m2 eribulin and 1,000 mg/m2 gemcitabine on days 1 and 8 every 3 weeks. The primary endpoint was progression-free survival rate at 12 weeks (PFSR12wks), with null and alternative hypotheses of PFSR12wks ≤20.0% and ≥40.0%, respectively. Exploratory biomarker analyses with next-generation sequencing (NGS) were performed on pretreatment tumor samples. Among the 37 patients included, the overall PFSR12wks was 73.0%, achieving the primary endpoint. The objective response rate, disease control rate, median progression-free survival, and median overall survival were 16.2%, 78.4%, 5.6 months, and 31.9 months, respectively, without differences according to histologic type. New safety signals and treatment-related deaths were not documented. NGS-based transcriptome analysis revealed that functional enrichment in the TGFβ pathway was mostly associated with a poor outcome, whereas single genetic alterations largely failed to predict treatment outcome. Eribulin-gemcitabine combination therapy showed promising activity and an acceptable safety profile in patients with liposarcoma or leiomyosarcoma. Gene expression profiling with pathway enrichment analysis would have possibilities to have predictive value for survival outcome, necessitating further investigation to confirm.


Patency tests of the maxillary ostium in health and disease.
Three different maxillary ostial function tests (simultaneous pressure recording in nose and sinus; simultaneous differential pressure and flow recording across the ostium; pressure recording at a constant artificial air-flow into the sinus) have been evaluated on healthy subjects and used in clinical materials. No quantitative evaluation of the ostial function was possible when using the simultaneous nasosinusal pressure recording method. The ostial function can be expressed as ostial resistance or as corresponding equivalent ostial diameter with the pressure-flow and the constant artificial air-flow methods. An isolated determination of the ostial function in both sinu-nasal and naso-sinusal directions during nasal breathing was only possible with the pressure-flow method. No impaired ostial function was found in cases with maxillary pain and intrasinusal cysts, but in chronic sinusitis the ostial function was considerably deteriorated. In cases with impaired ostial function the constant artificial air-flow method does not mirror the true functional state due to the non-physiological air-flow level used. Thus, the pressure-flow method seems to be the most physiological one.


Protein kinase C activation increases transepithelial transport of biologically active insulin.
Protein kinase C activation leads to tight junctional leakiness and, consequently, to increased transepithelial (paracellular) solute flux across epithelial barriers. This leakiness is shown here to result in as much as a 20-fold increase in the transepithelial flux of insulin. Using an epithelial/fibroblast coculture model, this transepithelially transported insulin is shown to be biologically active. The 3T3 fibroblasts situated on one side of the epithelial barrier exhibited increased insulin binding and resulting DNA synthesis when the epithelial junctions were made leaky to insulin on the opposite side of the epithelial barrier. The dramatically enhanced permeability of macromolecules across epithelial cell layers undergoing protein kinase C activation may play a significant role in epithelial cancer, immunology, and drug delivery.


Molecular evidence supports the distinction between Xanthoria parietina and X. aureola (Teloschistaceae, lichenized Ascomycota).
This study aims to clarify taxonomic relationships within the current concept of Xanthoria parietina in northern Europe. For comparison, X. calcicola was also included in the study. Morphological as well as molecular data were utilized. Morphology indicated the presence of three species, Xanthoria parietina, X. calcicola, and X. aureola, the latter of which is resurrected here from synonymy. The most important separating characters involve colour and thickness of the thallus, lobe width, morphology of laminar structures, and the texture of the upper surface. X. aureola, as recognized here, mostly occurs on seashore rocks. Part of the IGS region as well as the complete ITS were sequenced in 70 individual thalli representing ten geographical regions in Europe. In total, 19 different IGS haplotypes and 20 different ITS haplotypes were present in the data set. Owing to indications of possible recombination between the IGS and the ITS, the two data sets were analyzed separately. Haplotype networks were estimated, both of which indicate that X. parietina is distinct from X. aureola and X. calcicola. In our sample, the two latter do not share haplotypes, but are only separated by a few mutational steps.


Adult scoliosis. Current concepts.
Back pain and increasing deformity of the spine can develop in adults with idiopathic scoliosis. It is known that scoliosis can continue to progress after skeletal maturity and cause problems in adults. Conservative treatment is effective in most cases, but occasionally surgery is necessary. Recent advances in spinal instrumentation have resulted in improved results and fewer complications.


Improved cryotolerance and developmental potential of in vitro and in vivo matured mouse oocytes by supplementing with a glutathione donor prior to vitrification.
Can supplementation of media with a glutathione (GSH) donor, glutathione ethyl ester (GEE), prior to vitrification protect the mouse oocyte from oxidative damage and critical changes in redox homeostasis, and thereby improve cryotolerance? GEE supplementation supported redox regulation, rapid recovery of spindle and chromosome alignment after vitrification/warming and improved preimplantation development of mouse metaphase II (MII) oocytes. Cryopreservation may affect mitochondrial functionality, induce oxidative stress, and thereby affect spindle integrity, chromosome segregation and the quality of mammalian oocytes. GEE is a membrane permeable GSH donor that promoted fertilization and early embryonic development of macaque and bovine oocytes after IVM. Two experimental groups consisted of (i) denuded mouse germinal vesicle (GV) oocytes that were matured in vitro in the presence or absence of 1 mM GEE (IVM group 1) and (ii) in vivo ovulated (IVO) MII oocytes that were isolated from the ampullae and exposed to 1 mM GEE for 1 h prior to vitrification (IVO group 2). Recovery of oocytes from both groups was followed after CryoTop vitrification/warming for up to 2 h and parthenogenetic activation. Reactive oxygen species (ROS), spindle morphology and chromosome alignment were analyzed by confocal laser scanning microscopy (CLSM) and polarization microscopy in control and GEE-supplemented MII oocytes. The relative overall intra-oocyte GSH content was assessed by analysis of monochlorobimane (MBC)-GSH adduct fluorescence in IVM MII oocytes. The GSH-dependent intra-mitochondrial redox potential (EmGSH) of IVM MII oocytes was determined after microinjection with specific mRNA at the GV stage to express a redox-sensitive probe within mitochondria (mito-Grx1-roGFP2). The absolute negative redox capacity (in millivolts) was determined by analysis of fluorescence of the oxidized versus the reduced form of sensor by CLSM and quantification according to Nernst equation. Proteome analysis was performed by quantitative 2D saturation gel electrophoresis (2D DIGE). Since microinjection and expression of redox sensor mRNA required removal of cumulus cells, and IVM of denuded mouse oocytes in group 1 induces zona hardening, the development to blastocysts was not assessed after IVF but instead after parthenogenetic activation of vitrified/warmed MII oocytes from both experimental groups. IVM of denuded mouse oocytes in the presence of 1 mM GEE significantly increased intra-oocyte GSH content. ROS was not increased by CryoTop vitrification but was significantly lower in the IVM GEE group compared to IVM without GEE before vitrification and after recovery from vitrification/warming (P < 0.001). Vitrification alone significantly increased the GSH-dependent intra-mitochondrial redox capacity after warming (EmGSH, P < 0.001) in IVM oocytes, presumably by diffusion/uptake of cytoplasmic GSH into mitochondria. The presence of 1 mM GEE during IVM increased the redox capacity before vitrification and there was no further increase after vitrification/warming. None of the reproducibly detected 1492 spots of 2D DIGE separated proteins were significantly altered by vitrification or GEE supplementation. However, IVM of denuded oocytes significantly affected spindle integrity and chromosome alignment right after warming from vitrification (0 h) in group 1 and spindle integrity in group 2 (P < 0.05). GEE improved recovery in IVM group as numbers of oocytes with unaligned chromosomes and aberrant spindles was not significantly increased compared to unvitrified controls. The supplementation with GEE for 1 h before vitrification also supported more rapid recovery of spindle birefringence. GEE improved significantly development to the 2-cell stage for MII oocytes that were activated directly after vitrification/warming in both experimental groups, and also the blastocyst rate in the IVO GEE-supplemented group compared to the controls (P < 0.05). None LIMITATIONS, REASONS FOR CAUTION: The studies were carried out in a mouse model, in IVM denuded rather than cumulus-enclosed oocytes, and in activated rather than IVF MII oocytes. Whether the increased GSH-dependent intra-mitochondrial redox capacity also improves male pronuclear formation needs to be studied further experimentally. The influence of GEE supplementation requires also further examination and optimization in human oocytes before it can be considered for clinical ART. Although GEE supplementation did not alter the proteome at MII, the GSH donor may support cellular homeostasis and redox regulation and, thus, increase developmental competence. While human MII oocyte vitrification is an established procedure, GEE might be particularly beneficial for oocytes that suffer from oxidative stress and reduced redox capacity (e.g. aged oocytes) or possess low GSH due to a reduced supply of GSH from cumulus. It might also be of relevance for immature human oocytes that develop without cumulus to MII in vitro (e.g. in ICSI cycles) for ART. The study has been supported by the German Research Foundation (DFG FOR 1041; EI 199/3-2). There are no conflict of interests.


Migraine prevention: what patients want from medication and their physicians (a headache specialty clinic perspective).
To document the results of a migraine patients survey, from a headache specialty clinic, in which patients were asked to rank, in order of importance, certain characteristics of migraine preventive treatment. A 10-question survey was completed by 150 patients (114 females and 36 males) with a history of migraine who presented to the Michigan Head Pain & Neurological Institute. The patients were asked to rank, in order of importance, characteristics of migraine preventive treatment. Each characteristic was rated individually on a 1 to 10 scale (1 being of little importance and 10 being extremely important). The mean rating of each characteristic was then calculated and the results analyzed. From this migraine preventive treatment survey, the most important thing to migraineurs, from a headache specialty clinic population, is that the prescribing physician involves them in the decision making of choosing a preventive agent. The physician taking time to explain the possible medication side effects is the second most highly ranked characteristic. Migraine preventives with published efficacy in the medical literature are also deemed very important. Migraineurs do not mind using more than 1 preventive agent at one time if greater efficacy can be achieved. Agents that may affect weight and/or cause sedation may be important factors as to why patients (especially females) may not want to take a preventive medication. Natural therapies and once-daily dosing are ranked lower overall but still are important characteristics of preventive treatment. Some gender differences are noted in the ranking of migraine preventive characteristics.


Building the pipeline: the creation of a residency training pathway for future physician leaders in health care quality.
Many health care organizations seek physicians to lead quality improvement (QI) efforts, yet struggle to find individuals with the necessary expertise. Although most residency programs incorporate QI and patient safety principles into their curricula, few provide a specialized training program for residents exploring careers as physician leaders in quality. Recognizing this training void, the authors designed and implemented the Healthcare Leadership in Quality (HLQ) track for residents at the University of Pennsylvania Health System in 2010. This longitudinal, two-year graduate medical education (GME) track aligns with the quality goals of the University of Pennsylvania Health System and includes a core curriculum, integration into an interprofessional health care leadership team that is accountable for quality and safety outcomes on a hospital unit, a capstone QI project, and mentorship. Early evaluation has demonstrated the feasibility and efficacy of the track diverse graduate medical education training programs. Using Yardley and Dornan's interpretation of the Kirkpatrick framework, the authors have demonstrated the track's impact on four levels of educational and organizational outcomes. Building on their early experiences, the authors are integrating project and time management skills into the core curriculum, and they are focusing more effort on faculty development in QI mentorship. Additionally, the authors plan to follow HLQ track graduates to determine whether they seek leadership roles in quality and safety and to assess the influence of the program on their careers.


Elucidation of the mechanism by which homocysteine potentiates the anti-vaccinia virus effects of the S-adenosylhomocysteine hydrolase inhibitor 9-(trans-2',trans-3'-dihydroxycyclopent-4'-enyl)-adenine.
9-(trans-2',trans-3'-dihydroxycyclopent-4'-enyl)-adenine (DHC), a specific inhibitor of S-adenosyl-L-homocysteine (AdoHcy) hydrolase, has been used in this study to elucidate the mechanism by which DL-homocysteine (Hcy) potentiates the antiviral effects of AdoHcy hydrolase inhibitors as reported by De Clercq [Biochem. Pharmacol. 36:2567-2575 (1987)]. The potentiating effects of Hcy on the antiviral effects of DHCA were determined using murine L929 cells infected with vaccinia virus. When virus-infected cells were incubated with DHCA alone or in combination with various concentrations of Hcy, the following IC50 values (concentrations of the drug required to reduce by 50% viral plaque formation) were observed: 0.30 microM (0 mM Hcy), 0.15 microM (0.3 mM Hcy), 0.09 microM (1.0 mM Hcy), and 0.04 microM (3.0 mM Hcy). In the drug combination studies, increased cellular toxicity, compared with DHCA alone, was observed only at the highest concentration of Hcy (3.0 mM); thus, at lower concentrations Hcy increased the antiviral effectiveness [ID50 (concentration of the drug required to reduce the increase in cell number by 50%)/IC50] of DHCA. For example the following ID50/IC50 values were observed for DHCA alone or in combination with Hcy: 64 (0 mM Hcy), 113 (0.3 mM Hcy), 151 (1.0 mM Hcy), and 88 (3.0 mM Hcy). In these studies, Hcy was also observed to potentiate the increase in cellular levels of AdoHcy and the ratio of AdoHcy/S-adenosyl-L-methionine (AdoMet) in DHCA-treated cells. In earlier studies, our laboratory has shown that antiviral effects of DHCA are caused by only slight elevations in intracellular levels of AdoHcy [from 50 pmol/mg of protein (controls) to 100-200 pmol/mg of protein (drug-treated)] and slight elevations in the ratios of AdoHcy/AdoMet [from 0.05-0.1 (control) to 0.15-0.20 (drug-treated)]. Thus, in the presence of Hcy, lower concentrations of DHCA are needed to increase the intracellular concentration of AdoHcy and the AdoHcy/AdoMet ratio to levels that suppress replication of vaccinia virus. Murine L929 cells were shown to contain DHCA-sensitive and DHCA-insensitive forms of AdoHcy hydrolase. Based on the results of labeling experiments using [2,8-3H]adenosine and [35S]methionine, the elevated levels of AdoHcy were shown to arise from the reaction of [2,8-3H]adenosine and Hcy, catalyzed by the DHCA-insensitive form of AdoHcy hydrolase.


Cells bearing mutations causing Leber's hereditary optic neuropathy are sensitized to Fas-Induced apoptosis.
Three prevalent mitochondrial DNA pathogenic mutations at positions 11778, 3460, and 14484, which affect different subunits of Complex I, cause retinal ganglion cell death and optic nerve atrophy in Leber's hereditary optic neuropathy (LHON). The cell death is painless and without inflammation, consistent with an apoptotic mechanism. We have investigated the possibility that the LHON mutation confers a pro-apoptotic stimulus and have tested the sensitivity of osteosarcoma-derived cybrid cells carrying the most common and severe mutations (11778 and 3460) to cell death induced by Fas. We observed that LHON cybrids were sensitized to Fas-dependent death. Control cells that bear the same mitochondrial genetic background (the J haplogroup) without the pathogenic 11778 mutation are no more sensitive than other controls, indicating that increased Fas-dependent death in LHON cybrids was induced by the LHON pathogenic mutations. The type of death was apoptotic by several criteria, including induction by Fas, inhibition by the caspase inhibitor zVAD-fmk (zVal-Ala-Asp-fluoro-methyl ketone), activation of DEVDase activity (Asp-Glu-Val-Asp protease), specific cleavage of caspase-3, DNA fragmentation, and increased Annexin-V labeling. These data indicate that the most common and severe LHON pathogenic mutations 11778 and 3460 predispose cells to apoptosis, which may be relevant for the pathophysiology of cell death in LHON, and potential therapy.


Natural resistance to worms exacerbates bovine tuberculosis severity independently of worm coinfection.
Pathogen interactions arising during coinfection can exacerbate disease severity, for example when the immune response mounted against one pathogen negatively affects defense of another. It is also possible that host immune responses to a pathogen, shaped by historical evolutionary interactions between host and pathogen, may modify host immune defenses in ways that have repercussions for other pathogens. In this case, negative interactions between two pathogens could emerge even in the absence of concurrent infection. Parasitic worms and tuberculosis (TB) are involved in one of the most geographically extensive of pathogen interactions, and during coinfection worms can exacerbate TB disease outcomes. Here, we show that in a wild mammal natural resistance to worms affects bovine tuberculosis (BTB) severity independently of active worm infection. We found that worm-resistant individuals were more likely to die of BTB than were nonresistant individuals, and their disease progressed more quickly. Anthelmintic treatment moderated, but did not eliminate, the resistance effect, and the effects of resistance and treatment were opposite and additive, with untreated, resistant individuals experiencing the highest mortality. Furthermore, resistance and anthelmintic treatment had nonoverlapping effects on BTB pathology. The effects of resistance manifested in the lungs (the primary site of BTB infection), while the effects of treatment manifested almost entirely in the lymph nodes (the site of disseminated disease), suggesting that resistance and active worm infection affect BTB progression via distinct mechanisms. Our findings reveal that interactions between pathogens can occur as a consequence of processes arising on very different timescales.


Preclinical models of arthritis for studying immunotherapy and immune tolerance.
Increasingly earlier identification of individuals at high risk of rheumatoid arthritis (RA) (eg, with autoantibodies and mild symptoms) improves the feasibility of preventing or curing disease. The use of antigen-specific immunotherapies to reinstate immunological self-tolerance represent a highly attractive strategy due to their potential to induce disease resolution, in contrast to existing approaches that require long-term treatment of underlying symptoms.Preclinical animal models have been used to understand disease mechanisms and to evaluate novel immunotherapeutic approaches. However, models are required to understand critical processes supporting disease development such as the breach of self-tolerance that triggers autoimmunity and the progression from asymptomatic autoimmunity to joint pain and bone loss. These models would also be useful in evaluating the response to treatment in the pre-RA period.This review proposes that focusing on immune processes contributing to initial disease induction rather than end-stage pathological consequences is essential to allow development and evaluation of novel immunotherapies for early intervention. We will describe and critique existing models in arthritis and the broader field of autoimmunity that may fulfil these criteria. We will also identify key gaps in our ability to study these processes in animal models, to highlight where further research should be targeted.


Galleria mellonella as a model system to study Cryptococcus neoformans pathogenesis.
Evaluation of Cryptococcus neoformans virulence in a number of nonmammalian hosts suggests that C. neoformans is a nonspecific pathogen. We used the killing of Galleria mellonella (the greater wax moth) caterpillar by C. neoformans to develop an invertebrate host model system that can be used to study cryptococcal virulence, host immune responses to infection, and the effects of antifungal compounds. All varieties of C. neoformans killed G. mellonella. After injection into the insect hemocoel, C. neoformans proliferated and, despite successful phagocytosis by host hemocytes, killed caterpillars both at 37 degrees C and 30 degrees C. The rate and extent of killing depended on the cryptococcal strain and the number of fungal cells injected. The sequenced C. neoformans clinical strain H99 was the most virulent of the strains tested and killed caterpillars with inocula as low as 20 CFU/caterpillar. Several C. neoformans genes previously shown to be involved in mammalian virulence (CAP59, GPA1, RAS1, and PKA1) also played a role in G. mellonella killing. Combination antifungal therapy (amphotericin B plus flucytosine) administered before or after inoculation was more effective than monotherapy in prolonging survival and in decreasing the tissue burden of cryptococci in the hemocoel. The G. mellonella-C. neoformans pathogenicity model may be a substitute for mammalian models of infection with C. neoformans and may facilitate the in vivo study of fungal virulence and efficacy of antifungal therapies.


Comparative Genomics of Clinical Isolates of the Emerging Tick-Borne Pathogen Neoehrlichia mikurensis.
Tick-borne 'Neoehrlichia (N.) mikurensis' is the cause of neoehrlichiosis, an infectious vasculitis of humans. This strict intracellular pathogen is a member of the family Anaplasmataceae and has been unculturable until recently. The only available genetic data on this new pathogen are six partially sequenced housekeeping genes. The aim of this study was to advance the knowledge regarding 'N. mikurensis' genomic relatedness with other Anaplasmataceae members, intra-species genotypic variability and potential virulence factors explaining its tropism for vascular endothelium. Here, we present the de novo whole-genome sequences of three 'N. mikurensis' strains derived from Swedish patients diagnosed with neoehrlichiosis. The genomes were obtained by extraction of DNA from patient plasma, library preparation using 10× Chromium technology, and sequencing by Illumina Hiseq-4500. 'N. mikurensis' was found to have the next smallest genome of the Anaplasmataceae family (1.1 Mbp with 27% GC contents) consisting of 845 protein-coding genes, every third of which with unknown function. Comparative genomic analyses revealed that 'N. mikurensis' was more closely related to Ehrlichia chaffeensis than to Ehrlichia ruminantium, the opposite of what 16SrRNA sequence-based phylogenetic analyses determined. The genetic variability of the three whole-genome-sequenced 'N. mikurensis' strains was extremely low, between 0.14 and 0.22‰, a variation that was associated with geographic origin. No protein-coding genes exclusively shared by N. mikurensis and E. ruminantium were identified to explain their common tropism for vascular endothelium.


Community perceptions and personal accounts of HIV discordance in rural western Kenya.
Among HIV-discordant couples, the literature is sparse regarding issues related to stigma, relationships and coping. Objectives were to explore: 1) perceptions about discordant HIV status; 2) understanding of HIV discordancy; 3) effects of discordancy on couples; and 4) adaptation and coping strategies for discordant HIV status. A survey was administered to 202 members of heterosexual discordant couples in rural western Kenya. In addition, to understand the community perspective, in-depth interviews (IDI) (n = 26) and focus group discussions (FGD) (n = 10) were conducted with community opinion leaders, healthcare workers and members of discordant couples. More than 70% of men (73.4%) and women (80.4%) surveyed agreed that their relationship changed for the worse when they disclosed their HIV status to their partner. Participants of IDIs and FGDs provided several explanations for discordancy including the perception that discordancy is a lie, the negative partner has "thick blood", HIV infection is a punishment for sexual promiscuity or cultural disobedience, and that HIV is a punishment from God. Members of discordant couples reported experiencing tension and fear, stigma and rejection, and changes in partner support. Adaptation and coping strategies included counselling, sero-sorting and pursuing concordancy with the uninfected partner. HIV discordancy in a relationship can potentially cause long-term negative emotional and physical consequences. There is an acute need to develop and disseminate locally sensitive HIV-discordant couple counselling messages, and to provide couple-centred HIV care and treatment. Communication can help couples rebuild and rebalance their relationship and adjust to a new normal.


Regulation of bacterial drug export systems.
The active transport of toxic compounds by membrane-bound efflux proteins is becoming an increasingly frequent mechanism by which cells exhibit resistance to therapeutic drugs. This review examines the regulation of bacterial drug efflux systems, which occurs primarily at the level of transcription. Investigations into these regulatory networks have yielded a substantial volume of information that has either not been forthcoming from or complements that obtained by analysis of the transport proteins themselves. Several local regulatory proteins, including the activator BmrR from Bacillus subtilis and the repressors QacR from Staphylococcus aureus and TetR and EmrR from Escherichia coli, have been shown to mediate increases in the expression of drug efflux genes by directly sensing the presence of the toxic substrates exported by their cognate pump. This ability to bind transporter substrates has permitted detailed structural information to be gathered on protein-antimicrobial agent-ligand interactions. In addition, bacterial multidrug efflux determinants are frequently controlled at a global level and may belong to stress response regulons such as E. coli mar, expression of which is controlled by the MarA and MarR proteins. However, many regulatory systems are ill-adapted for detecting the presence of toxic pump substrates and instead are likely to respond to alternative signals related to unidentified physiological roles of the transporter. Hence, in a number of important pathogens, regulatory mutations that result in drug transporter overexpression and concomitant elevated antimicrobial resistance are often observed.


Multiparametric Cardiovascular Magnetic Resonance in Acute Myocarditis: Comparison of 2009 and 2018 Lake Louise Criteria With Endomyocardial Biopsy Confirmation.
Background: Cardiac magnetic resonance (CMR) has been shown to improve the diagnosis of myocarditis, but no systematic comparison of this technique is currently available. The purpose of this study was to compare the 2009 and 2018 Lake Louise Criteria (LLC) for the diagnosis of acute myocarditis using 3.0 T MRI with endomyocardial biopsy (EMB) as a reference and to provide the cutoff values for multiparametric CMR techniques. Methods: A total of 73 patients (32 ± 14 years, 71.2% men) with clinically suspected myocarditis undergoing EMB and CMR with 3.0 T were enrolled in the study. Patients were divided into two groups according to EMB results (EMB-positive and -negative groups). The CMR protocol consisted of cine-SSFP, T2 STIR, T2 mapping, early and late gadolinium enhancement (EGE, LGE), and pre- and post-contrast T1 mapping. Their potential diagnostic ability was assessed with receiver operating characteristic curves. Results: The myocardial T1 and T2 relaxation times were significantly higher in the EMB-positive group than in the EMB-negative group. Optimal cutoff values were 1,228 ms for T1 relaxation times and 58.5 ms for T2 relaxation times with sensitivities of 86.0 and 83.7% and specificities of 93.3 and 93.3%, respectively. The 2018 LLC had a better diagnostic performance than the 2009 LLC in terms of sensitivity, specificity, positive predictive value, and negative predictive value. T1 mapping + T2 mapping had the largest area under the curve (0.95) compared to other single or combined parameters (2018 LLC: 0.91; 2009 LLC: 0.76; T2 ratio: 0.71; EGEr: 0.67; LGE: 0.73; ). The diagnostic accuracy for the 2018 LLC was the highest (91.8%), followed by T1 mapping (89.0%) and T2 mapping (87.7%). Conclusion: Emerging technologies such as T1/ T2 mapping have significantly improved the diagnostic performance of CMR for the diagnosis of acute myocarditis. The 2018 LLC provided the overall best diagnostic performance in acute myocarditis compared to other single standard CMR parameters or combined parameters. There was no significant gain when 2018LLC is combined with the EGE sequence.


Organic complexation of U(VI) in reducing soils at a natural analogue site: Implications for uranium transport.
Understanding the long-term fate, stability, and bioavailability of uranium (U) in the environment is important for the management of nuclear legacy sites and radioactive wastes. Analysis of U behavior at natural analogue sites permits evaluation of U biogeochemistry under conditions more representative of long-term equilibrium. Here, we have used bulk geochemical and microbial community analysis of soils, coupled with X-ray absorption spectroscopy and μ-focus X-ray fluorescence mapping, to gain a mechanistic understanding of the fate of U transported into an organic-rich soil from a pitchblende vein at the UK Needle's Eye Natural Analogue site. U is highly enriched in the Needle's Eye soils (∼1600 mg kg-1). We show that this enrichment is largely controlled by U(VI) complexation with soil organic matter and not U(VI) bioreduction. Instead, organic-associated U(VI) seems to remain stable under microbially-mediated Fe(III)-reducing conditions. U(IV) (as non-crystalline U(IV)) was only observed at greater depths at the site (>25 cm); the soil here was comparatively mineral-rich, organic-poor, and sulfate-reducing/methanogenic. Furthermore, nanocrystalline UO2, an alternative product of U(VI) reduction in soils, was not observed at the site, and U did not appear to be associated with Fe-bearing minerals. Organic-rich soils appear to have the potential to impede U groundwater transport, irrespective of ambient redox conditions.


A decade of sustained selection pressure on two surface sites of the VP1 protein of Enterovirus A71 suggests that immune evasion may be an indirect driver for virulence.
Enterovirus A71 (EV-A71) is an emerging pathogen in the Enterovirus A species group. EV-A71 causes hand, foot and mouth disease (HFMD), with virulent variants exhibiting polio-like acute flaccid paralysis and other central nervous system manifestations. We analysed all enterovirus A71 complete genomes with collection dates from 2008 to mid-2018. All sub-genotypes exhibit a strong molecular clock with omega (dN/dS) suggesting strong purifying selection. In sub-genotypes B5 and C4, positive selection can be detected at two surface sites on the VP1 protein, also detected in positive selection studies performed prior to 2008. Toggling of a limited repertoire of amino acids at these positively selected residues over the last decade suggests that EV-A71 may be undergoing a sustained frequency-dependent selection process for immune evasion, raising issues for vaccine development. These same sites have also been previously implicated in virus-host binding and strain-associated severity of HFMD, suggesting that immune evasion may be an indirect driver for virulence (154 words).


Multivariate method to identify inequalities in oral healthcare access.
The aim of this study is to apply a multivariate method for municipality's classification according access to oral health in adults. This is a cross-sectional epidemiological study. Were used multivariate classification called nonhierarchical cluster analysis K-means. The strategy brings together municipalities' similarity as access to oral health, where the most similar are next and the most different from getting further away. In addition, it allows reducing the intragroup variance and maximizing intergroup variance. It was assumed the number of four groups. Among adults, 3,185 (52.63%) visited the dentist less than a year, and 357 (5.90%) have never been to the dentist. Homogeneous groups showed differences in the time since the last visit to the dentist for adults. The analysis of variance by the F statistic rejected the hypothesis that the variances are equal for the variables related to the time since the last visit to the dentist. It was possible to identify the inequalities in the access to oral healthcare services for adults and locate spatially municipalities whose subjects take longer to visit the dentist.


Direct electrochemistry and electrocatalysis of hemoglobin in nafion/carbon nanochip film on glassy carbon electrode.
The use of heat treated carbon nanofibers, known as carbon nanochips (CNCs) for the studies of the direct electrochemistry and electrocatalytic properties of heme proteins, is demonstrated. A glassy carbon electrode (GCE) was modified with CNCs, and hemoglobin (Hb) was immobilized on the modified electrode surface by casting a film of Hb. Nafion was employed to fix the CNCs and Hb tightly on the surface of the GCE. The modified electrode was characterized by scanning electron microscopy. Ultraviolet-visible and Fourier transform infrared spectroscopy showed that Hb immobilized in the CNC film remained in its native structure. Electrochemical impedance spectroscopy and cyclic voltammetry (CV) were employed for electrochemical studies. The results showed that the presence of CNCs in the film can greatly enhance the electrochemical response of Hb. A pair of well-defined reversible CV peaks was observed, and the formal potential of the heme Fe(III)/Fe(II) redox couple was found to be -253 mV [vs Ag/AgCl (saturated KCl)]. The apparent heterogeneous electron-transfer rate constant (k(s)) was estimated as 2.54 s(-1). The modified electrode showed excellent electrocatalytic behavior to hydrogen peroxide (H(2)O(2)), trichloroacetic acid, and sodium nitrite. H(2)O(2) had a linear current response from 0.5 to 30 microM (R(2) = 0.9997; n = 5) with a detection limit of 0.05 microM when the signal-to-noise ratio was 3 and the apparent Michaelis-Menten constant (K(m)(app)) was 21.55 microM. These values suggest that CNCs are the best matrix described so far for the development of biosensors, far superior to untreated carbon nanofibers. The direct immobilization of proteins onto the surface of CNCs is shown to be a highly efficient method for the development of a new class of very sensitive, stable, and reproducible electrochemical biosensors.