Quality, Non-clinical and Clinical Considerations for Biosimilar Monoclonal Antibody Development: EU, WHO, USA, Canada, and BRICS-TM Regulatory Guidelines.
Objective: The aim was to critically evaluate well-established regulatory agencies mAb biosimilar guidelines for development and marketing authorization about quality, efficacy and safety and compare to BRICS-TM regulations to identify challenges. Materials and Methods: The current valid guidelines of EMA, WHO, USFDA, BGTD/HC, ICH, and BRICS-TM were obtained from official websites and comparative qualitative review was performed. Results: The review revealed that Health Canada uses mAb specific guidelines from EMA or USFDA when necessary. The BRICS agencies (except Russia) have incorporated some or most of the WHO SBP TRS and related annexes in similar national biotechnological/biological guidelines; however, gaps or insufficient information have been identified. The Russian Federation has issued general product registration guideline/s with very brief information about mAbs. The TMMDA (Turkey) has published an updated biosimilar guideline which parallels those of the EMA and the ones from WHO; however, no mAb specific guidelines are published. COFEPRIS (Mexico) has published a biotechnological/biological product registration guideline with no information about mAb. The SAHPRA biosimilar guideline has an annex on mAbs which focuses on non-clinical and clinical aspects. The comparative evaluation of BRICS-TM agencies indicates a gap pertaining to clarification for physico-chemical characterization, manufacturing process, overages and compatibility requirements between biological substances and excipients specifically on mAbs. In vitro assay requirements seem quite aligned with those of WHO, whereas in vivo studies mostly have disparity in terms of necessity, type of studies as well as design and criteria. Clinical safety and efficacy studies are indicated in emerging regulatory agencies, however detailed information pertaining to design, size of populations, requirements for primary and secondary endpoints, clarity and evaluation criteria differ. In general, BRICS-TM agencies allow extrapolation of indications provided that pre-defined conditions are met. Interchangeability, switching and substitution of biosimilars are not defined in most of BRIC-TM guidelines whereas South Africa, by law, allows neither interchangeability nor substitution. Pediatric research remains questionable across BRICS-TM. Conclusions: EMA, USFDA guidelines are broadly aligned with WHO and in addition, they also contain specific requirements pertaining to their own region. BRICS-TM has considerably less defined mAb specific biosimilar development and comparability parameters in their published guidelines.


Reversal of P-glycoprotein-mediated multidrug resistance by the murine double minute 2 antagonist nutlin-3.
Murine double minute 2 (MDM2) negatively regulates the activity of the tumor suppressor protein p53. Nutlin-3 is a MDM2 inhibitor under preclinical investigation as nongenotoxic activator of the p53 pathway for cancer therapy. Here, nutlin-3 was evaluated for its activity alone or in combination with established chemotherapeutic drugs for antitumor action in chemosensitive and chemoresistant neuroblastoma and rhabdomyosarcoma cell lines. Effects of nutlin-3 single treatment were much more pronounced in p53 wild-type cell lines (IC(50)s <3 micromol/L) than in p53-mutated cell lines (IC(50)s >17 micromol/L). In sharp contrast to the expectations, nutlin-3 concentrations that did not affect viability of p53-mutated cell lines strongly increased the efficacy of vincristine in p53-mutated, P-glycoprotein (P-gp)-overexpressing cell lines (decrease in IC(50)s 92- to 3,434-fold). Similar results were obtained for other P-gp substrates. Moreover, nutlin-3 reduced efflux of rhodamine 123 and other fluorescence dyes that are effluxed by P-gp. Investigation of Madin-Darby canine kidney (MDCK) II cells stably transfected with plasmids encoding for P-gp (MDCKII MDR1) or multidrug resistance protein 1 (MRP-1, MDCKII MRP1) revealed that nutlin-3 not only interferes with P-gp but also affects MRP-1-mediated efflux. Kinetic studies and investigation of P-gp-ATPase activity showed that nutlin-3 is likely to act as a P-gp transport substrate. Examination of the nutlin-3 enantiomers nutlin-3a and nutlin-3b revealed that, in contrast to MDM2-inhibitory activity that is limited to nutlin-3a, both enantiomers similarly interfere with P-gp-mediated drug efflux. In conclusion, nutlin-3-induced inhibition of P-gp and MRP-1 was discovered as a novel anticancer mechanism of the substance in this report.


High-performance and site-directed in utero electroporation by a triple-electrode probe.
In utero electroporation is a powerful tool to transfect and manipulate neural-precursor cells of the rodent parietal cortex and their progeny in vivo. Although this technique can potentially target numerous brain areas, reliability of transfection in some brain regions is low or physical access is limited. Here we present a new in utero electroporation configuration based on the use of three electrodes, the relative position and polarities of which can be adjusted. The technique allows easy access and exceedingly reliable monolateral or bilateral transfection at brain locations that could only be sporadically targeted before. By improvement in the efficiency of the electrical field distribution, demonstrated here by a mathematical simulation, the multi-electrode configuration also extends the developmental timeframe for reliable in utero electroporation, allowing for the first time specific transfection of Purkinje cells in the rat cerebellum.


Bringing toxicology into the 21st century: a global call to action.
Conventional toxicological testing methods are often decades old, costly and low-throughput, with questionable relevance to the human condition. Several of these factors have contributed to a backlog of chemicals that have been inadequately assessed for toxicity. Some authorities have responded to this challenge by implementing large-scale testing programmes. Others have concluded that a paradigm shift in toxicology is warranted. One such call came in 2007 from the United States National Research Council (NRC), which articulated a vision of "21st century toxicology" based predominantly on non-animal techniques. Potential advantages of such an approach include the capacity to examine a far greater number of chemicals and biological outcomes at more relevant exposure levels; a substantial reduction in testing costs, time and animal use; and the grounding of regulatory decisions on human rather than rodent biology. In order for the NRC's and similar proposals to make a significant impact on regulatory toxicology in the foreseeable future, they must be translated into sustained multidisciplinary research programmes that are well co-ordinated and funded on a multinational level. The Humane Society is calling for a "big biology" project to meet this challenge. We are in the process of forging an international, multi-stakeholder consortium dedicated to implementing the NRC vision.


Killer-cell immunoglobulin-like receptors and falciparum malaria in southwest Nigeria.
Killer-cell immunoglobulin-like receptors (KIRs) are a group of natural killer cell receptors (NKRs) that regulate NK-cell-mediated production of interferon gamma (IFN-γ) in response to infection. These receptors have recently been suggested to influence the severity of clinical Plasmodium falciparum malaria infection. We examined the KIR locus in relation to malaria in children from southwest Nigeria. Sequence specific priming (SSP)-PCR was used to detect the KIR genes. The presence or absence of fifteen different KIR genes was determined in each individual and the proportions compared across 3 clinical groups; asymptomatic malaria, uncomplicated clinical malaria and severe clinical malaria. The genes KIR2DL5, KIR2DS3 and KIR2DS5 were present in a significantly higher proportion of individuals in the asymptomatic control group than in the malaria cases. Furthermore, KIR2DS3 and KIR2DS5 were present in a higher proportion of uncomplicated malaria cases than severe malaria cases. Carriage c-AB2 genotype (which comprises all centromeric KIR genes including KIR2DL5, KIR2DS3 and KIR2DS5) decreases with severity of the disease suggesting that the KIR AB profile might be associated with protection from severe malaria infection in this population in Nigeria.


Geriatric experience following cardiac arrest at six interventional cardiology centers in the United States 2006-2011: interplay of age, do-not-resuscitate order, and outcomes.
It is not known if aggressive postresuscitation care, including therapeutic hypothermia and percutaneous coronary intervention, benefits cardiac arrest survivors more than 75 years old. We compared treatments and outcomes of patients at six regional percutaneous coronary intervention centers in the United States to determine if aggressive care of elderly patients was warranted. Retrospective evaluation of registry data. Six interventional cardiology centers in the United States. Six hundred and twenty-five unresponsive cardiac arrest survivors aged 18-75 were compared with 129 similar patients aged more than 75. None. Cardiac arrest survivors aged more than 75 had more comorbidities (3.0 ± 1.6 vs 2.0 ± 1.6, p < 0.001), but were matched to younger patients in initial heart rhythm, witnessed arrests, bystander cardiopulmonary resuscitation, and total ischemic time. Patients aged more than 75 frequently underwent therapeutic hypothermia (97.7%), urgent coronary angiography (44.2%), and urgent percutaneous coronary intervention (24%). They had more sustained hyperglycemia (70.5% vs 59%, p = 0.015), less postcooling fever (25.2% vs 35.2%, p = 0.03), were more likely to have do-not-resuscitate orders (65.9% vs 48.2%, p < 0.001), and undergo withdrawal of life support (61.2% vs 47.5%, p = 0.005). Good functional outcome at 6 months (Cerebral Performance Category 1-2) was seen in 27.9% elderly versus 40.4% younger patients overall (p = 0.01) and in 44% versus 55% (p = 0.13) of patients with an initial shockable rhythm. Of 35 survivors more than 75 years old, 33 (94.8%) were classified as Cerebral Performance Category 1 or 2 at (mean) 6.5-month follow-up. In multivariable logistic regression modeling, age more than 75 was significantly associated with outcome only when the presence of a do-not-resuscitate order was excluded from the model. Elderly patients were more likely to have do-not-resuscitate orders and to undergo withdrawal of life support. Age was independently associated with outcome only when correction for do-not-resuscitate status was excluded, and functional outcomes of elderly survivors were similar to younger patients. Exclusion of patients more than 75 years old from aggressive care is not warranted on the basis of age alone.


BOLD activity during mental rotation and viewpoint-dependent object recognition.
We measured brain activity during mental rotation and object recognition with objects rotated around three different axes. Activity in the superior parietal lobe (SPL) increased proportionally to viewpoint disparity during mental rotation, but not during object recognition. In contrast, the fusiform gyrus was preferentially recruited in a viewpoint-dependent manner in recognition as compared to mental rotation. In addition, independent of the effect of viewpoint, object recognition was associated with ventral areas and mental rotation with dorsal areas. These results indicate that the similar behavioral effects of viewpoint obtained in these two tasks are based on different neural substrates. Such findings call into question the hypothesis that mental rotation is used to compensate for changes in viewpoint during object recognition.


Tumor microsatellite-instability status as a predictor of benefit from fluorouracil-based adjuvant chemotherapy for colon cancer.
Colon cancers with high-frequency microsatellite instability have clinical and pathological features that distinguish them from microsatellite-stable tumors. We investigated the usefulness of microsatellite-instability status as a predictor of the benefit of adjuvant chemotherapy with fluorouracil in stage II and stage III colon cancer. Tumor specimens were collected from patients with colon cancer who were enrolled in randomized trials of fluorouracil-based adjuvant chemotherapy. Microsatellite instability was assessed with the use of mononucleotide and dinucleotide markers. Of 570 tissue specimens, 95 (16.7 percent) exhibited high-frequency microsatellite instability. Among 287 patients who did not receive adjuvant therapy, those with tumors displaying high-frequency microsatellite instability had a better five-year rate of overall survival than patients with tumors exhibiting microsatellite stability or low-frequency instability (hazard ratio for death, 0.31 [95 percent confidence interval, 0.14 to 0.72]; P=0.004). Among patients who received adjuvant chemotherapy, high-frequency microsatellite instability was not correlated with increased overall survival (hazard ratio for death, 1.07 [95 percent confidence interval, 0.62 to 1.86]; P=0.80). The benefit of treatment differed significantly according to the microsatellite-instability status (P=0.01). Adjuvant chemotherapy improved overall survival among patients with microsatellite-stable tumors or tumors exhibiting low-frequency microsatellite instability, according to a multivariate analysis adjusted for stage and grade (hazard ratio for death, 0.72 [95 percent confidence interval, 0.53 to 0.99]; P=0.04). By contrast, there was no benefit of adjuvant chemotherapy in the group with high-frequency microsatellite instability. Fluorouracil-based adjuvant chemotherapy benefited patients with stage II or stage III colon cancer with microsatellite-stable tumors or tumors exhibiting low-frequency microsatellite instability but not those with tumors exhibiting high-frequency microsatellite instability.


VISTA-Rehab: a resource for stroke rehabilitation trials.
Stroke rehabilitation is a complex intervention. Many factors influence the interaction between the patient and the elements of the intervention. Rehabilitation interventions are aimed at altering different domains of patient outcome including body functions, activity and participation. As a consequence, randomised clinical trials in this area are difficult to design. We developed an archive of stroke rehabilitation trials (VISTA-Rehab) to act as a resource to help trialists model and design future rehabilitation studies. We developed specific eligibility criteria for the entry of stroke rehabilitation trials into the archive. We established a Steering Committee to oversee projects and publications and commenced the recruitment of rehabilitation trials into this resource. As of August 2009, VISTA-Rehab contains data from 23 stroke rehabilitation trials (>3400 patients). Demographic data, including age [median=73, interquartile range (63,79)], gender (male=53%) and initial dependency [median baseline Barthel index score=6, interquartile range (9,19)], are available for all patients. Outcome measures include the modified Rankin Scale, Barthel Index, Rivermead Motor Assessment, Fugl-Meyer Assessment, General Health Questionnaire and Nottingham Extended Activities of Daily Living Scale. VISTA-Rehab expands the Virtual International Stroke Trials Archive to include rehabilitation trials. Anonymised data can be used to examine questions specific to stroke rehabilitation and to generate novel hypotheses.


ESIPT and FRET probes for monitoring nanoparticle polymer coating stability.
Coating strategies of inorganic nanoparticles (NPs) can provide properties unavailable to the NP core alone, such as targeting, specific sensing, and increased biocompatibility. Non-covalent amphiphilic NP capping polymers function via hydrophobic interactions with surface ligands and are extensively used to transfer NPs to aqueous media. For applications of coated NPs as actuators (sensors, markers, or for drug delivery) in a complex environment, such as biological systems, it is important to achieve a deep understanding of the factors affecting coating stability and behavior. We have designed a system that tests the coating stability of amphiphilic polymers through a simple fluorescent readout using either polarity sensing ESIPT (excited state intramolecular proton transfer) dyes or NP FRET (Förster resonance energy transfer). The stability of the coating was determined in response to changes in polarity, pH and ionic strength in the medium. Using the ESIPT system we observed linear changes in signal up to ∼20-25% v/v of co-solvent addition, constituting a break point. Based on such data, we propose a model for coating instability and the important adjustable parameters, such as the electrical charge distribution. FRET data provided confirmatory evidence for the model. The ESIPT dyes and FRET based methods represent new, simple tools for testing NP coating stability in complex environments.


Dimenhydrinate in children with infectious gastroenteritis: a prospective, RCT.
Vomiting is a common symptom in children with infectious gastroenteritis. It contributes to fluid loss and is a limiting factor for oral rehydration therapy. Dimenhydrinate has traditionally been used for children with gastroenteritis in countries such as Canada and Germany. We investigated the efficacy and safety of dimenhydrinate in children with acute gastroenteritis. We performed a prospective, randomized, placebo-controlled, multicenter trial. We randomly assigned 243 children with presumed gastroenteritis and vomiting to rectal dimenhydrinate or placebo. Children with no or mild dehydration were included. All children received oral rehydration therapy. Primary outcome was defined as weight gain within 18 to 24 hours after randomization. Secondary outcomes were number of vomiting episodes, fluid intake, parents' assessment of well-being, number of diarrheal episodes, and admission rate to hospital. We recorded potential adverse effects. Change of weight did not differ between children who received dimenhydrinate or placebo. The mean number of vomiting episodes between randomization and follow-up visit was 0.64 in the dimenhydrinate group and 1.36 in the placebo group. In total, 69.6% of the children in the dimenhydrinate group versus 47.4% in the placebo group were free of vomiting between randomization and the follow-up visit. Hospital admission rate, fluid intake, general well-being of the children, and potential adverse effects, including the number of diarrhea episodes, were similar in both groups. Dimenhydrinate reduces the frequency of vomiting in children with mild dehydration; however, the overall benefit is low, because it does not improve oral rehydration and clinical outcome.


Signatures of positive selection at hemopexin (PEX) domain of matrix metalloproteinase-9 (MMP-9) gene.
Matrix metalloproteinases-9 (MMP-9) is an important cancer-associated, zinc-dependent endopeptidase. To investigate the natural selection hypothesis of MMP-9, the orthologous sequences from 12 vertebrates were compared and a molecular evolution analysis was performed. Results suggest that amino acid residues present in the middle region of the protein are more selectively constrained, whereas amino acid residues in the C-terminal region of the MMP-9 protein including exon 13 showed lowest conservation level in non-primate species, suggesting that it is an exon with fast evolving rate compared to the others analyzed. InterProScan analysis shows that exon 13 was located in hemopexin (PEX) domain of MMP-9. Positive selection was detected in PEX domain of MMP-9 protein between human and other species, which indicates that selective pressure may play a role in shaping the function of MMP-9 in the course of evolution.


Distribution of monocyte chemoattractant protein-1 (MCP-1 A-2518G) and chemokine receptor (CCR2-V64Ι) gene variants in hyperbilirubinemic newborns.
Hyperbilirubinemia is one of the most crucial syndromes, which is characterized by high levels of bilirubin, especially when it occurs in newborns. Bilirubin has cytoprotective properties with an antioxidant function and plays several major roles in the inflammation process with its members such as chemokines. The monocyte chemoattractant protein-1 (MCP-1) is a member of the C-C chemokine family and it has been associated with the inflammatory process. There are no data on the chemokine and its receptor genotypes in hyperbilirubinemic newborns to show their distribution. The aim of this study is to investigate the genotypic relationship of MCP-1 and its receptor CCR2-V64Ι with hyperbilirubinemia in Turkish newborns. A total of 85 newborns were included in the study: 20 infants with hyperbilirubinemia (hyperbilirubinemic group) and 65 infants without hyperbilirubinemia (non-hyperbilirubinemic group). Genotyping of MCP-1 A-2518G and CCR2-V64Ι gene polymorphisms were detected by PCR-RFLP, respectively. MCP-1 GG genotype in patients was higher than the controls and this genotype had 2.69 times higher risk for hyperbilirubinemic neonates (P: 0.20). The frequency of MCP-1 A-2518G G+ genotype in patients was higher than the controls (55.0% and 38.5%, respectively). The results of our preliminary study suggest that MCP-1 G+ genotype has the ability to increase the hyperbilirubinemia risk of newborns. These results should be focused on to research on a larger scale to confirm the findings.


Effect of lysine acetylsalicylate on biliary lipid secretion in dogs.
1. The influence of lysine acetylsalicylate on bile flow, erythritol clearance and bile salt, phospholipid and cholesterol secretion in bile was studied in unanaesthetized dogs fitted with a Thomas duodenal cannula. 2. Lysine acetylsalicylate induced a marked increase in bile flow and a parallel increase in erythritol clearance although the bile salt secretion remained unchanged; this suggests that the compound stimulated the formation of the canalicular (hepatocytic) bile salt-independent fraction of bile flow. 3. Lysine acetylsalicylate induced a significant decrease in biliary phospholipid and cholesterol secretion and the cholesterol saturation of bile was significantly reduced. 4. It is postulated that the decrease in phospholipid and cholesterol secretion resulted from the dilution of intracanalicular bile salts. This effect of lysine acetylsalicylate, and possibly of other bile salt-independent choleretics, may be of value in the treatment of cholesterol gallstones in man.


PAK2-c-Myc-PKM2 axis plays an essential role in head and neck oncogenesis via regulating Warburg effect.
The histone modifiers (HMs) are crucial for chromatin dynamics and gene expression; however, their dysregulated expression has been observed in various abnormalities including cancer. In this study, we have analyzed the expression of HMs in microarray profiles of head and neck cancer (HNC), wherein a highly significant overexpression of p21-activated kinase 2 (PAK2) was identified which was further validated in HNC patients. The elevated expression of PAK2 positively correlated with enhanced cell proliferation, aerobic glycolysis and chemoresistance and was associated with the poor clinical outcome of HNC patients. Further, dissection of molecular mechanism revealed an association of PAK2 with c-Myc and c-Myc-dependent PKM2 overexpression, wherein we showed that PAK2 upregulates c-Myc expression and c-Myc thereby binds to PKM promoter and induces PKM2 expression. We observed that PAK2-c-Myc-PKM2 axis is critical for oncogenic cellular proliferation. Depletion of PAK2 disturbs the axis and leads to downregulation of c-Myc and thereby PKM2 expression, which resulted in reduced aerobic glycolysis, proliferation and chemotherapeutic resistance of HNC cells. Moreover, the c-Myc complementation rescued PAK2 depletion effects and restored aerobic glycolysis, proliferation, migration and invasion in PAK2-depleted cells. The global transcriptome analysis of PAK2-depleted HNC cells revealed the downregulation of various genes involved in active cell proliferation, which indicates that PAK2 overexpression is critical for HNC progression. Together, these results suggest that the axis of PAK2-c-Myc-PKM2 is critical for HNC progression and could be a therapeutic target to reduce the cell proliferation and acquired chemoresistance and might enhance the efficacy of standard chemotherapy which will help in better management of HNC patients.


Catecholamine effects upon rat hypothalamic corticotropin-releasing hormone secretion in vitro.
To further our understanding of the functional role of catecholaminergic systems in regulating hypothalamic corticotropin-releasing hormone (CRH) secretion, we assessed the direct effects of a multiplicity of catecholamine agonists and antagonists on hypothalamic CRH secretion. To accomplish this, we used an in vitro rat hypothalamic organ culture system in which CRH secretion from single explants was evaluated by a specific RIA (IR-rCRH). Norepinephrine (NE) stimulated IR-rCRH secretion dose dependently, with peak effects in the nanomolar range. The effect of NE was antagonized by the mixed alpha antagonist phentolamine, the alpha 1 antagonist prazosin, and the alpha 2 antagonist yohimbine, but not by the beta blocker, L-propanolol. Compatible with these data were the findings that the alpha 1 agonist phenylephrine and the alpha 2 agonist clonidine both stimulated IR-rCRH secretion in a dose-dependent fashion. On the other hand, whereas the beta agonist, isoproterenol, caused a weak, non-dose-dependent increase in IR-rCRH secretion, this effect could not be antagonized by L-propanolol. Despite pretreatment with serotonin and acetylcholine antagonists, the effect of NE upon IR-rCRH secretion was undiminished, suggesting that NE-induced CRH secretion is not mediated by either neurotransmitter. On the other hand, pretreatment with gamma-aminobutyric acid (GABA) attenuated NE-induced IR-rCRH secretion. Whereas epinephrine (E) stimulated IR-rCRH secretion, this occurred only at higher concentrations, and was antagonized by phentolamine, but not by L-propanolol. Dopamine (DA) had a weak stimulatory effect that could be antagonized by the DA1 receptor antagonist, SCH 23390, but not by phentolamine. We conclude that NE and E stimulate hypothalamic IR-rCRH secretion via alpha 1 and alpha 2 receptors. The effect of NE upon IR-rCRH secretion is not apparently mediated by serotonergic or cholinergic interneurons, but is modulated by the inhibitory neurotransmitter, GABA. These data support the idea that the central catecholaminergic systems are excitatory rather than inhibitory upon CRH secretion when acting directly at the hypothalamic level.


Activation of persulfate/copper by hydroxylamine via accelerating the cupric/cuprous redox couple.
Cuprous copper [Cu(I)] reacts with sodium persulfate (PDS) to generate sulfate radical SO4(-)•, but it has been seldom investigated owing to its instability and difficulty in dissolving it. This study proposes a new method to regenerate Cu(I) from cupric copper [Cu(II)] by addition of hydroxylamine (HA) to induce the continuous production of radicals through active PDS, and investigates the resulting enhanced methyl orange (MO) degradation efficiency and mechanism in the new system. HA accelerated the degradation of MO markedly in the pH range from 6.0 to 8.0 in the HA/Cu(II)/PDS process. Both SO4(-)• and hydroxyl radicals (•OH) were considered as the primary reactive radicals in the process. The MO degradation in the HA/Cu(II)/PDS process can be divided into three stages: the fast stage, the transitory stage, and the low stage. MO degradation was enhanced with increased dosage of PDS. Although high dosage of HA could accelerate the transformation of the Cu(II)/Cu(I) cycle to produce more reactive radicals, excess HA can quench the reactive radicals. This study indicates that through a copper-redox cycling mechanism by HA, the production of SO4(-)• and •OH can be strongly enhanced, and the effective pH range can be expanded to neutral conditions.


Template for the description of cell-based toxicological test methods to allow evaluation and regulatory use of the data.
Only few cell-based test methods are described by Organisation for Economic Co-operation and Development (OECD) test guidelines or other regulatory references (e.g., the European Pharmacopoeia). The majority of toxicity tests still falls into the category of non-guideline methods. Data from these tests may nevertheless be used to support regulatory decisions or to guide strategies to assess compounds (e.g., drugs, agrochemicals) during research and development if they fulfill basic requirements concerning their relevance, reproducibility and predictivity. Only a method description of sufficient clarity and detail allows interpretation and use of the data. To guide regulators faced with increasing amounts of data from non-guideline studies, the OECD formulated Guidance Document 211 (GD211) on method documentation for the purpose of safety assessment. As GD211 is targeted mainly at regulators, it leaves scientists less familiar with regulation uncertain as to what level of detail is required and how individual questions should be answered. Moreover, little attention was given to the description of the test system (i.e., cell culture) and the steps leading to it being established in the guidance. To address these issues, an annotated toxicity test method template (ToxTemp) was developed (i) to fulfill all requirements of GD211, (ii) to guide the user concerning the types of answers and detail of information required, (iii) to include acceptance criteria for test elements, and (iv) to define the cells sufficiently and transparently. The fully annotated ToxTemp is provided here, together with reference to a database containing exemplary descriptions of more than 20 cell-based tests.


The Development of the strain in dementia care scale (SDCS).
Though many staff gain satisfaction from working with people with dementia in residential facilities, they also experience significant stress. This is a serious issue because this in turn can affect the quality of care. There is, however, a lack of instruments to measure staff strain in the dementia-specific residential care environment, and the aim of this study, accordingly, was to develop the "Strain in Dementia Care Scale." The instrument was developed in three steps. In the first step, items were derived from six focus group discussions with 35 nurses in the United Kingdom, Australia, and Sweden concerning their experience of strain. In the second step, a preliminary 64-item scale was distributed to 927 dementia care staff in Australia and Sweden, which, based on exploratory factor analysis, resulted in a 29-item scale. In the final step, the 29-item scale was distributed to a new sample of 346 staff in Sweden, and the results were subjected to confirmatory factor analysis. The final scale comprised the following 27 items producing a five-factor solution: Frustrated empathy; difficulties understanding and interpreting; balancing competing needs; balancing emotional involvement; and lack of recognition. The scale can be used (a) as an outcome measurement in residential care intervention studies; (b) to help residential facilities identify interventions needed to improve staff well-being, and, by extension, those they care for; and


Decreased expression of neurofibromin contributes to epithelial-mesenchymal transition in neurofibromatosis type 1.
Plexiform and/or dermal neurofibromas are nerve sheath tumors of the peripheral nervous system that are usually present in individuals with neurofibromatosis type 1 (NF1). Neurofibromas arise from Schwann cells with biallelic inactivation of NF1, the gene that encodes neurofibromin. This protein is responsible for regulation of the Ras-mediated pathway, which has been shown to play a crucial role in epithelial-to-mesenchymal transition (EMT). EMT is a biological process that occurs during embryogenesis and wound healing and is involved in pathological processes such as organ fibrosis and cancer metastasis. However, the relationship between neurofibromin and EMT has not been elucidated. We investigated whether the EMT-related signaling pathway was upregulated in NF1-associated neurofibromas and Schwann cells by assessing the expression levels of the EMT-related transcription factors Snail, Slug, Twist, ZEB1 and ZEB2. Immunohistochemical studies and quantitative reverse transcription polymerase chain reaction revealed an increase in the expression levels of EMT-related transcription factors in neurofibroma specimens and NF1-derived Schwann cells (sNF96.2). In addition, the silencing of NF1 by siRNA induced the expression of EMT-related transcription factors in normal human Schwann cells and in epithelial-like breast cancer cells. Our findings suggest that the loss of neurofibromin activated the EMT-related signaling pathway and that the excessive mesenchymal reaction may play a key role in the development of NF1-associated neurofibromas.


Structural role of conserved Asn179 in the short-chain dehydrogenase/reductase scaffold.
Short-chain dehydrogenases/reductases (SDR) constitute a large family of enzymes found in all forms of life. Despite a low level of sequence identity, the three-dimensional structures determined display a nearly superimposable alpha/beta folding pattern. We identified a conserved asparagine residue located within strand betaF and analyzed its role in the short-chain dehydrogenase/reductase architecture. Mutagenetic replacement of Asn179 by Ala in bacterial 3beta/17beta-hydroxysteroid dehydrogenase yields a folded, but enzymatically inactive enzyme, which is significantly more resistant to denaturation by guanidinium hydrochloride. Crystallographic analysis of the wild-type enzyme at 1.2-A resolution reveals a hydrogen bonding network, including a buried and well-ordered water molecule connecting strands betaE to betaF, a common feature found in 16 of 21 known three-dimensional structures of the family. Based on these results, we hypothesize that in mammalian 11beta-hydroxysteroid dehydrogenase the essential Asn-linked glycosylation site, which corresponds to the conserved segment, displays similar structural features and has a central role to maintain the SDR scaffold.


P Granules Protect RNA Interference Genes from Silencing by piRNAs.
P granules are perinuclear condensates in C. elegans germ cells proposed to serve as hubs for self/non-self RNA discrimination by Argonautes. We report that a mutant (meg-3 meg-4) that does not assemble P granules in primordial germ cells loses competence for RNA-interference over several generations and accumulates silencing small RNAs against hundreds of endogenous genes, including the RNA-interference genes rde-11 and sid-1. In wild type, rde-11 and sid-1 transcripts are heavily targeted by piRNAs and accumulate in P granules but maintain expression. In the primordial germ cells of meg-3 meg-4 mutants, rde-11 and sid-1 transcripts disperse in the cytoplasm with the small RNA biogenesis machinery, become hyper-targeted by secondary sRNAs, and are eventually silenced. Silencing requires the PIWI-class Argonaute PRG-1 and the nuclear Argonaute HRDE-1 that maintains trans-generational silencing of piRNA targets. These observations support a "safe harbor" model for P granules in protecting germline transcripts from piRNA-initiated silencing.


A two-step Notch-dependant mechanism controls the selection of the polar cell pair in Drosophila oogenesis.
Organisers control the patterning and growth of many tissues and organs. Correctly regulating the size of these organisers is crucial for proper differentiation to occur. Organiser activity in the epithelium of the Drosophila ovarian follicle resides in a pair of cells called polar cells. It is known that these two cells are selected from a cluster of equivalent cells. However, the mechanisms responsible for this selection are still unclear. Here, we present evidence that the selection of the two cells is not random but, by contrast, depends on an atypical two-step Notch-dependent mechanism. We show that this sequential process begins when one cell becomes refractory to Notch activation and is selected as the initial polar cell. This cell then produces a Delta signal that induces a high level of Notch activation in one other cell within the cluster. This Notch activity prevents elimination by apoptosis, allowing its selection as the second polar cell. Therefore, the mechanism used to select precisely two cells from among an equivalence group involves an inductive Delta signal that originates from one cell, itself unable to respond to Notch activation, and results in one other cell being selected to adopt the same fate. Given its properties, this two-step Notch-dependent mechanism represents a novel aspect of Notch action.


Spatial patterns of neutral and functional genetic variations reveal patterns of local adaptation in raccoon (Procyon lotor) populations exposed to raccoon rabies.
Local adaptation is necessary for population survival and depends on the interplay between responses to selective forces and demographic processes that introduce or retain adaptive and maladaptive attributes. Host-parasite systems are dynamic, varying in space and time, where both host and parasites must adapt to their ever-changing environment in order to survive. We investigated patterns of local adaptation in raccoon populations with varying temporal exposure to the raccoon rabies virus (RRV). RRV infects approximately 85% of the population when epizootic and has been presumed to be completely lethal once contracted; however, disease challenge experiments and varying spatial patterns of RRV spread suggest some level of immunity may exist. We first assessed patterns of local adaptation in raccoon populations along the eastern seaboard of North America by contrasting spatial patterns of neutral (microsatellite loci) and functional, major histocompatibility complex (MHC) genetic diversity and structure. We explored variation of MHC allele frequencies in the light of temporal population exposure to RRV (0-60 years) and specific RRV strains in infected raccoons. Our results revealed high levels of MHC variation (66 DRB exon 2 alleles) and pronounced genetic structure relative to neutral microsatellite loci, indicative of local adaptation. We found a positive association linking MHC genetic diversity and temporal RRV exposure, but no association with susceptibility and resistance to RRV strains. These results have implications for landscape epidemiology studies seeking to predict the spread of RRV and present an example of how population demographics influence the degree to which populations adapt to local selective pressures.


Effects of a high-fat diet exposure in utero on the metabolic syndrome-like phenomenon in mouse offspring through epigenetic changes in adipocytokine gene expression.
The links between obesity in parents and their offspring and the role of genes and a shared environment are not completely understood. Adipocytokines such as leptin and adiponectin play important roles in glucose and lipid metabolism. Therefore, we examined whether the offspring from dams exposed to a high-fat diet during pregnancy (OH mice) exhibited hypertension, insulin resistance, and hyperlipidemia along with epigenetic changes in the expression of adipocytokine genes. OH mice were significantly heavier than the offspring of dams exposed to a control diet during pregnancy (OC mice) from 14 wk of age after an increased caloric intake from 8 wk. OH mice exhibited higher blood pressure and worse glucose tolerance than the OC mice at 24 wk. Total triglyceride and leptin levels were significantly higher and the adiponectin level was significantly lower in OH compared with OC mice at 12 wk of age. This was associated with changes in leptin and adiponectin expression in white adipose tissue. There were lower acetylation and higher methylation levels of histone H3 at lysine 9 of the promoter of adiponectin in adipose tissues of OH mice at 2 wk of age as well as at 12 and 24 wk of age compared with OC mice. In contrast, methylation of histone 4 at lysine 20 in the leptin promoter was significantly higher in OH compared with OC mice. Thus, exposure to a high-fat diet in utero might cause a metabolic syndrome-like phenomenon through epigenetic modifications of adipocytokine, adiponectin, and leptin gene expression.


Interests of the Non-Human Primate Models for HIV Cure Research.
Non-human primate (NHP) models are important for vaccine development and also contribute to HIV cure research. Although none of the animal models are perfect, NHPs enable the exploration of important questions about tissue viral reservoirs and the development of intervention strategies. In this review, we describe recent advances in the use of these models for HIV cure research and highlight the progress that has been made as well as limitations using these models. The main NHP models used are (i) the macaque, in which simian immunodeficiency virus (SIVmac) infection displays similar replication profiles as to HIV in humans, and (ii) the macaque infected by a recombinant virus (SHIV) consisting of SIVmac expressing the HIV envelope gene serving for studies analyzing the impact of anti-HIV Env broadly neutralizing antibodies. Lessons for HIV cure that can be learned from studying the natural host of SIV are also presented here. An overview of the most promising and less well explored HIV cure strategies tested in NHP models will be given.


Clavicular hook plate may induce subacromial shoulder impingement and rotator cuff lesion--dynamic sonographic evaluation.
Clavicular hook plates are effective fixation devices for distal clavicle fractures and severe acromioclavicular joint dislocations. However, increasing number of studies has revealed that subacromial portion of the hook may induce acromial bony erosion, shoulder impingement, or even rotator cuff damage. By sonographic evaluation, we thus intended to determine whether the presence of hook plate may induce subacromial shoulder impingement and its relationship relative to surrounding subacromial structures. We prospectively followed 40 patients with either distal clavicle fracture or acromioclavicular joint dislocation that had surgery using the Arbeitsgemeinschaft für Osteosynthesefragen (AO) clavicular hook plate. All patients were evaluated by monthly clinical and radiographic examinations. Static and dynamic musculoskeletal sonography examinations were performed at final follow-up before implant removal. Clinical results for pain, shoulder function, and range of motion were evaluated using Constant-Murley and Disability of Arm, Shoulder, and Hand (DASH) scores. Clinically, 15 out of 40 patients (37.5%) presented with subacromial impingement syndrome and their functional scores were poorer than the non-impinged patients. Among them, six patients were noted to have rotator cuff lesion. Acromial erosion caused by hook pressure developed in 20 patients (50%). We demonstrated by musculoskeletal sonography that clavicular hook plate caused subacromial shoulder impingement and rotator cuff lesion. The data also suggest an association between hardware-induced impingement and poorer functional scores. To our knowledge, the only solution is removal of the implant after bony consolidation/ligamentous healing has taken place. Thus, we advocate the removal of the implant as soon as bony union and/or ligamentous healing is achieved.


Nonalcoholic fatty liver disease and the renin-angiotensin system blockers in the patients with chronic kidney disease.
Recent data suggest that the renin-angiotensin-aldosteron system (RAAS) may be of importance in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). We were interested to investigate whether the therapy with RAAS blockers in patients with different stages of chronic kidney disease (CKD) has any effect on steatosis and fibrosis grade; NAFLD documented by transient elastography (TE) (Fibroscan(®)-CAP). Of 191 patients with various stages of CKD there were 61 patients with CKD grade III and IV, 62 patients with end-stage renal disease treated with chronic hemodialysis and 68 renal transplant recipients. Liver stiffness was selected as the parameter to quantify liver fibrosis. Furthermore, the Controlled Attenuation Parameter (CAP) was used to detect and quantify liver steatosis with the help of TE. CKD patients (p = 0.005) and CKD-NAFLD patients (p = 0.0005) with angiotensin-converting enzyme inhibitors (ACE-I) or angiotensin receptor blockers (ARBs) had statistically significant lower degree of liver stiffness in comparison to those without these medications (p = 0.005). Also, we were interested to explore is there any difference in fibrosis and steatosis grade due to use of ACE-I or ARBs. We did not find statistically significant differences between those two subgroups of patients with respect to liver steatosis/fibrosis. Based on our results, RAAS blockers could be an attractive option for the management of NAFLD. We believe that TE provides the opportunity of noninvasive screening of NAFLD in CKD patients. In further prospective analysis, we believe that by using TE as noninvasive method we could investigate are ACE-I/ARBs really effective medications for the treatment of NAFLD in CKD patients.


Telemedicine Use for Movement Disorders: A Global Survey.
Telemedicine is increasingly used to care for patients with movement disorders, but data regarding its global use are limited. To obtain baseline international data about telemedicine use among movement disorder clinicians. An online survey was sent to all 6,056 Movement Disorder Society members in 2015. Scope, reimbursement, and perceived quality of telemedicine were assessed. There were 549 respondents (9.1% overall response rate) from 83 countries. Most (85.8%) were physicians, and most (70.9%) worked in an academic or university practice. Half of respondents (n = 287, from 57 countries) used telemedicine for clinical care; activities included e-mail (63.2%), video visits (follow-up [39.7%] and new [35.2%]), and video-based education (35.2%). One hundred five respondents personally conducted video visits, most frequently to outpatient clinics (53.5%), patient homes (30.8%), and hospital inpatients (30.3%). The most common challenges were a limited neurological examination (58.9%) and technological difficulties (53.3%), and the most common benefits were reduced travel time (92.9%) and patient costs (60.1%). The most frequent reimbursements were none (39.0%), public insurance (24.5%), and patient payment (9.3%). Half of respondents planned to use telemedicine in the future, and three-quarters were interested in telemedicine education. More than 250 respondents around the world engage in telemedicine for movement disorders; most perceived benefit for patients, despite challenges and reimbursement for clinicians. Formal instruction on telemedicine is highly desired. Although the survey response was low and possibly biased to over represent those with telemedicine experience, the study provides baseline data for future comparison and to improve telemedicine delivery.


Impediments to comprehensive research on climate change and health.
During every climatic era Life on Earth is constrained by a limited range of climatic conditions, outside which thriving and then surviving becomes difficult. This applies at both planetary and organism (species) levels. Further, many causal influences of climate change on human health entail changes-often disruptive, sometimes irreversible-in complex system functioning. Understanding the diverse health risks from climate change, and their influence pathways, presents a challenge to environmental health researchers whose prior work has been in a more definable, specific and quantitative milieu. Extension of the research agenda and conceptual framework to assess present and future health risks from climate change may be constrained by three factors: (i) lack of historically-informed understanding of population-health sensitivity to climatic changes; (ii) an instinctual 'epidemiologising' tendency to choose research topics amenable to conventional epidemiological analysis and risk estimation; and (iii) under-confidence in relation to interdisciplinary collaborative scenario-based modeling of future health risks. These constraints must be recognized and remedied. And environmental researchers must argue for heightened public attention to today's macro-environmental threats to present and future population health-emphasising the ecological dimension of these determinants of long-term health that apply to whole populations and communities, not just to individuals and social groupings.


Parental Flooding During Conflict: A Psychometric Evaluation of a New Scale.
Parents who are overwhelmed by the intensity and aversive nature of child negative affect - those who are experiencing flooding - may be less likely to react effectively and instead may focus on escaping the aversive situation, disciplining either overly permissively or punitively to escape quickly from child negative affect. However, there are no validated self-report measures of the degree to which parents experience flooding, impeding the exploration of these relations. Thus, we created and evaluated the Parent Flooding scale (PFS), assessing the extent to which parents believe their children's negative affect during parent-child conflicts is unexpected, overwhelming and distressing. We studied its factorial validity, reliability, and concurrent validity in a community sample of 453 couples with 3- to 7-year-old children (51.9 % girls) recruited via random digit dialing. Confirmatory factor analyses indicated a one-factor solution with excellent internal consistency. Test-retest stability over an average of 5.6 months was high. Concurrent validity was suggested by the associations of flooding with parents' aggression toward their children, overreactive and lax discipline, parenting satisfaction, and parents' anger, as well as with child externalizing behavior and negative affect. Incrementally concurrent validity analyses indicated that flooding was a unique predictor of mothers' and fathers' overreactive discipline and fathers' parent-child aggression and lax discipline, over and above the contributions of parents' anger and children's negative affect. The present results support the psychometric validity of the PFS.


The contribution of insects to global forest deadwood decomposition.
The amount of carbon stored in deadwood is equivalent to about 8 per cent of the global forest carbon stocks1. The decomposition of deadwood is largely governed by climate2-5 with decomposer groups-such as microorganisms and insects-contributing to variations in the decomposition rates2,6,7. At the global scale, the contribution of insects to the decomposition of deadwood and carbon release remains poorly understood7. Here we present a field experiment of wood decomposition across 55 forest sites and 6 continents. We find that the deadwood decomposition rates increase with temperature, and the strongest temperature effect is found at high precipitation levels. Precipitation affects the decomposition rates negatively at low temperatures and positively at high temperatures. As a net effect-including the direct consumption by insects and indirect effects through interactions with microorganisms-insects accelerate the decomposition in tropical forests (3.9% median mass loss per year). In temperate and boreal forests, we find weak positive and negative effects with a median mass loss of 0.9 per cent and -0.1 per cent per year, respectively. Furthermore, we apply the experimentally derived decomposition function to a global map of deadwood carbon synthesized from empirical and remote-sensing data, obtaining an estimate of 10.9 ± 3.2 petagram of carbon per year released from deadwood globally, with 93 per cent originating from tropical forests. Globally, the net effect of insects may account for 29 per cent of the carbon flux from deadwood, which suggests a functional importance of insects in the decomposition of deadwood and the carbon cycle.


The Fra-2 transgenic mouse model of systemic sclerosis.
In systemic sclerosis, microvascular injury often precedes the development of fibrosis. Whereas the development of digital ulcers and skin fibrosis causes high morbidity, the affection of internal organs, in particular complications such as interstitial lung disease and pulmonary (arterial) hypertension, account for the high disease-associated mortality of these patients. Vascular animal models of systemic sclerosis are of utmost importance to study pathophysiological aspects, to identify molecular key players, and to perform interventional proof of concept-studies. So far, animal models of systemic sclerosis have mainly reflected the pro-fibrotic features of the human disease. The Fra-2 (Fos-related antigen-2) transgenic mouse model simultaneously displays both pro-fibrotic and vascular characteristics of human systemic sclerosis.


Climate, duration, and N placement determine N2 O emissions in reduced tillage systems: a meta-analysis.
No-tillage and reduced tillage (NT/RT) management practices are being promoted in agroecosystems to reduce erosion, sequester additional soil C and reduce production costs. The impact of NT/RT on N2 O emissions, however, has been variable with both increases and decreases in emissions reported. Herein, we quantitatively synthesize studies on the short- and long-term impact of NT/RT on N2 O emissions in humid and dry climatic zones with emissions expressed on both an area- and crop yield-scaled basis. A meta-analysis was conducted on 239 direct comparisons between conventional tillage (CT) and NT/RT. In contrast to earlier studies, averaged across all comparisons, NT/RT did not alter N2 O emissions compared with CT. However, NT/RT significantly reduced N2 O emissions in experiments >10 years, especially in dry climates. No significant correlation was found between soil texture and the effect of NT/RT on N2 O emissions. When fertilizer-N was placed at ≥5 cm depth, NT/RT significantly reduced area-scaled N2 O emissions, in particular under humid climatic conditions. Compared to CT under dry climatic conditions, yield-scaled N2 O increased significantly (57%) when NT/RT was implemented <10 years, but decreased significantly (27%) after ≥10 years of NT/RT. There was a significant decrease in yield-scaled N2 O emissions in humid climates when fertilizer-N was placed at ≥5 cm depth. Therefore, in humid climates, deep placement of fertilizer-N is recommended when implementing NT/RT. In addition, NT/RT practices need to be sustained for a prolonged time, particularly in dry climates, to become an effective mitigation strategy for reducing N2 O emissions.


Erythropoietin and small molecule agonists of the tissue-protective erythropoietin receptor increase FXN expression in neuronal cells in vitro and in Fxn-deficient KIKO mice in vivo.
Friedreich's ataxia (FA) is a progressive neurodegenerative disease caused by reduced levels of the mitochondrial protein frataxin (FXN). Recombinant human erythropoietin (rhEPO) increased FXN protein in vitro and in early clinical studies, while no published reports evaluate rhEPO in animal models of FA. STS-E412 and STS-E424 are novel small molecule agonists of the tissue-protective, but not the erythropoietic EPO receptor. We find that rhEPO, STS-E412 and STS-E424 increase FXN expression in vitro and in vivo. RhEPO, STS-E412 and STS-E424 increase FXN by up to 2-fold in primary human cortical cells and in retinoic-acid differentiated murine P19 cells. In primary human cortical cells, the increase in FXN protein was accompanied by an increase in FXN mRNA, detectable within 4 h. RhEPO and low nanomolar concentrations of STS-E412 and STS-E424 also increase FXN in normal and FA patient-derived PBMC by 20%-40% within 24 h, an effect that was comparable to that by HDAC inhibitor 4b. In vivo, STS-E412 increased Fxn mRNA and protein in wild-type C57BL6/j mice. RhEPO, STS-E412, and STS-E424 increase FXN expression in the heart of FXN-deficient KIKO mice. In contrast, FXN expression in the brains of KIKO mice increased following treatment with STS-E412 and STS-E424, but not following treatment with rhEPO. Unexpectedly, rhEPO-treated KIKO mice developed severe splenomegaly, while no splenomegaly was observed in STS-E412- or STS-E424-treated mice. RhEPO, STS-E412 and STS-E424 upregulate FXN expression in vitro at equal efficacy, however, the effects of the small molecules on FXN expression in the CNS are superior to rhEPO in vivo.


Neglect and failures of human security in humanitarian settings: challenges and recommendations.
The lack of attention to basic safety and security standards by the humanitarian community is endemic. The tragic bombing and loss of life of the United Nations Assistance Mission in Iraq in 2003 is but one of many unfortunate examples; these incidents are increasing in number. Tools for establishing an organizational security framework are readily available. Capacity to implement this framework requires understanding the culture of safety and security and individual and organizational leadership. This report outlines the essential steps and components necessary to meet this requirement.


Pretreatment serum albumin as a predictor of cancer survival: a systematic review of the epidemiological literature.
There are several methods of assessing nutritional status in cancer of which serum albumin is one of the most commonly used. In recent years, the role of malnutrition as a predictor of survival in cancer has received considerable attention. As a result, it is reasonable to investigate whether serum albumin has utility as a prognostic indicator of cancer survival in cancer. This review summarizes all available epidemiological literature on the association between pretreatment serum albumin levels and survival in different types of cancer. A systematic search of the literature using the MEDLINE database (January 1995 through June 2010) to identify epidemiologic studies on the relationship between serum albumin and cancer survival. To be included in the review, a study must have: been published in English, reported on data collected in humans with any type of cancer, had serum albumin as one of the or only predicting factor, had survival as one of the outcome measures (primary or secondary) and had any of the following study designs (case-control, cohort, cross-sectional, case-series prospective, retrospective, nested case-control, ecologic, clinical trial, meta-analysis). Of the 29 studies reviewed on cancers of the gastrointestinal tract, all except three found higher serum albumin levels to be associated with better survival in multivariate analysis. Of the 10 studies reviewed on lung cancer, all excepting one found higher serum albumin levels to be associated with better survival. In 6 studies reviewed on female cancers and multiple cancers each, lower levels of serum albumin were associated with poor survival. Finally, in all 8 studies reviewed on patients with other cancer sites, lower levels of serum albumin were associated with poor survival. Pretreatment serum albumin levels provide useful prognostic significance in cancer. Accordingly, serum albumin level could be used in clinical trials to better define the baseline risk in cancer patients. A critical gap for demonstrating causality, however, is the absence of clinical trials demonstrating that raising albumin levels by means of intravenous infusion or by hyperalimentation decreases the excess risk of mortality in cancer.


Intestinal response to myeloablative chemotherapy in piglets.
Chemotherapy-induced myeloablation prior to allogeneic hematopoietic stem cell transplantation (HSCT) may be associated with severe toxicity. The current understanding of the pathophysiology of oral and gastrointestinal (GI) toxicity is largely derived from studies in rodents and very little is known from humans, especially children. We hypothesized that milk-fed piglets can be used as a clinically relevant model of GI-toxicity related to a standard conditioning chemotherapy (intravenous busulfan, Bu plus cyclophosphamide, Cy) used prior to HSCT. In study 1, dose-response relationships were investigated in three-day-old pigs (Landrace × Yorkshire × Duroc, n = 6). Pigs were given one of three different dose combinations of Bu and Cy (A: 4 days Bu, 2 × 1.6 mg/kg plus 2 days Cy, 60 mg/kg; B: 4 days Bu, 2 × 0.8 mg/kg plus 2 days Cy, 30 mg/kg; C: 2 days Bu at 2 × 1.6 mg/kg plus 1 day Cy, 60 mg/kg) and bone marrow was collected on day 11. Histology of bone marrow samples showed total aplasia after treatment A. Using this treatment in study 2, Bu-Cy pigs showed lowered spleen and intestinal weights and variable clinical signs of dehydration, sepsis, and pneumonia at tissue collection. Oral mucositis was evident as ulcers in the soft palate in 4/9 Bu-Cy pigs and villus height and brush-border enzyme activities were reduced, especially in the proximal intestine. There were no consistent effects on tissue cytokine levels (IL-8, IL-6, IL-1β, TNF-α) or blood chemistry values (electrolytes, liver transaminases, bilirubin, alkaline phosphatase), except that blood iron levels were higher in Bu-Cy pigs. We conclude that a myeloablative Bu-Cy regimen to piglets results in clinical signs comparable to those seen in pediatric patients subjected to myeloablative treatment prior to HSCT. Piglets may be used as a model for investigating chemotherapy-induced toxicity and dietary and medical interventions.


Mechanisms generating modification of benthos following tidal flat invasion by a Spartina hybrid.
Many coastal habitats are being substantially altered by introduced plants. In San Francisco Bay, California, USA, a hybrid form of the eastern cordgrass Spartina alterniflora is rapidly invading open mudflats in southern and central sections of the Bay, altering habitat, reducing macrofaunal densities, and shifting species composition. The invasion has resulted in significant losses of surface-feeding amphipods, bivalves, and cirratulid polychaetes, while subsurface feeding groups such as tubificid oligochaetes and capitellid polychaetes have been unaffected. In the present paper, we document the causes and mechanisms underlying the changes observed. Through a series of in situ manipulative experiments we examined the influence of hybrid Spartina canopy on a range of physical, chemical, and biological properties. The hybrid Spartina canopy exerted a strong influence on the hydrodynamic regime, triggering a series of physical, chemical, and biological changes in the benthic system. Relative to tidal flats, water velocity was reduced in hybrid patches, promoting deposition of fine-grained, organic-rich particles. The resulting changes in the sediment environment included increased porewater sulfide concentrations and anoxia, which led to poor survivorship of surface feeders such as bivalves, amphipods, and polychaetes. These are key taxa that support higher trophic levels including migratory shorebirds that feed on tidal flats. Altered flow in the Spartina canopy further contributed to changes in barnacle recruitment and resuspension of adult benthic invertebrates. Increased crab-induced predation pressure associated with Spartina invasion also contributed to changes in benthic invertebrate communities. Our results suggest that multiple physical, chemical, biotic, and trophic impacts of the Spartina invasion have resulted in substantial changes in benthic communities that are likely to have important effects on the entire ecosystem.


Influence of body mass index on revision rates after primary total knee arthroplasty.
Studies demonstrate that revision rates after primary total knee arthroplasty (TKA) tend to be higher in obese patients. However, the existence of a body mass index (BMI) threshold remains unexplored. We conducted a prospective cohort study of 2442 primary TKAs in 2035 patients (69.1 % women; mean age 72 years; mean follow-up 93 months, range 38-203). We evaluated the influence of BMI in five categories on all-cause revision after TKA using incidence rates (IR), hazard ratios (HR), and Kaplan-Meier survival analysis. Adjustment for baseline imbalances was performed using Cox regression analysis. Over the study period, 71 revisions occurred. Revision rates were 3.2 cases/1000 patient-years for patients of normal weight, 3.4/1000 for overweight patients and 3.0/1000 for patients classified as obese class I. At BMI ≥ 35, a significant increase in revision was noted. Comparing BMI ≥ 35 vs. < 35, there were 6.4 vs. 3.2 /1000. Crude HR was 2.0 [95 % confidence interval (CI) 1.2-3.3, p = 0.009], and the adjusted HR was 2.1 (95 % CI 1.2-3.6, p = 0.008). All-cause revision rates after primary TKA doubled in patients with a BMI of 35 but were similar in those with a BMI <35.


Continuous subcutaneous insulin infusion versus multiple daily injections in individuals with type 1 diabetes: a systematic review and meta-analysis.
The relative efficacy of continuous subcutaneous insulin infusion and multiple daily injections in individuals with type 1 diabetes is unclear. We sought to synthesize the existing evidence about the effect of continuous subcutaneous insulin infusion on glycosylated hemoglobin, hypoglycemic events, and time spent in hypoglycemia compared to multiple daily injections. We searched MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials and Database of Systematic Reviews, and Scopus from January 2008 through November 2015 for randomized controlled trials that enrolled children or adults with type 1 diabetes. Trials identified in a previous systematic review and published prior to 2008 were also included. We included 25 randomized controlled trials at moderate risk of bias. Meta-analysis showed a significant reduction in glycosylated hemoglobin in patients treated with continuous subcutaneous insulin infusion compared to multiple daily injections (mean difference 0.37; 95 % confidence interval, 0.24-0.51). This effect was demonstrated in both children and adults. There was no significant difference in minor or severe hypoglycemic events. Continuous subcutaneous insulin infusion was associated with lower incidence of nocturnal hypoglycemia. There was no significant difference in the time spent in hypoglycemia. In children and adults with type 1 diabetes and compared to multiple daily injections, continuous subcutaneous insulin infusion is associated with a modest reduction in glycosylated hemoglobin. There was no difference in severe or minor hypoglycemia, but likely a lower incidence of nocturnal hypoglycemia with continuous subcutaneous insulin infusion.


Eccentricity effects on blur and depth perception.
Foveation and (de)focus are two important visual factors in designing near eye displays. Foveation can reduce computational load by lowering display details towards the visual periphery, while focal cues can reduce vergence-accommodation conflict thereby lessening visual discomfort in using near eye displays. We performed two psychophysical experiments to investigate the relationship between foveation and focus cues. The first study measured blur discrimination sensitivity as a function of visual eccentricity, where we found discrimination thresholds significantly lower than previously reported. The second study measured depth discrimination threshold where we found a clear dependency on visual eccentricity. We discuss the study results and suggest further investigation.


Analysis of depth-of-focus of synthetic aperture antenna arrays.
Interferometric synthetic aperture arrays (SAA) can be refocused at any range via digital processing of the raw data, called the visibility function. Such systems are sensitive, however, to ranging errors in digital refocusing. They, therefore, exhibit a limited depth of focus, because these errors are equivalent to introducing defocus in the system. We derive an analytical expression for the Strehl ratio of synthetic aperture arrays, which accounts for both the antenna voltage pattern and the focal shift factor encountered in imaging systems with a low Fresnel number. Assessment of the depth of focus of short-range imaging arrays is allowed and is illustrated for common array designs.


Possible pitfalls in the identification of glycophorin-binding proteins of Plasmodium falciparum.
Plasmodium falciparum proteins that bind to the putative erythrocyte receptor (glycophorin) have been identified in several laboratories by their ability to bind to glycophorin immobilized on aminoethyl-BioGel (AE-BioGel). We here report that several parasite proteins bind to AE-BioGel in the absence of coupled glycophorin. Binding is apparently due to the strong ion-exchange properties of the matrix, and is sensitive to ionic conditions such as the degree of equilibration of the matrix and the pH. The parasite proteins that bind to the blank column under appropriate conditions include proteins with the serological activities of S-antigen and Ag 23, which also bind to glycophorin-coupled AE-BioGel. In the light of these results, the glycophorin-binding specificity of these and other proteins reported to bind to glycophorin-coupled AE-BioGel will have to be reevaluated, preferably using a different support matrix.


Overexpression of HER2/HER3 and clinical feature of ovarian cancer.
Human epidermal growth factor receptor-2 (HER2) and 3 (HER3) belong to the epidermal growth factor receptor (EGFR) family of transmembrane receptor tyrosine kinases. In this study, we assessed HER2/HER3 expression levels in specimens of epithelial ovarian cancer and determined their correlation with clinical features of ovarian cancer. Tissue microarrays (TMAs) were prepared from paraffin blocks of 105 ovarian tumour samples. HER2, HER3, PI3K, Akt, p-Akt, mTOR, p-mTOR, S6, and p-S6 expression levels were investigated using immunohistochemistry (IHC). HER2 and HER3 amplifications were determined using in situ hybridization (ISH). The correlation between HER2/3 expression and disease outcome of the patients including surgical outcome, progression-free survival (PFS) and overall survival (OS) was analysed. HER2 positivity was 3.8% by IHC and 5.7% by ISH, whereas that of HER3 was 12.4% and 8.6%, respectively. HER2 status by either IHC or ISH was not related to PFS (p=0.128, 0.168, respectively) and OS (p=0.245, 0.164, respectively). However, the HER3 status determined using fluorescence ISH was associated with poor PFS (p=0.035 on log rank test), which was a significant risk factor even after adjusting other possible risk factors in multivariate analysis (hazard ratio=2.377 [1.18-7.49], p=0.021). Expressions of Akt, p-mTOR, and S6 were also related with poor progression (p=0.008, 0.049, 0.014, respectively). HER3 is possibly an independent marker for poor prognosis in individuals with ovarian cancer, as the HER3 signalling pathway is distinct from that of HER2. The possibility of targeted therapy for patients with HER3 alteration in ovarian cancer should be evaluated.


From Haemobartonella to hemoplasma: molecular methods provide new insights.
Hemotropic mycoplasmas (aka hemoplasmas) are the causative agents of infectious anemia in numerous mammalian species. Originally known as Haemobartonella and Eperythrozoon species, these organisms have been reclassified within the genus Mycoplasma. The development of new molecular assays has expanded our knowledge of this heterogeneous group of agents and allowed us to study their epidemiology and pathogenesis. The present review summarizes recently gained insights into feline hemotropic mycoplasmas, formerly known as Haemobartonella felis. Besides the two initially identified feline hemoplasma species, Mycoplasma haemofelis and Candidatus Mycoplasma haemominutum, we discovered a third novel hemoplasma in a Swiss pet cat; preliminary results suggest that the pathogenic potential of the latter agent depends on cofactors. In applying PCR-based assays, feline hemoplasma infections have been documented in domestic cats and wild felids worldwide. Differences between the three hemoplasmas in regard to response to antibiotic treatment and establishment of a carrier status have been reported. Additionally, besides an ostensible vector-borne transmission, direct transmission by aggressive interaction of cats or interspecies transmission might play a role in the epidemiology of these organisms. Based on a potential vector-borne and interspecies transmission, a zoonotic potential of hemoplasmas should be further investigated.


A prospective review of the use of ketamine to facilitate endotracheal intubation in the helicopter emergency medical services (HEMS) setting.
Ketamine is a dissociative agent used for sedation and intubation in various clinical settings. Despite its proven haemodynamic safety, ketamine has not been widely used in prehospital medicine. This study examined the use of ketamine in helicopter emergency medical services (HEMS). This prospective cohort study enrolled all patients transported by a single HEMS program in whom ketamine was used to facilitate intubation. Data were collected using standard forms by two independent trained research staff. Demographics, medical condition, intubation conditions, vital signs (pre and post drug administration) and complications were recorded. Proportions, medians with IQR, change scores and CIs are reported; differences were compared using paired t tests. During the 2-year study period, 71 patients received ketamine to facilitate endotracheal intubation. Ketamine was used most often in men (52 (73%)), and the median age was 49 years (IQR: 31, 69). Most patients were adults (70 (99%)) with medical illnesses (42 (59%)); 37 (52%) intubations were performed at the sending hospital, and 30 (42%) were performed on scene. A paramedic performed the intubation in 58 cases (82%). The median ketamine dose was 80 mg (IQR: 60, 100; ~ 1mg/kg); 53 (75%) patients also received a paralytic agent. Mean arterial pressure (2.3 mmHg; 95% CI: -8.0 to 3.3) and heart rate (0.45 beats/min, 95% CI: -4.9 to 4.0) changes failed to reach statistical or clinical significance. No differences were found between patients with suspected concomitant head injury and other patients with respect to ketamine dose, changes in vital signs and complications. Complications included: one (1.4%) interstitial IV, five (7%) failed intubations, five (7%) hypotension and four (6%) hypertension episodes, one (1%) bradycardia, two (3%) tachycardia and five (7%) deaths. Ketamine is an effective agent in facilitating intubation in a HEMS environment. Complications are similar to use in the controlled Emergency Department setting.


US Army split forward surgical team management of mass casualty events in Afghanistan: surgeon performed triage results in excellent outcomes.
U.S. Army "split"forward surgical teams (FST) currently provide most of the resuscitative surgical care for combat patients in Afghanistan. These small units typically comprised 10 personnel and two surgeons each, who frequently encounter mass casualty (MASCAL) situations in geographically isolated regions. This article evaluates the effectiveness of one split FST managing 43 MASCAL situations in two separate locations for more than a 14-month period in Afghanistan. An Institutional Review Board-approved review of all admission data of the 541st FST was conducted. Comparison was made between patients treated in MASCAL situations to those of patients treated in non-MASCAL events. Split-based US Army forward surgical elements in a combat environment in Afghanistan. Two hundred eighty-two patients were treated during MASCAL events and 479 in non-MASCAL situations. The primary endpoint was survival outcomes among trauma patients when 5 or more patients arrived simultaneously or if3 or more patients required immediate surgery. Four patients (1.70 percent) died in the MASCAL group compared with 12 (3.30 percent) in the non-MASCAL group. The mortality of patients receiving surgery at the FST was 2.73 percent and the mortality was 0.93 percent in those transferred without surgery. In the MASCAL group, 41 patients (14.5 percent) were critically injured and the critical mortality rate was 6.25 percent. In MASCAL events, 39 percent of patients required surgery compared with 44.9 percent in the non-MASCAL group. The average Injury Severity Score (ISS) of the most severely injured patient was 21.19 and ISS rapidly decreased to scores consistent with mild injury suggesting over triage at the scene. Despite very limited resources, the split FST can achieve, with appropriate triage, acceptable mortality outcomes in MASCAL situations. Over triage at the wounding scene is common and surgical intervention is frequently required.


Ascorbic acid for the common cold. A prophylactic and therapeutic trial.
Three hundred eleven employees of the National Institutes of Health volunteered to take 1 gm of ascorbic acid or lactose placebo in capsules three times a day for nine months. At the onset of a cold, the volunteers were given an additional 3 gm daily of either a placebo or ascorbic acid. One hundred ninety volunteers completed the study. Dropouts were defined as those who missed at least one month of drug ingestion. They represented 44% of the placebo group and 34% of those taking ascorbic acid. Analysis of these data showed that ascorbic acid had at best only a minor influence on the duration and severity of colds, and that the effects demonstrated might be explained equally well by a break in the double blind.


Basic number processing in children with specific learning disorders: Comorbidity of reading and mathematics disorders.
As well as being the hallmark of mathematics disorders, deficits in number processing have also been reported for individuals with reading disorders. The aim of the present study was to investigate separately the components of numerical processing affected in reading and mathematical disorders within the framework of the Triple Code Model. Children with reading disorders (RD), mathematics disorders (MD), comorbid deficits (RD + MD), and typically developing children (TD) were tested on verbal, visual-verbal, and nonverbal number tasks. As expected, children with MD were impaired across a broad range of numerical tasks. In contrast, children with RD were impaired in (visual-)verbal number tasks but showed age-appropriate performance in nonverbal number skills, suggesting their impairments were domain specific and related to their reading difficulties. The comorbid group showed an additive profile of the impairments of the two single-deficit groups. Performance in speeded verbal number tasks was related to rapid automatized naming, a measure of visual-verbal access in the RD but not in the MD group. The results indicate that deficits in number skills are due to different underlying cognitive deficits in children with RD compared to children with MD: a phonological deficit in RD and a deficit in processing numerosities in MD.


Inference of transposable element ancestry.
Most common methods for inferring transposable element (TE) evolutionary relationships are based on dividing TEs into subfamilies using shared diagnostic nucleotides. Although originally justified based on the "master gene" model of TE evolution, computational and experimental work indicates that many of the subfamilies generated by these methods contain multiple source elements. This implies that subfamily-based methods give an incomplete picture of TE relationships. Studies on selection, functional exaptation, and predictions of horizontal transfer may all be affected. Here, we develop a Bayesian method for inferring TE ancestry that gives the probability that each sequence was replicative, its frequency of replication, and the probability that each extant TE sequence came from each possible ancestral sequence. Applying our method to 986 members of the newly-discovered LAVA family of TEs, we show that there were far more source elements in the history of LAVA expansion than subfamilies identified using the CoSeg subfamily-classification program. We also identify multiple replicative elements in the AluSc subfamily in humans. Our results strongly indicate that a reassessment of subfamily structures is necessary to obtain accurate estimates of mutation processes, phylogenetic relationships and historical times of activity.


Glutamine: mode of action in critical illness.
A recent editorial in Critical Care Medicine was titled "Glutamine, a life-saving nutrient, but why?" (2003; 31:2555-2556). This review will attempt to utilize new understanding of gene-nutrient interactions and molecular medicine to address potential mechanisms by which glutamine may be lifesaving after critical illness and injury. Recent meta-analysis data reveal that glutamine seems to exert a beneficial effect on mortality in critically ill patients. However, this effect seems to be dose and route dependent. The questions that remain to be answered are in what settings and via what method of administration does this phamaconutrient show optimal benefit? It is likely that examination of the molecular mechanisms by which glutamine exerts its effects will lead to an understanding of how best to utilize glutamine as both a pharmacologic and a nutritional agent. Clearly, clinical critical illness leads to a marked deficiency in glutamine that is correlated with mortality in the intensive care unit setting. It makes obvious sense that the deficiency of this vital stress nutrient should be replaced. In addition, recent laboratory data reveal glutamine may act via mechanisms independent of its role as a metabolic fuel. These include enhanced stress protein response, attenuation of the inflammatory response, improved tissue metabolic function, and attenuation of oxidant stress. Present data indicate that glutamine functions as a metabolic fuel and "stress-signaling molecule" after illness and injury. Thus, deficiencies observed in critical illness demand replacement for both pharmacologic and metabolic optimization. Presently, randomized, multicenter, clinical trials utilizing glutamine as a pharmacologic and a nutritional agent are ongoing.


Novel pyridazinone inhibitors for vascular adhesion protein-1 (VAP-1): old target-new inhibition mode.
Vascular adhesion protein-1 (VAP-1) is a primary amine oxidase and a drug target for inflammatory and vascular diseases. Despite extensive attempts to develop potent, specific, and reversible inhibitors of its enzyme activity, the task has proven challenging. Here we report the synthesis, inhibitory activity, and molecular binding mode of novel pyridazinone inhibitors, which show specificity for VAP-1 over monoamine and diamine oxidases. The crystal structures of three inhibitor-VAP-1 complexes show that these compounds bind reversibly into a unique binding site in the active site channel. Although they are good inhibitors of human VAP-1, they do not inhibit rodent VAP-1 well. To investigate this further, we used homology modeling and structural comparison to identify amino acid differences, which explain the species-specific binding properties. Our results prove the potency and specificity of these new inhibitors, and the detailed characterization of their binding mode is of importance for further development of VAP-1 inhibitors.


NaCl adaptation in Rana ridibunda and a comparison with the euryhaline toad Bufo viridis.
The physiological adaptation of the frog Rana ridibunda to saline environment was studied. It was found that blood was always hypertonic to the external solution, but at the highest salinity tolerated (i.e. 300 mOsM) the osmotic gradient across the skin was nearly abolished. Water uptake by the living frog remained unchanged, whereas sodium transport across the skin decreased markedly. Neurohypophyseal hormone increased water uptake and sodium transport to levels similar to those in tap water frogs. Water content of the tissues was not affected by saline adaptation, although it varied appreciably under acute conditions. Oxygen consumption increased in dehydrated frogs, but not in adapted ones. The results are discussed and compared to the euryhaline toad Bufo viridis; the importance of high urea levels for high salt adaptation is stressed.


The uncertain timing of reaching 8 billion, peak world population, and other demographic milestones.
We present new probabilistic forecasts of the timing of the world's population reaching 8 billion, the world's peak population, and the date at which one-third or more of the world's population would be 60+ years old. The timing of these milestones, as well as the timing of the Day of 7 Billion, is uncertain. We compute that the 60 percent prediction interval for the Day of 8 Billion is between 2024 and 2033. Our figures show that there is around a 60 percent chance that one-third of the world's population would be 60+ years old in 2100. In the UN 2010 medium variant, that proportion never reaches one-third. As in our past forecasts (Lutz et al. 2001, 2008), we find the chance that the world's population will peak in this century to be around 84 percent and the timing of that peak to be highly uncertain. Focal days, like the Day of 7 Billion, play a role in raising public awareness of population issues, but they give a false sense of the certainty of our knowledge. The uncertainty of the timing of demographic milestones is not a constant of nature. Understanding the true extent of our demographic uncertainty can help motivate governments and other agencies to make the investments necessary to reduce it.


Soft and persistent-The influence of sand-flooring and calves on the resting behavior of a zoo-kept African elephant (Loxodonta africana) group.
Caring for all aspects of zoo elephants' well-being is considered a major challenge. Providing an appropriate flooring substrate to facilitate lying rest presents a meaningful part of a holistic management concept. Investigating the impact of a new sand flooring on the nocturnal resting behavior of a breeding group of seven African elephants living at one zoo revealed more total lying rest, longer bouts of lying rest and a reduced side preference in the adult females. With an average total daily lying rest of about 1.5-2.0 hrs, the investigated zoo elephants expressed longer lying rest compared to recently reported data from free-ranging individuals in Botswana. In addition, the presence of nursing calves in the observed elephant group seemed to impact the resting pattern of all group members, with around 60% of all lying bouts being discontinued after interruption by the youngsters. With respect to observed nursing during leaning rest, we encourage the installation of appropriate horizontal structures in breeding facilities to support leaning rest behavior of their female elephants. In doing so, zoos may improve rest quality of nursing females, and, in general, the welfare aspect of sleep for their elephants.


Exploring callous and unemotional traits in youth via general personality traits: An eye toward DSM-5.
The current study aimed at better understanding callous-unemotional (CU) traits in youth within a traditional personality trait/temperament framework as well as in relation to current proposals for the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5). Participants were 174 mothers and their sons age 11-16 years. Mothers and youth reported on youth CU traits and general personality trait/ temperament dimensions. Overall, analyses revealed significant unique associations of personality trait/temperament dimensions with CU total and subscale scores. Personality trait/temperament dimensions explained 36% to 58% of the variance in CU subscales and total score. Furthermore, specific personality dimensions differentially and uniquely predicted various CU subscales, indicating marked specificity in association such that these traits should be considered separately rather than as a single unit. Taken together, these results confirm the importance of considering traditional personality trait models to understand "callous and unemotional" traits and risk for psychopathy more fully. Additionally, our findings bear implications for the conceptualization and operationalization of these traits in DSM-5.


Medial temporal lobe atrophy, apolipoprotein genotype, and plasma homocysteine in Sri Lankan patients with Alzheimer's disease.
The authors studied the association of Alzheimer's disease (AD) with total plasma homocysteine (tHcy) and apolipoprotein E (apoE) genotype, and the usefulness of measuring medial temporal lobe thickness (MTL) thickness for the diagnosis of AD in Sri Lankan patients. Using criteria of the NINCDS-ADRDA, 23 AD patients and 21 controls were recruited. All underwent MTL-oriented computed tomographic (CT) scans, measurement of plasma tHcy, and apoE genotyping. Mean plasma tHcy was significantly higher in AD patients than controls (p=.001). This association was independent of age, sex, body mass index (BMI), serum folate and vitamin B12, and serum creatinine. The frequency of apoE4 allele was significantly higher (p=.003) in AD patients, and the adjusted odds ratio of AD for the presence of one or more apoE4 alleles compared with none was 10.39 (95% CI 1.77-61.10; p=.010). The mean minimum MTL thickness was significantly higher in control subjects compared to that of AD patients (p<.001). This first report of apoE4, plasma tHcy, MTL thickness, and AD from Sri Lanka shows that high plasma tHcy, the presence of apoE4 allele, and MTL atrophy are associated with AD.


MC-12, an annexin A1-based peptide, is effective in the treatment of experimental colitis.
Annexin A1 (ANXA1) inhibits NF-κB, a key regulator of inflammation, the common pathophysiological mechanism of inflammatory bowel diseases (IBD). MC-12, an ANXA1-based tripeptide, suppresses NF-κB activation. Here, we determined the efficacy of MC-12 in the control of IBD. Mice with colitis induced by dextran sodium sulfate (DSS) or 2,4,6-trinitro benzene sulfonic acid (TNBS) were treated with various doses of MC-12 administered intraperitoneally, orally or intrarectally. We determined colon length and the histological score of colitis, and assayed: in colon tissue the levels of TNF-α, IFN-γ, IL-1β, IL-6 and IL-10 by RT-PCR; prostaglandin E(2) (PGE(2)), cytoplasmic phospholipase A(2) (cPLA(2)) and myeloperoxidase by immunoassay; and COX-2 and NF- κB by immunohistochemistry; and in serum the levels of various cytokines by immunoassay. In both models MC-12: reversed dose-dependently colonic inflammation; inhibited by up to 47% myeloperoxidase activity; had a minimal effect on cytoplasmic phospholipase A(2); reduced significantly the induced levels of TNF-α, IFN-γ, IL-1β, IL-6 and IL-10, returning them to baseline. DSS and TNBS markedly activated NF-κB in colonic epithelial cells and MC-12 decreased this effect by 85.8% and 72.5%, respectively. MC-12 had a similar effect in cultured NCM460 normal colon epithelial cells. Finally, MC-12 suppressed the induction of COX-2 expression, the level of PGE(2) in the colon and PGE(2) metabolite in serum. In conclusion, MC-12, representing a novel class of short peptide inhibitors of NF-κB, has a strong effect against colitis in two preclinical models recapitulating features of human IBD. Its mechanism of action is complex and includes pronounced inhibition of NF-κB. MC-12 merits further development as an agent for the control of IBD.


Water induces a structural conversion and accelerates the oxygenation of carboxylate-bridged non-heme diiron enzyme synthetic analogues.
Recently, we reported the synthesis of a carboxylate-rich non-heme diiron enzyme model compound [Fe2(mu-O2CAr(Tol))4(4-CNPy)2] (1), where (-)O(2)CAr(Tol) is 2,6-di-p-tolylbenzoate and 4-CNPy is 4-cyanopyridine (Yoon, S.; Lippard, S. J. J. Am. Chem. Soc. 2005, 127, 8386-8397). A metal-to-ligand charge-transfer band in the visible region of the optical absorption spectrum involving the nitrogen-donor ligand endowed this complex with a distinctive red color that facilitated analysis of its chemistry. Following this strategy, we prepared and characterized two related isomeric complexes, windmill (3) and paddlewheel (4) species having the formula [Fe2(O2CAr(Tol))4(4-AcPy)2], where 4-AcPy is 4-acetylpyridine. In anhydrous solvents, 1 and 4 adopt paddlewheel structures, but upon the addition of water, they convert to aquated forms, windmill structures having the composition [Fe2(mu-O2CAr(Tol))2(O2CAr(Tol))2(4-RPy)2(H2O)2]. This conversion is favored at low temperature and was studied by NMR spectroscopy. A kinetic analysis of the aquation reaction was undertaken by stopped-flow measurements between 198 and 223 K for both 1 and 4, which revealed a first-order dependence on both the diiron compound and water. The oxygenation rates for the water-containing complexes are much faster than those for the corresponding anhydrous complexes, being 20-fold faster for 4 and 10-fold more rapid for 1. The presence or absence of water had little effect on the activation enthalpies, suggesting that the loss of water may not be necessary prior to dioxygen binding in the transition state.


α2A -Adrenergic receptor polymorphisms and mRNA expression levels are associated with delay discounting in cocaine users.
Cocaine users characteristically display preferences for smaller immediate rewards over larger delayed rewards, and this delay discounting (DD) has been proposed as an endophenotype of cocaine addiction. Recent evidence suggests that the norepinephrine system and more specifically the α2A -adrenergic receptor (ADRA2A) are impacted by chronic cocaine use while also being potentially involved in the neural mechanisms underlying DD. Hence, we investigated the effects of ADRA2A polymorphisms and ADRA2A mRNA expression levels on DD of cocaine users and stimulant-naïve controls. Two hundred and twenty-three participants (129 cocaine users and 94 stimulant-naïve healthy controls) completed a computerized DD paradigm and were genotyped for three single nucleotide polymorphisms (SNPs; rs1800544, rs521674 and rs602618) in the ADRA2A gene, while their peripheral ADRA2A mRNA expression was quantified in whole blood samples. The three SNPs were in near-perfect linkage disequilibrium. Accordingly, significant group*genotype interactions were found for all three ADRA2A variants revealing steeper DD in cocaine users (but not in controls) carrying the G-allele of SNP rs1800544, the T-allele of rs521674 and the C-allele of rs602618. Similarly, high ADRA2A mRNA expression levels were significantly associated with a reduced tendency to choose smaller more immediate rewards (over larger delayed rewards) in cocaine users but not in controls. As the relationship between DD and cocaine use was moderated by ADRA2A SNPs and by peripheral ADRA2A gene expression, we propose that the norepinephrine system is involved in DD deficits observed in cocaine using individuals. Consequently, pharmacological compounds targeting ADRA2As might be considered for the symptom-specific treatment of delay aversion in stimulant addiction.


Chemical structure of steroids in relation to promotion of growth of the vagina and uterus of the hypophysectomized rat.
In the hypophysectomized albino rat which is protected from contact with steroids in the ration and environment the uterus and vagina are highly atrophic but are sensitive indicators of activity of substances which promote their growth. Both the pituitary growth hormone and certain steroids have the common property of inducing growth of these tissues. The vaginal epithelium consists of 2 layers of cells which differ profoundly in their growth in response to steroids, depending on the molecular structure of these compounds. The differential response to modifications of chemical structures of steroids permits evaluation of the importance of the intramolecular components for the process of growth. The number and site of functional groups, the geometry of the molecule and the state of oxidation are of high importance in determining physiologic activity of steroids in the androstane series; these features are less specific in the estrane series. Side groups at positions C(3) and C(17) are of importance in the promotion of growth by steroids in the androstane series, but these active centers are not equivalent in their physiological influence. As a generalization, hydrogenation of the oxygen function at C(17) (but not at C(3)) and dehydrogenation at critical areas of the ring structure increase the quantitative efficacy of steroids in promoting growth. The position of double bonds and the state of oxidation at both C(3) and C(17) determine the qualitative type of growth-cellular pattern, which a compound in the androstane series induces in the vaginal epithelium.


Kinetics of the a-C3H5 + O2 reaction, investigated by photoionization using synchrotron radiation.
The kinetics of the combustion-relevant reaction of the allyl radical, a-C3H5, with molecular oxygen has been studied in a flow tube reactor at the vacuum ultraviolet (VUV) beamline of the Swiss Light Source storage ring, using the CRF-PEPICO (Combustion Reactions Followed by Photoelectron Photoion Coincidence Spectroscopy) setup. The ability to measure threshold photoelectron spectra enables a background-free detection of reactive species as well as an isomer-specific analysis of reaction products. Allyl was generated by direct photodissociation of allyl iodide at 266 nm and 213 nm and indirectly by the reaction of propene with Cl atoms, which were generated by photolysis from oxalyl chloride at 266 nm. Experiments were conducted at room temperature at low pressures between 0.8 and 3 mbar using Ar as the buffer gas and with excess O2 to maintain nearly pseudo-first-order reaction conditions. Whereas allyl was detected by photoionisation using synchrotron radiation, the main reaction product allyl peroxy was not observed due to dissociative ionisation of this weakly bound species. From the concentration-time profiles of the allyl signal, second-order rate constants between 1.35 × 1011 cm3 mol-1 s-1 at 0.8 mbar and 1.75 × 1011 cm3 mol-1 s-1 at 3 mbar were determined. The rates obtained for the different allyl radical generation schemes agree well with each other, but are about a factor of 2 higher than the ones reported previously using He as a buffer gas. The discrepancy is partly attributed to the higher collision efficiency of Ar causing a varying fall-off behavior. When allyl is produced by the reaction of propene with Cl atom, an unexpected product is observed at m/z = 68, which was identified as 1,3-butadienal in the threshold photoelectron spectrum. It is formed in a secondary reaction of allyl with the OCCl radical, which is generated in the 266 nm photolysis of oxalyl chloride.