Functional display of proteins, mutant proteins, fragments of proteins and peptides on the surface of filamentous (bacterio) phages: A review.
Cytoplasmic expression of complex eukaryotic proteins inEscherichia coli usually yields inactive protein preparations. In some cases, (part) of the biological activity can be recovered by rather inefficient denaturation-renaturation procedures. Recently, novel concepts have been developed for the expression of fully functional eukaryotic proteins inE. coli. Essential to the success of these procedures is the transport of such proteins across the inner membrane to the periplasmic space, allowing proper folding and the establishment of disulfide bonding. Subsequently, fully functional proteins can be exposed on the surface of filamentous (bacterio)phages, provided a system is employed that consists of a cloning vector (e.g. the phagemid pComb3, Barbas et al., 1991) that generates phage particles in the presence of a helper phage. The main advantage of surface display of recombinant proteins is to facilitate the screening of very large numbers of different molecules by simple selection methods ("panning"). In addition, periplasmic expression yields relatively large quantities (e.g. 1 mg l(-1) of culture) soluble protein. In this review, the principle aspects of this novel expression system based on the phagemid pComb3 will be discussed. Two examples for functional periplasmic expression of human proteins inE. coli will be presented, namely i) the antigen-binding moiety (Fab fragment) of human immunoglobulins (IgGs) and ii) the human plasminogen activator inhibitor 1, an essential regulator of the plasminogen activation system. Finally, perspectives for the application of this system to express mutant proteins, fragments of proteins and peptides are indicated.


The effects of l-thyroxine and dexamethasone on steroid dynamics in male cynomologous monkeys.
Male cynomologous monkeys (M. fascicularis) were infused with [3H]androgens, [14C]estrogens and [3H]cortisol before and after the administration of l-thyroxine, (l-T4) 150 micrograms/day for 6 wk, dexamethasone 8 mg every 8 h for 3 doses and dexamethasone 1.0 mg/day for 8 days. Blood samples were obtained before each of the infusions and analyzed for endogenous T, A, E1, E2 and F concentrations, % free T and % free E2, sex hormone-binding globulin (SHBG) and cortisol binding globulin (CBG) capacity. When l-T4 was being administered, T4 and triiodothyronine (T3) concentrations were also measured. Blood samples were obtained during the infusions and analyzed for radioactivity as testosterone (T), androstenedione (A), dihydrotestosterone (DHT), estradiol (E1), estrone (E2), and cortisol (F). All urine was collected for 96 h and an aliquot of the pooled urine was analyzed for radioactivity as estrone and estradiol glucuronide. The administration of l-T4 for 6 wk to 3 monkeys resulted in a marked rise in T4 and T3 levels, from 4.8 +/- 0.4 micrograms/dl and from 136 +/- 6 to 515 +/- 71 ng/dl, respectively. MCRT, MCRE2 and MCRE1 did not change, but MCRA values increased slightly and MCRF increased 2-3 fold. [rho]T.E2 did not change but [rho]A.E1BM showed a slight but significant increase. The inter-conversions between the androgens and between the estrogens were not altered. There was a 2-3-fold increase in SHBG and a decrease in %FT but no change in %FE2 or CBG. The concentrations of T, A and DHT rose but there was no trend in the levels of the estrogens. The administration of dexamethasone 8 mg every 8 h for 3 doses or 1 mg/day for 8 days caused no changes in the MCRs for T, A, E1 and E2 but did cause a significant decrease in MCRF. Measurement of splanchnic and peripheral tissue extractions before and after acute dexamethasone administration in 1 monkey showed that the decrease in MCRF was the result of a marked decrease, 11-2%, in splanchnic extraction of F. The extractions of T and E2 were relatively unaffected. The concentrations of T and F fell but E2 remained the same. % FT and % FE2 rose slightly and the concentrations of SHBG and CBG were unchanged. The androgen interconversions and estrogen interconversions were not affected but [rho]T,E2BM and [rho]A,E1BM showed slight decreases.(ABSTRACT TRUNCATED AT 400 WORDS)


Evidence of diffusion artefacts in diaminobenzidine immunocytochemistry revealed during immune electron microscope studies of the early interactions between influenza virus and cells.
Anti-haemagglutinin-labelled antibodies have been used to search for influenza entry into cells by fusion of viral and plasma membranes. The plasma membranes of infected cells were stained by immunoperoxidase but not by immunoferritin reagents. It is suggested that the staining obtained with the peroxidase conjugate was due to diffusion of the diaminobenzidine reaction product away from the enzymic site. Immunoferritin labelling provided no evidence for entry of influenza by fusion of viral and plasma membranes under conditions of physiological pH.


Spatial and non-spatial learning in turtles: the role of medial cortex.
In mammals and birds, hippocampal processing is crucial for allocentric spatial learning. In these vertebrate groups, lesions to the hippocampal formation produce selective impairments in spatial tasks that require the encoding of relationships among environmental features, but not in tasks that require the approach to a single cue or simple non-spatial discriminations. In reptiles, a great deal of anatomical evidence indicates that the medial cortex (MC) could be homologous to the hippocampus of mammals and birds; however, few studies have examined the functional role of this structure in relation to learning and memory processes. The aim of this work was to study how the MC lesions affect spatial strategies. Results of Experiment 1 showed that the MC lesion impaired the performance in animals pre-operatively trained in a place task, and although these animals were able to learn the same task after surgery, probe test revealed that learning strategies used by MC lesioned turtles were different to that observed in sham animals. Experiment 2 showed that the MC lesion did not impair the retention of the pre-operatively learned task when a single intramaze visual cue identified the goal. These results suggest that the reptilian MC and hippocampus of mammals and birds function in quite similar ways, not only in relation to those spatial functions that are impaired, but also in relation to those learning processes that are not affected.


Intra-tumoral cytolytic cells: pattern of distribution in B-cell non Hodgkin s lymphoma.
Non-Hodgkin s lymphomas (NHLs) constitute a heterogeneous group of lymphoid neoplasms and a majority of them in India are of B-cell phenotype. Varying numbers of T lymphocytes and natural killer (NK) cells are consistently present within the lymph nodes (LNs). The role of these reactive cells is becoming understood. TIA-1 is a cytotoxic granule associated RNA binding protein, the expression of which is restricted to cytotoxic T lymphocytes (CTLs) and NK cells. Snap frozen lymph node biopsies obtained from 41 B-cell NHLs were localized for intra-tumoral TIA-1 + cytolytic cells by immunohistochemistry. Distribution of T cell subsets and NK cells were also quantified. Cells expressing TIA-1 antigen was observed in all the cases, seen as a strong granular cytoplasmic signal. Results indicate significantly higher number of TIA-1 cytolytic cells outside (periphery of the follicle and interfollicular areas) than within the neoplastic follicle in follicular lymphomas (p<0.001). In small lymphocytic lymphomas, cytolytic cells were mainly seen as uniformly scattered single cells, distributed throughout the tumor environment. In mantle cell and diffuse large B-cell lymphomas these were most often seen as small clusters and less frequently as singly scattered cells. Higher numbers of CD4 + than the CD8 + T cells were observed in most cases. Contrary to the follicles in follicular hyperplasia, CD57 + NK cells were predominantly observed outside the neoplastic follicle in follicular lymphomas (FLs). These results outline specific interactions between the potential anti-tumoral cytolytic and the malignant cells of B-cell NHLs.


Monoliths and Millennials.
Typifying all members of a particular age group as having specific characteristics is as deleterious as doing it with members of any other population, such as racial, ethnic, gender, and geographic groups.


Diagnostic value of endoscopic ultrasound-guided directional eFLOW in solid pancreatic lesions.
Ultrasound using microbubble-based contrast agents is useful for vascular imaging. Directional eFLOW (D-eFLOW) is a novel technology for vascular assessment that provides high spatial and temporal resolution. The purpose of this study was to investigate the utility of endoscopic ultrasound (EUS)-guided D-eFlow before and after administration of an ultrasound contrast agent (USCA) for assessing the vascularity of solid pancreatic lesions. D-eFlow was compared to power Doppler EUS (PD-EUS) or color Doppler EUS (CD-EUS) before and after USCA injection. We also evaluated the Visual Vascular Assessment (ViVA) scale for the estimation of vascularity and investigated its reliability using the interclass correlation coefficient (ICC). From January 2007 to March 2007, 35 patients (mean age, 64.5 years old; age range, 28-81 years) underwent EUS followed by D-eFLOW EUS, PD-EUS, and CD-EUS before and after administration of USCA. The pancreatic parenchymal ViVA score, pancreatic vascular pattern, and ICC were evaluated for all lesions. Concerning the sensitivity for detection of the hypovascular pattern in pancreatic adenocarcinoma, D-eFLOW (before and after USCA) had similar sensitivity to PD-EUS (before and after USCA) and CD-EUS (before and after USCA). D-eFLOW after contrast showed the highest accuracy (82.3 %) and negative predictive value (53.8 %) among all the modalities investigated. There was a good correlation among the ViVA scores for D-eFLOW before contrast, those for D-eFLOW EUS, and those for PD-EUS and CD-EUS. The reliability of the ViVA scale was excellent with an ICC of 0.81. In conclusion, D-eFLOW EUS is a sensitive, reliable, and highly accurate method of assessment of pancreatic vascularity. D-eFLOW EUS with contrast was more sensitive than PD-EUS and CD-EUS for assessment of pancreatic vascularity.


Neutralizing antibody responses in vaccinated and unvaccinated individuals infected with Omicron BA.1 variant.
The Omicron variant, with numerous mutations in the spike protein, reduces vaccine-induced immunity, leading to breakthrough infections. However, vaccine protection after infection with the Omicron variant is unclear. To compare the neutralizing antibody responses between unvaccinated and vaccinated individuals infected with the Omicron variant, we have collected serial plasma samples from five unvaccinated and four vaccinated individuals with Omicron variant infection, including the first Omicron breakthrough infection case in the Republic of Korea. We evaluated neutralization antibody titers against D614G, Delta, and Omicron using live virus neutralizing assay, and calculated the plaque reduction neutralizing test value. In patients with two-dose vaccinations, neutralizing antibodies against Omicron variant were detected in plasma collected 4-9 days post symptom onset. However, in the plasma from unvaccinated patients and those vaccinated with one dose, neutralizing antibodies against the Omicron variant at the same time point were undetectable. Next, the 1- or 2-dose vaccinated infected groups showed potent cross-neutralizing activity against D614G and Delta variants after 11-14 days. In contrast, the neutralizing antibody titers in the unvaccinated group were low or undetectable. The major limitation of this study is the small sample size due to the limited samples targeting the first reported cases of Omicron BA.1 variant infection in the Republic of Korea (n = 9). Nevertheless, we found that vaccinated individuals rapidly produced neutralizing antibodies against Omicron, and potent cross-neutralizing antibodies against D614G and Delta upon infection with Omicron.


Evidence for two K+ currents activated upon hyperpolarization of Paramecium tetraurelia.
Hyperpolarization of voltage-clamped Paramecium tetraurelia in K+ solutions elicits a complex of Ca2+ and K+ currents. The tail current that accompanies a return to holding potential (-40 mV) contains two K+ components. The tail current elicited by a step to -110 mV of greater than or equal to 50-msec duration contains fast-decaying (tau approximately 3.5 msec) and slow-decaying (tau approximately 20 msec) components. The reversal potential of both components shifts by 55-57 mV/10-fold change in external [K+], suggesting that they represent pure K+ currents. The dependence of the relative amplitudes of the two tail currents on duration of hyperpolarization suggests that the slow K+ current activates slowly and is sustained, whereas the fast current activates rapidly during hyperpolarization and then rapidly inactivates. Iontophoretic injection of a Ca2+ chelator, EGTA, specifically reduces slow tail-current amplitude without affecting the fast tail component. Both K+ currents are inhibited by extracellular TEA+ in a concentration-dependent, noncooperative manner, whereas the fast K+ current alone is inhibited by 0.7 mM quinidine.


Patient functional recovery after a 23-h surgery - a prospective, follow-up study.
We evaluated patients' functional outcomes 2 weeks after a 23-h surgery model in a tertiary care hospital. This prospective study comprised data on 993 consecutive adult patients who underwent a 23-h surgery. Patients were interviewed before surgery and at 14 days after surgery by telephone with a multidimensional structural survey including closed- and open-ended questions. Regarding functional outcomes, the patients were asked to assess their general wellbeing, energy levels and activities of daily living on a 5-point numeric rating scale (1 = poor to 5 = excellent). Data on patient characteristics, medical history, alcohol use, smoking status and pre-, peri- and postoperative pain and satisfaction with the care received were collected and analysed to determine whether these factors contributed to their recovery. The primary outcome measure was patient functional recovery at 14 days after surgery. Most patients reported moderate to excellent functional outcomes: 93.6% (95% CI, 92.1--95.1) of the patients showed a score ≥ 3 on the 5-point numeric scale. One out of four patients (23%) scored all three domains as excellent. A weak inverse correlation was noted between functional recovery and most pain in the 23-h postanaesthesia care unit as well as pain at 2 weeks after surgery. A weak positive correlation was noted between functional recovery and patient satisfaction with the instructions at discharge. Most patients showed ample functional recovery at 14 days after the 23-h surgery. Higher pain scores in the postanaesthesia care unit and 2 weeks after surgery predicted poor functional outcomes, and satisfaction with postoperative counselling predicted better outcomes. ClinicalTrials.gov NCT04142203.


Neurite outgrowth mediated by the heat shock protein Hsp90α: a novel target for the antipsychotic drug aripiprazole.
Aripiprazole is an atypical antipsychotic drug approved for the treatment of psychiatric disorders such as schizophrenia, bipolar disorder, major depressive disorder and autism. The drug shows partial agonistic activity at dopamine D(2) receptors and 5-hydroxytryptamine (5-HT) 5-HT(1A) receptors, and antagonistic activity at 5-HT(2A) receptors. However, the precise mechanistic pathways remain unclear. In this study, we examined the effects of aripiprazole on neurite outgrowth. Aripiprazole significantly potentiated nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells, in a concentration-dependent manner. The 5-HT(1A) receptor antagonist WAY-100635, but not the dopamine D(2) receptor antagonist sulpiride, blocked the effects of aripiprazole, although, only partially. Specific inhibitors of inositol 1,4,5-triphosphate (IP(3)) receptors and BAPTA-AM, a chelator of intracellular Ca(2+), blocked the effects of aripiprazole. Moreover, specific inhibitors of several common signaling pathways phospholipase C-γ (PLC-γ), phosphatidylinositol-3 kinase (PI3K), mammalian target of rapamycin, p38 MAPK, c-Jun N-terminal kinase, Akt, Ras, Raf, ERK, MAPK) also blocked the effects of aripiprazole. Using proteomic analysis, we found that aripiprazole significantly increased levels of the heat shock protein Hsp90α in cultured cells. The effects of aripiprazole on NGF-induced neurite outgrowth were significantly attenuated by treatment with Hsp90α RNA interference, but not by the negative control of Hsp90α. These findings suggest that both 5-HT(1A) receptor activation and Ca(2+) signaling via IP(3) receptors, as well as their downstream cellular signaling pathways play a role in the promotion of aripiprazole-induced neurite outgrowth. Furthermore, aripiprazole-induced increases in Hsp90α protein expression may form part of the therapeutic mechanism for this drug.


Porous graphitic carbon shows promise for the rapid chromatographic analysis of polar drug metabolites.
A rapid and simple HPLC method has been developed and used to separate the polar metabolic conjugates of AZT, chloramphenicol, and beta-estradiol based upon the use of porous graphitic carbon. The HPLC system is sufficiently selective to resolve the polar drug conjugates from their parent compounds and from endogenous material present in urine. The compounds are separated, without the need for sample pretreatment or gradient elution, on a porous graphitic carbon (Hypercarb) column using aqueous trifluoroacetic acid modified with tetrahydrofuran as the mobile phase. Porous graphitic carbon exhibits a novel mechanism of retention towards these very polar substances, which are unretained under reversed-phase conditions on alkyl-bonded silica phases.


[Calcium pros and cons: long term use of bisphosphonates. Bisphosphonates should be used for postmenopausal osteoporosis for a long term].
Only bisphosphonates have evidence that show to reduce the risk of vertebral, nonvertebral, and hip fractures in postmenopausal osteoporosis. From the viewpoint of risk benefit, it may be said that a utility is far superior to problems of the long-term use. Long-term use of bisphosphonates is necessary for the patients at elevated risk for the hip fracture.


Unusual multicentric angiomyolipoma of knee joint and soft tissue foot.
To report unusual occurrence of angiomyolipoma at intraarticular location with another lesion in the same side foot. A 12-year-old girl was referred to us after initial inconclusive work-up done elsewhere for swelling of left knee joint. There was a 15 × 12 cm swelling in the knee joint partially encasing patella while also a similar hourglass shaped swelling measuring 9 × 4 cm was noted in the same side foot. After clinical and radiological evaluation an excision biopsy was planned for both sites. The specimen sent for histopathological evaluation revealed angiomyolipoma with identical characteristics in the two locations and was HMB45 immunostain negative. Patient was evaluated for possibility of tuberous sclerosis but there was no contributory evidence. Angiomyolipoma is typically a solitary renal tumor with rare occurrence at musculoskeletal sites. Multicentric variety is still infrequent. Intraarticular occurrence of angiomyolipoma presents a diagnostic challenge not only in terms of unfamiliarity but also unusual presentation with absence of characteristic immunostaining and multicentricity requiring careful exclusion of other lesions that may require a more radical approach for treatment.


Compositional aspects of lipid hydration.
The effect of various lipids such as cerebrosides, gangliosides and dipalmitoyl lecithin (DPPC) on the lowering of the melting temperature of water, was determined by differential scanning calorimetry (DSC). The lowering of the melting temperature, and the number of water molecules per lipid molecule which apparently do not undergo melting, increase with the increase of the size and charge of the polar group and with the unsaturation of the hydrocarbon chains. Freezing curves show supercooling to about -20 degrees C. It was found that the number of apparently unfreezable water molecules is about four for glucocerebroside from Gaucher's spleen and about eight or nine for galactocerebroside from bovine brain. In gangliosides from bovine brain the following number of water molecules/lipid molecule are apparently unfreezable: 22-30 in GM1, 33-40 in GD1a + GD1b while a fraction of gangliosides containing 75% GQ1b and 25% GT1b affects up to 60 molecules of water/molecule of lipid. A zwitterionic DPPC molecule removes apparently six to seven water molecules from freezing. There is no indication that the apparently unfreezable water molecules are in a distinct state. It is suggested that they freeze at very low temperatures producing a flat tail preceding the transition peak which cannot be discerned from the base line.


Mammary homeostasis in the mother-offspring dyad.
Homeostasis during lactation is a special case in which the unit for regulation is a dyad comprising the mother and her currently nursing offspring (the mother-offspring dyad). This arrangement is not a trivial. A litter of mice can have a mass greater than the mother and nutrient demands that far exceed her. Homeostasis for milk secretion, appetite, and calcium metabolism must come under integrated control, responding seamlessly to the needs of the mother and the offspring. Serotonin (5-HT) is a primary local regulator of mammary homeostasis. 5-HT synthesis in the mammary epithelium is high during lactation and increases during milk stasis. Two important functions are attributed to the 5-HT system. Firstly, when alveolar spaces are filled with milk 5-HT inhibits milk secretion and opens tight junctions. This feedback induces early phases of involution. Secondly, 5-HT induces synthesis and secretion of parathyroid hormone-related peptide (PTHrP).


Loss of Adenylyl Cyclase 6 in Leptin Receptor-Expressing Stromal Cells Attenuates Loading-Induced Endosteal Bone Formation.
Bone marrow stromal/stem cells represent a quiescent cell population that replenish the osteoblast bone-forming cell pool with age and in response to injury, maintaining bone mass and repair. A potent mediator of stromal/stem cell differentiation in vitro and bone formation in vivo is physical loading, yet it still remains unclear whether loading-induced bone formation requires the osteogenic differentiation of these resident stromal/stem cells. Therefore, in this study, we utilized the leptin receptor (LepR) to identify and trace the contribution of bone marrow stromal cells to mechanoadaptation of bone in vivo. Twelve-week-old Lepr-cre;tdTomato mice were subjected to compressive tibia loading with an 11 N peak load for 40 cycles, every other day for 2 weeks. Histological analysis revealed that Lepr-cre;tdTomato+ cells arise perinatally around blood vessels and populate bone surfaces as lining cells or osteoblasts before a percentage undergo osteocytogenesis. Lepr-cre;tdTomato+ stromal cells within the marrow increase in abundance with age, but not following the application of tibial compressive loading. Mechanical loading induces an increase in bone mass and bone formation parameters, yet does not evoke an increase in Lepr-cre;tdTomato+ osteoblasts or osteocytes. To investigate whether adenylyl cyclase-6 (AC6) in LepR cells contributes to this mechanoadaptive response, Lepr-cre;tdTomato mice were further crossed with AC6 fl/fl mice to generate a LepR+ cell-specific knockout of AC6. These Lepr-cre;tdTomato;AC6 fl/fl animals have an attenuated response to compressive tibia loading, characterized by a deficient load-induced osteogenic response on the endosteal bone surface. This, therefore, shows that Lepr-cre;tdTomato+ cells contribute to short-term bone mechanoadaptation. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.


Serum Trough Concentration and Effects of Mycophenolate Mofetil Based on Pathologic Findings in Infants After Liver Transplantation.
Mycophenolate mofetil (MMF) is mainly used in conjunction with calcineurin inhibitors as an additional immunosuppressive for renal sparing after liver transplantation. However, few reports about MMF use in infants after living donor liver transplantation (LDLT) are available. The purpose of this study was to examine the efficacy and safety of MMF in infants. This study enrolled infants younger than 1 year of age who received LDLT at our institution. Patients received oral MMF twice daily. The initial dose was 40 to 50 mg/kg/d, which was increased to a target mycophenolic acid (MPA) trough level of 2 mg/L. Body weight, height, MMF dose, MPA trough level, acute cellular rejection (ACR) episodes, pathologic findings, and adverse effects were analyzed. Allograft fibrosis was graded using the Meta-analysis of Histological Data in Viral Hepatitis score. Patients received MMF for refractory ACR (n = 2), fulminant hepatitis (n = 2), and pre-existing antibodies (n = 1). Original diseases were biliary atresia (n = 3) and fulminant hepatitis (n = 2). Mean age at transplant was 8 months (range 3-10 months). The last available mean trough level was 2.7 mg/L. The mean dose was 66 mg/kg/d or 1429 mg/m2/d at the time of the last available through level. The regression line for MMF dose and MPA trough level was y = 1.8 × 10-3x. The correlation coefficient was 0.65. All allografts showed F1 to F2 fibrosis. Two patients discontinued MMF because of infection and bone marrow suppression, respectively. Two patients converted to everolimus. One patient continued on MMF. After LDLT, infants require a higher MMF dose than older patients based on trough levels, but allograft fibrosis can progress.


Retinoid reversal of squamous metaplasia in organ cultures of tracheas derived from hamsters fed on vitamin A-deficient diet.
Cytokinetic and ultrastructural studies were carried out to elucidate mechanisms involved in the reversal of squamous metaplasia (SM) by beta-retinoic acid in organ cultures of tracheas derived from vitamin A-deficient hamsters. Tracheal cultures exhibiting focal areas of SM were treated with the retinoid for up to 7 days. The retinoid significantly inhibited [3H]-thymidine labeling indices in the basal cells and stimulated the labeling indices in mucous cells. At the ultrastructural level the retinoid induced marked remodeling alterations in the metaplastic epithelium that included: (a) disruption of desmosomes and widening of intercellular spaces; (b) extensive vacuolation and degeneration of the metaplastic cells; (c) extrusion of the degenerated cells; (d) aggregation of keratin filaments; and (e) differentiation of certain basal cells into secretory cells. Consequently most degenerated metaplastic cells were extruded and the epithelium repopulated as a result of differentiation of basal cells into mucous cells and hyperplasia of the pre-existing mucous cells. The degenerative effects of the retinoid were limited to the metaplastic foci since the uninvolved epithelium adjoining metaplastic foci were not significantly altered. The results suggest that the restoration of normal tracheal epithelium following the retinoid treatment of explants exhibiting focal areas of squamous metaplasia is associated with the enhanced proliferation of the mucous cells. The inhibition of proliferation of basal cells further prevented hyperplasia and restored cell replication within the normal range.


Photosynthetic accumulation of carbon storage compounds under CO₂ enrichment by the thermophilic cyanobacterium Thermosynechococcus elongatus.
The growth characteristics of Thermosynechococcus elongatus on elevated CO₂ were studied in a photobioreactor. Cultures were able to grow on up to 20% CO₂. The maximum productivity and CO₂ fixation rates were 0.09 ± 0.01 and 0.17 ± 0.01 mg ml⁻¹ day⁻¹, respectively, for cultures grown on 20% CO₂. Three major carbon pools--lipids, polyhydroxybutyrates (PHBs), and glycogen--were measured. These carbon stores accounted for 50% of the total biomass carbon in cultures grown on atmospheric CO₂ (no supplemental CO₂), but only accounted for 30% of the total biomass carbon in cultures grown on 5-20% CO₂. Lipid content was approximately 20% (w/w) under all experimental conditions, while PHB content reached 14.5% (w/w) in cultures grown on atmospheric CO₂ and decreased to approximately 2.0% (w/w) at 5-20% CO₂. Glycogen levels did not vary significantly and remained about 1.4% (w/w) under all test conditions. The maximum amount of CO₂ sequestered over the course of the nine-day chemostat experiment was 1.15 g l⁻¹ in cultures grown on 20% CO₂.


A sphingosine-dependent protein kinase that specifically phosphorylates 14-3-3 (SDK1) is identified as the kinase domain of PKCdelta: a preliminary note.
A specific protein kinase that phosphorylates Ser60, Ser59, or Ser58 of 14-3-3beta, eta, or zeta, respectively, only in the presence of sphingosine (Sph) or N,N-dimethyl-Sph (DMS), was termed "sphingosine-dependent protein kinase-1" (SDK1) [J. Biol. Chem. 273(34) (1998) 21834]. We have now identified SDK1 as a protein having the same amino acid sequence as in the C-terminal-half kinase domain of PKCdelta, with approximately 40 kDa molecular mass, based on large-scale purification of a protein from rat liver, and partial sequence using three different combinations of LC-MS or LC-MS/MS with respective search engine. PKCdelta did not display any SDK1 activity and PKCdelta activity was inhibited by Sph and DMS. However, strong SDK1 activity, only in the presence of Sph or DMS, became detectable when PKCdelta was incubated with caspase-3, which releases the approximately 40 kDa kinase domain.


Tolerability of high cumulative doses of the HFA modulite beclomethasone dipropionate/formoterol combination inhaler in asthmatic patients.
The corticosteroid beclomethasone dipropionate (BDP) has been formulated with the long acting beta agonist formoterol (BDP/formoterol 100 microg/6 microg, Foster) in a single inhaler using Modulite technology. We have investigated the acute tolerability of high, cumulative doses of BDP/formoterol compared to formoterol alone and placebo. This was a double blind, 3-way cross-over comparison of 10 puffs of BDP/formoterol 100 microg/6 microg or formoterol 6 microg or placebo during maintenance treatment with BDP/formoterol two puffs per day. Pharmacokinetics over 12h during maintenance treatment was measured on day 7. High cumulative doses were then administered on three separated days. Eighteen patients with asthma were recruited (mean FEV(1) 65% predicted). The primary endpoint was serum potassium over the 12h period after high doses. QTc, blood pressure and heart rate over 12h, and plasma lactate and glucose over 3h following dosing were assessed. Formoterol caused a significantly greater decrease in serum potassium than BDP/formoterol or placebo (difference in mean minimum concentrations; 0.11 and -0.15 mmol/l, respectively, p<0.05 for both comparisons). No significant differences in serum potassium parameters were found between BDP/formoterol and placebo. QTc, plasma lactate and vital signs values observed with the combination were not statistically different from those with formoterol alone. For glucose, the mean maximum increase after formoterol treatment was 0.4 mmol/l (p<0.01 compared to placebo), while BDP/formoterol treatment caused a maximum increase of 0.7 mmol/l (p<0.01 compared to formoterol and placebo). The active metabolite of BDP is beclomethasone-17-monopropriate (B17MP), which reached Cmax at 0.25 h, with an elimination half-life of 3.7 h. Formoterol also reached Cmax at 0.25 h, and concentrations were measurable up to 12 h. High doses of BDP/formoterol did not significantly reduce serum potassium, while formoterol alone did to a greater extent. The BDP/formoterol combination was well tolerated, and exhibited a safety profile generally similar to formoterol alone when administered in high doses to stable asthmatic patients.


[Infection of the prostate by herpesviruses and HPV in patients with prostate cancer].
The exploration of the presence of the HCMV, EBV, HHV8 and HPV-infection in the prostatic tissue of the prostate cancer patients was conducted. The methods of PCR and in situ hybridization (ISH) were used and the results were compared. Authors revealed high degree of virus infection in prostate. Using of PCR and ISH resulted in following frequencies of the viruses in prostate tissue: HCMV-87 and 77%, EBV-53 and 54%, HHV8--0 and 50%, HPV--0 and 23%. The question of the nature of discrepancies is discussed.


Management of Slipped Capital Femoral Epiphysis (SCFE) on Top of Fixed Fracture Neck of Femur (Case Report).
The incidence of Slipped capital femoral epiphysis (SCFE) after management of femoral neck fracture in pediatrics is very rare. In this case report, a nine-year-old female child sustained left sided femur neck fracture after a motor car accident. The fracture was fixed by two cannulated screws and healing with mild varus occurred after six weeks. However, progressive slippage of femoral epiphysis was observed. This was treated by subtrochanteric valgus osteotomy and fixed by tension band and Wagner technique. Better radiological and functional outcomes were obtained at the short term follow up.


Genetic determinism of bone and mineral metabolism in meat-type chickens: A QTL mapping study.
Skeletal integrity in meat-type chickens is affected by many factors including rapid growth rate, nutrition and genetics. To investigate the genetic basis of bone and mineral metabolism, a QTL detection study was conducted in an intercross between two lines of meat-type chickens divergently selected for their high (D +) or low (D -) digestive efficiency. Tibia size (length, diameter, volume) and ash content were determined at 3 weeks of age as well as phosphorus (P) retention and plasma concentration. Heritability of these traits and their genetic correlations with digestive efficiency were estimated. A QTL mapping study was performed using 3379 SNP markers. Tibia size, weight, ash content and breaking strength were highly heritable (0.42 to 0.61). Relative tibia diameter and volume as well as P retention were strongly and positively genetically correlated with digestive efficiency (0.57 to 0.80). A total of 35 QTL were identified (9 for tibia weight, 13 for tibia size, 5 for bone strength, 5 for bone mineralization, 2 for plasma P concentration and 1 for P retention). Six QTL were genome-wide significant, and 3 QTL for tibia relative volume, weight and ash weight on chromosome 6 were fixed, the positive allele coming from the D-line. For two QTL for ash content on chromosome 18 and relative tibia length on chromosome 26, the confidence intervals were small enough to identify potential candidate genes. These findings support the evidence of multiple genetic loci controlling bone and mineral metabolism. The identification of candidate genes may provide new perspectives in the understanding of bone regulation, even beyond avian species.


Organophosphorus insecticides extraction and heterogeneous oxidation on column for analysis with an acetylcholinesterase (AChE) biosensor.
This paper presents an analysis method for organophosphorus insecticides based on AChE biosensors coupled with a preconcentration and oxidation on a solid phase column. Three organic solvents, acetonitrile (ACN), ethanol and methanol were tested for their influence on AChE activity, insecticide inhibition and their ability to elute the adsorbed insecticides. Our results showed that ACN in a concentration of 5% (v/v) had the less negative effect on biosensor analysis and was the most appropriate organic solvent for the column elution. The presence of the organic solvent in the incubation media of the biosensor was found to induce a reduction of the inhibition percentages. The inhibition of the biosensors was performed in phosphate buffer with 5% (v/v) ACN, while the initial and remaining response of the biosensors were measured in PBS. In these conditions, the LODs of paraoxon and dichlorvos were measured with or without a preconcentration step. The LODs of the AChE biosensor without sample preconcentration were 8 x 10(-8) M for paraoxon and 1 x 10(-7) M dichlorvos and the LOD obtained after the preconcentration step were 2.5 x 10(-8) M for paraoxon and 2.5 x 10(-8) M for dichlorvos. Moreover, the use of the column allowed the heterogeneous oxidation of organophosphorus insecticides for improved LOD.


Transition of average drug-to-antibody ratio of trastuzumab deruxtecan in systemic circulation in monkeys using a hybrid affinity capture liquid chromatography-tandem mass spectrometry.
Trastuzumab deruxtecan (T-DXd, DS-8201a) is an antibody-drug conjugate, comprising an anti-HER2 antibody at a drug-to-antibody ratio of 7-8 with the topoisomerase I inhibitor DXd. In this study, the concentrations of antibody-conjugated DXd and total antibody were determined and observed to decrease over time following intravenous administration of T-DXd to monkeys. The drug-to-antibody ratio of T-DXd also decreased in a time-dependent manner, which reached approximately 2.5 in 21 days after administration. It was suggested that antibody-conjugated DXd of T-DXd was relatively stable in vivo compared with that of other reported antibody-drug conjugates.


Radical and non-radical cooperative degradation in metal-free electro-Fenton based on nitrogen self-doped biochar.
To achieve sustainable metal-free electron-Fenton, N self-doped biochar air-cathode (BCAC) was prepared by pyrolyzing coffee residues. During the pyrolysis process, the endogenous N transformed from edge-doping to graphite-doping. Particularly, N vacancies started to evolve when the peak temperature exceeded 700 °C. A high Tetracycline removal rate of 70.42% was obtained on the BCAC at the current density of 4 mA cm-2. Quenching tests incorporated with ESR spectroscopy were adopted to identify the specific oxidants produced on the cathode. The results showed that •OH (37.36%), •O2- (29.67%) and 1O2 (24.17%) played comparable role in the tetracycline removal, suggesting the coexist of radical and non-radical oxidants in our electro-Fenton system. According to the structure characterization and the DFT calculation, graphitic N was suggested as the critical site for H2O2 generation, and both graphitic N and pyridinic N were electroactive sites for H2O2 activation to •OH. Graphitic N and N vacancies with stronger capabilities in O2 adsorption and electron-trapping were proposed as the electroactive sites for 1O2 and •O2- formation. This work predicts a novel electro-Fenton process with cooperative radical and non-radical degradation on N self-doped carbonaceous catalysts at a mild condition, which is extremely meaningful for boosting sustainable electro-Fenton technology.


In situ electrochemical reductive construction of metal oxide/metal-organic framework heterojunction nanoarrays for hydrogen peroxide sensing.
Transition metal oxide/metal-organic framework heterojunctions (TMO@MOF) that combine the large specific surface area of MOFs with TMOs' high catalytic activity and multifunctionality, show excellent performances in various catalytic reactions. Nevertheless, the present preparation approaches of TMO@MOF heterojunctions are too complex to control, stimulating interests in developing simple and highly controllable methods for preparing such heterojunction. In this study, we propose an in situ electrochemical reduction approach to fabricating Cu2O nanoparticle (NP)@CuHHTP heterojunction nanoarrays with a graphene-like conductive MOF CuHHTP (HHTP is 2,3,6,7,10,11-hexahydroxytriphenylene). We have discovered that size-controlled Cu2O nanoparticles could be in situ grown on CuHHTP by applying different electrochemical reduction potentials. Also, the obtained Cu2O NP@CuHHTP heterojunction nanoarrays show high H2O2 sensitivity of 8150.6 μA·mM-1·cm2 and satisfactory detection performances in application of measuring H2O2 concentrations in urine and serum samples. This study offers promising guidance for the synthesis of MOF-based heterojunctions for early cancer diagnosis.


Size-dependent sex allocation in hermaphroditic plants: the effects of resource pool and self-incompatibility.
The effects of the resource pool and resource obtained during a season for seed maturation and self-incompatibility on the size-dependency of evolutionarily stable sex allocation were analysed theoretically. In hermaphroditic plants, reproductive resources allocated between male and female function may not be paid from a single resource pool, because plants can mature seeds using not only reserved resources but also newly gained resources after flowering. But the resource investment to male function is limited to the flowering stage. Under the assumption of constant reserve efficiency and diminishing resource return per investment to leaves, large plants should use both reserved and newly gained resources for seed maturation, while small plants should use only new resources. When both reserved and new resources are used, the optimal allocation for self-compatible species is to invest a constant amount of resources into male function irrespective of resource size, because the female fitness curve increases linearly and the male curve decelerates due to local mate competition. In self-incompatible species, on the other hand, fitness gain per investment through male function and the optimal amount of resources invested in male function decrease with size. Thus a decrease in maleness with size should be emphasized more in self-incompatible species than in self-compatible one. When only new resources are used for seed growth, the female fitness curve as well as male one decelerates with investment. Consequently, the investment in both male and female functions should increase with size, in both self-compatible and self-incompatible species. The magnitude of reserve efficiency relative to efficiency of resource gain after flowering affects size-dependent pattern of sex allocation, while the cost of seed maturation relative to ovule production has little effect on it. The plant size variation in a population emphasizes size-dependency of sex allocation. When size variation is large enough, it is possible that large plants become complete female in self-incompatible species, but it is not in self-compatible species.


Risk factors for the development of retroprosthetic membranes with Boston keratoprosthesis type 1: multicenter study results.
The purpose of this study was to identify possible risk factors for retroprosthetic membrane (RPM) development in a large, multicenter cohort of patients receiving a Boston type 1 keratoprosthesis. Cohort study. The final analysis included 265 eyes of 265 patients who underwent implantation of a Boston keratoprosthesis type I device between January 2003 and July 2008 by 1 of 19 surgeons at 18 medical centers. Forms reporting preoperative, intraoperative, and postoperative parameters were prospectively collected and subsequently analyzed at a central data collection site. The primary outcome was the presence or absence of an RPM during the follow-up period. The average age of patients was 63.3±19.1 years, 48.5% of the patients were female, and 52.5% of procedures were performed on the right eye. The mean follow-up time was 17.8±14.9 months. The majority (85.4%; n = 222) had undergone an average of 2.2±1.2 (range, 1-8) penetrating keratoplasties before keratoprosthesis implantation, and 38 eyes (14.6%) received a primary keratoprosthesis. The overall RPM formation rate was 31.7% (n = 84). The most significant risk factor for RPM development was infectious keratitis (as a surgical indication for keratoprosthesis surgery itself), resulting in a rate of RPM formation of 70.6%. As an independent risk factor, the hazard ratio (HR) of RPM development in these eyes was 3.20 (95% confidence interval, 1.66-6.17). Aniridia was also an independent risk factor for RPM development (HR, 3.13; 95% confidence interval, 1.10-8.89). Formation of RPM is a common complication of keratoprosthesis surgery, occurring in approximately one-third of cases. Eyes at the highest risk of RPM development are those receiving corneal replacement for infectious keratitis and aniridia.


Structure and Conformation of Individual Molecules upon Adsorption of a Mixture of Benzoporphyrins on Ag(111), Cu(111), and Cu(110) Surfaces.
We investigated the adsorption behavior of a mixture of six 2H-tetrakis-(3, 5-di-tert-butylphenyl)(x)benzoporphyrins (2H-diTTBP(x)BPs, x=0, 1, 2-cis, 2-trans, 3, and 4) on Ag(111), Cu(111) and Cu(110) at room temperature by scanning tunneling microscopy (STM) under ultra-high vacuum conditions. On Ag(111), we observe an ordered two-dimensional square phase, which is stable up to 400 K. On Cu(111), the same square phase coexists with a stripe phase, which disappears at 400 K. In contrast, on Cu(110), 2H-diTTBP(x)BPs adsorb as immobile isolated molecules or dispersed short chains along the [1 1 ‾ ${\bar{1}}$ 0] substrate direction, which remain intact up to 450 K. The stabilization of the 2D supramolecular structures on Ag(111) and Cu(111), and of the 1D short chains on Cu(110) is attributed to van der Waals interactions between the tert-butyl and phenyl groups of neighboring molecules. From high-resolution STM, we can assign all six 2H-diTTBP(x)BPs within the ordered structures. Moreover, we deduce a crown shape quadratic conformation on Ag(111) and Cu(111), an additional saddle-shape on Cu(111), and an inverted structure and a quadratic appearance on Cu(110). The different conformations are attributed to the different degree of interaction of the iminic nitrogen atoms of the isoindole and pyrrole groups with the substrate atoms.


The Roles of Type I Interferon in Bacterial Infection.
Type I interferons (IFNs) are pleiotropic cytokines well recognized for their role in the induction of a potent antiviral gene program essential for host defense against viruses. They also modulate innate and adaptive immune responses. However, the role of type I IFNs in host defense against bacterial infections is enigmatic. Depending on the bacterium, they exert seemingly opposite and capricious functions. In this review, we summarize the effect of type I IFNs on specific bacterial infections and highlight the effector mechanisms regulated by type I IFNs in an attempt to elucidate new avenues to understanding their role.


Layer-by-layer DNA films incorporating highly transfecting bioreducible poly(amido amine) and polyethylenimine for sequential gene delivery.
The layer-by-layer (LbL) assembly method offers a molecular level control of the amount and spatial distribution of bioactive molecules. However, successful clinical translation of LbL film technology will most certainly require a better understanding and control of not only the film assembly process, but also film disassembly kinetics in physiologic conditions. This work focuses on the understanding and control of degradation properties of LbL films for localized gene delivery. Bioreducible poly(amido amine)s (PAAs) containing cystaminebisacrylamide (CBA), methylenebisacrylamide, and 5-amino-1-pentanol (APOL) were synthesized by Michael addition polymerization for the construction of bioreducible LbL films capable of sequential gene delivery. The synthesized PAAs were screened for desirable buffering capacity, cell transfection, and cytotoxicity characteristics together with 25 kDa branched polyethylenimine (PEI) and cross-linked 800 Da PEI. By screening the various polycations we were able to identify a copolymer of CBA and APOL for the subsequent construction of the LbL films. By incorporating a highly transfecting polycation and a nondiffusing polycation we were able to improve the overall transfection of HEK293 and MC3T3 cells from the bioreducible LbL films. We also demonstrated the dual-stage release and transfection of two different DNAs from the LbL films. The results indicate that LbL films consisting of bioreducible PAAs and non-diffusing polyelectrolytes have excellent degradation properties for the development of LbL coating technology for localized gene delivery applications.


A rare case of clear cell sarcoma with 4 types of EWSR1-ATF1 fusions detected not in primary site but in metastatic site.
Clear cell sarcoma is a unique tumor which has EWSR1-ATF1 or EWSR1-CREB1 fusion. Several patterns of EWSR1-ATF1 fusion are observed in clear cell sarcoma. Since type 5-7 fusions were reported recently, they are classified as type 1-7. We examined EWSR1-ATF1 and EWSR1-CREB1 fusions in a single case of clear cell sarcoma with lung metastasis in a 36-year-old Japanese man. As a result, we found only type 1 EWSR1-ATF1 fusion in the primary site, but 4 types of EWS-ATF1 fusion (type 1, 2, 5, 6) were detected in the metastatic site. These 4 types of fusion were completely identical to the recent report, but the case had the same fusion patterns in both primary and metastatic sites. In our case, increased splicing activity in the EWSR1-ATF1 fusion might be acquired at the metastatic site. There is another possibility that metastasis might develop through the increased splicing activity in the fusion.


Genetic architecture of the inflammatory bowel diseases across East Asian and European ancestries.
Inflammatory bowel diseases (IBDs) are chronic disorders of the gastrointestinal tract with the following two subtypes: Crohn's disease (CD) and ulcerative colitis (UC). To date, most IBD genetic associations were derived from individuals of European (EUR) ancestries. Here we report the largest IBD study of individuals of East Asian (EAS) ancestries, including 14,393 cases and 15,456 controls. We found 80 IBD loci in EAS alone and 320 when meta-analyzed with ~370,000 EUR individuals (~30,000 cases), among which 81 are new. EAS-enriched coding variants implicate many new IBD genes, including ADAP1 and GIT2. Although IBD genetic effects are generally consistent across ancestries, genetics underlying CD appears more ancestry dependent than UC, driven by allele frequency (NOD2) and effect (TNFSF15). We extended the IBD polygenic risk score (PRS) by incorporating both ancestries, greatly improving its accuracy and highlighting the importance of diversity for the equitable deployment of PRS.


Evaluation of the Expression Level and Hormone Receptor Association of miR-126 in Breast Cancer.
Breast cancer is a major cause of cancer-related death in women worldwide. miRNAs are new players of breast tumorigenesis, used as diagnostic and prognostic biomarkers. Among various miRNAs, miR-126 has been proposed to have a tumor suppressive role in HER2 positive cancer. However, to have a better understanding of its role, further validation is required. The aim of this study was evaluating miR-126 expression level in breast cancer tissues and investigating its potential association with HER2, estrogen and progesterone receptors. miR-126 expression level was measured in 108 specimens including 78 malignant and 30 normal samples using RT-qPCR. The outcome was statistically analyzed. In silico studies were performed to find the potential mechanism of action, through which miR-126 imposes its function. Down-regulation of miR-126 was observed in tumor samples, as compared to the matched normal tissues. Down-regulation of miR-126 was also associated significantly with the absence of estrogen receptor in malignant samples. No association between miR-126 expression and HER2 status was observed. Our in silico analyses showed the possible role of Crk, PI3K and Ras proto-oncogenes in breast cancer tumorigenesis. miR-126 is significantly down-regulated in breast cancer tissues. Statistically, it showed no correlation with HER2 positivity. However, the association between lower miR-126 and estrogen receptor negativity was observed.


Modeling the intrathymic pathogenesis of myasthenia gravis.
Myasthenia gravis is (MG) a prototypic autoimmune disease; the immune effector mechanisms and autoantigenic target have been delineated. However, the events that lead to the abrogation of self-tolerance to neuromuscular acetylcholine receptors (nAChRs) remain a mystery. The thymus gland has long been considered to hold the key to solving this mystery, although the nature of its involvement remains to be elucidated. The nAChR was one of the first self-proteins associated with a defined autoimmune disease that was found to be expressed on thymic stromal populations. The studies described herein represent our efforts to determine how this "promiscuous" autoantigen expression may be involved in the immunopathogenesis of MG. We review our work, characterizing the expression of the nAChR alpha subunit in the thymus, and advance a hypothesis and experimental model, which explore how intrathymic expression of this autoantigen may contribute to the immunopathogenesis of this autoimmune disease.


Attempted transmission of Brucella ovis between red deer stags by successive grazing or adjacent-paddock grazing.
To determine whether Brucella ovis can be transmitted from stag to stag by successive grazing of infected and noninfected stags in the same paddock, or by grazing infected and non-infected stags in adjacent paddocks. Six red deer stags (Cervus elaphus) were artificially infected with B. ovis and 5 were confirmed to be shedding the organism in their semen. Infected stags alternated paddocks, and therefore grazing and wallows (successive grazing), once or twice weekly with 6 non-infected stags from 3 March to 18 August, 1999. Direct contact between the 2 groups of animals was prevented. The 2 groups alternated paddocks 32 times. Six other non-infected stags were grazed in a paddock adjacent to the infected stags throughout this period, separated by a standard deer fence. Non-infected stags were blood sampled at 2to 6-week intervals to test for B. ovis antibodies using a complement fixation test and an enzyme linked immunosorbent assay. No stag from either non-infected group became infected with B. ovis. The risk of stags becoming infected with B. ovis by successive grazing of the same paddock as, or by grazing in paddocks adjacent to, infected stags appears to be low. We conclude from this result, and similar experimental evidence and experience of this disease in sheep, that transmission of B. ovis requires animals to be grazed or confined together in a way that allows direct contact between animals. It is likely that infected and non-infected stags can be managed on the same property without transmission occurring between the groups, provided that they do not come into direct contact with one another.


A novel modified method for the efficient removal of Pb and Cd from wastewater by biochar: Enhanced the ion exchange and precipitation capacity.
The purpose of this research was to improve the sorption ability of Pb and Cd by promoting the ion exchange and precipitation capacity of biochar. The adsorption performance and mechanisms of Pb and Cd in wastewater using coconut shell biochar modified with magnesium were investigated. After modification, the total adsorption capacity (Qt) of Pb and Cd on Mg-coated biochar (MgBC400) increased by 20 and 30 times compared with the unmodified biochar (BC400), respectively. The removal of Pb and Cd to biochar was attributed to ion exchange (Qe), mineral precipitation (Qp), interaction with oxygen functional groups (OFGs) [(Qf)], and metal-π electron coordination (Qπ). Compared with the BC400, the adsorption capacity of the four fractions of MgBC400 increased especially the ion exchange and precipitation. The Qe values of MgBC400 were almost 49 and 59 times that of BC400 in the adsorption of Pb and Cd, respectively. The Qp values of MgBC400 increased by 214.4 and 81.7 mg/g, respectively. Ion exchange and mineral precipitation dominated the adsorption of Pb and Cd by MgBC400.


Assessment of diagnostic reference levels for paediatric cardiac computed tomography in accordance with European guidelines.
Recently, paediatric cardiac computed tomography (CCT) has caused concerns that diagnostic image quality and dose reduction may require further improvement. Consequently, this study aimed to establish institutional (local) diagnostic reference levels (LDRLs) for CCT for paediatric patients, and assess the impact of tube voltage on proposed DRLs in terms of the volume computed tomography index (CTDIvol) and dose length product (DLP). In addition, effective doses (EDs) of exposure were estimated. A population of 453 infants, whose mass and age were less than 12 kg and 2 years, respectively, were considered from January 2018 to August 2021. Based on previous studies, this number of patients was considered to be sufficient for establishing LDRLs. A group of 245 patients underwent CCT examinations at 70 kVp tube voltage with an average scan range of 23.4 cm. Another set of 208 patients underwent CCT examinations at 100 kVp tube voltage with an average scan range of 15.8 cm. The observed CTDIvol and DLP values were 2.8 mGy and 54.8 mGy.cm, respectively. The mean effective dose (ED) was 1.2 mSv. It is concluded that provisional establishment and use of DRLs for cardiac computed tomography in children are crucial, and further research is needed to develop regional and international DRLs.


Inhibition of neurite outgrowth by reduced level of NDRG4 protein in antisense transfected PC12 cells.
NDRG4, a member of the new NDRG gene family, was originally cloned as a gene that was expressed predominantly in the early postnatal rat brain. To determine whether the NDRG4 protein contributes to differentiation of neural cells, the effect of lowering the cellular NDRG4 protein level on the nerve growth factor (NGF)-induced neurite formations and transcription factor activations in PC12 cells was examined. An antisense construct of rat NDRG4 cDNA was made and transfected to PC12 cells, which constitutively express a basal level of the NDRG4 protein. Of the stably transfected antisense cell clones that expressed exogenous NDRG4 antisense RNA, six clones showed reduced levels of the NDRG4 protein, but unexpectedly two clones showed quite higher levels of NDRG4 protein than the control cells. The clones having decreased levels of the NDRG4 protein extended shorter neurites than control cells in response to NGF or dibutyryl cAMP. In contrast, the NDRG4 protein-highly expressing clones did not show suppressed neurite outgrowth induced by NGF. NGF-mediated activation of the transcription factor AP-1 was found to be suppressed in the NDRG4 protein-diminished clone and enhanced in the NDRG4 protein-upregulated clone as compared with those in the control cells. These results suggest that NDRG4 plays a role in neurite outgrowth and has an influence on an NGF-stimulated AP-1 activation by an undefined mechanism in PC12 cells.


A new method using simple solid phase extraction for the rapid gas-chromatographic mass-spectrometric determination of 11-dehydro-thromboxane B2 in urine.
A new gas-chromatographic mass-spectrometric method for the rapid determination of 11-dehydro-thromboxane B2 in urine, the major metabolite of systemic thromboxane formation, has been developed. Excellent sample clean-up was obtained in a single step by adsorption of 11-dehydro-TXB2 on phenylboronate cartridges, vigorous polar and organic washing and elution with an acidic methanol mixture. Then the pentafluorobenzylester trimethylsilylether derivative of 11-dehydro-TXB2 was formed and quantified in isotope dilution technique by negative chemical ionisation gas-chromatography tandem mass-spectrometry. The daughter fragments m/z 243/247 of the parent ion m/z 511/515 were monitored. Recovery was linear and quantitative over a wide range, accuracy was 95 + 7% and precision was 11% down to the very low pg range in biologic samples. Independent validation of this very fast extraction method with a reference method applying extensive sample purification with consecutive reversed and straight phase extraction, precolumn derivatisation, reversed phase high pressure liquid chromatography and tandem gas-chromatography mass-spectrometry gave excellent agreement of values. Values for 11-dehydro-TXB2 excretion in 8 healthy controls were 501 + 298 (range 231 to 1141) ng/g creatinin. Excretion was suppressed by aspirin, moderately elevated in heavy smokers (range 680 to 1540) and increased in patients with venous thrombosis or pulmonary embolism (2370 to 13350 ng/g creatinin). This rapid extraction method is useful for the highly specific and sensitive determination of 11-dehydro-TXB2 in large sample numbers.


Methyltransferase-like proteins in cancer biology and potential therapeutic targeting.
RNA modification has recently become a significant process of gene regulation, and the methyltransferase-like (METTL) family of proteins plays a critical role in RNA modification, methylating various types of RNAs, including mRNA, tRNA, microRNA, rRNA, and mitochondrial RNAs. METTL proteins consist of a unique seven-beta-strand domain, which binds to the methyl donor SAM to catalyze methyl transfer. The most typical family member METTL3/METTL14 forms a methyltransferase complex involved in N6-methyladenosine (m6A) modification of RNA, regulating tumor proliferation, metastasis and invasion, immunotherapy resistance, and metabolic reprogramming of tumor cells. METTL1, METTL4, METTL5, and METTL16 have also been recently identified to have some regulatory ability in tumorigenesis, and the rest of the METTL family members rely on their methyltransferase activity for methylation of different nucleotides, proteins, and small molecules, which regulate translation and affect processes such as cell differentiation and development. Herein, we summarize the literature on METTLs in the last three years to elucidate their roles in human cancers and provide a theoretical basis for their future use as potential therapeutic targets.


Influence of culture conditions on lipopeptide production by Bacillus subtilis.
Bacillus subtilis produces various families of lipopeptides with different homologous compounds. To produce "new molecules" with improved activities and to select strains that produced a reduced number of homologs or isomers, we studied the effects of different media on the nature of the synthesis of fatty acid chains for each lipopeptide family. This study focused on two B. subtilis strains cultivated in flasks. Optimized medium for lipopeptide production and Landy medium modified by replacing glutamic acid with other alpha-amino acids were used. We found that the intensity of production of homologous compounds depends on the strain and the culture medium. Analysis of these lipopeptides by high-performance liquid chromatography showed that the strain B. subtilis NT02 yielded various homologous compounds when cultivated in Landy medium (L-Glu), but primarily one homologous product in high relative amounts when cultivated in the optimized medium. Mass spectrometric analysis and determination of the amino acid composition of this molecule enabled us to identify it as Bacillomycine L c15.


Graphita gen. nov., a New Genus for Neonympha griphe C. Felder & R. Felder, 1867 (Lepidoptera, Nymphalidae, Satyrinae).
A new genus is described for Neonympha griphe C. Felder & R. Felder, 1867, to contribute toward a revision of the polyphyletic genus Euptychoides Forster, 1964. Based on DNA sequence data, Graphita Nakahara, Marín & Barbosa, gen. nov. is strongly supported as a member of a clade of predominantly southeastern Brazilian taxa, in which it is weakly supported as sister to a well-supported clade containing Pharneuptychia Forster, 1964, Moneuptychia Forster, 1964 and the E. castrensis (Schaus, 1902) species complex. The data show that Graphita griphe comb. nov. is not related to other Euptychoides and not very closely related to any other sampled euptychiines, and thus support the description of this new genus. In addition, we provide morphological illustrations and a distribution map for this taxon based on museum specimens.


Injection Drug Use Quality of Life scale (IDUQOL): a validation study.
Existing measures of injection drug users' quality of life have focused primarily on health and health-related factors. Clearly, however, quality of life among injection drug users is impacted by a range of unique cultural, socioeconomic, medical, and geographic factors that must also be considered in any measure. The Injection Drug User Quality of Life (IDUQOL) scale was designed to capture the unique and individual circumstances that determine quality of life among injection drug users. The overall purpose of the present study was to examine the validity of inferences made from the IDUQOL by examining the (a) dimensionality, (b) reliability of scores, (c) criterion-related validity evidence, and (d) both convergent and discriminant validity evidence. An exploratory factor analysis using principal axis factoring in SPSS 12.0 was conducted to determine whether the use of a total score on the IDUQOL was advisable. Reliability of scores from the IDUQOL was obtained using internal consistency and one-week test-retest reliability estimates. Criterion-related validity evidence was gathered using variables such as stability of housing, sex trade involvement, high-risk injection behaviours, involvement in treatment programs, emergency treatment or overdose over the previous six months, hospitalization and emergency treatment over the subsequent six month period post data collection. Convergent and discriminant validity evidence was gathered using measures of life satisfaction, self-esteem, and social desirability. The sample consisted of 241 injection drug users ranging in age from 19 to 61 years. Factor analysis supports the use of a total score. Both internal consistency (alpha = .88) and one-week test-retest reliability (r = .78) for IDUQOL total scores were good. Criterion-related, convergent, and discriminant validity evidence supports the interpretation of IDUQOL total scores as measuring a construct consistent with quality of life. The findings from this study provide initial evidence to support the use of the IDUQOL total score. The results of the study also suggest the IDUQOL could be further strengthened with additional attention to how some IDUQOL domains are described and satisfaction is measured.


Bacterial acid stress response: from cellular changes to antibiotic tolerance and phenotypic heterogeneity.
Most bacteria are neutralophiles but can survive fluctuations in pH in their environment. Herein, we provide an overview of the adaptation of several human, soil, and food bacteria to acid stress, mainly based on next-generation sequencing studies, highlighting common and specific strategies. We also discuss the interplay between acid stress response and antibiotic tolerance, as well as the response of individual cells.


Appropriateness of Non-vitamin K Antagonist Oral Anticoagulants Dosing According to Different Prescription Guides Used in Belgian Ambulatory Care.
Non-vitamin K antagonist oral anticoagulants (NOACs) are the preferred choice of anticoagulants to prevent stroke in most patients with atrial fibrillation (AF). NOAC's dosing algorithms are defined in the respective Summary of Product Characteristics (SmPC) but the European Heart Rhythm Association (EHRA) Practical Guide can also be used as it considers more complex clinical scenarios. Nevertheless, suboptimal dosing of NOACs compromises the efficacy and safety of this commonly prescribed therapy in the AF population. Clearer objectification of inappropriate dosing and its influencing factors is needed to optimise management of AF patients. The primary aim of this study was to investigate whether there is a difference in the perceived appropriateness of NOAC dosing with respect to the SmPC or the 2018 EHRA Practical Guide in AF patients criteria and influencing factors. The secondary aim was to explore if there were differences in appropriateness of NOAC dosing between primary care and specialist care, and when using different renal function formulas. This retrospective study included AF patients treated with a NOAC in primary or in ambulatory specialist care in Antwerp (Belgium). Appropriateness of the NOAC dose was assessed according to the SmPC and 2018 EHRA recommendations. Univariate/multivariate analyses were performed to explore influencing factors for under- and overdosing of NOACs. Of the included 294 AF patients, 19.4% and 15.6% received an inappropriate dose according to the SmPC and the 2018 EHRA Practical Guide respectively (p = 0.003). Perceived frailty and higher weight were associated with underdosing relative to the SmPC, while a higher body mass index and the use of drugs/alcohol were associated with underdosing relative to the EHRA 2018 recommendations. Lower renal function and treatment with other NOACs than apixaban were associated with relative overdosing compared to both standards. Inappropriate NOAC dosing is present in almost twenty percent of AF patients according to the SmPC and requires further education of health care professionals and frequent reassessment of NOAC dosing. However, a significant lower prevalence of underdosing was present when judged by the 2018 EHRA criteria, likely reflecting decision making in complex AF patients. Perceived frailty, weight, renal function and type of NOAC are the main determinants of deviated dosing.


Preservation of alveolar ridge height through mechanical memory: A novel dental implant design.
Irreversible marginal bone loss can hinder recovery around dental implants. Insufficient alveolar osteogenesis and stress concentration during chewing contribute to marginal bone resorption and can result in implant failure. A biomaterial with a micropore-channel structure was developed using 3D printing technology. This design facilitated bony ingrowth and provided similar mechanical stimulation at the implant neck during mastication to a natural tooth. The micropore channels provided a guiding structure for bone mesenchymal stem cell proliferation and differentiation without the need for growth factors. Specifically, this was achieved through mechanical transduction by F-actin remodeling and the activation of Yes-associated protein (YAP). The implants were verified in a canine dental implant surgery model, which demonstrated the promising use of biomaterial-based dental implants in future clinical applications.


Data-driven electron-diffraction approach reveals local short-range ordering in CrCoNi with ordering effects.
The exceptional mechanical strength of medium/high-entropy alloys has been attributed to hardening in random solid solutions. Here, we evidence non-random chemical mixing in a CrCoNi alloy, resulting from short-range ordering. A data-mining approach of electron nanodiffraction enabled the study, which is assisted by neutron scattering, atom probe tomography, and diffraction simulation using first-principles theory models. Two samples, one homogenized and one heat-treated, are observed. In both samples, results reveal two types of short-range-order inside nanoclusters that minimize the Cr-Cr nearest neighbors (L12) or segregate Cr on alternating close-packed planes (L11). The L11 is predominant in the homogenized sample, while the L12 formation is promoted by heat-treatment, with the latter being accompanied by a dramatic change in dislocation-slip behavior. These findings uncover short-range order and the resulted chemical heterogeneities behind the mechanical strength in CrCoNi, providing general opportunities for atomistic-structure study in concentrated alloys for the design of strong and ductile materials.


Undergraduate Placements in Geriatric Care Facilities: Students Gaining Experience With Challenging/Responsive Behaviors.
Undergraduate nursing students may experience challenging behaviors in residents during their clinical placement in geriatric long-term care (GLTC) facilities. Bachelor of Nursing students participated in an anonymous online questionnaire (n = 116). The students witnessed a wide range of challenging behaviors, also referred to as responsive behaviors, in GLTC residents. The most frequent behaviors included agitation/restlessness, repetitive talk, and wandering. Although behaviors such as physical aggression and disinhibited sexual behavior were experienced less frequently, students found these behaviors the most distressing. Students felt ill-prepared to manage these behaviors, which was associated with higher levels of distress. The students demonstrated good theoretical knowledge about responsive behaviors, but the lack of personal experience in managing such behaviors left the students feeling ill-prepared and distressed. Incorporating the opportunity to experience behaviors in a supported environment, such as in simulation, could reduce student distress and increase their sense of competency. [J Nurs Educ. 2017;56(10):623-627.].


Endoscopic repair of combined orbital floor and medial wall fractures involving the inferomedial strut.
To evaluate the safety, efficacy, and feasibility of an endoscopic transconjunctival inferior fornix and precaruncular approach for repair of combined orbital medial wall and floor fractures involving the inferomedial strut. A retrospective study was performed in 84 patients with combined orbital medial wall and floor fractures involving the inferomedial strut. All patients underwent surgery by endoscopic transconjunctival inferior fornix and precaruncular approach. A large polyester mesh plate was implanted to cover the fracture defect. The enophthalmos, diplopia, and surgical complications were evaluated pre- and postoperatively. Patients were followed for at least 6 months. A total of 84 patients (55 men and 29 women) with a mean age of 36.88 ± 12.95 years were included in this study. Preoperatively, all 84 patients had an enophthalmos >2 mm, and the mean exophthalmometry measurement was 11.6 ± 2.14 mm. Diplopia was presented in 33 patients. Postoperatively, good symmetry was acquired in 81 of 84 patients, with a mean improvement of 3.02 ± 0.99 mm in the enophthalmos (P < 0.05). Recovery from diplopia occurred in 32 of 33 patients. A new onset of diplopia occurred postoperatively in 13 of the remaining 51 patients, only to recover spontaneously after 1 month. One patient suffered retrobulbar hemorrhage after surgery and was cured by conservative therapy. No other serious complications occurred. The endoscopic transconjunctival inferior fornix and precaruncular approach is a promising management technique for combined orbital medial wall and floor fractures involving the inferomedial strut.


Role of signal transduction and actin in G1 phase progression.
Progression through the cell cycle of mammalian cells is dependent upon external factors such as growth- and ECM factors. These factors exert their effect predominantly during the G1 phase of the cell cycle. When cells are cultured in suspension or when growth factors are withdrawn from the medium, cells will stop cell cycle progression and enter a quiescent state. Cells will remain in this quiescent state until extracellular conditions change and cells are stimulated to re-enter the cell cycle. This stimulation is mediated by various signal transduction cascades such as the mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositol 3-kinase (PI3-kinase) pathway. In Chinese hamster ovary cells at least two serum-dependent points exist during G1 phase that lead to diffent cellular responses. The first point is located immediately after mitosis and is suggested to link with apoptosis. The second point is located in late G1 phase and probably corresponds with cellular differentiation. Signal transduction is mutually related to the cytoskeleton, especially the actin microfilament system. The actin microfilament system influences signal transduction and several signal transduction pathways influence the actin structure. Here we describe the role of the MAPK and PI3-kinase activities and of actin microfilaments in progression through the cell cycle and their role in the two G1 checkpoints.


Effect of respiratory muscle endurance training in patients with COPD undergoing pulmonary rehabilitation.
Respiratory muscle endurance training (hyperpnea training) has been shown to have beneficial effects in patients with COPD. The purpose of this study was to determine whether hyperpnea training, when added to an endurance exercise training program, would lead to additional benefits compared with endurance training alone in patients with COPD. Patients with COPD entering an 8-week outpatient pulmonary rehabilitation program. Fifteen patients (mean [+/- SE] FEV1, 45 +/- 6% predicted) were randomized to combined therapy, and 14 patients (mean FEV1, 44 +/- 4% predicted) were randomized to endurance training. Peak exercise capacity, exercise endurance time during constant workload cycle exercise, 6-min walk distance, quality of life as measured by the chronic respiratory questionnaire, respiratory muscle strength and endurance, and quadriceps fatigability were measured before and after endurance or combined training. After rehabilitation, peak exercise capacity, exercise endurance time, 6-min walk distance, and quality of life all increased in both groups, but there was no significant difference in the extent of improvement between groups. Mean respiratory muscle endurance increased to a significantly greater extent in the combined therapy group (17.5 +/- 2.7 vs 8.5 +/- 2.5 min, respectively; p = 0.02). Respiratory muscle strength was significantly increased, and quadriceps fatigability was significantly reduced after rehabilitation in the combined therapy group but not in the endurance training group, but the difference between groups did not reach statistical significance. The endurance of the respiratory muscles can be improved by specific training beyond that achieved by endurance training alone in patients with COPD. However, this improvement did not translate into additional improvement in quality of life or exercise performance.


Hypoxia-driven miR-1307-3p promotes hepatocellular carcinoma cell proliferation and invasion by modulating DAB2 interacting protein.
Hypoxia is a common feature of the solid tumor microenvironment that is presented as poor clinical outcomes in multiple tumor types, including HCC. Hypoxia stabilizes HIF-1α/HIF-2α, which then moves into the nucleus and binds with HIF-1β to form a transcription complex, thereby promoting the transcription of target genes, including mRNAs, miRNAs and lncRNAs to exert their biological functions. Here, through a series of functional assay, including hypoxia culture, MTT, colony-formation, Transwell, qRT-PCR and western blot, we confirmed that miR-1307-3p, as a novel hypoxia-responsive factor, can be directly transcribed by HIF-1α rather than HIF-2α. Hypoxia-driven miR-1307-3p facilitated proliferation and invasion of HCC cells via repressing DAB2IP. Moreover, under hypoxia microenvironment, DAB2IP, as a direct target of miR-1307-3p, was down-regulated to activate AKT/mTOR signaling to further maintain the expression level of HIF-1α, thereby forming a feedback loop between HIF-1α/miR-1307-3p and DAB2IP. Targeting miR-1307-3p/DAB2IP axis also modulated tumor growth and metastasis in vivo. In summary, there exists a feedback loop between HIF-1α/miR-1307-3p and DAB2IP in HCC. Targeting a vicious feedback loop between HIF-1α/miR-1307-3p and DAB2IP may be a promising strategy to combat HCC.


Dechlorination of atrazine using zero-valent iron (Fe0) under neutral pH conditions.
Zero-valent iron (Fe0) is frequently used for the dechlorination of pesticides, because it is economical, easily acquired and stable. The kinetics of dechlorination by Fe0 are improved at low pH, but this requires additional acid addition, while dechlorination hardly occurs under basic conditions. Due to the buffer capacity of geological materials such as clay and sediment, however, the addition of acid to obtain a low pH may not be effective. In this research, the dechlorination constants of atrazine by Fe0 were measured with the addition of buffer solution to simulate the buffer capacity of sediment. In the presence of the buffer solution, the pH values remained neutral, while dechlorination occurred more slowly than that observed under acid additions but faster than that without any buffer. When the initial concentrations of atrazine were 10mg/L, 30 mg/L, and 50mg/L, its dechlorination was explained using pseudo-first order reaction kinetics. The pseudo-first order constants were 3.01 x 10(-2)d(-1) at 10 mg/L, 3.23 x 10(-2)d(-1) at 30 mg/L and 3.38 x 10(-2)d(-1) at 50mg/L. In addition, the half-lives of atrazine were 8.91 d at 10mg/L, 9.32 d at 30 mg/L, and 10.00 d at 50mg/L. Acid addition may not be omitted to obtain acidic pH conditions when dechlorination is necessary in geologic materials.


Health Equity Tourism: Reckoning with Medical Mistrust.
In this editorial, we further discuss the effects that health equity tourism has had on Black, Native, and Latinx marginalized communities. We identify the consequences of the resulting medical mistrust within these communities as well as implications for data collection in research. Throughout, solutions are proposed that may eventually empower these communities to become actively engaged with the research and initiatives that influence their health outcomes, as well as improve the quality and quantity of data extracted from these communities.


Biomagnetic functional localization of a peripheral nerve in man.
The first detection of the magnetic field of a stimulated peripheral nerve in man is presented. The measurement was performed noninvasively and in vivo on a healthy subject. The spatio-temporal field distribution is utilized to calculate the location of bioelectric activity on the basis of the equivalent current dipole model. The localization of the active nerve tissue is confirmed by a computer tomography image of the upper arm cross-section. Furthermore, a calculation of the total current distribution in the nerve explains the observed morphology of the signal.


Effects on in vivo and in vitro administration of vinblastine on the perfused rat liver--identification of crinosomes.
Livers of nonstarved rats were perfused for up to 4 hr in a recirculating system. Bile production, transaminases, and the lactate/pyruvate ratio remained at normal values. The ultrastructure of the hepatocytes was also well preserved even after the 4-hr perfusion. When vinblastine was given either in vivo or in vitro by addition to the perfusion fluid, it caused a conspicuous expansion of the autophagic-lysosomal compartment. Initially, nascent autophagic vacuoles developed, followed by the appearance of more mature ones and finally an increase in dense bodies was observed. In addition, administration of vinblastine in vivo gave rise to an increased occurrence of a subpopulation of lysosomes laden with VLDL-like particles. The term crinosomes seems appropriate for these lysosomal vesicles, since they apparently evolve by means of fusion between retained secretory granules and preexisting lysosomes (dense bodies). Addition of vinblastine of the perfusion fluid decreased the rate of proteolysis whether four times the serum concentration of amino acids were added or not. However, when vinblastine was given in vivo, proteolysis as measured in the perfusate decreased during the initial 3 hr of VBL treatment, whereas by longer times of pretreatment protein degradation exceeded the control value, constituting an example of catch-up proteolysis. Autophagic vacuoles isolated after short exposure to vinblastine in vivo exhibited high rates of protein degradation when incubated at acid pH. Insufficient proton pumping rather than lack of hydrolytic enzymes seems to be the most plausible explanation for this prompt pH effect.


Suppressor mechanisms in tumor immunity.
There are many parallels between T cell-mediated suppression of tumor immunity and suppression of immune responses to haptens and polypeptides. We propose a cell interaction model which takes this into account and outlines a regulatory pathway for suppression of immunity to tumor antigens. Free antigen or antigen/antibody complexes trigger an inducer T cell subset, Tsi, which is tumor-specific. This cell activates a non-immune T cell population, pre Tse, to generate effector suppressor cells, Tse. The Tse are specific for either the idiotype of Tsi or for antigen complexed with a soluble factor made by the Tsi, but the suppression they mediate is antigenically nonspecific. Tumor antigen-specific suppressor factors, TsF, play a major role in the communication between different suppressor cells. Characterization of polyclonal and monoclonal factors produced by Tsi, called TsFi, indicates that they both bind to tumor antigen and contain tumor-specific (idiotypic?) determinants.


Scarless Total Breast Reconstruction with a Fat-augmented Latissimus Dorsi Flap.
Total breast reconstruction with a fat-augmented latissimus dorsi flap (F-LDF) is a breakthrough approach that surmounts the shortcomings of the latissimus dorsi flap (LDF), such as volume insufficiency. Unlike the abdominal flap, the LDF can be harvested as a sole muscle flap without a skin paddle. This makes it possible to perform breast reconstruction with no donor-site scar when breast skin replacement is not required (eg, nipple-sparing mastectomy, two-stage reconstruction using a tissue expander). Here we describe a new approach for total breast reconstruction, namely scarless F-LDF reconstruction. First, the dorsal and ventral planes of the LDF are widely dissected through an inferolateral incision with monopolar electrocautery. The origin of the muscle is then separated using an energy-based device inserted through a stab incision, and immediate fat grafting is performed concurrently to the LDF and pectoralis major muscle. This new method was used in five cases, with a mean specimen weight of 285 g (range, 181-420), mean flap weight of 174 g (125-230), mean total fat graft volume of 214 ml (126-335), and mean duration of reconstruction surgery of 213 minutes (161-260). In all cases, sufficient volume was obtained postoperatively with satisfactory esthetic results. In addition to avoiding a donor-site scar, this method could reduce postoperative pain and donor-site seroma. The scarless F-LDF can be used for total breast reconstruction in certain populations, especially in cases requiring no skin replacement and for small- to medium-sized breasts.


Intellectual property rights and challenges for development of affordable human papillomavirus, rotavirus and pneumococcal vaccines: Patent landscaping and perspectives of developing country vaccine manufacturers.
The success of Gavi, the Vaccine Alliance depends on the vaccine markets providing appropriate, affordable vaccines at sufficient and reliable quantities. Gavi's current supplier base for new and underutilized vaccines, such as the human papillomavirus (HPV), rotavirus, and the pneumococcal conjugate vaccine is very small. There is growing concern that following globalization of laws on intellectual property rights (IPRs) through trade agreements, IPRs are impeding new manufacturers from entering the market with competing vaccines. This article examines the extent to which IPRs, specifically patents, can create such obstacles, in particular for developing country vaccine manufacturers (DCVMs). Through building patent landscapes in Brazil, China, and India and interviews with manufacturers and experts in the field, we found intense patenting activity for the HPV and pneumococcal vaccines that could potentially delay the entry of new manufacturers. Increased transparency around patenting of vaccine technologies, stricter patentability criteria suited for local development needs and strengthening of IPRs management capabilities where relevant, may help reduce impediments to market entry for new manufacturers and ensure a competitive supplier base for quality vaccines at sustainably low prices.


Immunoglobulin surface-binding kinetics studied by total internal reflection with fluorescence correlation spectroscopy.
An experimental application of total internal reflection with fluorescence correlation spectroscopy (TIR/FCS) is presented. TIR/FCS is a new technique for measuring the binding and unbinding rates and surface diffusion coefficient of fluorescent-labeled solute molecules in equilibrium at a surface. A laser beam totally internally reflects at the solid-liquid interface, selectively exciting surface-adsorbed molecules. Fluorescence collected by a microscope from a small, well-defined surface area approximately 5 micron2 spontaneously fluctuates as solute molecules randomly bind to, unbind from, and/or diffuse along the surface in chemical equilibrium. The fluorescence is detected by a photomultiplier and autocorrelated on-line by a minicomputer. The shape of the autocorrelation function depends on the bulk and surface diffusion coefficients, the binding rate constants, and the shape of the illuminated and observed region. The normalized amplitude of the autocorrelation function depends on the average number of molecules bound within the observed area. TIR/FCS requires no spectroscopic or thermodynamic change between dissociated and complexed states and no extrinsic perturbation from equilibrium. Using TIR/FCS, we determine that rhodamine-labeled immunoglobulin and insulin each nonspecifically adsorb to serum albumin-coated fused silica with both reversible and irreversible components. The characteristic time of the most rapidly reversible component measured is approximately 5 ms and is limited by the rate of bulk diffusion. Rhodamine-labeled bivalent antibodies to dinitrophenyl (DNP) bind to DNP-coated fused silica virtually irreversibly. Univalent Fab fragments of these same antibodies appear to specifically bind to DNP-coated fused silica, accompanied by a large amount of nonspecific binding. TIR/FCS is shown to be a feasible technique for measuring absorption/desorption kinetic rates at equilibrium. In suitable systems where nonspecific binding is low, TIR/FCS should prove useful for measuring specific solute-surface kinetic rates.


[Results of physiological and psychological studies of different body types. A study of 210 patients with cardiovascular diseases and 100 students (author's transl)].
210 male patients with myocardial infarction, functional cardiovascular diseases, and angina pectoris hospitalized in the course of a cardiac rehabilitation program had been studied. Missing data in some anthropometric variables reduced the sample to 199 patients. The body of data included various anthropometric variables and indices, parameters of the circulatory and respiratory systems, biochemical measures, personality questionnaires, and information about the psychosocial background. In order to replicate the findings a sample of 100 students was studied, too. The measures and indices of body type are influenced by age, which leads to age-dependent spurious correlations between body type and other variables if this dependency is not corrected. Different statistical approaches proved the superiority of a factor-analytic typisation based on a "body-breadth" and "body-size" factor. There is a low correlation between body type and disposition for certain diseases showing that patients with "athletic" or "pycnic" body build suffer preferably from coronary infarction. Significant differences between body types exist for blood pressure, physical fitness, several psychosocial data, and the number of previous courses of treatment. Correlations of body type with personality dimensions and bodily complaints did not occur.


Expert Elicitation of Population-Level Effects of Disturbance.
Expert elicitation is a rigorous method for synthesizing expert knowledge to inform decision making and is reliable and practical when field data are limited. We evaluated the feasibility of applying expert elicitation to estimate population-level effects of disturbance on marine mammals. Diverse experts estimated parameters related to mortality and sublethal injury of North Atlantic right whales (Eubalaena glacialis). We are now eliciting expert knowledge on the movement of right whales among geographic regions to parameterize a spatial model of health. Expert elicitation complements methods such as simulation models or extrapolations from other species, sometimes with greater accuracy and less uncertainty.


Safety and Efficacy of an Early Home-Based Walking Program After Receipt of an Initial Implantable Cardioverter-Defibrillator.
To assess the safety and efficacy of an early home-based walking program for first-time implantable cardioverter-defibrillator (ICD) recipients. Pre-post intervention trial. Institutional and private practice. Cardiac patients (N=301) with an initial ICD implantation for primary or secondary prevention; able to read, speak, and write English; and having access to telephone. Early home-based walking protocol implemented 1-month post-ICD implant. Exercise tolerance monitored by study nurses via telephone. Safety assessment was based on the frequency of ICD therapies and hospitalizations, and efficacy assessment was based on pedometer measures and self-report of ICD self-efficacy and physical activity. ICD recipients were on average 64.1±11.9 years old, predominantly men, and white, with an ejection fraction of <35% and a mean Charlson comorbidity score of 2.3±1.5. Nineteen individuals (6.3%) received 28 ICD shocks; 15 (53.6%) were appropriate and 13 (46.4%) inappropriate. Antitachycardia pacing therapies were delivered 72 times in 18 individuals (6%), with 61 (84.7%) being appropriate and 11 (15.3%) inappropriate. Five ICD shocks (2 appropriate and 3 inappropriate) and 2 antitachycardia pacing therapies occurred during walking. Five participants (2%) were hospitalized for an ICD shock, none of which was associated with walking. Average steps per day increased by 806 over 3 months. Perceived exercise self-efficacy improved significantly as did weekly exercise. Predictors of receiving any ICD shock were younger age (P<.0001), moderate to severe renal disease (P=.001), and lymphoma (P=.024). Early ambulation after an initial ICD was safe and effective, with few ICD shocks and improved efficacy.


Monoubiquitination of nuclear RelA negatively regulates NF-κB activity independent of proteasomal degradation.
Termination and resolution of inflammation are tightly linked to the inactivation of one of its strongest inducers, NF-κB. While canonical post-stimulus inactivation is achieved by upregulation of inhibitory molecules that relocate NF-κB complexes to the cytoplasm, termination of the NF-κB response can also be accomplished directly in the nucleus by posttranslational modifications, e.g., ubiquitination of the RelA subunit. Here we reveal a functional role for RelA monoubiquitination in regulating NF-κB activity. By employing serine-to-alanine mutants, we found that hypo-phosphorylated nuclear RelA is monoubiquitinated on multiple lysine residues. Ubiquitination was reversed by IκBα expression and was reduced when nuclear translocation was inhibited. RelA monoubiquitination decreased NF-κB transcriptional activity despite prolonged nuclear presence and independently of RelA degradation, possibly through decreased CREB-binding protein (CBP) co-activator binding. Polyubiquitin-triggered proteasomal degradation has been proposed as a model for RelA inactivation. However, here we show that proteasomal inhibition, similar to RelA hypo-phosphorylation, resulted in nuclear translocation and monoubiquitination of RelA. These findings indicate a degradation-independent mechanism for regulating the activity of nuclear RelA by ubiquitination.


Pathophysiology and treatment of systemic amyloidosis.
Amyloid is an abnormal extracellular fibrillar protein deposit in the tissues. In humans, more than 25 different proteins can adopt a fibrillar conformation in vivo that results in the pathognomonic tinctorial property of amyloid (that is, green birefringence when an affected tissue specimen is stained with Congo red dye and viewed by microscopy under cross-polarized light). Amyloid deposition is associated with disturbance of organ function and causes a wide variety of clinical syndromes that are classified according to the respective fibril protein precursor. Systemic amyloidosis, in which amyloid deposits are widespread and typically accumulate gradually, continues to be fatal and is responsible for about one in 1,500 deaths per year in the UK. Advances in our understanding of the pathogenesis of systemic amyloidosis have resulted in the identification of new therapeutic targets, and several drugs with novel mechanisms of action are currently under development. Meanwhile, an increased awareness of amyloidosis coupled with enhancements to existing diagnostic techniques and therapeutic strategies have already resulted in better outcomes for patients with the disease.


PDZ/PDZ interaction between PSD-95 and nNOS neuronal proteins: A thermodynamic analysis of the PSD95-PDZ2/nNOS-PDZ interaction.
N-Methyl-D-aspartate (NMDA) receptors are key components in synaptic communication and are highly relevant in central nervous disorders, where they trigger excessive calcium entry into the neuronal cells causing harmful overproduction of nitric oxide by the neuronal nitric oxide synthase (nNOS) protein. Remarkably, NMDA receptor activation is aided by a second protein, postsynaptic density of 95 kDa (PSD95), forming the ternary protein complex NMDA/PSD95/nNOS. To minimize the potential side effects derived from blocking this ternary complex or either of its protein components, a promising approach points to the disruption of the PSD-95/nNOS interaction which is mediated by a PDZ/PDZ domain complex. Since the rational development of molecules targeting such protein-protein interaction relies on energetic and structural information herein, we include a thermodynamic and structural analysis of the PSD95-PDZ2/nNOS-PDZ. Two energetically relevant events are structurally linked to a "two-faced" or two areas of recognition between both domains. First, the assembly of a four-stranded antiparallel β-sheet between the β hairpins of nNOS and of PSD95-PDZ2, mainly enthalpic in nature, contributes 80% to the affinity. Second, binding is entropically reinforced by the hydrophobic interaction between side chains of the same nNOS β-hairpin with the side chains of α2-helix at the binding site of PSD95-PDZ2, contributing the remaining 20% of the total affinity. These results suggest strategies for the future rational design of molecules able to disrupt this complex and constitute the first exhaustive thermodynamic analysis of a PDZ/PDZ interaction.