Omics-based approaches to methionine side chain elongation in Arabidopsis: characterization of the genes encoding methylthioalkylmalate isomerase and methylthioalkylmalate dehydrogenase.
Glucosinolates (GSLs) are secondary metabolites in Brassicaceae plants synthesized from amino acids. Methionine-derived GSLs (Met-GSLs) with diverse side chains of various lengths are the major GSLs in Arabidopsis. Methionine chain elongation enzymes are responsible for variations in chain length in Met-GSL biosynthesis. The genes encoding methionine chain elongation enzymes are considered to have been recruited from the leucine biosynthetic pathway in the course of evolution. Among them, the genes encoding methylthioalkylmalate synthases and aminotransferases have been identified; however, the remaining genes that encode methylthioalkylmalate isomerase (MAM-I) and methylthioalkylmalate dehydro-genase (MAM-D) remain to be identified. In a previous study based on transcriptome co-expression analysis, we identified candidate genes for the large subunit of MAM-I and MAM-D. In this study, we confirmed their predicted functions by targeted GSL analysis of the knockout mutants, and named the respective genes MAM-IL1/AtleuC1 and MAM-D1/AtIMD1. Metabolic profiling of the knockout mutants of methionine chain elongation enzymes, conducted by means of widely targeted metabolomics, implied that these enzymes have roles in controlling metabolism from methionine to primary and methionine-related secondary metabolites. As shown here, an omics-based approach is an efficient strategy for the functional elucidation of genes involved in metabolism.


Hearts deficient in both Mfn1 and Mfn2 are protected against acute myocardial infarction.
Mitochondria alter their shape by undergoing cycles of fusion and fission. Changes in mitochondrial morphology impact on the cellular response to stress, and their interactions with other organelles such as the sarcoplasmic reticulum (SR). Inhibiting mitochondrial fission can protect the heart against acute ischemia/reperfusion (I/R) injury. However, the role of the mitochondrial fusion proteins, Mfn1 and Mfn2, in the response of the adult heart to acute I/R injury is not clear, and is investigated in this study. To determine the effect of combined Mfn1/Mfn2 ablation on the susceptibility to acute myocardial I/R injury, cardiac-specific ablation of both Mfn1 and Mfn2 (DKO) was initiated in mice aged 4-6 weeks, leading to knockout of both these proteins in 8-10-week-old animals. This resulted in fragmented mitochondria (electron microscopy), decreased mitochondrial respiratory function (respirometry), and impaired myocardial contractile function (echocardiography). In DKO mice subjected to in vivo regional myocardial ischemia (30 min) followed by 24 h reperfusion, myocardial infarct size (IS, expressed as a % of the area-at-risk) was reduced by 46% compared with wild-type (WT) hearts. In addition, mitochondria from DKO animals had decreased MPTP opening susceptibility (assessed by Ca(2+)-induced mitochondrial swelling), compared with WT hearts. Mfn2 is a key mediator of mitochondrial/SR tethering, and accordingly, the loss of Mfn2 in DKO hearts reduced the number of interactions measured between these organelles (quantified by proximal ligation assay), attenuated mitochondrial calcium overload (Rhod2 confocal microscopy), and decreased reactive oxygen species production (DCF confocal microscopy) in response to acute I/R injury. No differences in isolated mitochondrial ROS emissions (Amplex Red) were detected in response to Ca(2+) and Antimycin A, further implicating disruption of mitochondria/SR tethering as the protective mechanism. In summary, despite apparent mitochondrial dysfunction, hearts deficient in both Mfn1 and Mfn2 are protected against acute myocardial infarction due to impaired mitochondria/SR tethering.


Radiation-induced transformations of matrix-isolated formic acid: evidence for the HCOOH → HOCO + H channel.
The effect of X-ray irradiation on the isolated formic acid molecules (HCOOH) in solid noble gas matrices (Xe, Kr, Ar, and Ne) at very low temperatures (6 K) was first studied by FTIR spectroscopy. Carbon oxides (CO and CO2) and hydrocarboxyl radicals (HOCO) have been detected as the principal degradation products. The formation of HOCO radicals represents a primary dissociation channel for formic acid, which was not reported previously under UV photolysis in solids. This reaction can be explained by the involvement of the recombination-induced excited states, which are not populated in photolysis. The effects of the matrix and the absorbed dose on the product formation were studied in detail and possible mechanisms are discussed with particular attention to the difference between radiolysis and UV-photolysis of the matrix-isolated formic acid. The results obtained provide a new insight into the effects of high-energy impact on the simplest carboxylic acid with possible implications to the astrochemical problems, in particular, the prebiotic evolution in the interstellar medium.


The mechanics of translocation: a molecular "spring-and-ratchet" system.
The translation of genetic information into proteins is a fundamental process of life. Stepwise addition of amino acids to the growing polypeptide chain requires the coordinated movement of mRNA and tRNAs through the ribosome, a process known as translocation. Here, we review current understanding of the kinetics and mechanics of translocation, with particular emphasis on the structure of a functional mammalian ribosome stalled during translocation by an mRNA pseudoknot. In the context of a pseudoknot-stalled complex, the translocase EF-2 is seen to compress a hybrid-state tRNA into a strained conformation. We propose that this strain energy helps overcome the kinetic barrier to translocation and drives tRNA into the P-site, with EF-2 biasing this relaxation in one direction. The tRNA can thus be considered a molecular spring and EF-2 a Brownian ratchet in a "spring-and-ratchet" system within the translocation process.


Aging of the liver: proteolysis of oxidatively modified glutamine synthetase.
During aging cells accumulate altered forms of proteins, notably oxidatively modified proteins. The multicatalytic protease selectively degrades oxidized proteins, suggesting that the age-related accumulation of oxidized proteins might be a consequence of decreased activity of this protease. The protease activity of liver homogenates was assayed with an improved fluorimetric method, using oxidatively modified glutamine synthetase as substrate. Application of this assay to extracts from liver of Fischer 344 rats from both Japan and the United States demonstrated a marked preference for the oxidized substrates, as expected. Extracts from animals ages 8 to 26 months maintain both total proteolytic activity and the ability to distinguish between native and oxidized substrates. Oxidatively modified hepatocyte extracts were also employed as substrate, and older animals again maintained proteolytic activity. The multicatalytic protease was purified from liver of young and old rats, and the specific activity of the preparations were comparable when assayed with oxidatively modified glutamine synthetase. We conclude that the intrinsic neutral or alkaline proteolytic activity of rat liver is maintained during aging.


Evaluation of metabolic factors on the prognosis of patients undergoing resection of hepatocellular carcinoma.
The metabolic factors including obesity, diabetes, and hypertension have been implicated as risk factors of hepatocellular carcinoma (HCC) in patients with chronic hepatitis. The effects of metabolic factors were investigated on the prognosis of patients undergoing resection of HCC. A total of 469 HCC patients were classified into three groups; hepatitis B virus (HBV)-, hepatitis C virus (HCV)-, and non-HBV/HCV (NBC)-related HCC. Further, the patients with HCV-related HCC were sub-classified into three groups; the patients who did not have documented hypertension, hypertensive patients who received angiotensin II-blocking agents (ABA), and hypertensive patients who received no ABA. There were no significant difference of survival in the HBV-HCC and NBC-HCC patients with or without obesity, diabetes, and hypertension. In the patients with HCV-related HCC, however, hypertensive patients were significantly worse on both disease-free and overall survivals than non-hypertensive patients. Among the HCV-HCC patients with chronic hepatitis, hypertensive patients with ABA had significantly better preoperative liver function, and hypertensive patients without ABA were significantly worse on both disease-free and overall survivals than those of hypertensive patients with ABA and non-hypertensive patients. Results suggest that hypertension is a risk factor for a poor prognosis after resection of HCV-related HCC. Angiotensin II blockade may improve the prognosis of hypertensive patients with early hepatic fibrosis after resection in HCV-related HCC.


Specific binding and internalization: an investigation of fluorescent aptamer-gold nanoclusters and cells with fluorescence lifetime imaging microscopy.
Fluorescent gold nanoclusters show promising properties for biological applications. We biofunctionalized fluorescent 11-mercaptoundecanoic-acid stabilized gold nanoclusters (AuNCs) with an aptamer to target the interleukin-6-receptor expressed on BaF3 cells specifically. Although the fluorescence emission of the AuNCs (535 nm) is in the same wavelength region as the autofluorescence of the cell, we are able to distinguish between nanoclusters and cells using the fluorescence decay time, which is much longer for the AuNCs (100 ns) than for the autofluorescence. After a first short incubation period we detected AuNCs specifically bound to the cell membrane by using two fluorescence lifetime imaging microscopy (FLIM) methods: gated and direct FLIM. After a second incubation period the previously bound AuNCs are internalized by the cells, as could be resolved solely by the direct FLIM. This proves the superior sensitivity of this method compared to gated FLIM. We find that the optical properties of AuNCs do not change upon binding to the cells, but exhibit a change when internalized into the cells, induced by an interaction between the AuNCs and cells.


Association Between Results of a Gene Expression Signature Assay and Recurrence-Free Interval in Patients With Stage II Colon Cancer in Cancer and Leukemia Group B 9581 (Alliance).
Conventional staging methods are inadequate to identify patients with stage II colon cancer (CC) who are at high risk of recurrence after surgery with curative intent. ColDx is a gene expression, microarray-based assay shown to be independently prognostic for recurrence-free interval (RFI) and overall survival in CC. The objective of this study was to further validate ColDx using formalin-fixed, paraffin-embedded specimens collected as part of the Alliance phase III trial, C9581. C9581 evaluated edrecolomab versus observation in patients with stage II CC and reported no survival benefit. Under an initial case-cohort sampling design, a randomly selected subcohort (RS) comprised 514 patients from 901 eligible patients with available tissue. Forty-nine additional patients with recurrence events were included in the analysis. Final analysis comprised 393 patients: 360 RS (58 events) and 33 non-RS events. Risk status was determined for each patient by ColDx. The Self-Prentice method was used to test the association between the resulting ColDx risk score and RFI adjusting for standard prognostic variables. Fifty-five percent of patients (216 of 393) were classified as high risk. After adjustment for prognostic variables that included mismatch repair (MMR) deficiency, ColDx high-risk patients exhibited significantly worse RFI (multivariable hazard ratio, 2.13; 95% CI, 1.3 to 3.5; P < .01). Age and MMR status were marginally significant. RFI at 5 years for patients classified as high risk was 82% (95% CI, 79% to 85%), compared with 91% (95% CI, 89% to 93%) for patients classified as low risk. ColDx is associated with RFI in the C9581 subsample in the presence of other prognostic factors, including MMR deficiency. ColDx could be incorporated with the traditional clinical markers of risk to refine patient prognosis.


The insulin-like growth factor 1 receptor induces physiological heart growth via the phosphoinositide 3-kinase(p110alpha) pathway.
Insulin-like growth factor 1 (IGF1) was considered a potential candidate for the treatment of heart failure. However, some animal studies and clinical trials have questioned whether elevating IGF1 chronically is beneficial. Secondary effects of increased serum IGF1 levels on other tissues may explain these unfavorable results. The aim of the current study was to examine the role of IGF1 in cardiac myocytes in the absence of secondary effects, and to elucidate downstream signaling pathways and transcriptional regulatory effects of the IGF1 receptor (IGF1R). Transgenic mice overexpressing IGF1R in the heart displayed cardiac hypertrophy, which was the result of an increase in myocyte size, and there was no evidence of histopathology. IGF1R transgenics also displayed enhanced systolic function at 3 months of age, and this was maintained at 12-16 months of age. The phosphoinositide 3-kinase (PI3K)-Akt-p70S6K1 pathway was significantly activated in hearts from IGF1R transgenics. Cardiac hypertrophy induced by overexpression of IGF1R was completely blocked by a dominant negative PI3K(p110alpha) mutant, suggesting IGF1R promotes compensated cardiac hypertrophy in a PI3K(p110alpha)-dependent manner. This study suggests that targeting the cardiac IGF1R-PI3K(p110alpha) pathway could be a potential therapeutic strategy for the treatment of heart failure.


Polyamide as a Denture Base Material: A Literature Review.
The purpose of this article was to review the biocompatibility, physical, and mechanical properties of the polyamide denture base materials. An electronic search of scientific papers from 1990-2014 was carried out using PubMed, Scopus and Wiley Inter Science engines using the search terms "nylon denture base" and "polyamide denture base". Searching the key words yielded a total of 82 articles. By application of inclusion criteria, the obtained results were further reduced to 24 citations recruited in this review. Several studies have evaluated various properties of polyamide (nylon) denture base materials. According to the results of the studies, currently, thermo-injectable, high impact, flexible or semi-flexible polyamide is thought to be an alternative to the conventional acrylic resins due to its esthetic and functional characteristics and physicochemical qualities. It would be justifiable to use this material for denture fabrication in some cases such as severe soft/ hard tissue undercuts, unexplained repeated fracture of denture, in aesthetic-concerned patients, those who have allergy to other denture base materials, and in patients with microstomia. Although polyamide has some attractive advantages, they require modifications to produce consistently better properties than the current polymethyl methacrylate (PMMA) materials. Moreover, since there is a very limited knowledge about their clinical performance, strict and careful follow-up evaluation of the patients rehabilitated with polyamide prosthesis is recommended.


Minimally invasive endoscopic pericranial flap: a new method for endonasal skull base reconstruction.
One of the major challenges of cranial base surgery is reconstruction of the dural defect. Following a craniofacial resection, the standard reconstructive technique is direct suture repair of the dural defect with a fascial graft and rotation of an anteriorly based pericranial scalp flap to cover the dura. The introduction of endoscopic techniques and an endonasal approach to the ventral skull base has created new challenges for reconstruction. The nasoseptal flap has become the workhorse for vascularized endoscopic skull base reconstruction; however at times, the septal mucosal flap may be unavailable for reconstruction. This can be due to prior surgical resection or involvement of the nasal septum by sinonasal cancer. We have developed a minimally invasive endoscopic pericranial flap for endoscopic skull base reconstruction. The use of a pericranial scalp flap for reconstruction during endonasal skull base surgery using minimally invasive techniques has not been previously reported. We performed cadaveric studies to illustrate feasibility of an endoscopic pericranial flap for endonasal skull base reconstruction, then applied this novel technique to an elderly patient after endonasal skull base and dural resection of an esthesioneuroblastoma. The technical report of the minimally invasive pericranial flap is outlined and the advantages and limitations during endonasal skull base reconstruction are discussed. The patient had excellent healing of her skull base and had no evidence of any postoperative cerebrospinal fluid leak. The minimally invasive endoscopic pericranial flap provides another option for endonasal reconstruction of cranial base defects. There is minimal donor site morbidity, and it provides a large flap that can cover the entire ventral skull base. The issues of intranasal tissue tumor involvement and the need for radiotherapy make the endoscopic pericranial flap an ideal reconstruction for anterior cranial base defects resulting from endonasal sinonasal and skull base cancer resections.


Transmission versus transflection mode in FTIR analysis of blood plasma: is the electric field standing wave effect the only reason for observed spectral distortions?
Fourier transform infrared (FTIR) microspectroscopy is assessed in terms of two techniques (i.e., transmission and transflection) as a method for rapid measurements of blood plasma. Apart from the expected effect of the electric field standing wave (EFSW), we also noticed that second-derivative IR spectra recorded in transflection mode exhibited a significant shift in the amide I band (up to 1667 cm(-1)) in comparison to the one recorded in transmission (1658 cm(-1)). This has not been reported thus far in studies of the EFSW distortion of IR spectra of biological material. The thinner the sample deposited on the low-e microscope slide, the lower the position of the amide I band found in FTIR spectra, suggesting various plasma compositions after stratification or certain changes in secondary protein conformations due to chemical and/or physical effects. There are potentially several phenomena that can occur at the surface of both IR substrates affecting the protein profile, including changes in optical properties (refractive index), variation in water content in the sample, and segregation of plasma components. All three hypotheses are discussed here, with the help of atomic force microscopy (AFM).


The maize NCS2 abnormal growth mutant has a chimeric nad4-nad7 mitochondrial gene and is associated with reduced complex I function.
The molecular basis of the maternally inherited, heteroplasmic NCS2 mutant of maize was investigated. Analysis of the NCS2 mtDNA showed that it closely resembles the progenitor cmsT mitochondrial genome, except that the mutant genome contains a fused nad4-nad7 gene and is deleted for the small fourth exon of nad4. The rearrangement has occurred at a 16-bp repeat present in the third intron of the nad4 gene and in the second intron of the nad7 gene. Transcripts containing exon 4 of the nad4 gene are greatly reduced in mtRNA preparations from heteroplasmic NCS2 plants; larger transcripts are associated with the first three nad4 exons. Identical 5' ends of the nad4 transcripts have been mapped 396 and 247 bp upstream of the start codon in mtRNAs from both NCS2 and related non-NCS plants. The putative transcription termination signal of nad4 is deleted in mutant DNA, resulting in the production of the unique longer transcripts. The complex transcript pattern associated with nad7 is also altered in the mutant. Both nad4 and nad7 encode subunits of complex I (NADH dehydrogenase) of the mitochondrial electron transfer chain. Oxygen uptake experiments show that the functioning of complex I is specifically reduced in mitochondria isolated from NCS2 mutant plants.


The demography of primary biliary cirrhosis in Ontario, Canada.
The demographics of primary biliary cirrhosis in Ontario, Canada, are described. Two hundred and twenty-five primary biliary cirrhosis patients were identified by 85 of 502 gastroenterologists (or internists) practicing in Ontario acute care hospitals that have 150 or more beds. Two hundred and six patients were verified as being antimitochondrial antibody-positive, resulting in an incidence of 3.26 per million per year and a prevalence of 22.39 per million. Questionnaire data were obtained on 88.5% of these patients. Twenty-nine percent of the patients were found to be asymptomatic. Geographical clustering and racial predisposition were not seen. No increase in breast cancer prevalence was noted. By the time the diagnosis of primary biliary cirrhosis was established, the patients had consulted a median number of 3.5 physicians. Fatigue was reported as the most disabling symptom. The diagnosis of primary biliary cirrhosis in patients referred from across the province of Ontario was independently confirmed by us, using standard criteria (antimitochondrial antibody testing and liver biopsy), and was found to be reliable.


Effects of radiographic techniques on the low-contrast detail detectability performance of digital radiography systems.
To evaluate the effects of the radiation exposure factors kilovolt peak and tube current time (milliampere seconds) on the low-contrast detail detectability performance of 3 types of planar digital radiography systems. Detectability performance of an imaging system refers to its ability to detect and present the low-contrast details of organs in the acquired image. The authors also compare detectability performance between computed radiography, indirect digital radiography, and direct digital radiography by evaluating low-contrast details of the obtained images. A low-contrast detail phantom was inserted within 10-cm thicknesses of Perspex plastic sheets. The images were obtained with various kilovolt peak and milliampere second settings for each of the 3 digital radiography systems. Artinis CDRAD Analyser software was used to score the images and calculate the inverse image quality figure (IQFinv). The higher milliampere second levels in each kilovolt peak selection resulted in higher IQFinv in computed radiography and indirect and direct digital radiography. IQFinv values significantly increased in indirect digital radiography with increasing kilovolt peak in only 1 and 2 mAs. There were insignificant differences in IQFinv values when altering kilovolt peak in each milliampere second level in direct digital radiography. The indirect digital radiography system generally demonstrated better detectability performance than computed radiography and direct digital radiography. However, direct digital radiography demonstrated better detectability performance than indirect digital radiography at lower kilovolt peak and milliampere second settings, as did computed radiography at lower kilovolt peak settings. Higher milliampere second settings increase photon count, which results in a higher signal-to-noise ratio and thus increased detectability. Lower milliampere second settings increase noise level on images, which increases the risk of diagnostic detail loss. Changing the kilovolt peak at the different milliampere second settings essentially did not affect the IQFinv of the different digital radiography systems. Increasing milliampere seconds in all digital imaging systems generally improves detectability performance. However, altering the kilovolt peak setting does not significantly change the IQFinv and detectability of objects in a digital radiograph. Imaging system selection should be based on typical radiographic examinations. Indirect digital radiography systems are better for studies that require higher kilovolt peak, such as large organs, and direct digital radiography is better for studies that require low kilovolt peak, such as small organs and mammography, which is used to examine fine tissue details.


The impacts of COVID-19 hospitalizations on non-COVID-19 deaths and hospitalizations: A panel data analysis using Brazilian municipalities.
The COVID-19 pandemic in Brazil has brought many challenges, particularly regarding the management of hospital capacity, and a new demand for healthcare that added to the preexisting demands, such as neoplasms, cardiovascular diseases and births. In this paper, we estimate the impact of the pandemic on the number of deaths and hospitalizations for other diseases. We construct a monthly panel data of deaths and hospitalizations for various causes by the municipality of residence and relate them to COVID-19 hospitalizations using regression models that control for municipalities fixed-effects and interactions between State and month fixed-effects. The standard errors are clustered at the municipality level. Our estimates imply that 100 more hospitalizations by COVID-19 is associated with a drop of 49 non-COVID-19 hospitalizations and an additional four deaths for other reasons (all measured per 100,000 pop.). The impact of intensive care units COVID-19 hospitalizations on mortality is larger. The groups most affected are the African Brazilians, less-educated and the elderly. Additional deaths occurred both at households and at hospitals. The main causes of additional deaths were diseases related to the circulatory and endocrine system. The decline in hospitalizations for other causes seems to be related to the overcrowding of hospitals in periods of surge in the COVID-19, alongside with the fall in the demand for care by the citizens who were afraid of COVID-19 infection. These mechanisms affected more strongly the vulnerable groups of the population. Our results highlight the importance of promoting the awareness of heightened risk of non-communicable chronic diseases during a health emergency context. This should be done preferably through already established channels with community outreach, such as the Family Health Program in Brazil.


Increased precision using countermatching in nested case-control studies.
Nested case-control studies in occupational cohorts are often used to estimate exposure effects when development of detailed exposure estimates for all cohort members is too costly. Duration of exposure, which can act as a surrogate for cumulative exposure, is often readily available for all cohort members. Langholz and others have recently proposed a method of control selection called countermatching, which uses data on the surrogate to determine which controls are selected from the risk set for a given case. This method may increase precision relative to the usual random sampling of the risk set. We compare countermatching with random sampling in a nested case-control study of silicosis among miners. Data on cumulative exposure were in fact available for all cohort members, enabling estimation of the parameter of interest in the full cohort. We conducted nested case-control analyses using 100, 20, 10, and 3 controls per case using random sampling and additional analyses using 3 controls per case with two different methods of countermatching. All analyses were replicated 50 times to explore the statistical properties of the estimated exposure parameter. We found that one of the countermatching methods markedly increased efficiency compared with random sampling. Countermatching using 3 controls per case yielded an approximate 25% increase in relative efficiency compared with random sampling; it was approximately equivalent to random sampling using 10 controls.


Bile acids regulate intestinal antigen presentation and reduce graft-versus-host disease without impairing the graft-versus-leukemia effect.
Acute graft-versus-host disease causes significant mortality in patients undergoing allogeneic hematopoietic cell transplantation. Immunosuppressive treatment for graft-versus-host disease can impair the beneficial graft-versus-leukemia effect and facilitate malignancy relapse. Therefore, novel approaches that protect and regenerate injured tissues without impeding the donor immune system are needed. Bile acids regulate multiple cellular processes and are in close contact with the intestinal epithelium, a major target of acute graft-versus-host disease. Here, we found that the bile acid pool is reduced following graft-versus-host disease induction in a preclinical model. We evaluated the efficacy of bile acids to protect the intestinal epithelium without reducing anti-tumor immunity. We observed that application of bile acids decreased cytokine-induced cell death in intestinal organoids and cell lines. Systemic prophylactic administration of tauroursodeoxycholic acid, the most potent compound in our in vitro studies, reduced graft-versus-host disease severity in three different murine transplantation models. This effect was mediated by decreased activity of the antigen presentation machinery and subsequent prevention of apoptosis of the intestinal epithelium. Moreover, bile acid administration did not alter the bacterial composition in the intestine suggesting that its effects are cell-specific and independent of the microbiome. Treatment of human and murine leukemic cell lines with tauroursodeoxycholic acid did not interfere with the expression of antigen presentation-related molecules. Systemic T cell expansion and especially their cytotoxic capacity against leukemic cells remained intact. This study establishes a role for bile acids in the prevention of acute graft-versus-host disease without impairing the graft-versus-leukemia effect. In particular, we provide a scientific rationale for the systematic use of tauroursodeoxycholic acid in patients undergoing allogeneic hematopoietic cell transplantation.


Increased mortality, hypoactivity, and hypoalgesia in cannabinoid CB1 receptor knockout mice.
Delta9-Tetrahydrocannabinol (Delta9-THC), the major psychoactive ingredient in preparations of Cannabis sativa (marijuana, hashish), elicits central nervous system (CNS) responses, including cognitive alterations and euphoria. These responses account for the abuse potential of cannabis, while other effects such as analgesia suggest potential medicinal applications. To study the role of the major known target of cannabinoids in the CNS, the CB1 cannabinoid receptor, we have produced a mouse strain with a disrupted CB1 gene. CB1 knockout mice appeared healthy and fertile, but they had a significantly increased mortality rate. They also displayed reduced locomotor activity, increased ring catalepsy, and hypoalgesia in hotplate and formalin tests. Delta9-THC-induced ring-catalepsy, hypomobility, and hypothermia were completely absent in CB1 mutant mice. In contrast, we still found Delta9-THC-induced analgesia in the tail-flick test and other behavioral (licking of the abdomen) and physiological (diarrhea) responses after Delta9-THC administration. Thus, most, but not all, CNS effects of Delta9-THC are mediated by the CB1 receptor.


IGF-1 receptor regulates dynamic changes in neuronal polarity during cerebral cortical migration.
During cortical development, neurons undergo polarization, oriented migration and layer-type differentiation. The biological and biochemical mechanisms underlying these processes are not completely understood. In neurons in culture we showed that IGF-1 receptor activation is important for growth cone assembly and axonal formation. However, the possible roles of the insulin like growth factor-1 receptor (IGF-1R) on neuronal differentiation and polarization in vivo in mammals have not yet been studied. Using in utero electroporation, we show here that the IGF-1R is essential for neocortical development. Neurons electroporated with a shRNA targeting IGF-1 receptor failed to migrate to the upper cortical layers and accumulated at the ventricular/subventricular zones. Co-electroporation with a constitutively active form of PI3K rescued migration. The change of the morphology from multipolar to bipolar cells was also attenuated. Cells lacking the IGF-1 receptor remain arrested as multipolar forming a highly disorganized tissue. The typical orientation of the migrating neurons with the Golgi complex oriented toward the cortical upper layers was also affected by electroporation with shRNA targeting IGF-1 receptor. Finally, cells electroporated with the shRNA targeting IGF-1 receptor were unable to form an axon and, therefore, neuron polarity was absent.


Interaction between Reelin and Notch signaling regulates neuronal migration in the cerebral cortex.
Neuronal migration is a fundamental component of brain development whose failure is associated with various neurological and psychiatric disorders. Reelin is essential for the stereotypical inside-out sequential lamination of the neocortex, but the molecular mechanisms of its action still remain unclear. Here we show that regulation of Notch activity plays an important part in Reelin-signal-dependent neuronal migration. We found that Reelin-deficient mice have reduced levels of the cleaved form of Notch intracellular domain (Notch ICD) and that loss of Notch signaling in migrating neurons results in migration and morphology defects. Further, overexpression of Notch ICD mitigates the laminar and morphological abnormalities of migrating neurons in Reeler. Finally, our in vitro biochemical studies show that Reelin signaling inhibits Notch ICD degradation via Dab1. Together, our results indicate that neuronal migration in the developing cerebral cortex requires a Reelin-Notch interaction.


Chromosome mapping of repetitive sequences in Rachycentron canadum (Perciformes: Rachycentridae): implications for karyotypic evolution and perspectives for biotechnological uses.
The cobia, Rachycentron canadum, a species of marine fish, has been increasingly used in aquaculture worldwide. It is the only member of the family Rachycentridae (Perciformes) showing wide geographic distribution and phylogenetic patterns still not fully understood. In this study, the species was cytogenetically analyzed by different methodologies, including Ag-NOR and chromomycin A(3) (CMA(3))/DAPI staining, C-banding, early replication banding (RGB), and in situ fluorescent hybridization with probes for 18S and 5S ribosomal genes and for telomeric sequences (TTAGGG)(n). The results obtained allow a detailed chromosomal characterization of the Atlantic population. The chromosome diversification found in the karyotype of the cobia is apparently related to pericentric inversions, the main mechanism associated to the karyotypic evolution of Perciformes. The differential heterochromatin replication patterns found were in part associated to functional genes. Despite maintaining conservative chromosomal characteristics in relation to the basal pattern established for Perciformes, some chromosome pairs in the analyzed population exhibit markers that may be important for cytotaxonomic, population, and biodiversity studies as well as for monitoring the species in question.


Evaluation of Commercial Molecular Diagnostic Methods for Detection and Determination of Macrolide Resistance in Mycoplasma pneumoniae.
We evaluated six commercial molecular tests targeting Mycoplasma pneumoniae, namely, the BioFire FilmArray respiratory panel (RP), the Meridian Alethia Mycoplasma Direct, the GenMark ePlex respiratory pathogen panel (RPP), the Luminex NxTAG RPP, the ELITech ELITe InGenius Mycoplasma MGB research use only (RUO) PCR, and the SpeeDx Resistance Plus MP assays. Laboratory-developed PCR assays at the University of Alabama at Birmingham and the Centers for Disease Control and Prevention were used as reference standards. Among 428 specimens, 212 were designated confirmed positives for M. pneumoniae The highest clinical sensitivities were found with the InGenius PCR (99.5%) and the FilmArray RP (98.1%). The Resistance Plus MP identified 93.3% of the confirmed-positive specimens, whereas 83.6, 64.6, and 55.7% were identified by the ePlex RPP, NxTAG RPP, and Mycoplasma Direct assays, respectively. There was no significant difference between the sensitivity of the reference methods and that of the FilmArray RP and InGenius assays, but the remaining four assays detected significantly fewer positive specimens (P < 0.05). Specificities of all assays were 99.5 to 100%. The Resistance Plus MP assay detected macrolide resistance in 27/33 specimens, resulting in a sensitivity of 81.8%. This study provides the first large-scale comparison of commercial molecular assays for detection of M. pneumoniae in the United States and identified clear differences among their performance. Additional studies are necessary to explore the impact of various test performances on patient outcome.


Increased peptide YY blood concentrations, not decreased acyl-ghrelin, are associated with reduced hunger and food intake in healthy older women: Preliminary evidence.
With ageing there is frequently a loss of appetite, termed anorexia of ageing, which can result in under-nutrition. We do not know how appetite control alters with ageing. The objective of this study was to investigate whether differences in the release of, and response to, gastrointestinal appetite hormones is altered in young compared to old healthy volunteers. We hypothesised that an increase in PYY and GLP-1 or a decrease ghrelin may result in a decreased appetite. A comparative experimental design, using a cross-sectional sample of ages from a healthy population, matched for sex and BMI was used. The study compared total ghrelin, acyl-ghrelin, PYY, GLP-1 and subjective appetite responses to ingestion of a standardised 2781kj (660 kcal) test meal. 31 female volunteers aged between 21 and 92yrs took part. Multiple linear regression showed that both age and sex had an independent effect on energy intake. Subjective appetite scores showed that hunger, pleasantness to eat, and prospective food intake were significantly lower in the older age groups. PYY incremental area under the curve (IAUC) was greater in the oldest old compared to younger ages f(3,27) = 2.9, p = 0.05. No differences in GLP-1, ghrelin or acyl-ghrelin were observed in the older compared to younger age groups. Our data suggest that there may be increases in postprandial PYY(3-36) levels in female octogenarians, potentially resulting in reduced appetite. There does not appear to be any change in ghrelin or acyl-ghrelin concentrations with ageing.


Association of Hemoglobin A1c and Wound Healing in Diabetic Foot Ulcers.
This study evaluated the association between hemoglobin A1c (A1C) and wound outcomes in patients with diabetic foot ulcers (DFUs). We conducted a retrospective analysis of an ongoing prospective, clinic-based study of patients with DFUs treated at an academic institution during a 4.7-year period. Data from 270 participants and 584 wounds were included in the analysis. Cox proportional hazards regression was used to assess the incidence of wound healing at any follow-up time in relation to categories of baseline A1C and the incidence of long-term (≥90 days) wound healing in relation to tertiles of nadir A1C change and mean A1C change from baseline, adjusted for potential confounders. Baseline A1C was not associated with wound healing in univariate or fully adjusted models. Compared with a nadir A1C change from baseline of -0.29 to 0.0 (tertile 2), a nadir A1C change of 0.09 to 2.4 (tertile 3) was positively associated with long-term wound healing in the subset of participants with baseline A1C <7.5% (hazard ratio [HR] 2.07; 95% CI 1.08-4.00), but no association with wound healing was seen with the mean A1C change from baseline in this group. Neither nadir A1C change nor mean A1C change were associated with long-term wound healing in participants with baseline A1C ≥7.5%. There does not appear to be a clinically meaningful association between baseline or prospective A1C and wound healing in patients with DFUs. The paradoxical finding of accelerated wound healing and increase in A1C in participants with better baseline glycemic control requires confirmation in further studies.


Biallelic ATM inactivation significantly reduces survival in patients treated on the United Kingdom Leukemia Research Fund Chronic Lymphocytic Leukemia 4 trial.
The prognostic significance of ATM mutations in chronic lymphocytic leukemia (CLL) is unclear. We assessed their impact in the context of a prospective randomized trial. We analyzed the ATM gene in 224 patients treated on the Leukemia Research Fund Chronic Lymphocytic Leukemia 4 (LRF-CLL4) trial with chlorambucil or fludarabine with and without cyclophosphamide. ATM status was analyzed by denaturing high-performance liquid chromatography and was related to treatment response, survival, and the impact of TP53 alterations for the same patient cohort. We identified 36 ATM mutations in 33 tumors, 16 with and 17 without 11q deletion. Mutations were associated with advanced disease stage and involvement of multiple lymphoid sites. Patients with both ATM mutation and 11q deletion showed significantly reduced progression-free survival (median, 7.4 months) compared with those with ATM wild type (28.6 months), 11q deletion alone (17.1 months), or ATM mutation alone (30.8 months), but survival was similar to that in patients with monoallelic (6.7 months) or biallelic (3.4 months) TP53 alterations. This effect was independent of treatment, immunoglobulin heavy chain variable gene (IGHV) status, age, sex, or disease stage. Overall survival for patients with biallelic ATM alterations was also significantly reduced compared with those with ATM wild type or ATM mutation alone (median, 42.2 v 85.5 v 77.6 months, respectively). The combination of 11q deletion and ATM mutation in CLL is associated with significantly shorter progression-free and overall survival following first-line treatment with alkylating agents and purine analogs. Assessment of ATM mutation status in patients with 11q deletion may influence the choice of subsequent therapy.


Systemic Assessment of Patients Undergoing Dental Implant Surgeries: A Trans- and Post-operative Analysis.
Procedure-related and patient-related factors influence the prognosis of dental implants to a major extent. Hence, we aimed to evaluate and analyze various systemic factors in patients receiving dental implants. Fifty-one patients were included in the study, in which a total of 110 dental implants were placed. Complete examination of the subjects was done before and after placement of dental implants. Implant surgery was planned, and osseointegrated dental implants were placed in the subjects. Postoperative evaluation of the dental implant patients was done after 3 weeks. Anxiety levels were determined using State-Trait Anxiety Inventory (STAI) questionnaire on the surgery day and after 1 week of surgery. The participant describes how they feel at the moment by responding to twenty items as follows: (1) absolutely not, (2) slightly, (3) somewhat, or (4) very much. All the results were recorded and statistical analyzed by SPSS software. Out of 51, 29 patients were males while 22 were females, with ratio of 1.32:1. Female patients' mean age was 50.18 years while male patients' mean age was 52.71 years, with statistically nonsignificant difference between them. Functional rehabilitation was the main purpose of choosing dental implants in more than 90% of the subjects. Diameter of 3.75 mm was the shortest implants to be placed in the present study, whereas in terms of length, 8.5 mm was the shortest length of dental implant used in the present study. Tooth area in which maximum implants were placed in our study was 36 tooth region. Maximum implants were placed in Type II bone quality (n = 38). Implants installed in the mandible were clamped more efficiently than implants placed in the maxilla (P < 0.001). The difference of average STAI-State subscore before and after the surgery was statistically significant (P < 0.05; significant). Mandibular dental implants show more clamping (torque) than maxillary dental implants.


An age, period and cohort analysis of pleural cancer mortality in Europe.
Death certification data from pleural cancer in eight European countries providing data to the World Health Organization database over the period 1970-1994 were analysed using a log-linear Poisson model to disentangle the effects of age, birth cohort and period of death. The age effect reached values between 10 and 15/100,000 males at age 80-84 in most countries, except Hungary (6.7), Switzerland (18.0), France (20.6) and the Netherlands (36.5). Cohort effects were steadily and appreciably upwards in all countries up to the generations born in 1940 or 1945, and levelled off for the 1950 cohort, except in Hungary, where persistent rises were observed. Thus, most rises in pleural cancer mortality in Europe were on a cohort of birth basis. Since most pleural cases were asbestos-related mesotheliomas, and since asbestos has an early-stage effect on subsequent mesothelioma risk, exposure early in life is important for determining the subsequent mesothelioma risk of each generation. Consequently, the data indicate that the peak mortality from pleural cancer in most western European countries will be reached in the first decades of the 21st century, i.e. around 2010-2020, when the generations born between 1940 and 1950 will reach the peak age for mesothelioma incidence and mortality. This contrasts with US data, where the peak of pleural cancer incidence has been reached at the end of the 20th century, and reflects a delay in adopting adequate prevention measures since the 1940-1945 generations entered the workforce in the 1960s, when cancer risk from asbestos exposure was already recognized.


Environmental factors drive language density more in food-producing than in hunter-gatherer populations.
Linguistic diversity is a key aspect of human population diversity and shapes much of our social and cognitive lives. To a considerable extent, the distribution of this diversity is driven by environmental factors such as climate or coast access. An unresolved question is whether the relevant factors have remained constant over time. Here, we address this question at a global scale. We approximate the difference between pre- versus post-Neolithic populations by the difference between modern hunter-gatherer versus food-producing populations. Using a novel geostatistical approach of estimating language and language family densities, we show that environmental-chiefly climate factors-have driven the language density of food-producing populations considerably more strongly than the language density of hunter-gatherer populations. Current evidence suggests that the population dynamics of modern hunter-gatherers is very similar to that of what can be reconstructed from the Palaeolithic record. Based on this, we cautiously infer that the impact of environmental factors on language densities underwent a substantial change with the transition to agriculture. After this transition, the environmental impact on language diversity in food-producing populations has remained relatively stable since it can also be detected-albeit in slightly weaker form-in models that capture the reduced linguistic diversity during large-scale language spreads in the Mid-Holocene.


Targeted delivery of human recombinant superoxide dismutase by chemical modification with mono- and polysaccharide derivatives.
Four types of superoxide dismutase (SOD) derivatives such as SOD-carboxymethyl dextran conjugate, SOD-diethylaminoethyl dextran conjugate, galactosylated SOD and mannosylated SOD were synthesized and their potential for selective targeting to organs or cells was evaluated in mice by pharmacokinetic analysis. All SOD derivatives retained 50 to 80% of the original enzymatic activity and were stable during incubation with mouse serum retaining enzymatic activity greater than 80% for 3 hr. After intravenous injection, native SOD was rapidly excreted into urine and no significant accumulation was observed in the organs except the kidney. SOD-carboxymethyl dextran conjugate gave a long plasma half-life because of impaired glomerular filtration and tissue interaction. By contrast, galactosylated-SOD and mannosylated-SOD were very rapidly eliminated from the circulation and taken up by parenchymal and nonparenchymal cells of the liver, respectively, via receptor-mediated endocytosis. These uptake processes were nonlinear and hepatic uptake clearance decreased as the dose increased, although almost complete extraction was obtained at a dose of 0.1 mg/kg. Furthermore, the accumulation in kidney of both glycosylated SODs was drastically decreased due to reduced renal proximal tubular reabsorption and also enhanced hepatic clearance. SOD-diethylaminoethyl dextran conjugate also rapidly disappeared from plasma and distributed into liver, but its accumulation occurred due to electrostatic interaction and was nonspecific in cellular distribution. These results suggest the possibility of controlling the in vivo fate of SOD at a cellular level by chemical modification utilizing sugar moieties with varied physicochemical and/or biological characteristics.


[["Conflicting Right": A "Bottom-up" Approach to Map the Problem of Abortion].]
The paper examines the particular nature of the rights involved in disciplines related to the termination of pregnancy. It focuses on the ways by which these rights must be appropriately balanced with constitutional rights and values. It also stresses the need of a "botton-up" approach rather than a more restrictive and dangerous "top-down" approach. Only in this way it seems possible to tackle and appreciate the problem in all its actual complexity.


Structural basis of cholesterol binding by a novel clade of dendritic cell modulators from ticks.
Two crystal structures of Japanin, an 18 kDa immune-modulatory lipocalin from the Brown Ear Tick (Rhipicephalus appendiculatus), have been determined at 2.2 and 2.4 Å resolution. In both crystal forms the protein is in complex with cholesterol, which sits in a closed pocket at the centre of the lipocalin barrel. Both crystal forms are dimers, which are also observed in solution. Molecular modelling suggests that previously-described members of a tick protein family bearing high sequence homology to Japanin are also likely to bind cholesterol or cholesterol derivatives.


Ratio of C-Reactive Protein to Albumin Predicts Muscle Mass in Adult Patients Undergoing Hemodialysis.
Recent studies have indicated that the ratio of C-reactive protein to albumin (CRP-Alb ratio) is associated with clinical outcomes in patients with disease. We examined the predictive value of this ratio in patients undergoing hemodialysis (HD). In this cross-sectional study, 91 eligible adult HD patients were analyzed, and the correlation between the CRP-Alb ratio and skeletal muscle mass normalized for body weight (SMM/wt; estimated using a bioelectrical impedance analyzer) was investigated. The mean age of the study participants was 54.9 ± 6.6 years (ranging from 27 to 64 years); 43 (47.2%) were men. The mean values for the SMM/wt were 39.1% ± 5.4%. The CRP-Alb ratio was found to be negatively correlated with SMM/wt (r = -0.33, P = 0.002) and creatinine (r = -0.20, P = 0.056). All the univariate significant and nonsignificant relevant covariates were selected for multivariable stepwise regression analysis. We determined that the homeostasis model assessment-estimated insulin resistance and CRP-Alb ratio were independent risk determinants for SMM/wt (βHOMA-IR = -0.18 and βCRP-Alb ratio = -3.84, adjusted R2 = 0.32). This study indicated that the CRP-Alb ratio may help clinicians in predicting muscle mass in adult patients undergoing HD.


Glaucomatous cell derived matrices differentially modulate non-glaucomatous trabecular meshwork cellular behavior.
Ocular hypertension is a causal risk-factor to developing glaucoma. This is associated with stiffening of the trabecular meshwork (TM), the primary site of resistance to aqueous-humor-outflow. The mechanisms underlying this stiffening or how pathologic extracellular matrix (ECM) affects cell function are poorly understood. It is recognized that mechanotransduction systems allow cells to sense and translate the intrinsic biophysical properties of ECM into intracellular signals to control gene transcription, protein expression, and cell behavior. Using an anterior segment perfusion model, we document that there are significantly more low flow regions that are much stiffer, and fewer high flow regions that are less stiff in glaucomatous TM (GTM) when compared to non-glaucomatous TMs (NTM). GTM tissue also has fewer cells overall when compared with NTM tissue. In order to study the role of pathologic ECM in glaucoma disease progression, we conducted studies using cell derived matrices (CDM). First, we characterized the mechanics, composition and organization of fibronectin in ECM deposited by GTM and NTM cells treated with glucocorticosteroids. Then, we determined that these GTM-derived ECM are able to induce stiffening of normal NTM cells, and alter their gene/protein expression to resemble that of a glaucomatous phenotype. Further, we demonstrate that GTM-derived ECM causes endoplasmic reticular stress in NTM. They also became resistant to being reorganized by these NTM cells. These phenomena were exacerbated by ECMs obtained from steroid treated glaucoma model groups. Collectively, our data demonstrates that CDMs represent a novel tool for the study of bidirectional interactions between TM cells and their immediate microenvironment. Extracellular matrix (ECM) changes are prevalent in a number of diseases. The precise mechanisms by which changes in the ECM contribute to disease progression is unclear, primarily due to absence of appropriate models. Here, using glaucoma as a disease model, we document changes in cell derived matrix (CDM) and tissue mechanics that contribute to the pathology. Subsequently, we determine the effect that ECMs from diseased and healthy individuals have on healthy cell behaviors. Data emanating from this study demonstrate that CDMs are a potent tool for the study of cell-ECM interactions.


Overexpression of eNOS in the RVLM causes hypotension and bradycardia via GABA release.
In this study, we examine the role of NO located in the rostral ventrolateral medulla (RVLM) in the control of blood pressure and the activity of the sympathetic nervous system. To determine the effect of an increase in NO production in the RVLM on blood pressure in conscious rats, adenovirus vectors encoding either endothelial NO synthase (AdeNOS) or beta-galactosidase (Adbetagal) were transfected into the bilateral RVLM. The local expression of endothelial NO synthase (eNOS) protein in the RVLM was confirmed by immunohistochemical staining for the eNOS protein and by Western blot analysis. Mean arterial blood pressure (MAP) and heart rate, which were monitored using a radio-telemetry system, were significantly decreased in the AdeNOS-treated group from day 5 to day 10 after the gene transfer. Urinary norepinephrine excretion was decreased on day 7 after the gene transfer in the AdeNOS-treated group. Microinjection of either N(G)-monomethyl-L-arginine (L-NMMA) or bicuculine, a gamma-amino butyric acid (GABA) receptor antagonist, into the RVLM at day 7 after the gene transfer increased MAP to significantly greater levels in the AdeNOS-treated group. However, microinjection of kynurenic acid into the RVLM on day 7 after the gene transfer did not alter MAP levels in either group. GABA and glutamate levels in the RVLM, when measured by in vivo microdialysis, were significantly increased in the AdeNOS-treated group. These results suggest that the increase in NO production caused by the overexpression of eNOS in the bilateral RVLM decreases blood pressure, heart rate, and sympathetic nerve activity in conscious rats. Furthermore, these responses may be mediated by an increased release of GABA in the RVLM.


Corticosteroids in the treatment of community-acquired pneumonia in adults: a meta-analysis.
The benefit of corticosteroids in community-acquired pneumonia (CAP) remains controversial. We did a meta-analysis to include all the randomized controlled trials (RCTs) which used corticosteroids as adjunctive therapy, to examine the benefits and risks of corticosteroids in the treatment of CAP in adults. Databases including Pubmed, EMBASE, the Cochrane controlled trials register, and Google Scholar were searched to find relevant trials. Randomized and quasi-randomized trials of corticosteroids treatment in adult patients with CAP were included. Effects on primary outcome (mortality) and secondary outcomes (adverse events) were accessed in this meta-analysis. Nine trials involving 1001 patients were included. Use of corticosteroids did not significantly reduce mortality (Peto odds ratio [OR] 0.62, 95% confidence interval [CI] 0.37-1.04; P = 0.07). In the subgroup analysis by the severity, a survival benefit was found among severe CAP patients (Peto OR 0.26, 95% CI 0.11-0.64; P = 0.003). In subgroup analysis by duration of corticosteroids treatment, significant reduced mortality was found among patients with prolonged corticosteroids treatment (Peto OR 0.51, 95% CI 0.26-0.97; P = 0.04; I(2) = 37%). Corticosteroids increased the risk of hyperglycemia (Peto OR 2.64, 95% CI 1.68-4.15; P<0.0001), but without increasing the risk of gastroduodenal bleeding (Peto OR 1.67, 95% CI 0.41-6.80; P = 0.47) and superinfection (Peto OR 1.36, 95% CI 0.65-2.84; P = 0.41). Results from this meta-analysis did not suggest a benefit for corticosteroids treatment in patients with CAP. However, the use of corticosteroids was associated with improved mortality in severe CAP. In addition, prolonged corticosteroids therapy suggested a beneficial effect on mortality. These results should be confirmed by future adequately powered randomized trials.


Influence of EPICardial adipose tissue in HEART diseases (EPICHEART) study: Protocol for a translational study in coronary atherosclerosis.
Accumulation of epicardial adipose tissue (EAT) is associated with coronary artery disease (CAD) and increased risk of coronary events in asymptomatic subjects and low-risk patients, suggesting that EAT promotes atherosclerosis in its early stage. Recent studies have shown that the presence of CAD affects the properties of adjacent EAT, leading to dynamic changes in the molecular players involved in the interplay between EAT and the coronary arteries over the history of the disease. The role of EAT in late-stage CAD has not been investigated. In a comparative analysis with mediastinal and subcutaneous adipose tissue, we aim to investigate whether the volume of EAT assessed by computed tomography and its proteome assessed by SWATH-MS mass spectrometry are associated with late stages of CAD in an elderly cohort of severe aortic stenosis patients. The EPICHEART study (NCT03280433) is a prospective study enrolling patients with severe degenerative aortic stenosis referred for elective aortic valve replacement, whose protocol includes preoperative clinical, nutritional, echocardiographic, cardiac computed tomography and invasive coronary angiographic assessments. During cardiac surgery, samples of EAT and mediastinal and subcutaneous thoracic adipose tissue are collected for proteomics analysis by SWATH-MS. In addition, pericardial fluid and peripheral and coronary sinus blood samples are collected to identify circulating and local adipose tissue-derived biomarkers of CAD. We designed a translational study to explore the association of EAT quantity and quality with advanced CAD. We expect to identify new biochemical factors and biomarkers in the crosstalk between EAT and the coronary arteries that are involved in the pathogenesis of late coronary atherosclerosis, especially coronary calcification, which might be translated into new therapeutic targets and imaging tools by biomedical engineering.


Adolescent girls with emotional disorders have a lower end-tidal CO2 and increased respiratory rate compared with healthy controls.
Hyperventilation has been linked to emotional distress in adults. This study investigates end-tidal carbon dioxide (ETCO2 ), respiratory rate (RR), and heart rate variability (HRV) in adolescent girls with emotional disorders and healthy controls. ETCO2 , RR, HRV, and ratings of emotional symptom severity were collected in adolescent female psychiatric patients with emotional disorders (n = 63) and healthy controls (n = 62). ETCO2 and RR differed significantly between patients and controls. ETCO2, HR, and HRV were significant independent predictors of group status, that is, clinical or healthy, while RR was not. ETCO2 and RR were significantly related to emotional symptom severity and to HRV in the total group. ETCO2 and RR were not affected by use of selective serotonin reuptake inhibitors. It is concluded that emotional dysregulation is related to hyperventilation in adolescent girls. Respiratory-based treatments may be relevant to investigate in future research.


Comparative Genomic and Metabolic Analysis of Streptomyces sp. RB110 Morphotypes Illuminates Genomic Rearrangements and Formation of a New 46-Membered Antimicrobial Macrolide.
Morphotype switches frequently occur in Actinobacteria and are often associated with disparate natural product production. Here, we report on differences in the secondary metabolomes of two morphotypes of a Streptomyces species, including the discovery of a novel antimicrobial glycosylated macrolide, which we named termidomycin A. While exhibiting an unusual 46-member polyene backbone, termidomycin A (1) shares structural features with the clinically important antifungal agents amphotericin B and nystatin A1. Genomic analyses revealed a biosynthetic gene cluster encoding for a putative giant type I polyketide synthase (PKS), whose domain structure allowed us to propose the relative configuration of the 46-member macrolide. The architecture of the biosynthetic gene cluster was different in both morphotypes, thus leading to diversification of the product spectrum. Given the high frequency of genomic rearrangements in Streptomycetes, the metabolic analysis of distinct morphotypes as exemplified in this study is a promising approach for the discovery of bioactive natural products and pathways of diversification.


Damage Patterns, Monitoring, and Management of Procontarinia mangiferae (Diptera: Cecidomyiidae) in Pakistan.
Since 2005, mango has been damaged by a group of new pests, the gall midges, in all mango-growing areas of Pakistan. Little is known about these pests in Pakistan. In this report, we present information on the occurrence, damage patterns, methods for monitoring, and management of Procontarinia mangiferae (Felt), a mango gall midge found in the country. At the study site (Rahim Yar Khan, a district of the province Punjab, Pakistan), the pest was active from January/February to April as eggs, larvae, and adults on mango inflorescence buds, branches (axillaries), and immature fruits. Females of P. mangiferae oviposited in inflorescence tissues, and larvae, after feeding on plant tissues, dropped to the soil under the mango trees for pupation from February to April. Mango trees in commercial orchards were more heavily damaged by P. mangiferae than were isolated trees in farmer fields (66.7%). The adults of P. mangiferae were captured on sticky traps of all tested colors, and were in flight from January to May. Captures per trap were highest on yellow traps, followed by green, blue, and clear traps. Control of mango gall midge was effectively provided by the synthetic insecticide bifenthrin (Talstar 10 EC) and also by application of neem seed kernel extract on the tree canopy if integrated with raking the soil under the mango tree canopy.


Effect of decoyinine on the regulation of alpha-amylase synthesis in Bacillus subtilis.
Decoyinine, an inhibitor of GMP synthetase, allows sporulation in Bacillus subtilis to initiate and proceed under otherwise catabolite-repressing conditions. The effect of decoyinine on alpha-amylase synthesis in B. subtilis, an event which exhibits regulatory features resembling sporulation initiation, was examined. Decoyinine did not overcome catabolite repression of alpha-amylase synthesis in a wild-type strain of B. subtilis but did cause premature and enhanced synthesis in a mutant strain specifically blocked in catabolite repression of alpha-amylase synthesis. Decoyinine had no effect on alpha-amylase enzymatic activity. Thus, it appears that the catabolite control mechanisms governing alpha-amylase synthesis and sporulation in B. subtilis differ in their responses to decoyinine and hence must consist at least partially of separate components.


The Saccharomyces cerevisiae SEC14 gene encodes a cytosolic factor that is required for transport of secretory proteins from the yeast Golgi complex.
We have obtained and characterized a genomic clone of SEC14, a Saccharomyces cerevisiae gene whose product is required for export of yeast secretory proteins from the Golgi complex. Gene disruption experiments indicated that SEC14 is an essential gene for yeast vegetative growth. Nucleotide sequence analysis revealed the presence of an intron within the SEC14 structural gene, and predicted the synthesis of a hydrophilic polypeptide of 35 kD in molecular mass. In confirmation, immunoprecipitation experiments demonstrated SEC14p to be an unglycosylated polypeptide, with an apparent molecular mass of some 37 kD, that behaved predominantly as a cytosolic protein in subcellular fractionation experiments. These data were consistent with the notion that SEC14p is a cytosolic factor that promotes protein export from yeast Golgi. Additional radiolabeling experiments also revealed the presence of SEC14p-related polypeptides in extracts prepared from the yeasts Kluyveromyces lactis and Schizosaccharomyces pombe. Furthermore, the K. lactis SEC14p was able to functionally complement S. cerevisiae sec14ts defects. These data suggested a degree of conservation of SEC14p structure and function in these yeasts species.


Ablation of the calpain-targeted site in cardiac myosin binding protein-C is cardioprotective during ischemia-reperfusion injury.
Cardiac myosin binding protein-C (cMyBP-C) phosphorylation is essential for normal heart function and protects the heart from ischemia-reperfusion (I/R) injury. It is known that protein kinase-A (PKA)-mediated phosphorylation of cMyBP-C prevents I/R-dependent proteolysis, whereas dephosphorylation of cMyBP-C at PKA sites correlates with its degradation. While sites on cMyBP-C associated with phosphorylation and proteolysis co-localize, the mechanisms that link cMyBP-C phosphorylation and proteolysis during cardioprotection are not well understood. Therefore, we aimed to determine if abrogation of cMyBP-C proteolysis in association with calpain, a calcium-activated protease, confers cardioprotection during I/R injury. Calpain is activated in both human ischemic heart samples and ischemic mouse myocardium where cMyBP-C is dephosphorylated and undergoes proteolysis. Moreover, cMyBP-C is a substrate for calpain proteolysis and cleaved by calpain at residues 272-TSLAGAGRR-280, a domain termed as the calpain-target site (CTS). Cardiac-specific transgenic (Tg) mice in which the CTS motif was ablated were bred into a cMyBP-C null background. These Tg mice were conclusively shown to possess a normal basal structure and function by analysis of histology, electron microscopy, immunofluorescence microscopy, Q-space MRI of tissue architecture, echocardiography, and hemodynamics. However, the genetic ablation of the CTS motif conferred resistance to calpain-mediated proteolysis of cMyBP-C. Following I/R injury, the loss of the CTS reduced infarct size compared to non-transgenic controls. Collectively, these findings demonstrate the physiological significance of calpain-targeted cMyBP-C proteolysis and provide a rationale for studying inhibition of calpain-mediated proteolysis of cMyBP-C as a therapeutic target for cardioprotection.


Substrate specificities of SR proteins in constitutive splicing are determined by their RNA recognition motifs and composite pre-mRNA exonic elements.
We report striking differences in the substrate specificities of two human SR proteins, SF2/ASF and SC35, in constitutive splicing. beta-Globin pre-mRNA (exons 1 and 2) is spliced indiscriminately with either SR protein. Human immunodeficiency virus tat pre-mRNA (exons 2 and 3) and immunoglobulin mu-chain (IgM) pre-mRNA (exons C3 and C4) are preferentially spliced with SF2/ASF and SC35, respectively. Using in vitro splicing with mutated or chimeric derivatives of the tat and IgM pre-mRNAs, we defined specific combinations of segments in the downstream exons, which mediate either positive or negative effects to confer SR protein specificity. A series of recombinant chimeric proteins consisting of domains of SF2/ASF and SC35 in various combinations was used to localize trans-acting domains responsible for substrate specificity. The RS domains of SF2/ASF and SC35 can be exchanged without effect on substrate specificity. The RNA recognition motifs (RRMs) of SF2/ASF are active only in the context of a two-RRM structure, and RRM2 has a dominant role in substrate specificity. In contrast, the single RRM of SC35 can function alone, but its substrate specificity can be influenced by the presence of an additional RRM. The RRMs behave as modules that, when present in different combinations, can have positive, neutral, or negative effects on splicing, depending upon the specific substrate. We conclude that SR protein-specific recognition of specific positive and negative pre-mRNA exonic elements via one or more RRMs is a crucial determinant of the substrate specificity of SR proteins in constitutive splicing.


High sensitive determination of laser-induced frequency shifts of ultracold cesium molecules.
We experimentally present a technique for sensitively determining the laser-induced frequency shifts of the long-range molecular vibrational and rotational levels. The scheme relies on an optical frequency shifter, leading to two laser beams with a precise and adjustable frequency interval. A series of photoassociation spectra are recorded with both beams inducing molecular lines, whose peak separation provides an accurate frequency ruler to measure the frequency shifts of cesium molecular levels, which have not yet been observed in previous reports. The data are compared to theoretical predictions and show a good agreement.


Cyclin D3 is essential for intestinal epithelial cell proliferation.
Intestinal epithelium is a complex organ that undergoes continuous proliferation. D-type cyclins bind cyclin-dependent kinases (Cdk4 and Cdk6) and are expressed during the transition from G0 into the S phase. Previously, we reported that cyclins D1 and D3 are induced by growth factors in two rat intestinal epithelial cell lines, IEC-6 and RIE-1. However, transforming growth factor beta induces G1 arrest in both intestinal cell lines without inhibiting cyclin D3, suggesting that cyclin D3 may not have essential functions in the gut. In the present study, we determined whether cyclin D3 is required for the transition from G0 into the S phase in intestinal epithelial cells. Microinjection of anti-cyclin D3 antiserum inhibited quiescent IEC-6 and RIE-1 cells from entering the S phase, while cells microinjected with a nonspecific mouse immunoglobin G continued to progress into the S phase. We also examined the expression of cyclin D3 in rat jejunal mucosa after fasting and refeeding. Cyclin D3 levels were not altered by fasting and refeeding; however, Cdk4 expression was suppressed by fasting and returned to control levels after refeeding. Our results suggest that cyclin D3 is essential for intestinal epithelial cell proliferation, although its expression is not regulated by dietary restriction.


Coexistence and survival in conservative Lotka-Volterra networks.
Analyzing coexistence and survival scenarios of Lotka-Volterra (LV) networks in which the total biomass is conserved is of vital importance for the characterization of long-term dynamics of ecological communities. Here, we introduce a classification scheme for coexistence scenarios in these conservative LV models and quantify the extinction process by employing the Pfaffian of the network's interaction matrix. We illustrate our findings on global stability properties for general systems of four and five species and find a generalized scaling law for the extinction time.


Effect of Mobile Phone Radiofrequency Electromagnetic Fields on.
Since cell phones emit radiofrequency electromagnetic fields (EMFs), this study tested the hypothesis that cell phones placed near the heart may interfere with the electrical rhythm of the heart or affect the blood pressure. Following informed consent, eighteen randomly selected apparently healthy male volunteers aged 21.44 ± 0.53 years had their blood pressure, pulse rates and ECG measured before and after acute exposure to a cell phone. The ECG parameters obtained were: heart rate (HR), QRS complex duration (QRS), PR interval (PR) and Corrected QT interval (QTc). Results are presented as mean ± SEM. Statistical analyses were done using two-tailed paired t test for blood pressure and pulse rate data and one way ANOVA with a post hoc Tukey test for the ECG data. P<0.05 was considered statistically significant. The blood pressure and pulse rates before and after exposure to the cell phone showed no significant difference. The ECG parameters (HR: beats/min, QRS:ms, PR:ms and QTc respectively) did not differ before (66.33 ± 2.50, 91.78 ± 1.36, 151.67 ± 5.39 and 395.44 ± 4.96), during (66.33 ± 2.40, 91.11 ± 1.61, 153.67 ± 5.06 and 394.33 ± 4.05) and after calls (67.22 ± 2.77, 91.11 ± 1.67, 157.44 ± 4.46 and 396.56 ± 4.93) compared to baseline (67.17 ± 2.19, 94.33 ± 1.57, 150.56 ± 4.93 and 399.56 ± 3.88). These results suggest that acute exposure to EMFs from cell phones placed near the heart may not interfere with the electrical activity of the heart or blood pressure in healthy individuals.


Consequences of the crosstalk between monocytes/macrophages and natural killer cells.
The interaction between natural killer (NK) cells and different other immune cells like T cells and dendritic cells is well-described, but the crosstalk with monocytes or macrophages and the nature of ligands/receptors implicated are just emerging. The macrophage-NK interaction is a major first-line defense against pathogens (bacteria, viruses, fungi, and parasites). The recruitment and the activation of NK cells to perform cytotoxicity or produce cytokines at the sites of inflammation are important to fight infections. The two main mechanisms by which macrophages can prime NK cells are (1) activation through soluble mediators such as IL-12, IL-18, and (2) stimulation through direct cell-to-cell contact. We will discuss the progress in matters of modulation of NK cell functions by monocytes and macrophages, in the steady state and during diseases.


The World Health Organization Recommendations for Trachoma Surveillance, Experience in Nepal and Added Benefit of Testing for Antibodies to Chlamydia trachomatis pgp3 Protein: NESTS Study.
The World Health Organization (WHO) now requires a second surveillance survey for trachoma after an impact assessment has found follicular trachoma (TF) <5% to determine if re-emergence has occurred. Using new WHO guidelines, we undertook surveillance surveys, and determined the prevalence of infection and antibody positivity, in two districts in Nepal. 20 clusters were randomly selected within each district, 15 were randomly selected for antibody testing. In each cluster, we randomly selected 50 children ages 1-9 years and 100 adults ≥15 years. TF and trachomatous trichiasis (TT) were evaluated. Conjunctival swabs to test for chlamydial infection using GenXpert platform were obtained, and dried blood spots were collected to test for antibodies to Chlamydia Trachomatis pgp3 using the Luminex platform. 3 cases of TF were found in the two districts, and one case of infection. Pgp3 antibody positivity was 2·4% (95% confidence interval: 1·4%, 3·7%), and did not increase with age (P = 0.24). No clustering of antibody positivity within communities was found. TT prevalence was <1/1,000 population. The surveillance surveys, as proposed by WHO, showed no evidence for re-emergence of trachoma in two districts of Nepal. The low level and no significant increase by age in seroprevalence of antibodies to C trachomatis pgp3 antigen deserve further investigation as a marker of interruption of transmission.


Effects of histone deacetylase inhibitor on extracellular matrix production in human nasal polyp organ cultures.
Nasal polyposis is associated with a chronic inflammatory condition of the sinonasal mucosa and involves myofibroblast differentiation and extracellular matrix (ECM) accumulation. Epigenetic modulation by histone deacetylase (HDAC) inhibitors including trichostatin A (TSA) has been reported to have inhibitory effects on myofibroblast differentiation in lung and renal fibroblasts. The purpose of this study was to investigate the inhibitory effect of TSA on myofibroblast differentiation and ECM production in nasal polyp organ cultures. Nasal polyp tissues from 18 patients were acquired during endoscopic sinus surgery. After organ culture, nasal polyps were stimulated with TGF-beta1 and then treated with TSA. Alpha-smooth muscle actin (α-SMA), fibronectin, and collagen type I expression levels were examined by reverse transcription-polymerase chain reaction (PCR), real-time PCR, Western blot, and immunofluorescent staining. HDAC2, HDAC4, and acetylated H4 expression levels were assayed by Western blot. Cytotoxicity was analyzed by the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay. The expression levels of α-SMA, fibronectin, and collagen type 1 were increased in nasal polyp after transforming growth factor (TGF) beta1 treatment. TSA-inhibited TGF-beta1 induced these gene and protein expression levels. Furthermore, TSA suppressed protein expression levels of HDAC2 and HDAC4. However, TSA induced hyperacetylation of histones H4. Treatment with TGF-beta1 with or without TSA did not have cytotoxic effect. These findings provide novel insights into the epigenetic regulation in myofibroblast differentiation and ECM production of nasal polyp. TSA could be a candidate of a therapeutic agent for reversing the TGF-beta1-induced ECM synthesis that leads to nasal polyp development.


Acute caffeine administration effect on brain activation patterns in mild cognitive impairment.
Previous studies showed that acute caffeine administration enhances task-related brain activation in elderly individuals with preserved cognition. To explore the effects of this widely used agent on cognition and brain activation in early phases of cognitive decline, we performed a double-blinded, placebo-controlled functional magnetic resonance imaging (fMRI) study during an n-back working memory task in 17 individuals with mild cognitive impairment (MCI) compared to 17 age-matched healthy controls (HC). All individuals were regular caffeine consumers with an overnight abstinence and given 200 mg caffeine versus placebo tablets 30 minutes before testing. Analyses included assessment of task-related activation (general linear model), functional connectivity (tensorial-independent component analysis, TICA), baseline perfusion (arterial spin labeling, ASL), grey matter density (voxel-based morphometry, VBM), and white matter microstructure (tract-based spatial statistics, TBSS). Acute caffeine administration induced a focal activation of the prefrontal areas in HC with a more diffuse and posteromedial activation pattern in MCI individuals. In MCI, TICA documented a significant caffeine-related enhancement in the prefrontal cortex, supplementary motor area, ventral premotor and parietal cortex as well as the basal ganglia and cerebellum. The absence of significant group differences in baseline ASL perfusion patterns supports a neuronal rather than a purely vascular origin of these differences. The VBM and TBSS analyses excluded potentially confounding differences in grey matter density and white matter microstructure between MCI and HC. The present findings suggest a posterior displacement of working memory-related brain activation patterns after caffeine administration in MCI that may represent a compensatory mechanism to counterbalance a frontal lobe dysfunction.


Activity of Moxifloxacin against Mycobacterium tuberculosis in Acid Phase and Nonreplicative-Persister Phenotype Phase in a Hollow-Fiber Infection Model.
A major goal for improving tuberculosis therapy is to identify drug regimens with improved efficacy and shorter treatment durations. Shorter therapies improve patient adherence to the antibiotic regimens, which, in turn, decreases resistance emergence. Mycobacterium tuberculosis exists in multiple metabolic states. At the initiation of therapy, the bulk of the population is in log-phase growth. Consequently, it is logical to focus initial therapy on those organisms. Moxifloxacin has good early bactericidal activity against log-phase bacteria and is a logical component of initial therapy. It would be optimal if this agent also possessed activity against acid-phase and nonreplicative-persister (NRP) phenotype organisms. In our hollow-fiber infection model, we studied multiple exposures to moxifloxacin (equivalent to 200 mg to 800 mg daily) against strain H37Rv in the acid phase and against strain 18b in streptomycin starvation, which is a model for NRP-phase organisms. Moxifloxacin possesses good activity against acid-phase organisms, generating cell killing of 3.75 log10(CFU/ml) (200 mg daily) to 5.16 log10(CFU/ml) (800 mg daily) over the 28 days of the experiment. Moxifloxacin also has activity against streptomycin-starved strain 18b. The 400- to 800-mg daily regimens achieved extinction at day 28, while the no-treatment control still had 1.96 log10(CFU/ml) culturable. The lowest dose (200 mg daily) still had 0.7 log10(CFU/ml) measurable at day 28, a net kill of 1.26 log10(CFU/ml). Moxifloxacin is an attractive agent for early therapy, because it possesses activity against three metabolic states of M. tuberculosis.


Emerging Pharmacologic Targets in Cerebral Cavernous Malformation and Potential Strategies to Alter the Natural History of a Difficult Disease: A Review.
Cerebral cavernous malformations (CCMs) are vascular lesions of the brain that may lead to hemorrhage, seizures, and neurologic deficits. Most are linked to loss-of-function mutations in 1 of 3 genes, namely CCM1 (originally called KRIT1), CCM2 (MGC4607), or CCM3 (PDCD10), that can either occur as sporadic events or are inherited in an autosomal dominant pattern with incomplete penetrance. Familial forms originate from germline mutations, often have multiple intracranial lesions that grow in size and number over time, and cause an earlier and more severe presentation. Despite active preclinical research on a few pharmacologic agents, clinical translation has been slow. Open surgery and, in some cases, stereotactic radiosurgery remain the only effective treatments, but these options are limited by lesion accessibility and are associated with nonnegligible rates of morbidity and mortality. We discuss the limits of CCM management and introduce findings from in vitro and in vivo studies that provide insight into CCM pathogenesis and indicate molecular mechanisms as potential therapeutic targets. These studies report dysregulated cellular pathways shared between CCM, cardiovascular diseases, and cancer. They also suggest the potential effectiveness of proper drug repurposing in association with, or as an alternative to, targeted interventions. We propose methods to exploit specific molecular pathways to design patient-tailored therapeutic approaches in CCM, with the aim to alter its natural progression. In this scenario, the lack of effective pharmacologic options remains a critical barrier that poses an unfulfilled and urgent medical need.


A Dedicated Type II NADPH Dehydrogenase Performs the Penultimate Step in the Biosynthesis of Vitamin K1 in Synechocystis and Arabidopsis.
Mutation of Arabidopsis thaliana NAD(P)H DEHYDROGENASE C1 (NDC1; At5g08740) results in the accumulation of demethylphylloquinone, a late biosynthetic intermediate of vitamin K1. Gene coexpression and phylogenomics analyses showed that conserved functional associations occur between vitamin K biosynthesis and NDC1 homologs throughout the prokaryotic and eukaryotic lineages. Deletion of Synechocystis ndbB, which encodes for one such homolog, resulted in the same defects as those observed in the cyanobacterial demethylnaphthoquinone methyltransferase knockout. Chemical modeling and assay of purified demethylnaphthoquinone methyltransferase demonstrated that, by virtue of the strong electrophilic nature of S-adenosyl-l-methionine, the transmethylation of the demethylated precursor of vitamin K is strictly dependent on the reduced form of its naphthoquinone ring. NDC1 was shown to catalyze such a prerequisite reduction by using NADPH and demethylphylloquinone as substrates and flavine adenine dinucleotide as a cofactor. NDC1 displayed Michaelis-Menten kinetics and was markedly inhibited by dicumarol, a competitive inhibitor of naphthoquinone oxidoreductases. These data demonstrate that the reduction of the demethylnaphthoquinone ring represents an authentic step in the biosynthetic pathway of vitamin K, that this reaction is enzymatically driven, and that a selection pressure is operating to retain type II NAD(P)H dehydrogenases in this process.


The effects of literacy interventions on single-word reading for individuals who use aided AAC: a systematic review.
The purpose of this systematic review was to investigate the effects of instruction on single-word reading of individuals who use aided augmentative and alternative communication (AAC). A systematic search identified nine single-case experimental design studies that involved 24 individuals who used aided AAC. Overall, the evidence indicated that instruction had positive effects on reading at the single-word level for individuals across ages and diagnostic categories (i.e., autism spectrum disorder (ASD), cerebral palsy (CP), Down syndrome, and intellectual disability). The studies revealed that these effects were consistent across a range of participant, intervention, and outcome measure characteristics. Phonological approaches, sight-word approaches, and a combination of these two approaches yielded very large effects. Despite the large effects, the findings must be viewed with caution due to limitations in the number of studies and participants and limitations in the reporting of detailed participant and intervention characteristics across the studies. In order to determine which interventions are most effective for which individuals, future research directions are discussed, including the need for greater specificity in describing participant and intervention characteristics, investigations into how to best measure intervention outcomes without requiring spoken responses, and investigations into longer-term interventions targeting a wider range of reading skills.


Notch-induced rat and human bone marrow stromal cell grafts reduce ischemic cell loss and ameliorate behavioral deficits in chronic stroke animals.
Gene transfection with Notch 1 intracellular domain and subsequent growth factor treatment stimulate neuron-like differentiation of bone marrow stromal cells (BMSCs). Here, we examined the potential of transplanting Notch-induced BMSCs to exert therapeutic effects in a rat model of chronic ischemic stroke. In experiment 1, Notch-induced rat BMSCs were intrastriatally transplanted in rats at 1 month after being subjected to transient occlusion of middle cerebral artery (MCAo). Compared to post-stroke/pretransplantation level, significant improvements in locomotor and neurological function were detected in stroke rats that received 100 k and 200 k BMSCs, but not in those that received 40 k BMSCs. Histological results revealed 9%-15% graft survival, which dose-dependently correlated with behavioral recovery. At 5 weeks post-transplantation, some grafted BMSCs were positive for the glial marker GFAP (about 5%), but only a few cells (2-5 cells per brain) were positive for the neuronal marker NeuN. However, at 12 weeks post-transplantation, where the number of GFAP-positive BMSCs was maintained (5%), there was a dramatic increase in NeuN-positive BMSCs (23%). In experiment 2, Notch-induced human BMSCs were intrastriatally transplanted in rats at 1 month following the same MCAo model. Improvements in both locomotor and neurological function were observed from day 7 to day 28 post-transplantation, with the high dose (180 k) displaying significantly better behavioral recovery than the low dose (90 k) or vehicle. There were no observable adverse behavioral effects during this study period that also involved chronic immunosuppression of all animals. Histological analyses revealed a modest 5%-7% graft survival, with few (<1%) cells expressing an intermediate MAP2 neuronal marker, but not glial or oligodendroglial markers. In addition, striatal peri-infarct cell loss was significantly reduced in transplanted stroke animals compared to vehicle-treated stroke animals. The present study demonstrates the potential of Notch-induced BMSC cell therapy for patients presenting with fixed ischemic stroke.


Functional selectin ligands mediating human CD34(+) cell interactions with bone marrow endothelium are enhanced postnatally.
Hematopoietic progenitor cells (HPCs) can home to the bone marrow (BM) after a simple intravenous injection, but the adhesive mechanisms mediating the initial interactions of human HPCs with the BM endothelium have not been evaluated in vivo. Using fluorescence intravital microscopy and homing assays in NOD/SCID mice, we show that endothelial selectins are necessary for human adult CD34(+) cell homing, since rolling on BM endothelium and retention in the BM compartment are drastically reduced (>90%) in endothelial selectin-deficient NOD/SCID mice. Comparative analyses of CD34(+) cells collected from adults and from cord blood (CB) reveal that neonatal cells display reduced rolling fractions compared with adult CD34(+) cells obtained from peripheral blood or BM, suggesting abnormal selectin ligand function on neonatal progenitors. Flow cytometric and intravital microscopy studies suggest that this defect results from nonfunctional P-selectin ligand on a subset ( approximately 30%) of neonatal CD34(+) cells. Further analyses indicate that P-selectin glycoprotein ligand-1 (PSGL-1) is expressed in a nonfunctional form among neonatal CD34(+) cells that do not bind P-selectin and that this subset is enriched in primitive CD34(+)CD38(lo/-) progenitors. These results underscore the potential to improve homing of CB CD34(+) cells to the BM by manipulation of selectins and their ligands.


Rasburicase-Induced Methemoglobinemia in a Patient with Aggressive Non-Hodgkin's Lymphoma.
Rasburicase is a recombinant urate oxidase enzyme that converts uric acid to allantoin (an inactive and soluble metabolite that is readily excreted in urine). It is used for the management of tumor lysis syndrome (TLS) in cancer patients receiving chemotherapy. Although rasburicase is a generally safe and effective treatment, it can be associated with the rare and potentially severe complication of methemoglobinemia. Here, we report a case of rasburicase-induced methemoglobinemia in a patient who was diagnosed with aggressive non-Hodgkin's lymphoma. A 74-year-old man with aggressive non-Hodgkin's lymphoma was admitted for initiation of chemotherapy. Upon admission, the patient was found to have hyperkalemia, hyperuricemia, hyperphosphatemia, elevated LDH levels, and acute renal failure. As a result, he was diagnosed with TLS. Rasburicase 6 mg was administered intravenously over a period of 30 min to treat TLS. Later, methemoglobinemia developed, with requirements for oxygen supplementation. Multiple units of packed red blood cells were transfused for recurrent significant anemia secondary to his cancer co-morbidity. The patient was tested for glucose-6 phosphate dehydrogenase (G6PD) deficiency, which returned negative; therefore, methylene blue was considered. After transfusion, the methemoglobin level normalized over the course of a few days, and the oxygen saturation improved without the use of methylene blue. However, during his hospitalization, the patient also developed a pulmonary embolism and had evidence of acute coronary syndrome. Later, the patient died of multiple complications related to his cancer co-morbidity on day 12 of admission. Blood transfusion and supplemental oxygen, without the use of methylene blue, may be an appropriate therapeutic alternative in rasburicase-induced methemoglobinemia treatment.


Ending the neglect of global oral health: time for radical action.
Oral diseases are a major global public health problem affecting over 3·5 billion people. However, dentistry has so far been unable to tackle this problem. A fundamentally different approach is now needed. In this second of two papers in a Series on oral health, we present a critique of dentistry, highlighting its key limitations and the urgent need for system reform. In high-income countries, the current treatment-dominated, increasingly high-technology, interventionist, and specialised approach is not tackling the underlying causes of disease and is not addressing inequalities in oral health. In low-income and middle-income countries (LMICs), the limitations of so-called westernised dentistry are at their most acute; dentistry is often unavailable, unaffordable, and inappropriate for the majority of these populations, but particularly the rural poor. Rather than being isolated and separated from the mainstream health-care system, dentistry needs to be more integrated, in particular with primary care services. The global drive for universal health coverage provides an ideal opportunity for this integration. Dental care systems should focus more on promoting and maintaining oral health and achieving greater oral health equity. Sugar, alcohol, and tobacco consumption, and their underlying social and commercial determinants, are common risk factors shared with a range of other non-communicable diseases (NCDs). Coherent and comprehensive regulation and legislation are needed to tackle these shared risk factors. In this Series paper, we focus on the need to reduce sugar consumption and describe how this can be achieved through the adoption of a range of upstream policies designed to combat the corporate strategies used by the global sugar industry to promote sugar consumption and profits. At present, the sugar industry is influencing dental research, oral health policy, and professional organisations through its well developed corporate strategies. The development of clearer and more transparent conflict of interest policies and procedures to limit and clarify the influence of the sugar industry on research, policy, and practice is needed. Combating the commercial determinants of oral diseases and other NCDs should be a major policy priority.


Microindentation for in vivo measurement of bone tissue mechanical properties in humans.
Bone tissue mechanical properties are deemed a key component of bone strength, but their assessment requires invasive procedures. Here we validate a new instrument, a reference point indentation (RPI) instrument, for measuring these tissue properties in vivo. The RPI instrument performs bone microindentation testing (BMT) by inserting a probe assembly through the skin covering the tibia and, after displacing periosteum, applying 20 indentation cycles at 2 Hz each with a maximum force of 11 N. We assessed 27 women with osteoporosis-related fractures and 8 controls of comparable ages. Measured total indentation distance (46.0 +/- 14 versus 31.7 +/- 3.3 microm, p = .008) and indentation distance increase (18.1 +/- 5.6 versus 12.3 +/- 2.9 microm, p = .008) were significantly greater in fracture patients than in controls. Areas under the receiver operating characteristic (ROC) curve for the two measurements were 93.1% (95% confidence interval [CI] 83.1-100) and 90.3% (95% CI 73.2-100), respectively. Interobserver coefficient of variation ranged from 8.7% to 15.5%, and the procedure was well tolerated. In a separate study of cadaveric human bone samples (n = 5), crack growth toughness and indentation distance increase correlated (r = -0.9036, p = .018), and scanning electron microscope images of cracks induced by indentation and by experimental fractures were similar. We conclude that BMT, by inducing microscopic fractures, directly measures bone mechanical properties at the tissue level. The technique is feasible for use in clinics with good reproducibility. It discriminates precisely between patients with and without fragility fracture and may provide clinicians and researchers with a direct in vivo measurement of bone tissue resistance to fracture.


Controlling In Vivo, Human Pulp Temperature Rise Caused by LED Curing Light Exposure.
The objective of this study was to evaluate the in vivo effectiveness of air spray to reduce pulp temperature rise during exposure of intact premolars to light emitted by a high-power LED light-curing unit (LCU). After local Ethics Committee approval (#255945), intact, upper first premolars requiring extraction for orthodontic reasons from five volunteers received infiltrative and intraligamental anesthesia. The teeth (n=9) were isolated using rubber dam, and a minute pulp exposure was attained. The sterile probe from a wireless, NIST-traceable, temperature acquisition system was inserted directly into the coronal pulp chamber. Real-time pulp temperature (PT) (°C) was continuously monitored, while the buccal surface was exposed to a polywave LED LCU (Bluephase 20i, Ivoclar Vivadent) for 30 seconds with simultaneous application of a lingually directed air spray (30s-H/AIR) or without (30s-H), with a seven-minute span between each exposure. Peak PT values were subjected to one-way, repeated-measures analysis of variance, and PT change from baseline (ΔT) during exposure was subjected to paired Student's t-test (α=0.05). Peak PT values of the 30s-H group were significantly higher than those of 30s-H/AIR group and those from baseline temperature (p<0.001), whereas peak PT values in the 30s-H/AIR group were significantly lower than the baseline temperature (p=0.003). The 30s-H/AIR group showed significantly lower ΔT values than did the 30s-H group (p<0.001). Applying air flow simultaneously with LED exposure prevents in vivo pulp temperature rise.