Fabrication and characterization of self-folding thermoplastic sheets using unbalanced thermal shrinkage.
Self-folding or micro-origami technologies are actively investigated as a novel manufacturing process to fabricate three-dimensional macro/micro-structures. In this paper, we present a simple process to produce a self-folding structure with a biaxially oriented polystyrene sheet (BOPS) or Shrinky Dinks. A BOPS sheet is known to shrink to one-third of its original size in plane, when it is heated above 160 °C. A grid pattern is engraved on one side of the BOPS film with a laser engraver to decrease the thermal shrinkage of the engraved side. The thermal shrinkage of the non-engraved side remains the same and this unbalanced thermal shrinkage causes folding of the structure as the structure shrinks at high temperature. We investigated the self-folding mechanism and characterized how the grid geometry, the grid size, and the power of the laser engraver affect the bending curvature. The developed fabrication process to locally modulate thermomechanical properties of the material by engraving the grid pattern and the demonstrated design methodology to harness the unbalanced thermal shrinkage can be applied to develop complicated self-folding macro/micro structures.


Recent advances in pneumococcal vaccination of children.
Pneumococcal disease is a major cause of childhood morbidity and mortality worldwide. However, there is a lack of epidemiological data to describe the vaccine-preventable burden of disease. New pneumococcal conjugate vaccines offer hope of preventing infant pneumococcal disease. The efficacy of the 7- and 9-valent pneumococcal conjugate vaccine (PCV) against invasive vaccine-type pneumococcal disease in young children is between 77 and 97%. The PCV vaccine efficacy against radiological pneumonia in HIV-negative infants for the 7- or 9-valent PCV is 23-30%. The vaccine efficacy in HIV-positive infants is lower--65% against invasive vaccine-type pneumococcal disease and no significant efficacy against radiological pneumonia. The 7-valent PCV showed modest efficacy against acute otitis media (7%) but seems to be more effective in preventing recurrent or severe disease. The high cost of these new vaccines is a barrier to their widespread introduction. The development of other pneumococcal vaccine candidates with wider serotype coverage should be encouraged. These vaccines should be affordable for all countries, particularly those with the highest burden of disease. In addition, other vaccination strategies such as maternal and neonatal immunisation and combinations of fewer doses of the PCV combined with an early dose of the cheaper pneumococcal polysaccharide vaccine need to be assessed further.


Concomitant changes in cross-sectional area and water content in skeletal muscle after resistance exercise.
This study investigated how one bout (1EX) and three bouts (3EX) of strenuous resistance exercise affected the cross-sectional area (CSA) and water content (WC) of the quadriceps muscle and patella tendon (PT), 4 h and 52 h after the last exercise bout. Ten healthy untrained male subjects performed 1EX with one leg and 3EX with the other leg. CSA and WC were measured with magnetic resonance imaging 10, 20 and 30 cm proximal to the tibia plateau (TP) for the muscle, and at the proximal, central and distal site for the PT prior to exercise, and 4 h and 52 h after the last exercise bout. Ten centimeter above the TP, muscle CSA was significantly increased at 4 h (1EX: 13 ± 5%; 3EX: 13 ± 4%) and 52 h (1EX: 16 ± 5%; 3EX: 16 ± 5%) compared with baseline. Muscle WC was significantly increased at 4 h (1EX: 7 ± 1%; 3EX: 6 ± 2%) and 52 h (1EX: 8 ± 2%; 3EX: 8 ± 3%) compared to baseline. PT central CSA was significantly reduced at 52 h (3EX: 14 ± 2%) compared with baseline and (3EX: 13 ± 1%) compared with 4 h. Present data demonstrate that strenuous resistance exercise results in an acute increase in muscle WC and underlines the importance of ensuring sufficient time between the last exercise bout and the determination of anatomical dimensions in muscles.


Effect of pulse depletion in a Brillouin optical time-domain analysis system.
Energy transfer between the interacting waves in a distributed Brillouin sensor can result in a distorted measurement of the local Brillouin gain spectrum, leading to systematic errors. It is demonstrated that this depletion effect can be precisely modelled. This has been validated by experimental tests in an excellent quantitative agreement. Strict guidelines can be enunciated from the model to make the impact of depletion negligible, for any type and any length of fiber.


Electron transfer reactions of glucose oxidase at Au111 electrodes modified with phenothiazine derivatives.
The catalytic reaction of glucose oxidase (GOx) mediated by 3-(10-phenothiazyl)propionic acid (PT-PA) and phenothiazine-labeled poly(ethylene oxide) (PT-PEO1000) that are covalently bonded to Au(111) electrodes has been investigated. The PT-PA and PT-PEO1000 are reacted with 2-aminoethanethiol (AET), followed by the formation of a self-assembled monolayer (SAM) onto the Au surface. The PT group immobilized on the SAM of AET acts as an effective mediator for the electron transfer (ET) between the electrode and the FAD center of freely diffusing GOx in solution. The ET rate constant estimated from the catalytic current using a newly derived equation is larger by 1 order of magnitude for the PT-PA-modified system (1.1 x 10(5) dm(3) mol(-1) s(-1)) than for the PT-PEO1000 system (1.4 x 10(4) dm(3) mol(-1) s(-1)). The order of the magnitude of the ET rate constant clearly contrasts with the GOx hybrid systems that we previously investigated (Anal. Chem. 2003, 75, 910-917), in which the presence of the PEO spacer enhances the ET reaction rate. The reduction in the apparent PT concentration at the electrode interface due to the high mobility of the PEO chain, leading to low efficiency in the formation of an enzyme-mediator complex, is a possible reason for the lower mediation ability of PT-PEO1000 than that of PT-PA for the ET between the FAD group and PT(+) immobilized on the electrode. Inhibition of the penetration of GOx molecules into the monolayer and of the accessibility of some part of PT groups to GOx molecules could also be reasons for the lower mediation ability of PT-PEO1000 thickly modified on the electrode.


THz coherent lensless imaging.
Imaging with THz radiation has proved an important tool for both fundamental science and industrial use. Here we review a class of THz imaging implementations, named coherent lensless imaging, that reconstruct the coherent response of arbitrary samples with a minimized experimental setup based only on a coherent source and a camera. After discussing the appropriate sources and detectors to perform them, we detail the fundamental principles and implementations of THz digital holography and phase retrieval. These techniques owe a lot to imaging with different wavelengths, yet innovative concepts are also being developed in the THz range and are ready to be applied in other spectral ranges. This makes our review useful for both the THz and imaging communities, and we hope it will foster their interaction.


Persistent domestic circulation of African swine fever virus in Tanzania, 2015-2017.
African swine fever (ASF) is a highly fatal viral hemorrhagic disease of domestic pigs that threatens livelihoods and food security. In Africa, ASF virus (ASFV) circulates in sylvatic (transmission between warthogs and soft argasid ticks) and domestic (transmission between domestic pigs) cycles, with outbreaks resulting from ASFV spill-over from sylvatic cycle. A number of outbreaks were reported in different parts of Tanzania between 2015 and 2017. The present study investigated ASFV transmission patterns through viral DNA sequencing and phylogenetic analysis. A total of 3120 tissue samples were collected from 2396 domestic pigs during outbreaks at different locations in Tanzania between 2015 and 2017. Partial sequencing of the B646L (p72) gene was conducted for diagnostic confirmation and molecular characterization of ASFV. Phylogenetic analysis to study the relatedness of current ASFV with those that caused previous outbreaks in Tanzania and representatives of all known 24 ASFV was performed using the Maximum Composite Likelihood model with 1000 bootstrap replications in MEGA 6.0. ASFV was confirmed to cause disease in sampled domestic pigs. ASFV genotypes II, IX, and X were detected from reported outbreaks in 2015-2017. The current ASFV isolates were similar to those recently documented in the previous studies in Tanzania. The similarities of these isolates suggests for continuous circulation of ASFV with virus maintenance within the domestic pigs. Genetic analysis confirmed the circulation of ASFV genotypes II, IX, and X by partial B646L (p72) gene sequencing. The similarities of current isolates to previously isolated Tanzanian isolates and pattern of disease spread suggest for continuous circulation of ASF with virus' maintenance in the domestic pigs. Although certain viral genotypes seem to be geographically restricted into certain zones within Tanzania, genotype II seems to expand its geographical range northwards with the likelihood of spreading to other states of the East African Community. The spread of ASFV is due to breach of quarantine and transportation of infected pigs via major highways. Appropriate control measures including zoosanitary measures and quarantine enforcement are recommended to prevent ASF domestic circulation in Tanzania.


Proliferation of ASC-derived endothelial cells in a 3D electrospun mesh: impact of bone-biomimetic nanocomposite and co-culture with ASC-derived osteoblasts.
Fractures with a critical size bone defect are associated with high rates of delayed- and non-union. The treatment of such complications remains a serious issue in orthopaedic surgery. Adipose derived stem cells (ASCs) combined with biomimetic materials can potentially be used to increase fracture healing. Nevertheless, a number of requirements have to be fulfilled; in particular, the insufficient vascularisation of the bone constructs. Here, the objectives were to study the impact of ASC-derived osteoblasts on ASC-derived endothelial cells in a 3D co-culture and the effect of 40wt% of amorphous calcium phosphate nanoparticles on the proliferation and differentiation of ASC-derived endothelial cells when present in PLGA. Five primary ASC lines were differentiated towards osteoblasts (OBs) and endothelial cells (ECs) and two of them were chosen based on quantitative PCR results. Either a mono-culture of ASC-derived EC or a co-culture of ASC-derived EC with ASC-derived OB (1:1) was seeded on an electrospun nanocomposite of poly-(lactic-co-glycolic acid) and amorphous calcium phosphate nanoparticles (PLGA/a-CaP; reference: PLGA). The proliferation behaviour was determined histomorphometrically in different zones and the expression of von Willebrand Factor (vWF) was quantified. Independently of the fat source (biologic variability), ASC-derived osteoblasts decelerated the proliferation behaviour of ASC-derived endothelial cells in the co-culture compared to the mono-culture. However, expression of vWF was clearly stronger in the co-culture, indicating further differentiation of the ASC-derived EC into the EC lineage. Moreover, the presence of a-CaP nanoparticles in the scaffold slowed the proliferation behaviour of the co-culture cells, too, going along with a further differentiation of the ASC-derived OB, when compared to pure PLGA scaffolds. This study revealed significant findings for bone tissue-engineering. Co-cultures of ASC-derived EC and ASC-derived OB stimulate each other's further differentiation. A nanocomposite with a-CaP nanoparticles offers higher mechanical stability, bioactivity and osteoconductivity compared to mere PLGA and can easily be seeded with pre-differentiated EC and OB.


2-[N-(2,4-Dimeth-oxy-phen-yl)acetamido]-1,3-thia-zol-4-yl acetate.
The title compound, C15H16N2O5S, is a product of the reaction of 2-(2,4-dimeth-oxy-phenyl-amino)-1,3-thia-zol-4(5H)-one with acetic anhydride. The presence of the acetyl and acet-oxy groups in the mol-ecule indicates that the starting thia-zole exists as a tautomer in the reaction mixture with exocyclic amino and enol moieties. The acetyl group is tilted slightly from the heterocyclic ring plane [dihedral angle = 4.46 (11)°], while the acet-oxy group is almost perpendicular to this ring [dihedral angle = 88.14 (12)°]. An intra-molecular acet-yl-meth-oxy C-H⋯O inter-action is noted. In the crystal, mol-ecules are connected into a three-dimensional architecture by C-H⋯O inter-actions.


Susceptibility of podocytes to palmitic acid is regulated by stearoyl-CoA desaturases 1 and 2.
Type 2 diabetes mellitus is characterized by dyslipidemia with elevated free fatty acids (FFAs). Loss of podocytes is a hallmark of diabetic nephropathy, and podocytes are highly susceptible to saturated FFAs but not to protective, monounsaturated FFAs. We report that patients with diabetic nephropathy develop alterations in glomerular gene expression of enzymes involved in fatty acid metabolism, including induction of stearoyl-CoA desaturase (SCD)-1, which converts saturated to monounsaturated FFAs. By IHC of human renal biopsy specimens, glomerular SCD-1 induction was observed in podocytes of patients with diabetic nephropathy. Functionally, the liver X receptor agonists TO901317 and GW3965, two known inducers of SCD, increased Scd-1 and Scd-2 expression in cultured podocytes and reduced palmitic acid-induced cell death. Similarly, overexpression of Scd-1 attenuated palmitic acid-induced cell death. The protective effect of TO901317 was associated with a reduction of endoplasmic reticulum stress. It was lost after gene silencing of Scd-1/-2, thereby confirming that the protective effect of TO901317 is mediated by Scd-1/-2. TO901317 also shifted palmitic acid-derived FFAs into biologically inactive triglycerides. In summary, SCD-1 up-regulation in diabetic nephropathy may be part of a protective mechanism against saturated FFA-derived toxic metabolites that drive endoplasmic reticulum stress and podocyte death.


Glucose tolerance test periodicity as a descriptor of glucose tolerance abnormality.
The periodicity of the standard 100-g glucose tolerance test (GTT) was examined in a prospective study of 194 pregnant patients to determine how well gestational diabetes could be identified. A simplified formula, the GTT periodicity, was used to estimate the time for the GTT curve to return to the fasting level. One hundred one study subjects had all normal glucose values by the National Diabetes Data Group criteria (0-abnormal group), 47 had one value greater than normal (1-abnormal group), and 46 had more than one value abnormal or gestational diabetes. The 0-abnormal patients had a significantly shorter GTT periodicity than did 1-abnormal or gestational diabetic mothers (3.6 versus 4.8 versus 6.6 hours, respectively; P less than .04). Calculating the periodicity for the corresponding insulin excursions yielded significantly increasing values in a graduated fashion for each group (5.2 versus 6.9 versus 9.6 hours, respectively; P less than .05). Examination of the oscillation of the GTT curve about the fasting level allows a physiologic description of normal and abnormal glucose responses in pregnancy. Furthermore, our findings suggest that glucose and insulin periodicities are useful predictors of gestational diabetes in patients with positive screening.


Effect of human rotavirus vaccine on severe diarrhea in African infants.
Rotavirus is the most common cause of severe gastroenteritis among young children worldwide. Data are needed to assess the efficacy of the rotavirus vaccine in African children. We conducted a randomized, placebo-controlled, multicenter trial in South Africa (3166 infants; 64.1% of the total) and Malawi (1773 infants; 35.9% of the total) to evaluate the efficacy of a live, oral rotavirus vaccine in preventing severe rotavirus gastroenteritis. Healthy infants were randomly assigned in a 1:1:1 ratio to receive two doses of vaccine (in addition to one dose of placebo) or three doses of vaccine - the pooled vaccine group - or three doses of placebo at 6, 10, and 14 weeks of age. Episodes of gastroenteritis caused by wild-type rotavirus during the first year of life were assessed through active follow-up surveillance and were graded with the use of the Vesikari scale. A total of 4939 infants were enrolled and randomly assigned to one of the three groups; 1647 infants received two doses of the vaccine, 1651 infants received three doses of the vaccine, and 1641 received placebo. Of the 4417 infants included in the per-protocol efficacy analysis, severe rotavirus gastroenteritis occurred in 4.9% of the infants in the placebo group and in 1.9% of those in the pooled vaccine group (vaccine efficacy, 61.2%; 95% confidence interval, 44.0 to 73.2). Vaccine efficacy was lower in Malawi than in South Africa (49.4% vs. 76.9%); however, the number of episodes of severe rotavirus gastroenteritis that were prevented was greater in Malawi than in South Africa (6.7 vs. 4.2 cases prevented per 100 infants vaccinated per year). Efficacy against all-cause severe gastroenteritis was 30.2%. At least one serious adverse event was reported in 9.7% of the infants in the pooled vaccine group and in 11.5% of the infants in the placebo group. Human rotavirus vaccine significantly reduced the incidence of severe rotavirus gastroenteritis among African infants during the first year of life. (ClinicalTrials.gov number, NCT00241644.).


Loss of ALS2 function is insufficient to trigger motor neuron degeneration in knock-out mice but predisposes neurons to oxidative stress.
Amyotrophic lateral sclerosis (ALS), the most common motor neuron disease, is caused by a selective loss of motor neurons in the CNS. Mutations in the ALS2 gene have been linked to one form of autosomal recessive juvenile onset ALS (ALS2). To investigate the pathogenic mechanisms of ALS2, we generated ALS2 knock-out (ALS2(-/-)) mice. Although ALS2(-/-) mice lacked obvious developmental abnormalities, they exhibited age-dependent deficits in motor coordination and motor learning. Moreover, ALS2(-/-) mice showed a higher anxiety response in the open-field and elevated plus-maze tasks. Although they failed to recapitulate clinical or neuropathological phenotypes consistent with motor neuron disease by 20 months of age, ALS2(-/-) mice or primary cultured neurons derived from these mice were more susceptible to oxidative stress compared with wild-type controls. These observations suggest that loss of ALS2 function is insufficient to cause major motor deficits or motor neuron degeneration in a mouse model but predisposes neurons to oxidative stress.


Low pH increases the yield of exosome isolation.
Exosomes are the extracellular vesicles secreted by various cells. Exosomes mediate intercellular communication by delivering a variety of molecules between cells. Cancer cell derived exosomes seem to be related with tumor progression and metastasis. Tumor microenvironment is thought to be acidic and this low pH controls exosome physiology, leading to tumor progression. Despite the importance of microenvironmental pH on exosome, most of exosome studies have been performed without regard to pH. Therefore, the difference of exosome stability and yield of isolation by different pH need to be studied. In this research, we investigated the yield of total exosomal protein and RNA after incubation in acidic, neutral and alkaline conditioned medium. Representative exosome markers were investigated by western blot after incubation of exosomes in different pH. As a result, the concentrations of exosomal protein and nucleic acid were significantly increased after incubation in the acidic medium compared with neutral medium. The higher levels of exosome markers including CD9, CD63 and HSP70 were observed after incubation in an acidic environment. On the other hand, no exosomal protein, exosomal RNA and exosome markers have been detected after incubation in an alkaline condition. In summary, our results indicate that the acidic condition is the favorable environment for existence and isolation of exosomes.


Anaphylactic reaction after intramuscular injection of cyanocobalamin (vitamin B12): a case report.
We report a case of anaphylactic reaction to intramuscular injection of cyanocobalamin. This 52-year-old lady was diagnosed as a case of megaloblastic anemia secondary to dietary vitamin B12 deficiency. She had severe anaphylactic reaction after the parenteral administration of cyanocobalamine. Later she received oral vitamin B12 with no adverse effects. The purpose of this case report is to draw attention to the hypersensitive reaction to injectable vitamin B12, which is rarely seen. This could be due to sensitization to the vitamin B12 molecule itself or an IgE mediated reaction. We concluded that anaphylactic reaction to vitamin B12 is a rare but serious side effect and it should be kept in mind while the drug is being administered to the patient, especially via the parenteral route.


Correlation between morphology and anisotropic transport properties of diblock copolymers melts.
Molecular simulations of coarse-grained diblock copolymers (DBP) were conducted to study the effect of segregation strength and morphology on transport properties. It was found that in the strong segregation limit (i.e., high χN, where χ is the Flory-Huggins parameter and N is the degree of polymerization), the presence of the DBP interfaces imposes topological constraints similar to those of entanglements as manifested in the rheological signature of the polymer (i.e., a plateau modulus). Furthermore, compared to the behavior of isotropic melts, the crossover from Rouse to reptation scaling of the self-diffusion coefficient (D) parallel to the DBP interface takes place at a smaller N, an effect that depends on temperature and is more pronounced in the Lamellae morphology than in the hexagonal cylinder morphology. Additionally, it is shown that for an entangled melt (i.e., N ≫ Ne where Ne is the entanglement length) block retraction is instrumental for chains to diffuse parallel to the interface of lamellar layers. Lastly, it is found that the anisotropic viscosity of different morphologies is mostly affected by the orientation of the chains relative to the shear flow direction, exhibiting reduced values when chains align in the neutral or flow directions.


Differences in the prevalence of colorectal polyps in patients undergoing endoscopic removal of gastric adenoma or early gastric cancer and in healthy individuals.
We compared the prevalence of adenomatous and cancerous colon polyps in patients who underwent endoscopic removal of gastric neoplasms and in healthy controls. This retrospective study reviewed the medical records of 186 patients with gastric neoplasms and 186 healthy subjects from January 2002 to October 2008. The gastric neoplasm group was comprised of patients undergoing endoscopic removal of gastric adenomas or early gastric cancers and serial fiberoptic colonoscopy (FCS) for checkups. The control group was comprised of subjects undergoing fiberoptic esophagogastroduodenoscopy (FEGD) and FCS for general checkup and was matched for age and sex with the gastric neoplasm group. Advanced colonic neoplasm was defined by any of the following: (1) the presence of three or more polyps; (2) polyp size at least 1.0 cm; (3) high-grade dysplasia or adenocarcinoma confirmed by histopathologic examination. Of the 372 persons, colorectal polyps were detected in 124 (33.3 %), advanced colonic neoplasms in 44 (11.8 %), and adenocarcinomas in 10 (2.7 %). The overall prevalence of adenomatous or cancerous polyps ("all polyps") and the prevalence of advanced colonic neoplasms were significantly higher in the gastric neoplasm group than in the control group (all polyps: 40.9 % in the gastric neoplasm group vs. 25.8 % in the control group, P = 0.002; advanced colonic neoplasms: 15.6 % vs. 8.1 %, P = 0.025). The risk factors for all polyps were age, male sex, diabetes mellitus, and being assigned to the gastric neoplasm group, and those for advanced colonic neoplasms were age and being assigned to the gastric neoplasm group. Confining the analysis to the gastric neoplasm group, the risk factors for all polyps were identical with those for the total group; however, those for advanced colonic neoplasm were different (age vs. diabetes and hypertriglyceridemia). Endoscopists should consider performing routine FCS in patients undergoing endoscopic removal of gastric neoplasms.


Renal oxidative stress, oxygenation, and hypertension.
Hypertension is closely associated with progressive kidney dysfunction, manifested as glomerulosclerosis, interstitial fibrosis, proteinuria, and eventually declining glomerular filtration. The postulated mechanism for development of glomerulosclerosis is barotrauma caused by increased capillary pressure, but the reason for development of interstitial fibrosis and the subsequently reduced kidney function is less clear. However, it has been hypothesized that tissue hypoxia induces fibrogenesis and progressive renal failure. This is very interesting, since recent reports highlight several different mechanisms resulting in altered oxygen handling and availability in the hypertensive kidney. Such mechanisms include decreased renal blood flow due to increased vascular tone induced by ANG II that limits oxygen delivery and increases oxidative stress, resulting in increased mitochondrial oxygen usage, increased oxygen usage for tubular electrolyte transport, and shunting of oxygen from arterial to venous blood in preglomerular vessels. It has been shown in several studies that interventions to prevent oxidative stress and to restore kidney tissue oxygenation prevent progression of kidney dysfunction. Furthermore, inhibition of ANG II activity, by either blocking ANG II type 1 receptors or angiotensin-converting enzyme, or by preventing oxidative stress by administration of antioxidants also results in improved blood pressure control. Therefore, it seems likely that tissue hypoxia in the hypertensive kidney contributes to progression of kidney damage, and perhaps also persistence the high blood pressure.


Aggregation-induced emission and the working mechanism of 1-benzoyl and 1-benzyl pyrene derivatives.
Over the past decade, research studies on solid-state luminescent materials featuring aggregation-induced emission (AIE) have achieved great success. It has been proved that lots of planar ACQ (aggregation-caused quenching) chromophores can be converted to AIE luminogens (AIEgens) by combining with an AIE-active unit such as tetraphenylethene (TPE). In this work, we present a new method to create AIE luminogens just by introducing benzoyl or benzyl to a planar chromophore, pyrene. The generated 1-benzoyl and 1-benzyl pyrene derivatives exhibit weak emission when molecularly dissolved in good solvents but strong emission from pyrene dimers when aggregated in poor solvent or the solid state. Their crystal structure analysis and theoretical calculations are performed to depict the working mechanism of these new AIEgens. The results show that the structural rigidification of these 1-benzoyl pyrene derivatives is the major cause for their AIE effect. This new AIE system along with a clear working mechanism will contribute to the development of AIE-related functional materials and theories.


DEMONSTRATION OF MOUSE ISOANTIGENS AT THE CELLULAR LEVEL BY THE FLUORESCENT ANTIBODY TECHNIQUE.
The fluorescent antibody technique has been applied for the demonstration of mouse isoantigens at the cellular level. Specific reactions were obtained by the indirect or "sandwich" technique with a variety of living normal and neoplastic cells. Isoantigens of the H-2 system and of other systems could be demonstrated as well and appeared to be localized at the cell membrane. As far as the H-2 system was concerned, the membrane localization could be confirmed on histological sections. Different types of non-specific staining reactions have been identified and described. Pinocytosis and cell injury led to such reactions that were morphologically distinguishable from the specific "ring" reaction and as far as pinocytosis is concerned, could be easily avoided by reducing the incubation time. In addition, a non-specific staining reaction morphologically indistinguishable from the specific "ring" reaction could be seen in a small proportion of bone marrow and lymph node cells but in no other cell type studied. The possible nature of this reaction is discussed.


Cell replacement therapies to promote remyelination in a viral model of demyelination.
Persistent infection of the central nervous system (CNS) of mice with the neuroadapted JHM strain of mouse hepatitis (MHV) is characterized by ongoing demyelination mediated by inflammatory T cells and macrophages that is similar both clinically and histologically with the human demyelinating disease multiple sclerosis (MS). Although extensive demyelination occurs in mice persistently infected with MHV there is only limited remyelination. Therefore, the MHV model of demyelination is a relevant model for studying disease and evaluating therapeutic approaches to protect cells of the oligodendrocyte lineage and promote remyelination. This concept is further highlighted as the etiology of MS remains enigmatic, but viruses have long been considered as potential triggering agents in initiating and/or maintaining MS symptoms. As such, understanding mechanisms associated with promoting repair within the CNS in the context of a persistent viral infection is critical given the possible viral etiology of MS. This review focuses on recent studies using either mouse neural stem cells (NSCs) or human oligodendrocyte progenitor cells (OPCs) derived from human embryonic stem cell (hESC) to promote remyelination in mice persistently infected with MHV. In addition, the potential role for chemokines in positional migration of transplanted cells is addressed.


Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion.
Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging techniques require propagation of light through glass, but on glass macrophage fusion is a rare event. This protocol presents the fabrication of several optical-quality glass surfaces where adsorption of compounds containing long-chain hydrocarbons transforms glass into a fusogenic surface. First, preparation of clean glass surfaces as starting material for surface modification is described. Second, a method is provided for the adsorption of compounds containing long-chain hydrocarbons to convert non-fusogenic glass into a fusogenic substrate. Third, this protocol describes fabrication of surface micropatterns that promote a high degree of spatiotemporal control over MGC formation. Finally, fabricating glass bottom dishes is described. Examples of use of this in vitro cell system as a model to study macrophage fusion and MGC formation are shown.


Heterogeneity in search strategies among Cochrane acupuncture reviews: is there room for improvement?
Given the international focus and rigorous literature searches employed in Cochrane systematic reviews, this study was undertaken to evaluate strategies employed in Cochrane reviews and protocols assessing acupuncture as a primary or secondary intervention. The Cochrane Collaboration of systematic reviews was searched in February 2009 for all reviews and protocols including information on acupuncture. Information was abstracted from all retrieved articles on review status, type and number of English and Chinese language databases searched, participation of at least one Chinese speaking author and language restriction. Frequencies were calculated and bivariate analyses were performed stratifying on interventions of interest to assess differences in search strategy techniques, language restrictions and results. The search retrieved 68 titles, including 48 completed reviews, 17 protocols and three previously withdrawn titles. Acupuncture was the primary intervention of interest in 44/65 (67.7%) of the retrieved reviews and protocols. While all articles searched at least one English language database, only 26/65 (40.0%) articles searched Chinese language databases. Significantly more articles where acupuncture was the primary intervention of interest searched Chinese language databases (53% vs 9%, p<0.01). Inconclusive findings as to the effectiveness of acupuncture were found in 28/48 (58.3%) of all completed reviews; this type of finding was more common in reviews which did not search any Chinese language databases. It is important for reviews assessing the effectiveness of acupuncture to search Chinese language databases. The Cochrane Collaboration should develop specific criteria for Chinese language search strategies to ensure the continued publication of high-quality reviews.


Application of permeability-limited physiologically-based pharmacokinetic models: part I-digoxin pharmacokinetics incorporating P-glycoprotein-mediated efflux.
A prerequisite for the prediction of the magnitude of P-glycoprotein (P-gp)-mediated drug-drug interactions between digoxin and P-gp inhibitors (e.g. verapamil and its metabolite norverapamil) or P-gp inducers (e.g. rifampicin) is a predictive pharmacokinetic model for digoxin itself. Thus, relevant in vitro metabolic, transporter and inhibitory data incorporated into permeability-limited models, such as the "advanced dissolution, absorption and metabolism" (ADAM) module and the permeability-limited liver (PerL) module, integrated with a mechanistic physiologically-based pharmacokinetic (PBPK) model such as that of the Simcyp Simulator (version 12.2) are necessary. Simulated concentration-time profiles of digoxin generated using the developed model were consistent with observed data across 31 independent studies [13 intravenous single dose (SD), 12 per oral SD and six multiple dose studies]. The fact that predicted tmax (time of maximum plasma concentration observed) and Cmax (maximum plasma concentration observed) of oral digoxin were similar to observed values indicated that the relative contributions of permeation and P-gp-mediated efflux in the model were appropriate. There was no indication of departure from dose proportionality over the dose range studied (0.25-1.5 mg). All dose normalised area under the plasma concentration-time curve profiles (AUCs) for the 0.25, 0.5, 0.75 and 1 mg doses resembled each other. Thus, PBPK modelling in conjunction with mechanistic absorption and distribution models and reliable in vitro transporter data can be used to assess the impact of dose on P-gp-mediated efflux (or otherwise).


A novel immunoregulatory role for NK-cell cytotoxicity in protection from HLH-like immunopathology in mice.
The impairment of cytotoxic activity of lymphocytes disturbs immune surveillance and leads to the development of hemophagocytic lymphohistiocytic syndrome (HLH). Although cytotoxic T lymphocyte (CTL) control of HLH development is well documented, the role for natural killer (NK)-cell effector functions in the pathogenesis of this immune disorder remains unclear. In this study, we specifically targeted a defect in cytotoxicity to either CTL or NK cells in mice so as to dissect the contribution of these lymphocyte subsets to HLH-like disease severity after lymphocytic choriomeningitis virus (LCMV) infection. We found that NK-cell cytotoxicity was sufficient to protect mice from the fatal outcome that characterizes HLH-like disease and was also sufficient to reduce HLH-like manifestations. Mechanistically, NK-cell cytotoxicity reduced tissue infiltration by inflammatory macrophages and downmodulated LCMV-specific T-cell responses by limiting hyperactivation of CTL. Interestingly, the critical protective effect of NK cells on HLH was independent of interferon-γ secretion and changes in viral load. Therefore our findings identify a crucial role of NK-cell cytotoxicity in limiting HLH-like immunopathology, highlighting the important role of NK cytotoxic activity in immune homeostasis.


Metabolite releasing polymers control dendritic cell function by modulating their energy metabolism.
Metabolites control immune cell functions, and delivery of these metabolites in a sustained manner may be able to modulate function of the immune cells. In this study, alpha-ketoglutarate (aKG) and diol based polymeric-microparticles (termed paKG MPs) were synthesized to provide sustained release of aKG and promote an immunosuppressive cellular phenotype. Notably, after association with dendritic cells (DCs), paKG MPs modulated the intracellular metabolic-profile/pathways, and decreased glycolysis and mitochondrial respiration in vitro. These metabolic changes resulted in modulation of MHC-II, CD86 expression in DCs, and altered the frequency of regulatory T cells (Tregs), and T-helper type-1/2/17 cells in vitro. This unique strategy of intracellular delivery of key-metabolites in a sustained manner provides a new direction in immunometabolism field-based immunotherapy with potential applications in different diseases associated with immune disorders.


Synthesis and Biological Evaluation of Macrocyclized Betulin Derivatives as a Novel Class of Anti-HIV-1 Maturation Inhibitors.
A macrocycle provides diverse functionality and stereochemical complexity in a conformationally preorganized ring structure, and it occupies a unique chemical space in drug discovery. However, the synthetic challenge to access this structural class is high and hinders the exploration of macrocycles. In this study, efficient synthetic routes to macrocyclized betulin derivatives have been established. The macrocycle containing compounds showed equal potency compared to bevirimat in multiple HIV-1 antiviral assays. The synthesis and biological evaluation of this novel series of HIV-1 maturation inhibitors will be discussed.


Emodin suppresses angiogenesis and metastasis in anaplastic thyroid cancer by affecting TRAF6‑mediated pathways in vivo and in vitro.
Emodin has been recognized to be an anti‑cancer agent against a number of types of human cancer. It was demonstrated that TNF receptor‑associated factor 6 (TRAF6) was correlated with cancer angiogenesis and metastasis. The present study confirmed the association between TRAF6 and the angiogenesis/metastasis of anaplastic thyroid cancer (ATC). The anti‑angiogenesis and metastatic effects of emodin, in addition to its molecular mechanisms in ATC, were investigated. A total of two ATC cell lines, namely 8505c and SW1736, were studied. ATC cells were implanted into nude mice to form xenografts or to establish lung metastasis models. Emodin was used to incubate ATC cells or to treat animals orally. An MTT assay was used to assess cell proliferation. A wound healing assay was employed to evaluate cell migration. ELISA analysis was used to detect the vascular endothelial growth factor (VEGF) content. Western blotting was used to determine the protein expression levels. In the in vivo study, cancer angiogenesis was assessed by micro vascular density measurement. The lung metastatic rate was the criterion for cancer metastasis. The results of the present study demonstrated that the proliferation of ATC was inhibited by emodin. The activation of the TRAF6/hypoxia inducible factor (HIF)‑1α/VEGF and TRAF6/basigin (CD147)/matrix metalloproteinase‑9 (MMP9) pathways were associated with the angiogenesis and metastasis of ATC. In a concentration‑dependent manner, emodin inhibited the TRAF6/HIF‑1α/VEGF and TRAF6/CD147/MMP9 signaling pathways to suppress angiogenesis and metastasis. In conclusion, emodin exerted anti‑angiogenic and anti‑metastatic activities in ATC by affecting TRAF6‑mediated pathways.


Nicotine gum as a substitute for cigarettes: a behavioral economic analysis.
The present experiment attempted to identify a substitute for cigarette smoking in a laboratory analog of the behavioral economics of drug dependence. The interaction between cigarette consumption and nicotine gum consumption was examined with eight human smokers by increasing the price of cigarettes (i.e. the number of responses required to obtain puffs) across sessions, while the price of concurrently available nicotine gum remained constant. In another phase, the price of nicotine gum was increased while the price of concurrently available cigarettes remained constant. To determine whether the presence of concurrently available nicotine gum influenced cigarette consumption, we also examined the effect of increasing the price of cigarettes on cigarette consumption without available nicotine gum. When cigarettes and nicotine gum were concurrently available, increases in the price of cigarettes increased consumption of nicotine gum. When the price of nicotine gum increased while the price of cigarettes remained constant, smokers nearly exclusively consumed cigarettes. The presence or absence of nicotine gum did not affect the relation between cigarette consumption and cigarette price. The results suggest that nicotine gum can maintain operant behavior of smokers in the laboratory and can function, in a behavioral economic sense, as a weak substitute for cigarette puffs. As a result, nicotine gum may be useful in human laboratory studies of the behavioral economics of reinforcer interactions and their role in drug dependence.


Cost and outcome of behavioural activation versus cognitive behaviour therapy for depression (COBRA): study protocol for a randomised controlled trial.
Cognitive behaviour therapy (CBT) is an effective treatment for depression. However, CBT is a complex therapy that requires highly trained and qualified practitioners, and its scalability is therefore limited by the costs of training and employing sufficient therapists to meet demand. Behavioural activation (BA) is a psychological treatment for depression that may be an effective alternative to CBT and, because it is simpler, might also be delivered by less highly trained and specialised mental health workers. COBRA is a two-arm, non-inferiority, patient-level randomised controlled trial, including clinical, economic, and process evaluations comparing CBT delivered by highly trained professional therapists to BA delivered by junior professional or para-professional mental health workers to establish whether the clinical effectiveness of BA is non-inferior to CBT and if BA is cost effective compared to CBT. Four hundred and forty patients with major depressive disorder will be recruited through screening in primary care. We will analyse for non-inferiority in per-protocol and intention-to-treat populations. Our primary outcome will be severity of depression symptoms (Patient Health Questionnaire-9) at 12 months follow-up. Secondary outcomes will be clinically significant change and severity of depression at 18 months, and anxiety (General Anxiety Disorder-7 questionnaire) and health-related quality of life (Short-Form Health Survey-36) at 12 and 18 months. Our economic evaluation will take the United Kingdom National Health Service/Personal Social Services perspective to include costs of the interventions, health and social care services used, plus productivity losses. Cost-effectiveness will explored in terms of quality-adjusted life years using the EuroQol-5D measure of health-related quality of life. The clinical and economic outcomes of this trial will provide the evidence to help policy makers, clinicians and guideline developers decide on the merits of including BA as a first-line treatment of depression. Current Controlled Trials ISRCTN27473954.


nm23 as a prognostic marker in primary cutaneous melanoma: evaluation using tissue microarray in a patient group with long-term follow-up.
The accurate estimation of prognosis in patients with melanoma is of increasing importance with novel adjuvant therapies on the horizon. The current prediction of prognosis employs techniques involving sentinel lymph node biopsy, which carries an associated morbidity and is of little use in patients who develop direct distant metastases or direct in-transit metastases. New strategies or factors are therefore needed to improve the accuracy of determination of prognosis. nm23 is a putative metastasis suppressor; however, conflicting data exist as to its role in melanoma progression and its use as a potential prognostic marker. The purpose of this study was to use the technique of tissue microarray to study a cohort of melanoma patients with long-term follow-up data in order to ascertain its potential use as a prognostic marker. One hundred and twenty patients with primary cutaneous melanoma were included in the tissue microarray and a commercially available immunohistochemical marker for nm23 was used for protein detection. nm23 expression was strongly correlated with Clark's level (P<0.001), Breslow depth (P=0.002) and patient age (P=0.014). nm23 expression was significantly associated with a poor patient outcome (chi2=7.2219, P=0.0072). Further analysis revealed that the intensity of nm23 expression also correlated with patient outcome (chi2=11.3281, P=0.0035). However, on multivariate analysis, nm23 was shown not to be an independent marker of prognosis. The results of this study, when taken with the existing literature, suggest a role for nm23 in melanoma disease progression. However, its use as a prognostic marker in routine practice does not appear to be justified.


Cognition and neuropsychiatry in behavioral variant frontotemporal dementia by disease stage.
To characterize the cognitive and neuropsychiatric symptoms of patients with behavioral variant frontotemporal dementia (bvFTD) over the natural course of the disease. We examined the initial and subsequent neuropsychological test performance and neuropsychiatric symptoms in a large cohort of patients with bvFTD (n = 204) across progressive stages of disease as measured by the Clinical Dementia Rating (CDR). We also compared cognitive and neuropsychiatric impairments of patients with bvFTD to those of an age-matched cohort with Alzheimer disease (AD) dementia (n = 674). At the earliest stage (CDR = 0.5), patients with bvFTD had profound neuropsychiatric disturbances, insensitivity to errors, slower response times, and poor naming, with intact attention span, memory, and facial affect naming. Tests continuing to show progressive, statistically significant stepwise declines after the CDR = 1 stage included free recall, visuoconstruction, set-shifting, error insensitivity, semantic fluency, design fluency, emotion naming, calculations, confrontation naming, syntax comprehension, and verbal agility. At CDR = 0.5, patients with bvFTD significantly outperformed patients with AD in episodic memory and were faster in set-shifting, while scoring quantitatively worse in lexical fluency, emotion naming, and error sensitivity. The overall rate of disease progression in bvFTD was more rapid than in AD. There are distinct patterns of cognitive deficits differentiating the earlier and later disease stages in bvFTD, with the pattern of cognitive decline revealing in greater detail the natural history of the disease. These cognitive symptoms are readily apparent clinical markers of dysfunction in the principal brain networks known to undergo molecular and anatomical changes in bvFTD, thus are important indicators of the evolving pathology in individual patients.


Observation of the developing optical continuum along a nonlinear waveguide.
We describe what is to our knowledge the first nondestructive measurement of the evolution of an optical continuum as a function of distance along a nonlinear waveguide. Spectral mapping is achieved on a subwavelength scale by utilizing near-field microscopy to probe the waveguide's evanescent field. The measured continuum broadening along the waveguide agrees in general form with predictions of broadening from theoretical calculations, but differs in some important details. Subwavelength resolution measurements are made both along and across the waveguide to reveal spectral variations not seen before by other techniques.


Functional characterization of bacteria isolated from ancient arctic soil exposes diverse resistance mechanisms to modern antibiotics.
Using functional metagenomics to study the resistomes of bacterial communities isolated from different layers of the Canadian high Arctic permafrost, we show that microbial communities harbored diverse resistance mechanisms at least 5,000 years ago. Among bacteria sampled from the ancient layers of a permafrost core, we isolated eight genes conferring clinical levels of resistance against aminoglycoside, β-lactam and tetracycline antibiotics that are naturally produced by microorganisms. Among these resistance genes, four also conferred resistance against amikacin, a modern semi-synthetic antibiotic that does not naturally occur in microorganisms. In bacteria sampled from the overlaying active layer, we isolated ten different genes conferring resistance to all six antibiotics tested in this study, including aminoglycoside, β-lactam and tetracycline variants that are naturally produced by microorganisms as well as semi-synthetic variants produced in the laboratory. On average, we found that resistance genes found in permafrost bacteria conferred lower levels of resistance against clinically relevant antibiotics than resistance genes sampled from the active layer. Our results demonstrate that antibiotic resistance genes were functionally diverse prior to the anthropogenic use of antibiotics, contributing to the evolution of natural reservoirs of resistance genes.


An igf-I gene polymorphism modifies the risk of diabetic retinopathy.
The role of IGF-I in the pathogenesis of diabetic retinopathy is unclear. We studied, prospectively, the relationship between an IGF-I gene polymorphism, retinal vessel diameters, and incident diabetic retinopathy in subjects with impaired glucose tolerance (IGT) or type 2 diabetes. In all 5,505 participants of the population-based Rotterdam Study (775 with IGT, 394 with type 2 diabetes, and 4,336 control subjects), fundus color transparencies were taken at baseline (between 1990 and 1993) and at follow-up (from 1997 to 1999). The wild-type genotype (i.e., carriers of the 192- or 194-bp alleles) was present in 72.7% of the participants, while 27.3% were variant carriers. Variant carriers with IGT or type 2 diabetes appeared to have larger retinal arteriolar and venular diameters at baseline than individuals with the wild-type genotype, but these differences did not reach statistical significance. This trend was especially observed in subjects who developed retinopathy at follow-up. In variant carriers with IGT/diabetes, an increase (odds ratio 1.8 [95% CI 1.0-3.2]; P = 0.04) in the risk of retinopathy was observed compared with participants with the wild-type genotype. In conclusion, our findings suggest that this IGF-I gene polymorphism is associated with an increased risk of diabetic retinopathy.


Paediatric and congenital heart disease in South America: an overview.
Congenital heart diseases are one of the most common structural defects present at birth, with an approximate incidence of 8 per 1000 live births. As most countries in South America have a high birth rate, they are a significant public health concern. This paper provides a brief overview of the burden of congenital heart disease in South America, focusing on its local prevalence, facilities for treatment and outcomes after medical, surgical or catheter intervention for the most common diseases.


Analyzing real-time PCR data by the comparative C(T) method.
Two different methods of presenting quantitative gene expression exist: absolute and relative quantification. Absolute quantification calculates the copy number of the gene usually by relating the PCR signal to a standard curve. Relative gene expression presents the data of the gene of interest relative to some calibrator or internal control gene. A widely used method to present relative gene expression is the comparative C(T) method also referred to as the 2 (-DeltaDeltaC(T)) method. This protocol provides an overview of the comparative C(T) method for quantitative gene expression studies. Also presented here are various examples to present quantitative gene expression data using this method.


A wearable ECG acquisition system with compact planar-fashionable circuit board-based shirt.
A wearable electrocardiogram (ECG) acquisition system implemented with planar-fashionable circuit board (P-FCB)-based shirt is presented. The proposed system removes cumbersome wires from conventional Holter monitor system for convenience. Dry electrodes screen-printed directly on fabric enables long-term monitoring without skin irritation. The ECG monitoring shirt exploits a monitoring chip with a group of electrodes around the body, and both the electrodes and the interconnection are implemented using P-FCB to enhance wearability and to lower production cost. The characteristics of P-FCB electrode are shown, and the prototype hardware is implemented to successfully verify the proposed concept.


Direct genotyping of Toxoplasma gondii from amniotic fluids based on B1 gene polymorphism using minisequencing analysis.
Because some Toxoplasma gondii genotypes may be more virulent in pregnant women, discriminating between them appears valuable. Currently, the main genotyping method is based on single copy microsatellite markers, which limit direct genotyping from amniotic fluids (AFs) to samples with a high parasitic load. We investigated whether the multicopy gene B1 could type the parasite with a higher sensitivity. To estimate the amplifiable DNA present in AFs, we first compared three different PCR assays used for Toxoplasma infection diagnosis: the P30-PCR, targeting the single copy gene P30; the B1-PCR, targeting the repeated B1 gene; and RE-PCR, targeting the repeated element. Of the 1792 AFs analyzed between 2008 and 2011, 73 were RE-PCR positive. Of those, 49 (67.1%) were P30-PCR and B1-PCR positive, and 14 (19.2%) additional AFs were B1-PCR positive only.All 63 BI-positive AFs (France n = 49; overseas n = 14) could be genotyped based on an analysis of eight nucleotide polymorphisms (SNPs) located within the B1 gene. Following high-resolution melting (HRM) analysis, minisequencing was carried out for each of the eight SNPs. DNA from six reference strains was included in the study, and AFs were assigned to one of the three major lineages (Types I, II, and III). In total, 26 genotypes were observed, and the hierarchical clustering distinguished two clades in lineages II (IIa, n = 30 and IIb, n = 4) and III (IIIa n = 23 and IIIb n = 6). There was an overrepresentation of overseas isolates in Clade IIb (4/4, 100%) and Clade IIIa (8/22; 36.4%) (p <0.0001), whereas medical interruption and fetal death were overrepresented in Clade IIb (2/4, 50%) and Clade IIIa (4/23, 17.4%) (p = 0.049). Although the current genotyping system cannot pretend to replace multilocus typing, we clearly show that targeting the multicopy B1 gene yields a genotyping capacity of AFs around 20% better than when single copy targets are used. The present genotyping method also allows clear identification of genotypes of potential higher virulence.


Glucocorticoid-induced effects on pattern formation and epithelial cell differentiation in early embryonic rat lungs.
In this study, we examined the effects of dexamethasone (DEX) on airway branching and subsequent lung maturation. DEX treatment of fetal rat lung explants was initiated during the early pseudoglandular stage of development. Day 14 fetal lung explants were cultured with and without DEX for 4 d. Explants treated with 10 nM or higher concentrations of DEX showed features of both distorted and accelerated maturation. DEX-treated lungs had growth retardation, distorted branching, dilated proximal tubules, and suppressed proliferation of epithelial cells of the distal tubules. Several biochemical and morphologic features of accelerated maturation were also observed: 1) the epithelial cells lining the distal tubules (prospective respiratory airways) were generally cuboidal or flattened; 2) the cuboidal cells often contained lamellar bodies and abundant glycogen; 3) rudimentary septa and large airspace were present; 4) mesenchymal tissue was attenuated and compressed between adjacent epithelial tubules; 5) the distribution of SP-C mRNA in distal tubules was more mature, with individual and clusters of cells expressing SP-C transcripts; and 6) the transcript levels of several genes related to epithelial growth [keratinocyte growth factor (KGF), KGF receptor, and hepatocyte growth factor receptor] and differentiation [surfactant proteins, SP-A, SP-B and SP-C and the Clara cell secretory protein, CC10] were precociously increased. These results show that DEX treatment of the lung during the early pseudoglandular stage accelerates the acquisition of several features of advanced maturation that normally accompany late stages of fetal development. We postulate that KGF mediates at least some effects of DEX on lung maturation and gene expression.


Public health management of perinatal hepatitis B virus.
Infants who are born to hepatitis B surface antigen (HbsAg)-positive women are at high risk for contracting perinatal hepatitis B virus (HBV) infection. When maternal status is known, postexposure prophylaxis (PEP) consisting of a birth dose of (HBV) vaccine and Hepatitis B Immune Globulin may be administered within 12 hr of birth to provide 90% protection. Providers' reporting of maternal HBsAg positivity or perinatal HBV should prompt public health nurses to initiate nurse case management (NCM). NCM is the most successful way to ensure that at-risk infants receive PEP and follow-up serology. Unfortunately, reporting laws vary greatly by state and the Centers for Disease Control and Prevention estimate that perinatal HBV is significantly under-reported nationally. This article discusses public health measures for preventing perinatal HBV and presents a case study that used a novel method to assess the extent of under-reporting. We discuss barriers to public health NCM and the importance of a universal HBV vaccine birth dose to protect undetected and unreported cases. Finally, we suggest implications for public health nursing practice.


Genomic and proteomic analysis reveals a threshold level of MYC required for tumor maintenance.
MYC overexpression has been implicated in the pathogenesis of most types of human cancers. MYC is likely to contribute to tumorigenesis by its effects on global gene expression. Previously, we have shown that the loss of MYC overexpression is sufficient to reverse tumorigenesis. Here, we show that there is a precise threshold level of MYC expression required for maintaining the tumor phenotype, whereupon there is a switch from a gene expression program of proliferation to a state of proliferative arrest and apoptosis. Oligonucleotide microarray analysis and quantitative PCR were used to identify changes in expression in 3,921 genes, of which 2,348 were down-regulated and 1,573 were up-regulated. Critical changes in gene expression occurred at or near the MYC threshold, including genes implicated in the regulation of the G(1)-S and G(2)-M cell cycle checkpoints and death receptor/apoptosis signaling. Using two-dimensional protein analysis followed by mass spectrometry, phospho-flow fluorescence-activated cell sorting, and antibody arrays, we also identified changes at the protein level that contributed to MYC-dependent tumor regression. Proteins involved in mRNA translation decreased below threshold levels of MYC. Thus, at the MYC threshold, there is a loss of its ability to maintain tumorigenesis, with associated shifts in gene and protein expression that reestablish cell cycle checkpoints, halt protein translation, and promote apoptosis.


The Influence of In-Season Training Loads on Injury Risk in Professional Rugby Union.
To explore the association between in-season training-load (TL) measures and injury risk in professional rugby union players. This was a 1-season prospective cohort study of 173 professional rugby union players from 4 English Premiership teams. TL (duration × session-RPE) and time-loss injuries were recorded for all players for all pitch- and gym-based sessions. Generalized estimating equations were used to model the association between in-season TL measures and injury in the subsequent week. Injury risk increased linearly with 1-wk loads and week-to-week changes in loads, with a 2-SD increase in these variables (1245 AU and 1069 AU, respectively) associated with odds ratios of 1.68 (95% CI 1.05-2.68) and 1.58 (95% CI 0.98-2.54). When compared with the reference group (<3684 AU), a significant nonlinear effect was evident for 4-wk cumulative loads, with a likely beneficial reduction in injury risk associated with intermediate loads of 5932-8651 AU (OR 0.55, 95% CI 0.22-1.38) (this range equates to around 4 wk of average in-season TL) and a likely harmful effect evident for higher loads of >8651 AU (OR 1.39, 95% CI 0.98-1.98). Players had an increased risk of injury if they had high 1-wk cumulative loads (1245 AU) or large week-to-week changes in TL (1069 AU). In addition, a U-shaped relationship was observed for 4-wk cumulative loads, with an apparent increase in risk associated with higher loads (>8651 AU). These measures should therefore be monitored to inform injury-risk-reduction strategies.


Improving precise counting of mitotic cells in mantle cell lymphoma using phosphohistone H3 (PHH3) antibody.
Mantle cell lymphoma (MCL) has a highly heterogeneous clinical course ranging from indolent, to aggressive and rapidly progressive disease. Proliferation is a strong predictor for disease outcome. In routine clinical practice, Ki-67 expression is used as a measure of proliferation. However, several studies have documented a high degree of inter-laboratory and inter-observer variation with Ki-67 immunohistochemistry. Phosphorylation of histone H3 occurs specifically during mitosis and hence serves as a specific marker for cells in mitosis. We investigated phosphohistone H3 (PHH3) immunohistochemistry as a proliferation maker in 28 tissue biopsies of MCL and compared the PHH3 results (as evaluated by direct microscopic visualisation and image analysis-aided scoring) with morphological subtyping, mitotic counts and Ki-67 index. We found PHH3-mitotic count was about sixfold higher than H&E-mitotic count (mitoses in 10 high power fields). Furthermore, PHH3-mitotic count in aggressive morphological variants of MCL was significantly higher than in usual MCL. The PHH3-mitotic count showed a strong linear correlation with PHH3-mitotic index (percentage positive cells). We found PHH3 immunohistochemistry, a reliable mitosis-specific marker, in MCL. Performing precise counts and evaluating precise proliferation indices is easier with PHH3 immunohistochemistry. This contrasts with the conventional estimation of Ki-67 percentages by 'eye-balling'.


Hexathioalkyl sumanenes: an electron-donating buckybowl as a building block for supramolecular materials.
Unlike planar aromatic compounds, bowl-shaped sumanene, which has concave and convex faces with different electrostatic potentials, tends to form a one-dimensional columnar assembly without causing slip-stacking in the crystal. Here we report the first successful synthesis of liquid-crystalline (LC) sumanenes, which was brought about by the incorporation of six thioalkyl groups (R = SC6H13 or SC12H25) into the aromatic part of sumanene. In contrast to the case of the mesophase formation of corannulene, which requires the presence of many dendritic side chains, sumanene derivatives with simple alkyl chains can exhibit a remarkably high-order columnar LC mesophase over a wide temperature range. While non-substituted sumanene inherently behaves as an electron acceptor, hexathioalkyl versions, such as hexathiomethyl sumanene, show electron-donating properties, resulting in complexation with C60. Considering its unique shape, electronic properties, and self-assembly behavior, the electron-donating sumanene may represent a new building block for constructing supramolecular materials, both by itself and in combination with fullerene derivatives.


Pain persists in DAS28 rheumatoid arthritis remission but not in ACR/EULAR remission: a longitudinal observational study.
Disease remission has become a feasible goal for most rheumatoid arthritis (RA) patients; however, patient-reported symptoms, such as pain, may persist despite remission. We assessed the prevalence of pain in RA patients in remission according to the Disease Activity Score (DAS28-CRP4) and the American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) remission criteria. Data were analyzed from RA patients in the Brigham Rheumatoid Arthritis Sequential Study with data at baseline and 1 year. DAS28 remission was defined as DAS28-CRP4 <2.6. The ACR/EULAR remission criteria included (a) one or more swollen joints, (b) one or more tender joints, (c) C-reactive protein ≤1 mg/dl, and (d) patient global assessment score ≤1. Pain severity was measured by using the pain score from the Multi-Dimensional Health Assessment Questionnaire (MDHAQ). The associations between baseline clinical predictors and MDHAQ pain at baseline and 1 year were assessed by using multivariable linear regression. Among the 865 patients with data at baseline and 1 year, 157 (18.2%) met DAS28-CRP4 remission criteria at both time points. Thirty-seven (4.3%) met the ACR/EULAR remission criteria at baseline and 1 year. The prevalence of clinically significant pain (MDHAQ pain ≥4) at baseline ranged from 11.9% among patients meeting DAS28-CRP4 remission criteria to none among patients meeting ACR/EULAR remission criteria. Patient global assessment, MDHAQ function, MDHAQ fatigue, MDHAQ sleep, and arthritis self-efficacy were significantly associated with MDHAQ pain in cross-sectional (P ≤ 0.0005) and longitudinal analyses (P ≤ 0.03). Low swollen-joint counts were associated with high MDHAQ pain in longitudinal analyses (P = 0.02) but not cross-sectional analyses. Other measures of inflammatory disease activity and joint damage were not significantly associated with MDHAQ pain at baseline or at 1 year. Clinically significant pain continues among a substantial proportion of patients in DAS28 remission but not among those in ACR/EULAR remission. Among patients in DAS28 remission, patient global assessment, disability, fatigue, sleep problems, and self-efficacy are strongly associated with pain severity at baseline and 1 year, whereas inflammatory disease activity and joint damage are not significantly associated with elevated pain severity at either baseline or 1 year.


Electronically-coupled tungsten-tungsten quadruple bonds: comparisons of electron delocalization in 3,6-dioxypyridazine and oxalate-bridged compounds.
The preparation of the 3,6-dioxypyridazine-bridged tungsten complex, [W(2)(O(2)C(t)Bu)(3)](2)(mu-H(2)C(4)N(2)O(2)), I, is described, along with its single-electron oxidized cation, I(+), formed in the reaction between I and Ag(+)PF(6)(-). Compound I has been structurally characterized as a PPh(3) adduct, and I(+)PF(6)(-) as a THF solvate, by single-crystal X-ray studies. The geometric parameters of these compounds compare well with those calculated for the model compounds [W(2)(O(2)CH)(3)](2)(mu-H(2)C(4)N(2)O(2)) and [W(2)(O(2)CH)(3)](2)(micro-H(2)C(4)N(2)O(2))(+) by density functional theory employing the Gaussian 98 and 03 suite of programs. The calculations indicate that the two W(2) centers are strongly coupled by M(2) delta-to-bridge pi-bonding, and further coupled by direct M(2)...M(2) bonding. Compound I is purple and shows an intense absorption in the visible region due to a metal-to-bridge charge transfer and, with excitation within this absorption, compound I exhibits pronounced resonance Raman bands associated with symmetric vibrations of the bridge and the M(4) unit. The cyclic voltammogram of I in THF, the EPR spectrum of I(+)PF(6) in 2-MeTHF and the electronic absorption spectrum of I(+)PF(6)(-) in THF are consistent with electron delocalization over both W(2) units. These new data are compared with previous data for the molybdenum analogue, related oxalate-bridged compounds and closely related cyclic polyamidato-bridged Mo(4)-containing compounds. It is proposed that, while the electronic coupling occurs principally by an electron-hopping mechanism for oxalate-bridged compounds, hole-hopping contributes significantly in the cases of the amidate bridges and that this is more important for M = Mo than for M = W. Furthermore, for Class III fully delocalized mixed-valence compounds, the magnitude of K(c), determined from electrochemical methods, is not necessarily a measure of the extent of electron delocalization.


Severity detection tool for patients with infectious disease.
Hand foot and mouth disease (HFMD) and tetanus are serious infectious diseases in low- and middle-income countries. Tetanus, in particular, has a high mortality rate and its treatment is resource-demanding. Furthermore, HFMD often affects a large number of infants and young children. As a result, its treatment consumes enormous healthcare resources, especially when outbreaks occur. Autonomic nervous system dysfunction (ANSD) is the main cause of death for both HFMD and tetanus patients. However, early detection of ANSD is a difficult and challenging problem. The authors aim to provide a proof-of-principle to detect the ANSD level automatically by applying machine learning techniques to physiological patient data, such as electrocardiogram waveforms, which can be collected using low-cost wearable sensors. Efficient features are extracted that encode variations in the waveforms in the time and frequency domains. The proposed approach is validated on multiple datasets of HFMD and tetanus patients in Vietnam. Results show that encouraging performance is achieved. Moreover, the proposed features are simple, more generalisable and outperformed the standard heart rate variability analysis. The proposed approach would facilitate both the diagnosis and treatment of infectious diseases in low- and middle-income countries, and thereby improve patient care.


Plasma brain natriuretic peptide levels and blood pressure tracking in the Framingham Heart Study.
Increased brain natriuretic peptide (BNP) expression in the ventricles antedates elevated blood pressure (BP) in experimental studies. We hypothesized that higher plasma BNP levels in nonhypertensive individuals may be associated with a greater likelihood of future BP increase and/or hypertension. We evaluated the relations of plasma BNP to longitudinal BP tracking and incidence of hypertension in 1801 nonhypertensive Framingham Heart Study participants (mean age, 56 years; 57% women) by using gender-specific multivariable logistic regression. Progression of BP stage was defined as an increment of one or more BP categories, as classified by the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC VI). Hypertension was defined as a systolic BP > or =140 or diastolic BP > or =90 mm Hg or use of antihypertensive medications. On follow-up 4 years from baseline, progression of BP category was observed in 36.2% of men and 33.1% of women; hypertension developed in 16.4% of men and 15.5% of women. In multivariable models adjusting for known risk factors, elevated plasma BNP level was associated with increased risk of BP progression in men (odds ratio of 1.15 for trend across categories, P=0.046) but not in women (P=0.82). There were no significant trends of increasing incidence of hypertension across BNP categories in men or women. In our community-based sample, higher plasma BNP levels were associated with increased risk of BP progression in men but not women. Additional investigations are warranted to confirm these findings and elucidate the basis for these gender-related differences.


Kite-string injuries: A case series.
Kites are very popular in India. Over the years, both kite-flying and kite-making skills have evolved. The conventional cotton threads that were used as kite string (manja) have been replaced by much cheaper and stronger Chinese manja, which is based on nonbiodegradable synthetic fibers. It is hard to break and has caused a sudden surge in dangerous kite string-related injuries. There are a lot of injuries usually sustained by kite-flyers, two-wheeler riders, and pedestrians. Very few case reports and case series have shown injuries related to flying a kite, which range from laceration of hand to fatal throat injuries. Secondary impact injuries attributed to kite string (manja) are rarely reported in the medical literature. We present a series of four cases with special emphasis on a patient, who sustained secondary impact injury with fatal outcome. Emergency physician should know that these trivial looking injuries can be associated with significant neck injuries. They can also cause significant secondary impact injuries.


Intrinsic ssDNA annealing activity in the C-terminal region of WRN.
Werner syndrome (WS) is a rare autosomal recessive disorder in humans characterized by premature aging and genetic instability. WS is caused by mutations in the WRN gene, which encodes a member of the RecQ family of DNA helicases. Cellular and biochemical studies suggest that WRN plays roles in DNA replication, DNA repair, telomere maintenance, and homologous recombination and that WRN has multiple enzymatic activities including 3' to 5' exonuclease, 3' to 5' helicase, and ssDNA annealing. The goal of this study was to map and further characterize the ssDNA annealing activity of WRN. Enzymatic studies using truncated forms of WRN identified a C-terminal 79 amino acid region between the RQC and the HRDC domains (aa1072-1150) that is required for ssDNA annealing activity. Deletion of the region reduced or eliminated ssDNA annealing activity of the WRN protein. Furthermore, the activity appears to correlate with DNA binding and oligomerization status of the protein.


The Great Disrupter: Relationship of Alexithymia to Emotion Regulation Processes and Smoking among Pregnant Women.
Background: Many women continue to smoke during pregnancy, despite known risks, often in response to negative affect. Recent scholarship has begun to examine factors that decrease the success of behavioral treatments for smoking cessation in pregnancy, which are the preferred interventions. Alexithymia is one factor that may interfere with smoking cessation interventions. Alexithymia restricts access to emotional information and increases propensity toward maladaptive behaviors, including smoking. However, mechanisms underlying such effects are largely unknown. Objectives: Using data from a longitudinal treatment study, the present research examined difficulties with emotion regulation as a potential mechanism linking alexithymia and smoking. Pregnant smokers (n = 73; mean age = 24.78; SD = 4.50) completed measures related to alexithymia, smoking, emotion regulation, depression, anxiety, and anger at baseline and then again following eight sessions of Cognitive-Behavioral Smoking Cessation Treatment. Results: Nearly 40% of the sample met the criteria for alexithymia. The alexithymia group reported higher depression, anxiety, and anger. They also reported more difficulties with emotion regulation. In a path analysis, baseline alexithymia had a significant positive indirect effect on number of cigarettes smoked at the end of treatment through difficulties with emotion regulation. Conclusions/Importance: Similar to other studies, alexithymia limits the understanding of emotional information necessary for selection and implementation of adaptive coping responses. Our results extend the literature by suggesting that smoking may be an attempt to manage undifferentiated and unpleasant sensations created by alexithymia.


Selection of spectro-temporal patterns in multichannel MEG with support vector machines for schizophrenia classification.
We present a new framework for the diagnosis of schizophrenia based on the spectro-temporal patterns selected by a support vector machine from multichannel magnetoencephalogram (MEG) recordings in a verbal working memory task. In the experimental paradigm, five letters appearing sequentially on a screen were memorized by subjects. The letters constituted a word in one condition and a pronounceable nonword in the other. Power changes were extracted as features in frequency subbands of 248 channel MEG data to form a rich feature dictionary. A support vector machine has been used to select a small subset of features with recursive feature elimination technique (SVM-RFE) and the reduced subset was used for classification. We note that the discrimination between patients and controls in the word condition was higher than in the non-word condition (91.8% vs 83.8%). Furthermore, in the word condition, the most discriminant patterns were extracted in delta (1-4 Hz), theta (4-8Hz) and alpha (12-16 Hz) frequency bands. We note that these features were located around the left frontal, left temporal and occipital areas, respectively. Our results indicate that the proposed approach can quantify discriminative neural patterns associated to a functional task in spatial, spectral and temporal domain. Moreover these features provide interpretable information to the medical expert about physiological basis of the illness and can be effectively used as a biometric marker to recognize schizophrenia in clinical practice.


Versatility of the Anterolateral Thigh Flap: An AKUH experience.
Anterolateral thigh flap is used for reconstruction of various soft tissue defects. We planned the study to evaluate the versatility of the anterolateral thigh flap as it may be used for reconstruction of head and neck, torso and extremities with minimal donor-site morbidity. The retrospective study was conducted at Aga Khan University Hospital, Karachi, and comprised date from October 2012 to December 2015. Of the 75 patients in which anterolateral thigh flap was used for reconstruction, 6(8%) flaps did not survive. The overall flap survival was 69(92%). Anterolateral thigh flap was versatile enough to provide soft tissue coverage to defects of various soft tissue and bulk requirements. Adequate outcomes were achieved pertaining to the reconstruction of the soft tissue defects. Anterolateral thigh flap can be used to reconstruct soft tissue defects almost anywhere and almost of any complexity.


Recognition of nuclear export signals by CRM1 carrying the oncogenic E571K mutation.
The E571K mutation of CRM1 is highly prevalent in some cancers, but its mechanism of tumorigenesis is unclear. Glu571 of CRM1 is located in its nuclear export signal (NES)-binding groove, suggesting that binding of select NESs may be altered. We generated HEK 293 cells with either monoallelic CRM1WT/E571K or biallelic CRM1E571K/E571K using CRISPR/Cas9. We also combined analysis of binding affinities and structures of 27 diverse NESs for wild-type and E571K CRM1 with structure-based bioinformatics. While most NESs bind the two CRM1 similarly, NESs from Mek1, eIF4E-transporter, and RPS2 showed >10-fold affinity differences. These NESs have multiple charged side chains binding close to CRM1 position 571, but this feature alone was not sufficient to predict different binding to CRM1(E571K). Consistent with eIF4E-transporter NES binding weaker to CRM1(E571K), eIF4E-transporter was mislocalized in tumor cells carrying CRM1(E571K). This serves as proof of concept that understanding how CRM1(E571K) affects NES binding provides a platform for identifying cargoes that are mislocalized in cancer upon CRM1 mutation. Finally, we showed that large affinity changes seen with some NES peptides (of Mek1 and RPS2) do not always translate to the full-length cargoes, suggesting limitations with current NES prediction methods. Therefore, comprehensive studies like ours are imperative to identify CRM1 cargoes with real pathogenic potential.


Heavy metal immobilization through phosphate and thermal treatment of dredged sediments.
Disposal of dredged sediments is expensive and poses a major challenge for harbor dredging projects. Therefore beneficial reuse of these sediments as construction material is highly desirable assuming contaminants such as heavy metals are immobilized and organics are mineralized. In this research, the effect of the addition of 2.5% phosphate, followed by thermal treatment at 700 degrees C, was investigated for metal contaminants in dredged sediments. Specifically, Zn speciation was evaluated, using X-ray absorption spectroscopy (XAS), by applying principal component analysis (PCA), target transformation (TT), and linear combination fit (LCF) to identify the main phases and their combination from an array of reference compounds. In dredged sediments, Zn was present as smithsonite (67%) and adsorbed to hydrous manganese oxides (18%) and hydrous iron oxides (15%). Phosphate addition resulted in precipitation of hopeite (22%), while calcination induced formation of spinels, gahnite (44%), and franklinite (34%). Although calcination was previously used to agglomerate phosphate phases by sintering, we found that it formed sparingly soluble Zn phases. Results from the U.S. EPA toxicity characteristic leaching procedure (TCLP) confirmed both phosphate addition and calcination reduced leachability of heavy metals with the combined treatment achieving up to an 89% reduction.


Eliminating malignant contamination from therapeutic human spermatogonial stem cells.
Spermatogonial stem cell (SSC) transplantation has been shown to restore fertility in several species and may have application for treating some cases of male infertility (e.g., secondary to gonadotoxic therapy for cancer). To ensure safety of this fertility preservation strategy, methods are needed to isolate and enrich SSCs from human testis cell suspensions and also remove malignant contamination. We used flow cytometry to characterize cell surface antigen expression on human testicular cells and leukemic cells (MOLT-4 and TF-1a). We demonstrated via FACS that EpCAM is expressed by human spermatogonia but not MOLT-4 cells. In contrast, HLA-ABC and CD49e marked >95% of MOLT-4 cells but were not expressed on human spermatogonia. A multiparameter sort of MOLT-4-contaminated human testicular cell suspensions was performed to isolate EpCAM+/HLA-ABC-/CD49e- (putative spermatogonia) and EpCAM-/HLA-ABC+/CD49e+ (putative MOLT-4) cell fractions. The EpCAM+/HLA-ABC-/CD49e- fraction was enriched for spermatogonial colonizing activity and did not form tumors following human-to-nude mouse xenotransplantation. The EpCAM-/HLA-ABC+/CD49e+ fraction produced tumors following xenotransplantation. This approach could be generalized with slight modification to also remove contaminating TF-1a leukemia cells. Thus, FACS provides a method to isolate and enrich human spermatogonia and remove malignant contamination by exploiting differences in cell surface antigen expression.


Maternal and Newborn Outcomes Following Waterbirth: The Midwives Alliance of North America Statistics Project, 2004 to 2009 Cohort.
Data on the safety of waterbirth in the United States are lacking. We used data from the Midwives Alliance of North America Statistics Project, birth years 2004 to 2009. We compared outcomes of neonates born underwater waterbirth (n = 6534), neonates not born underwater nonwaterbirth (n = 10,290), and neonates whose mothers intended a waterbirth but did not have one intended waterbirth (n = 1573). Neonatal outcomes included a 5-minute Apgar score of less than 7, neonatal hospital transfer, and hospitalization or neonatal intensive care unit (NICU) admission in the first 6 weeks. Maternal outcomes included genital tract trauma, postpartum hospital transfer, and hospitalization or infection (uterine, endometrial, perineal) in the first 6 weeks. We used logistic regression for all analyses, controlling for primiparity. Waterbirth neonates experienced fewer negative outcomes than nonwaterbirth neonates: the adjusted odds ratio (aOR) for hospital transfer was 0.46 (95% confidence interval [CI], 0.32-0.68; P < .001); the aOR for infant hospitalization in the first 6 weeks was 0.75 (95% CI, 0.63-0.88; P < .001); and the aOR for NICU admission was 0.59 (95% CI, 0.46-0.76; P < .001). By comparison, neonates in the intended waterbirth group experienced more negative outcomes than the nonwaterbirth group, although only 5-minute Apgar score was significant (aOR, 2.02; 95% CI, 1.40-2.93; P < 0001). For women, waterbirth (compared to nonwaterbirth) was associated with fewer postpartum transfers (aOR, 0.65; 95% CI, 0.50-0.84; P = .001) and hospitalizations in the first 6 weeks (aOR, 0.72; 95% CI, 0.59-0.87; P < 0.001) but with an increased odds of genital tract trauma (aOR, 1.11; 95% CI, 1.04-1.18; P = .002). Waterbirth was not associated with maternal infection. Women in the intended waterbirth group had increased odds for all maternal outcomes compared to women in the nonwaterbirth group, although only genital tract trauma was significant (aOR, 1.67; 95% CI, 1.49-1.87; P < .001). Waterbirth confers no additional risk to neonates; however, waterbirth may be associated with increased risk of genital tract trauma for women.


Genome-Wide Analysis of Mycoplasma bovirhinis GS01 Reveals Potential Virulence Factors and Phylogenetic Relationships.
Mycoplasma bovirhinis is a significant etiology in bovine pneumonia and mastitis, but our knowledge about the genetic and pathogenic mechanisms of M. bovirhinis is very limited. In this study, we sequenced the complete genome of M. bovirhinis strain GS01 isolated from the nasal swab of pneumonic calves in Gansu, China, and we found that its genome forms a 847,985 bp single circular chromosome with a GC content of 27.57% and with 707 protein-coding genes. The putative virulence determinants of M. bovirhinis were then analyzed. Results showed that three genomic islands and 16 putative virulence genes, including one adhesion gene enolase, seven surface lipoproteins, proteins involved in glycerol metabolism, and cation transporters, might be potential virulence factors. Glycerol and pyruvate metabolic pathways were defective. Comparative analysis revealed remarkable genome variations between GS01 and a recently reported HAZ141_2 strain, and extremely low homology with others mycoplasma species. Phylogenetic analysis demonstrated that M. bovirhinis was most genetically close to M. canis, distant from other bovine Mycoplasma species. Genomic dissection may provide useful information on the pathogenic mechanisms and genetics of M. bovirhinis.


Tacit consent: the Church and birth control in northern Italy.
This article employs novel documentation to examine ways in which the Church's moral rules on contraception were (or were not) communicated to parishioners in a predominantly Catholic context in a period of rapid fertility decline: the diocese of Padua, in the northeastern Italian region of Veneto, during the first half of the twentieth century. The account is based on documents that have until now been overlooked: the moral cases discussed during the periodic meetings among Padua priests in the years 1916–58, and the written answers provided by priests in response to a question asked of them concerning their efforts to combat the limiting of births. This documentation reveals the limited effect on the reproductive behavior of the position of the Catholic Church against birth control.


Rapid depletion of mutant eukaryotic initiation factor 5A at restrictive temperature reveals connections to actin cytoskeleton and cell cycle progression.
Eukaryotic initiation factor 5A (eIF5A) is the only protein in nature that contains hypusine, an unusual amino acid derived from the modification of lysine by spermidine. Two genes, TIF51A and TIF51B, encode eIF5A in the yeast Saccharomyces cerevisiae. In an effort to understand the structure-function relationship of eIF5A, we have generated yeast mutants by introducing plasmid-borne tif51A into a double null strain where both TIF51A and TIF51B have been disrupted. One of the mutants, tsL102A strain (tif51A L102A tif51aDelta tif51bDelta) exhibits a strong temperature-sensitive growth phenotype. At the restrictive temperature, tsL102A strain also exhibits a cell shape change, a lack of volume change in response to temperature increase and becomes more sensitive to ethanol, a hallmark of defects in the PKC/WSC cell wall integrity pathway. In addition, a striking change in actin dynamics and a complete cell cycle arrest at G1 phase occur in tsL102A cells at restrictive temperature. The temperature-sensitivity of tsL102A strain is due to a rapid loss of mutant eIF5A with the half-life reduced from 6 h at permissive temperature to 20 min at restrictive temperature. Phenylmethyl sulfonylfluoride (PMSF), an irreversible inhibitor of serine protease, inhibited the degradation of mutant eIF5A and suppressed the temperature-sensitive growth arrest. Sorbitol, an osmotic stabilizer that complement defects in PKC/WSC pathways, stabilizes the mutant eIF5A and suppresses all the observed temperature-sensitive phenotypes.


Comparison of techniques for handling missing covariate data within prognostic modelling studies: a simulation study.
There is no consensus on the most appropriate approach to handle missing covariate data within prognostic modelling studies. Therefore a simulation study was performed to assess the effects of different missing data techniques on the performance of a prognostic model. Datasets were generated to resemble the skewed distributions seen in a motivating breast cancer example. Multivariate missing data were imposed on four covariates using four different mechanisms; missing completely at random (MCAR), missing at random (MAR), missing not at random (MNAR) and a combination of all three mechanisms. Five amounts of incomplete cases from 5% to 75% were considered. Complete case analysis (CC), single imputation (SI) and five multiple imputation (MI) techniques available within the R statistical software were investigated: a) data augmentation (DA) approach assuming a multivariate normal distribution, b) DA assuming a general location model, c) regression switching imputation, d) regression switching with predictive mean matching (MICE-PMM) and e) flexible additive imputation models. A Cox proportional hazards model was fitted and appropriate estimates for the regression coefficients and model performance measures were obtained. Performing a CC analysis produced unbiased regression estimates, but inflated standard errors, which affected the significance of the covariates in the model with 25% or more missingness. Using SI, underestimated the variability; resulting in poor coverage even with 10% missingness. Of the MI approaches, applying MICE-PMM produced, in general, the least biased estimates and better coverage for the incomplete covariates and better model performance for all mechanisms. However, this MI approach still produced biased regression coefficient estimates for the incomplete skewed continuous covariates when 50% or more cases had missing data imposed with a MCAR, MAR or combined mechanism. When the missingness depended on the incomplete covariates, i.e. MNAR, estimates were biased with more than 10% incomplete cases for all MI approaches. The results from this simulation study suggest that performing MICE-PMM may be the preferred MI approach provided that less than 50% of the cases have missing data and the missing data are not MNAR.


Impact of TIEG1 Deletion on the Passive Mechanical Properties of Fast and Slow Twitch Skeletal Muscles in Female Mice.
As transforming growth factor (TGF)-β inducible early gene-1 is highly expressed in skeletal muscle, the effect of TIEG1 gene deletion on the passive mechanical properties of slow and fast twitch muscle fibers was analyzed. Twenty five muscle fibers were harvested from soleus (Sol) and extensor digitorum longus (EDL) muscles from TIEG1-/- (N = 5) and control (N = 5) mice. Mechanical tests were performed on fibers and the dynamic and static stresses were measured. A viscoelastic Hill model of 3rd order was used to fit the experimental relaxation test data. In parallel, immunohistochemical analyses were performed on three serial transverse sections to detect the myosin isoforms within the slow and fast muscles. The percentage and the mean cross sectional area of each fiber type were calculated. These tests revealed a significant increase in the mechanical stress properties for the TIEG1-/- Sol fibers while a significant decrease appeared for the TIEG1-/- EDL fibers. Hill model tracked the shape of the experimental relaxation curve for both genotypes and both fiber types. Immunohistochemical results showed hypertrophy of all fiber types for TIEG1-/- muscles with an increase in the percentage of glycolytic fibers (IIX, and IIB) and a decrease of oxidative fibers (I, and IIA). This study has provided new insights into the role of TIEG1, known as KLF10, in the functional (SoltypeI: more resistant, EDLtypeIIB: less resistant) and morphological (glycolytic hypertrophy) properties of fast and slow twitch skeletal muscles. Further investigation at the cellular level will better reveal the role of the TIEG1 gene in skeletal muscle tissue.


Type IV pili promote early biofilm formation by Clostridium difficile.
Increasing morbidity and mortality from Clostridium difficile infection (CDI) present an enormous challenge to healthcare systems. Clostridium difficile express type IV pili (T4P), but their function remains unclear. Many chronic and recurrent bacterial infections result from biofilms, surface-associated bacterial communities embedded in an extracellular matrix. CDI may be biofilm mediated; T4P are important for biofilm formation in a number of organisms. We evaluate the role of T4P in C. difficile biofilm formation using RNA sequencing, mutagenesis and complementation of the gene encoding the major pilin pilA1, and microscopy. RNA sequencing demonstrates that, in comparison to other growth phenotypes, C. difficile growing in a biofilm has a distinct RNA expression profile, with significant differences in T4P gene expression. Microscopy of T4P-expressing and T4P-deficient strains suggests that T4P play an important role in early biofilm formation. A non-piliated pilA1 mutant forms an initial biofilm of significantly reduced mass and thickness in comparison to the wild type. Complementation of the pilA1 mutant strain leads to formation of a biofilm which resembles the wild-type biofilm. These findings suggest that T4P play an important role in early biofilm formation. Novel strategies for confronting biofilm infections are emerging; our data suggest that similar strategies should be investigated in CDI.


The Transmembrane Domain of a Bicomponent ABC Transporter Exhibits Channel-Forming Activity.
Pseudomonas aeruginosa is an opportunistic pathogen that expresses two unique forms of lipopolysaccharides (LPSs) on its bacterial surface, the A- and B-bands. The A-band polysaccharides (A-band PSs) are thought to be exported into the periplasm via a bicomponent ATP-binding cassette (ABC) transporter located within the inner membrane. This ABC protein complex consists of the transmembrane (TMD) Wzm and nucleotide-binding (NBD) Wzt domain proteins. Here, we were able to probe ∼1.36 nS-average conductance openings of the Wzm-based protein complex when reconstituted into a lipid membrane buffered by a 200 mM KCl solution, demonstrating the large-conductance, channel-forming ability of the TMDs. In agreement with this finding, transmission electron microscopy (TEM) imaging revealed the ring-shaped structure of the transmembrane Wzm protein complex. As hypothesized, using liposomes, we demonstrated that Wzm interacts with Wzt. Further, the Wzt polypeptide indeed hydrolyzed ATP but exhibited a ∼75% reduction in the ATPase activity when its Walker A domain was deleted. The distribution and average unitary conductance of the TMD Wzm protein complex were altered by the presence of the NBD Wzt protein, confirming the regulatory role of the latter polypeptide. To our knowledge, the large-conductance, channel-like activity of the Wzm protein complex, although often hypothesized, has not previously been demonstrated. These results constitute a platform for future structural, biophysical, and functional explorations of this bicomponent ABC transporter.


Creatine monohydrate increases bone mineral density in young Sprague-Dawley rats.
Creatine kinase, found in osteoblasts, is an enzyme that is upregulated in response to interventions that enhance bone mass accretion. Creatine monohydrate supplementation can increase fat-free mass in young healthy men and women and can reduce markers of bone breakdown in boys with Duchenne muscular dystrophy. The objective of this study was to determine the influence of supplementation with creatine monohydrate on bone structure and function in growing rats, to establish a therapeutic model. Creatine monohydrate (2% w.w.) (CR; N = 16) or standard rat chow (CON; N = 16) was fed to Sprague-Dawley rats beginning at 5 wk of age, for 8 wk. Bone mineral density (BMD) and content (BMC) were assessed using dual-energy x-ray absorptiometry at the beginning and end of the protocol. The rats were sacrificed, and one femur was removed for the determination of mechanical properties. The CR-treated rats showed greater lumbar BMD and femoral bending load at failure compared with the CON rats (P < 0.05). Together, these data suggest that creatine monohydrate potentially has a beneficial influence on bone function and structure; further investigation is warranted into its effect on bone functional properties and its effects in disorders associated with bone loss.