CRISPR-Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome.
Large genome-mapping consortia and thousands of genome-wide association studies have identified non-protein-coding elements in the genome as having a central role in various biological processes. However, decoding the functions of the millions of putative regulatory elements discovered in these studies remains challenging. CRISPR-Cas9-based epigenome editing technologies have enabled precise perturbation of the activity of specific regulatory elements. Here we describe CRISPR-Cas9-based epigenomic regulatory element screening (CERES) for improved high-throughput screening of regulatory element activity in the native genomic context. Using dCas9KRAB repressor and dCas9p300 activator constructs and lentiviral single guide RNA libraries to target DNase I hypersensitive sites surrounding a gene of interest, we carried out both loss- and gain-of-function screens to identify regulatory elements for the β-globin and HER2 loci in human cells. CERES readily identified known and previously unidentified regulatory elements, some of which were dependent on cell type or direction of perturbation. This technology allows the high-throughput functional annotation of putative regulatory elements in their native chromosomal context.


Elimination of Escherichia coli O157:H7 from Alfalfa seeds through a combination of high hydrostatic pressure and mild heat.
Escherichia coli O157:H7 has been associated with contaminated seed sprout outbreaks. The majority of these outbreaks have been traced to sprout seeds contaminated with low levels of pathogens. Sanitizing sprout seeds presents a unique challenge in the arena of produce safety in that even a low residual pathogen population remaining on contaminated seed after treatments appears capable of growing to very high levels during sprouting. In this study, the effectiveness of high-pressure treatment in combination with low and elevated temperatures was assessed for its ability to eliminate E. coli O157:H7 on artificially contaminated alfalfa seeds. Inoculated seed samples were treated at 600 MPa for 2 min at 4, 20, 25, 30, 35, 40, 45, and 50 degrees C. The pressure sensitivity of the pathogenic bacteria was strongly dependent on the treatment temperature. At 40 degrees C, the process was adequate in eliminating a 5-log-unit population on the seeds with no adverse effect on seed viability. Three treatments carried out at reduced pressure levels and/or extended treatment time, 550 MPa for 2 min at 40 degrees C, 300 MPa for 2 min at 50 degrees C, and 400 MPa for 5 min at 45 degrees C, were equally lethal to the pathogen. When all three treatments were compared in terms of their impact on seed viability, the process of 550 MPa for 2 min at 40 degrees C was the most desirable, achieving final germination percentages and sprout sizes statistically similar to those of control untreated seeds (P > 0.05).


Vecuronium requirement during liver transplantation under sevoflurane anesthesia.
In liver transplantation patients under intravenous anesthesia, the vecuronium dose is known to be reduced, especially during the anhepatic phase. Volatile anesthetics potentiate a muscle relaxation effect of neuromuscular blocking agents, so the vecuronium dose is supposed to further decrease if sevoflurane is used during liver transplantation. The purpose of this study was to determine the appropriate dose of vecuronium at each phase of liver transplantation under sevoflurane anesthesia. Thirty-five patients scheduled for living donor liver transplantation because of liver cirrhosis were enrolled in this study. They were anesthetized with 1 MAC of sevoflurane and intermittent administration of fentanyl. Continuous infusion of vecuronium (0.5 mg/ml) was used for muscle relaxation, which was adjusted every 15 min for consistent muscle relaxation aimed at T1/Tc of 0.1 monitored by ulnar nerve stimulation. Vecuronium infusion was stopped after hepatic artery anastomosis was finished. The infusion rate of each operative phase-dissection, anhepatic, and neohepatic-was calculated and analyzed by one-way analysis of variance. The recovery time from train-of-four (TOF) count 1 to TOF ratio 25% was also measured. The vecuronium infusion rate of each operation phase for adequate muscle relaxation was as follows: 0.033 ± 0.009 mg/kg/h during dissection phase, 0.031 ± 0.009 mg/kg/h during anhepatic phase, and 0.026 ± 0.006 mg/kg/h during early neohepatic phase. There was a statistically significant difference between doses at each phase (P = 0.033). The recovery time from TOF count 1 to TOF ratio 25% was 103 ± 29 min. The required vecuronium dose in all phases was less than the known dose in the anhepatic phase (0.036 mg/kg/h) under midazolam-fentanyl anesthesia. In addition, the vecuronium infusion dose was not reduced in the anhepatic phase compared to the dissection phases.


Metabolism of 4 beta -hydroxycholesterol in humans.
One of the major oxysterols in the human circulation is 4 beta-hydroxycholesterol formed from cholesterol by the drug-metabolizing enzyme cytochrome P450 3A4. Deuterium-labeled 4 beta-hydroxycholesterol was injected into two healthy volunteers, and the apparent half-life was found to be 64 and 60 h, respectively. We have determined earlier the half-lives for 7 alpha-, 27-, and 24-hydroxycholesterol to be approximately 0.5, 0.75, and 14 h, respectively. Patients treated with certain antiepileptic drugs have up to 20-fold increased plasma concentrations of 4 beta-hydroxycholesterol. The apparent half-life of deuterium-labeled 4 beta-hydroxycholesterol in such a patient was found to be 52 h, suggesting that the high plasma concentration was because of increased synthesis rather than impaired clearance. 4 beta-Hydroxycholesterol was converted into acidic products at a much slower rate than 7 alpha-hydroxycholesterol in primary human hepatocytes, and 4 beta-hydroxycholesterol was 7 alpha-hydroxylated at a slower rate than cholesterol by recombinant human CYP7A1. CYP7B1 and CYP39A1 had no activity toward 4 beta-hydroxycholesterol. These results suggest that the high plasma concentration of 4 beta-hydroxycholesterol is because of its exceptionally slow elimination, probably in part because of the low rate of 7 alpha-hydroxylation of the steroid. The findings are discussed in relation to a potential role of 4 beta-hydroxycholesterol as a ligand for the nuclear receptor LXR.


A pre-post evaluation of a knowledge translation capacity-building intervention.
Knowledge translation (KT) aims to reduce research-practice gaps. Few studies have investigated methods to build clinicians' capacity for KT. To: (i) evaluate the impact of a multifaceted KT capacity-building intervention; (ii) understand barriers and enablers to clinicians' use of KT; and (iii) identify useful strategies. A pre-post study to develop KT capacity amongst occupational therapy clinicians (n = 46) in a large metropolitan hospital. A customised questionaire (baseline and 18 months) identified KT-related behaviours and barriers and enablers guided by the Theoretical Domains Framework (TDF). McNemar's tests and Wilcoxon signed rank tests were completed on matched data (n = 20). At follow-up additional items explored perceptions of change and usefulness of strategies. At follow-up, participants had read more clinical guidelines (10 vs. 17) and more participants reported using strategies to increase the use of recommended clinical practices (P = 0.006). The main barriers at baseline were from the TDF domains of 'attention, memory and decision processes', 'knowledge' and 'environmental context and resources', while main enablers were from 'social/professional role and identity', 'reinforcement', 'social influence' and 'beliefs about consequences' domains. At follow-up, significant improvements were seen in 'knowledge' (p < 0.001), 'environmental context & resources' (P < 0.001), 'skills' (P = 0.008) 'beliefs about consequences' (P = 0.011), 'beliefs about capabilities' (P = 0.018), and 'memory, attention & decision processes' (P = 0.048) and participants agreed that KT had become part of the departments' culture. Strategies perceived most useful included working as a team, having a dedicated staff member, mentoring meetings, department leader support, learning about KT over time, and training sessions. The KT capacity-building intervention changed one clinician-reported behaviour and perceived impact of barriers across six domains. Clinicians reported perceived improvement in understanding of - and confidence in - KT, and changes in the culture to one of engaging with KT as part of clinical practice. Further research into KT capacity building is needed.


Resveratrol partially prevents rotenone-induced neurotoxicity in dopaminergic SH-SY5Y cells through induction of heme oxygenase-1 dependent autophagy.
Parkinson disease (PD) is a complex neurodegenerative disorder characterized by a progressive loss of dopaminergic neurons. Mitochondrial dysfunction, oxidative stress or protein misfolding and aggregation may underlie this process. Autophagy is an intracellular catabolic mechanism responsible for protein degradation and recycling of damaged proteins and cytoplasmic organelles. Autophagic dysfunction may hasten the progression of neuronal degeneration. In this study, resveratrol promoted autophagic flux and protected dopaminergic neurons against rotenone-induced apoptosis. In an in vivo PD model, rotenone induced loss of dopaminergic neurons, increased oxidation of mitochondrial proteins and promoted autophagic vesicle development in brain tissue. The natural phytoalexin resveratrol prevented rotenone-induced neuronal apoptosis in vitro, and this pro-survival effect was abolished by an autophagic inhibitor. Although both rotenone and resveratrol promoted LC3-II accumulation, autophagic flux was inhibited by rotenone and augmented by resveratrol. Further, rotenone reduced heme oxygenase-1 (HO-1) expression, whereas resveratrol increased HO-1 expression. Pharmacological inhibition of HO-1 abolished resveratrol-mediated autophagy and neuroprotection. Notably, the effects of a pharmacological inducer of HO-1 were similar to those of resveratrol, and protected against rotenone-induced cell death in an autophagy-dependent manner, validating the hypothesis of HO-1 dependent autophagy in preventing neuronal death in the in vitro PD model. Collectively, our findings suggest that resveratrol induces HO-1 expression and prevents dopaminergic cell death by regulating autophagic flux; thus protecting against rotenone-induced neuronal apoptosis.


Imaging of formaldehyde in plants with a ratiometric fluorescent probe.
The fluorescence monitoring of formaldehyde in real environmental samples and live plant tissues is of great importance for physiological and pathological studies. However, there is a lack of suitable chemical tools to directly trace and measure the formaldehyde activity in bio-systems, and developing effective and, in particular, selective sensors for mapping formaldehyde in live tissues still remains a great challenge. Here, we demonstrate for the first time that the ratiometric fluorescence monitoring of formaldehyde in live plant tissues is achieved with a newly developed ratiometric fluorescent probe, FAP, which effectively eliminated interference from other comparative analytes. Live tissue analyses reveal that FAP can potentially detect exogenous and endogenous formaldehyde in live Arabidopsis thaliana tissues, exposing a potential application for biological and pathological studies of formaldehyde.


Heterogeneous catalytic oxidation of glycerol over a UiO-66-derived ZrO2@C supported Au catalyst at room temperature.
The catalytic conversion of biomass-derived glycerol into high-value-added products, such as glyceric acid (GLYA), using catalyst-supported Au nanoparticles (Au NPs) at room temperature presents a significant challenge. In this study, we constructed a series of supported Au catalysts, including Au/ZrO2@C, Au/C, Au/ZrO2, and Au/ZrO2-C, and investigated their effectiveness in selectively catalytic oxidizing glycerol to GLYA at room temperature. Among these catalysts, the Au/ZrO2@C catalyst exhibited the best catalytic performance, achieving a glycerol conversion rate of 73% and a GLYA selectivity of 79% under the optimized reaction conditions (reaction conditions: 30 mL 0.1 M glycerol, glycerol/Au = 750 mol mol-1, T = 25 °C, p(O2) = 10 bar, stirring speed = 600 rpm, time = 6 h). Physical adsorption, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM), and other characterization methods were employed to analyze the texture properties of the catalyst. The findings indicated that the support structure, the strong metal-support interactions between Au NPs and the support, and the presence of small metallic Au NPs were the primary factors contributing to the catalyst's high activity and selectivity. Moreover, the reusability of the Au/ZrO2@C catalyst was investigated, and a probable reaction mechanism for the oxidation of glycerol was proposed.


Imaging in Retinopathy of Prematurity.
Retinopathy of prematurity (ROP) is a leading cause of preventable childhood blindness worldwide. Barriers to ROP screening and difficulties with subsequent evaluation and management include poor access to care, lack of physicians trained in ROP, and issues with objective documentation. Digital retinal imaging can help address these barriers and improve our knowledge of the pathophysiology of the disease. Advancements in technology have led to new, non-mydriatic and mydriatic cameras with wider fields of view as well as devices that can simultaneously incorporate fluorescein angiography, optical coherence tomography (OCT), and OCT angiography. Image analysis in ROP is also being employed through smartphones and computer-based software. Telemedicine programs in the United States and worldwide have utilized imaging to extend ROP screening to infants in remote areas and have shown that digital retinal imaging can be reliable, accurate, and cost-effective. In addition, tele-education programs are also using digital retinal images to increase the number of healthcare providers trained in ROP. Although indirect ophthalmoscopy is still an important skill for screening, digital retinal imaging holds promise for more widespread screening and management of ROP.


Effect of Revtech thermal processing on volatile organic compounds and chemical characteristics of split yellow pea (Pisum sativum L.) flour.
Yellow pea (Pisumsativum L.) is an economically rich source of nutrients with health-promoting effects. However, the consumption of pea ingredients is minimal due to their off-flavor characteristics. The present study investigated the effect of Revtech heat treatment on the chemical profile and volatile compounds in split yellow pea flour. Revtech treatment (RT) was applied at 140°C with a residence time of 4 min in dry condition (RT 0%) and in the presence of 10% steam (RT 10%). Both thermal treatments resulted in a significant reduction (p < 0.05) in lipoxygenase activity and the concentration of key beany-related odors such as heptanal, (E)-2-heptenal, 1-octen-3-ol, octanal, and (E)-2-octenal. In addition, RT 10% resulted in a significant reduction in pentanal, 1-penten-3-ol, hexanal, and 1-hexanol compared to untreated flour. The content of known precursors of lipoxygenase such as linoleic and linolenic acids was found in higher concentrations in heat-treated flours, indicating the efficacy of Revtech technology in minimizing the degradation of polyunsaturated fatty acids. No significant changes in the amino acid composition or the 29 selected phenolic compounds in pea flours were observed with Revtech processing except for two compounds, caffeic acid and gallocatechin, which were found at higher concentrations in RT 0%. PRACTICAL APPLICATION: Thermal processing of split yellow pea flours at 140°C using Revtech technology successfully decreased the concentrations of volatile compounds responsible for beany off-flavor while improving the nutritional quality of studied yellow pea flours. These results provide valuable information to the food industry for developing novel pulse-based products with enhanced sensory characteristics.


High prevalence rates of antibody to three sandfly fever viruses (Sicilian, Naples, and Toscana) among Cypriots.
Neutralizing antibodies to sandfly fever Naples, sandfly fever Sicilian and Toscana viruses were investigated among 479 sera collected from a normal human population in Cyprus. Antibody prevalence rates of 57%, 32% and 20% were found to Naples, Sicilian and Toscana viruses, respectively. The observed frequency of dual and triple infections was higher than would be expected with a random chance of infection. Antibody prevalence rates were similar for men and women for all three viruses tested, but one of two study sites had significantly higher antibody prevalence to Naples and Sicilian viruses than the other. Individuals with antibodies to both Naples and Toscana viruses had higher antibody levels to Naples virus than those with antibodies to Naples virus only. If the antibody prevalence rates found in this study reflect a history of clinical disease as described in the literature, sandfly fever poses a significant public health problem in Cyprus.


Characterization of genomic single nucleotide polymorphism via colorimetric detection using a single gold nanoprobe.
Identification of specific nucleic acid sequences mediated by gold nanoparticles derivatized thiol-modified oligonucleotides (Au-nanoprobes) has been proven to be a useful tool in molecular diagnostics. Here, we demonstrate that, on optimization, detection may be simplified via the use of a single Au-nanoprobe to detect a single nucleotide polymorphism (SNP) in homo- or heterozygote condition. We validated this non-cross-linking approach through the analysis of 20 clinical samples using a single specific Au-nanoprobe for an SNP in the FTO (fat mass and obesity-associated) gene against direct DNA sequencing. Sensitivity, specificity, and limit of detection (LOD) were determined, and statistical differences were calculated by one-way analysis of variance (ANOVA) and a post hoc Tukey's test to ascertain whether there were any differences between Au-nanoprobe genotyped groups. For the first time, we show that the use of a single Au-nanoprobe can detect SNP for each genetic status (wild type, heterozygous, or mutant) with high degrees of sensitivity (87.50%) and specificity (91.67%).


[Should recurrent Cytomegalovirus infections in patients with liver transplantation be treated?].
Cytomegalovirus (CMV) is the single most important viral pathogen in organ transplantation. Treatment strategy for CMV infection and disease is not well established in transplantation. We report a case of primary CMV infection and two relapses in a woman with a liver transplant in whom spontaneous clearing of the second CMV relapse was seen. A 23 year-old CMV-seronegative woman received a liver transplant with a CMV-negative organ. Six weeks after transplantation she had her primary CMV infection proved by seroconversion and virus isolation. She had no clinical symptoms. Treatment with ganciclovir for five weeks resulted in declining CMV-antigen positive cells from 300/200.000 PMNs to CMV-antigen negativity. Only a slight antibody response was seen. At week 13 the first relapse occurred evidenced by antigenaemia. Ganciclovir was reinstituted for six weeks resulting in reduced antigenaemia. At week 22 liver biopsy was performed due to slightly elevated ALAT. The biopsy showed evidence of focal CMV hepatitis and blood analysis showed 120 CMV-antigen positive cells/200.000 PMNs. In spite of this, ganciclovir was not reinstituted, but the immunosuppressive treatment was reduced to a minimum to stimulate the patient's immune response to CMV. During the following months the patient gradually developed IgG antibody, cleared the antigen and levels of liver enzymes returned to normal. We suggest that ganciclovir treatment, may be omitted in cases of relapse with minimal clinical symptoms, slight antigenaemia and a beginning antibody response and that, the immunosuppressive treatment should be reduced instead. Such an approach requires careful clinical monitoring of the patient.


GPIbα regulates platelet size by controlling the subcellular localization of filamin.
Interaction between the cytoplasmic domain of GPIbα with its cytoskeletal binding partner, filamin, is a major determinant of platelet size, and deficiency of either protein results in macrothrombocytopenia. To clarify the mechanism by which GPIbα-filamin interactions regulate platelet production, we manipulated the expression levels of filamin and GPIb in cultured embryonic stem cells (ESCs) that were subsequently differentiated into platelets. Knocking down filamins A and B resulted in the production of ESC-derived proplatelets with abnormally large swellings and proplatelet shafts that generated giant platelets in culture. Large platelets could also be generated by overexpressing GPIbα in ESCs, or by overexpressing in vivo a transgene encoding a chimeric protein containing the cytoplasmic domain of GPIbα. To identify the mechanism by which the GPIb:filamin ratio regulates platelet size, we manipulated filamin and GPIbα levels in HEK293T cells and examined the effects of overexpressing either protein on their ability to traffic to the cell periphery. Accumulation of either protein within the endoplasmic reticulum resulted in trapping of the other. Taken together, these data demonstrate that coordinated expression of GPIbα and filamin is required for efficient trafficking of either protein to the cell surface, and for production of normal-sized platelets.


Establishing a framework to implement 4D XCAT phantom for 4D radiotherapy research.
To establish a framework to implement the 4D integrated extended cardiac torso (XCAT) digital phantom for 4D radiotherapy (RT) research. A computer program was developed to facilitate the characterization and implementation of the 4D XCAT phantom. The program can (1) generate 4D XCAT images with customized parameter files; (2) review 4D XCAT images; (3) generate composite images from 4D XCAT images; (4) track motion of selected region-of-interested (ROI); (5) convert XCAT raw binary images into DICOM format; (6) analyse clinically acquired 4DCT images and real-time position management (RPM) respiratory signal. Motion tracking algorithm was validated by comparing with manual method. Major characteristics of the 4D XCAT phantom were studied. The comparison between motion tracking and manual measurements of lesion motion trajectory showed a small difference between them (mean difference in motion amplitude: 1.2 mm). The maximum lesion motion decreased nearly linearly (R 2 = 0.97) as its distance to the diaphragm (DD) increased. At any given DD, lesion motion amplitude increased nearly linearly (R 2 range: 0.89 to 0.95) as the inputted diaphragm motion increased. For a given diaphragm motion, the lesion motion is independent of the lesion size at any given DD. The 4D XCAT phantom can closely reproduce irregular breathing profile. The end-to-end test showed that clinically comparable treatment plans can be generated successfully based on 4D XCAT images. An integrated computer program has been developed to generate, review, analyse, process, and export the 4D XCAT images. A framework has been established to implement the 4D XCAT phantom for 4D RT research.


Rupture of pluronic micelles by di-methylated β-cyclodextrin is not due to polypseudorotaxane formation.
Spectroscopic measurements (uv/vis absorbance and fluorescence) and time-resolved small-angle neutron scattering experiments (TR-SANS) were used to follow the breakdown of Pluronic micelles by heptakis(2,6-di-O-methyl)-β-cyclodextrin (DIMEB) over time in order to elucidate the mechanism of micellar rupture, generally attributed to polypseudotorotaxane (PR) formation between the cyclodextrin and the central hydrophobic PPO block. The spectroscopic measurements with two different probes (methyl orange and nile red) suggest that very rapid changes (on the order of seconds) take place when mixing DIMEB with F127 Pluronic and that no displacement of the probe from the cyclodextrin cavity occurs, which is in disagreement with PR formation. TR-SANS measurements demonstrate for the first time that the micelles are broken down in less than 100 ms, which categorically rules out PR formation as the mechanism of rupture. In addition, the same mechanism is demonstrated with other Pluronics, P85 and P123. In the latter case, after micellar rupture, lamellar structures are seen to form over a longer period of time, thus suggesting that after the instantaneous micellar disruption, further, longer-scale rearrangements are not excluded.


Adrenergic drug effects on aqueous outflow facility following ciliary muscle retrodisplacement in the cynomolgus monkey.
Cynomolgus monkeys with untouched, totally iridectomized, or totally iridectomized and ciliary muscle-disinserted eyes underwent two-level constant-pressure perfusion of the anterior chamber to determine the effect of intracameral mock aqueous humor, l-epinephrine bitartrate (10 microgram), l-norepinephrine bitartrate (10 microgram), ergotamine tartrate (1 and 20 microgram), and angiotensin II amide (1 and 100 microgram) on total outflow facility. Ergotamine and angiotensin both tended to decrease facility. Epinephrine and norepinephrine significantly increased facility, approximately 25% and 40%, respectively, beyond the effect of mock aqueous in all three types of eyes. The percentage increases in facility produced by epinephrine and norepinephrine were virtually identical in disinserted and nondisinserted eyes and were independent of starting facility in the individual eyes. Our results prove that the epinephrine- and norepinephrine-induced facility increase in cynomolgus eyes is not mediated by the iris or ciliary muscle and suggest that the increase in total facility is due largely to increased facility across the trabecular meshwork.


Wafer-Scale Synthesis of Semiconducting SnO Monolayers from Interfacial Oxide Layers of Metallic Liquid Tin.
Atomically thin semiconductors are one of the fastest growing categories in materials science due to their promise to enable high-performance electronic and optical devices. Furthermore, a host of intriguing phenomena have been reported to occur when a semiconductor is confined within two dimensions. However, the synthesis of large area atomically thin materials remains as a significant technological challenge. Here we report a method that allows harvesting monolayer of semiconducting stannous oxide nanosheets (SnO) from the interfacial oxide layer of liquid tin. The method takes advantage of van der Waals forces occurring between the interfacial oxide layer and a suitable substrate that is brought into contact with the molten metal. Due to the liquid state of the metallic precursor, the surface oxide sheet can be delaminated with ease and on a large scale. The SnO monolayer is determined to feature p-type semiconducting behavior with a bandgap of ∼4.2 eV. Field effect transistors based on monolayer SnO are demonstrated. The synthetic technique is facile, scalable and holds promise for creating atomically thin semiconductors at wafer scale.


Glyco-functionalized dinuclear rhenium(i) complexes for cell imaging.
The design, synthesis and photophysical characterization of four new luminescent glycosylated luminophores based on dinuclear rhenium complexes, namely Glyco-Re, are described. The derivatives have the general formula [Re2(μ-Cl)2(CO)6(μ-pydz-R)] (R-pydz = functionalized 1,2-pyridazine), where a sugar residue (R) is covalently bound to the pyridazine ligand in the β position. Different synthetic pathways have been investigated including the so-called neo-glycorandomization procedure, affording stereoselectively glyco-conjugates containing glucose and maltose in a β anomeric configuration. A multivalent dinuclear rhenium glycodendron bearing three glucose units is also synthesized. All the Glyco-Re conjugates are comprehensively characterized and their photophysical properties and cellular internalization experiments on human cervical adenocarcinoma (HeLa) cells are reported. The results show that such Glyco-Re complexes display interesting bio-imaging properties, i.e. high cell permeability, organelle selectivity, low cytotoxicity and fast internalization. These findings make the presented Glyco-Re derivatives efficient phosphorescent probes suitable for cell imaging application.


Glutamylation on alpha-tubulin is not essential but affects the assembly and functions of a subset of microtubules in Tetrahymena thermophila.
Tubulin undergoes glutamylation, a conserved posttranslational modification of poorly understood function. We show here that in the ciliate Tetrahymena, most of the microtubule arrays contain glutamylated tubulin. However, the length of the polyglutamyl side chain is spatially regulated, with the longest side chains present on ciliary and basal body microtubules. We focused our efforts on the function of glutamylation on the alpha-tubulin subunit. By site-directed mutagenesis, we show that all six glutamates of the C-terminal tail domain of alpha-tubulin that provide potential sites for glutamylation are not essential but are needed for normal rates of cell multiplication and cilium-based functions (phagocytosis and cell motility). By comparative phylogeny and biochemical assays, we identify two conserved tubulin tyrosine ligase (TTL) domain proteins, Ttll1p and Ttll9p, as alpha-tubulin-preferring glutamyl ligase enzymes. In an in vitro microtubule glutamylation assay, Ttll1p showed a chain-initiating activity while Ttll9p had primarily a chain-elongating activity. GFP-Ttll1p localized mainly to basal bodies, while GFP-Ttll9p localized to cilia. Disruption of the TTLL1 and TTLL9 genes decreased the rates of cell multiplication and phagocytosis. Cells lacking both genes had fewer cortical microtubules and showed defects in the maturation of basal bodies. We conclude that glutamylation on alpha-tubulin is not essential but is required for efficiency of assembly and function of a subset of microtubule-based organelles. Furthermore, the spatial restriction of modifying enzymes appears to be a major mechanism that drives differential glutamylation at the subcellular level.


Cetylpyridinium chloride inhibits human breast tumor cells growth in a no-selective way.
Analyze the antitumor capacity of cetylpyridinium chloride (CPC) on human breast tumor cells, and the possible action mechanism. The human breast tumor cells MCF-7 and no-tumor breast cells MCF-10A were exposed to CPC under various condition (concentration and duration). Cell viability was measured with MTT assay, the LIVE/DEAD assay, and fluorescence microscopy. Membrane permeability after CPC exposure was evaluated by Calcein AM assay, mitochondrial morphology with a MitoView staining, and genotoxicity with the comet assay and fluorescence microscopy. CPC was cytotoxic to both MCF-7 and MCF-10A as of a 24-h exposure to 0.1 µM. Cytotoxicity was dose-dependent and reached 91% for MCF-7 and 78% for MCF-10A after a 24-h exposure to 100 µM CPC, which outperformed the positive control doxorubicin in effectiveness and selectivity. The LD50 of CPC on was 6 µM for MCF-7 and 8 µM for MCF-10A, yielding a selectivity index of 1.41. A time response analysis revealed 64% dead cells after only 5 min of exposure to 100 µM CPC. With respect to the action mechanisms, the comet assay did not reveal genome fragmentation. On the other hand, membrane damage was dose-dependent and may also affect mitochondrial morphology. Cetylpyridinium chloride inhibits MCF-7 cell growing in a non-selective way as of 5 min of exposure. The action mechanism of CPC on tumor cells involves cell membrane damage without change neither mitochondrial morphology nor genotoxicity.


Reported and Unreported Potentially Important Incidental Findings in Urgent Nonenhanced Abdominal CT for Renal Colic.
The aim of the study was to retrospectively evaluate the prevalence of reported and unreported potentially important incidental findings (PIFs) in consecutive nonenhanced abdominal CTs performed specifically for renal colic in the urgent setting. One radiologist, blinded to the finalized report, retrospectively re-evaluated nonenhanced abdominal CTs performed from January to December 2017 on adult patients from the emergency department with the specific request of urgent evaluation for renal colic, searching for PIFs. The CTs of 312 patients were included in the study. Thirty-eight findings were reported in 38 different CTs, whereas the re-evaluation added 47 unreported findings in 47 different CTs, adding to total of 85 findings (27%). The difference in the proportion of reported and unreported PIFs between the original report and re-evaluation was significant (p < 0.001). No significant difference was found between the age of patients with and without reported findings. The proportion of potentially important findings did not vary significantly among the 3 shifts in the original report and in re-evaluation. The most frequent findings, both reported and unreported, were pleural effusion, lymphadenopathies, and liver nodules. Potentially important additional findings are frequently present in urgent nonenhanced abdominal CTs performed for renal colic, and many are not described in the finalized reports. Radiologists should take care not to underreport PIFs even in the urgent setting because of the possible consequences on the patient's health and in order to avoid legal issues, while at the same time satisfying the need for timely and efficient reporting.


Ginsenoside Rg3 enhances the inhibitory effects of chemotherapy on esophageal squamous cell carcinoma in mice.
The present study was conducted in order to investigate the inhibitory effects of ginsenoside Rg3 combined with chemotherapy on Eca-109 esophageal squamous cell carcinoma (ESCC) in mice. Tumor xenograft models were established in the right forelimb of 20 BALB/c nude mice by subcutaneous injection. The tumor-bearing mice were randomly assigned to 4 treatment groups (n=5 per group) as follows: the control group (saline), the ginsenoside Rg3 alone group (6 mg/kg/day, once a day for 3 weeks), the chemotherapy alone group (paclitaxel 10 mg/kg/day + cisplatin 5 mg/kg/day on days 1, 7, 14 and 21) and the chemotherapy + Rg3 group (combined treatment). The length and width of the tumor were directly measured with calipers at different time points and the tumor volume (cm3) was calculated using the formula 0.52 × length × width2 every other day. The mice were sacrificed by cervical dislocation following completion of therapy, the tumors were removed and weighed and the expression levels of Ki-67 were determined by immunohistochemistry. The results indicated that the coadministration of ginsenoside Rg3 significantly enhanced the inhibitory effects of chemotherapy on tumor growth. In addition, the expression levels of Ki-67 in the chemotherapy + Rg3 group were significantly lower compared to those in the other 3 groups. The chemotherapy + Rg3 group also exhibited the lowest microvascular density among all four groups. These findings suggested that ginsenoside Rg3 may improve the antitumor efficacy of chemotherapy in Eca-109 ESCC in mice.


Protocol for development and validation of postpartum cardiovascular disease (CVD) risk prediction model incorporating reproductive and pregnancy-related candidate predictors.
Cardiovascular disease (CVD) is a leading cause of death among women. CVD is associated with reduced quality of life, significant treatment and management costs, and lost productivity. Estimating the risk of CVD would help patients at a higher risk of CVD to initiate preventive measures to reduce risk of disease. The Framingham risk score and the QRISK® score are two risk prediction models used to evaluate future CVD risk in the UK. Although the algorithms perform well in the general population, they do not take into account pregnancy complications, which are well known risk factors for CVD in women and have been highlighted in a recent umbrella review. We plan to develop a robust CVD risk prediction model to assess the additional value of pregnancy risk factors in risk prediction of CVD in women postpartum. Using candidate predictors from QRISK®-3, the umbrella review identified from literature and from discussions with clinical experts and patient research partners, we will use time-to-event Cox proportional hazards models to develop and validate a 10-year risk prediction model for CVD postpartum using Clinical Practice Research Datalink (CPRD) primary care database for development and internal validation of the algorithm and the Secure Anonymised Information Linkage (SAIL) databank for external validation. We will then assess the value of additional candidate predictors to the QRISK®-3 in our internal and external validations. The developed risk prediction model will incorporate pregnancy-related factors which have been shown to be associated with future risk of CVD but have not been taken into account in current risk prediction models. Our study will therefore highlight the importance of incorporating pregnancy-related risk factors into risk prediction modeling for CVD postpartum.


Experience of Racial Discrimination was Associated with Psychological Distress and Worsening Sex Life Among Adult Americans During COVID-19.
The recent escalation of racism in the U.S. during the COVID-19 pandemic points to the importance of examining the association between experienced racism and sexual health. Based on data from a nationally representative survey conducted in the U.S. in October 2020 (n = 1,915), Chi-square tests and multivariable logistic regressions were estimated to examine the association between experience of racism and changes in sex life during the pandemic. We further performed a causal mediation analysis using the bootstrap technique to assess the mediating role of psychological distress in the observed association between the experience of racism and changes in sex life. Among the respondents, the proportions reporting better, worse, or no change in sex life were, respectively, 15%, 21%, and 64%. Experiencing racial discrimination during COVID-19 was significantly associated with worsening sex life (adjusted odd ratio [AOR] = 1.53; 95% confidence interval [CI] = 1.04, 2.25). Respondents with experienced racism were also more likely to report psychological distress (AOR = 1.68; 95% CI = 1.09, 2.59). About one-third (32.66%) of the observed association between experienced racism and worsening sex life was mediated through psychological distress. Addressing racism and its association with psychological distress has the potential to improve sexual health and reduce related racial and ethnic disparities.


Fine needle aspiration cytology of pilomatrixoma.
To delineate and characterize the cytomorphologic features of pilomatrixoma (PMX) helpful in correct diagnosis of the lesion on fine needle aspiration cytology (FNAC). Archival records of clinical findings, FNAC and histopathological reports of PMX cases were analyzed. Aspirate findings in 14 cases of PMX were correlated with detailed clinical data and subsequent histopathology on excisional biopsy. Different morphological findings were graded semiquantitatively from 0 to 3+. The series showed female preponderance, with head and neck being the commonest site. Majority patients had a single tumor with mean size of 1.6 cm. Out of 14 biopsy proven cases of PMX, cytology findings revealed PMX in 7 cases on the basis of ghost cells, groups of basaloid cells, squamous cells in combination with multinucleated giant cells and calcium deposits in a background of debris. The main reasons for erroneous diagnosis were predominance of one component over the others and non-representative aspirated material. The cytological features of PMX are characteristic and allow a conclusive diagnosis provided the smears are examined keenly bearing in mind the diagnostic traps that can mislead a cytopathologist.


[Treatment of whiplash-associated disorders].
Treatment of whiplash-associated disorders starts with a thorough clinical examination, which may be repeated after 1-3 weeks. For optimal results it is essential that the patient receives clear information about the condition and that any pain is treated effectively with analgesics. Risk factors for persistent symptoms can often be identified early and should be addressed adequately. If symptoms persist and conservative treatments are chosen, these should be active and they should focus on sustaining or regaining usual activities.


Hypoglycemia is more common among type 2 diabetes patients with limited health literacy: the Diabetes Study of Northern California (DISTANCE).
Little is known about the frequency of significant hypoglycemic events in actual practice. Limited health literacy (HL) is common among patients with type 2 diabetes, may impede diabetes self-management, and thus HL could increase the risk of hypoglycemia. To determine the proportion of ambulatory, pharmacologically-treated patients with type 2 diabetes reporting > or =1 significant hypoglycemic events in the prior 12 months, and evaluate whether HL is associated with hypoglycemia. Cross-sectional analysis in an observational cohort, the Diabetes Study of Northern California (DISTANCE). The subjects comprised 14,357 adults with pharmacologically-treated, type 2 diabetes who are seen at Kaiser Permanente Northern California (KPNC), a non-profit, integrated health care delivery system. Patient-reported frequency of significant hypoglycemia (losing consciousness or requiring outside assistance); patient-reported health literacy. At least one significant hypoglycemic episode in the prior 12 months was reported by 11% of patients, with the highest risk for those on insulin (59%). Patients commonly reported limited health literacy: 53% reported problems learning about health, 40% needed help reading health materials, and 32% were not confident filling out medical forms by themselves. After adjustment, problems learning (OR 1.4, CI 1.1-1.7), needing help reading (OR 1.3, CI 1.1-1.6), and lack of confidence with forms (OR 1.3, CI 1.1-1.6) were independently associated with significant hypoglycemia. Significant hypoglycemia was a frequent complication in this cohort of type 2 diabetes patients using anti-hyperglycemic therapies; those reporting limited HL were especially vulnerable. Efforts to reduce hypoglycemia and promote patient safety may require self-management support that is appropriate for those with limited HL, and consider more vigilant surveillance, conservative glycemic targets or avoidance of the most hypoglycemia-inducing medications.


Variation in the dynamic stiffness index of different types of medical elastic compression stockings.
To calculate the dynamic stiffness index (DSI) of 18 different brands of medical elastic compression stockings (MECS). In all, 18 different brands of MECS that were divided into five categories (class II round-knitted, class II flat-knitted, class III round-knitted, class III flat-knitted and class IV flat-knitted MECS) were tested. The static pressure and dynamic pressure pulsations at the B1 level were measured with a newly developed dynamic pressure-determining device. The DSI was calculated. The DSI of all 18 brands of MECS showed higher values compared with the static stiffness. A wide range of dynamic stiffness indices was observed not only between all brands of MECS, but also within the five categories. The DSI of MECS is independent of the compression class and the type of knit. The variation in the DSIs between MECS is not because of any measurement error and would indicate that different therapeutic effectiveness may be expected within one compression class. Therefore, a refinement in the current classification system for MECS with other characteristics such as the DSI is warranted.


Sex Differences in Prevalence, Symptoms, Impact, and Psychiatric Comorbidities in Migraine and Probable Migraine: A Population-Based Study.
This study was conducted to investigate sex differences in the prevalence and clinical presentation of migraine and probable migraine in a general population-based sample. While there is research on sex differences in clinical characteristics and their impact on migraine headache, only few studies have investigated sex differences in probable migraine in population-based settings. Moreover, compared with Western countries, the prevalence of probable migraine in Asia is relatively high. This cross-sectional study was designed to investigate sex differences in the prevalence and clinical presentation of migraine and probable migraine in a general population-based sample. We used the data of the Korean Headache-Sleep Study, which is a nationwide survey on headache and sleep. We interviewed 7430 people, and 3114 of them agreed to participate in our study (rejection rate, 58.1%). Among these people, 419 withdrew their participation during the interview. Ultimately, 2695 people completed our survey (cooperation rate, 36.3%). The prevalence of overall migraine and probable migraine was 350/1350 (25.9%) for women and 172/1345 (12.8%) for men (P < .001, respectively). The prevalence of migraine (107/1350 [7.9%] vs 36/1345 [2.7%], P < .001) and probable migraine (243/1350 [18.0%] vs 136/1345 [10.1%], P < .001) was significantly higher among women than among men. Headache frequency per month (median [interquartile range]) (1.0 [0.3-3.0] vs 0.8 [0.3-2.0], P = .037), the visual analog scale score for headache intensity (5.0 [4.0-7.0] vs 5.0 [3.0-6.0], P = .019), and the impact of headache {Headache Impact Test-6 score (47.0 [42.0-54.0] vs 44.0 [42.0-51.8], P = .013)} were significantly higher among women with probable migraine than men. Headache frequency per month (2.0 [0.4-4.0] vs 1.0 [0.3-2.0], P = .073), headache intensity (6.0 [5.0-8.0] vs 6.0 [4.2-7.0], P = .281), and the impact of headache (55.0 [48.0-61.0] vs 49.0 [46.3-60.8], P = .225) were not significantly different between women and men with migraine. Other comorbidities or associated symptoms, such as anxiety and depression, were not significantly different between women and men with migraine and probable migraine, except for nausea in probable migraine. Women experience more severe symptoms and a higher impact of headache than men among participants with probable migraine. Our findings suggest that women with PM need a more intensive evaluation and treatment than men with PM.


[Angiodynography: technical principles and clinical uses].
Angiodynography is a newly developed system that enables Doppler sonography to be used to obtain a colour-coded real-time image of the blood vessels in the familiar ultrasound slice image. A new computerised technique allows both the familiar pulse echo signal and the Doppler shift of the signal resulting from movement in the body to be displayed for each point on the ultrasound slice plane. Stationary tissue structures are shown as the familiar grey scale image, while movement such as of the blood is shown in colour. 453 patients were examined. Normal and pathological flow measurements were obtained in the carotid artery, the jugular vein, in renal transplants, in the thyroid, the testis and in the urethra. Pathological flow velocities were visualised in stenosis, tumours and diffuse alterations of parenchyma.


[Sleeping disorders rarely come singly].
Four patients, a woman aged 60 and three men aged 61, 53 and 56 years, presented with hypersomnia during the day. The cause was determined by polygraphic registration and was a variable combination of sleep apnoea syndrome, narcolepsy and periodic movements of the limbs in sleep syndrome (PMLS). Such a combination may lead to a therapeutic paradox as the treatment of one disturbance may lead to aggravation of another one. Therapy is possible but only when it is completely clear which component of the disorder prevails. Whole night polygraphy is indispensable for this approach.


Basal joint osteoarthritis of the thumb: a prospective trial of steroid injection and splinting.
There have been few prospective studies evaluating the results of nonsurgical treatment of a well-defined patient cohort with symptomatic basal joint osteoarthritis of the thumb. This prospective study uses a validated outcome instrument to examine the effectiveness of a single steroid injection and 3 weeks of splinting in patients with osteoarthritis in Eaton stages 1 to 4 with a minimum of 18 months of follow-up evaluation. Thirty consecutive patients (30 thumbs) were studied prospectively to evaluate the efficacy of a single injection of corticosteroid into the trapeziometacarpal joint, followed by immobilization in a thumb spica splint for 3 weeks. All patients answered an outcome-based questionnaire (Disabilities of the Arm, Shoulder, and Hand) and were examined before injection, 6 weeks after injection, and at final follow-up examination (minimum, 18 months). Eaton radiographic stage was recorded by 3 independent observers. At 6 weeks 13 patients had improvement in pain intensity and 17 patients reported no symptomatic improvement. Twelve of those with relief at 6 weeks continued to have relief at long term follow-up evaluation (mean, 25 months). Of patients with long-term relief average grip strength of the affected thumb was 95% of contralateral side, whereas those without relief had grip strength values that were 60% of contralateral side. For those patients without relief at 6 weeks there was no improvement seen at later follow-up evaluation. Five patients with Eaton stage 1 disease had an average of 23 months of relief with nonsurgical treatment. In stage 2 and stage 3 disease 7 thumbs improved at 6 weeks after injection and 6 thumbs had long-term relief. In stage 4 disease, 6 thumbs had neither short-term nor long-term relief with the injection. Disease side, handedness, and smoking did not affect outcomes. At final follow-up evaluation 12 thumbs had had surgical treatment. Steroid injection with splinting for the treatment of basal joint arthritis of the thumb provided reliable long-term relief in thumbs with Eaton stage 1 disease but provided long-term relief in only 7 of 17 thumbs with Eaton stage 2 and stage 3 basal joint arthritis.


An Overview on Antimalarial Peptides: Natural Sources, Synthetic Methodology and Biological Properties.
Peptide compounds play a significant role in medicinal chemistry as they can inhibit the activity of species that cause malaria. This literature review summarizes the isolation of antimalarial peptides, the synthesis method with the detailed structure and sequences of each peptide, and discusses the biological activity of the isolated and synthesized compounds. The synthetic routes and reactions for cyclic and linear antimalarial peptides are systematically highlighted in this review including preparing building blocks, protection and deprotection, coupling and cyclization reactions until the target compound is obtained. Based on the literature data and the results, this review's aim is to provide information to discover and synthesize more antimalarial peptide for future research.


Polyethylene glycol repairs membrane damage and enhances functional recovery: a tissue engineering approach to spinal cord injury.
The integrity of the neuronal membrane is crucial for its function and cellular survival; thus, ineffective repair of damaged membranes may be one of the key elements underlying the neuronal degeneration and overall functional loss that occurs after spinal cord injury (SCI). it has been shown that polyethylene glycol (PEG) can reseal axonal membranes following various injuries in multiple in vitro and in vivo injury models. in addition, PEG may also directly prevent the effects of mitochondria-derived oxidative stress on intracellular components. Thus, PEG repairs mechanically injured cells by at least two distinct pathways: resealing of the disrupted plasma membrane and direct protection of mitochondria. Besides repairing primary membrane damage, PEG treatment also results in significant attenuation of oxidative stress, likely due to its capacity to reseal the membrane, thereby breaking the cycle of cellular damage and free-radical production. Based on this, in addition to the practicality of its application, we expect that PEG may be established as an effective treatment for SCI where membrane disruption and mitochondrial damage are implicated.


Classification of blood pressure during sleep impacts designation of nocturnal nondipping.
The identification of nocturnal nondipping blood pressure (< 10% drop in mean systolic blood pressure from awake to sleep periods), as captured by ambulatory blood pressure monitoring, is a valuable element of risk prediction for cardiovascular disease, independent of daytime or clinic blood pressure measurements. However, capturing measurements, including determination of wake/sleep periods, is challenging. Accordingly, we sought to evaluate the impact of different definitions and algorithms for defining sleep onset on the classification of nocturnal nondipping. Using approaches based upon participant self-reports, applied definition of a common sleep period (12 am -6 am), manual actigraphy, and automated actigraphy we identified changes to the classification of nocturnal nondipping, and conducted a secondary analysis on the potential impact of an ambulatory blood pressure monitor on sleep. Among 61 participants in the Eastern Caribbean Health Outcomes Research Network hypertension study with complete ambulatory blood pressure monitor and sleep data, the concordance for nocturnal nondipping across methods was 0.54 by Fleiss' Kappa (depending on the method, 36 to 51 participants classified as having nocturnal nondipping). Sleep quality for participants with dipping versus nondipping was significantly different for total sleep length when wearing the ambulatory blood pressure monitor (shorter sleep duration) versus not (longer sleep duration), although there were no differences in sleep efficiency or disturbances. These findings indicate that consideration of sleep time measurements is critical for interpreting ambulatory blood pressure. As technology advances to detect blood pressure and sleep patterns, further investigation is needed to determine which method should be used for diagnosis, treatment, and future cardiovascular risk.


Metabolic changes in rats during developing thiamin deficiency.
Determinations of rectal temperature, blood glucose, plasma free fatty acids, liver acetyl-CoA and carcass fat of thiamin-deficient rats indicated that during the ensuing anorexia they metabolized their fat reserves more rapidly than did pair-fed normal controls. Their lower metabolic rate indicates that the reserves mobilized are utilized inefficiently.


Relationship between vertical facial morphology and dental arch measurements in class II malocclusion: a retrospective study.
To evaluate the relationship between dental arch measurements and the vertical facial pattern determined in skeletal Class II untreated patients. Lateral cephalograms and plaster models were obtained from 124 untreated female adults (average age: 17.6 ± 3.8 years). Class I (CI), Class II Division 1 (CII/1) and Class II Division 2 (CII/2) malocclusions were divided into three subgroups according to their vertical morphology as hypodivergent, normodivergent and hyperdivergent. The multivariate variance analysis (MANOVA) method was used in the comparison of measurement values according to vertical and sagittal morphology. The relationship between both A point-Nasion-B point (ANB) and Frankfurt-mandibular plane (FMA) angles and dental arch measurements was examined by Pearson correlation analysis. The significance level was received as p < 0.05. While vertical morphology has a statistically significant effect on mandibular arch length, sagittal morphology affects maxillary arch depth. The parameters influenced by both morphologies are maxillary and mandibular arch length, as well as maxillary intermolar width. The mandibular arch length was significantly shorter in hyperdivergent-CII-2 malocclusion (50.5 ± 7.4 mm). Larger values were obtained in both mandibular arch length and maxillary arch depth measurements in CII-1 malocclusion compared to CII-2 malocclusion. The maxillary intermolar width was significantly shorter in hypodivergent-CII-1 malocclusion (46.8 ± 3.4 mm), while it was higher in hypodivergent-CI malocclusion (51.1 ± 3.4 mm). The maxillary arch length was the lowest in hyperdivergent-CI malocclusion (63.1 ± 13.3 mm) and the highest in hypodivergent-CI malocclusion (72.8 ± 7.6 mm). Additionally, a positive but weak correlation was found between ANB and FMA angles. Dental arch measurements have been found to be affected by both vertical facial morphology and skeletal sagittal relationship. A positive correlation was found between ANB and FMA angles.


Acyline: the first study in humans of a potent, new gonadotropin-releasing hormone antagonist.
Acyline is a novel GnRH antagonist found in animal studies to be a potent suppressor of circulating gonadotropin and testosterone (T) levels. We conducted the first study of acyline administration to humans. Eight healthy, eugonadal young men were administered a series of acyline injections (0, 2.5, 7.5, 25, and 75 microg/kg), each injection separated by at least 10 d. Serum FSH, LH, and T levels were measured for 7 d after injections. Acyline suppressed FSH, LH, and T levels in a dose-dependent fashion. Maximal suppression occurred after injection of 75 microg/kg acyline, which suppressed FSH to 46.9 +/- 2.5%, LH to 12.4 +/- 2.2%, and T to 13.4 +/- 1.4% of baseline levels, maintaining suppression for over 48 h. Serum acyline levels peaked at 1 h at 18.9 +/- 0.9 ng/ml, remained significantly elevated above background 7 d after injection, and returned to background levels by 14-17 d after injection. Side-effects at the site of injection were limited to infrequent blush and pruritus that resolved within 90 min of injection. Higher doses of acyline might be effective as depot injections for long-lasting gonadotropin suppression in hormone-dependent diseases and for use in male hormonal contraception regimens.


Small compounds mimicking the adhesion molecule L1 improve recovery in a zebrafish demyelination model.
Demyelination leads to a loss of neurons, which results in, among other consequences, a severe reduction in locomotor function, and underlies several diseases in humans including multiple sclerosis and polyneuropathies. Considerable clinical progress has been made in counteracting demyelination. However, there remains a need for novel methods that reduce demyelination while concomitantly achieving remyelination, thus complementing the currently available tools to ameliorate demyelinating diseases. In this study, we used an established zebrafish demyelination model to test selected compounds, following a screening in cell culture experiments and in a mouse model of spinal cord injury that was aimed at identifying beneficial functions of the neural cell adhesion molecule L1. In comparison to mammalian nervous system disease models, the zebrafish allows testing of potentially promotive compounds more easily than what is possible in mammals. We found that our selected compounds tacrine and duloxetine significantly improved remyelination in the peripheral and central nervous system of transgenic zebrafish following pharmacologically induced demyelination. Given that both molecules are known to positively affect functions other than those related to L1 and in other disease contexts, we propose that their combined beneficial function raises hope for the use of these compounds in clinical settings.


Cordycepin Isolated from Cordyceps militaris: Its Newly Discovered Herbicidal Property and Potential Plant-Based Novel Alternative to Glyphosate.
There is currently much interest in finding new phytochemicals among plants and fungi as nature-based alternatives to replace problematic herbicides such as glyphosate, which are preferentially used in agricultural production n. We discovered striking herbicidal potency in Cordyceps militaris (L.) and identified cordycepin as its principal plant growth inhibitor. Cordycepin obtained as an ethyl acetate extract was subjected to column chromatography and evaluated for its bioassay-guided phytotoxic capacity against Raphanus sativus (radish), showing a maximum inhibition on germination and growth of radish (IC50 = 0.052-0.078 mg/mL). Gas chromatography-mass spectrometry (GC-MS) (m/z: 251.2) and liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) ([M + Na]+ m/z: 274.1; [M + H]+ m/z: 252.1) analyses confirmed cordycepin as the major component of the tested column fraction (55.38%). At 0.04 mg/mL, cordycepin showed 3.8-5.9- and 3.3-3.7-fold greater inhibition of the germination and growth of radish than benzoic acid (BA) and glyphosate, respectively. Compared with BA, isolated cordycepin reduced plant chlorophyll and carotenoid contents (2.0-9.5 -fold), while proline, total phenolic and total flavonoid contents were increased 1.2-1.8-fold. Finally, cordycepin promoted electrolyte leakage and malondialdehyde accumulation in radish aerial parts. Thus, cordycepin successfully isolated from Cordyceps militaris is a highly potent plant growth inhibitor with pending worldwide patent and may become a potential plant-based novel alternative to the disputed glyphosate.


Modified peptides as potent inhibitors of the postsynaptic density-95/N-methyl-D-aspartate receptor interaction.
The protein-protein interaction between the NMDA receptor and its intracellular scaffolding protein, PSD-95, is a potential target for treatment of ischemic brain diseases. An undecapeptide corresponding to the C-terminal of the NMDA was used as a template for finding lead candidates for the inhibition of the PSD-95/NMDA receptor interaction. Initially, truncation and alanine scan studies were carried out, which resulted in a pentapeptide with wild-type affinity, as examined in a fluorescence polarization assay. Further examination was performed by systematic substitutions with natural and unnatural amino acids, which disclosed a tripeptide with micromolar affinity and N-methylated tetrapeptides with improved affinities. Molecular modeling studies guided further N-terminal modifications and introduction of a range of N-terminal substitutions dramatically improved affinity. The best compound, N-cyclohexylethyl-ETAV (56), demonstrated up to 19-fold lower K i value ( K i = 0.94 and 0.45 microM against PDZ1 and PDZ2 of PSD-95, respectively) compared to wild-type values, providing the most potent inhibitors of this interaction reported so far. These novel and potent inhibitors provide an important basis for development of small molecule inhibitors of the PSD-95/NMDA receptor interaction.


Correlation between refractory periods and activation-recovery intervals from electrograms: effects of rate and adrenergic interventions.
Unipolar electrograms from ventricular epicardium in dogs were analyzed for the timing of local excitation and repolarization with computer assistance. The most rapid decrease in voltage in the QRS (dV/dt min) was used to determine local excitation time, and the maximum rate of voltage increase (dV/dt max) near the peak of the T wave was used to time local repolarization. The difference between dV/dt min and dV/dt max, the activation-recovery interval, is theoretically related to the net effect of the durations of the action potentials at that site. Paired data for refractory periods and activation-recovery intervals obtained from the same electrodes during fixed activation orders were obtained before and during repolarization changes induced by changes in cycle length, infusion of norepinephrine, and cardiac sympathetic nerve stimulation. Correlation coefficients were close to 1.00 and standard errors were 2.0 to 4.3 msec for changes at individual sites. Pooling of data from multiple sites increased standard errors and reduced correlation coefficients. Results provide quantification of errors in the use of unipolar electrograms to time local repolarization changes induced by variations in rate and adrenergic tone. They should increase the practical usefulness of the unipolar electrogram as a tool for assessing the time course and spatial distribution of repolarization changes.


Muscular dystrophy in adults: a five-year follow-up.
The aim was to describe the natural history of adults with hereditary muscular dystrophies, including myotonic dystrophy, with respect to muscular function, ventilation and electrocardiogram. In a prospective study, 46 subjects were followed over a period of five years. In 1991 and 1996, their muscle function was assessed according to an observation scheme and their lung vital capacity was measured by spirometer. Electrocardiograms were obtained in 1991, 1993 and 1996. Deterioration of muscular function was seen with regard to both the functional muscle tests and the vital capacity. The proportion of pathological electrocardiograms increased from 38% in 1991 to 54% in 1996 in the 26 patients with myotonic dystrophy without an increase in clinically detected cardiac abnormalities. Timely examinations using standard methods can reveal medically important information on deterioration, which often passes clinically unnoticed because of the insidious progress of the diseases.


Comparative development of thoracic intervertebral discs and intra-articular ligaments in the human, monkey, mouse, and cat.
Developmental features of thoracic intervertrebral discs and their association in the adult with other vertebral structures were investigated in four species. The human anulus fibrosus, nucleus pulposus, and intra-articular ligaments were compared to those of the fetal rhesus monkey, mouse, and kitten. Photomicrographs of transverse sections of intervertebral discs document the presence of intra-articular ligaments in fetuses of these four species. Both transverse and sagittal sections of kittens were used to identify the intercapital ligament as it differentiated from the dorsal part of the intra-articular ligament. Relatively frequent dorsal herniation of the thoracic nucleus pulposus in humans may be due to the vestigial nature of the human intra-articular ligament. Quadrupeds have well-developed intra-articular ligaments, which explains anatomically the paucity of dorsal protrusions of the nucleus pulposus into the vertebral canal in the thoracic region of the cat and mouse when compared to the human. The intra-articular ligament was closely associated with the developing prenatal mammalian intervertebral disc in the four species studied, and this relationship and its surgical importance are described.


Risk factors for post-operative periprosthetic fractures following primary total hip arthroplasty with a proximally coated double-tapered cementless femoral component.
The aim of this study was to identify patient- and surgery-related risk factors for sustaining an early periprosthetic fracture following primary total hip arthroplasty (THA) performed using a double-tapered cementless femoral component (Bi-Metric femoral stem; Biomet Inc., Warsaw, Indiana). A total of 1598 consecutive hips, in 1441 patients receiving primary THA between January 2010 and June 2015, were retrospectively identified. Level of pre-operative osteoarthritis, femoral Dorr type and cortical index were recorded. Varus/valgus placement of the stem and canal fill ratio were recorded post-operatively. Periprosthetic fractures were identified and classified according to the Vancouver classification. Regression analysis was performed to identify risk factors for early periprosthetic fracture. The mean follow-up was 713 days (1 to 2058). A total of 48 periprosthetic fractures (3.0%) were identified during the follow-up and median time until fracture was 16 days, (interquartile range 10 to 31.5). Patients with femoral Dorr type C had a 5.2 times increased risk of post-operative periprosthetic fracture compared with type B, while female patients had a near significant two times increased risk over time for post-operative fracture. Dorr type C is an independent risk factor for early periprosthetic fracture, following THA using a double tapered cementless stem such as the Bi-Metric. Surgeons should take bone morphology into consideration when planning for primary THA and consider using cemented femoral components in female patients with poor bone quality. Cite this article: Bone Joint J 2017;99-B:451-7.


Folic Acid-Conjugated pH and Redox-Sensitive Ellipsoidal Hybrid Magnetic Nanoparticles for Dual-Triggered Drug Release.
Stimuli-sensitive and multifunctional nanoparticles are highly desirable biomedical materials for triggered and targeted drug delivery applications. Here, we have designed pH- and redox-triggered magnetic lipid-polymer hybrid nanoparticles (MHNPs) with a core-shell structure. This design is composed of a silica-/mesoporous silica-coated ellipsoidal magnetic nanoparticle with multifunctionality: carrying the anticancer drug (doxorubicin, DOX), the cancer cell targeting ligand (folic acid-conjugated poly(ethylene glycol), FA-PEG) polymer, and being coated with a biocompatible pH-/redox-responsive (poly-l-histidine-poly(ethylene glycol)-lipoic acid; PLH-PEG-LA) polymer. The lipoic acid units of the PLH-PEG-LA shell of the FA-MHNPs were cross-linked using 1,4-dithiothreitol (DTT). When the FA-MHNPs-DOX were exposed to an endolysosomal pH of 5.5 and 10 mM glutathione (GSH), the particles exhibited a very efficient DOX release profile within 24 h. In addition, cytotoxicity, uptake, and apoptosis assays were performed against breast cancer cell lines. These results showed that FA-MHNPs-DOX promote an enhanced uptake and cell morbidity compared to the nontargeted MHNPs-DOX against tested cell lines. Moreover, the FA-MHNPs-DOX exhibited very effective cytotoxicity and also decreased the cell viability through apoptosis against breast cancer cell lines. In conclusion, it can be said that the pH and redox dual-responsive hybrid FA-MHNPs-DOX has a great potential for controlled drug release.


Corticotropin-releasing factor in the ovine fetus and pregnant ewe: role of placenta.
In the sheep, maternal plasma adrenocorticotropic hormone and cortisol are increased in late pregnancy, and fetal plasma cortisol and adrenocorticotropic hormone rise precipitously in late gestation. To test whether the ovine placenta secretes corticotropin-releasing factor (CRF) into either the maternal or fetal circulation, pregnant ewes and their fetuses were prepared with femoral arterial catheters and uterine and umbilical venous catheters. Samples were taken from all sites before and during hypoxia. There was no difference in CRF concentration across the placenta in the mothers or the fetuses under resting or hypoxemic conditions, but maternal and fetal arterial plasma CRF concentrations increased between 128 and 145 days. In a second study, maternal and fetal femoral venous plasma CRF concentrations were measured 1-19 days before spontaneous parturition. The mean concentration increased 8.6 +/- 0.6 pg/ml 11-19 days before parturition to 13.0 +/- 1.0 and 13.2 +/- 1.4 pg/ml in fetuses 4-8 and 1-3 days before parturition, respectively. Maternal plasma concentrations did not significantly increase in the days closer to parturition. These studies demonstrate that there are low but measurable CRF concentrations in fetal and maternal sheep plasma but that these are not the result of tonic placental secretion of CRF.


Systematic Heuristic Evaluation of Computerized Consultation Order Templates: Clinicians' and Human Factors Engineers' Perspectives.
We assessed the usability of consultation order templates and identified problems to prioritize in design efforts for improving referral communication. With a sample of 26 consultation order templates, three evaluators performed a usability heuristic evaluation. The evaluation used 14 domain-independent heuristics and the following three supplemental references: 1 new domain-specific heuristic, 6 usability goals, and coded clinicians' statements regarding ease of use for 10 sampled templates. Evaluators found 201 violations, a mean of 7.7 violations per template. Minor violations outnumbered major violations almost twofold, 115 (57%) to 62 (31%). Approximately 68% of violations were linked to 5 heuristics: aesthetic and minimalist design (17%), error prevention (16%), consistency and standards (14%), recognition rather than recall (11%), and meet referrers' information needs (10%). Severe violations were attributed mostly to meet referrers' information needs and recognition rather than recall. Recorded violations yielded potential negative consequences for efficiency, effectiveness, safety, learnability, and utility. Evaluators and clinicians demonstrated 80% agreement in usability assessment. Based on frequency and severity of usability heuristic violations, the consultation order templates reviewed may impede clinical efficiency and risk patient safety. Results support the following design considerations: communicate consultants' requirements, facilitate information seeking, and support communication. While the most frequent heuristic violations involved interaction design and presentation, the most severe violations lacked information desired by referring clinicians. Violations related to templates' inability to support referring clinicians' information needs had the greatest potential negative impact on efficiency and safety usability goals. Heuristics should be prioritized in future design efforts.


The fatty acid composition of 1,2-diacylglycerol and polyphosphoinositides from human erythrocyte membranes.
The fatty acid compositions of 1,2-diacylglycerol and polyphosphoinositides have been determined in human erythrocyte membranes that have been incubated in the presence or in the absence of Ca2+. The results show that the diacylglycerol that is formed in Ca2+-treated membranes has a fatty acid composition closely similar to that of the inositides, consistent with previous indications that Ca2+ stimulates the activity of a polyphosphoinositide phosphodiesterase in the membranes. In contrast with some previous results, it appears that these plasma-membrane inositides and their derived diacylglycerols are rich in stearic acid and arachidonic acid.


[Health and disability in the elderly: old paradigms and future prospects].
The projections regarding the ageing of the Italian population are cause for great concern; however, the ageing scenario may actually be interpreted in a more optimistic way. Theories formulated in the 80s envisaging a decline of mortality, morbidity and disability in the elderly are now confirmed and prefigure an unexpected decrease in disability rates in the elderly population. The aim of this review is to attempt to explain the reasons for this by analyzing the role played by the various determinants of health, in particular social isolation, which are likely to play an important role in the future as well.


Coverage bias and sensitivity of variant calling for four whole-genome sequencing technologies.
The emergence of high-throughput, next-generation sequencing technologies has dramatically altered the way we assess genomes in population genetics and in cancer genomics. Currently, there are four commonly used whole-genome sequencing platforms on the market: Illumina's HiSeq2000, Life Technologies' SOLiD 4 and its completely redesigned 5500xl SOLiD, and Complete Genomics' technology. A number of earlier studies have compared a subset of those sequencing platforms or compared those platforms with Sanger sequencing, which is prohibitively expensive for whole genome studies. Here we present a detailed comparison of the performance of all currently available whole genome sequencing platforms, especially regarding their ability to call SNVs and to evenly cover the genome and specific genomic regions. Unlike earlier studies, we base our comparison on four different samples, allowing us to assess the between-sample variation of the platforms. We find a pronounced GC bias in GC-rich regions for Life Technologies' platforms, with Complete Genomics performing best here, while we see the least bias in GC-poor regions for HiSeq2000 and 5500xl. HiSeq2000 gives the most uniform coverage and displays the least sample-to-sample variation. In contrast, Complete Genomics exhibits by far the smallest fraction of bases not covered, while the SOLiD platforms reveal remarkable shortcomings, especially in covering CpG islands. When comparing the performance of the four platforms for calling SNPs, HiSeq2000 and Complete Genomics achieve the highest sensitivity, while the SOLiD platforms show the lowest false positive rate. Finally, we find that integrating sequencing data from different platforms offers the potential to combine the strengths of different technologies. In summary, our results detail the strengths and weaknesses of all four whole-genome sequencing platforms. It indicates application areas that call for a specific sequencing platform and disallow other platforms. This helps to identify the proper sequencing platform for whole genome studies with different application scopes.


Validation of a pan-orthopox real-time PCR assay for the detection and quantification of viral genomes from nonhuman primate blood.
In 1980, smallpox disease was eradicated from nature and Variola virus, the etiological agent of smallpox, was confined to two laboratories, one located in Russia (Moscow) later moved to VECTOR (Novosibirsk, Siberia) and one in the United States (CDC Atlanta). Vaccinations among the general public ceased shortly after the successful eradication campaign, resulting in an increasingly immunologically susceptible population. Because of the possibility of intentional reintroduction of Variola virus and the emergence of other pathogenic poxviruses, there is a great need for the development of medical countermeasures to treat poxvirus disease. It is highly likely that the U.S. FDA "animal rule" will be necessary for regulatory approval of these interventions. Therefore, relevant animal models and the associated supporting assays will require development to stand up to regulatory scrutiny. An optimized real time PCR assay for the detection of orthopoxviruses has been developed by researchers at the United States Army Research Institute of Infectious Diseases (USAMRIID). To support animal studies that will be used to support approval of medical countermeasures by the U.S. FDA, the assay was designed to quantitate poxvirus genomic DNA in a nonhuman primate (cynomolgus macaque) blood matrix as a measurement of viremia. This manuscript describes the validation of the process, including DNA extraction from whole blood anticoagulated with EDTA, for obtaining and quantitating monkeypox genomes by evaluating precision, accuracy, the standard curve, specificity, robustness and stability of the assay and/or components of the assay. The assay had a lower limit of quantitation of 50 genome copies/5 uL sample, upper limit of quantitation of 5 × 107 GC/5uL sample and a limit of detection of 2.5 genome copies /5uL sample. The assay was specific for orthopoxvirus. Matrix effects were detected and suggest the presence of PCR inhibitor(s) that was co-extracted with the target DNA. The assay has been validated for the purpose of quantitating monkeypox viral load in blood from cynomolgus macaques. This assay has and will continue to support submissions to the FDA for approval of antiviral therapeutics for smallpox.


Metabolism of vitamin D3 by cytochromes P450.
The vitamin D3 25-hydroxylase (CYP27A1), 25-hydroxyvitamin D3 1alpha-hydroxylase (CYP27B1) and 1alpha,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1) are members of the cytochrome P450 superfamily, and key enzymes of vitamin D3 metabolism. Using the heterologous expression in E. coli, enzymatic properties of the P450s were recently investigated in detail. Upon analyses of the metabolites of vitamin D3 by the reconstituted system, CYP27A1 surprisingly produced at least seven forms of minor metabolites including 1alpha,25(OH)2D3 in addition to the major metabolite 25(OH)D3. These results indicated that human CYP27A1 catalyzes multiple reactions involved in the vitamin D3 metabolism. In contrast, CYP27B1 only catalyzes the hydroxylation at C-1alpha position of 25(OH)D3 and 24R,25(OH)2D3. Enzymatic studies on substrate specificity of CYP27B1 suggest that the 1alpha-hydroxylase activity of CYP27B1 requires the presence of 25-hydroxyl group of vitamin D3 and is enhanced by 24-hydroxyl group while the presence of 23-hydroxyl group greatly reduced the activity. Eight types of missense mutations in the CYP27B1 gene found in vitamin D-dependent rickets type I (VDDR-I) patients completely abolished the 1alpha-hydroxylase activity. A three-dimensional model of CYP27B1 structure simulated on the basis of the crystal structure of rabbit CYP2C5 supports the experimental data from mutagenesis study of CYP27B1 that the mutated amino acid residues may be involved in protein folding, heme-propionate binding or activation of molecular oxygen. CYP24A1 expressed in E. coli showed a remarkable metabolic processes of 25(OH)D3 and 1alpha,25(OH)2D3. Rat CYP24A1 catalyzed six sequential monooxygenation reactions that convert 1alpha,25(OH)2D3 into calcitroic acid, a known final metabolite of C-24 oxidation pathway. In addition to the C-24 oxidation pathway, human CYP24A1 catalyzed also C-23 oxidation pathway to produce 1alpha,25(OH)2D3-26,23-lactone. Surprisingly, more than 70 % of the vitamin D metabolites observed in a living body were found to be the products formed by the activities of CYP27A1, CYP27B1 and CYP24A1. The species-based difference was also observed in the metabolism of vitamin D analogs by CYP24A1, suggesting that the recombinant system for human CYP24A1 may be of great use for the prediction of the metabolism of vitamin D analogs in humans.


[In search of politics in knowledge production. A plea for a historical-political epistemology].
Knowledge production has an intrinsic political dimension. Starting from this presupposition, it is argued that the systematic integration of and reflection on the political dimension is necessary for an adequate understanding of historical processes of knowledge production in the sciences. The consecutive plea for a historical-political epistemology proceeds in two steps: First, it is illustrated that in a number of recent historical science study cases, the political dimension is frequently marginal, or even absent. After a short discussion of previous theoretical concepts to describe the impact of politics for the production of scientific knowledge, an approach is sketched which builds on Hans-Jörg Rheinberger's historical epistemology and Bruno Latour's symmetrical anthropology. It is argued that in addition to Rheinberger's program to describe epistemic systems, the political dimension is intrinsic to three stages of the process of data production: First to an initial phase which consists in the arrangement socio-technical configurations to produce new evidence. Here, factors such as the culturally shaped perception and evaluation of "relevant" problems, as well as the perception of career resources have to be taken into account. Second, the political dimension is relevant in view of the continuous re-adjustments of the configuration of epistemic systems, e.g., towards newly available financial, technical, or intellectual resources and "relevant" challenges from outside the system. Thirdly, the data produced and represented by epistemic systems--"evidence"--are yet in need of interpretation. This process is in itself imbued with continuously shifting mechanisms of selecting and creating hierarchies amongst the pool of available data.


[Physico-chemical properties and functional peculiarities of mRNP of different subcellular location of Nicotiana rustica plants affected by potato curly dwarf virus].
mRNP of different subcellular location of N. rustica plants affected by PCDV contain virus-specific proteins. RNA-replicase, poly (A)-polymerase and protein kinase have been found in mRNP under study. The most expressed replicase, poly(A)-polymerase and protein kinase activity has been noticed in the systems containing membrane-bound non-polysomal mRNP.


Female genitalia concealment promotes intimate male courtship in a water strider.
Violent coercive mating initiation is typical for animals with sexual conflict over mating. In these species, the coevolutionary arms-race between female defenses against coercive mating and male counter-adaptations for increased mating success leads to coevolutionary chases of male and female traits that influence the mating. It has been controversial whether one of the sexes can evolve traits that allow them to "win" this arms race. Here, we use morphological analysis (traditional and scanning electron micrographs), laboratory experiments and comparative methods to show how females of a species characterized by typical coercive mating initiation appear to "win" a particular stage of the sexual conflict by evolving morphology to hide their genitalia from direct, forceful access by males. In an apparent response to the female morphological adaptation, males of this species added to their typically violent coercive mounting of the female new post-mounting, pre-copulatory courtship signals produced by tapping the water's surface with the mid-legs. These courtship signals are intimate in the sense that they are aimed at the female, on whom the male is already mounted. Females respond to the signals by exposing their hidden genitalia for copulatory intromission. Our results indicate that the apparent victory of coevolutionary arms race by one sex in terms of morphology may trigger evolution of a behavioral phenotype in the opposite sex.


Optimization and implementation of the integer wavelet transform for image coding.
This paper deals with the design and implementation of an image transform coding algorithm based on the integer wavelet transform (IWT). First of all, criteria are proposed for the selection of optimal factorizations of the wavelet filter polyphase matrix to be employed within the lifting scheme. The obtained results lead to the IWT implementations with very satisfactory lossless and lossy compression performance. Then, the effects of finite precision representation of the lifting coefficients on the compression performance are analyzed, showing that, in most cases, a very small number of bits can be employed for the mantissa keeping the performance degradation very limited. Stemming from these results, a VLSI architecture is proposed for the IWT implementation, capable of achieving very high frame rates with moderate gate complexity.


Transition metal complexes as imaging or therapeutic agents for neurodegenerative diseases.
Neurodegenerative diseases are the result of neurodegeneration, which is the process of losing neuronal functions gradually due to the irreversible damage and death of neurons. Metal complexes have attracted intense interest over recent decades as probes or inhibitors of biomolecules. In this review, we discuss the application of transition metal complexes in the diagnosis and therapy of different neurodegenerative diseases. We first briefly introduce conventional approaches for the diagnosis and treatment of neurodegenerative diseases. Then, the characteristics and mechanisms of transition metal complexes as luminescent probes and drug candidates are discussed. Representative examples of the use of metal complexes in targeted detection and treatment of neurodegenerative diseases are highlighted. Finally, the challenges and future perspectives of this field are presented.


Insulin degradation. XVI. Evidence for the sequential degradative pathway in isolated liver cells.
Isolated liver cells contain insulin-degrading activity. Examination by chromatography on Sephadex G-75 of the products formed from 125I-insulin (1 nM or 1 muM) upon incubation with suspensions of hepatocytes for various time periods showed that there is at first a transient accumulation of the intermediate product, A chain, which is further hydrolyzed by protease(s) to low-molecular-weight components. These results indicate that the sequential degradative pathway is operative, both at low and high concentrations of insulin, in isolated liver cells, i.e., the insulin is first split at the disulfide bonds by glutathione-insulin transhydrogenase (GIT) into A and B chains, followed by proteolysis of the resultant polypeptides, and that this system might be used for well-defined studies of factors controlling insulin metabolism. The chelating agent ethylenediamine tetraacetic acid (EDTA) accelerated the accumulation of A chain (i.e., enhanced the activity of GIT), which is in keeping with its effect with purified GIT. In contrast to previous studies with tissue homogenates in which EDTA caused a marked inhibition of the proteolytic stage, EDTA had little or no effect with intact liver cells. Since EDTA does not appreciably penetrate the cell membrane, these data suggest that GIT activity either occurs on the cell surface or is readily available at the cell surface, whereas the proteolytic activity either occurs inside the cell or is inaccessible at the cell surface.


Identifying Small Bowel Gastrointestinal Stromal Tumor as the Culprit Lesion in Obscure Gastrointestinal Bleeding: Emphasis on Angiographic Findings.
Gastrointestinal stromal tumors (GISTs) are not uncommon and often cause gastrointestinal bleeding. GISTs occurring in the small intestine are occasionally difficult to identify by endoscopy and CT. In this case, the patient underwent CT three times before surgery, and the lesion was found to be located in a different area of the abdominal cavity on each CT scan. Moreover, the lesion was missed in the first two CT images because it was difficult to distinguish it from the nearby collapsed small intestine. The lesion was eventually detected through angiography; however, the correct diagnosis and treatment were delayed for 3 years because it was mistaken for a vascular malformation, which is the most common cause of obscure GI bleeding in elderly patients. This report emphasizes the need for interventional radiologists to be updated and vigilant of the angiographic features of GISTs to make an accurate diagnosis and establish a management strategy.


Responses of human adipose-derived mesenchymal stem cells to chemical microenvironment of the intervertebral disc.
Human adipose-derived mesenchymal stem cells (ADMSCs) may be ideal source of cells for intervertebral disc (IVD) regeneration, but the harsh chemical microenvironment of IVD may significantly influence the biological and metabolic vitality of ADMSCs and impair their repair potential. This study aimed to investigate the viability, proliferation and the expression of main matrix proteins of ADMSCs in the chemical microenvironment of IVD under normal and degeneration conditions. ADMSCs were harvested from young (aged 8-12 years, n = 6) and mature (aged 33-42 years, n = 6) male donors and cultured under standard condition and IVD-like conditions (low glucose, acidity, high osmolarity, and combined conditions) for 2 weeks. Cell viability was measured by annexin V-FITC and PI staining and cell proliferation was measured by MTT assay. The expression of aggrecan and collagen-I was detected by real-time quantitative polymerase chain reaction and Western blot analysis. IVD-like glucose condition slightly inhibited cell viability, but increased the expression of aggrecan. In contrast, IVD-like osmolarity, acidity and the combined conditions inhibited cell viability and proliferation and the expression of aggrecan and collagen-I. ADMSCs from young and mature donors exhibited similar responses to the chemical microenvironments of IVD. IVD-like low glucose is a positive factor but IVD-like high osmolarity and low pH are deleterious factors that affect the survival and biological behaviors of ADMSCs. These findings may promote the translational research of ADMSCs in IVD regeneration for the treatment of low back pain.


Primary squamous cell carcinoma of endometrium: case report and literature review.
In this paper, we report a case of primary squamous cell carcinoma of the endometrium (PSCCE) with a literature review. A 64-year-old woman was admitted because of postmenopausal bleeding. The gynecological exam found bleeding from the endocervix. The pelvic ultrasound objectified uterine regular contours, endometrial thickened was 10 mm, the presence of an intra cavitary lesion measuring 56/70 mm. The diagnostic hysteroscopy revealed a whitish appearance taking all the uterine cavity making evoke a tumor of the endometrium. Pelvic MRI showed a tumor limited to the uterine corpus endometrium (invasion by more than 50% of the myometrium) without invasion of the cervix. Radical hysterectomy, bilateral salpingo-oophorectomy, and lymph nodes dissection were performed. Grossly, the endometrial carcinoma was polypoid tumor occupying the entire uterine cavity. Histologically, the diagnosis of SCC was retained. No adenocarcinoma element was recognized. Neither squamous metaplasia nor dysplasia was recognized. No ectopic cervical tissue was found. The SCC was found to invade into deeper one half of the myometrium. No tumor cells were seen in other sites including the cervix, ovaries, parameters, and lymph nodes. The patients was FIGO 2009 stage IB (pT1B, N0), and was treated with adjuvant radiation. The patient had a disease progression in the pelvis 3 months after the irradiation. We reported a case of PSCCE which can help to enrich the literature for the treatment and prognosis of this disease.


A New Bioactive Fibrin Formulation Provided Superior Cartilage Regeneration in a Caprine Model.
The effective and long-term treatment of cartilage defects is an unmet need among patients worldwide. In the past, several synthetic and natural biomaterials have been designed to support functional articular cartilage formation. However, they have mostly failed to enhance the terminal stage of chondrogenic differentiation, leading to scar tissue formation after the operation. Growth factors substantially regulate cartilage regeneration by acting on receptors to trigger intracellular signaling and cell recruitment for tissue regeneration. In this study, we investigated the effect of recombinant insulin-like growth factor 1 (rIGF-1), loaded in fibrin microbeads (FibIGF1), on cartilage regeneration. rIGF-1-loaded fibrin microbeads were injected into full-thickness cartilage defects in the knees of goats. The stability, integration, and quality of tissue repair were evaluated at 1 and 6 months by gross morphology, histology, and collagen type II staining. The in vivo results showed that compared to plain fibrin samples, particularly at 6 months, FibIGF1 improved the functional cartilage formation, confirmed through gross morphology, histology, and collagen type II immunostaining. FibIGF1 could be a promising candidate for cartilage repair in the clinic.


Unilateral sacral erector spinae plane block for lower limb surgery in children.
The erector spinae plane block is a versatile regional anaesthesia technique used for a variety of truncal surgeries. A novel variation is the sacral erector spinae plane block which is gaining popularity for perineal and anorectal procedures. Local anaesthetic injected at this level blocks the sacral dermatomes and has the potential for more proximal spread to involve lumbar nerve fibres via spread to the lumbar plexus or the epidural space. The advantage of a sacral erector spinae plane block over a neuraxial block is that it is less invasive and may have a better safety profile: there is a reduced risk of epidural haematoma, epidural abscess, haemodynamic instability and motor weakness of the lower extremities. Until now, this approach has been used for midline surgical procedures when the local anaesthetic was injected bilaterally. Its application for lower limb procedures with a single unilateral injection has not been described. We report the use of a unilateral sacral erector spinae plane block at the level of the S2 median crest in two children undergoing lower limb procedures of the hip and thigh. The block was found to provide effective peri-operative analgesia with minimal need for any opioid analgesics and without any significant adverse effects.


The Medial Femoral Condyle Free Flap for Reconstruction of Recalcitrant Defects in the Head and Neck.
Small recalcitrant defects of the mandible and maxilla may be secondary to tumor, trauma, infection, and congenital origin. Vascularized bone grafting has been shown to effectively manage these defects; however, donor sites are limited. The vascularized medial femoral condyle (MFC) provides adequate cortical cancellous bone with the option of a skin island, consistent anatomy, and minimal donor site morbidity. This article outlines the use of the MFC flap for maxillomandibular reconstruction. A retrospective chart review of patients who required segmental maxillomandibular reconstruction with the MFC flap was conducted. A total of 9 patients (5 men and 5 women) with an average age of 45.3 years were identified. The etiology of the defects, flap sizes, and postoperative outcomes were recorded. Three patients had osteoradionecrosis of the neomandible after irradiation of the free fibula reconstruction, 3 patients had defects after cancer extirpation (1 mandible, 2 maxillary), 1 patient had a maxillary defect from trauma, and 2 patients had a residual cleft palate defect. All defects failed initial treatment with nonvascularized bone grafts. The average dimensions of the MFC flaps were 1.2 × 2.5 × 4 cm. Two of 9 flaps included a skin island. Eight flaps survived completely, but 1 patient suffered from flap failure requiring debridement and resulted in an oroantral fistula. Four patients received endosseous dental implants. Average time to union was 6.7 months, and average time to implant was 6.75 months. The average follow-up time was 24.9 months. The MFC flap is useful in the reconstruction of small segmental maxillomandibular defects and for the salvage of a neomandible after osteoradionecrosis. The MFC flap provides a reliable platform for endosseous dental implants and serves as an alternative source of vascularized bone reconstruction in the head and neck.