[Bortezomib-based regimen for the treatment of 110 multiple myeloma patients].
To analyze the outcomes and adverse effects of bortezomib-based regimen for the treatment of multiple myeloma (MM) patients. A total of 110 MM patients were treated with a bortezomib-based regimen at our hospital from January 2006 to February 2010. The patients over 65 years old received bortezomib-prednisone ± thalidomide (PD±T) regimen or velcade-melphalan-prednisone (VMP) regimen therapy and the patients under 65 years old or resistant to PD±T regimen received bortezomib-doxorubicin-prednisone ± thalidomide (PAD±T) regimen therapy. The outcomes and adverse effects of bortezomib-based regimen were retrospectively evaluated. There were 47 newly-diagnosed MM patients and 63 relapsing/refractory MM patients. The overall remission (OR) rate was 76.4% (84/110) and the OR rate of newly-diagnosed MM patients was statistically higher than that of relapsing/refractory MM patients (83.0% vs 71.4%, P<0.05). The complete remission (CR)+very good partial remission (VGPR) rate in group bortezomib 1.0 mg/m2 was lower than that in group bortezomib 1.3 mg/m2 (newly-diagnosed 53.6% vs 73.7%, relapsing/refractory 28.9% vs 40.0%, both P<0.05). The OR rate of ISS III stage patients was as better as that of ISS I and II stage patients (newly-diagnosed 82.1% vs 83.6%, relapsing/refractory 69.2% vs 72.2%, both P>0.05). Thirteen newly-diagnosed MM patients underwent autologous stem cell transplantation (ASCT) after induced therapy and achieved a VGPR or above. The median follow-up time was 13.0 (6.0-20.0) months. Their conditions were stable except two patients with extramedullary plasmacytoma after ASCT. Thirteen relapsing/refractory MM patients were retreated with a bortezomib-based regimen. The CR rate was 15.4% (2/13), VGPR rate was 23.1% (3/13), partial remission (PR) rate was 23.1% (3/13), OR rate 61.5% (8/13) and the median duration of remission (DOR) was 6.7 (3.0-21.0) months. Six MM patients with extramedullary plasmacytoma were treated with a bortezomib-based regimen and all of them achieved a PR or above. The median DOR was 4.5 (2.0-10.0) months. The main adverse effects were peripheral neuropathy, thrombocytopenia, neutropenia, fatigue, gastrointestinal symptoms, anemia, etc. The bortezomib-based combination regimen is the front-line therapy for newly-diagnosed and relapsing/refractory MM patients.


Lewis acid catalyzed cascade annulation of alkynols with α-ketoesters: a facile access to γ-spiroketal-γ-lactones.
A novel Lewis acid catalyzed intermolecular cascade annulation of alkynols with α-ketoesters has been developed. This simple and efficient cascade annulation proceeds through a 5-exo-dig cyclization of alkynols followed by annulation with α-ketoester to provide a wide variety of unsaturated γ-spiroketal-γ-lactones (1,6-dioxaspiro[4.4]non-3-en-2-ones) related to many natural products.


A new sparse optimization scheme for simultaneous beam angle and fluence map optimization in radiotherapy planning.
[Formula: see text]-minimization-based sparse optimization was employed to solve the beam angle optimization (BAO) in intensity-modulated radiation therapy (IMRT) planning. The technique approximates the exact BAO formulation with efficiently computable convex surrogates, leading to plans that are inferior to those attainable with recently proposed gradient-based greedy schemes. In this paper, we alleviate/reduce the nontrivial inconsistencies between the [Formula: see text]-based formulations and the exact BAO model by proposing a new sparse optimization framework based on the most recent developments in group variable selection. We propose the incorporation of the group-folded concave penalty (gFCP) as a substitution to the [Formula: see text]-minimization framework. The new formulation is then solved by a variation of an existing gradient method. The performance of the proposed scheme is evaluated by both plan quality and the computational efficiency using three IMRT cases: a coplanar prostate case, a coplanar head-and-neck case, and a noncoplanar liver case. Involved in the evaluation are two alternative schemes: the [Formula: see text]-minimization approach and the gradient norm method (GNM). The gFCP-based scheme outperforms both counterpart approaches. In particular, gFCP generates better plans than those obtained using the [Formula: see text]-minimization for all three cases with a comparable computation time. As compared to the GNM, the gFCP improves both the plan quality and computational efficiency. The proposed gFCP-based scheme provides a promising framework for BAO and promises to improve both planning time and plan quality.


How do family physicians provide anticipatory guidance during well-child visits?
Anticipatory guidance is an important part of well-child care, yet little is known about the way family physicians provide this guidance. This study describes the methods that family physicians use to provide anticipatory guidance during well-child visits. A questionnaire was mailed to 1000 family physicians. Respondents rated 6 anticipatory guidance methods on frequency of use. The questionnaire addressed method of documentation, use of forms or guidance prompts, visit frequency for total, well-child, and other pediatric visits, and demographic information; t tests and ANOVA were applied (P < .05 significant). There were 495 questionnaires returned for a response rate of 49.5%. Respondents were more likely to provide anticipatory guidance verbally than by handout (Likert scale where 1 = never and 9 = always, mean 7.8 vs 4.2, P < .0001). Physicians using well visit forms more commonly initiate guidance discussions (7.6 vs 6.8, P = .0002), address concerns (8.2 vs 7.5, P = .0001), and provide handouts (4.3 vs 3.3, P = .0002). Physicians in academic or multispecialty practices used handouts more often than private practitioners (5.0 vs 3.6, P = .0003, 5.1 vs 3.6, P = .0002, respectively) as did those with > or = 7 well-child visits per week compared with < 7 visits per week (4.8 vs 3.8, P = .0028). Family physicians primarily provide anticipatory guidance verbally. Use of well visit forms or other prompts are associated with better provision of anticipatory guidance.


Medical Care of the Patient With Compensated Cirrhosis.
The prevalence of liver disease and its complications is rapidly increasing in the United States. Gastroenterologists and hepatologists provide most of the medical care for patients with chronic liver disease and cirrhosis. While most gastroenterologists and hepatologists are comfortable managing the serious complications related to cirrhosis, many fail to provide the necessary education, prevention, and treatment for non-life-threatening problems associated with cirrhosis. Health-related quality of life has been found to strongly correlate with how well cirrhosis-related problems are addressed in the outpatient setting. This paper will review the outpatient management of patients with stable cirrhosis, including the surveillance and health maintenance recommendations necessary to monitor, prevent, or delay the onset of serious complications.


MiR-7a is an important mediator in Fas-associated protein with death domain (FADD)-regulated expression of focal adhesion kinase (FAK).
Fas-associated protein with death domain (FADD), a classical adaptor protein mediating apoptotic stimuli-induced cell death, has been reported to engage in several non-apoptotic processes such as T cell and cardiac development and tumorigenesis. Recently, there are several reports about the FADD's involvement in cell migration, however the underlying mechanism remains elusive. Here, we present a new finding that FADD could regulate the expression of FAK, a non-receptor protein tyrosine kinase overexpressed in many cancers, and played an important role in cell migration in murine MEF and melanoma cells with different metastatic potential, B16F10 and B16F1. Moreover, miR-7a, a tumor suppressor which prohibits cell migration and invasion, was up-regulated in FADD-deficient cells. And FAK was verified to be the direct target gene of miR-7a in B16F10 cells. Furthermore, we demonstrate that miR-7a was a necessary mediator in FADD-regulated FAK expression. In contrast to its classical apoptotic role, FADD interference could reduce the rate of cell migration, which could be rescued by inhibiting miR-7a expression. Taken together, our data provide a novel explanation regarding how FADD regulates cell migration in murine melanoma cells.


Park environment and moderate-to-vigorous physical activity in parks among adolescents in a high-density city: the moderating role of neighbourhood income.
Urban parks are critical environmental resources in which adolescents engage in physical activity (PA). Evidence on the associations between park environmental characteristics and park-based PA in adolescents is mixed, particularly for high-density cities. Evidence is also lacking concerning the moderating role of neighbourhood socioeconomic status on the park-PA relationships. The current study aimed to examine the associations between park environmental characteristics and moderate-to-vigorous physical activity (MVPA) in parks among adolescents in Hong Kong and the moderating effect of neighbourhood income on these associations. A cross-sectional study involving direct observations of adolescents was conducted in 32 randomly selected urban parks in Hong Kong. Park environmental characteristics were measured using the Community Park Audit Tool. Park-based MVPA among adolescents was measured using the System for Observation Play and Recreation in Communities. Neighbourhood income was extracted from the 2011 Hong Kong Population Census data on median household income. There was a significant positive association between the quality of amenities and park-based MVPA (metabolic equivalents per observation) in adolescents. However, the associations between the diversity of active facilities, greenness and adolescents' park-based MVPA were not significant. Neighbourhood income moderated the association between adolescents' park-based MVPA and park safety, where the relationship between park safety and park-based MVPA was significantly positive in low-income neighbourhoods but not significant in high-income neighbourhoods. An income-by-environment interaction was also observed concerning park aesthetics, with a negative relationship between park aesthetics and park-based MVPA in high-income neighbourhoods but not in low-income neighbourhoods. Our findings provide evidence regarding how park environment and neighbourhood income impact adolescents' park-based MVPA in Hong Kong. These findings can inform urban planning and policymakers who seek to improve urban park development in high-density cities.


Left-handed Z-DNA and in vivo supercoil density in the Escherichia coli chromosome.
A system for studying Z-DNA formation in the Escherichia coli chromosome was developed. Prior investigations in recombinant plasmids showed that alternating (Pur-Pyr) sequences can adopt a left-handed Z-DNA conformation both in vitro and in vivo. We constructed mobile, transposon-based cassettes carrying cloned (Pur-Pyr) sequences containing an EcoRI site in the center. These cassettes were subsequently inserted into different locations in the E. coli chromosome in a random fashion. A number of stable insertions were characterized by Southern analysis and pulsed-field gel electrophoresis mapping. A cloned temperature-sensitive MEcoRI methylase was expressed in trans as the probe to study Z-DNA formation in vivo. In this system, the control EcoRI sites were quickly methylated when cells were placed at the permissive temperature. Strong inhibition of the methylation was observed, however, only for the EcoRI sites embedded in a 56-bp run of (C-G). In contrast, the shorter sequence of 32 bp did not show this behavior. Prior in vitro determinations revealed that the longer tract required less energy to stabilize the Z-helix than the shorter block. We conclude that the observed inhibition of methylation is due to Z-DNA formation in the E. coli chromosome. In vitro, these sequences undergo the B- to Z-DNA transition at a supercoil density of -0.026 for the 56-bp insert and -0.032 for the 32-bp block. Since only the longer (C-G) tract but not the shorter run adopted the left-handed conformation in the chromosome, we propose that these densities establish the boundaries in the different chromosomal loci investigated; these boundaries are in good agreement with the extremes found in plasmids.


A Qualitative Study of a Maintenance Support Program for Women at Risk of Homelessness: Part 2: Situational Factors.
People who are homeless tend to have a number of complex needs. A housing maintenance support program (MSP) for women from the perspectives of clients, case managers and health professionals within the program was explored in this qualitative descriptive study. Interviews were conducted, and data were analysed using thematic analysis. The overarching theme that emerged from the data was "A life-changing event: I have the power to change." This theme was supported by three sub-themes: personal, situational and societal dimensions. In this article, the sub-theme - situational factors, is presented and refers to poverty, resources and services, as well as social support systems. These aspects all impinged on the client's ability to face life changes with optimism towards a better future. Their experiences of disconnection with the community changed for the clients after being part of the MSP. The MSP enabled the clients to feel part of society again, and empowered them to participate in the world around them. Key aspects of inclusion in the world are relationships based on acceptance, connecting with others, being involved and creating a sense of home/community.


[EFFECT OF Zn²⁺ ON SYNTHESIS OFACINETOBACTER CALCOACETICUS IMV B-7241 SURFACTANTS WITH ANTIMICROBIAL AND ANTIADHESIVE PROPERTIES].
To study the effect of zinc cations in the composition of ethanol and n-hexadecane containing medium on the antiadhesive and antimicrobial activity of Acinetobacter calcoaceticus IMV B-7241 surfactants. Surfactants were extracted from supernatant of cultural liquid by mixture of chloroform and methanol (2:1). The number of attached cells was determined spectrophotometrically, antimicrobial properties - by index of the minimum inhibitory concentration (MIC). Adding Zn²⁺ (38 mmol/l) into medium with ethanol and n-hexadecane containing copper sulphate and iron sulphate, was accompanied by the formation of surfactant with higher antimicrobial and antiadhesive activity, as well as increasing activity of NADP⁺-dependent glutamate dehydrogenase - a key enzyme of aminolipids biosynthesis. The minimum inhibitory concentration against Escherichia coli IEM- 1, Enterobacter cloaceae C-8, Staphylococcus aureus EMC- 1 and Proteus vulgaris IIA- 12 of surfactants, synthesized in the presence of Zn²⁺, and the adhesion of E. coli IEM-1 on abiotic surfaces treated with such surfactants, were respectively in 1.6-3.3 times and 10-19 % lower than those of the preparations obtained under cultivation of IMV B-7241 strain in medium without zinc cations. The activity of NADP⁺-dependent glutamate dehydrogenase at the end of exponential phase of A. calcoaceticus IMV B-7241 growth in medium with ethanol (n-hexadecane), copper, zinc and iron sulfate; was 1739±87 (8333±416) nmol-min⁻¹ mg⁻¹ protein that in 2 and 15 times higher than under the same conditions cultivation on ethanol and n-hexadecane without Zn²⁺. The obtained data suggest the possibility of biosynthesis regulation of A. calcoaceticus IMV B-7241 surfactants with antimicrobial and antiadhesive properties, when zinc cations (activator NADP⁺-dependent glutamate dehydrogenase - a key enzyme of aminolipids synthesis) were added into medium with ethanol (n-hexadecane), as well as the possibility of regulating the biological properties of the surfactants during cultivation of producer.


Co-segregation of the malignant hyperthermia and the Arg615-Cys615 mutation in the skeletal muscle calcium release channel protein in five European Landrace and Pietrain pig breeds.
A total of 392 pigs of European Landrace and Pietrain origin segregating for malignant hyperthermia (MH) were genotyped using a polymerase chain reaction (PCR)/restriction endonuclease test for the C-T mutation at nucleotide (nt) 1843 in the skeletal muscle ryanodine receptor (RYR1) gene, earlier identified as the causal mutation for MH. All pigs had been halothane tested and genotyped at linked polymorphic marker loci. There was complete correlation between MH status of the 392 animals, as diagnosed by a combination of the halothane challenge test with S, GPI, H, A1BG, PGD haplotyping, and the DNA-based test. DNA-based detection of the MH status in 238 MH-susceptible heterozygous (N/n) and homozygous (n/n) pigs was shown to be accurate, eliminating the 2% diagnostic error that is associated with the halothane challenge test. The mutation was also associated with an allele of a polymorphic microsatellite (ETH5 001) at the RYR1 locus.


Run-on of germline apoptosis promotes gonad senescence in C. elegans.
Aging (senescence) includes causal mechanisms (etiologies) of late-life disease, which remain poorly understood. According to the recently proposed hyperfunction theory, based on the older theory of antagonistic pleiotropy, senescent pathologies can arise from futile, post-reproductive run-on of processes that in early life promote fitness. Here we apply this idea to investigate the etiology of senescent pathologies in the reproductive system of Caenorhabditis elegans hermaphrodites, particularly distal gonad degeneration and disintegration. Hermaphrodite germ cells frequently undergo "physiological" (non-damage-induced) apoptosis (PA) to provision growing oocytes. Run-on of such PA is a potential cause of age-related gonad degeneration. We document the continuation of germline apoptosis in later life, and report that genetically blocking or increasing PA retards or accelerates degeneration, respectively. In wild-type males, which lack germ line apoptosis, gonad disintegration does not occur. However, mutational induction of PA in males does not lead to gonad disintegration. These results suggest that as germ-cell proliferation rate declines markedly in aging hermaphrodites (but not males), run-on of PA becomes a pathogenic mechanism that promotes gonad degeneration. This illustrates how hyperfunction, or non-adaptive run-on in later life of a process that promotes fitness in early life, can promote atrophic senescent pathology in C. elegans.


Acid ceramidase, an emerging target for anti-cancer and anti-angiogenesis.
Sphingolipid metabolism plays an important role in determining the fate of a cell. Among several sphingolipid metabolites, ceramide is a key player in intracellular signal transduction. Ceramide is usually converted to various metabolites such as sphingomyelin, sphingosine, ceramide-1-phosphate, and glucosylceramide. If ceramide is accumulated in the cell, it induces apoptosis. On the other hand, its metabolite sphingosine is converted to sphingosine-1-phosphate (S1P), which promotes angiogenesis via G protein coupled receptor signaling. Therefore, the equilibrium in ceramide and S1P levels in cells plays an important role in angiogenesis as well as cell death. Acid ceramidase (AC) is a promising target protein in the development of multi-targeted anticancer drugs as its inhibition can simultaneously inhibit angiogenesis via the Akt and ERK 1/2 pathway and limit cancer growth through ceramide-induced apoptosis. Although some inhibitors of AC have been reported, they have not been proven effective for human therapy. Recent advancement in the elucidation of AC structure will facilitate the development of better inhibitors for treating human diseases.


Glutamate containing neurons in the cat superior colliculus revealed by immunocytochemistry.
Glutamate is the probable neurotransmitter of both retinal and cortical afferents to the cat superior colliculus (SC). The present study shows that glutamate is also contained in many postsynaptic neurons in SC. The distribution, morphology, and ultrastructure of neurons in SC were examined using glutamate antibody immunocytochemistry. Labeled cells were widely distributed throughout, but a specific laminar pattern was evident. Relatively few cells were found in the zonal and upper superficial gray layers (SGL). A dense band of intensely labeled neurons was found within the deep superficial gray and upper optic layers. Many cells were also labeled in the deeper layers. Labeled cells had varied sizes and morphologies. Soma diameters ranged from 9-67 microns, with a mean of 22 microns. Cells with stellate, vertical fusiform, and multipolar morphologies were labeled. Cells in the deep subdivision all had morphologies and sizes typical of projection neurons. To determine if labeled cells in the dense band were also projection neurons, WGA-HRP was injected into the lateral posterior nucleus and these sections were double-labeled with the glutamate antibody. Over one-half of cells in the dense band that were labeled by HRP were also obviously labeled by antibody. At the electron-microscope level, both medium- and large-sized neurons were also labeled by glutamate antibodies. These cells had different but characteristic morphologies.


The Relationship Between Mental Health and Psychosocial Stress of Reservoir Migrants in the Middle Route of China's South-to-North Water Diversion Project (SNWDP): The Mediating Role of Social Support.
South-North Water Diversion Project (SNWDP) migrants were vulnerable to developing mental health problems due to post-migration stresses and lack of social support. However, the evidence on the mental health, psychosocial stress and social support of SNWDP migrants and their interrelationships are limited and inconclusive. We aimed to explore the mechanism of influence of social support on the relationship between mental health and social psychosocial stress of SNWDP migrants. We performed a cross-sectional analysis of the data of 983 SNWDP migrants. The mental health status, psychosocial stress and social support of the participants were evaluated by Symptom Checklist-90-Revised (SCL-90-R), Psychosocial Stress Survey for Groups (PSSG) and Social Support Rating Scale (SSRS). Multiple stepwise regression model was used to analyze the data. Among the 983 individuals, the average SCL-90-R score was 1.47, the PSSG score was 30.50, and the SSRS score was 40.30. The SCL-90-R was positively correlated with PSSG (r=0.483, P<0.001) and negatively correlated with SSRS (r=-0.257, P<0.001), PSSG was negatively correlated with SSRS (r=-0.516, P<0.001). Multiple regression analysis showed that PSSG and SSRS had significant effects on SCL-90-R prediction and that SSRS played a partial intermediary role in SCL-90-R and PSSG (46.87%). The SNWDP migrants performed a strong correlation between social support, mental health and psychosocial stress. Social support plays a part role in mediating mental health and psychosocial stress.


Long-term administration of tacrolimus and everolimus prevents high cholesterol-high fructose-induced steatosis in C57BL/6J mice by inhibiting de-novo lipogenesis.
To investigate the effects of tacrolimus (TC) and everolimus (EV) on non-alcoholic steatohepatitis (NASH) induced by high fat, high cholesterol and fructose (fast food) diet in C57BL/6J mice. C57BL/6J mice were divided into four groups (n=8). 1) Standard Chow (SC); 2) Fast food (FF) diet; 3) FF + Tacrolimus (TC, 1mg/kg) and; 4) FF + Everolimus (EV, 1mg/kg) and treated for 16 weeks. Serum and tissue samples were analyzed for evidence of inflammation, fibrosis, lipogenesis, and apoptosis. TC and EV treatments significantly reduced the hepatic lipid accumulation, improved liver-body weight ratio, blood biochemistry, and insulin resistance in mice fed with FF diet. However, inflammation, enlarged portal tracts, and fibrosis were pronounced in EV treated group. The lipogenic parameters, Peroxisome proliferator-activated receptor gamma (PPAR-γ), Sterol regulatory element-binding protein 1(SREBP-1), mammalian target of rapamycin (m-TOR), Stearoyl-CoA desaturase-1 (SCD-1) and fatty acid translocase (CD36) were significantly down-regulated in livers of TC and EV treated groups as compared to FF group. TC improved Bcl2/Bax ratio, decreased apoptosis, CYP2E1 protein expression and liver fibrosis levels, however, EV offered no such protection. Further, in an In-vitro model of lipotoxicity using the mouse hepatocyte (AML-12) cell line, treatment with TC and EV significantly reduced lipid accumulation and lipogenic and apoptotic markers induced with palmitic acid. In FF diet induced model of NASH, both TC and EV inhibited hepatic lipid accumulation and improved metabolic parameters such as insulin resistance and dyslipidemia. However, mice administered with EV exhibited inflammatory and fibrotic responses despite reduced hepatic steatosis.


Role of cation interactions in the reduction process in plutonium-americium mixed oxides.
The oxygen to metal ratio (O/M) is directly related to oxygen potential, which strongly influences the sintering and irradiation performance of nuclear fuels. A better understanding of these two parameters is therefore of major interest. To further ascertain the correlation between O/M ratio and oxygen potential in Am-bearing MOX, several thermodynamic descriptions are being developed. Despite their differences, they all involve the valence of actinide cations (e.g., U, Pu, and Am) as essential parameters. However, as no experimental data on their valence are available, these models rely on assumptions. In the present work, we coupled X-ray diffraction and X-ray absorption spectroscopy to follow the behavior of Pu and Am in three hypo-stoichiometric, U-free Pu(1-y)Am(y)O(2-x) compounds. We provide for the first time a quantitative determination of Pu and Am valences, demonstrating that plutonium reduction from Pu(4+) to Pu(3+) starts only when americium reduction from Am(4+) to Am(3+) is completed. This result fills in an important gap in experimental data, thereby improving the thermodynamic description of nuclear fuels. At last, we suggest that the O/M ratio may evolve at room temperature, especially for high Am content, which is of main concern for the fabrication of Am-loaded MOX and their storage prior to irradiation.


SDS agarose gels for analysis of proteins.
A new agarose-based protein electrophoresis gel system is described. The system consists of a highly resolving agarose, MetaPhor XR (FMC BioProducts, Rockland, ME, USA) dissolved in urea and TBE buffer and a stacking gel composed of a high gel-strength agarose, SeaKem Gold (FMC BioProducts). TBE containing sodium dodecyl sulfate (SDS) is used as electrophoresis buffer. The disadvantages of traditional agarose gels have been overcome, and several advantages over polyacrylamide gels have been demonstrated. The system is capable of high-resolution separation of small proteins and has a dynamic separation range equivalent to a 4%-20% gradient polyacrylamide gel. Furthermore, the staining of protein bands by Coomassie Brilliant Blue is very uniform in this gel, and depending on the protein, higher detection sensitivity can be obtained compared to SDS polyacrylamide gels. In Western blotting, proteins are more efficiently transferred to the membrane from the agarose gel than from polyacrylamide gels. Finally, the exceptional stability of agarose allows for gels to be precast and stored for a year.


Trends associated with debt loads among oral and maxillofacial surgery chief residents.
This survey-based study was undertaken to investigate how total debt loads are impacting the personal and professional decisions made by graduating oral and maxillofacial surgery (OMS) residents. The aim of this study was to evaluate differences in total debt load on graduating residents and analyze the effects of this debt on career, family, and lifestyle choices after graduation. This study was a cross-sectional, web-based survey of all graduating OMS residents in accredited OMS residency programs in the United States. Participation in the survey was optional, and all responses were anonymously collected and the data analyzed by using Qualtrics software. The respondents were analyzed as a collective, with the predictor of the study being program training length and the outcome being total debt load, with independent analysis of select other financial variables. For the 246 deliverable emails, there were 120 respondents (48.7% response rate). The average graduating OMS resident was a Caucasian male (median age 32 years), living with a significant other or spouse who independently earned money, and had no dependents. The average range of accumulated debt of graduating residents was between $300,000 and $350,000, with 50.83% of the respondents having $350,000 or less in overall debt and 49.17% of the respondents having $350,000 or greater in accumulated debt. For those respondents completing 4-year programs, the average range of accumulated debt was between $250,000 and $300,000, and for those respondents completing 6-year programs, the average range of accumulated debt was between $400,000 and $450,000 (P < .08). Graduating OMS residents carry with them a significant amount of debt whether graduating from a 4-year program or a 6-year program. However, when subjectively queried, most of these residents stated they would again choose OMS as a career choice.


Elucidating Sequence-Assembly Relationships for Bilingual PNA Biopolymers.
Nucleic acids and proteins possess encoded "languages" that can be used for information storage or to direct function. However, each biopolymer is limited to encoding its respective "language." Using a peptide nucleic acid (PNA) scaffold, nucleobase and amino acid residues can be installed on a singular backbone, enabling a single biopolymer to encode both languages. Our laboratory previously reported the development of a "bilingual" PNA biopolymer that incorporates a sequence-specific nucleic acid code interspersed with hydrophobic (alanine) and hydrophilic (lysine) amino acid residues at defined positions to produce amphiphilic character. We observed the amphiphilic amino acid residues directing the biopolymer to undergo self-assembly into micelle-like structures, while the nucleic acid recognition was harnessed for disassembly. Herein, we report a series of bilingual PNA sequences having amino acid residues with varying lengths, functional group charges, hydrophobicities, and spacings to elucidate the effect of these parameters on micelle assembly and nucleic acid recognition. Negative charges in the hydrophilic block or increased bulkiness of the hydrophobic side chains led to assembly into similarly sized micelles; however, the negative charge additionally led to increased critical micelle concentration. Upon PNA sequence truncation to decrease the spacing between side chains, the biopolymers remained capable of self-assembling but formed smaller structures. Characterization of disassembly revealed that each variant retained sequence recognition capabilities and stimuli-responsive disassembly. Together, these data show that the amino acid and nucleic acid sequences of amphiphilic bilingual biopolymers can be customized to finely tune the assembly and disassembly properties, which has implications for applications such as the encapsulation and delivery of cargo for therapeutics.


Estolides synthesis catalyzed by immobilized lipases.
Estolides are vegetable-oil-based lubricants obtained from oleic acid or any source of hydroxy fatty acids. In this work, the estolides synthesis from oleic acid and methyl ricinoleate (biodiesel from castor oil), using immobilized commercial lipases (Novozym 435, Lipozyme RM-IM, and Lipozyme TL-IM) in a solvent-free medium was investigated. Acid value was used to monitor the reaction progress by determining the consumption of acid present in the medium. Novozym 435 showed the best performance. Water removal improved the conversion. Novozym 435 was more active at atmospheric pressure. Novozym 435 was reused four times with conversion reaching 15% after the fourth reaction at 80°C. Estolides produced under the reaction conditions used in this work presented good properties, such as, low temperature properties as pour point (-24°C), viscosity (23.9 cSt at 40°C and 5.2 cSt at 100°C), and viscosity index (153).


A nationwide study on prevalence and factors associated with dynapenia in older adults: ELSI-Brazil.
This study aimed to assess the prevalence and factors associated with dynapenia in a nationally representative sample of Brazilians aged 50 years and older. A cross-sectional study was performed with baseline data from the Brazilian Longitudinal Study of Aging (ELSI-Brazil). Dynapenia was defined as low muscle strength (< 27kg for men and < 16kg for women). Explanatory variables were sociodemographic characteristics, health conditions, health behaviors and physical performance. Analyses were based on multivariate logistic regression and population attributable fractions. Among the 8,396 participants, the prevalence of dynapenia was 17.2% (16.6% among men and 17.7% among women); for those aged 65 years and older, the prevalence was 28.2% (29.1% and 27.5% among men and women, respectively). Dynapenia was positively associated with age, low gait speed, limitations in performing two or more basic daily activities, falls and self-reported chronic diseases; and negatively associated with education level, physical activity and body mass index (overweight/obese, OR = 0.26). Prevalence of dynapenia is high in Brazilian older adults. Educational skills and physical activity improvement present greater potential to reduce dynapenia in this population.


Development of an Autonomous Endoscopically Implantable Submucosal Microdevice Capable of Neurostimulation in the Gastrointestinal Tract.
Gastric dysmotility can be a sign of common diseases such as longstanding diabetes mellitus. It is known that the application of high-frequency low-energetic stimulation can help to effectively moderate and alleviate the symptoms of gastric dysmotility. The goal of our research was the development of a miniature, endoscopically implantable device to a submucosal pocket. The implantable device is a fully customized electronics package which was specifically designed for the purpose of experiments in the submucosa. The device was endoscopically inserted into the submucosal pocket of a pig stomach and partially severed pig side in order to adequately simulate a live animal model. The experiment confirmed that the designed device can be implanted into the submucosa and is capable of the measurement of sensor data and the transmission of this data wirelessly in real time to a computer outside of the body. After proving that the device can be implanted submucosally and transmit data, further experiments can now be performed, primarily with an electrogastrography (EGG) instrument and implantable device with tissue stimulation capability.


Single colony genetic analysis of epilithic stream algae of the genus Chamaesiphon spp.
In order to understand Chamaesiphon spp. evolution and ecological diversification, we investigated the phylogenetic differentiation of three morphospecies from field samples by means of single colony genetics. Individual colonies of three different morphospecies (C. starmachii, C. polonicus, C. geitleri,) were isolated from lotic gravel streams and their 16S rDNA nucleotide variability was analyzed. For a number of individual colonies, microscopical and ultrastructural analysis was also performed. A phylogenetic tree of all major lineages of the phylum of Cyanobacteria assigned all Chamaesiphon genotypes (1149-1176 bp) most closely with the family of Gomontiellaceae of the order Oscillatoriales. The sequences obtained from colonies assigned to C. starmachii (n = 21), C. polonicus (n = 9), and C. geitleri (n = 17) were found to reveal high average (3.5%) nucleotide diversity. No phylogenetic sub-branching in correspondence with morphology was observed suggesting that the three Chamaesiphon morphospecies did not represent monophyletic taxa. We could not attribute specific thylakoid ultrastructure to phylogenetic sub-branches; however, the observed parietally and loosely arranged thylakoids indicate that for the genus Chamaesiphon, the variability in thylakoid ultrastructure might have been underestimated. In summary, the high nucleotide diversity of the 16S rDNA gene implies phylogenetic diversity that corresponds little to morphological classification.


Current status and perspective of EBUS-TBNA.
In 2002, a new bronchoscope equipped with a convex type ultrasound probe on the tip was introduced into clinical practice. This convex probe endobronchial ultrasound (CP-EBUS) provides a long axis image of surrounding structures of the major airway. The CP-EBUS combined with a dedicated biopsy needle allows real-time endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) of mediastinal and hilar lymph nodes. Samples obtained from EBUS-TBNA can be used for pathological diagnosis including immunohistochemistry. EBUS-TBNA is now being performed worldwide for several indications including mediastinal staging, mediastinal pathology and for benign diseases. For mediastinal staging in lung cancer, the diagnostic yield of EBUS-TBNA is comparable to surgical staging in patients with enlarged lymph nodes. In addition, EBUS-TBNA can be used for molecular analysis of tumor cells obtained during the procedure. DNA mutation testing using EBUS-TBNA samples was first reported in 2007 for a chondrosarcoma case. Also, isolated DNA from EBUS-TBNA samples was applied for epidermal growth factor receptor mutation testing and aberrant methylation analysis in patients with lung cancer. RNA was also isolated from properly stored EBUS-TBNA samples and used for gene expression research and aberrant fusion gene detection in lung cancer patients. Optimization of tissue banking methods of EBUS-TBNA specimens was also investigated. Currently, EBUS-TBNA samples can be used for comprehensive mRNA and miRNA expression analysis. EBUS-TBNA will become an important modality for bronchoscopists in the era of personalized medicine in lung cancer.


A novel prophage lysin Ply5218 with extended lytic activity and stability against Streptococcus suis infection.
Streptococcus suis (S. suis) is an emerging zoonotic agent that exhibits high level resistance to classic antibiotics and a heavy burden in the swine industry. Therefore alternative antibacterial agents need to be developed. A novel endolysin derived from the S. suis temperate phage phi5218, termed Ply5218, was identified. The minimum inhibitory concentration (MIC) of Ply5218 was 2.5 μg ml(-1) against S. suis strain HA9801, an activity many times greater than the lysins reported previously (MIC of LY7917 and Ply30 against HA9801 were 80 and 64 μg ml(-1), respectively). Ply5218 at 10 μg ml(-1) in vitro exerted broad antibacterial activities against S. suis strains with OD600 ratios decreased from 1 to <0.2 within 1 h. Moreover, Ply5218 showed favorable thermal stability. It was stable at 50°C >30 min, 4°C >30 days, -80°C >7 months, and >60% of the enzyme activity remained after 5 min pre-incubation at 70°C. In vivo, a 0.2 mg dose of Ply5218 protected 90% (9/10) of mice after infection with S. suis HA9801. Finally, Ply5218 maintained high antibacterial activity in some bio-matrices, such as culture media and milk. The data indicate that Ply5218 has all the characteristics to be an effective therapeutic agent against multiple S. suis infections.


New measures for new roles: defining and measuring the current practices of health sciences librarians.
The roles of academic health sciences librarians are continually evolving as librarians initiate new programs and services in response to developments in computer technology and user demands. However, statistics currently collected by libraries do not accurately reflect or measure these new roles. It is essential for librarians to document, measure, and evaluate these new activities to continue to meet the needs of users and to ensure the viability of their professional role. To determine what new measures should be compiled, the authors examined current statistics, user demands, professional literature, and current activities of librarians as reported in abstracts of poster sessions at Medical Library Association annual meetings. Three new categories of services to be measured are proposed. The first, consultation, groups activities such as quality filtering and individual point-of-need instruction. The second, outreach, includes activities such as working as liaisons, participating in grand rounds or morning report, and providing continuing education. The third area, Web authoring, encompasses activities such as designing Web pages, creating online tutorials, and developing new products. Adding these three measures to those already being collected will provide a more accurate and complete depiction of the services offered by academic health sciences librarians.


Elusive implementation: an ethnographic study of intersectoral policymaking for health.
For more than 30 years policy action across sectors has been celebrated as a necessary and viable way to affect the social factors impacting on health. In particular intersectoral action on the social determinants of health is considered necessary to address social inequalities in health. However, despite growing support for intersectoral policymaking, implementation remains a challenge. Critics argue that public health has remained naïve about the policy process and a better understanding is needed. Based on ethnographic data, this paper conducts an in-depth analysis of a local process of intersectoral policymaking in order to gain a better understanding of the challenges posed by implementation. To help conceptualize the process, we apply the theoretical perspective of organizational neo-institutionalism, in particular the concepts of rationalized myth and decoupling. On the basis of an explorative study among ten Danish municipalities, we conducted an ethnographic study of the development of a municipal-wide implementation strategy for the intersectoral health policy of a medium-sized municipality. The main data sources consist of ethnographic field notes from participant observation and interview transcripts. By providing detailed contextual description, we show how an apparent failure to move from policy to action is played out by the ongoing production of abstract rhetoric and vague plans. We find that idealization of universal intersectoralism, inconsistent demands, and doubts about economic outcomes challenge the notion of implementation as moving from rhetoric to action. We argue that the 'myth' of intersectoralism may be instrumental in avoiding the specification of action to implement the policy, and that the policy instead serves as a way to display and support good intentions and hereby continue the process. On this basis we expand the discussion on implementation challenges regarding intersectoral policymaking for health.


Laparoscopic versus open selective lateral pelvic lymph node dissection following total mesorectal excision for locally advanced low rectal cancer.
Lateral pelvic lymph node (LPLN) dissection represents a technically challenging procedure with a high potential risk of surgical morbidity. The purpose of this study was to compare the technical feasibility, safety, and oncological efficacy of laparoscopic LPLN dissection (LPLD) following total mesorectal excision (TME) with open LPLD for locally advanced low rectal cancer (LALRC). Between January 2010 and December 2016, consecutive patients with LALRC and swollen LPLNs who underwent laparoscopic or open TME with LPLD at our institution were enrolled in this retrospective observational study. Data regarding patient demographics, perioperative characteristics, and oncological outcomes were analyzed and compared. A total of 64 patients met the inclusion criteria. Thirty-four patients underwent open procedure, and 30 underwent laparoscopic procedure. The mean blood loss volume was significantly less in the laparoscopic group than in the open group (165 vs. 422 mL; P = 0.012). The mean operative time was not significantly different between the laparoscopic and the open groups (354 ± 91 vs. 315 ± 78 min; P = 0.522). The overall postoperative complication rates were 30.0% and 35.3% for the laparoscopic and open groups (P = 0.428), respectively. Postoperative urinary retention was significantly less in the laparoscopic group than in the open group (14.7 vs. 0%; P = 0.036).The duration of postoperative hospital stay was significantly shorter in the laparoscopic group (8.5 ± 3.8 vs. 13.6 ± 6.5 days; P = 0.025). The numbers of harvested lymph nodes and positive resection margin rates showed no significant differences. Pathological LPLN metastases were confirmed in 10 patients (29.4%) in the open group and 11 (36.7%) in the laparoscopic group (P = 0.537). The median follow-up duration was 41.5 months (range 3-98). The laparoscopic and open groups also showed a similar 3-year overall survival rate (88.2% vs. 85.3%; P = 0.577), relapse-free survival rate (73.3% vs. 67.6%; P = 0.889), and local recurrence rate (3.3 vs. 5.9%; P = 0.653). Laparoscopic TME with LPLD is technically feasible and safe in selected patients with LALRC and is associated with similar oncological outcomes as open approach.


Middle-Ear Resonance Frequency and Eustachian Tube Function in Players of Wind Instruments.
The effect of the continuous forced expiration action of players of wind instruments to produce sound, on the eustachian tube functions and the middle-ear resonance frequency (RF), has not been investigated in the literature to date. The aim of this study is to evaluate eustachian tube functions and the middle-ear RF of players of wind instruments. In this prospective case-control clinical study, a study group of 28 players of wind instruments in the orchestra (28 participants, 56 ears) and a control group of 34 volunteers (34 participants, 68 ears) were included. The eustachian function of wind instrument players in a symphony orchestra was measured using an automatic eustachian tube function test in acoustic tympanometry and the RF of the middle ear was determined in multifrequency tympanometry. There was a statistically significant difference among the musicians, especially in players of woodwind instruments, in terms of dysfunction of the eustachian tubes (p = 0.048). In the musicians, the pre- and postperformance RF mean values for all ears were 925 and 1,020 Hz, respectively, and these were significantly different (p = 0.004). This is the first study to uses multifrequency tympanometry to examine the middle-ear RF and eustachian tube function of wind instrument musicians in an orchestra. Eustachian tube dysfunction was found to be more prominent and a higher RF of the middle ear was seen after a performance, especially in players of wood wind instruments. However, the effect of these on the professional performance of players of wind instruments should be investigated in future work.


Local anaesthetic techniques for post-caesarean delivery analgesia.
In this narrative review we summarise pertinent data from published studies investigating the use of local anaesthetic techniques as adjuncts for managing post-caesarean delivery pain. Based on currently available evidence, ultrasound-guided transversus abdominis plane (TAP), quadratus lumborum (QL) and ilio-inguinal and iliohypogastric (ILIH) blocks are preferable to landmark techniques. When intrathecal morphine is used for caesarean delivery analgesia, TAP blocks do not confer any additional benefit. In the absence of intrathecal morphine, TAP blocks have been shown to reduce pain scores and opioid consumption in the first 24 h postoperatively. In the absence of intrathecal morphine, single-dose local anaesthetic wound infiltration also results in a moderate reduction in opioid consumption postoperatively. If a wound catheter is to be incorporated into a multimodal analgesic regimen, a position below the fascia and a continuous infusion of low-concentration local anaesthetic solutions should be considered. Intraperitoneal local anaesthetic instillation may be of benefit in patients who undergo peritoneal closure but larger studies are still needed. Quadratus lumborum and ILIH blocks show promising results but the data are limited, so recommendations for routine use cannot be made. In summary, evidence supports the use of local anaesthetic techniques for post-caesarean delivery pain but additional research is required to determine the optimum dosing regimens, and the potential role of liposomal local anaesthetics. Further studies are required to compare techniques and determine their role in conjunction with low-dose long-acting neuraxial opioids.


Response of the diatom Phaeodactylum tricornutum to photooxidative stress resulting from high light exposure.
The response of microalgae to photooxidative stress resulting from high light exposure is a well-studied phenomenon. However, direct analyses of photosystem II (PSII) D1 protein (the main target of photoinhibition) in diatoms are scarce. In this study, the response of the diatom model species Phaeodactylum tricornutum to short-term exposure to high light was examined and the levels of D1 protein determined immunochemically. Low light (LL) acclimated cells (40 µmol photons m(-2) s(-1)) subjected to high light (HL, 1,250 µmol photons m(-2) s(-1)) showed rapid induction of non-photochemical quenching (NPQ) and ca. 20-fold increase in diatoxanthin (DT) concentration. This resulted from the conversion of diadinoxanthin (DD) to DT through the activation of the DD-cycle. D1 protein levels under LL decreased about 30% after 1 h of the addition of lincomycin (LINC), a chloroplast protein synthesis inhibitor, showing significant D1 degradation and repair under low irradiance. Exposure to HL lead to a 3.2-fold increase in D1 degradation rate, whereas average D1 repair rate was 1.3-x higher under HL than LL, leading to decreased levels of D1 protein under HL. There were significant effects of both HL and LINC on P. tricornutum maximum quantum yield of PSII (F(v)/F(m)), showing a reduction of active PSII reaction centres. Partial recovery of F(v)/F(m) in the dark demonstrates the photosynthetic resilience of this diatom to changes in the light regime. P. tricornutum showed high allocation of total protein to D1 and an active D1-repair cycle to limit photoinhibition.


Projections for antiinfective drug shortages and time to actual resolution.
The results of a national study of projected versus actual times to resolution of temporary U.S. shortages of antiinfective drugs are presented. Descriptive data on antiinfective drug shortages, including differences between manufacturer-estimated and actual times to shortage resolution, were collected over a one-year period via regular monitoring of the websites of ASHP and the Food and Drug Administration. Inventory data from one large hospital in the Midwest were collected in order to characterize realized shortages (i.e., those for which mitigation was required). During the study period, there were 47 transient shortages of antiinfective medications involving 381 unique products or formulations, of which 40% (n = 19) were emergent shortages. Generic-only and brand-only medications accounted for 53% (n = 25) and 21% (n = 10) of the evaluated shortages, respectively; the median shortage duration was 40 days (interquartile range [IQR], 22-91 days). The reasons for shortages most frequently cited by manufacturers were product or formulation discontinuation (21%), increased demand (12%), and raw material shortfalls (8%). Some shortages were resolved sooner than originally projected, but overall, actual shortage durations exceeded manufacturer-projected durations by a median of 17 days (IQR, 0-52.5 days). Ten realized shortages occurred at the hospital study site, compelling the antimicrobial stewardship team to recommend alternative therapies or restrict the use of drugs in short supply. The actual durations of evaluated antiinfective drug shortages during the study period were longer than the manufacturer-projected durations by a median of 17 days.


Use of lung clearance index to assess the response to intravenous treatment in cystic fibrosis.
Progressive lung disease is the main cause of clinical deterioration and mortality in cystic fibrosis (CF) patients. Being able to assess the effectiveness of interventions is very significant. To assess the response to intravenous (IV) treatment among CF patients, using forced expired volume in 1(st) second (FEV1) and Lung Clearance Index (LCI) as outcome measures and to compare the effect of IV treatment on lung function and LCI between CF children being treated on a regular basis, or in case of a pulmonary exacerbation. Thirty-two CF patients (15 males) with mean age 9.90 (range: 2-23) years, performed spirometry and multiple breath washout (MBW) before and one month after IV antibiotic treatment. Nineteen patients received a course of elective treatment (group A) and 13 received IV antibiotic regimens for an acute exacerbation (group B). Statistically significant differences after treatment were seen in LCI (p≤0.001), and Forced Expiratory Flow (FEF) z-scores (p<0.05). FEV1 did not change significantly after drug intervention. Dividing patients into two groups, only LCI showed significant change (p<0.05), when treatment was administered on a regular basis. Patients being treated for an acute pulmonary exacerbation showed significant improvement in most of the lung function parameters: LCI (p=0.0001), FEV1% (p=0.05), FEV1 z-score (p=0.033) and FEF25-75 (p=0.046). The mean LCI difference was significantly greater in group B compared to group A (p=0.001). LCI is more sensitive marker than FEV1 to assess the effect of IV antibiotic treatment among CF children. IV antibiotics are more effective on lung function parameters, when they are administered for an acute exacerbation, than when they are given on a regular basis. Hippokratia 2015, 19 (1): 47-52.


Biliary strictures and recurrence after liver transplantation for primary sclerosing cholangitis: A retrospective multicenter analysis.
Liver transplantation (LT) is the only definitive treatment for patients with end-stage liver disease due to primary sclerosing cholangitis (PSC), but a high rate of biliary strictures (BSs) and of recurrent primary sclerosing cholangitis (recPSC) has been reported. In this multicenter study, we analyzed a large patient cohort with a long follow-up in order to evaluate the incidence of BS and recPSC, to assess the impact on survival after LT, and to identify risk factors. We collected clinical, surgical, and laboratory data and records on inflammatory bowel disease (IBD), immunosuppression, recipient and graft outcome, and biliary complications (based on cholangiography and histology) of all patients who underwent LT for PSC in 10 German transplant centers between January 1990 and December 2006; 335 patients (68.4% men; mean age, 38.9 years; 73.5% with IBD) underwent transplantation 8.8 years after PSC diagnosis with follow-up for 98.8 months. The 1-, 5-, and 10-year recipient and graft survival was 90.7%, 84.8%, 79.4% and 79.1%, 69.0%, 62.4%, respectively. BS was diagnosed in 36.1% after a mean time of 3.9 years, and recPSC was diagnosed in 20.3% after 4.6 years. Both entities had a significant impact on longterm graft and recipient survival. Independent risk factors for BS were donor age, ulcerative colitis, chronic ductopenic rejection, bilirubin, and international normalized ratio (INR) at LT. Independent risk factors for recPSC were donor age, IBD, and INR at LT. These variables were able to categorize patients into risk groups for BS and recPSC. In conclusion, BS and recPSC affect longterm graft and patient survival after LT for PSC. Donor age, IBD, and INR at LT are independent risk factors for BS and recPSC and allow for risk estimation depending on the recipient-donor constellation.


Chemical Peels as Field Therapy for Actinic Keratoses: A Systematic Review.
Actinic keratoses (AKs) are a common premalignant cutaneous neoplasm and can progress to squamous cell carcinoma. A variety of treatment options are available for field therapy of diffuse AKs. This review systematically analyzes the use of chemical peels for treatment of AKs. A systematic review of PubMed was performed searching from 1946 to March 2020 to identify the literature on chemical peels for AKs. Of the 151 articles identified, 5 met inclusion criteria for review. Four of the reviewed articles demonstrated the efficacy of chemical peels in reducing AK count and minimal adverse effects. In some studies, chemical peels exhibited potential to prevent additional AK formation and development of keratinocyte carcinomas. Chemical peels are an efficacious and affordable treatment option for field treatment of AKs. With improved patient tolerance and adherence, chemical peels are an attractive option for field therapy of AKs for both dermatologists and patients.


Inhibition of the osteoclast activity with the application of recombinant murine RANK protein.
To investigate the effects of recombinant Murine RANK on the osteoclast activity. Osteoclast was observed with soluble RANK. Female Wistar rats were bilaterally ovariectomized, and intra-abdominally injected with 5 mg/Kg soluble RANK. The bone metabolism, bone density, and bone histomorphology were observed. Compared with the control group, the quantity of TRAP-positive osteoclasts and bone resorption pit counting in the rest groups were significantly reduced. The bone density of the dosed groups was significantly increased and TRAP-stained osteoclasts in bone tissue sections were almost inhibited. rh-Murine RANK was able to inhibit osteoclast differentiation and prevent ovariectomy-induced bone loss.


Effect of Osmotic Strength on 5'-Inosinic Acid Fermentation in Mutants of Corynebacterium ammoniagenes.
The effect of osmotic strength on 5'-inosinic acid (IMP) fermentation was examined with a mutant, Corynebacterium ammoniagenes KY10895. The growth of KY10895 was inhibited by increased osmotic strength in the medium, but the specific IMP production (IMP produced/cell density) was promoted. The concentration of L-proline increased greatly under osmotic stress. Mutants resistant to growth inhibition by osmotic strength were derived from KY10895. Strain H-4415 had the highest IMP productivity. The intracellular concentration of L-proline in strain H-4415 was higher than in the parental strain. The addition of an osmoprotectant, betaine, in the production medium did not increase IMP production. These results suggested that the derivation of an osmotolerant strain is an effective method to obtain a hyperproducer of IMP, and that an increase in intracellular L-proline is related to increased IMP production.


Analysis in vitro of the enzyme CRTISO establishes a poly-cis-carotenoid biosynthesis pathway in plants.
Most enzymes in the central pathway of carotenoid biosynthesis in plants have been identified and studied at the molecular level. However, the specificity and role of cis-trans-isomerization of carotenoids, which occurs in vivo during carotene biosynthesis, remained unresolved. We have previously cloned from tomato (Solanum lycopersicum) the CrtISO gene, which encodes a carotene cis-trans-isomerase. To study the biochemical properties of the enzyme, we developed an enzymatic in vitro assay in which a purified tomato CRTISO polypeptide overexpressed in Escherichia coli cells is active in the presence of an E. coli lysate that includes membranes. We show that CRTISO is an authentic carotene isomerase. Its catalytic activity of cis-to-trans isomerization requires redox-active components, suggesting that isomerization is achieved by a reversible redox reaction acting at specific double bonds. Our data demonstrate that CRTISO isomerizes adjacent cis-double bonds at C7 and C9 pairwise into the trans-configuration, but is incapable of isomerizing single cis-double bonds at C9 and C9'. We conclude that CRTISO functions in the carotenoid biosynthesis pathway in parallel with zeta-carotene desaturation, by converting 7,9,9'-tri-cis-neurosporene to 9'-cis-neurosporene and 7'9'-di-cis-lycopene into all-trans-lycopene. These results establish that in plants carotene desaturation to lycopene proceeds via cis-carotene intermediates.


Serum-mediated suppression of nonspecific B cell activation. I. Description of an inhibitory capacity in normal mouse serum and characterization of the inhibitory component.
Normal mouse serum (NMS) in relatively small amounts was demonstrated to inhibit in vitro splenic B cell activation by several B cell mitogens, polyclonal activators, and T-independent antigens. Proliferative and polyclonal responses elicited by bacterial endotoxin (ET), PPD, or Fc fragment of Ig were suppressed routinely 90% or greater when 0.5% homologous NMS was present in the culture medium. Similar suppression of T-independent antibody responses to TNP-ET and TNP-Ficoll was also observed. In contrast, activation by the T cell mitogen, Con A, and a T-dependent antigen response to SRBC was not inhibited by NMS. Suppression of B cell responses by NMS was not dependent on T cells. Preliminary characterization of NMS-In revealed a m.w. of approximately 200,000, an isoelectric point of 5, and solubility in 40% saturated ammonium sulfate. Both functionally and physically, NMS-In appears to be different from other currently relatively well characterized regulatory substances in serum. NMS-In was found in plasma as well as in serum, suggesting that NMS-In may function naturally in vivo to minimize nonspecific activation of B cells.


Chemical Composition, Antiprotozoal and Cytotoxic Activities of Indole Alkaloids and Benzofuran Neolignan of Aristolochia cordigera.
This is a comparative study on the intraspecific chemical variability of Aristolochia cordigera species, collected in two different regions of Brazil, Biome Cerrado (semiarid) and Biome Amazônia (coastal). The use of GC-MS and statistical methods led to the identification of 56 compounds. A higher percentage of palmitone and germacrene-D in the hexanes extracts of the leaves of plants from these respective biomes was observed. Phytochemical studies on the extracts led to the isolation and identification of 19 known compounds, including lignans, neolignans, aristolochic acids, indole-β-carboline, and indole alkaloids. In addition, two new indole alkaloids, 3,4-dihydro-hyrtiosulawesine and 6-O-(β-glucopyranosyl)hyrtiosulawesine, were isolated and a new neolignan, cis-eupomatenoid-7, was obtained in a mixture with its known isomer eupomatenoid-7. Their structures were determined by spectroscopic methods, mainly by 1D- and 2D-NMR. The occurrence of indole alkaloids is being described for the first time in the Aristolochiaceae family. Moreover, the in vitro susceptibility of intracellular amastigote and promastigote forms of Leishmania amazonensis to the alkaloids and eupomatenoid-7 were evaluated. This neolignan exhibited low activity against promastigotes (IC50 = 46 µM), while the alkaloids did not show inhibitory activity. The new alkaloid 6-O-(β-glucopyranosyl)hyrtiosulawesine exhibited activity in the low micromolar range against Plasmodium falciparum, with an IC50 value of 5 µM and a selectivity index higher than 50.


Update on Gadolinium Based Contrast Agent Safety, From the AJR Special Series on Contrast Media.
Since its introduction 35 years ago, gadolinium-enhanced MRI has fundamentally changed medical practice. While extraordinarily safe, gadolinium-based contrast agents (GBCAs) may have side effects. Four distinct safety considerations include: acute allergic-like reactions, nephrogenic systemic fibrosis (NSF), gadolinium deposition, and symptoms associated with gadolinium exposure. Acute reactions after GBCA administration are uncommon-far less than with iodinated contrast agents-and, while rare, serious reactions can occur. NSF is a rare, but serious, scleroderma-like condition occurring in patients with kidney failure after exposure to American College of Radiology (ACR) Group 1 GBCAs. Group 2 and 3 GBCAs are considered lower risk, and, through their use, NSF has largely been eliminated. Unrelated to NSF, retention of trace amounts of gadolinium in the brain and other organs has been recognized for over a decade. Deposition occurs with all agents, although linear agents appear to deposit more than macrocyclic agents. Importantly, to date, no data demonstrate any adverse biologic or clinical effects from gadolinium deposition, even with normal kidney function. This article summarizes the latest safety evidence of commercially available GBCAs with a focus on new agents, discusses updates to the ACR NSF GBCA safety classification, and describes approaches for strengthening the evidence needed for regulatory decisions.


Effect of rate of body weight gain of steers during the stocker phase. II. Visceral organ mass and body composition of growing-finishing beef cattle.
Two experiments were conducted to examine the effect of rate of BW gain during the stocker phase on visceral organ mass and body composition of growing-finishing cattle that had grazed dormant native range (DNR) or winter wheat pasture (WP). In each experiment, fall-weaned steers were allotted randomly to 1 of these stocker production programs: 1) control, 1.02 kg · steer(-1) · d(-1) of a 40% CP cottonseed meal-based supplement during grazing of DNR (CON); 2) corn/soybean meal-based supplement fed at 1% of BW during grazing of DNR (CORN); 3) grazing WP at a high stocking rate to achieve a reduced rate of BW gain (LGWP); and 4) grazing WP at a low stocking rate to achieve an increased rate of BW gain (HGWP). In Exp. 1, 3 steers per treatment were harvested after winter grazing (138 d). The remaining WP steers were transitioned into a finishing phase and DNR steers were allowed to graze the same pastures for another 115 d before entering a feedyard. In Exp. 2, steers grazed respective pastures until each treatment reached an estimated HCW of 200 kg (262, 180, 142, and 74 d, respectively, for CON, CORN, LGWP, and HGWP treatments), at which time 4 steers per treatment were randomly selected for intermediate harvest before finishing. At the end of the finishing period, 4 additional steers from each treatment were randomly selected for final carcass measurements. All steers were fed to a common 12th rib fat thickness of 1.27 cm. After winter grazing in Exp. 1, HGWP steers had the greatest (P < 0.01) mesenteric/omental fat, total viscera, total splanchnic tissue mass, and carcass and empty body fat, compared with the other treatments. In Exp. 2 at intermediate harvest, WP steers had greater (P < 0.03) mesenteric/omental fat, total viscera, and total splanchnic tissue mass, compared with CORN steers, with CON steers being intermediate. Also, the WP steers had greater (P < 0.02) carcass and empty body fat, compared with CORN steers, with CON steers being intermediate. At final harvest in Exp. 2, LGWP steers had the least total viscera and total splanchnic tissue mass, compared with the other treatments. However, there were no differences (P > 0.53) among treatments for carcass or empty body fat. Stocker systems using WP or DNR result in cattle with differences in body fat and visceral organ mass before finishing; this may influence feedlot efficiency, even though there were no differences in body fat and visceral organ mass at the end of the finishing period.


Evaluation of humoral immune function in patients with chronic idiopathic thrombocytopenic purpura.
Coincidence of autoimmune diseases such as immune thrombocytopenic purpura (ITP) with immunodeficiencies has been reported previously in patients who suffered from primary antibody deficiency (PAD). But there is no original study on immunological profiles of ITP patients to find out their probable immune deficiency. In this case-control study, ITP patients' humoral immunity was investigated for diagnosis of PAD in comparison with normal population. To evaluate the humoral immune system against polysaccharide antigens, patients' serum immunoglobulin levels were measured and a 23-valent pneumococcal capsular polysaccharide vaccine (PPV23) was administrated to evaluate the antibody response to vaccination. In this study, 14 out of 36 patients (39%) were diagnosed with antibody mediated immune deficiency including 2 patients (5.5%) with immunoglobulin class deficiency and 4 (11%) with IgG subclass deficiency. The remaining patients suffered from specific antibody deficiency. The most frequent deficiency in ITP patients was specific antibody deficiency.Therefore, immunological survey on ITP patients may be important especially for those who have undergone splenectomy.


Prevalence of Shiga toxin-producing Escherichia coli in ground beef and cattle feces from King County, Washington.
Shiga toxin-producing Escherichia coli (STEC) is increasingly recognized as a common cause of diarrhea. STEC infection is a major public health threat because of its ability to cause serious and potentially life-threatening illnesses. The main reservoirs of STEC are believed to be the intestinal tracts of animals. Several studies have investigated the prevalence of STEC in various food items. The objective of this study was to determine the prevalence of STEC in the Seattle ground beef supply. In addition, the relative amount of STEC contamination between stores was compared, and possible differences between types of ground beef based on fat content (9, 16, and 23%) were investigated. A survey of Stx-I and/or Stx-II genes in fecal samples from cattle at a local slaughterhouse was also conducted. Of 296 ground beef samples tested from area retail grocery stores, 16.8% were positive for the presence of the toxin genes. Our data showed that there was no statistically significant difference (P > 0.05) in the prevalence of STEC between the ground beef samples of different fat contents and between grocery store chains. Of the 103 cattle fecal samples tested, 19 (18.4%) were found positive for the presence of Stx-I and/or Stx-II genes. The presence of a rather high percentage of STEC in the food supply in the absence of large number of cases suggests that not all STEC lineages are pathogenic for humans.


Genetic diversity in populations of plants with different breeding and dispersal strategies in a free-flowing boreal river system.
We have studied the genetic diversity of three plant species: Angelica archangelica (Apiacieae), Bistorta vivipara (Polygonaceae) and Viscaria alpina (Caryophyllaceae) along the free-flowing Vindel River in northern Sweden. The plants differ in reproductive strategy. A archangelica and V. alpina are insect pollinated outbreeders while B. vivipara reproduces with apomixis through bulbils. The seeds of A. archangelica may float for over a year, while the propagules (seeds and bulbils, respectively) of V. alpina and B. vivipara float for less than two days. Genetic diversity was assessed using starch gel electrophoresis. The clonal diversity of B. vivipara measured by Simpson's index (D) ranged between 0.78 and 0.99. Only a few clones were shared between localities. The average percentages polymorphic loci and mean He based on polymorphic loci for V. alpina over all localities were 23.1 and 0.15, respectively. Wright's F-statistics showed a significant overall deficit of heterozygotes. The diversity of A. archangelica was found to increase downstream. Genetic diversity of each species is sufficiently high to be used in studies on hydrochory. Dispersal appears to be related to the floating ability of progagules.


Response to PCV13 vaccination in patients with multiple myeloma versus healthy controls.
Infections are a major cause of morbidity and mortality in individuals with multiple myeloma (MM). These individuals exhibit humoral dysfunction and show a suboptimal response to pneumococcal polysaccharide vaccine (PPV23). Since pneumococcal conjugate vaccine (PCV13) elicits a T cell dependent response, it is recommended in patients with multiple myeloma. This study compares the initial response to PCV13 and durability of the response at 6 months in patients with multiple myeloma versus normal controls. Seven patients with multiple myeloma and 18 control patients were enrolled in the study. Streptococcal pneumonia serotype IgG titers were drawn at baseline, day 30, and day 180 after MM patients and controls received PCV13. Although vaccination with PCV13 produced a similar initial response in patients with multiple myeloma compared to control subjects, the duration of response may have waned in patients with multiple myeloma as compared to control subjects.


GOOD GENES AND DIRECT SELECTION IN THE EVOLUTION OF MATING PREFERENCES.
A model is used to study quantitatively the impact of a good genes process and direct natural selection on the evolution of a mating preference. The expression of a male display trait is proportional to genetic quality, which is determined by the number of deleterious mutations a male carries throughout his genome. Genetic variances and covariances, including the covariance between the preference and male trait that drives the good genes process, are allowed to evolve under an infinitesimal model. Results suggest that the good genes process generates only weak indirect selection on preferences, with an effective selection intensity of a few percent or less. If preferences are subject to direct natural selection of the intensity observed for other characters, the good genes process alone is not expected to exaggerate the male trait by more than a few phenotypic standard deviations, contrary to what is observed in highly sexually selected species. Good genes can, however, cause substantial exaggeration if preference genes are nearly selectively neutral. Alternatively, direct selection on preference genes, acting on mating behavior itself or on the genes' pleiotropic effects, can cause mating preferences and male display traits to be exaggerated by any degree. Direct selection of preference genes may therefore play an important role in species that show extreme sexual selection.


Thyroid carcinomas that express telomerase follow a more aggressive clinical course in children and adolescents.
With each cell division, DNA is lost from the telomeres, limiting the number of divisions, and leading to senescence. Malignant tumors maintain immortality by expressing a specific DNA repair enzyme, telomerase, that replaces this DNA. We hypothesized that tumors which express telomerase would have the highest recurrence risk and we tested this by determining telomerase expression in 27 papillary thyroid carcinomas (PTC), 5 follicular thyroid carcinomas (FTC) and 13 benign thyroid lesions from children and adolescents. Patients were 6-21 yr of age (mean+/-SE=16.6+/-4.1 yr) and followed from 0-14.1 yr (mean+/-SE=4.71+/-3.5 yr). Original tumors were sectioned, and immunostained for telomerase. Telomerase-specific staining was determined by two independent, blind examiners and graded from absent (Grade 0) to intense (Grade 3). Telomerase was detected in a similar majority of benign (11/13, 85%) and malignant tumors (24/32, 75%). However, the intensity of telomerase expression was greater among FTC (mean+/-SE=2.4+/-0.5 relative intensity) followed by PTC (mean+/-SE=1.9+/-1.0 relative intensity) and benign tumors (mean+/-SE=1.8+/-1.0 relative intensity). Autoimmune lesions had lower telomerase expression (mean+/-SE=1.25+/-0.5 relative intensity) compared to FTC (p=0.01), PTC (p=0.06) and benign lesions (p=0.15). Among PTC, 19 (70%) expressed telomerase, and 8 (30%) did not. Direct invasion (no.=4, 21%), distant metastasis (no.=2, 10%) and recurrence (no.=7, 37%) developed exclusively in PTC that expressed telomerase (p=0.02). Disease-free survival was also shorter for PTC that expressed telomerase (p=0.06). Recurrence developed in 1/2 (50%) FTC that expressed telomerase. We conclude that childhood thyroid cancers which express telomerase have an increased risk of tissue invasion, metastasis, and recurrence.


Autophagic flux regulates microglial phenotype according to the time of oxygen-glucose deprivation/reperfusion.
Microglial phenotype alternation is a potential novel pathogenic mechanism for cerebral ischemia. Cerebral ischemia induced autophagy aggravates inflammation and neural injury. However, the effect of autophagy in the modulation of microglial phenotype is still unknown. In this study, we investigated the role of autophagic flux in the alternation of microglial phenotype following oxygen glucose deprivation/reperfusion (OGD/R) in BV-2 cells. Inhibition of autophagic flux by NH4Cl exposure significantly increased the level of microtubule-associated protein 1 light chain 3 (LC3)-II and p62 in control and OGD/R (12h, 24h and 48h) groups, but did not change their expression in OGD/R 72h group, indicating that autophagic flux was inhibited at OGD/R 72h. Once autophagic flux was inhibited at OGD/R 72h or at OGD/R 24h (with NH4Cl), BV-2 cells mainly showed M1 phenotype with increased tumor necrosis factor alpha (TNF-α), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and decreased M2 markers including interleukin-10 (IL-10), Arginase 1 (Arg-1), and brain derived neurotrophic factor (BDNF). Further study indicated that inhibition of autophagic flux activated NF-κB pathway and decreased the activity of cAMP-response element binding protein (CREB), which contributed to the alternation of microglial phenotype. Therefore, inhibition of autophagic flux regulated the alternation of microglial phenotype by modulating the balance between NF-κB and CREB.


Hahb-10, a sunflower homeobox-leucine zipper gene, is regulated by light quality and quantity, and promotes early flowering when expressed in Arabidopsis.
Homeodomain-leucine zipper proteins constitute a family of transcription factors found only in plants. Expression patterns of the sunflower homeobox-leucine zipper gene Hahb-10 (Helianthus annuus homeobox-10), that belongs to the HD-Zip II subfamily, were analysed. Northern blots showed that Hahb-10 is expressed primarily in mature leaves, although expression is clearly detectable in younger leaves and also in stems. Considerably higher expression levels were detected in etiolated seedlings compared with light-grown seedlings. Induction of Hahb-10 expression was observed when seedlings were subjected to treatment with gibberellins. Transgenic Arabidopsis thaliana plants that express Hahb-10 under the 35S cauliflower mosaic virus promoter show special phenotypic characteristics such as darker cotyledons and planar leaves. A reduction in the life cycle of about 25% allowing earlier seed collection was also observed, and this phenomenon is clearly related to a shortened flowering time. When the number of plants per pot increased, the difference in developmental rate between transgenic and non-transformed individuals became larger. After gibberellin treatment, the relative difference in life cycle duration was considerably reduced. Several light-regulated genes have been tested as possible target genes of Hahb-10. One of them, PsbS, shows a different response to illumination conditions in transgenic plants compared with the response in wild-type plants while the other genes behave similarly in both genotypes. We propose that Hahb-10 functions in a signalling cascade(s) that control(s) plant responses to light quality and quantity, and may also be involved in gibberellin transduction pathways.


Process optimization for production and purification of a thermostable, organic solvent tolerant lipase from Acinetobacter sp. AU07.
The purpose of this study was to isolate, purify and optimize the production conditions of an organic solvent tolerant and thermostable lipase from Acinetobacter sp. AU07 isolated from distillery waste. The lipase production was optimized by response surface methodology, and a maximum production of 14.5U/mL was observed at 30°C and pH 7, using a 0.5% (v/v) inoculum, 2% (v/v) castor oil (inducer), and agitation 150rpm. The optimized conditions from the shake flask experiments were validated in a 3L lab scale bioreactor, and the lipase production increased to 48U/mL. The enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography and the overall yield was 36%. SDS-PAGE indicated a molecular weight of 45kDa for the purified protein, and Matrix assisted laser desorption/ionization time of flight analysis of the purified lipase showed sequence similarity with GDSL family of lipases. The optimum temperature and pH for activity of the enzyme was found to be 50°C and 8.0, respectively. The lipase was completely inhibited by phenylmethylsulfonyl fluoride but minimal inhibition was observed when incubated with ethylenediaminetetraacetic acid and dithiothreitol. The enzyme was stable in the presence of non-polar hydrophobic solvents. Detergents like SDS inhibited enzyme activity; however, there was minimal loss of enzyme activity when incubated with hydrogen peroxide, Tween 80 and Triton X-100. The kinetic constants (Km and Vmax) revealed that the hydrolytic activity of the lipase was specific to moderate chain fatty acid esters. The Vmax, Km and Vmax/Km ratio of the enzyme were 16.98U/mg, 0.51mM, and 33.29, respectively when 4-nitrophenyl palmitate was used as a substrate.


Defucosylated anti-CCR4 monoclonal antibody exercises potent ADCC-mediated antitumor effect in the novel tumor-bearing humanized NOD/Shi-scid, IL-2Rgamma(null) mouse model.
There are no suitable small animal models to evaluate human antibody-dependent cellular cytotoxicity (ADCC) in vivo, due to species incompatibilities. Thus, the first aim of this study was to establish a human tumor-bearing mouse model in which human immune cells can engraft and mediate ADCC, but where the endogenous mouse immune cells cannot mediate ADCC. The second aim was to evaluate ADCC mediated in these humanized mice by the defucosylated anti-CC chemokine receptor 4 (CCR4) monoclonal antibody (mAb) which we have developed and which is now in phase I clinical trials. NOD/Shi-scid, IL-2Rgamma(null) (NOG) mice were the recipients of human immune cells, and CCR4-expressing Hodgkin lymphoma (HL) and cutaneous T-cell lymphoma (CTCL) cell lines were used as target tumors. Humanized mice have been established using NOG mice. The chimeric defucosylated anti-CCR4 mAb KM2760 showed potent antitumor activity mediated by robust ADCC in these humanized mice bearing the HL or CTCL cell lines. KM2760 significantly increased the number of tumor-infiltrating CD56-positive NK cells which mediate ADCC, and reduced the number of tumor-infiltrating FOXP3-positive regulatory T (Treg) cells in HL-bearing humanized mice. Anti-CCR4 mAb could be an ideal treatment modality for many different cancers, not only to directly kill CCR4-expressing tumor cells, but also to overcome the suppressive effect of Treg cells on the host immune response to tumor cells. In addition, using our humanized mice, we can perform the appropriate preclinical evaluation of many types of antibody based immunotherapy.


Interferon-alpha Induced Sarcoidosis with Cutaneous Involvement along the Lines of Venous Drainage.
Sarcoidosis is a systemic inflammatory disease of an unknown origin and it is characterized by the presence of noncaseating epitheloid cell granulomas in multiple organs. Herein we report on a case of cutaneous and pulmonary sarcoidosis that was associated with interferon alpha treatment for hepatitis C. A 39-year-old man, a former intravenous drug user, presented with several erythematous papules on both antecubital areas. The histopathologic finding of a skin biopsy showed noncaseating granuloma. The mediastinal and axillary lymph nodes were enlarged on chest X-ray and computed tomography. To the best of our knowledge, this is the first report of cutaneous and pulmonary sarcoidosis that was associated with interferon treatment in the Korean dermatologic literature.


Cefuroxime axetil.
Cefuroxime axetil is a orally active prodrug formulation of cefuroxime, which upon absorption undergoes immediate deesterification to free cefuroxime. Cefuroxime axetil offers an in vitro antibacterial spectrum against many gram-positive and some gram-negative organisms. Its beta-lactamase stability makes it useful in treating a variety of infections caused by beta-lactamase-producing strains of Haemophilus influenzae, Branhamella catarrhalis, and Staphylococcus aureus. Cefuroxime axetil has good activity against the Enterobacteriaceae and moderate activity against non-Bacteroides fragilis anaerobes. Clinical studies suggest it is at least as effective as ampicillin, amoxicillin, amoxicillin/clavulanic acid, penicillin V, or cefaclor in the treatment of uncomplicated urinary tract infections, acute otitis media, upper respiratory infections, skin and soft tissue infections, and uncomplicated gonorrhea.


Corynebacterium striatum: a diphtheroid with pathogenic potential.
Although nondiphtherial corynebacteria are ubiquitous in nature and commonly colonize the skin and mucous membranes of humans, they rarely account for clinical infection. Corynebacterium striatum has rarely been reported to be a pathogen, causing pleuropulmonary infections and bacteremia in only immunocompromised or anatomically altered patients. We noted C. striatum to be the infecting pathogen or copathogen in six patients. To our knowledge, this report describes the first cases of C. striatum causing infection of exist sites of central venous catheters, thrombophlebitis associated with central venous catheters, conjunctivitis, and chorioamnionitis as well as a possible pathogen contributing to peritonitis and pyogenic granuloma. Unlike Corynebacterium jeikeium, which is highly resistant to beta-lactam agents, aminoglycosides, and quinolones, all strains of C. striatum isolated from the patients described in this report were susceptible to vancomycin and aminoglycosides, and all strains except one were susceptible to penicillin G, imipenem, and ciprofloxacin. C. striatum should be recognized as a potential pathogen in both immunocompromised and normal hosts in the appropriate circumstances, and appropriate antimicrobial therapy can quickly lead to resolution of infection.


Erythrocyte Saturation with IgG Is Required for Inducing Antibody-Mediated Immune Suppression and Impacts Both Erythrocyte Clearance and Antigen-Modulation Mechanisms.
Anti-D prevents hemolytic disease of the fetus and newborn, and this mechanism has been referred to as Ab-mediated immune suppression (AMIS). Anti-D, as well as other polyclonal AMIS-inducing Abs, most often induce both epitope masking and erythrocyte clearance mechanisms. We have previously observed that some Abs that successfully induce AMIS effects could be split into those that mediate epitope masking versus those that induce erythrocyte clearance, allowing the ability to analyze these mechanisms separately. In addition, AMIS-inducing activity has recently been shown to induce Ag modulation (Ag loss from the erythrocyte surface). To assess these mechanisms, we immunized mice with transgenic murine RBCs expressing a single Ag protein comprising a recombinant Ag composed of hen egg lysozyme, OVA sequences comprising aa 251-349, and the human Duffy transmembrane protein (HOD-Ag) with serial doses of polyclonal anti-OVA IgG as the AMIS-inducing Ab. The anti-OVA Ab induced AMIS in the absence of apparent epitope masking. AMIS occurred only when the erythrocytes appeared saturated with IgG. This Ab was capable of inducing HOD-RBC clearance, as well as loss of the OVA epitope at doses of Ab that caused AMIS effects. HOD-RBCs also lost reactivity with Abs specific for the hen egg lysozyme and Duffy portions of the Ag consistent with the initiation of Ag modulation and/or trogocytosis mechanisms. These data support the concept that an AMIS-inducing Ab that does not cause epitope masking can induce AMIS effects in a manner consistent with RBC clearance and/or Ag modulation.


Loading of nitrate into the xylem: apoplastic nitrate controls the voltage dependence of X-QUAC, the main anion conductance in xylem-parenchyma cells of barley roots.
We report here that NO(3)(-) in the xylem exerts positive feedback on its loading into the xylem through a change in the voltage dependence of the Quickly Activating Anion Conductance, X-QUAC. Properties of this conductance were investigated on xylem-parenchyma protoplasts prepared from roots of Hordeum vulgare by applying the patch-clamp technique. Chord conductances were minimal around -40 mV and increased with plasma membrane depolarisation as well as with hyperpolarisation. Two gates with opposite voltage dependences were postulated. When 30 mM Cl- in the bath was replaced by NO(3)(-), a shift in the midpoint potential of the depolarisation-activated gate by about -60 mV from 43 to -16 mV occurred (K(m) = 3.4 mM). No such effect was seen when chloride was replaced by malate. Addition of 10 mM NO(3)(-)to the pipette solution and reduction of [Cl-] from 124 to 4 mM (to simulate cytoplasmic concentrations) did not interfere with the voltage dependence of X-QUAC activation, nor was it affected by changes in external [K+]. If only the NO(3)(-) effect on gating was considered, an increase of the NO(3)(-) concentration in the xylem sap to 5 mM would result in an enhancement of NO(3)(-) efflux by about 30%. Although the driving force for NO(3)(-) efflux would be reduced simultaneously, NO(3)(-) efflux into the xylem through X-QUAC would be maintained with high NO(3)(-) concentrations in the xylem sap; a situation which occurs for instance during the night.


Characterization of an antiserum to guinea pig antithrombin III (AT III). I. Reactivity with guinea pig T lymphocytes.
In the course of studies investigating the effects of antisera prepared against a variety of guinea pig proteins on lymphocyte function, a goat antiserum prepared against a guinea pig gamma-globulin preparation was found to react with guinea pig T lymphocytes. This antiserum, serum 592, contained a significant titer of antibodies that were cytotoxic for a subpopulation of lymph node cells and thymocytes, and mitogenic for lymph node T cells. Immunoelectrophoretic analysis and selective absorptions of the antiserum demonstrated that the antigen recognized on thymocytes was also present on an alpha 2 globulin in guinea pig plasma, which, on the basis of physiochemical characteristics and heparin-binding affinity, appeared to be guinea pig antithrombin III (AT III). Although the antiserum was shown to contain antibodies to both protein and carbohydrate determinants on the AT III molecule, studies comparing the effects of 7 M guanidine and periodate oxidation on the antigenicity of the AT III determinant also recognized on the thymocytes indicated that this shared antigenic determinant was carbohydrate in nature. The thymocyte membrane molecule bearing this determinant was also isolated and was found to be a 210,000-dalton macromolecule that was very sensitive to proteolytic and/or autolytic degradation. These data raise the interesting possibility that guinea pig lymphoid cells may have a membrane-associated protease inhibitor related to plasma AT III.


Engineering a Corneal Stromal Equivalent Using a Novel Multilayered Fabrication Assembly Technique.
To overcome the serious shortage of donor corneas for transplantation, alternatives based on tissue engineering need to be developed. Decellularized corneas are one potential alternative, but their densely packed collagen architecture inhibits recellularization in vitro. Therefore, a new rapid method of recellularizing these constructs to ensure high cellularity throughout the collagen scaffold is needed. In this study, we developed a novel method for fabricating corneal constructs by using decellularized porcine corneal sheets assembled using a bottom-up approach by layering multiple sheets between cell-laden collagen I hydrogel. Corneal lenticules were cut from porcine corneas by cryosectioning, then decellularized with detergents and air-dried for storage as sheets. Human corneal stromal cells were encapsulated in collagen I hydrogel and cast between the dried sheets. Constructs were cultured in serum-free medium supplemented with ascorbic acid and insulin for 2 weeks. Epithelial cells were then seeded on the surface and cultured for an additional week. Transparency, cell viability, and phenotype were analyzed by qPCR, histology, and immunofluorescence. Constructs without epithelial cells were sutured onto an ex vivo porcine cornea and cultured for 1 week. Lenticules were successfully decellularized, achieving dsDNA values of 13 ± 1.2 ng/mg dry tissue, and were more resistant to degradation than the collagen I hydrogels. Constructs maintained high cell viability with a keratocyte-like phenotype with upregulation of keratocan, decorin, lumican, collagen I, ALDH3A1, and CD34 and the corneal epithelial cells stratified with a cobblestone morphology. The construct was amenable to surgical handling and no tearing occurred during suturing. After 7 days ex vivo, constructs were covered by a neoepithelium from the host porcine cells and integration into the host stroma was observed. This study describes a novel approach toward fabricating anterior corneal substitutes in a simple and rapid manner, obtaining mature and suturable constructs using only tissue-derived materials.


Spontaneous Formation of an Air-Stable Radical upon the Direct Fusion of Diphenylmethane to a Triarylporphyrin.
The direct fusion of a diphenylmethane segment to a Ni(II) 5,10,15-triarylporphyrin with three linkages furnished an air- and moisture-stable neutral radical through unexpected and spontaneous oxidation. This radical was demetalated by treatment with H2 SO4 and CF3 CO2 H to provide the corresponding free-base radical. These porphyrin radicals are very stable owing to spin delocalization and have been fully characterized through UV/Vis/NIR absorption spectroscopy, X-ray crystallographic analysis, magnetic susceptibility measurements, electrochemical studies, laser-based ultrafast spectroscopic studies, and theoretical calculations. They were chemically oxidized and reduced to the corresponding cation and anion but did not react with hydrogen-atom donors.


Quantifying thiol ligand density of self-assembled monolayers on gold nanoparticles by inductively coupled plasma-mass spectrometry.
Gold nanoparticles (GNPs) are often used as colloidal carriers in numerous applications owing to their low-cost and size-controlled preparation as well as their straightforward surface functionalization with thiol containing molecules forming self-assembling monolayers (SAM). The quantification of the ligand density of such modified GNPs is technically challenging, yet of utmost importance for quality control in many applications. In this contribution, a new method for the determination of the surface coverage of GNPs with thiol containing ligands is proposed. It makes use of the measurement of the gold-to-sulfur (Au/S) ratio by inductively coupled plasma mass spectrometry (ICP-MS) and its dependence on the nanoparticle diameter. The simultaneous ICP-MS measurement of gold and sulfur was carefully validated and found to be a robust method with a relative standard uncertainty of lower than 10%. A major advantage of this method is the independence from sample preparation; for example, sample loss during the washing steps is not affecting the results. To demonstrate the utility of the straightforward method, GNPs of different diameters were synthesized and derivatized on the surface with bifunctional (lipophilic) ω-mercapto-alkanoic acids and (hydrophilic) mercapto-poly(ethylene glycol) (PEG)(n)-carboxylic acids, respectively, by self-assembling monolayer (SAM) formation. Thereby, a size-independent but ligand-chain length-dependent ligand density was found. The surface coverage increases from 4.3 to 6.3 molecules nm⁻² with a decrease of ligand chain length from 3.52 to 0.68 nm. Furthermore, no significant difference between the surface coverage of hydrophilic and lipophilic ligands with approximately the same ligand length was found, indicating that sterical hindrance is of more importance than, for example, intermolecular strand interactions of Van der Waals forces as claimed in other studies.


Following immunization antigen becomes concentrated in a limited number of APCs including B cells.
Immunization with the hen egg-white lysozyme (HEL) protein induces T cells to various of its peptide determinants. The distribution of such T cells, however, does not correlate with the peptide level of each epitope on class II molecules. For this reason, we sought information on the cells responsible for Ag presentation following immunization, hoping to understand the lack of immunodominance in this system. By tracking HEL, and the ensuing peptide/MHC complexes, we find the following: 1) that HEL in the draining lymph node gets concentrated in a limited number of APC, particularly in dendritic cells and macrophages, 2) that these APC are functionally capable of presenting both major and minor determinants of HEL over a 100-fold range of Ag dose, and 3) that B cells present Ag gained at early times after immunization, but only following higher dose immunization. These data indicate that the breadth of a response is maintained over a wide dosage range by concentration of Ag in a limited number of cells presenting high levels and a great diversity of epitopes.