Progressive Massive Fibrosis Resurgence Identified in U.S. Coal Miners Filing for Black Lung Benefits, 1970-2016.
There has been a resurgence of progressive massive fibrosis (PMF) in the United States, particularly among central Appalachian miners. We characterized the proportion of PMF among former U.S. coal miners applying for Federal Black Lung Program benefits, 1970-2016. Data from the U.S. Department of Labor were used to characterize trends in proportion of PMF cases, defined as an approved black lung claim with a determination of PMF, among all miners who filed for federal benefits between January 1, 1970, and December 31, 2016. Joinpoint, logistic, and linear regression models were used to identify changes in the proportion of claimants with PMF over time. There were 4,679 unique PMF cases among claimants for federal black lung benefits between 1970 and 2016, with 2,474 miners determined to have PMF since 1996. The number of PMF cases among Federal Black Lung Program claimants fell from 404 (0.5% of claimants) in 1978 to a low of 18 cases (0.6%) in 1988, and then increased to 353 cases (8.3%) in 2014. The proportion of federal black lung benefits claimants with PMF has been increasing since 1978 (0.06% annual percent change [APC]; 95% confidence interval [CI], 0.05-0.07%; P < 0.0001), and began increasing at a significantly increased rate after 1996 (0.26% APC; 95% CI, 0.25-0.28%; P < 0.0001). Most miners with PMF (84%) last mined in West Virginia, Kentucky, Pennsylvania, or Virginia. Since 1970, the proportion of claimants with PMF has increased significantly among miners who last worked in Kentucky (16.6% APC; 95% CI, 16.5-16.7%), Pennsylvania (4.7% APC; 95% CI, 4.6-4.8%), Tennessee (16.1% APC; 95% CI, 15.7-16.4%), West Virginia (16.8% APC; 95% CI, 16.6-16.9%), and most sharply among miners last working in Virginia (31.5% APC; 95% CI, 31.2-31.7%), where in 2009, more than 17% of claimants received a PMF determination. The proportion of PMF determinations for the rest of the United States has not exceeded 4%. There has been a resurgence of PMF, particularly in central Appalachian miners. The resurgence of this preventable disease points to the need for improved primary and secondary prevention of dust-related lung disease in U.S. coal miners.


Inhibition of macrophage migration by nucleotide-containing streptococcal preparations.
Certain extracts of streptococcal cell walls are known to inhibit macrophage migration in vitro. In this study, we attempted to identify the streptococcal components responsible for this phenomenon. Trypsinized cell walls and cytoplasm from groups A and B streptococci were extracted with hot formamide followed by acetone precipitation. Subsequent gel filtration in aqueous solutions yielded a fraction devoid of C-carbohydrate and containing mostly oligonucleotides, apparently derived from streptococcal cytoplasm. This fraction significantly inhibited the migration of peritoneal exudate cells from rats sensitized to groups A and B streptococci. It was noteworthy that no inhibition of migration was observed with cells from nonsensitized animals or control rats injected with BCG or complete Freund adjuvant. Similarly, no inhibition was obtained with formamide extracts of calf thymus RNA. Although the inhibition does not show specificity for streptococcal groups, it seems to have immunological specificity since prior sensitization with streptococci is required for migration inhibition.


[Resistance of Biomphalaria schrammi of Arcos, Minas Gerais, Brazil, to infection with 2 strains of Schistosoma mansoni].
The descendants of planorbid snail Biomphalaria schrammi Crosse, 1864, collected in the region of Arcos, State of Minas Gerais, Brazil, were exposed to miracidia of two strains of Schistosoma mansoni: the "LE" strain from Belo Horizonte, Minas Gerais State and the "SJ" strain from São José dos Campos, State of São Paulo. Of the 172 snails exposed to miracidia of both strains, none were infected. On the other hand, 100 Biomphalaria glabrata snails of the control group showed infection rates of 88% ("LE" strain) and 40% ("SJ" strain). The mortality rates of B. schrammi and B. glabrata were 40% and 10%, respectively.


Philadelphia chromosome-positive acute myeloid leukemia: cytoimmunologic and cytogenetic features.
Little is known about the morphological and clinical features of the minority of acute myeloid leukemias (AML) that carry the t(9;22)(q34;q11) translocation. Cytologic, cytogenetic and clinical features were studied at diagnosis and during disease evolution in 11 patients presenting with de novo AML. Diagnoses according to the FAB criteria were AML-M2 (3 cases), AML-MO and AML-M4 (2 cases each), AML-M4eos, AML-M5, AML-M6, AML-M7 (1 case each). Immunophenotyping disclosed positivity for CD33 or CD13 and for the CD34 stem cell antigen in all cases tested. Lymphoid-associated markers (LM) were detected in 7/9 patients. In 5 cases the expression of at least 2 LM was seen. In addition, evidence of clonal rearrangement of the immunoglobulin (Ig) and/or T-cell receptor (TCR) genes was documented in 3/4 evaluable cases. The Ph chromosome was found as the sole change in 5/11 cases; the karyotype reverted to normal in 2/4 patients who achieved complete remission. Rearrangement in the M-bcr region was detected by Southern blotting in 2/7 cases. This infrequent cytogenetic subset of de novo AML appears to be characterized by heterogeneous cytologic features, with frequent expression of lymphoid markers, and by unfavorable prognosis. The combination of clinical, cytologic and cytogenetic studies is important in distinguishing de novo AML with the t(9;22) from chronic myelogenous leukemia blast crisis.


Mutants in the Exo I motif of Escherichia coli dnaQ: defective proofreading and inviability due to error catastrophe.
The Escherichia coli dnaQ gene encodes the proofreading 3' exonuclease (epsilon subunit) of DNA polymerase III holoenzyme and is a critical determinant of chromosomal replication fidelity. We constructed by site-specific mutagenesis a mutant, dnaQ926, by changing two conserved amino acid residues (Asp-12-->Ala and Glu-14-->Ala) in the Exo I motif, which, by analogy to other proofreading exonucleases, is essential for the catalytic activity. When residing on a plasmid, dnaQ926 confers a strong, dominant mutator phenotype, suggesting that the protein, although deficient in exonuclease activity, still binds to the polymerase subunit (alpha subunit or dnaE gene product). When dnaQ926 was transferred to the chromosome, replacing the wild-type gene, the cells became inviable. However, viable dnaQ926 strains could be obtained if they contained one of the dnaE alleles previously characterized in our laboratory as antimutator alleles or if it carried a multicopy plasmid containing the E. coli mutL+ gene. These results suggest that loss of proofreading exonuclease activity in dnaQ926 is lethal due to excessive error rates (error catastrophe). Error catastrophe results from both the loss of proofreading and the subsequent saturation of DNA mismatch repair. The probability of lethality by excessive mutation is supported by calculations estimating the number of inactivating mutations in essential genes per chromosome replication.


Theory and methodology of vitreous fluorophotometry.
The technique of vitreous fluorophotometry is discussed with particular reference to the uncertainty in the measurements resulting from instrumental and biological factors. The methods proposed for reducing the interference from these factors are explained. Various optical systems for carrying out the measurements are described. The suggestion is made that the technique could be much more accurate and safe if fluorescein was replaced by a dye which fluoresced at longer wavelengths and was more rapidly eliminated from the blood.


Interaction between functionalized gold nanoparticles in physiological saline.
The interactions between functionalized noble-metal particles in an aqueous solution are central to applications relying on controlled equilibrium association. Herein, we obtain the potentials of mean force (PMF) for pair-interactions between functionalized gold nanoparticles (AuNPs) in physiological saline. These results are based upon >1000 ns experiments in silico of all-atom model systems under equilibrium and non-equilibrium conditions. Four types of functionalization are built by coating each globular Au144 cluster with 60 thiolate groups: GS-AuNP (glutathionate), PhS-AuNP (thiophenol), CyS-AuNP (cysteinyl), and p-APhS-AuNP (para-amino-thiophenol), which are, respectively, negatively charged, hydrophobic (neutral-nonpolar), hydrophilic (neutral-polar), and positively charged at neutral pH. The results confirm the behavior expected of neutral (hydrophilic or hydrophobic) particles in a dilute aqueous environment, however the PMF curves demonstrate that the charged AuNPs interact with one another in a unique way-mediated by H2O molecules and an electrolyte (Na(+), Cl(-))-in a physiological environment. In the case of two GS-AuNPs, the excess, neutralizing Na(+) ions form a mobile (or 'dynamic') cloud of enhanced concentration between the like-charged GS-AuNPs, inducing a moderate attraction (∼25 kT) between them. Furthermore, to a lesser degree, for a pair of p-APhS-AuNPs, the excess, neutralizing Cl(-) ions (less mobile than Na(+)) also form a cloud of higher concentration between the two like-charged p-APhS-AuNPs, inducing weaker yet significant attractions (∼12 kT). On combining one GS- with one p-APhS-AuNP, the direct, attractive Coulombic force is completely screened out while the solvation effects give rise to moderate repulsion between the two unlike-charged AuNPs.


Green Pea (Pisum sativum L.) Hull Polyphenol Extract Alleviates NAFLD through VB6/TLR4/NF-κB and PPAR Pathways.
Green pea hull is a processing byproduct of green pea and rich in polyphenols. Nonalcoholic fatty liver disease (NAFLD) is a chronic metabolic disease characterized by accumulation of lipids in the liver for which there are no effective treatment strategies. Here, a mouse model of NAFLD induced by a DSS+high-fat diet (HFD) was established to investigate the effect of green pea hull polyphenol extract (EGPH). The results show that EGPH relief of NAFLD was a combined effect, including reducing hepatic fat accumulation, improving antioxidant activity and blood lipid metabolism, and maintaining glucose homeostasis. Increased intestinal permeability aggravated NAFLD. Combined metabolomics and transcriptomic analysis showed that vitamin B6 is the key target substance for EGPH to alleviate NAFLD, and it may be the intestinal flora metabolite. After EGPH intervention, the level of vitamin B6 in mice was significantly increased, and more than 60% in the blood enters the liver, which activated or inhibited PPAR and TLR4/NF-κB signaling pathways to relieve NAFLD. Our research could be a win-win for expanding the use of green pea hull and the search for NAFLD prophylactic drugs.


Ethylene levels are regulated by a plant encoded 1-aminocyclopropane-1-carboxylic acid deaminase.
Control of the levels of the plant hormone ethylene is crucial in the regulation of many developmental processes and stress responses. Ethylene production can be controlled by altering endogenous levels of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor to ethylene or by altering its conversion to ethylene. ACC is known to be irreversibly broken down by bacterial or fungal ACC deaminases (ACDs). Sequence analysis revealed two putative ACD genes encoded for in the genome of Arabidopsis thaliana (A. thaliana) and we detected ACD activity in plant extracts. Expression of one of these A. thaliana genes (AtACD1) in bacteria indicated that it had ACD activity. Moreover, transgenic plants harboring antisense constructs of the gene decreased ACD activity to 70% of wild-type (WT) levels, displayed an increased sensitivity to ACC and produced significantly more ethylene. Taken together, these results show that AtACD1 can act as a regulator of ACC levels in A. thaliana.


Coccygeal glomus tumors: a case of mistaken identity?
We undertook an anatomical and histological study to differentiate glomus-cell tumors of the pericoccygeal tissues from the normal coccygeal body. Removal of the coccyx was performed on five consecutive autopsy specimens from patients with no history of coccygeal symptoms. In each specimen, the coccygeal body (glomus coccygeum) was identified grossly and histologically. The histological appearance was indistinguishable from that of photomicrographs published in case reports of patients with glomus tumors of the coccyx. It is likely that the so-called tumors reported previously were in actuality normal glomus bodies.


Combining experimental and mathematical modeling to reveal mechanisms of macrophage-dependent left ventricular remodeling.
Progressive remodeling of the left ventricle (LV) following myocardial infarction (MI) can lead to congestive heart failure, but the underlying initiation factors remain poorly defined. The objective of this study, accordingly, was to determine the key factors and elucidate the regulatory mechanisms of LV remodeling using integrated computational and experimental approaches. By examining the extracellular matrix (ECM) gene expression and plasma analyte levels in C57/BL6J mice LV post-MI and ECM gene responses to transforming growth factor (TGF-β₁) in cultured cardiac fibroblasts, we found that key factors in LV remodeling included macrophages, fibroblasts, transforming growth factor-β₁, matrix metalloproteinase-9 (MMP-9), and specific collagen subtypes. We established a mathematical model to study LV remodeling post-MI by quantifying the dynamic balance between ECM construction and destruction. The mathematical model incorporated the key factors and demonstrated that TGF-β₁ stimuli and MMP-9 interventions with different strengths and intervention times lead to different LV remodeling outcomes. The predictions of the mathematical model fell within the range of experimental measurements for these interventions, providing validation for the model. In conclusion, our results demonstrated that the balance between ECM synthesis and degradation, controlled by interactions of specific key factors, determines the LV remodeling outcomes. Our mathematical model, based on the balance between ECM construction and destruction, provides a useful tool for studying the regulatory mechanisms and for predicting LV remodeling outcomes.


Mislocalization of CDK11/PITSLRE, a regulator of the G2/M phase of the cell cycle, in Alzheimer disease.
Post-mitotic neurons are typically terminally differentiated and in a quiescent status. However, in Alzheimer disease (AD), many neurons display ectopic re-expression of cell cycle-related proteins. Cyclin-dependent kinase 11 (CDK11) mRNA produces a 110-kDa protein (CDK11(p110)) throughout the cell cycle, a 58-kDa protein (CDK11(p58)) that is specifically translated from an internal ribosome entry site and expressed only in the G(2)/M phase of the cell cycle, and a 46-kDa protein (CDK11(p46)) that is considered to be apoptosis specific. CDK11 is required for sister chromatid cohesion and the completion of mitosis. In this study, we found that the expression patterns of CDK11 vary such that cytoplasmic CDK11 is increased in AD cellular processes, compared to a pronounced nuclear expression pattern in most controls. We also investigated the effect of amyloid precursor protein (APP) on CDK11 expression in vitro by using M17 cells overexpressing wild-type APP and APP Swedish mutant phenotype and found increased CDK11 expression compared to empty vector. In addition, amyloid-β(25-35) resulted in increased CDK11 in M17 cells. These data suggest that CDK11 may play a vital role in cell cycle re-entry in AD neurons in an APP-dependent manner, thus presenting an intriguing novel function of the APP signaling pathway in AD.


CEA/CD3-bispecific T cell-engaging (BiTE) antibody-mediated T lymphocyte cytotoxicity maximized by inhibition of both PD1 and PD-L1.
Bispecific T cell-engaging (BiTE) antibodies recruit polyclonal cytotoxic T cells (CTL) to tumors. One such antibody is carcinoembryonic antigen (CEA) BiTE that mediates T cell/tumor interaction by simultaneously binding CD3 expressed by T cells and CEA expressed by tumor cells. A widely operative mechanism for mitigating cytotoxic T cell-mediated killing is the interaction of tumor-expressed PD-L1 with T cell-expressed PD-1, which may be partly reversed by PD-1/PD-L1 blockade. We hypothesized that PD-1/PD-L1 blockade during BiTE-mediated T cell killing would enhance CTL function. Here, we determined the effects of PD-1 and PD-L1 blockade during initial T cell-mediated killing of CEA-expressing human tumor cell lines in vitro, as well as subsequent T cell-mediated killing by T lymphocytes that had participated in tumor cell killing. We observed a rapid upregulation of PD-1 expression and diminished cytolytic function of T cells after they had engaged in CEA BiTE-mediated killing of tumors. T cell cytolytic activity in vitro could be maximized by administration of anti-PD-1 or anti-PD-L1 antibodies alone or in combination if applied prior to a round of T cell killing, but T cell inhibition could not be fully reversed by this blockade once the T cells had killed tumor. In conclusion, our findings demonstrate that dual blockade of PD-1 and PD-L1 maximizes T cell killing of tumor directed by CEA BiTE in vitro, is more effective if applied early, and provides a rationale for clinical use.


Selective vagal innervation of sinoatrial and atrioventricular nodes in canine heart.
Parasympathetic pathways mediating chronotropic and dromotropic responses to cervical vagal stimulation were determined from sequential, restricted, intrapericardial dissection around major cardiac vessels. Although right cervical vagal input evoked significantly greater bradycardia, supramaximal electrical stimulation of either vagus produced similar ventricular rates, both with and without simultaneous atrial pacing. Dissection of the triangular fat pad at the junction of the inferior vena cava-inferior left atrium (IVC-ILA) invariably eliminated all vagal input to the atrioventricular (AV) nodal region. Yet IVC-ILA dissection had minimal influence on evoked-chronotropic responses to either cervical vagal or stellate ganglia stimulation. Respective intrapericardial projection pathways, from either right or left vagi, are sufficiently distinct to allow unilateral parasympathetic denervation of the sinoatrial (SA) and atrioventricular (AV) nodal regions. Left vagal projections to the SA and AV nodal regions course primarily along and between the right pulmonary artery and left superior pulmonary vein. Right vagal projections to the SA and AV nodal regions are somewhat more diffuse but concentrate around the right pulmonary vein complex and adjacent segments of the right pulmonary artery. We conclude there are parallel, yet functionally distinct, inputs from right and left vagi to the SA and AV nodal regions.


Comparative Generalized Logic Modeling Reveals Differential Gene Interactions during Cell Cycle Exit in Drosophila Wing Development.
A comparative interaction detection paradigm is proposed to study the complex gene regulatory networks that control cell proliferation during development. Instead of attempting to reconstruct the entire cell cycle regulatory network from temporal transcript data, differential interactions - represented by generalized logic - are detected directly from time course transcript data under two distinct conditions. This comparative approach is scale- and shift-invariant and is capable of detecting nonlinear differential interactions. Simulation studies on E. coli circuits demonstrated that the proposed comparative method has substantially increased statistical power over the intuitive reconstruct-then-compare approach. This method was therefore applied to a microarray experiment, profiling gene expression in the fruit fly wing as cells exit the cell cycle, and under a condition which delays this exit, over-expression of the cell cycle regulator E2F. One statistically significant differential interaction was identified between two gene clusters that is strongly influenced by E2F activity, and suggests the involvement of the Hippo signaling pathway in response to E2F, a finding that may provide additional insights on cell cycle control mechanisms. Furthermore, the comparative modeling can be applied to both static and dynamic gene expression data, and is extendible to deal with more than two conditions, useful in many biological studies.


Nerve ultrasound: A useful screening tool for peripheral nerve sheath tumors in NF1?
To determine ultrasonographic peripheral nerve involvement in patients with asymptomatic neurofibromatosis type 1 (NF1). Thirteen asymptomatic and 4 minimally symptomatic patients with NF1 were included in this cross-sectional pilot study to detect asymptomatic abnormalities of the brachial plexus and upper and lower extremity nerves. Patients underwent clinical examination, nerve conduction studies (NCS), and high-resolution ultrasonography (HRUS). HRUS showed abnormalities in 16 patients (94.1%). Neurofibromas were identified in 10 patients (58.8%): localized neurofibromas were found in 3 patients (17.6%), plexiform neurofibromas in 3 (17.6%), and both in 4 (23.5%). In 6 patients (35.3%), only nerve enlargement without an abnormal fascicular pattern was observed. Severe involvement of the peripheral nervous system with multiple plexiform neurofibromas was observed in 7 patients (41.2%), while 4 patients (23.5%) had no or only minor involvement. Both NCS and HRUS were performed on 73 individual nerve segments. In 5.5%, abnormalities were found with both tests; in 50.7%, only with HRUS; and in 1.4%, only with NCS. HRUS frequently showed subclinical involvement of the peripheral nerves in NF1, also when NCS were normal. HRUS findings ranged from normal to widespread peripheral nerve involvement. Because the presence of plexiform neurofibromas and the benign tumor load are risk factors for the development of a malignant peripheral nerve sheath tumor, HRUS may be a useful tool to identify a subgroup of patients who could benefit from regular follow-up.


Direct comparison of demyelinating disease induced by the Daniel's strain and BeAn strain of Theiler's murine encephalomyelitis virus.
We compared CNS disease following intracerebral injection of SJL mice with Daniel's (DA) and BeAn 8386 (BeAn) strains of Theiler's murine encephalomyelitis virus (TMEV). In tissue culture, DA was more virulent then BeAn. There was a higher incidence of demyelination in the spinal cords of SJL/J mice infected with DA as compared to BeAn. However, the extent of demyelination was similar between virus strains when comparing those mice that developed demyelination. Even though BeAn infection resulted in lower incidence of demyelination in the spinal cord, these mice showed significant brain disease similar to that observed with DA. There was approximately 100 times more virus specific RNA in the CNS of DA infected mice as compared to BeAn infected mice. This was reflected by more virus antigen positive cells (macrophages/microglia and oligodendrocytes) in the spinal cord white matter of DA infected mice as compared to BeAn. There was no difference in the brain infiltrating immune cells of DA or BeAn infected mice. However, BeAn infected mice showed higher titers of TMEV specific antibody. Functional deficits as measured by Rotarod were more severe in DA infected versus BeAn infected mice. These findings indicate that the diseases induced by DA or BeAn are distinct.


Use of interactive live digital imaging to enhance histology learning in introductory level anatomy and physiology classes.
Histology is one of the main subjects in introductory college-level Human Anatomy and Physiology classes. Institutions are moving toward the replacement of traditional microscope-based histology learning with virtual microscopy learning amid concerns of losing the valuable learning experience of traditional microscopy. This study used live digital imaging (LDI) of microscopic slides on a SMART board to enhance Histology laboratory teaching. The interactive LDI system consists of a digital camera-equipped microscope that projects live images on a wall-mounted SMART board via a computer. This set-up allows real-time illustration of microscopic slides with highlighted key structural components, as well as the ability to provide the students with relevant study and review material. The impact of interactive LDI on student learning of Histology was then measured based on performance in subsequent laboratory tests before and after its implementation. Student grades increased from a mean of 76% (70.3-82.0, 95% CI) before to 92% (88.8-95.3, 95% CI) after integration of LDI indicating highly significant (P < 0.001) enhancement in students' Histology laboratory performance. In addition, student ratings of the impact of the interactive LDI on their Histology learning were strongly positive, suggesting that a majority of students who valued this learning approach also improved learning and understanding of the material as a result. The interactive LDI technique is an innovative, highly efficient and affordable tool to enhance student Histology learning, which is likely to expand knowledge and student perception of the subject and in turn enrich future science careers.


Use of plasma human herpesvirus-8 viral load measurement: evaluation of practice in three UK HIV treatment centres.
A retrospective audit of plasma human herpesvirus-8 (HHV-8) viral load testing was performed in three HIV treatment centres over 24 months. Reasons for testing (360 tests) were: symptoms of systemic inflammatory response syndrome (SIRS) (fever, lymphadenopathy and raised inflammatory markers); monitoring in known HHV-8 pathology other than Kaposi sarcoma (KS); investigation of known/suspected KS, and other/no reason. Of patients with multicentric Castleman disease (MCD), 14/16 (88%) had detectable plasma HHV-8, as did 27/45 (60%) with biopsy proven or clinically confirmed KS, and 6/19 (32%) with lymphoma. Neither of the two patients with MCD and no detectable HHV-8 had SIRS symptoms at the time of the test. There was wide variation between centres in the indications prompting HHV-8 testing, with a more conservative approach resulting in a higher proportion of positive results. Measuring plasma HHV-8 in the absence of SIRS symptoms, established HHV-8 disease monitoring, or confirmed/suspected KS is unlikely to yield detectable HHV-8 thus allowing potential cost savings.


Endoscopic extended transsphenoidal resection of craniopharyngiomas: nuances of neurosurgical technique.
Endoscopic approaches to the midline ventral skull base have been extensively developed and refined for resection of cranial base tumors over the past several years. As these techniques have improved, both the degree of resection and complication rates have proven comparable to those for transcranial approaches, while visual outcomes may be better via endoscopic endonasal surgery and hospital stays and recovery times are often shorter. Yet for all of the progress made, the steep learning curve associated with these techniques has hampered more widespread implementation and adoption. The authors address this obstacle by coupling a thorough description of the technical nuances for endoscopic endonasal craniopharyngioma resection with detailed illustrations of the important steps in the operation. Traditionally, transsphendoidal approaches to craniopharyngiomas have been restricted to lesions mostly confined to the sella. However, recently, endoscopic endonasal resections are more frequently employed for extrasellar and purely third ventricle craniopharyngiomas, whose typical retrochiasmatic location makes them ideal candidates for endoscopic transnasal surgery. The endonasal endoscopic approach offers many advantages, including direct access to the long axis of the tumor, early tumor debulking with minimal manipulation of the optic apparatus, more precise visualization of tumor planes, particularly along the undersurface of the chiasm and the roof of the third ventricle, and a minimal-access corridor that obviates the need for brain retraction. Although much emphasis has been placed on technical tenets of exposure and "how to get there," this article focuses on nuances of tumor resection "when you are there." Three operative videos illustrate our discussion of technical tenets.


Phytochrome B is involved in mediating red light-induced stomatal opening in Arabidopsis thaliana.
The stomatal pores of higher plants enable gaseous exchange into and out of leaves for photosynthesis and evaporation. Stomatal opening is induced by both blue and red lights. It is shown that blue light-induced stomatal opening is mediated by the blue light receptor phototropins (PHOT1 and PHOT2) and cryptochromes (CRY1 and CRY2). However, whether phytochrome B (phyB) is involved in red light regulation of stomatal opening remains largely unclear. Here, we report a positive role for Arabidopsis (Arabidopsis thaliana) phyB in the regulation of red light-induced stomatal opening. The phyB mutant stomata displayed a reduced red light response, whereas stomata of the phyB-overexpressing plants displayed a hypersensitive response to red light. In addition, stomata of the cry1 cry2 phyB, phot1 phot2 phyB, and cry1 phyA phyB triple mutant plants showed more reduced light response than those of the single or double mutant plants under white light, implying that phyB acts in concert with phyA, CRY, and PHOT in light regulation of stomatal opening. Stomata of phyB cop1 mutant opened less wide than those of the cop1 mutant, and stomata of the pif3 pif4 mutant opened wider than those of the wild-type, indicating that COP1, together with the PIFs (phytochrome interacting factors), may act downstream of PHYB in regulating stomatal opening. Furthermore, quantitative RT-PCR analysis showed that the expression of MYB60 was reduced in the cry1 cry2 and phyA phyB mutants under blue and red lights, respectively, but induced in the CRY1- and phyB-overexpressing plants. These results demonstrate that phyB and CRY might regulate stomatal opening, at least in part, by regulating MYB60 expression.


Methyl 3,6-di-O-pivaloyl-alpha-D-mannopyranoside.
The X-ray crystal structure analysis of the title compound, C(17)H(30)O(8), revealed a (4)C(1) conformation of the pyranosyl ring [Cremer-Pople puckering parameters of Q = 0.568 (2) A, theta = 5.1 (2) and phi = 218 (3) degrees ]. The structure shows no deviations from the geometric parameters of pyranoside carbohydrates. The hydroxyl groups participate in O-H.O hydrogen bonds, forming a two-dimensional pattern [O.O = 2.811 (3) and 2.995 (3) A].


Vestibulopathy and the age effects on protective stepping during unperturbed standing.
This study examined the prevalence of protective stepping and accompanying preparatory postural responses associated with lateral weight transfer (WT) while subjects attempted to sustain stationary standing. The subjects were 92 healthy young and older adults and persons with vestibular hypofunction. Force platform and whole-body-motion recordings were used to evaluate the prevalence of stepping and WT responses during stationary standing (eyes open or closed) using a semi-tandem foot position. WT components were also evaluated for volitionally requested step initiation, and as a function of support base configuration and direction of stepping among younger subjects. Only 10% of trials by subjects with bilateral vestibular hypofunction (BVH) during semi-tandem standing with eyes closed were completed without a step, while 31% of subjects with UVH, 69% of healthy elders, and all young healthy subjects were able to stand for the entire 7-sec trials. WT responses always preceded volitional steps from a standard feet-parallel orientation but occurred in only 13% of the spontaneous steps. The prevalence of WT was influenced by the direction of volitional stepping from semi-tandem standing, but not by the initial standing width. Spontaneous stepping to maintain standing balance is a naturally occurring and prevalent behavior among older adults and persons with vestibular hypofunction during tests of quasi-static standing. Differences between volitional and spontaneous step initiation involving the prevalence of preparatory lateral WT are a complex function of motor planning, mechanical constraints, and functional context.


Nurses' various ways of conceiving their learning process as doctoral students: a phenomenographic study.
The aim was to describe variations in how doctoral students conceive their learning process to become researchers in the light of their professional background as nurses. Nursing research is an emerging discipline and the number of nurses who acquire a doctor's degree is increasing. The study had a descriptive, qualitative design with a phenomenographic approach and was carried out by means of 20 interviews. Three different description categories emerged: (1) A learning process that provides a synthesis of different parts of the research process aimed at developing preparedness for action within the nursing profession. (2) A learning process where practical problems are integrated with and problematised in relation to scientific theories. (3) A learning process involving the transformation from nurse to researcher. The description categories revealed that the focus was on solving problems that occur in health care and synthesising them by means of research tools. Furthermore, the doctoral students explored different ways of understanding and developing their awareness of the nature of research. Focus was also on the nursing profession and practice and a shift towards the role of a researcher was evident.


[The rehabilitation of bronchial asthma patients at a polyclinic].
A study of 299 patients with bronchial asthma indicates that it is of importance to include breathing with positive pressure at expiration in the complex of the rehabilitation program. Broncholytics are recommended in patients with average severe and severe course during exacerbation with consideration of circadian rhythms of bronchial patency. Positive-pressure breathing allows to reduce the maintenance dose of berotek by 29% and theopek by 31%.


Recurrent lumbosacral herpes simplex in the bedridden hospitalized patient.
A clinical variant of genital herpes simplex virus (HSV) infection, recurrent lumbosacral HSV, occurs in bedridden hospitalized patients. We want to call attention to an uncommon pattern of HSV infection in the hospitalized bedridden patient seen by the dermatology consultation service in a large university hospital. HSV is characterized by a mixture of recurrent groups of herpetic vesicles in all stages of development, with multiple, persistent hyperpigmented patches bilaterally distributed over the lumbosacral (buttocks) area.


A randomized, controlled trial of foscarnet in the treatment of cytomegalovirus retinitis in patients with AIDS.
To evaluate foscarnet sodium in treating cytomegalovirus retinitis in patients with AIDS. Twenty-four previously untreated persons with AIDS and cytomegalovirus retinitis who were at low risk for loss of their visual acuity. PATIENTS were randomly assigned to receive either no therapy (delayed treatment, control group) or immediate treatment with intravenous foscarnet at a dose of 60 mg/kg body weight three times a day for 3 weeks (induction regimen) followed by a maintenance regimen of 90 mg/kg once a day. PATIENTS were examined weekly until they reached the primary clinical end point, defined as progression of their retinitis border by 750 microns or the development of a new retinal lesion due to cytomegalovirus. Progression was evaluated using retinal photographs by masked readers. Secondary evaluations included changes in visual acuity, cytomegalovirus shedding in the blood and urine, serum levels of human immunodeficiency virus type 1 (HIV-1) p24 antigen, and total CD4 T lymphocyte counts. The mean time to progression of retinitis was 3.2 weeks in the control group (n = 11) compared with 13.3 weeks in the treatment group (n = 13) (P less than 0.001). Nine of 13 patients in the treatment group had positive blood cultures for cytomegalovirus at entry and all nine cleared their blood of cytomegalovirus by the end of the induction period (P = 0.004) compared with one of six patients in the control group. No reductions in p24 levels were seen in the control patients compared with a reduction of more than 50% in p24 levels for all four patients on treatment for whom follow-up levels were available. The main adverse effects of foscarnet treatment were seizures (2 of 13 patients), hypomagnesemia (9 of 13), hypocalcemia (11 of 13), and elevations in serum creatinine above 176.8 mumol/L (2.0 mg/dL) (3 of 13). The control patients received an average of 0.2 units of blood per week compared with an average of 0.6 units of blood per week for the patients on treatment. The administration of foscarnet decreases the rate of progression of cytomegalovirus retinitis in persons with AIDS. Its judicious use is likely to prevent loss of vision in these patients. In this study, however, there was little change in visual acuity in patients in either the immediate or delayed treatment group because only patients with non-sight-threatening disease were selected.


Three-dimensional cluster analysis identifies interfaces and functional residue clusters in proteins.
Three-dimensional cluster analysis offers a method for the prediction of functional residue clusters in proteins. This method requires a representative structure and a multiple sequence alignment as input data. Individual residues are represented in terms of regional alignments that reflect both their structural environment and their evolutionary variation, as defined by the alignment of homologous sequences. From the overall (global) and the residue-specific (regional) alignments, we calculate the global and regional similarity matrices, containing scores for all pairwise sequence comparisons in the respective alignments. Comparing the matrices yields two scores for each residue. The regional conservation score (C(R)(x)) defines the conservation of each residue x and its neighbors in 3D space relative to the protein as a whole. The similarity deviation score (S(x)) detects residue clusters with sequence similarities that deviate from the similarities suggested by the full-length sequences. We evaluated 3D cluster analysis on a set of 35 families of proteins with available cocrystal structures, showing small ligand interfaces, nucleic acid interfaces and two types of protein-protein interfaces (transient and stable). We present two examples in detail: fructose-1,6-bisphosphate aldolase and the mitogen-activated protein kinase ERK2. We found that the regional conservation score (C(R)(x)) identifies functional residue clusters better than a scoring scheme that does not take 3D information into account. C(R)(x) is particularly useful for the prediction of poorly conserved, transient protein-protein interfaces. Many of the proteins studied contained residue clusters with elevated similarity deviation scores. These residue clusters correlate with specificity-conferring regions: 3D cluster analysis therefore represents an easily applied method for the prediction of functionally relevant spatial clusters of residues in proteins.


Growth of Aeromonas species on enteric agars.
The efficacy of eight routine enteric agars for supporting the growth of 32 strains of Aeromonas spp. (17 A. hydrophila strains, 8 A. sobria strains, and 7 A. caviae strains) was investigated. The plating efficiency of Aeromonas spp. on these media varied greatly (range, 0 to 100%), as did their colony size when compared with that on noninhibitory medium (5% sheep blood agar). Plating efficiency on seven of these eight media appeared to be strain- and not species dependent. Overall, eosin-methylene blue and Hektoen enteric agars showed low plating efficiencies for A. hydrophila, whereas both A. sobria and A. caviae were severely inhibited on brilliant green agar. When all these species are considered collectively, deoxycholate, MacConkey, and xylose lysine deoxycholate appeared to be the most satisfactory routine agars for Aeromonas spp. recovery when used in conjunction with blood agar.


Evaluation of radiation exposure dose at double-balloon endoscopy for the patients with small bowel disease.
Double-balloon endoscopy (DBE) is useful for the diagnosis and treatment of small bowel diseases. Although fluoroscopy is used to confirm the position of endoscope at DBE, the endoscopist does not have the knowledge with regard to the radiation exposure dose. In this study, we evaluated the absorbed dose during DBE in patients with suspected or established small bowel diseases. This was a retrospective study in which the estimated fluoroscopic radiation absorbed doses loaded on the small bowel and skin were determined according to the data of the referential X-ray experiment with a human body phantom. The subjects were 415 DBEs preformed in total. The mean small bowel absorbed doses on antegrade and retrograde DBEs were 42.2 and 53.8 mGy, respectively, showing that the organ dose applied in retrograde DBE was significantly higher (P<0.0001). The mean skin absorbed doses of them were 79.2 and 101.0 mGy, respectively, showing that the dose was also significantly higher on retrograde DBE (P<0.0001). Of 27 cases who were applied endoscopic balloon dilation, the mean fluoroscopy time was 16.0 minutes, and mean small bowel and skin absorbed doses were 121.9 and 228.9 mGy, respectively. In conclusion, endoscopist should be careful for reducing the organ exposure dose at DBE, particularly for the lower abdominal region. Double-balloon enteroscopy (DBE), endoscopic balloon dilation (EBD), endoscopic mucosal resection (EMR), double-balloon endoscopic retrograde cholangiopancreatography (DBERCP), percutaneous coronary intervention (PCI).


[Hygienic estimation a state of nutrition of infant and preschool children age of city of Murmansk].
The present research was directed on study of an actual meal and status of nutrition of children in the age of from birth till 5 years living in Murmansk (region of Far North). 998 children were surveyed. At an estimation of an actual meal of children the data about breast feeding are received, the basic nutrients misbalance of structure of diets of children are established, and their reasons are analyzed. On the basis of the received data the regional recommendations for organization of a healthy meal in children's preschool establishments and program of hygienic training of the parents to skills of a balanced diet of children of early and junior age were developed.


Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export.
Diverse molecules, from small antibacterial drugs to large protein toxins, are exported directly across both cell membranes of gram-negative bacteria. This export is brought about by the reversible interaction of substrate-specific inner-membrane proteins with an outer-membrane protein of the TolC family, thus bypassing the intervening periplasm. Here we report the 2.1-A crystal structure of TolC from Escherichia coli, revealing a distinctive and previously unknown fold. Three TolC protomers assemble to form a continuous, solvent-accessible conduit--a 'channel-tunnel' over 140 A long that spans both the outer membrane and periplasmic space. The periplasmic or proximal end of the tunnel is sealed by sets of coiled helices. We suggest these could be untwisted by an allosteric mechanism, mediated by protein-protein interactions, to open the tunnel. The structure provides an explanation of how the cell cytosol is connected to the external environment during export, and suggests a general mechanism for the action of bacterial efflux pumps.


Fence technique: guided bone regeneration for extensive three-dimensional augmentation.
This report describes a novel bone reconstructive technique based on guided bone regeneration for extensive three-dimensional hard tissue augmentation. This two-stage technique utilizes bioresorbable osteosynthesis plates, pins, and collagen membranes that form a physical barrier resembling a fence, which contains the bone graft biomaterials composed of a combination of deproteinized bovine bone matrix and autologous bone grafted from intraoral sites. This technique can result in significant bone regeneration with minimal patient discomfort. Four case reports are presented. Histologic analysis of specimens shows the presence of mature bone. This procedure yields favorable results for bone formation, implant placement, and patient satisfaction.


Kaposi's sarcoma (KS)-associated herpesvirus-like DNA sequences in peripheral blood mononuclear cells: association with KS and persistence in patients receiving anti-herpesvirus drugs.
Herpesvirus-like DNA sequences (KSHV/HHV-8) have recently been described in AIDS-associated Kaposi's sarcoma (KS) lesions. Many questions remain regarding the role of this virus in KS and the therapeutic implications of this finding. In the current study, KSHV/HHV-8 DNA was detected in peripheral blood mononuclear cells (PBMCs) from human immunodeficiency virus (HIV)-infected patients with KS (34/98) more often than in HIV-infected individuals without KS (12/64, P = .03). The detection of KSHV/HHV-8 DNA did not correlate with the CD4 lymphocyte count. Five patients demonstrated KSHV/HHV-8 DNA in their PBMCs during administration of intravenous foscarnet and/or ganciclovir. The continued detection of KSHV/HHV-8 DNA in the PBMCs of patients receiving these anti-herpesvirus drugs has potential implications regarding the virus-cell relationship of KSHV/HHV-8, as well as for the value of these drugs in treating or preventing KS, but additional studies are needed.


Improving the oral hygiene of institutionalized mentally retarded clients.
The quality of oral health care of persons with mental retardation has been reported in the literature to be less than that of their normal peers. The purpose of this study, set in a Midwest institution for persons with developmental retardation, was to determine if a change in policy followed by staff training and monitored by an interested third party would improve the oral hygiene of clients living on wards. Methods used to train staff in proper toothbrushing were developed. Three living units were randomly selected for study: control, training plus accountability (experimental group I), and training without accountability (experimental group II). The direct care staff of both experimental groups were trained in proper toothbrushing techniques. Staff of the control group received no training. A dental hygienist visited the living unit periodically to evaluate and provide feedback on the plaque index scores taken by the caregivers in both experimental groups. The plaque indexes of group I showed significant improvement over the control group and group II. The results of this pilot study support the other findings of higher plaque indexes for residents with mental retardation and that modifying toothbrushing policies and staff training are not as critical to the improvement of the clients' oral hygiene as is the presence of an involved, interested third party.


Telepsychiatry Use in U.S. Mental Health Facilities, 2010-2017.
The study examined the use of telepsychiatry in U.S. mental health facilities between 2010 and 2017. Changes over time in the availability of telepsychiatry were examined by using multiple waves of a national survey of mental health facilities. State and facility correlates of offering telepsychiatry in 2017 were examined. The proportion of state facilities that self-reported offering telepsychiatry increased significantly from 15.2% in 2010 to 29.2% in 2017, with wide variability among states.. In 2017, facilities with telepsychiatry were more commonly publicly owned than to have others forms of ownership (odds ratio [OR]=2.72, 95% confidence interval [CI]=2.47-2.99, p<0.001), although the percentage of privately owned facilities offering telepsychiatry has increased significantly since 2010 (OR=2.94, 95% CI=2.14-4.05, p<.001). Facilities offering telepsychiatry had lower odds of receiving funding from Medicaid (OR=0.86, 95% CI=0.75-0.98, p<0.001) but higher odds of receiving funding from all other sources. Facilities in states that did not fund telepsychiatry had lower odds of offering these services in 2017 (OR=0.57, 95% CI=0.51-0.62, p<0.001). Telepsychiatry was more commonly offered in states with higher proportions of rural population (OR=1.64, 95% CI=1.45-1.85, p<0.001) and designated medically underserved areas (OR=1.36, 95% CI=1.25-1.47, p<0.001), compared with other states. Nearly twice as many U.S. mental health facilities offered telepsychiatry in 2017 than in 2010. Medicaid funding lagged behind other funding sources, suggesting state administrative barriers. Telepsychiatry was commonly used by facilities in medically underserved and rural areas.


Multidisciplinary, early mobility approach to enhance functional independence in patients admitted to a cardiothoracic intensive care unit: a quality improvement programme.
Early mobilisation following cardiac surgery is vital for improved patient outcomes, as it has a positive effect on a patient's physical and psychological recovery following surgery. We observed that patients admitted to the cardiothoracic intensive care unit (CTICU) following cardiac surgery had only bed exercises and were confined to bed until the chest tubes were removed, which may have delayed patients achieving functional independence. Therefore, the CTICU team implemented a quality improvement (QI) project aimed at the early mobilisation of patients after cardiac surgery.A retrospective analysis was undertaken to define the current mobilisation practices in the CTICU. The multidisciplinary team identified various practice gaps and tested several changes that led to the implementation of a successful early mobility programme. The tests were carried out and reported using rapid cycle changes. A model for improvement methodology was used to run the project. The outcomes of the project were analysed using standard 'run chart rules' to detect changes in outcomes over time and Welch's t-test to assess the significance of these outcomes.This project was implemented in 2015. Patient compliance with early activity and mobilisation gradually reached 95% in 2016 and was sustained over the next 3 years. After the programme was implemented, the mean hours required for initiating out-of-bed-mobilisation was reduced from 22.77 hours to 11.74 hours. Similarly, functional independence measures and intensive care unit mobility scores also showed a statistically significant (p<0.005) improvement in patient transfers out of the CTICU.Implementing an early mobility programme for post-cardiac surgery patients is both safe and feasible. This QI project allowed for early activity and mobilisation, a substantial reduction in the number of hours required for initiating out-of-bed mobilisation following cardiac surgery, and facilitated the achievement of early ambulation and functional milestones in our patients.


Overcoming public resistance to carbon taxes.
Carbon taxes represent a cost-effective way to steer the economy toward a greener future. In the real world, their application has however been limited. In this paper, we address one of the main obstacles to carbon taxes: public opposition. We identify drivers of and barriers to public support, and, under the form of stylized facts, provide general lessons on the acceptability of carbon taxes. We derive our lessons from a growing literature, as well as from a combination of policy "failures" and "successes." Based on our stylized facts, we formulate a set of suggestions concerning the design of carbon taxes. We consider the use of trial periods, tax escalators, environmental earmarking, lump-sum transfers, tax rebates, and advanced communication strategies, among others. This paper contributes to the policy debate about carbon taxes, hopefully leading to more success stories and fewer policy failures. This article is categorized under: Climate Economics > Economics of Mitigation.


Deciphering Babesia-Vector Interactions.
Understanding host-pathogen-tick interactions remains a vitally important issue that might be better understood by basic research focused on each of the dyad interplays. Pathogens gain access to either the vector or host during tick feeding when ticks are confronted with strong hemostatic, inflammatory and immune responses. A prominent example of this is the Babesia spp.-tick-vertebrate host relationship. Babesia spp. are intraerythrocytic apicomplexan organisms spread worldwide, with a complex life cycle. The presence of transovarial transmission in almost all the Babesia species is the main difference between their life cycle and that of other piroplasmida. With more than 100 species described so far, Babesia are the second most commonly found blood parasite of mammals after trypanosomes. The prevalence of Babesia spp. infection is increasing worldwide and is currently classified as an emerging zoonosis. Babesia microti and Babesia divergens are the most frequent etiological agents associated with human babesiosis in North America and Europe, respectively. Although the Babesia-tick system has been extensively researched, the currently available prophylactic and control methods are not efficient, and chemotherapeutic treatment is limited. Studying the molecular changes induced by the presence of Babesia in the vector will not only elucidate the strategies used by the protozoa to overcome mechanical and immune barriers, but will also contribute toward the discovery of important tick molecules that have a role in vector capacity. This review provides an overview of the identified molecules involved in Babesia-tick interactions, with an emphasis on the fundamentally important ones for pathogen acquisition and transmission.


Cooperative activation of atrial naturetic peptide promoter by dHAND and MEF2C.
An intricate array of cell-specific multiprotein complexes participate in programs of cell-specific gene expression through combinatorial interaction with different transcription factors and cofactors. The dHAND basic helix-loop-helix (bHLH) transcription factor, which is essential for heart development and extra embryonic structures, is thought to regulate cardiomyocyte-specific gene expression through combinatorial interactions with other cardiac-restricted transcription factors such as GATA4 and NKX2.5. Here, we determine that dHAND also interacts with the myocyte enhancer binding factor-2c (MEF2C) protein, which belongs to MADS-box transcription factors and is essential for heart development. dHAND and MEF2C synergistically activated expression of the atrial naturetic peptide gene (ANP) in transfected HeLa cells. GST-pulldown and immunoprecipitation assay demonstrate that full-length MEF2C protein is able to interact with dHAND in vitro and in vivo, just like MEF2A and bHLH transcription factors MyoD in skeletal muscle cells. In addition, electrophoretic mobility shift assays (EMSAs) demonstrate that MEF2C and dHAND do not influence each other's DNA binding activity. Using chromatin immunoprecipitation (ChIP) analysis in H9c2 cells we show that dHAND interact with MEF2C to form protein complex and bind A/T sequence in promoter of ANP. Taken together with previous observations, these results suggest the existence of large multiprotein transcriptional complex with core DNA binding proteins that physically interact with other transcriptional factors to form favorable conformation to potentiate transcription.


Statistical modeling of the spatial variability of environmental noise levels in Montreal, Canada, using noise measurements and land use characteristics.
The availability of noise maps to assess exposure to noise is often limited, especially in North American cities. We developed land use regression (LUR) models for LAeq24h, Lnight, and Lden to assess the long-term spatial variability of environmental noise levels in Montreal, Canada, considering various transportation noise sources (road, rail, and air). To explore the effects of sampling duration, we compared our LAeq24h levels that were computed over at least five complete contiguous days of measurements to shorter sampling periods (20 min and 24 h). LUR models were built with General Additive Models using continuous 2-min noise measurements from 204 sites. Model performance (adjusted R2) was 0.68, 0.59, and 0.69 for LAeq24h, Lnight, and Lden, respectively. Main predictors of measured noise levels were road-traffic and vegetation variables. Twenty-minute non-rush hour measurements corresponded well with LAeq24h levels computed over 5 days at road-traffic sites (bias: -0.7 dB(A)), but not at rail (-2.1 dB(A)) nor at air (-2.2 dB(A)) sites. Our study provides important insights into the spatial variation of environmental noise levels in a Canadian city. To assess long-term noise levels, sampling strategies should be stratified by noise sources and preferably should include 1 week of measurements at locations exposed to rail and aircraft noise.


Ultrastructural analysis of throat dermal tissue and chromatophore components in the threespine stickleback (Gasterosteus aculeatus).
The threespine stickleback (Gasterosteus aculeatus) is an important model for studying the evolution of nuptial coloration, but histological analyses of color are largely lacking. Previous analyses of one nuptial coloration trait, orange-red coloration along the body, have indicated carotenoids are the main pigment producing this color. In addition, recent gene expression studies found variation in the correlates of throat coloration between the sexes and between populations, raising the possibility of variation in the mechanisms underlying superficially similar coloration. We used transmission electron microscopy (TEM) to investigate the histological correlates of color in the throat dermal tissue of threespine stickleback from Western North America, within and between sexes, populations, and ecotypes. Ultrastructural analysis revealed carotenoid-containing erythrophores to be the main chromatophore component associated with orange-red coloration in both males and females across populations. In individuals where some darkening of the throat tissue was present, with no obvious orange-red coloration, erythrophores were not detected. Melanophore presence was more population-specific in expression, including being the only chromatophore component detected in a population of darker fish. We found no dermal chromatophore units within colorless throat tissue. This work confirms the importance of carotenoids and the erythrophore in producing orange-red coloration across sexes, as well as melanin within the melanophore in producing darkened coloration, but does not reveal broad histological differences among populations with similar coloration.


Role of advanced glycation end products (AGEs) and oxidative stress in diabetic retinopathy.
Diabetic retinopathy is a common and potentially devastating microvascular complication in diabetes and is a leading cause of acquired blindness among the people of occupational age. However, current therapeutic options for the treatment of sight-threatening proliferative diabetic retinopathy such as photocoagulation and vitrectomy are limited by considerable side effects and far from satisfactory. Therefore, to develop novel therapeutic strategies that specifically target diabetic retinopathy is actually desired for most of the patients with diabetes. Chronic hyperglycemia is a major initiator of diabetic retinopathy. However, recent clinical study has substantiated the concept of 'hyperglycemic memory' in the pathogenesis of diabetic retinopathy. Indeed, the Diabetes Control and Complications Trial-Epidemiology of Diabetes Interventions and Complications (DCCT-EDIC) Research, has revealed that the reduction in the risk of progressive retinopathy resulting from intensive therapy in patients with type 1 diabetes persisted for at least several years after the DCCT trial, despite increasing hyperglycemia. These findings suggest a long-term beneficial influence of early metabolic control on clinical outcomes in type 1 diabetic patients. Among various biochemical pathways implicated in the pathogenesis of diabetic retinopathy, the process of formation and accumulation of advanced glycation end products (AGEs) and their mode of action are most compatible with the theory 'hyperglycemic memory'. Further, there is a growing body of evidence that AGEs-RAGE (receptor for AGEs) interaction-mediated oxidative stress generation plays an important role in diabetic retinopathy. This article summarizes the role of AGEs and oxidative stress in the development and progression of diabetic retinopathy and the therapeutic interventions that could prevent this devastating disorder. We also discuss here the pathological crosstalk between the AGEs-RAGE and the renin-angiotensin system in diabetic retinopathy and a potential clinical utility of telmisartan, an angiotensin II type 1 receptor blocker with peroxisome proliferator-activated receptor-gamma-modulating activity.


Biological and clinical effects of ozone. Has ozone therapy a future in medicine?
Although ozone therapy has been used as an alternative medical approach for four decades, it has encountered scepticism, if not outright objection, by orthodox medicine. This prejudice is not unjustified because ozone therapy often has been used without rational basis or appropriate controls. With the advent of precise medical ozone generators, it is now possible to evaluate some mechanisms of action and possible toxicity. In contrast with the respiratory tract, human blood exposed to appropriate ozone concentrations is able to tame its strong oxidant properties and neither acute nor chronic side effects have ensued in millions of patients treated with ozonated autohaemotherapy. This paper summarises studies aimed at clarifying biological effects, defining any possible damage, the therapeutic window, and suitable doses able to express therapeutic activity. Although an unfashionable and unpopular approach, it is hoped that orthodox medicine will help to critically assess the validity of ozone therapy.


[Peritoneal splenosis 26 years after traumatic splenic rupture--rare differential diagnosis of a subepithelial gastric mass--case report and review of the literature].
Differential diagnoses of subepithelial gastric masses include benign (leiomyoma, lipoma, haemangioma, lymphangioma, neurogenic tumours, glomus tumour) and malignant (leiomyosarcoma, gastric Kaposi's sarcoma, metastases) neoplastic lesions, gastrointestinal stromal tumours (GIST) and lesions of non-neoplastic origin (heterotopic pancreatic tissue, intramural pseudocysts, intramural haematoma). Occasionally, however, suspected gastric wall tumours are caused by extragastral lesions that are not always easily distinguished from genuine gastric wall lesions by endoscopy or radiological imaging. We report the case of a 77-year-old patient undergoing laparoscopy for suspected gastric GIST in our institution in whom splenectomy had been performed 26 years prior to presentation due to traumatic splenic rupture. The tumour revealed to be ectopic splenic tissue located at the parietal peritoneum of the ventral abdominal wall, thereby fulfilling the definition of splenosis. Epidemiology, pathogenesis, diagnostics and therapy of splenosis are discussed in the context of a review of the relevant literature.


Reduced affordability of cigarettes and socio-economic inequalities in smoking continuation in Stakhanov, Ukraine, 2009.
The recent tobacco excise tax increase and economic crisis reduced cigarette affordability in Ukraine dramatically. Using survey data from Stakhanov (n = 1691), eastern Ukraine, we employed logistic regression analysis to examine whether socio-economic status was associated with the continuation of smoking in this environment in 2009. Low education (in women) and ownership of household assets (in men) were negatively associated with smoking continuation, whereas a positive association was found for personal monthly income. Our findings suggest that in a low-income setting where efficient cessation services are absent, reduced cigarette affordability may have only a limited effect in cutting down smoking.


Dexmedetomidine Attenuates Lipopolysaccharide-Induced Sympathetic Activation and Sepsis via Suppressing Superoxide Signaling in Paraventricular Nucleus.
Sympathetic overactivity contributes to the pathogenesis of sepsis. The selective α2-adrenergic receptor agonist dexmedetomidine (DEX) is widely used for perioperative sedation and analgesia. We aimed to determine the central roles and mechanisms of DEX in attenuating sympathetic activity and inflammation in sepsis. Sepsis was induced by a single intraperitoneal injection of lipopolysaccharide (LPS) in rats. Effects of DEX were investigated 24 h after injection of LPS. Bilateral microinjection of DEX in the paraventricular nucleus (PVN) attenuated LPS-induced sympathetic overactivity, which was attenuated by the superoxide dismutase inhibitor DETC, cAMP analog db-cAMP or GABAA receptor antagonist gabazine. Superoxide scavenger tempol, NADPH oxidase inhibitor apocynin, adenylate cyclase inhibitor SQ22536 or PKA inhibitor Rp-cAMP caused similar effects to DEX in attenuating LPS-induced sympathetic activation. DEX inhibited LPS-induced superoxide and cAMP production, as well as NADPH oxidase, adenylate cyclase and PKA activation. The roles of DEX in reducing superoxide production and NADPH oxidase activation were attenuated by db-cAMP or gabazine. Intravenous infusion of DEX inhibited LPS-induced sympathetic overactivity, NOX activation, superoxide production, TNF-α and IL-1β upregulation in the PVN and plasma, as well as lung and renal injury, which were attenuated by the PVN microinjection of yohimbine and DETC. We conclude that activation of α2-adrenergic receptors with DEX in the PVN attenuated LPS-induced sympathetic overactivity by reducing NADPH oxidase-dependent superoxide production via both inhibiting adenylate cyclase-cAMP-PKA signaling and activating GABAA receptors. The inhibition of NADPH oxidase-dependent superoxide production in the PVN partially contributes to the roles of intravenous infusion of DEX in attenuating LPS-induced sympathetic activation, oxidative stress and inflammation.


A nuclear factor Y interacting protein of the GRAS family is required for nodule organogenesis, infection thread progression, and lateral root growth.
A C subunit of the heterotrimeric nuclear factor Y (NF-YC1) was shown to play a key role in nodule organogenesis and bacterial infection during the nitrogen fixing symbiosis established between common bean (Phaseolus vulgaris) and Rhizobium etli. To identify other proteins involved in this process, we used the yeast (Saccharomyces cerevisiae) two-hybrid system to screen for NF-YC1-interacting proteins. One of the positive clones encodes a member of the Phytochrome A Signal Transduction1 subfamily of GRAS (for Gibberellic Acid-Insensitive (GAI), Repressor of GAI, and Scarecrow) transcription factors. The protein, named Scarecrow-like13 Involved in Nodulation (SIN1), localizes both to the nucleus and the cytoplasm, but in transgenic Nicotiana benthamiana cells, bimolecular fluorescence complementation suggested that the interaction with NF-YC1 takes place predominantly in the nucleus. SIN1 is expressed in aerial and root tissues, with higher levels in roots and nodules. Posttranscriptional gene silencing of SIN1 using RNA interference (RNAi) showed that the product of this gene is involved in lateral root elongation. However, root cell organization, density of lateral roots, and the length of root hairs were not affected by SIN1 RNAi. In addition, the expression of the RNAi of SIN1 led to a marked reduction in the number and size of nodules formed upon inoculation with R. etli and affected the progression of infection threads toward the nodule primordia. Expression of NF-YA1 and the G2/M transition cell cycle genes Cyclin B and Cell Division Cycle2 was reduced in SIN1 RNAi roots. These data suggest that SIN1 plays a role in lateral root elongation and the establishment of root symbiosis in common bean.


Structural details of ribonuclease H from Escherichia coli as refined to an atomic resolution.
The crystal structure of RNase H from Escherichia coli has been determined by the multiple isomorphous replacement method, and refined by the stereochemically restrained least-squares procedure to a crystallographic R-factor of 0.196 at 1.48 A resolution. In the final structure, the root-mean-square (r.m.s.) deviation for bond lengths is 0.017 A, and for angle distances 0.036 A. The structure is composed of a five-stranded beta-sheet and five alpha-helices, and reveals the details of hydrogen bonding, electrostatic and hydrophobic interactions between intra- and intermolecular residues. The refined structure allows an explanation of the particular interactions between the basic protrusion, consisting of helix alpha III and the following loop, and the remaining major domain. The beta-sheet, alpha II, alpha III and alpha IV form a central hydrophobic cleft that contains all six tryptophan residues, and presumably serves to fix the orientation of the basic protrusion. Two parallel adjacent helices, alpha I and alpha IV, are associated with a few triads of hydrophobic interactions, including many leucine residues, that are similar to the repeated leucine motif. The well-defined electron density map allows detailed discussion of amino acid residues likely to be involved in binding a DNA/RNA hybrid, and construction of a putative model of the enzyme complexed with a DNA/RNA hybrid oligomer. In this model, a protein region, from the Mg(2+)-binding site to the basic protrusion, covers roughly two turns of a DNA/RNA hybrid double helix. A segment (11-23) containing six glycine residues forms a long loop between the beta A and beta B strands. This loop, which protrudes into the solvent region, lies on the interface between the enzyme and a DNA/RNA hybrid in the model of the complex. The mean temperature factors of main-chain atoms show remarkably high values in helix alpha III that constitutes the basic protrusion, suggesting some correlation between its flexibility and the nucleic acid binding function. The Mg(2+)-binding site, surrounded by four invariant acidic residues, can now be described more precisely in conjunction with the catalytic activity. The arrangement of molecules within the crystal appears to be dominated by the cancelling out of a remarkably biased charge distribution on the molecular surface, which is derived in particular from the separation between the acidic Mg(2+)-binding site and the basic protrusion.


Optimization of a high-performance liquid chromatography method for the analysis of complex polyphenol mixtures and application for sainfoin extracts (Onobrychis viciifolia).
A pentafluorophenylpropyl (PFP) stationary phase was tested for the simultaneous determination of several classes of phenolic compounds. The chromatographic results were compared with those obtained by using a bifunctional phase constituted of octadecyl and phenylpropyl bonded silica and three conventional C18 columns. The elution gradient was optimized with 5% formic acid and sodium acetate in combination with acetic acid as additives and methanol as solvents. For these evaluations, a complex phenolic extract of Onobrychis viciifolia (sainfoin) and test mixtures containing 54 standard substances including 2 simple phenolic compounds, 1 amino acid, 4 hydroxybenzoic acids (HBA), 6 hydroxycinnamic acids (HCA), 3 flavan-3-ols, 9 anthocyanins, 2 dihydroflavonols, 1 chalcone, 4 flavones, 1 isoflavone and 21 flavonols have been assayed. The perfluorinated column showed good resolution for the studied phenolic compounds which have the following elution order: HBA, HCA, flavan-3-ols, anthocyanins, dihydroflavonols, flavones, flavonols and isoflavones. Compared with other columns, it provides longer elution ranges for HBA, HCA and flavan-3-ols and increased retention times for all compound classes except anthocyanins which were similarly retained on a C18 column. Its selectivity is different from C18 and bifunctional phases. A high-performance liquid chromatography (HPLC) method with diode array detection (DAD) and post-column derivatization with p-dimethyl-aminocinnamic aldehyde (DMACA) has been validated for the analysis of individual phenolic compounds from a sainfoin plant extract (O. viciifolia).


The Vps13p-Cdc31p complex is directly required for TGN late endosome transport and TGN homotypic fusion.
Yeast VPS13 is the founding member of a eukaryotic gene family of growing interest in cell biology and medicine. Mutations in three of four human VPS13 genes cause autosomal recessive neurodegenerative or neurodevelopmental disease, making yeast Vps13p an important structural and functional model. Using cell-free reconstitution with purified Vps13p, we show that Vps13p is directly required both for transport from the trans-Golgi network (TGN) to the late endosome/prevacuolar compartment (PVC) and for TGN homotypic fusion. Vps13p must be in complex with the small calcium-binding protein Cdc31p to be active. Single-particle electron microscopic analysis of negatively stained Vps13p indicates that this 358-kD protein is folded into a compact rod-shaped density (20 × 4 nm) with a loop structure at one end with a circular opening ∼6 nm in diameter. Vps13p exhibits ATP-stimulated binding to yeast membranes and specific interactions with phosphatidic acid and phosphorylated forms of phosphatidyl inositol at least in part through the binding affinities of conserved N- and C-terminal domains.


Excitatory Amino Acid Pathway from the Hippocampus to the Prefrontal Cortex. Contribution of AMPA Receptors in Hippocampo-prefrontal Cortex Transmission.
Previous experiments in the rat have demonstrated that field CA1 and the subiculum project to the prefrontal cortex and that this direct unilateral pathway is excitatory. In the present study, anatomical and electrophysiological approaches were used to determine the transmitter mediating the excitatory responses in prefrontal cortex neurons to low-frequency stimulation of the hippocampus. The method of selective retrograde d-[3H]aspartate labelling was used to identify putative glutamatergic and/or aspartatergic hippocampal afferent fibres to the prefrontal cortex. Unilateral microinjection of d-[3H]aspartate into the prelimbic area of the prefrontal cortex resulted in the retrograde labelling of a fraction of hippocampal neurons. Some labelled cell bodies were distributed in field CA1 and the subiculum but larger numbers of neurons were detected in the ventral and intermediary subiculum. In a second series of experiments, the excitatory transmission from the hippocampus to the prefrontal cortex was pharmacologically analysed to provide further evidence for the involvement of glutamate and/or aspartate in the pathway. All prefrontal cortex neurons responding to the stimulation of the hippocampus were activated by selective agonists of the glutamate receptor subtypes alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) and N-methyl-d-aspartate (NMDA), and these effects were selectively antagonized by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2-amino-5-phosphonopentanoic acid (APV) respectively. Most of the excitatory responses of prefrontal cortex neurons to single and paired-pulse stimulation of the hippocampus were antagonized by CNQX. APV only affected the excitatory response in a few cells. These results suggest that the hippocampal input to the prefrontal cortex utilizes glutamate and/or aspartate as a transmitter. Even though prefrontal cortex neurons responding to the stimulation of the hippocampus appear to have both AMPA and NMDA receptors, low-frequency stimulation of the hippocampo-prefrontal cortex pathway activates cortical neurons mostly through AMPA receptors.


Oxidative Stress Induces HSP90 Upregulation on the Surface of Primary Human Endothelial Cells: Role of the Antioxidant 7,8-Dihydroxy-4-methylcoumarin in Preventing HSP90 Exposure to the Immune System.
We have previously demonstrated that human heat shock protein 90 (HSP90), an intracellular self protein, is the target of cellular and humoral autoimmune responses in patients with carotid atherosclerosis. In this study, we evaluated in vitro whether oxidative stress, a feature of atherosclerotic plaque, alters HSP90 expression in endothelial cells, thus inducing surface localization of this molecule and whether the antioxidant compound 7,8-dihydroxy-4-methylcoumarin (7,8-DHMC) is able to prevent oxidative stress-induced alterations of HSP90 localization. By the use of flow cytometry, immunofluorescence, enzyme-linked immunosorbent assay, and semiquantitative reverse-transcription polymerase chain reaction, we demonstrated that exposure of human umbilical vein endothelial cells (HUVEC) to the prooxidant compound H2O2 upregulated HSP90 surface expression and reduced its secretion without altering HSP90 gene expression and intracytoplasmic protein levels. Pretreatment of HUVEC with 7,8-DHMC prevented H2O2-induced alterations of HSP90 cellular distribution and secretion. Our results suggest that the strong oxidative conditions of atherosclerotic plaques promote the upregulation of HSP90 surface expression on endothelial cells, thus rendering the protein a possible target of autoimmune reactions. The antioxidant 7,8-DHMC, by preventing oxidative-stress-triggered HSP90 surface upregulation, may be useful to counteract possible autoreactive reactions to HSP90.


Recommendations from overweight youth regarding school-based weight control programs.
This study aimed to obtain recommendations from overweight youth on the development of school-based weight control programs; to determine their level of interest in participation; and to learn about the outcomes they desired from such a program. Because obesity is prevalent among youth of certain minority groups and low socioeconomic backgrounds, and since few programs address the needs of these youth, the study population was selected from inner city public schools with high percentages of youth from minority groups and low socioeconomic backgrounds. Sixty-one overweight adolescents participated in semi-structured, in-depth individual interviews, which were audiotaped, transcribed, coded, and analyzed. Findings indicated that many overweight adolescents were interested in participating in school-based weight control programs, provided they are conducted in a supportive manner; offer enjoyable activities; are informative; are sensitive to the needs of overweight youth; and do not conflict with other activities. Adolescents stressed the importance of a program leader who understands the difficulties that overweight youth face, and many expressed their preference for a leader who is currently overweight or had been overweight in the past.


CT of osteoid osteoma of the femoral neck: the value of oblique reformatting.
An osteoid osteoma is a benign bone lesion which may involve the femoral neck. The lesions are distinctively osteoblastic, containing a central fibrovascular and osteoid nidus which often evokes substantial periosteal reaction occasionally making the diagnosis by conventional radiography very difficult. Computed tomographic evaluation of these lesions at other sites has been previously reported. I wish to emphasize the unique value of CT multiplanar reformatting in a patient with an osteoid osteoma of the femoral neck.


Gene-boosted assembly of a novel bacterial genome from very short reads.
Recent improvements in technology have made DNA sequencing dramatically faster and more efficient than ever before. The new technologies produce highly accurate sequences, but one drawback is that the most efficient technology produces the shortest read lengths. Short-read sequencing has been applied successfully to resequence the human genome and those of other species but not to whole-genome sequencing of novel organisms. Here we describe the sequencing and assembly of a novel clinical isolate of Pseudomonas aeruginosa, strain PAb1, using very short read technology. From 8,627,900 reads, each 33 nucleotides in length, we assembled the genome into one scaffold of 76 ordered contiguous sequences containing 6,290,005 nucleotides, including one contig spanning 512,638 nucleotides, plus an additional 436 unordered contigs containing 416,897 nucleotides. Our method includes a novel gene-boosting algorithm that uses amino acid sequences from predicted proteins to build a better assembly. This study demonstrates the feasibility of very short read sequencing for the sequencing of bacterial genomes, particularly those for which a related species has been sequenced previously, and expands the potential application of this new technology to most known prokaryotic species.


Rationale for total nephrectomy for suspected renal cell carcinoma.
This article discusses the data surrounding a comparison of radical nephrectomy with nephron-sparing surgery in renal car carcinoma with an emphasis on patients with renal cell carcinoma who have normal renal function and contralateral normal anatomy and with special reference to the "case" presentation, which involves a small hilar lesion. The discussion argues in favor of radical nephrectomy in most of these cases.


Glia modulation of the extracellular milieu as a factor in central CO2 chemosensitivity and respiratory control.
We discuss the influence of astrocytes on respiratory function, particularly central CO2 chemosensitivity. Fluorocitrate (FC) poisons astrocytes, and studies in intact animals using FC provide strong evidence that disrupting astrocytic function can influence CO2 chemosensitivity and ventilation. Gap junctions interconnect astrocytes and contribute to K+ homeostasis in the extracellular fluid (ECF). Blocking gap junctions alters respiratory control, but proof that this is truly an astrocytic effect is lacking. Intracellular pH regulation of astrocytes has reciprocal effects on extracellular pH. Electrogenic sodium-bicarbonate transport (NBCe) is present in astrocytes. The activity of NBCe alkalinizes intracellular pH and acidifies extracellular pH when activated by depolarization (and a subset of astrocytes are depolarized by hypercapnia). Thus, to the extent that astrocytic intracellular pH regulation during hypercapnia lowers extracellular pH, astrocytes will amplify the hypercapnic stimulus and may influence central chemosensitivity. However, the data so far provide only inferential support for this hypothesis. A lactate shuttle from astrocytes to neurons seems to be active in the retrotrapezoid nucleus (RTN) and important in setting the chemosensory stimulus in the RTN (and possibly other chemosensory nuclei). Thus astrocytic processes, so vital in controlling the constituents of the ECF in the central nervous system, may profoundly influence central CO2 chemosensitivity and respiratory control.


Determination of serum gentamicin sulfate levels: ordering patterns and use as a guide to therapy.
Determination of serum gentamicin levels (SGLs) is helpful in monitoring therapy in patients receiving this drug because there is a narrow therapeutic margin and serum levels may vary greatly between patients receiving similar doses. During a ten-month period, definite indications for obtaining SGL determinations were present for 189 of 212 serum samples (89.2%) submitted to the laboratory. However, 110 samples (51.9%) were improperly drawn and results of 85 samples (40.1%) were ignored. Only 26 of the 62 correctly drawn and not ignored samples (41.9%) were appropriately acted on. At most, 42 of the 212 samples (19.8%) were appropriately used in making patient-care decisions. Thus, in this study, an inordinatley large number of SGLs were incorrectly drawn, apparently ignored, or inappropriately used in making patient-care decisions.


[THE INCONSISTENCIES OF REGULATION OF METABOLISM IN PHYLOGENESIS AT THREE LEVELS OF "RELATIVE BIOLOGICAL PERFECTION": ETIOLOGY OF METABOLIC PANDEMICS].
The regulation of metabolism in vivo can be comprehended by considering stages of becoming inphylogenesis of humoral, hormonal, vegetative regulators separately: at the level of cells; in paracrin-regulated cenosises of cells; organs and systems under open blood circulation and closed system of blood flow. The levels of regulations formed at different stages of phylogenesis. Their completion occurred at achievement of "relative biological perfection". Only this way need of cells in functional, structural interaction and forming of multicellular developed. The development of organs and systems of organs also completed at the level of "relative biological perfection". From the same level the third stage of becoming of regulation of metabolism at the level of organism started. When three conditions of "relative biological perfection" achieved consequently at level in vivo are considered in species Homo sapiens using system approach it is detected that "relative biological perfection" in vivo is accompanied by different inconsistencies of regulation of metabolism. They are etiologic factors of "metabolic pandemics ". The inconsistencies (etiological factors) are consider as exemplified by local (at the level of paracrin-regulated cenosises of cells) and system (at the level of organism) regulation of biological reaction metabolism-microcirculation that results in dysfunction of target organs and development of pathogenesis of essential metabolic arterial hypertension. The article describes phylogenetic difference between visceral fatty cells and adpocytes, regulation of metabolism by phylogenetically late insulin, reaction of albumin at increasing of content of unesterified fatty acids in blood plasma, difference of function of resident macrophage and monocytes-macrophages in pathogenesis of atherosclerosis, metabolic syndrome, insulin resistance, obesity, under diabetes mellitus and essential metabolic arterial hypertension.


All-optical flip-flop based on the bistability of injection locked Fabry-Perot laser diode.
We demonstrate the operation of a novel all-optical flip-flop. The flip-flop consists of a slave Fabry-Perot laser diode (FP-LD) and a specially designed master FP-LD which has a built-in external cavity and operates in single longitudinal mode oscillation. The set and reset pulses were generated by external modulators at 1 Gbit/s. The rising and falling times of the output signal in on-off operation of the flip-flop were about 50 ps. The required powers of both set and reset pulses were less than -9 dBm.


Are Members of Political Out-Groups More Morally or Physically Disgusting?
Recent research has found that Americans are disgusted by anonymous members of their political out-group. Determining whether the disgust elicited by political out-group members is more physical or moral may contribute to the understating of what enables its elicitation and regulation. Building on research showing the experience of moral disgust involves relatively abstract construal and the experience of physical disgust involves relatively concrete construal, we predicted that disgust experienced toward political out-group members is more moral than physical. Two preregistered experiments (total N=854) found that (a) the effect of level of construal on the intensity of disgust from political out-group members is more similar to the effect of level of construal on moral disgust than on physical disgust, and (b) the appraisal underlying disgust from political out-group members involves more abstract than concrete construal, similar to moral disgust. We discuss implications of these findings for intergroup relations and emotion regulation.


Derivation of Human Embryonic Stem Cells.
Embryonic stem cells (ESCs) represent a mainstay for pluripotent stem cell research and development (R&D) and provide tangible opportunities for clinical translation including cell therapies and drug discovery. Moreover, in spite of the discovery of induced pluripotent stem cells (iPSCs), ESCs are an essential reference point, against which other pluripotent cells are compared. Hence, there is an ongoing need to derive and bank quality-controlled research-grade and clinical-grade ESC lines using established and standardized methods. Here, we provide a concise, step-by-step protocol for the derivation of ESCs from human embryos. While largely based on previously reported method for clinical-grade human ESC (hESC) line derivation, the protocol is suitable for routine application, although adaptable for clinical-compliance.


Challenging script concordance test reference standard by evidence: do judgments by emergency medicine consultants agree with likelihood ratios?
We aimed to compare the clinical judgments of a reference panel of emergency medicine academic physicians against evidence-based likelihood ratios (LRs) regarding the diagnostic value of selected clinical and paraclinical findings in the context of a script concordance test (SCT). A SCT with six scenarios and five questions per scenario was developed. Subsequently, 15 emergency medicine attending physicians (reference panel) took the test and their judgments regarding the diagnostic value of those findings for given diseases were recorded. The LRs of the same findings for the same diseases were extracted from a series of published systematic reviews. Then, the reference panel judgments were compared to evidence-based LRs. To investigate the test-retest reliability, five participants took the test one month later, and the correlation of their first and second judgments were quantified using Spearman rank-order coefficient. In 22 out of 30 (73.3%) findings, the expert judgments were significantly different from the LRs. The differences included overestimation (30%), underestimation (30%), and judging the diagnostic value in an opposite direction (13.3%). Moreover, the score of a hypothetical test-taker was calculated to be 21.73 out of 30 if his/her answers were based on evidence-based LRs. The test showed an acceptable test-retest reliability coefficient (Spearman coefficient: 0.83). Although SCT is an interesting test to evaluate clinical decision-making in emergency medicine, our results raise concerns regarding whether the judgments of an expert panel are sufficiently valid as the reference standard for this test.


Diffusible component from the spore surface of the fungus Aspergillus fumigatus which inhibits the macrophage oxidative burst is distinct from gliotoxin and other hyphal toxins.
The fungus Aspergillus fumigatus, whose spores are present ubiquitously in the air, causes a range of diseases in the human lung. A small molecular weight (< 10 kD) heat stable toxin released from the spores of clinical and environmental isolates of A fumigatus within minutes of deposition in aqueous solution has previously been described. A key effect of the toxin was to inhibit the oxidative burst of macrophages as measured by superoxide anion release. It was hypothesised that the toxin was one of the commonly found A fumigatus hyphal toxins such as gliotoxin. This inhibitor may be an important factor which allows the fungus to colonise the lung. The spore derived inhibitor was shown to inhibit the respiratory burst of rat alveolar macrophages, as measured by the generation of superoxide anion. Samples of the spore diffusate were subject to reversed phase high performance liquid chromatography (HPLC), thin layer chromatography (TLC), high performance thin layer chromatography (HPTLC), or organic extraction followed by TLC or HPLC to identify the presence of gliotoxin, fumagillin, helvolic acid, fumigaclavine-C, and aurasperone-C. Commercially obtained preparations of the toxins gliotoxin, fumagillin and helvolic acid and extracts enriched for fumigaclavine-C and aurasperone-C were used as internal and external standards and in the respiratory burst measurements. Gliotoxin, fumagillin, helvolic acid, fumigaclavine-C, and aurasperone-C were not detected in spore derived diffusate using PHLC or TLC. Using extraction procedures with solvents known to extract gliotoxin from A fumigatus culture supernatants, no gliotoxin was detected in the spore derived diffusate. Commercial gliotoxin, fumagillin, and helvolic acid or extracts enriched for fumigaclavine-C and aurasperone-C did not inhibit the oxidative burst of macrophages. The hypothesis that the spore derived toxin is one of the toxins derived from hyphae such as gliotoxin, helvolic acid, fumagillin, fumigaclavine-C, or aurasperone-C is not proved. The spore toxin may exert its effect through its ability to diffuse rapidly into the lung lining fluid, diminish the macrophage oxidative burst, and play a part in allowing A fumigatus to persist in the lung and manifest its well known pathogenic effects.


Laminin 332 deposition is diminished in irradiated skin in an animal model of combined radiation and wound skin injury.
Skin exposure to ionizing radiation affects the normal wound healing process and greatly impacts the prognosis of affected individuals. We investigated the effect of ionizing radiation on wound healing in a rat model of combined radiation and wound skin injury. Using a soft X-ray beam, a single dose of ionizing radiation (10-40 Gy) was delivered to the skin without significant exposure to internal organs. At 1 h postirradiation, two skin wounds were made on the back of each rat. Control and experimental animals were euthanized at 3, 7, 14, 21 and 30 days postirradiation. The wound areas were measured, and tissue samples were evaluated for laminin 332 and matrix metalloproteinase (MMP) 2 expression. Our results clearly demonstrate that radiation exposure significantly delayed wound healing in a dose-related manner. Evaluation of irradiated and wounded skin showed decreased deposition of laminin 332 protein in the epidermal basement membrane together with an elevated expression of all three laminin 332 genes within 3 days postirradiation. The elevated laminin 332 gene expression was paralleled by an elevated gene and protein expression of MMP2, suggesting that the reduced amount of laminin 332 in irradiated skin is due to an imbalance between laminin 332 secretion and its accelerated processing by elevated tissue metalloproteinases. Western blot analysis of cultured rat keratinocytes showed decreased laminin 332 deposition by irradiated cells, and incubation of irradiated keratinocytes with MMP inhibitor significantly increased the amount of deposited laminin 332. Furthermore, irradiated keratinocytes exhibited a longer time to close an artificial wound, and this delay was partially corrected by seeding keratinocytes on laminin 332-coated plates. These data strongly suggest that laminin 332 deposition is inhibited by ionizing radiation and, in combination with slower keratinocyte migration, can contribute to the delayed wound healing of irradiated skin.


Defective phototransductive disk membrane morphogenesis in transgenic mice expressing opsin with a mutated N-terminal domain.
Retinitis pigmentosa is a heterogeneous group of inherited retinal disorders in which the photoreceptor cells degenerate. A line of transgenic mice expresses a mutant opsin gene that encodes three missense mutations near the amino terminus, including P23H, which is the basis for a common form of dominant retinitis pigmentosa. By studying the photoreceptor cells of these mice and their normal littermates, we found that: (1) opsin was routed correctly, (2) the concentration of opsin in the disk membranes appeared normal by freeze fracture analysis, (3) the amount of disk membrane shedding was normal, but (4) the basal disks of the outer segments were disorganized, indicating defective disk membrane morphogenesis. Defective disk membrane morphogenesis appears to result in the formation of fewer mature disks, thus accounting for observed gradual shortening of the photoreceptor outer segments with age. We suggest that abnormal disk membrane morphogenesis is the primary cellular defect that leads to blindness, and that it arises from the inability of nascent disk membranes, containing normal and mutant opsin, to interact normally with each other.


The use of clonidine for severe and intractable sleep problems in children with neurodevelopmental disorders--a case series.
This paper reports on the use of clonidine for the treatment of severe sleep problems associated with behavioural difficulties in children with neurodevelopmental disabilities. Data were obtained from reviewing the case notes of a series of six children with neurodevelopmental disorders of different nature and severity, presenting with problematic sleep. All children in this group showed maintained improvements in their sleep pattern following the use of clonidine with only mild side-effects reported.