Mechanisms of hydrogen bond formation between ionic liquids and cellulose and the influence of water content.
We study the dynamics of the formation of multiple hydrogen bonds between ionic liquid anions and cellulose using molecular dynamics simulations. We examine fifteen different ionic liquids composed of 1-alkyl-3-methylimidazolium cations ([Cnmim], n = 1, 2, 3, 4, 5) paired with either chloride, acetate or dimethylphosphate. We map the transitions of anions hydrogen bonded to cellulose into different bonding states. We find that increased tail length in the ionic liquids has only a very minor effect on these transitions, tending to slow the dynamics of the transitions and increasing the hydrogen bond lifetimes. Each anion can form up to four hydrogen bonds with cellulose. We find that this hydrogen bond "redundancy" leads to multiply bonded anions having lifetimes three to four times that of singly bound anions. Such redundant hydrogen bonds account for roughly half of all anion-cellulose hydrogen bonds. Additional simulations for [C2mim]Cl, [C2mim]Ac and [C2mim]DMP were performed at different water concentrations between 70 mol% and 90 mol%. It was found that water crowds the hydrogen bond-accepting sites of the anions, preventing interactions with cellulose. The more water that is present in the system, the more crowded these sites become. Thus, if a hydrogen bond between an anion and cellulose breaks, the likelihood that it will be replaced by a nearby water molecule increases as well. We show that the formation of these "redundant" hydrogen bonding states is greatly affected by the presence of water, leading to steep drops in hydrogen bonding between the anions and cellulose.


Valproic acid as a potential inhibitor of Plasmodium falciparum histone deacetylase 1 (PfHDAC1): an in silico approach.
A new Plasmodium falciparum histone deacetylase1 (PfHDAC1) homology model was built based on the highest sequence identity available template human histone deacetylase 2 structure. The generated model was carefully evaluated for stereochemical accuracy, folding correctness and overall structure quality. All evaluations were acceptable and consistent. Docking a group of hydroxamic acid histone deacetylase inhibitors and valproic acid has shown binding poses that agree well with inhibitor-bound histone deacetylase-solved structural interactions. Docking affinity dG scores were in agreement with available experimental binding affinities. Further, enzyme-ligand complex stability and reliability were investigated by running 5-nanosecond molecular dynamics simulations. Thorough analysis of the simulation trajectories has shown that enzyme-ligand complexes were stable during the simulation period. Interestingly, the calculated theoretical binding energies of the docked hydroxamic acid inhibitors have shown that the model can discriminate between strong and weaker inhibitors and agrees well with the experimental affinities reported in the literature. The model and the docking methodology can be used in screening virtual libraries for PfHDAC1 inhibitors, since the docking scores have ranked ligands in accordance with experimental binding affinities. Valproic acid calculated theoretical binding energy suggests that it may inhibit PfHDAC1.


Tyrosinase and tyrosinase related protein 1 alleles specify domestic cat coat color phenotypes of the albino and brown loci.
The genes encoding enzymes of the tyrosinase family are strong candidates for coat color variation in mammals. To investigate their influence in domestic cat coat color, we determined the complete nucleotide coding sequence of the domestic cat genes tyrosinase (TYR)--a plausible candidate gene for the albino (C) locus, and tyrosinase related protein 1 (TYRP1)--a candidate gene for the brown (B) locus. Sequence variants between individuals exhibiting variation in pigmentation were submitted to association studies. In TYR, two nonsynonymous substitutions encoding TYR-G301R and TYR-G227W were associated with the siamese and burmese phenotypes of the albino locus, respectively. TYRP1 was mapped on chromosome D4 within 5 cM of a highly polymorphic microsatellite, previously found to be fixed in a cat breed selected for the chocolate (b) allele of the B locus, which reinforced TYRP1 as a candidate gene for the B locus in the domestic cat. Two DNA polymorphisms, one leading to a TYRP1-A3G substitution in the signal peptide and another to an in-frame insertion TYRP1-421ins17/18 caused by a donor splice site mutation in intron 6, were associated with the chocolate (b) allele. A premature UAG stop codon at position 100 of TYRP1 was associated with a second allele of the B locus, cinnamon (b(l)). The results provide very strong evidence that the specific nucleotide variants of feline TYR (chromosome D1) are causative of the siamese (c(s)) and burmese (c(b)) alleles of the albino locus, as well as nucleotide variants of TYRP1 (chromosome D4) as specifying the chocolate (b) and cinnamon (b(l)) alleles of the B locus.


Sensitization, desensitization and stimulus-induced recovery of trigeminal neuronal responses to oral capsaicin and nicotine.
Repeated application of capsaicin at a 1-min interstimulus interval (ISI) to the tongue induces a progressively increasing irritant sensation (sensitization), followed after a rest period by reduced sensitivity to further capsaicin (desensitization). Sequential reapplication of capsaicin induces irritation that eventually increases to initial levels: stimulus-induced recovery (SIR). In contrast, repeated application of nicotine elicits a declining irritant sensation across trials. To investigate possible neural correlates of these phenomena, we recorded from single units in superficial laminae of the dorsomedial trigeminal subnucleus caudalis (Vc) that responded to noxious thermal (54 degrees C) and chemical (1 M pentanoic acid) stimulation of the tongue of anesthetized rats. We then recorded responses to either capsaicin (330 microM) or nicotine (0.6 M), delivered either once, repeatedly at 1-min ISI, or continually by constant flow. After the initial capsaicin application and a rest period, the capsaicin was reapplied in the identical manner to test for SIR. The mean response of 14 Vc units to sequential application of pentanoic acid did not vary significantly across trials, indicating lack of tachyphylaxis or sensitization. The averaged response of 11 Vc units to repeated capsaicin increased significantly across the first eight trials and then plateaued. Following the rest period, spontaneous firing had returned to the precapsaicin level. With capsaicin reapplication, the averaged response increased again after a significant delay (due to desensitization), but did not reattain the peak firing rate achieved in the initial series (partial SIR). Constant-flow application of capsaicin induced an identical sensitization followed by nearly complete SIR. A single application of capsaicin induced a significant rise in firing in eight other units, but the rate of rise and maximal firing rate were both much lower compared with repetitive or constant-flow capsaicin. When capsaicin was reapplied once after the rest period, there was no change in firing rate indicating absence of SIR. These results indicate that maintenance of the capsaicin concentration induces a progressive increase in neuronal response that parallels sensitization. With recurrent capsaicin application, desensitization can be overcome to result in a delayed recovery of Vc responses similar to SIR. In contrast, the averaged response of 17 Vc units to repeated or constant-flow application of nicotine increased only over the first 3 min, and then decreased to spontaneous levels even as nicotine was still being applied. These results are consistent with the decrease in the perceived irritation elicited by sequential application of nicotine in humans.


Threshold perimetry in tilted disc syndrome.
Twelve consecutive patients (17 eyes) with tilted disc syndrome underwent quantified visual field examination with the G1 Octopus program (Interzeag AG, Schlieren, Switzerland). Visual fields were abnormal in all cases. Although field loss was more pronounced in the superotemporal quadrant (P less than .001), it also involved the other three quadrants, thus demonstrating that optic nerve hypoplasia in tilted disc syndrome is apparently not entirely sectorial. Linear regression analysis showed that an increase in mean defect correlated with increase in myopic refractive error (P less than .05). However, the increase in myopic refractive error was found to be related to a decrease in corrected loss variance (P less than .05), indicating that in tilted discs with higher myopia, field loss is more homogeneous. With this series of patients, short-term fluctuation was within normal ranges in all visual fields, showing that this may be an additional clue for differential diagnosis from acquired disorders.


Surface immobilization of bio-functionalized cubosomes: sensing of proteins by quartz crystal microbalance.
A strategy for tethering lipid liquid crystalline submicrometer particles (cubosomes) to a gold surface for the detection of proteins is reported. Time-resolved quartz crystal microbalance (QCM-D) was used to monitor the cubosome-protein interaction in real time. To achieve specific binding, cubosomes were prepared from the nonionic surfactant phytantriol, block-copolymer, Pluronic F-127, and a secondary biotinylated lipid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000], which enabled attachment of the particles to a neutravidin (NAv)-alkanethiol monolayer at the gold surface of the QCM sensor chip. A second set of cubosomes was further functionalized with addition of the glycolipid (G(M1)) to facilitate a specific binding uptake of the protein, cholera toxin B subunit (CT(B)), from solution. QCM-D confirmed the specificity of the cubosome-NAv binding. The analysis of titration experiments, also performed with QCM, suggests that an optimal concentration of cubosomes is required for the efficient packing of the particles at the surface: high cubosome concentrations lead to chaotic cubosome binding onto the surface, sterically inhibiting surface attachment, or require significant reorganization to permit uniform cubosome coverage. The methodology enabled the straightforward preparation of a complex nanostructured edifice, which was then used to specifically capture analyte proteins (cholera toxin B subunit or free NAv) from solution, supporting the potential for development of this approach as a biosensing platform.


Activation of cAMP-responsive-element-binding protein by PI3 kinase and p38 MAPK is essential for elevated expression of transforming growth factor β2 in cancer cells.
Transforming growth factor β2 (TGFβ2) is highly expressed in a variety of different cancer cell lines. Using Z12 cells, a mutant of 293 cells with overexpression of TGFβ2, we found that the cyclic adenosine monophosphate (cAMP)-responsive element (CRE) sequence in the promoter of the TGFβ2 gene is crucial for its increased expression. Further, constitutive phosphorylation of CRE-binding protein (CREB) is increased in these cells. Treating Z12 cells with either the PI3 kinase inhibitor LY294002 or the p38 MAPK inhibitor SB203580 significantly inhibited both the phosphorylation of CREB and expression of TGFβ2. In addition, treating Z12 or cancer cell lines with either of these 2 inhibitors significantly decreased their secretion of TGFβ2. These data suggest that activated PI3 kinase and p38 MAPK play important roles in high expression of TGFβ2 in cancer cells by stimulating the phosphorylation of CREB, which activates the CRE in the promoter of the TGFβ2 gene. We have identified an important link between PI3 kinase, p38 MAPK, and TGFβ2, providing an additional rationale for using inhibitors of these kinases as therapeutic drugs in cancer.


Size structure and substitutability in an odonate intraguild predation system.
Interactions between different size classes of predator species have the potential to influence survival of prey species in intraguild predation (IGP) systems, but few studies test for these effects. Using a substitutive design in a field setting, I measured the effects of two size classes of IG predators (large and small larvae of the dragonfly Anax junius) on the mortality of IG prey (larvae of the dragonfly Pachydiplax longipennis). I also examined whether combinations of large A. junius and P. longipennis and small A. junius and P. longipennis had substitutable effects on shared prey (larvae of the damselfly Ischnura verticalis). The presence of both size classes of A. junius, when alone and in combination with P. longipennis, significantly increased mortality of I. verticalis. In the presence of P. longipennis, large and small A. junius had similar effects on the mortality of I. verticalis, and effects of size-structured assemblages of A. junius were similar to the effects of each size class alone at the same density. The effects of the two size classes of A. junius on P. longipennis differed, and P. longipennis mortality was lower when exposed to size structured assemblages of A. junius than when exposed to only large A. junius at the same density. Results were similar to those in a laboratory study, although the effect of P. longipennis on I. verticalis was much lower in the field setting. These results demonstrate that interactions between different size classes of IG predators promote the survival of IG prey and highlight the importance of within-species size structure as a characteristic that may promote the coexistence of predators in IGP systems.


VEGFR2 heterogeneity and response to anti-angiogenic low dose metronomic cyclophosphamide treatment.
Targeting tumor vasculature is a strategy with great promise in the treatment of many cancers. However, anti-angiogenic reagents that target VEGF/VEGFR2 signaling have met with variable results clinically. Among the possible reasons for this may be heterogeneous expression of the target protein. Double immunofluorescent staining was performed on formalin-fixed paraffin embedded sections of treated and control SW480 (colorectal) and WM239 (melanoma) xenografts, and tissue microarrays of human colorectal carcinoma and melanoma. Xenografts were developed using RAG1-/- mice by injection with WM239 or SW480 cells and mice were treated with 20 mg/kg/day of cyclophosphamide in their drinking water for up to 18 days. Treated and control tissues were characterized by double immunofluorescence using the mural cell marker α-SMA and CD31, while the ratio of desmin/CD31 was also determined by western blot. Hypoxia in treated and control tissues were quantified using both western blotting for HIF-1α and immunohistochemistry of CA-IX. VEGFR2 is heterogeneously expressed in tumor vasculature in both malignant melanoma and colorectal carcinoma. We observed a significant decrease in microvascular density (MVD) in response to low dose metronomic cyclophosphamide chemotherapy in both malignant melanoma (with higher proportion VEGFR2 positive blood vessels; 93%) and colorectal carcinoma (with lower proportion VEGFR2 positive blood vessels; 60%) xenografts. This reduction in MVD occurred in the absence of a significant anti-tumor effect. We also observed less hypoxia in treated melanoma xenografts, despite successful anti-angiogenic blockade, but no change in hypoxia of colorectal xenografts, suggesting that decreases in tumor hypoxia reflect a complex relationship with vascular density. Based on α-SMA staining and the ratio of desmin to CD31 expression as markers of tumor blood vessel functionality, we found evidence for increased stabilization of colorectal microvessels, but no such change in melanoma vessels. Overall, our study suggests that while heterogeneous expression of VEGFR2 is a feature of human tumors, it may not affect response to low dose metronomic cyclophosphamide treatment and possibly other anti-angiogenic approaches. It remains to be seen whether this heterogeneity is partly responsible for the variable clinical success seen to date with targeted anti-VEGFR2 therapy.


How to perform a successful drug-coated balloon angioplasty? Tips and tricks.
Drug-coated balloons (DCB) offer an excellent alternative to stents as the antiproliferative drugs are delivered via balloons and hence there is no permanent implant of metal or polymer. This rationale applies perfectly in in-stent restenosis (ISR) as we want to avoid another layer of metal in a previously failed stent. However, their use has also been extended to de novo lesions especially in patients and lesion subsets where stents are not ideal. There is an increased desire toward expanding this further and studies are now being done which are testing DCB in large-caliber vessels. As the use of DCB is escalating, we felt the importance of writing this article whereby we aim to provide important tips and tricks when using DCB especially for the operators who are in the early phase or have the desire of embarking this technology. From our experience, the DCB-angioplasty substantially differs on several aspects from DES-angioplasty. We have provided several case bases examples including algorithm when using DCB in ISR and de novo lesions.


Staging of intrahepatic cholangiocarcinoma.
Staging of intrahepatic cholangiocarcinoma (ICC) has historically mirrored that for hepatocellular carcinoma, largely due to the fact that ICC has been an uncommon disease. This review summarizes recent developments related to prognostication in ICC that have allowed the development of a distinct Western staging system for this disease. A large cancer registry study focused on identification of prognostic factors after resection of ICC with the aim of developing a distinct staging system for ICC. On the basis of this analysis and corroborative data from smaller institutional series, the American Joint Committee on Cancer (AJCC) 7th edition staging manual includes a distinct staging system for ICC that focuses on multiple tumors, vascular invasion, and lymph node metastasis. This system is simpler than the AJCC 6th edition staging yet preserves prognostic discrimination; the new system is also significantly superior to the Japanese staging system. Some evidence suggests that the number of lymph nodes involved by tumor may influence prognosis. Margin-negative resection is a major therapeutic determinant of outcome in ICC and should be pursued unless distant metastasis is present. For the first time, a distinct staging system for ICC has been adopted by the AJCC. Understanding of prognostic factors in ICC remains incomplete but is improving. This field is in evolution, and further refinements to the staging of ICC are likely as more data emerge on this increasingly common malignancy.


Preperitoneal collection after endoscopic extraperitoneal inguinal hernioplasty in a patient with malignant ascites.
Painful preperitoneal collection is a rare complication following endoscopic totally extraperitoneal inguinal hemioplasty. Here we present the case of a woman who underwent endoscopic extraperitoneal inguinal hernioplasty for a left inguinal hernia. Her past health was good. During the dissection of the extraperitoneal space, clear ascitic fluid was noted upon breaching the peritoneum near the round ligament. Endoscopic stapling was used to close the peritoneal tear, and the procedure was completed uneventfully. The patient complained of left iliac pain after the operation. A physical examination showed no swelling over the left iliac fossa. Contrast computed tomography of the abdomen revealed preperitoneal fluid collection over the hernioplasty site and a small amount of ascites. Expectant treatment with pain control by oral analgesics was adopted. A follow-up CT scan 4 months after the operation showed resolution of the preperitoneal fluid collection but with increased ascites. Abdominal paracentesis with peritoneal fluid for cytology analysis found adenocarcinoma cells. The patient succumbed to a terminal malignancy a year after surgery. Conversion of endoscopic extraperitoneal inguinal hernioplasty to open repair should be considered upon intraoperative discovery of ascites. Painful preperitoneal collection is a possible sequela following endoscopic extraperitoneal hernioplasty in patients with malignant ascites.


Comparative genomic hybridization analysis of adrenocortical tumors.
Comparative genomic hybridization (CGH) is a molecular cytogenetic technique that allows the entire genome of a tumor to be surveyed for gains and losses of DNA copy sequences. A limited number of studies reporting the use of this technique in adult adrenocortical tumors have yielded conflicting results. In this study we performed CGH analysis on 13 malignant, 18 benign, and 1 tumor of indeterminate malignant potential with the aim of identifying genetic loci consistently implicated in the development and progression of adrenocortical tumors. Tissue samples from 32 patients with histologically proven adrenocortical tumors were available for CGH analysis. CGH changes were seen in all cancers, 11 of 18 (61%) adenomas, and the 1 tumor of indeterminate malignant potential. Of the adrenal cancers, the most common gains were seen on chromosomes 5 (46%), 12 (38%), 19 (31%), and 4 (31%). Losses were most frequently seen at 1p (62%), 17p (54%), 22 (38%), 2q (31%), and 11q (31%). Of the benign adenomas, the most common change was gain of 4q (22%). Mann-Whitney analysis showed a highly significant difference between the cancer group (mean changes, 7.6) and the adenoma group (mean changes, 1.1) for the number of observed CGH changes (P < 0.01). Logistic regression analysis showed that the number of CGH changes was highly predictive of tumor type (P < 0.01). This study has identified several chromosomal loci implicated in adrenocortical tumorigenesis. Activation of a protooncogene(s) on chromosome 4 may be an early event, with progression from adenoma to carcinoma involving activation of oncogenes on chromosomes 5 and 12 and inactivation of tumor suppressor genes on chromosome arms 1p and 17p.


Benign cutaneous vascular tumors of infancy: when to worry, what to do.
To discuss the current knowledge regarding complicated hemangiomas (cervicofacial, periorbital, lumbosacral, and parotid), including the associated syndromes of diffuse neonatal hemangiomatosis and PHACES (posterior fossa malformations, most commonly of the Dandy-Walker variant; hemangiomas [especially large, plaquelike, facial lesions]; arterial anomalies; cardiac anomalies and coarctation of the aorta; eye abnormalities; and sternal cleft and/or supraumbilical raphe). To discuss 2 newly recognized entities that may be a source of diagnostic confusion with the common hemangioma, the kaposiform hemangioendothelioma and tufted angioma. To discuss the risks and benefits of current treatment options, including the use of systemic corticosteroids and interferon in necessary situations. The pertinent world literature was reviewed and incorporated into experience from our pediatric dermatology practice at the University of Texas Medical School at Houston. The common hemangioma, kaposiform hemangioendothelioma, and tufted angioma, though benign histologically, may cause serious consequences for children. Dermatologists should be able to recognize unique clinical presentations of these lesions and obtain further diagnostic evaluation accordingly. Dermatologists should also be aware of available treatment options, including the use of systemic chemotherapy in life-threatening situations.


Population-based mapping of pulmonary adenoma susceptibility 1 locus.
Pulmonary adenoma susceptibility 1 (Pas1), the major locus affecting inherited predisposition to lung tumor development in mice, maps near the Kras2 gene. We previously reported a significant association between a KRAS2/RsaI polymorphism and the risk and prognosis of lung adenocarcinoma (ADCA) in the Italian population. In the present case-control study, we examined 269 lung ADCA patients, 121 squamous cell lung carcinoma patients, and 632 healthy individuals (general population controls) in the Japanese population with genetic markers spanning approximately 1200 kb in the KRAS2 region. Allele-specific oligonucleotide hybridization revealed the same KRAS2/RsaI polymorphism associated with risk and prognosis as in Italian lung ADCA patients; the polymorphism was significantly associated with clinical stage (P < 0.001) and survival rate (log rank = 0.0014), confirming the mapping of PAS1 and pointing to the role of this locus in human lung cancer.


A Hierarchy-driven Multi-label Network with Label Constraints for Post-operative Complication Prediction of Lung Cancer.
Lung cancer is one of the most dangerous cancers all over the world. Surgical resection remains the only potentially curative option for patients with lung cancer. However, this invasive treatment often causes various complications, which seriously endanger patient health. In this study, we proposed a novel multi-label network, namely a hierarchy-driven multi-label network with label constraints (HDMN-LC), to predict the risk of complications of lung cancer patients. In this method, we first divided all complications into pulmonary and cardiovascular complication groups and employed the hierarchical cluster algorithm to analyze the hierarchies between these complications. After that, we employed the hierarchies to drive the network architecture design so that related complications could share more hidden features. And then, we combined all complications and employed an auxiliary task to predict whether any complications would occur to impose the bottom layer to learn general features. Finally, we proposed a regularization term to constrain the relationship between specific and combined complication labels to improve performance. We conducted extensive experiments on real clinical data of 593 patients. Experimental results indicate that the proposed method outperforms the single-label, multi-label baseline methods, with an average AUC value of 0.653. And the results also prove the effectiveness of hierarchy-driven network architecture and label constraints. We conclude that the proposed method can predict complications for lung cancer patients more effectively than the baseline methods.Clinical relevance-This study presents a novel multi-label network that can more accurately predict the risk of specific postoperative complications for lung cancer patients. The method can help clinicians identify high-risk patients more accurately and timely so that interventions can be implemented in advance to ensure patient safety.


Visualisation of chicken macrophages using transgenic reporter genes: insights into the development of the avian macrophage lineage.
We have generated the first transgenic chickens in which reporter genes are expressed in a specific immune cell lineage, based upon control elements of the colony stimulating factor 1 receptor (CSF1R) locus. The Fms intronic regulatory element (FIRE) within CSF1R is shown to be highly conserved in amniotes and absolutely required for myeloid-restricted expression of fluorescent reporter genes. As in mammals, CSF1R-reporter genes were specifically expressed at high levels in cells of the macrophage lineage and at a much lower level in granulocytes. The cell lineage specificity of reporter gene expression was confirmed by demonstration of coincident expression with the endogenous CSF1R protein. In transgenic birds, expression of the reporter gene provided a defined marker for macrophage-lineage cells, identifying the earliest stages in the yolk sac, throughout embryonic development and in all adult tissues. The reporter genes permit detailed and dynamic visualisation of embryonic chicken macrophages. Chicken embryonic macrophages are not recruited to incisional wounds, but are able to recognise and phagocytose microbial antigens.


The adenosine receptor blocker aminophylline increases anoxic ethanol excretion in crucian carp.
By depressing energy consumption, anoxia-tolerant animals are thought to compensate for a reduced ability to produce energy during anoxia. Adenosine is an inhibitory neuromodulator in vertebrates and, hence, has the potential ability to depress energy consumption. Ethanol is the main metabolic end product in anoxic Carassius, and the present study shows that the rate of ethanol excretion in anoxic crucian carp (Carassius carassius L.) can be increased threefold by treatment with the adenosine receptor antagonist aminophylline (75 mg/kg). By contrast, the same dose of aminophylline did not increase the rate of routine oxygen consumption during normoxia. It is hypothesized that adenosine acts as a metabolic depressant during anoxia in crucian carp.


Host defences against Giardia lamblia.
Giardia spp. is a protozoan parasite that inhabits the upper small intestine of mammals and other species and is the aetiological agent of giardiasis. It has been demonstrated that nitric oxide, mast cells and dendritic cells are the first line of defence against Giardia. IL-6 and IL-17 play an important role during infection. Several cytokines possess overlapping functions in regulating innate and adaptive immune responses. IgA and CD4(+) T cells are fundamental to the process of Giardia clearance. It has been suggested that CD4(+) T cells play a double role during the anti-Giardia immune response. First, they activate and stimulate the differentiation of B cells to generate Giardia-specific antibodies. Second, they act through a B-cell-independent mechanism that is probably mediated by Th17 cells. Several Giardia proteins that stimulate humoral and cellular immune responses have been described. Variant surface proteins, α-1 giardin, and cyst wall protein 2 can induce host protective responses to future Giardia challenges. The characterization and evaluation of the protective potential of the immunogenic proteins that are associated with Giardia will offer new insights into host-parasite interactions and may aid in the development of an effective vaccine against the parasite.


Protective effects of matrix metalloproteinase-12 following corneal injury.
Corneal scarring due to injury is a leading cause of blindness worldwide and results from dysregulated inflammation and angiogenesis during wound healing. Here we demonstrate that the extracellular matrix metalloproteinase MMP12 (macrophage metalloelastase) is an important regulator of these repair processes. Chemical injury resulted in higher expression of the fibrotic markers α-smooth muscle actin and type I collagen, and increased levels of angiogenesis in corneas of Mmp12(-/-) mice compared with corneas of wild-type mice. In vivo, we observed altered immune cell dynamics in Mmp12(-/-) corneas by confocal imaging. We determined that the altered dynamics were the result of an altered inflammatory response, with delayed neutrophil infiltration during the first day and excessive macrophage infiltration 6 days later, mediated by altered expression levels of chemokines CXCL1 and CCL2, respectively. Corneal repair returned to normal upon inhibition of these chemokines. Taken together, these data show that MMP12 has a protective effect on corneal fibrosis during wound repair through regulation of immune cell infiltration and angiogenesis.


Microtiter Array Diagonal Gel Electrophoresis (MADGE) for Population Scale Genotype Analyses.
Human populations display enormous genetic variation, evident both at the phenotypic level and at the DNA level. These variations include both single "mutations" causative of profound disease and variations that contribute to susceptibility to particular "polygenic" diseases. To date, genetic diagnostics have been applied mainly in the context of isolated diagnoses for individuals or families for single gene disorders. Population screening, for example, for carrier status for recessive disorders such as cystic fibrosis, has not yet advanced beyond feasibility studies. Such studies require satisfaction of the criteria of Wilson and Jungner (1), in that there must be the knowledge and resources (counseling, treatment, and so on) to confer benefit, and that there must be a viable and affordable means to undertake screening. For research into the genetic epidemiology of common disorders, such as cardiovascular disease and cancers, some of the feasibility shortfalls correspond with those for population screening for single gene mutations. First, tests for genetic variation are still very expensive, both in staff time and in reagents. Second, the combination of informed consent, collection of appropriate clinical detail, and collection of samples is expensive on a population scale. For these reasons, many associations, such as those between apolipoprotein E (APOE) genotype, hypercholesterolaemia and premature vascular disease (2), APOE genotype and late-onset Alzheimer disease (3), and angiotensinogen-converting enzyme (ACE) genotype and cardiovascular disease (4), have mainly been researched on groups of a few hundred individuals.


Rare gas isotopes in hawaiian ultramafic nodules and volcanic rocks: constraint on genetic relationships.
Differences in the rare gas isotopic ratios, especially the ratios of helium-3 to helium-4 and of argon-40 to argon-36, in Hawaiian ultramafic nodules and phenocrysts in volcanic rocks indicate that the nodules and phenocrysts were derived from different sources. The isotopic ratios in ultramafic nodules are similar to those in oceanic tholeiites. The phenocrysts seem to have formed in equilibrium with source materials richer in primordial components than those of the oceanic tholeiites. Mixing between the sources is quite likely.


Application of the Oral Health Impact Profile (OHIP) among Taiwanese elderly.
To validate the Chinese (Taiwan) version of the Oral Health Impact Profile (OHIP-49T) and develop a short form of the OHIP (OHIP-14T) for the elderly. A total of 1402 subjects, aged 65 and above, received a personal interview and oral examination. Internal consistency, measured by Cronbach's alpha, and test-retest reliability were assessed by intra-class correlation coefficient (ICC) during a 2-week interval. The criterion-related validity of OHIP-49T was evaluated through associations between the OHIP-49 score with prosthetic need and prosthetic status. A subset (OHIP-14T) questionnaire, derived through a controlled regression procedure, was compared with the original OHIP-14 by Slade (OHIP-14S). The Cronbach's alpha and ICC values were 0.97 and 0.98 for OHIP-49T and 0.90 and 0.93 for OHIP-14T. Mean scores of the OHIP-49T were significantly associated with prosthetic status (P = 0.0013) and prosthetic need (P = 0.0004), which were examined by dentists. The OHIP-14T score had stronger discriminatory ability than OHIP-14S. The OHIP-49T showed satisfactory reliability and validity for this Taiwanese elderly population. The OHIP-14T is more effective to measure OHRQoL than OHIP-14S.


Landscape and environmental influences on Mycobacterium ulcerans distribution among aquatic sites in Ghana.
Buruli ulcer, caused by Mycobacterium ulcerans, is highly endemic in West Africa. While the mode of transmission is unknown, many studies associate Buruli ulcer with different types of water exposure. We present results from the largest study to date to test for M. ulcerans in aquatic sites and identify environmental attributes associated with its presence. Environmental samples from 98 aquatic sites in the Greater Accra, Ashanti, and Volta regions of Ghana were tested for the presence of M. ulcerans DNA by polymerase chain reaction. The proportion of aquatic sites positive for M. ulcerans varied by region: Ashanti 66% (N = 39), Greater Accra 34% (N = 29), and Volta 0% (N = 30). We explored the spatial distribution of M. ulcerans positive and negative water bodies and found no significant clusters. We also determined both highly localized water attributes and broad scale remotely sensed land cover and terrain environmental characteristics associated with M. ulcerans presence through logistic regression. Our results concur with published results regarding conditions suitable for M. ulcerans growth and associations with Buruli ulcer disease burden with regards to water characteristics and disturbed environments, but differ from others with regards to spatial associations and topographic effects such as elevation and wetness. While our results suggest M. ulcerans is an environmental organism existing in a specific ecological niche, they also reveal variation in the elements defining this niche across the sites considered. In addition, despite the causal association between Buruli ulcer and M. ulcerans, we observed no significant statistical association between case reports of Buruli ulcer and presence of M. ulcerans in nearby waterbodies.


ICP-MS and fluorescence dual-mode detection of ZIKV-RNA based on quantum dot labeling with hybridization chain reaction.
The detection of Zika virus (ZIKV) is of great significance to human life and health. Herein, we presented an ICP-MS and fluorescent dual-mode sensor for quantitative analysis of Zika virus RNA fragments (ZIKV-RNA), which employed quantum dots (QDs) as signal tags and combined with hybridization chain reaction (HCR). The dual-mode sensor realized cross-checking of the analysis results and improved the assay accuracy. Firstly, the single-stranded DNA (ssDNA) was anchored on the surface of magnetic beads (MBs). Afterward, HCR was conducted with probe DNA-CdSe quantum dots conjugates (pDNA-QDs) and link DNA (lDNA), producing the MBs-ssDNA-[pDNA-QDs-lDNA]n conjugates. In the presence of target ZIKV-RNA, a strand displacement reaction occurred, leading to the dissociation of the [pDNA-QDs-lDNA]n labels from the conjugates into the solution. Finally, the signal intensity was detected using ICP-MS and fluorescence analysis, with achieved limits of detection of 131 pM and 152 pM, respectively. The inter-assay RSD values of fluorescence and ICP-MS were 3.94 % and 4.26 % at 10 nM level, respectively, showing that the method had good precision. This method showed high selectivity and was applied to the analysis of biological fluids. There was no significant difference between the results of ICP-MS modes and fluorescence mode. This method offers a new strategy for sensitivity analysis of ZIKV-RNA and exhibits promise in clinical applications for diagnosis.


Rates of protein synthesis are reduced in peripheral blood mononuclear cells (PBMCs) from fragile X individuals.
Fragile X syndrome (FXS) is the leading inherited cause of intellectual disability and is caused by the loss of expression of the Fragile X mental retardation protein (FMRP). In animal model of FXS, the absence of FMRP leads to an aberrant rate of neuronal protein synthesis, which in turn is believed to be at the origin of defects regarding spine morphology and synaptic plasticity. Normalisation of protein synthesis in these models has been associated with a rescue of FXS behavioral and biochemicals phenotype, thus establishing the rate of protein synthesis as one of the most promising monitoring biomarker for FXS. However, rate of protein synthesis alteration in fragile X individuals is not well characterized. We applied a robust radiolabeled assay to measure rate of protein synthesis in freshly extracted peripheral blood mononuclear cells (PBMCs) and blood platelets. We ultimately settle on PBMCs to measure and compare rate of protein synthesis in 13 males with fragile X and 14 matched controls individuals. Using this method, we measured a 26.9% decrease (p = 0,0193) in the rate of protein synthesis in fragile X individuals PBMCs. Furthermore, the rate of protein synthesis measurements obtained were highly reproducible, highlighting the robustness of the method. Our work presents the first evidence of a diminution of the rate of protein synthesis in a human peripheral model of fragile X. Our results also support the finding of previous studies using brain PET imaging in Fragile X individuals. Since our assay only requires a simple venous puncture, it could be used in other cases of intellectual disability in order to determine if an aberrant rate of protein synthesis is a common general mechanism leading to impairment in synaptic plasticity and to intellectual disability.


Isolation and characterization of Tarumizu tick virus: A new coltivirus from Haemaphysalis flava ticks in Japan.
During the course of tick-borne virus surveillance in Japan, three independent isolates of probably the same virus were obtained from three geographically distant populations of the hard tick Haemaphysalis flava. Genome analyses of the three isolates demonstrated that they were closely related but distinct strains of a novel virus, designated Tarumizu tick virus (TarTV), which has a genome of 12 double-stranded RNA segments. The development of the virus-induced cytopathic effects on BHK cells significantly varied according to virus strains. Ten out of 12 segments of TarTV appeared to encode putative orthologs or functional equivalents of viral proteins of Colorado tick fever virus (CTFV) and Eyach virus, suggesting that TarTV is the third member of the genus Coltivirus in the family Reoviridae. This was supported by the facts that the 5'- and 3'-terminal consensus sequences of coltivirus genomes were found also in TarTV genome, and segment 9 of TarTV had sequence and structural features that may mediate a stop codon read-through as observed in that of CTFV. However, segment 7 and 10 of TarTV had no significant sequence similarities to any other proteins of known coltiviruses. Electron microscopic analysis demonstrated that TarTV particle had a non-enveloped bilayer icosahedral structure, and viral inclusion bodies were formed in infected cells. TarTV could infect and replicate in several mammalian cell lines tested, but show no clinical symptoms in intracerebrally inoculated mice. Taken together, our findings provide new insights into genetic diversity and evolution of the genus Coltivirus.


Imaging changes after stereotactic radiosurgery of primary and secondary malignant brain tumors.
After radiosurgery of malignant tumors, it can be difficult to discriminate between transient treatment effects, radiation necrosis, and tumor progression on post-treatment imaging. Misinterpretation of an enlarging lesion may lead to inappropriate treatment and contribute to disagreements about treatment efficacy. In an effort to clarify this problem, we reviewed our experience with interpreting post-radiosurgical imaging in patients with malignant primary and secondary brain tumors. We reviewed results of radiosurgery of 30 malignant gliomas and 35 metastatic brain tumors with minimum follow up of 1 year or until death. Of 30 gliomas, 73% were larger a mean of 13 weeks after radiosurgery. Of 35 metatstatic tumors, 22% were larger a mean of 10 weeks after radiosurgery. Eleven had 18F-fluorodeoxyglucose-positron emission tomography (FDG-PET) of enlarging lesions. Eight showed increased activity with respect to brain; three decreased activity. Of the eight, six predicted incorrectly based upon the patients' subsequent courses (all alive, mean follow up of 27 months), and two correctly, with the patients dying from the imaged lesions 8 and 13 months later. Of the three with FDG uptake less than brain, one patient was alive with 32 weeks of follow up, and two patients died from the imaged lesion 13 and 21 months later. Radiographic enlargement after radiosurgery is common, especially for gliomas. Physicians caring for these patients should be aware of this phenomenon and be cautious in interpreting post-treatment images. MRI appearance may be useful for metastases. FDG-PET seems unreliable. Further evaluation of Tl-201 and HMPAO SPECT or MRS is warranted.


Sp3 is involved in the regulation of SOCS3 gene expression.
Cytokine-induced expression of SOCS (suppressor of cytokine signalling) molecules is important for the negative regulatory control of STAT (signal transduction and activators of transcription)-dependent cytokine signalling, e.g. for the signal transduction of IL-6 (interleukin-6)-type cytokines through the JAK (Janus kinase)/STAT cascade. STAT activation itself represents an important step in the transcriptional activation of SOCS3 gene expression. However, downstream of the STAT-responsive element, the SOCS3 gene contains a GC-rich element in its 5'-upstream region. The aim of the present study was to investigate the implications of this GC-rich element in the transcriptional control of SOCS3 gene expression. In the present study, we show that mutation of this GC-rich element abolishes IL-6-dependent transcriptional activation of the SOCS3 promoter and that Sp3 (specificity protein 3), a ubiquitously expressed transcription factor, but not Sp1 binds to this GC-rich motif, suggesting that Sp3 is involved in the regulation of SOCS3 expression. The results suggest that Sp3 is important for IL-6-induced transcriptional activation of the SOCS3 (gene) promoter and acts as an enhancer of basal as well as induced transcriptional activity, resulting in enhanced SOCS3 mRNA and protein expression. Mutation of Lys-483, a potential target for Sp3 acetylation, inhibited Sp3-mediated enhancement of SOCS3 mRNA expression and SOCS3 promoter activation, indicating that the acetylation of this lysine residue of Sp3 is important for the enhancing effect of Sp3 on SOCS3 expression.


Rare hereditary cause of chronic pancreatitis in a young male: SPINK1 mutation.
Hereditary chronic pancreatitis associated with a mutation in the serine protease inhibitor, Kazal Type-1 (SPINK-1 gene) is extremely rare. The SPINK1 mutation results in trypsinogen activation which predisposes to chronic pancreatitis predominately when combined with CFTR gene mutations. It presents as either chronic or recurrent acute pancreatitis. Symptom control and management of complications is important. Active surveillance with cross-sectional imaging for pancreatic malignancy in individuals with hereditary pancreatitis is advocated due to individuals being high risk. We present an unusual case of a young male who initially presented with renal colic and was incidentally diagnosed with severe chronic pancreatitis on abdominal imaging, with genetic testing confirming a homozygous SPINK1 mutation.


A rare cause of acute urinary retention in a young man: A median raphe penile cyst.
A penile median raphe cyst is an uncommon congenital lesion that is formed due to failure of the median raphe to close completely during embryo development. The majority of the cysts are asymptomatic and often go unnoticed. Here, we report acute urinary retention as a very rare complication of a penile median raphe cyst. A 21-year-old single man presented with a sudden onset of acute urinary retention of one days' duration. Urethral catheterization was done at the Emergency Department and this drained 800 ml of urine. On further questioning, he claimed that he had had a painless nodule at the glans penis since childhood. The swelling increased in size in the past week causing discomfort. He denied any history of genitalia trauma or recent sexual intercourse. On examination, there was a bluish lesion over the ventral aspect of the glans penis measuring about 3 x 2 cm. The lesion was later excised and histopathology revealed a median raphe cyst of the penis. A penile median raphe cyst is a rare lesion. Acute urinary retention caused by this lesion is very rare.


Complementary PET studies of striatal neuronal function in the differential diagnosis between multiple system atrophy and Parkinson's disease.
We used PET with the tracers [18F]fluorodeoxyglucose (FDG), [18F]fluorodopa (FDOPA) and [11C]raclopride (RACLO) to study striatal glucose and dopa metabolism, and dopamine D2 receptor binding, respectively, in nine patients with multiple system atrophy. Ten patients with classical Parkinson's disease were investigated with the same three PET tracers' and three separate groups, each of 10 healthy subjects, served as control populations. We found that striatal FDOPA values separated all healthy subjects from patients with parkinsonism but they were not useful in distinguishing multiple system atrophy from Parkinson's disease. Conversely, striatal RACLO as well as FDG values discriminated all multiple system atrophy from Parkinson's disease patients as well as from healthy control subjects. Metabolic and receptor binding decrements in the putamen of multiple system atrophy patients were significantly correlated. Stepwise regression analysis revealed that a linear combination of putamen RACLO and FDOPA values accurately predicted clinical measures of disease severity in the multiple system atrophy group. Our findings suggest that striatal FDG and particularly RACLO are sensitive and effective measures of striatal function and may help characterizing patients with multiple system atrophy. In contrast, FDOPA measurements are accurate in detecting abnormalities of the nigrostriatal dopaminergic system but may not distinguish among different forms of parkinsonism.


The solution structure of the domain from MeCP2 that binds to methylated DNA.
MeCP2 is an abundant mammalian protein that binds methylated CpG (mCpG) sequences within double-stranded DNA, represses transcription by recruiting histone deacetylases, and is essential for embryonic development. It is one of a family of proteins which mediate the biological consequences of DNA methylation. These proteins each possess a sequence motif of about 70 residues which, in MeCP2, form a domain necessary and sufficient for binding to mCpG. The solution structure of the mCpG-binding domain (MBD) from MeCP2 has been solved and the DNA-binding surface of the domain mapped using NMR spectroscopy. Residues 95-162 of MeCP2 adopt a novel fold forming a wedge-shaped structure. An N-terminal four-stranded antiparallel beta-sheet forms one face of the wedge, while the other face is formed mainly by a C-terminal helical region. The thin end of the wedge is extended by a long loop between beta-strands B and C containing many basic residues. The B-C loop together with residues in strands B, C and D, and at the N terminus of the alpha-helix, appears to form an interface with methylated DNA. Unstructured residues at the NH2 terminus of the domain are also involved in formation of the complex. The presence of numerous arginine and lysine side-chains on the DNA-binding surface of MBD is consistent with the requirement for the mCpG site to be flanked by non-specific sequences of base-pairs. The absence of symmetry in the domain implies that recognition does not exploit the symmetry of the binding site. A conserved hydrophobic pocket containing the side-chains of Tyr123 and Ile125 on the positively charged beta-sheet face is a candidate for the region of contact with the methyl-groups of the modified cytosine residues.


Anti-CD40 Antibody Fused to CD40 Ligand Is a Superagonist Platform for Adjuvant Intrinsic DC-Targeting Vaccines.
CD40 is a potent activating receptor expressed on antigen-presenting cells (APCs) of the immune system. CD40 regulates many aspects of B and T cell immunity via interaction with CD40L expressed on activated T cells. Targeting antigens to CD40 via agonistic anti-CD40 antibody fusions promotes both humoral and cellular immunity, but current anti-CD40 antibody-antigen vaccine prototypes require co-adjuvant administration for significant in vivo efficacy. This may be a consequence of dulling of anti-CD40 agonist activity via antigen fusion. We previously demonstrated that direct fusion of CD40L to anti-CD40 antibodies confers superagonist properties. Here we show that anti-CD40-CD40L-antigen fusion constructs retain strong agonist activity, particularly for activation of dendritic cells (DCs). Therefore, we tested anti-CD40-CD40L antibody fused to antigens for eliciting immune responses in vitro and in vivo. In PBMC cultures from HIV-1-infected donors, anti-CD40-CD40L fused to HIV-1 antigens preferentially expanded HIV-1-specific CD8+ T cells versus CD4+ T cells compared to analogous anti-CD40-antigen constructs. In normal donors, anti-CD40-CD40L-mediated delivery of Influenza M1 protein elicited M1-specific T cell expansion at lower doses compared to anti-CD40-mediated delivery. Also, on human myeloid-derived dendritic cells, anti-CD40-CD40L-melanoma gp100 peptide induced more sustained Class I antigen presentation compared to anti-CD40-gp100 peptide. In human CD40 transgenic mice, anti-CD40-CD40L-HIV-1 gp140 administered without adjuvant elicited superior antibody responses compared to anti-CD40-gp140 antigen without fused CD40L. In human CD40 mice, compared to the anti-CD40 vehicle, anti-CD40-CD40L delivery of Eα 52-68 peptide elicited proliferating of TCR I-Eα 52-68 CD4+ T cells producing cytokine IFNγ. Also, compared to controls, only anti-CD40-CD40L-Cyclin D1 vaccination of human CD40 mice reduced implanted EO771.LMB breast tumor cell growth. These data demonstrate that human CD40-CD40L antibody fused to antigens maintains highly agonistic activity and generates immune responses distinct from existing low agonist anti-CD40 targeting formats. These advantages were in vitro skewing responses towards CD8+ T cells, increased efficacy at low doses, and longevity of MHC Class I peptide display; and in mouse models, a more robust humoral response, more activated CD4+ T cells, and control of tumor growth. Thus, the anti-CD40-CD40L format offers an alternate DC-targeting platform with unique properties, including intrinsic adjuvant activity.


DNA as a powerful tool for morphology control, spatial positioning, and dynamic assembly of nanoparticles.
CONSPECTUS: Several properties of nanomaterials, such as morphologies (e.g., shapes and surface structures) and distance dependent properties (e.g., plasmonic and quantum confinement effects), make nanomaterials uniquely qualified as potential choices for future applications from catalysis to biomedicine. To realize the full potential of these nanomaterials, it is important to demonstrate fine control of the morphology of individual nanoparticles, as well as precise spatial control of the position, orientation, and distances between multiple nanoparticles. In addition, dynamic control of nanomaterial assembly in response to multiple stimuli, with minimal or no error, and the reversibility of the assemblies are also required. In this Account, we summarize recent progress of using DNA as a powerful programmable tool to realize the above goals. First, inspired by the discovery of genetic codes in biology, we have discovered DNA sequence combinations to control different morphologies of nanoparticles during their growth process and have shown that these effects are synergistic or competitive, depending on the sequence combination. The DNA, which guides the growth of the nanomaterial, is stable and retains its biorecognition ability. Second, by taking advantage of different reactivities of phosphorothioate and phosphodiester backbone, we have placed phosphorothioate at selective positions on different DNA nanostructures including DNA tetrahedrons. Bifunctional linkers have been used to conjugate phosphorothioate on one end and bind nanoparticles or proteins on the other end. In doing so, precise control of distances between two or more nanoparticles or proteins with nanometer resolution can be achieved. Furthermore, by developing facile methods to functionalize two hemispheres of Janus nanoparticles with two different DNA sequences regioselectively, we have demonstrated directional control of nanomaterial assembly, where DNA strands with specific hybridization serve as orthogonal linkers. Third, by using functional DNA that includes DNAzyme, aptamer, and aptazyme, dynamic control of assemblies of gold nanoparticles, quantum dots, carbon nanotubes, and iron oxide nanoparticles in response to one or more stimuli cooperatively have been achieved, resulting in colorimetric, fluorescent, electrochemical, and magnetic resonance signals for a wide range of targets, such as metal ions, small molecules, proteins, and intact cells. Fourth, by mimicking biology, we have employed DNAzymes as proofreading units to remove errors in nanoparticle assembly and further used DNAzyme cascade reactions to modify or repair DNA sequences involved in the assembly. Finally, by taking advantage of different affinities of biotin and desthiobiotin toward streptavidin, we have demonstrated reversible assembly of proteins on DNA origami.


Anserine and carnosine supplementation in the elderly: Effects on cognitive functioning and physical capacity.
The aim of this study was to investigate the potential beneficial effects of dietary anserine and carnosine (CRC) supplementation on cognitive functioning and physical activity of the elderly. The fifty-six subjects (65+) were allocated to the CRC group or placebo group at a 1:1 ratio. The double-blind procedure was used. Data were collected at the baseline and after 13-weeks of supplementation. In the follow up procedure fifty one subjects took part. Chicken meat extract (CME) containing 40% of CRC components (2:1 ratio of anserine to carnosine) was administered 2.5 g per day which allowed to rich the level of 1g CRC in dipeptides supplement. The cognitive function, physical capacity, body measurements, blood pressure and heart rate (HR) were assessed. After supplementation Body Mass Index (BMI) decreased significantly (p<0.05) in the CRC group performance comparing the placebo group. In two of six Senior Fitness Test the scores increased significantly (p<0.05) in CRC group comparing to the placebo group. The perceived exertion differed significantly (p<0.05) at the baseline and after follow up at the CRC group. The mean values of the Short Test of Mental Status (STMS) scores showed the significant (p<0.04) increase only in CRC group, in the subscores of construction/copying, abstraction and recall. Conducted anserine and carnosine supplementation in the elderly brings promising effects on cognitive functioning and physical capacity of participants. However, further studies are needed.


Design, synthesis and biological evaluation of quinoline-indole derivatives as anti-tubulin agents targeting the colchicine binding site.
A series of novel isocombretastatin A-4 (isoCA-4) analogs were designed and synthesized by replacing 3,4,5-trimethoylphenyl and isovanillin of isoCA-4 with quinoline and indole moieties, respectively. The structure activity relationships (SARs) of these synthesized quinoline-indole derivatives have been intensively investigated. Two compounds 27c and 34b exhibited the most potent activities against five cancer cell lines with IC50 values ranging from 2 to 11 nM, which were comparable to those of Combretastatin A-4 (CA-4, 1). Further mechanism investigations revealed that 34b effectively inhibited the microtubule polymerization by binding to the colchicine site of tubulin. Further cellular mechanism studies elucidated that 34b disrupted cell microtubule networks, arrested the cell cycle at G2/M phase, induced apoptosis and depolarized mitochondria of K562 cells. Moreover, 34b displayed potent anti-vascular activity in both wound healing and tube formation assays. Importantly, 27c and 34b significantly inhibited tumor growth in H22 xenograft models without apparent toxicity, suggesting that 27c and 34b deserve further research as potent antitumor agents for cancer therapy.


[A study of the tolerability and effectiveness of nicardipine retard in cognitive deterioration of vascular origin].
Nicardipine is a calcium antagonist which in previous trials has been shown to be effective in the prevention of stroke and the treatment of its sequelae, such as cognitive deterioration of vascular origin. We consider a phase IV study at primary care level to analyze the tolerability and efficacy of a retarded action formulation of 40 mg nicardipine. In this open, prospective, multicentric trial 6,375 patients took part, of whom 5,593 were evaluated (87.7%). All were diagnosed as having vascular-type dementia (Hachinski > 6) and were given treatment with nicardipine retard (40 mg/day/6 months). The patients were assessed on the Montorio (daily activity) and SPMSQ (cognitive function) scales at the first visit and after one, three and six months, together with a record of side effects. Only 0.9% of the patients recruited abandoned the study for problems of tolerability of the drug. The average improvement seen on Montorio's test was statistically significant from the first month, and reached 9% after six months. The 65.5% of the patients who started the study in seriously deteriorated condition showed improvement after six months. For the SPMSQ test, the average improvement was also statistically significant from the first visit and over 40% after six months. There was improvement in 64.4% of the patients who were severely deteriorated at the start of the study. Nicardipine retard is a drug which is safe and effective when used for the treatment of mental deterioration of vascular origin.


Pharmacodynamics of Cyclin D1 Degradation in Ovarian Cancer Xenografts with Repeated Oral SHetA2 Dosing.
SHetA2 is a promising, orally active small molecule with anticancer properties that target heat shock proteins. In this study, we aimed to investigate the pharmacodynamic (PD) effects of SHetA2 using preclinical in vitro and in vivo models of ovarian cancer and establish a physiologically based pharmacokinetic (PBPK)/PD model to describe their relationships with SHetA2 concentrations in mice. We found that daily oral administration of 60 mg/kg SHetA2 for 7 days resulted in consistent plasma PK and tissue distribution, achieving tumor drug concentrations required for growth inhibition in ovarian cancer cell lines. SHetA2 effectively induced cyclin D1 degradation in cancer cells in a dose-dependent manner, with up to 70% reduction observed and an IC50 of 4~5 µM. We identified cyclin D1 as a potential PD marker for SHetA2, based on a well-correlated time profile with SHetA2 PK. Additionally, we examined circulating levels of ccK18 as a non-invasive PD marker for SHetA2-induced apoptotic activity and found it unsuitable due to high variability. Using a PBPK/PD model, we depicted SHetA2 levels and their promoting effects on cyclin D1 degradation in tumors following multiple oral doses. The model suggested that twice-daily dosing regimens would be effective for sustained reduction in cyclin D1 protein. Our study provides valuable insights into the PK/PD of SHetA2, facilitating future clinical trial designs and dosing schedules.


Altered ROS production, NF-κB activation and interleukin-6 gene expression induced by electrical stimulation in dystrophic mdx skeletal muscle cells.
Duchenne muscular dystrophy is a fatal X-linked genetic disease, caused by mutations in the dystrophin gene, which cause functional loss of this protein. This pathology is associated with an increased production of reactive oxygen (ROS) and nitrogen species. The aim of this work was to study the alterations in NF-κB activation and interleukin-6 (IL-6) expression induced by membrane depolarization in dystrophic mdx myotubes. Membrane depolarization elicited by electrical stimulation increased p65 phosphorylation, NF-κB transcriptional activity and NF-κB-dependent IL-6 expression in wt myotubes, whereas in mdx myotubes it had the opposite effect. We have previously shown that depolarization-induced intracellular Ca2+ increases and ROS production are necessary for NF-κB activation and stimulation of gene expression in wt myotubes. Dystrophic myotubes showed a reduced amplitude and area under the curve of the Ca2+ transient elicited by electrical stimulation. On the other hand, electrical stimuli induced higher ROS production in mdx than wt myotubes, which were blocked by NOX2 inhibitors. Moreover, mRNA expression and protein levels of the NADPH oxidase subunits: p47phox and gp91phox were increased in mdx myotubes. Looking at ROS-dependence of NF-κB activation we found that in wt myotubes external administration of 50 μM H2O2 increased NF-κB activity; after administration of 100 and 200 μM H2O2 there was no effect. In mdx myotubes there was a dose-dependent reduction in NF-κB activity in response to external administration of H2O2, with a significant effect of 100 μM and 200 μM, suggesting that ROS levels are critical for NF-κB activity. Prior blockage with NOX2 inhibitors blunted the effects of electrical stimuli in both NF-κB activation and IL-6 expression. Finally, to ascertain whether stimulation of NF-κB and IL-6 gene expression by the inflammatory pathway is also impaired in mdx myotubes, we studied the effect of lipopolysaccharide on both NF-κB activation and IL-6 expression. Exposure to lipopolysaccharide induced a dramatic increase in both NF-κB activation and IL-6 expression in both wt and mdx myotubes, suggesting that the altered IL-6 gene expression after electrical stimulation in mdx muscle cells is due to dysregulation of Ca2+ release and ROS production, both of which impinge on NF-κB signaling. IL-6 is a key metabolic modulator that is released by the skeletal muscle to coordinate a multi-systemic response (liver, muscle, and adipocytes) during physical exercise; the alteration of this response in dystrophic muscles may contribute to an abnormal response to contraction and exercise.


Consumption of Two Healthy Dietary Patterns Restored Microbiota Dysbiosis in Obese Patients with Metabolic Dysfunction.
The consumption of two healthy diets (Mediterranean (MED) and low-fat (LF) diets) may restore the gut microbiome dysbiosis in obese patients depending on the degree of metabolic dysfunction. The differences in bacterial community at baseline and after 2 years of dietary intervention of 106 subjects from the CORDIOPREV study were analyzed, 33 of whom were obese patients with severe metabolic disease (5 criteria for metabolic syndrome) (MetS-OB), 32 obese patients without metabolic dysfunction (2 or less criteria for metabolic syndrome) (NonMetS-OB) and 41 non-obese subjects (NonMetS-NonOB). Our study showed a marked dysbiosis in people with severe metabolic disease (Met-OB), compared with obese people without MetS (NonMetS-OB) and non-obese people (NonMetS-NonOB). This disbiotic pattern was reversed by consumption of both MED (35% of calories as fat (22% MUFA fat, 6% PUFA fat and <10% saturated fat) or LF (<30% total fat (<10% saturated fat, 12%-14% MUFA fat and 6-8% PUFA fat) diets, whereas no significant microbiota changes were observed in NonMetS-NonOB and NonMetS-OB groups. Our results suggest that the chronic intake of two healthy dietary patterns partially restores the gut microbiome dysbiosis in obese patients with coronary heart disease, depending on the degree of metabolic dysfunction.


The Potential Physiological Role of γ-Tocopherol in Human Health: A Qualitative Review.
Chronic aging-related diseases result in the greatest burden to the health care system, yet there is little agreement on optimal levels of vitamins or the functional significance of many other dietary molecules in disease prevention. This review presents accumulated information regarding the role of γ-tocopherol in the prevention of nitrogen oxide-mediated damage and its impact on aging-related diseases. γ-Tocopherol is ubiquitous in the diet and levels appear to be physiologically regulated such that levels rise in response to inflammation and deficiencies in certain key vitamins. The unique antioxidant properties of γ-tocopherol, whereby DNA-damaging nitrogen dioxide is rapidly converted to nitric oxide, suggest a mechanistic justification for a functional role in the prevention of DNA damage over time. Data from cell, animal, and human studies indicate that γ-tocopherol appears to have significant beneficial effects, protecting cells from inflammatory damage; however, interpretation of epidemiologic studies is complex due to the paradoxical rise in levels of γ-tocopherol in response to known etiologic risk factors. Current knowledge of its antioxidant mechanism of action, apparent physiological regulation, and impact on various enzymatic pathways suggests γ-tocopherol may have a functional role in maintaining human health. Its utility as a biomarker and the consequences of its deficiency deserve further study.


The phylogenetic position of Serratia, Buttiauxella and some other genera of the family Enterobacteriaceae.
The phylogenetic relationships of the type strains of 38 species from 15 genera of the family Enterobacteriaceae were investigated by comparative 16S rDNA analysis. Several sequences of strains from the genera Citrobacter, Erwinia, Pantoea, Proteus, Rahnella and Serratia, analysed in this study, have been analysed previously. However, as the sequences of this study differ slightly from the published ones, they were included in the analysis. Of the 23 enterobacterial genera included in an overview dendrogram of relatedness, members of the genera Xenorhabdus, Photorhabdus, Proteus and Plesiomonas were used as a root. The other genera formed two groups which could be separated, although not exclusively, by signature nucleotides at positions 590-649 and 600-638. Group A contains species of Brenneria, Buttiauxella, Citrobacter, Escherichia, Erwinia, Klebsiella, Pantoea, Pectobacterium and Salmonella. All seven type strains of Buttiauxella share 16S rDNA similarities greater than 99%. Group B embraces two phylogenetically separate Serratia clusters, a lineage containing Yersinia species, Rahnella aquatica, Ewingella americana, and also the highly related pair Hafnia alvei and Obesumbacterium proteus.


[A study on physical symptom, activity of daily living, and health-related quality of life (HRQoL) in the community-dwelling older adults].
This study aimed to identify the relationships among physical symptoms, activities of daily living, and health-related quality of life (HRQoL) in community-dwelling older adults. A stratified random sampling method was conducted to recruit participants from May 10 to August 17, 2007. Physical symptoms were measured using the Physical Health Questionnaire (PHQ), activities of daily living using the Late-Life Functional and Disability Instrument (LLFDI), and HRQoL using the Medical Outcomes Short-Form Health Survey (SF-36) in 242 community-dwelling elderly Korean people. The HRQoL correlates with the physical symptoms (r=-.31) and the function component (r=.59). Of the two disability parts of the LLFDI, the limitation dimension correlates higher (r=.57) with HRQoL than the frequency dimension (r=.42). The HRQoL is significantly associated with the function component, and disability limitation in capability which explained 44.4% of variance in physical health. These results may contribute to a better understanding of physical symptoms, activities of daily living, and HRQoL in community-dwelling older adults. Therefore, health programs for prompting older adult's health should be planned based on results of the study.


Inhibition of phosphatidylinositol 3-kinase- and ERK MAPK-regulated protein synthesis reveals the pro-apoptotic properties of CD40 ligation in carcinoma cells.
CD40, a member of the tumor necrosis factor receptor superfamily, is frequently expressed in carcinomas where its stimulation results in induction of apoptosis when de novo protein synthesis is inhibited. The requirement of protein synthesis inhibition for efficient killing suggests that CD40 transduces potent survival signals capable of suppressing its pro-apoptotic effects. We have found that inhibition of CD40 signaling on the phosphatidylinositol 3-kinase (PI3K) and ERK MAPK but not on the p38 MAPK axis disrupts this balance and sensitizes carcinoma cells to CD40-mediated cell death. The CD40-mediated PI3K and ERK activities were found to converge on the regulation of protein synthesis in carcinoma cells via a pathway involving the activation of p90 ribosomal S6 kinase (p90Rsk) and p70S6 kinases, upstream of the translation elongation factor eEF2. In addition, CD40 ligation was found to mediate a PI3K- and mammalian target of rapamycin (mTOR)-dependent phosphorylation of 4E-BP1 and its subsequent dissociation from the mRNA cap-binding protein eIF4E as well as an ERK-dependent phosphorylation of eIF4E, thus promoting translation initiation. Concomitantly, the antiapoptotic protein cFLIP was found to be induced in CD40 ligand-stimulated carcinoma cells in a PI3K-, ERK-, and mammalian target of rapamycin (mTOR)-dependent manner and down-regulation of cFLIPS expression sensitized to CD40-mediated carcinoma cell death. These data underline the significance of the PI3K and ERK pathways in controlling the balance between CD40-mediated survival and death signals through the regulation of the protein synthesis machinery. Pharmacological agents that target this machinery or its upstream kinases could, therefore, be exploited for CD40-based tumor therapy.


Escherichia coli capsule bacteriophages. III. Fragments of bacteriophage 29.
A glycanase activity, catalyzing the depolymerization of host capsular polysaccharide, is associated with Escherichia coli capsule bacteriophage no. 29, a small virus with an isometric head, carrying a base plate with a set of spikes. The bacteriophage particles were disrupted by mild acid treatment (5 to 8 min at pH 3.5 and 37 C), and the enzymatically active fragments were isolated and subjected to sodium dodecyl sulfate-gel electrophoresis as well as to electron microscopy. Of the at least nine different polypeptide chains found in the complete virion, three (of 57,000 plus or minus 3,000, 29,500 plus or minus 2,000 and 13,500 plus or minus 1,000 daltons) were detected in detached base plates. They had the appearance of six-pointed stars of about 14 nm in outer diameter, with a central hole or prop, carrying six (or, possibly, a multiple thereof) spikes. Two sizes of polypeptide chains (57,000 and 29,500) were found in pure spikes, cylindrical particles of about 14.5 to 15 nm in length and 5 nm in diameter, and one (57,000) in -- still capsule depolymerizing -- spike subunits of roughly 5 nm in diameter. Phage 29 spike preparations, homogeneous in analytical ultracentrifugation and immunoelectrophoresis, were found to have a molecular weight of 245,000, as determined from the sedimentation equilibrium, and to contain equimolar amounts of the two polypeptides, probably three copies of each per organelle. The amino acid analysis of the isolated spikes revealed that aspartic acid, alanine, serine, and glycine are their dominant constituents; no amino sugars or other carbohydrates were detected in the preparations.


Clinical relevance of Fc gamma receptor polymorphisms.
Human IgG receptors are very heterogeneous and we currently distinguish three Fc gamma receptor classes specifying at least 12 receptor isoforms. On top of this complexity, Fc gamma R are further found to differ between different individuals. Polymorphisms have been identified for all three Fc gamma R classes. The best-studied ones represent allelic variation of Fc gamma RIIa (CD32) and Fc gamma RIIIb (CD16). The Fc gamma RIIa polymorphism is now considered to be a heritable risk factor for autoimmune and infectious diseases, and support for a relevant role of the IIIb polymorphism has also been obtained. A detailed analysis of the exact contribution of each of these Fc gamma R polymorphisms in relation to previously implicated risk factors should unravel the pathophysiological importance of Fc gamma R polymorphisms in the near future.


[Effect of sodium selenite and alpha-tocopherol on the microcirculation in massive blood loss in dogs].
Effect of little doses of sodium selenite combined with alpha-tocopherol on microcirculation was studied in experimental bloodletting in dogs. It was established that more rapid recovery, as compared to control, of blood flow in the visceral basin and more rapid elevation of arterial blood pressure after massive bloodletting occur following the pretreatment with sodium selenite at a dose of 30 micrograms/kg and alpha-tocopherol at a dose of 25 mg/kg. The blood flow gets recovered in small vessels (arterioles, vanules and capillaries) while in controls it recovers largely in greater vessels.


Correlation of prenatal and postnatal ultrasound findings with the incidence of vesicoureteral reflux in children with fetal renal pelvic dilatation.
Up to 1% of prenatal ultrasounds detect renal pelvic dilatation. This dilatation is associated with vesicoureteral reflux but its clinical significance and the necessity for vesicoureteral reflux detection have been questioned. We report an evaluation of fetal renal pelvic dilatation and postnatal sonographic features with the incidence of vesicoureteral reflux. Maximum fetal renal pelvic dilatation was prospectively measured at a single center between 1990 and 2003. Dilatation 4 mm or greater at less than 33 weeks of gestation, or 7 mm or greater at more than 33 weeks was the threshold for inclusion in the study. Postnatal evaluation included ultrasound and voiding cystourethrogram. Postnatal data included vesicoureteral reflux incidence and grade, and caliceal and ureteral dilatation on ultrasound. Of 215 neonates 46 (21%) had vesicoureteral reflux. Mean renal pelvic dilatation was 14.4 mm in those with reflux, which was not statistically different than the mean of 11.8 mm in 169 with a normal voiding cystourethrogram. ROC analysis revealed that fetal renal pelvic dilatation was a poor discriminator of reflux. Reflux was identified in a significantly greater number of neonates with vs without postnatal calicectasis (20% vs 9%, p <0.05). When fetal renal pelvic dilatation was combined with postnatal calicectasis, only 5% of infants with dilatation less than 10 mm and isolated renal pelvic dilatation had reflux, whereas reflux was identified in 25% with fetal renal pelvic dilatation 10 mm or greater and calicectasis (p <0.02). The magnitude of fetal renal pelvic dilatation is not reliably predictive of reflux and this measure alone cannot be used to direct postnatal cystography. However, postnatal calicectasis appears to be an important predictor of vesicoureteral reflux in children with fetal renal pelvic dilatation. Expectant management can be considered in the subset of newborns with minimal dilatation (less than 10 mm) and absent calicectasis.


Selection of LNA-containing DNA aptamers against recombinant human CD73.
LNA-containing DNA aptamers against CD73 (human ecto-5'-nucleotidase), a protein frequently overexpressed in solid tumours, were isolated by SELEX. A pre-defined stem-loop library, containing LNA in the forward primer region, was enriched with CD73 binding sequences through six rounds of SELEX with recombinant his-tagged CD73 immobilised on anti-his plates. Enriched pools isolated from rounds one, three and six were subjected to next-generation sequencing and analysed for enrichment using custom bioinformatics software. The software identified aptamer sequences via the primers and then performed several steps including sequence unification, clustering and alignment to identify enriched sequences. Three enriched sequences were synthesised for further analysis, two of which showed sequence similarities. These sequences exhibited binding to the recombinant CD73 with KD values of 10 nM and 3.5 nM when tested by surface plasmon resonance. Truncated variants of these aptamers and variants where the LNA nucleotides were substituted for the DNA equivalent also exhibited affinity for the recombinant CD73 in the low nanomolar range. In enzyme inhibition assays with recombinant CD73 the aptamer sequences were able to decrease the activity of the protein. However, the aptamers exhibited no binding to cellular CD73 by flow cytometry analysis likely since the epitope recognised by the aptamer was not available for binding on the cellular protein.


Ligand binding properties of human galanin receptors.
The galanin receptor family comprises of three members, GalR1, GalR2 and GalR3, all belonging to the G-protein-couple receptor superfamily. All three receptors bind the peptide hormone galanin, but show distinctly different binding properties to other molecules and effects on intracellular signaling. To gain insight on the molecular basis of receptor subtype specificity, we have generated a three-dimensional model for each of the galanin receptors based on its homologs in the same family. We found significant differences in the organization of the binding pockets among the three types of receptors, which might be the key for specific molecular recognition of ligands. Through docking of fragments of the galanin peptide and a number of ligands, we investigated the involvement of transmembrane and loop residues in ligand interaction.


Beta-agonist aerosol distribution in respiratory syncytial virus bronchiolitis in infants.
Bronchodilator aerosols are frequently administered to infants with bronchiolitis but with little success. The efficacy of aerosol treatments depends mainly on adequate targeting of the aerosol particles to the inflamed airways. This study evaluated the lower respiratory tract distribution characteristics of nebulized bronchodilators in infants with acute bronchiolitis. Twelve infants (mean age +/- SD, 8 mo +/- 4 mo) who were admitted for acute respiratory syncytial virus bronchiolitis were treated with (99m)Tc-albuterol aerosol. Gamma-scintigraphy was used to assess total body and lung deposition as well as pulmonary distribution of the medication. Of the total 6-min nebulized dose (i.e., drug aerosol dose leaving the nebulizer [not the nebulizer charge]), 1.5% +/- 0.7% reached the right lung, with only approximately one third of that (0.6%) penetrating to the peripheral lung zone. There was 7.8% +/- 4.9% deposition in the upper respiratory and gastrointestinal tracts and 10%-12% remained on the face. No correlation was found between any of the deposition indices and the clinical response data or any of the demographic parameters (e.g., height, weight, body surface area, or clinical score). Poor total aerosol deposition in infants may be related as much to their small conducting airways as to the disease state. There is considerable room for improvement in aerosol delivery in this age group, with greater emphasis on targeting narrowed peripheral airways with superfine aerosols.


Evolutionary algorithm based offline/online path planner for UAV navigation.
An evolutionary algorithm based framework, a combination of modified breeder genetic algorithms incorporating characteristics of classic genetic algorithms, is utilized to design an offline/online path planner for unmanned aerial vehicles (UAVs) autonomous navigation. The path planner calculates a curved path line with desired characteristics in a three-dimensional (3-D) rough terrain environment, represented using B-spline curves, with the coordinates of its control points being the evolutionary algorithm artificial chromosome genes. Given a 3-D rough environment and assuming flight envelope restrictions, two problems are solved: i) UAV navigation using an offline planner in a known environment, and, ii) UAV navigation using an online planner in a completely unknown environment. The offline planner produces a single B-Spline curve that connects the starting and target points with a predefined initial direction. The online planner, based on the offline one, is given on-board radar readings which gradually produces a smooth 3-D trajectory aiming at reaching a predetermined target in an unknown environment; the produced trajectory consists of smaller B-spline curves smoothly connected with each other. Both planners have been tested under different scenarios, and they have been proven effective in guiding an UAV to its final destination, providing near-optimal curved paths quickly and efficiently.


Calcium and magnesium concentrations in mature human milk: influence of calcium intake, age and socioeconomic level.
Concentrations of calcium and magnesium were measured in mature milk, collected between 30 and 90 days after childbirth, from a group of 90 mothers between 14 and 39 years of age, exclusively breastfeeding. The group was divided into three sub-groups: low socioeconomic-level adolescents (LSAd), low socioeconomic-level adults (LSA), and high socioeconomic-level adults (HSA). Each mother's nutritional status was determined using the body-mass index (BMI) and her eating habits, obtained on the basis of a 24-h dietary recall. Adolescent and adult mothers in the low socioeconomic-level group had lower average calcium intake (LSAd = 618.4 +/- 555.2 mg and LSA = 679.4 +/- 411.4 mg) than adult mothers in the higher socioeconomic-level group (853.6 +/- 415.5 mg). The average concentration of calcium in the adolescent mothers' milk (LSAd) was significantly lower (5.30 +/- 1.42 mmol Ca/L, P = 0.01) than that of the two adult groups (LSA = 5.82 +/- 1.55 mmol Ca/L and HSA = 6.40 mmol Ca/L). The average magnesium concentrations for all groups did not show significant differences (LSAd = 1.06 +/- 0.18, LSA = 1.16 +/- 0.23 and HSA = 1.11 +/- 0.23 mmol Mg/L, for P= 0.16). These results indicate that magnesium concentrations in mature human milk do not seem to depend on maternal nutritional status. The condition of adolescence, however, associated with lower calcium intake by the mother, resulted in lower calcium concentrations in the milk secreted when compared to that of adult mothers.


Ruptured mesenteric cyst: a rare presentation after trauma.
Mesenteric cysts are rare intraabdominal benign cystic lesions. These lesions are most commonly located in the ileal mesentery, without any sex predilection. Mesenteric cysts may be totally asymptomatic and discovered incidentally during routine radiologic examinations. Chronic abdominal pain or acute abdomen may be accompanying to these lesions. Ultrasonography, computed tomography and magnetic resonance imaging are valuable in the diagnosis. Rarely, these lesions may be presented with rupture after trauma. We present a patient with a ruptured ileal mesenteric cyst due to a blunt abdominal trauma and diagnosed by emergency laparotomy.


Oxidative and Nitrosative Stress in the Pathogenesis of Obstructive Lung Diseases of Increasing Severity.
The imbalance between increased oxidative agents and antioxidant defence mechanisms is central in the pathogenesis of obstructive lung diseases such as asthma and COPD. In these patients, there are increased levels of reactive oxygen species. Superoxide anions (O2 -), Hydrogen Peroxide (H2O2) and hydroxyl radicals (•OH) are critical for the formation of further cytotoxic radicals in the bronchi and lung parenchyma. Chronic inflammation, partly induced by oxidative stress, can further increase the oxidant burden through activated phagocytic cells (neutrophils, eosinophils, macrophages), particularly in severer disease states. Antioxidants and anti-inflammatory genes are, in fact, frequently downregulated in diseased patients. Nrf2, which activates the Antioxidant Response Element (ARE) leading to upregulation of GPx, thiol metabolism-associated detoxifying enzymes (GSTs) and stressresponse genes (HO-1) are all downregulated in animal models and patients with asthma and COPD. An exaggerated production of Nitric Oxide (NO) in the presence of oxidative stress can promote the formation of oxidizing reactive nitrogen species, such as peroxynitrite (ONO2 -), leading to nitration and DNA damage, inhibition of mitochondrial respiration, protein dysfunction, and cell damage in the biological systems. Protein nitration also occurs by activation of myeloperoxidase and H2O2, promoting oxidation of nitrite (NO2 -). There is increased nitrotyrosine and myeloperoxidase in the bronchi of COPD patients, particularly in severe disease. The decreased peroxynitrite inhibitory activity found in induced sputum of COPD patients correlates with pulmonary function. Markers of protein nitration - 3- nitrotyrosine, 3-bromotyrosine, and 3-chlorotyrosine - are increased in the bronchoalveolar lavage of severe asthmatics. Targeting the oxidative, nitrosative stress and associated lung inflammation through the use of either denitration mechanisms or new drug delivery strategies for antioxidant administration could improve the treatment of these chronic disabling obstructive lung diseases.


Sperm concentration and fertilization rate in Bufo arenarum (Amphibia: Anura).
The influence of sperm concentration upon the fertilization rate of Bufo arenarum oocytes was determinated. The experimental results were analysed according to the theories of Rothschild and Swann, and Hultin and Hagström for Psammechinus miliaris. The experimental results agreed with the predictions of the latter theory. Since Bufo arenarum and Psammechinus miliaris oocytes differ in both size and disposition of jelly envelopes, the postulations of Hultin and Hagström's theory appear to have a general validity.


Sublabial transseptal vs transnasal combined endoscopic microsurgery in patients with Cushing disease and MRI-depicted microadenomas.
To assess whether the type of surgical approach to the sella (sublabial transseptal vs transnasal) affects surgical outcome among patients with Cushing disease. Both procedures were performed at our institution from January 1, 1995, through January 31, 2003. From a total of 106 patients with Cushing disease who had had surgery, we identified 42 adults undergoing an initial surgery, with microadenoma (<1 cm) determined by magnetic resonance imaging and a minimal follow-up of 3 months. We identified 21 patients (4 male, 17 female) for sublabial transseptal transsphenoidal microsurgery. Mean+/-SD age was 45.0+/-12.9 years (range, 27.0-69.0 years); median duration of symptoms was 2.5 years (range, 1.5-7.5 years). Median follow-up was 1.0 year (range, 0.3-11.0 years). We identified another 21 patients (5 male, 16 female) for endoscopic transsphenoidal microsurgery whose mean+/-SD age was 43.8+/-14.1 years (range, 19.0-70.0 years); median duration of symptoms was 2.4 years (range, 0.2-6.0 years). Median follow-up was 2.5 years (range, 0.3-8.7 years). Complications (cerebrospinal fluid leak and transient diabetes insipidus) and cure (86% initial cure; 76% relapse-included cure) were equivalent between the 2 procedures. However, the endoscopic approach was associated with shorter anesthesia time, less blood loss, and shorter hospital stays. The endoscopic transnasal surgical approach led to shorter total anesthesia time, less blood loss, and shorter hospital stay when compared with the sublabial transseptal approach. However, we found no difference between the 2 surgical procedures with respect to cure or complications, suggesting that outcome is more closely related to the experience of the operating team.


Post-vaccine epidemiology of serotype 3 pneumococci identifies transformation inhibition through prophage-driven alteration of a non-coding RNA.
The respiratory pathogen Streptococcus pneumoniae (the pneumococcus) is a genetically diverse bacterium associated with over 101 immunologically distinct polysaccharide capsules (serotypes). Polysaccharide conjugate vaccines (PCVs) have successfully eliminated multiple targeted serotypes, yet the mucoid serotype 3 has persisted despite its inclusion in PCV13. This capsule type is predominantly associated with a single globally disseminated strain, GPSC12 (clonal complex 180). A genomic epidemiology study combined previous surveillance datasets of serotype 3 pneumococci to analyse the population structure, dynamics, and differences in rates of diversification within GPSC12 during the period of PCV introductions. Transcriptomic analyses, whole genome sequencing, mutagenesis, and electron microscopy were used to characterise the phenotypic impact of loci hypothesised to affect this strain's evolution. GPSC12 was split into clades by a genomic analysis. Clade I, the most common, rarely underwent transformation, but was typically infected with the prophage ϕOXC141. Prior to the introduction of PCV13, this clade's composition shifted towards a ϕOXC141-negative subpopulation in a systematically sampled UK collection. In the post-PCV13 era, more rapidly recombining non-Clade I isolates, also ϕOXC141-negative, have risen in prevalence. The low in vitro transformation efficiency of a Clade I isolate could not be fully explained by the ~100-fold reduction attributable to the serotype 3 capsule. Accordingly, prophage ϕOXC141 was found to modify csRNA3, a non-coding RNA that inhibits the induction of transformation. This alteration was identified in ~30% of all pneumococci and was particularly common in the unusually clonal serotype 1 GPSC2 strain. RNA-seq and quantitative reverse transcriptase PCR experiments using a genetically tractable pneumococcus demonstrated the altered csRNA3 was more effective at inhibiting production of the competence-stimulating peptide pheromone. This resulted in a reduction in the induction of competence for transformation. This interference with the quorum sensing needed to induce competence reduces the risk of the prophage being deleted by homologous recombination. Hence the selfish prophage-driven alteration of a regulatory RNA limits cell-cell communication and horizontal gene transfer, complicating the interpretation of post-vaccine population dynamics.


Novel insights into the pathogenesis of the Graffi murine leukemia retrovirus.
The Graffi murine leukemia virus (MuLV) was isolated in 1954 by Arnold Graffi, who characterized it as a myeloid leukemia-inducing retrovirus. He and his team, however, soon observed the intriguing phenomenon of hematological diversification, which corresponded to a decrease of myeloid leukemias and an increase of other types of leukemias. Recently, we derived two different molecular clones corresponding to ecotropic nondefective genomes that were named GV-1.2 and GV-1.4. The induced leukemias were classified as myeloid based on morphological analysis of blood smears. In this study, we further characterized the two variants of the Graffi murine retrovirus, GV-1.2 and GV-1.4, in three different strains of mice. We show that the Graffi MuLV is a multipotent retrovirus capable of inducing both lymphoid (T- and B-cell) and nonlymphoid (myeloid, erythroid, megakaryocytic) leukemia. Many of these are very complex with concomitant expression of different hematopoietic lineages. Interestingly, a high percentage of megakaryocytic leukemias, a type of leukemia rarely observed with MuLVs, arise in the FVB/n strain of mice. The genetic backgrounds of the different strains of mice influence greatly the results. Furthermore, the enhancer region, different for GV-1.2 and GV-1.4, plays a pivotal role in the disease specificity: GV-1.2 induces more lymphoid leukemias, and GV-1.4 induces more nonlymphoid ones.


[Pathomorphology of myocardium in the neuroleptic malignant syndrome].
It was performed morphometric analysis of 55 cases which formed 4 groups: 1 - control group - 10 patients without neuroleptic treatment and heart diseases; 2 - 12 patients without heart disease treated with antipsychotic drugs; 3 - 17 - without cardiaс pathology who died from neuroleptic malignant syndrome (NMS); 4 - 16 patients with neuroleptic cardiomyopathy (NCMP) who died from NMS. The damage of myocardium in NMS is a relatively acute process involving disturbances of microcirculation, interstitial edema and dystrophic-degenerative changes of cardiomyocytes (CMC). These changes worsen the previous abnormal state of the heart muscle (microfibrosis, CMC atrophy) caused by cardiotoxic side-effects of neuroleptics. The severity of the total myocardium damage in NMS is directly depended on the absence or presence of NCMP.


Comparison of left versus right atrial myocardium in patients with sinus rhythm or atrial fibrillation - an assessment of mitochondrial function and microRNA expression.
Several of the cellular alterations involved in atrial fibrillation (AF) may be linked to mitochondrial function and altered microRNA (miR) expression. A majority of studies on human myocardium involve right atrial (RA) tissue only. There are indications that AF may affect the two atria differentially. This study aimed to compare interatrial differences in mitochondrial respiration and miR expression in the RA versus left atrium (LA) within patients with sinus rhythm (SR) and AF. Thirty-seven patients with AF (n = 21) or SR (n = 16), undergoing coronary artery bypass surgery and/or heart valve surgery, were included. Myocardial biopsies were obtained from RA and LA appendages. Mitochondrial respiration was assessed in situ in permeabilized myocardium. MiR array and real-time quantitative polymerase chain reaction were performed to evaluate miR expression. Mitochondrial respiratory rates were similar in RA versus LA. Expression of miR-100, -10b, -133a, -133b, -146a, -155, -199a-5p, -208b, and -30b were different between the atria in both SR and AF patients. In contrast, differential expression was observed between RA versus LA for miR-93 in patients with SR only, and for miR-1, -125b, -142-5p, -208a, and -92b within AF patients only. These results indicate that mitochondrial respiratory capacity is similar in the RA and LA of patients with SR and AF. Differences in miR expressional profiles are observed between the RA versus LA in both SR and AF, and several interatrial differences in miR expression diverge between SR and AF. These findings may contribute to the understanding of how AF pathophysiology may affect the two atria differently.


Fluorescent and Colorimetric Dual-Readout Assay for Inorganic Pyrophosphatase with Cu(2+)-Triggered Oxidation of o-Phenylenediamine.
We demonstrate a rationally designed fluorescent and colorimetric dual-readout strategy for the highly sensitive, quantitative determination of inorganic pyrophosphatase (PPase) activity, a key hydrolytic enzyme involved in a variety of metabolic processes. Inspired by the selective oxidative and chromogenic reaction of o-phenylenediamine (OPD) with Cu(2+), the special inhibitory effects of pyrophosphate (PPi) on the oxidative ability of Cu(2+), and the specific hydrolysis of PPi into orthophosphate by PPase, a convenient small molecule OPD-based analytical system was developed for Cu(2+)/PPi recognition and PPase activity assay. We have confirmed that Cu(2+) acts as the oxidant in the reaction and the main chromogenic product of OPD is 2,3-diaminophenazine (usually called OPDox) in the assay by combining the ESI-MS, (1)H NMR, and XPS spectra analysis. Direct electrochemical insights into the Cu(2+)-triggered and PPi-inhibited mechanism were performed by cyclic voltammetry characterizations of the Cu(2+) in the absence and presence of PPi for the first time. Furthermore, the proposed analytical system with clear response mechanism exhibits a promising outlook for the PPase activity assay in real biological samples and inhibitor screening.