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Abstract Background The Moloney murine leukaemia virus (Mo-MLV) gag gene encodes three main structural proteins, matrix, capsid and nucleocapsid and a protein called p12. In addition to its role during the late stages of infection, p12 has an essential, but undefined, function during early post-entry events. As these stages of retroviral infection remain poorly understood, we set out to investigate the function of p12. Results Examination of the infectivity of Mo-MLV virus-like particles containing a mixture of wild type and mutant p12 revealed that the N- and C-terminal regions of p12 are sequentially acting domains, both required for p12 function, and that the N-terminal activity precedes the C-terminal activity in the viral life cycle. By creating a panel of p12 mutants in other gammaretroviruses, we showed that these domains are conserved in this retroviral genus. We also undertook a detailed mutational analysis of each domain, identifying residues essential for function. These data show that different regions of the N-terminal domain are necessary for infectivity in different gammaretroviruses, in stark contrast to the C-terminal domain where the same region is essential for all viruses. Moreover, chimeras between the p12 proteins of Mo-MLV and gibbon ape leukaemia virus revealed that the C-terminal domains are interchangeable whereas the N-terminal domains are not. Finally, we identified potential functions for each domain. We observed that particles with defects in the N-terminus of p12 were unable to abrogate restriction factors, implying that their cores were impaired. We further showed that defects in the C-terminal domain of p12 could be overcome by introducing a chromatin binding motif into the protein. Conclusions Based on these data, we propose a model for p12 function where the N-terminus of p12 interacts with, and stabilizes, the viral core, allowing the C-terminus of p12 to tether the preintegration complex to host chromatin during mitosis, facilitating integration.

Abstract Background Detection of a retrovirus, xenotropic murine leukaemia virus-related virus (XMRV), has recently been reported in 67% of patients with chronic fatigue syndrome. We have studied a total of 170 samples from chronic fatigue syndrome patients from two UK cohorts and 395 controls for evidence of XMRV infection by looking either for the presence of viral nucleic acids using quantitative PCR (limit of detection Results We have not identified XMRV DNA in any samples by PCR (0/299). Some serum samples showed XMRV neutralising activity (26/565) but only one of these positive sera came from a CFS patient. Most of the positive sera were also able to neutralise MLV particles pseudotyped with envelope proteins from other viruses, including vesicular stomatitis virus, indicating significant cross-reactivity in serological responses. Four positive samples were specific for XMRV. Conclusions No association between XMRV infection and CFS was observed in the samples tested, either by PCR or serological methodologies. The non-specific neutralisation observed in multiple serum samples suggests that it is unlikely that these responses were elicited by XMRV and highlights the danger of over-estimating XMRV frequency based on serological assays. In spite of this, we believe that the detection of neutralising activity that did not inhibit VSV-G pseudotyped MLV in at least four human serum samples indicates that XMRV infection may occur in the general population, although with currently uncertain outcomes.

Left ventricular assist devices (LVAD) are increasingly used for management of heart failure; infection remains a frequent complication. Phage therapy has been successful in a variety of antibiotic refractory infections and is of interest in treating LVAD infections. We performed a retrospective review of four patients that underwent five separate courses of intravenous (IV) phage therapy with concomitant antibiotic for treatment of endovascular Pseudomonas aeruginosa LVAD infection. We assessed phage susceptibility, bacterial strain sequencing, serum neutralization, biofilm activity, and shelf-life of phage preparations. Five treatments of one to four wild-type virulent phage(s) were administered for 14-51 days after informed consent and regulatory approval. There was no successful outcome. Breakthrough bacteremia occurred in four of five treatments. Two patients died from the underlying infection. We noted a variable decline in phage susceptibility following three of five treatments, four of four tested developed serum neutralization, and prophage presence was confirmed in isolates of two tested patients. Two phage preparations showed an initial titer drop. Phage biofilm activity was confirmed in two. Phage susceptibility alone was not predictive of clinical efficacy in P. aeruginosa endovascular LVAD infection. IV phage was associated with serum neutralization in most cases though lack of clinical effect may be multifactorial including presence of multiple bacterial isolates with varying phage susceptibility, presence of prophages, decline in phage titers, and possible lack of biofilm activity. Breakthrough bacteremia occurred frequently (while the organism remained susceptible to administered phage) and is an important safety consideration., Competing Interests: Saima Aslam: Research funding support from the Cystic Fibrosis Foundation, Armata Pharmaceuticals, Adaptive Phage Therapeutics, and Contrafect Inc. Consultant for BiomX and Phico Therapeutics. Medical advisory board for Pherecydes Pharma and Phiogen. Dwayne Roach: none. Mikeljon P. Nikolich: Royalty-bearing Biological Material License Agreement with Adaptive Phage Therapeutics exists now but work presented in this manuscript predated it. Patent PCT/US22/73852, METHOD OF TREATING DRUG RESISTANT ESKAPE PATHOGENS USING THERAPEUTIC BACTERIOPHAGES was filed, but work reported in this manuscript predated the filing. Biswajit Biswas: Navy Work Unit # A1417. Dr. Biswas has a patent "Bacteriophage compositions and methods of selection of components against specific bacteria" US patent #10357522, which was licensed before. Robert T Schooley: Consulting fees from GSK, LyseNtech. Kimberley A. Lilly-Bishop: none. Gregory K. Rice: none. Regina Z. Cer: none. Theron Hamilton: none. Mathew Henry: Mr. Henry has a patent 10357522 licensed. Tiffany Luong: none. Ann-Charlott Salabarria: none. Laura Sisk-Hackworth: none. Andrey A. Filippov: Pending Patent PCT/US22/73852, METHOD OF TREATING DRUG RESISTANT ESKAPE PATHOGENS USING THERAPEUTIC BACTERIOPHAGES Francois Lebreton: none. Lindsey Hall: none. Ran Nir-Paz: Consultant for BiomX; and has participated and served as a PI and on Data Safety Monitoring Board for a clinical trial by Technophage. Hadil Onalla: none. Gilat Livni: none. Eran Shostak: none. Anat Weider-Feinsod: none. Dafna Yahav: none. Ortal Yerushalmy: none. Sivan Alkalay-Oren: none. Leron Khalifa: none. Amit Rimon: none. Daniel Gelman: none. Ronen Hazan: none.

Purpose: The COVID-19 pandemic represents a consequential moment of disruption for medical training that has far-reaching implications for professional identity formation (PIF). To date, this has not been studied. As medical education grapples with a postpandemic era, it is essential to gain insight into how the pandemic has influenced PIF to better support its positive influences and mitigate its more detrimental effects. This study examined how PIF occurred during the COVID-19 pandemic to better adapt future medical training., Method: Constructivist grounded theory guided the iterative data collection and analyses. The authors conducted semistructured group interviews with 24 Ontario internal medicine residents in postgraduate years (PGYs) 1 to 3 between November 2020 and July 2021. Participants were asked to reflect on their day-to-day clinical and learning experiences during the pandemic., Results: Twenty-four internal medicine residents were interviewed (12 PGY-1 [50.0%], 9 PGY-2 [37.5%], and 3 PGY-3 [12.5%]). Participants described how navigating patient care and residency training through the pandemic consistently drew their attention to various system problems. How participants responded to these problems was shaped by an interplay among their personal values, their level of personal wellness or burnout, self-efficacy, institutional values, and the values of their supervisors and work community. As they were influenced by these factors, some were led toward acting on the problem(s) they identified, whereas others had a sense of resignation and deferred action. These interactions were evident in participants' experiences with communication, advocacy, and learning., Conclusions: Residents' professional identities are continuously shaped by how they perceive, reconcile, and address various challenges. As residents navigate tensions between personally held values and apparent system values, individuals in supervisory positions should be mindful of their influence as role models who empower values and practices that are recognized by participants to be important aspects of physician identity., (Copyright © 2023 the Association of American Medical Colleges.)

BACKGROUND. Incidental homogeneous renal masses are frequently encountered at portal venous phase CT. The American College of Radiology Incidental Findings Committee's white paper on renal masses recommends additional imaging for incidental homogeneous renal masses greater than 20 HU, but single-center data and the Bosniak classification version 2019 suggest the optimal attenuation threshold for detecting solid masses should be higher. OBJECTIVE. The purpose of this article is to determine the clinical importance of small (10-40 mm) incidentally detected homogeneous renal masses measuring 21-39 HU at portal venous phase CT. METHODS. We performed a 12-institution retrospective cohort study of adult patients who underwent portal venous phase CT for a nonrenal indication. The date of the first CT at each institution ranged from January 1, 2008, to January 1, 2014. Consecutive reports from 12,167 portal venous phase CT examinations were evaluated. Images were reviewed for 4529 CT examinations whose report described a focal renal mass. Eligible masses were 10-40 mm, well-defined, subjectively homogeneous, and 21-39 HU. Of these, masses that were shown to be solid without macroscopic fat; classified as Bosniak IIF, III, or IV; or confirmed to be malignant were considered clinically important. The reference standard was renal mass protocol CT or MRI, ultrasound of definitively benign cysts or solid masses, single-phase contrast-enhanced CT or unenhanced MRI showing no growth or morphologic change for 5 years or more, or clinical follow-up 5 years or greater. A reference standard was available for 346 masses in 300 patients. The 95% CIs were calculated using the binomial exact method. RESULTS. Eligible masses were identified in 4.2% of patients (514/12,167; 95% CI, 3.9-4.6%). Of 346 masses with a reference standard, none were clinically important (0%; 95% CI, 0-0.9%). Mean mass size was 17 mm; 72% (248/346) measured 21-30 HU, and 28% (98/346) measured 31-39 HU. CONCLUSION. Incidental small homogeneous renal masses measuring 21-39 HU at portal venous phase CT are common and highly likely benign. CLINICAL IMPACT. The change in attenuation threshold signifying the need for additional imaging from greater than 20 HU to greater than 30 HU proposed by the Bosniak classification version 2019 is supported.

Purpose: A discordance, predominantly towards overtreatment, exists between patients' expressed preferences for life-sustaining interventions and those documented at hospital admission. This quality improvement study sought to assess this discordance at our institution. Secondary objectives were to explore if internal medicine (IM) teams could identify patients who might benefit from further conversations and if the discordance can be reconciled in real-time., Methods: Two registered nurses were incorporated into IM teams at a tertiary hospital to conduct resuscitation preference conversations with inpatients either specifically referred to them (group I, n = 165) or randomly selected (group II, n = 164) from 1 August 2016 to 31 August 2018. Resuscitation preferences were documented and communicated to teams prompting revised resuscitation orders where appropriate. Multivariable logistic regression was used to determine potential risk factors for discordance., Results: Three hundred and twenty-nine patients were evaluated with a mean (standard deviation) age of 80 (12) and Charlson Comorbidity Index Score of 6.8 (2.6). Discordance was identified in 63/165 (38%) and 27/164 (16%) patients in groups I and II respectively. 42/194 patients (21%) did not want cardiopulmonary resuscitation (CPR) and 15/36 (41%) did not prefer intensive care unit (ICU) admission, despite these having been indicated in their initial preferences. 93% (84/90) of patients with discordance preferred de-escalation of care. Discordance was reconciled in 77% (69/90) of patients., Conclusion: Hospitalized patients may have preferences documented for CPR and ICU interventions contrary to their preferences. Trained nurses can identify inpatients who would benefit from further in-depth resuscitation preference conversations. Once identified, discordance can be reconciled during the index admission.

Objectives: To determine whether delay of initial anatomy ultrasound based on the maternal body mass index (BMI) reduces the rate of inadequate visualization compared to standard timing at 18 0/7 to 19 6/7 weeks., Methods: A retrospective study of singleton anatomy assessments was conducted at a tertiary care center in the 2-year period before (A, 2012-2014) and after (B, 2014-2016) protocol initiation. Assessments in period B were scheduled on the basis of the BMI in the first trimester: lower than 25 kg/m 2 , 18 0/7 to 19 6/7 weeks; 25 to 29.9 kg/m 2 , 19 0/7 to 20 6/7 weeks; 30 to 34.9 kg/m 2 , 20 0/7 to 21 6/7 weeks; 35 to 39.9 kg/m 2 , 21 0/7 to 22 6/7 weeks; and 40 kg/m 2 or higher, 22 0/7 to 23 6/7 weeks. In period A, assessments were scheduled between 18 0/7 and 19 6/7 weeks. The rate of inadequate visualization and repeated assessments in periods A and B were compared. Multivariable logistic regression, per-protocol, and BMI subgroup analyses were completed., Results: In total, 3491 pregnancies in period A and 3672 in period B were included. In period B, 74% were scheduled per protocol; however, this rate decreased for higher-BMI categories (52% for BMI ≥40 kg/m 2 ). The inadequate visualization rate was slightly higher in period B versus A (16.9% versus 15.0%; P = .03) and exceeded 35% for a BMI of 40 kg/m 2 or higher, with or without delay. After adjusting for maternal age and fetal presentation, period B had small increased odds of inadequate visualization versus period A (adjusted odds ratio, 1.2; 95% confidence interval, 1.02-1.38). Repeated assessment rates were similar in periods B and A (14.0% versus 13.1%; P = .25)., Conclusions: In pregnancies with obesity, a protocol delaying the initial assessment beyond 19 6/7 weeks based on the maternal BMI does not reduce the rate of inadequate visualization., (© 2020 by the American Institute of Ultrasound in Medicine.)

A lack of understanding of specific immune defects underlying canine immune-mediated diseases hampers optimal therapy. Failure to tailor treatment to an individual's immune abnormality can result in lack of efficacy, secondary complications, added expense, and drug-potentiated adverse effects. We adopted a small-volume whole-blood flow cytometric assay to determine the effect of immunosuppressant drugs on T-lymphocyte proliferation. Using healthy dogs in this proof-of-principle study, we hypothesized that there would be dose-dependent suppression of T-lymphocyte proliferation in response to dexamethasone, cyclosporine, mycophenolic acid, and the active metabolite of leflunomide (A77 1726). Whole blood was collected from 6 healthy pet dogs and incubated for 4 d with or without the mitogens concanavalin A and lipopolysaccharide and with increasing concentrations of immunosuppressant. Samples were subsequently stained with viability dye and with antibodies against the pan-T-lymphocyte marker CD5 and the cell proliferation marker Ki67. Percentages of proliferating T-lymphocytes were determined by flow cytometry, and the 50% inhibitory concentration (IC 50 ) was calculated. Inhibition of T-lymphocyte proliferation by the panel of immunosuppressants was shown to be dose-dependent, with marked variability among the dogs. The mean IC 50 was 394.8 ± 871 (standard deviation) μM for dexamethasone, 18.89 ± 36.2 ng/mL for cyclosporine, 106.3 ± 157.7 nM for mycophenolic acid, and 3.746 ± 6.8 μM for A77 1726. These results support the use of this assay for detecting the efficacy of individual immunosuppressants used to diminish T-lymphocyte proliferation. In future, the assay may be applied to pet dogs with spontaneous immune-mediated disease to help tailor individual treatment., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

Species have different strategies for loading sugars into the phloem, which vary in the route that sugars take to enter the phloem and the energetics of sugar accumulation. Species with passive phloem loading are hypothesized to have less flexibility in response to changes in some environmental conditions because sucrose export from mesophyll cells is dependent on fixed anatomical plasmodesmatal connections. Passive phloem loaders also have high mesophyll sugar content, and may be less likely to exhibit sugar-mediated down-regulation of photosynthetic capacity at elevated CO 2 concentrations. To date, the effect of phloem loading strategy on the response of plant carbon metabolism to rising atmospheric CO 2 concentrations is unclear, despite the widespread impacts of rising CO 2 on plants. Over three field seasons, five species with apoplastic loading, passive loading, or polymer-trapping were grown at ambient and elevated CO 2 concentration in free air concentration enrichment plots. Light-saturated rate of photosynthesis, photosynthetic capacity, leaf carbohydrate content, and anatomy were measured and compared among the species. All five species showed significant stimulation in midday photosynthetic CO 2 uptake by elevated CO 2 even though the two passive loading species showed significant down-regulation of maximum Rubisco carboxylation capacity at elevated CO 2 . There was a trend toward greater starch accumulation at elevated CO 2 in all species, and was most pronounced in passive loaders. From this study, we cannot conclude that phloem loading strategy is a key determinant of plant response to elevated CO 2 , but compelling differences in response counter to our hypothesis were observed. A phylogenetically controlled experiment with more species may be needed to fully test the hypothesis.

Lipoxygenases catalyze the oxygenation of polyunsaturated fatty acids and their derivatives to produce conjugated diene hydroperoxides. Soybean lipoxygenase-1 (SBLO-1) has been the subject of intensive structural and mechanistic study, but the manner in which this enzyme binds substrates is uncertain. Previous studies suggest that the fatty acyl group of the substrate binds in an internal cavity near the catalytic iron with the polar end at the surface of the protein or perhaps external to the protein. To test this model, we have investigated two pairs of enantiomeric N-linoleoylamino acids as substrates for SBLO-1. If the amino acid moiety binds external to the protein, the kinetics and product distribution should show little or no sensitivity to the stereochemical configuration of the amino acid moiety. Consistent with this expectation, N-linoleoyl-l-valine (LLV) and N-linoleoyl-d-valine (LDV) are both good substrates with k cat /K m values that are equal within error and about 40% higher than k cat /K m for linoleic acid. Experiments with N-linoleoyl-l-tryptophan (LLT) and N-linoleoyl-d-tryptophan (LDT) were complicated by the low critical micelle concentrations (CMC = 6-8 μM) of these substances. Below the CMC, LDT is a better substrate by a factor of 2.7. The rates of oxygenation of LDT and LLT continue to rise above the CMC, with modest stereoselectivity in favor of the d enantiomer. With all of the substrates tested, the major product is the 13(S)-hydroperoxide, and the distribution of minor products is not appreciably affected by the configuration of the amino acid moiety. The absence of stereoselectivity with LLV and LDV, the modest magnitude of the stereoselectivity with LLT and LDT, and the ability micellar forms of LLT and LDT to increase the concentration of available substrate are all consistent with the hypothesis that the amino acid moiety binds largely external to SBLO-1 and interacts with it only weakly., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Misty mice (m/m) have a loss of function mutation in Dock7 gene, a guanine nucleotide exchange factor, resulting in low bone mineral density, uncoupled bone remodeling and reduced bone formation. Dock7 has been identified as a modulator of osteoblast number and in vitro osteogenic differentiation in calvarial osteoblast culture. In addition, m/m exhibit reduced preformed brown adipose tissue innervation and temperature as well as compensatory increase in beige adipocyte markers. While the low bone mineral density phenotype is in part due to higher sympathetic nervous system (SNS) drive in young mice, it is unclear what effect aging would have in mice homozygous for the mutation in the Dock7 gene. We hypothesized that age-related trabecular bone loss and periosteal envelope expansion would be altered in m/m. To test this hypothesis, we comprehensively characterized the skeletal phenotype of m/m at 16, 32, 52, and 78wks of age. When compared to age-matched wild-type control mice (+/+), m/m had lower areal bone mineral density (aBMD) and areal bone mineral content (aBMC). Similarly, both femoral and vertebral BV/TV, Tb.N, and Conn.D were decreased in m/m while there was also an increase in Tb.Sp. As low bone mineral density and decreased trabecular bone were already present at 16wks of age in m/m and persisted throughout life, changes in age-related trabecular bone loss were not observed highlighting the role of Dock7 in controlling trabecular bone acquisition or bone loss prior to 16wks of age. Cortical thickness was also lower in the m/m across all ages. Periosteal and endosteal circumferences were higher in m/m compared to +/+ at 16wks. However, endosteal and periosteal expansion were attenuated in m/m, resulting in m/m having lower periosteal and endosteal circumferences by 78wks of age compared to +/+, highlighting the critical role of Dock7 in appositional bone expansion. Histomorphometry revealed that osteoblasts were nearly undetectable in m/m and marrow adipocytes were elevated 3.5 fold over +/+ (p=0.014). Consistent with reduced bone formation, osteoblast gene expression of Alp, Col1a1, Runx-2, Sp7, and Bglap was significantly decreased in m/m whole bone. Furthermore, markers of osteoclasts were either unchanged or suppressed. Bone marrow stromal cell migration and motility were inhibited in culture and changes in senescence markers suggest that osteoblast function may also be inhibited with loss of Dock7 expression in m/m. Finally, increased Oil Red O staining in m/m ear mesenchymal stem cells during adipogenesis highlights a potential shift of cells from the osteogenic to adipogenic lineages. In summary, loss of Dock7 in the aging m/m resulted in an impairment of periosteal and endocortical envelope expansion, but did not alter age-related trabecular bone loss. These studies establish Dock7 as a critical regulator of both cortical and trabecular bone mass, and demonstrate for the first time a novel role of Dock7 in modulating compensatory changes in the periosteum with aging., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Targeted gene mutation in the mouse is a primary strategy to understand gene function and relation to phenotype. The Knockout Mouse Project (KOMP) had an initial goal to develop a public resource of mouse embryonic stem (ES) cell clones that carry null mutations in all genes. Indeed, many useful novel mouse models have been generated from publically accessible targeted mouse ES cell lines. However, there are limitations, including incorrect targeting or cassette structure, and difficulties with germline transmission of the allele from chimeric mice. In our experience, using a small sample of targeted ES cell clones, we were successful ∼50% of the time in generating germline transmission of a correctly targeted allele. With the advent of CRISPR/Cas9 as a mouse genome modification tool, we assessed the efficiency of creating a conditional targeted allele in one gene, dedicator of cytokinesis 7 (Dock7), for which we were unsuccessful in generating a null allele using a KOMP targeted ES cell clone. The strategy was to insert loxP sites to flank either exons 3 and 4, or exons 3 through 7. By coinjecting Cas9 mRNA, validated sgRNAs, and oligonucleotide donors into fertilized eggs from C57BL/6J mice, we obtained a variety of alleles, including mice homozygous for the null alleles mediated by nonhomologous end joining, alleles with one of the two desired loxP sites, and correctly targeted alleles with both loxP sites. We also found frequent mutations in the inserted loxP sequence, which is partly attributable to the heterogeneity in the original oligonucleotide preparation., (Copyright © 2016 Bishop et al.)

Background: Interest in the use of digital game technologies by older adults is growing across disciplines from health and gerontology to computer science and game studies. The objective of this scoping review was to examine research evidence involving the oldest old (persons 85 years of age or greater) and digital game technology., Materials and Methods: PubMed, CINHAL, and Scopus were searched, and 46 articles were included in this review., Results: Results highlighted that 60 percent of articles were published in gerontological journals, whereas only 8.7 percent were published in computer science journals. No studies focused directly on the oldest old population. Few studies included sample sizes greater than 100 participants. Seven primary and 34 secondary themes were identified, of which Hardware Technology and Assessment were the most common., Conclusions: Existing evidence demonstrates the paucity of studies engaging older adults 85 years of age and above regarding the use of digital gaming and highlights a new understudied cohort for further research focus. Recommendations for future research include intentional recruitment and proportionate representation of participants ≥85 years of age, large sample sizes, and explicit mention of specific numbers of participants ≥85 years of age, which are necessary to advance knowledge in this area. Integrating a rigorous and robust mixed-methods approach including theoretical perspectives would lend itself to further in-depth understanding and knowledge generation in this field.

Background: We implemented oral fluid (OF) as an alternative specimen type to urine for detection of cocaine (COC) and opiate abuse in outpatient addiction medicine clinics., Methods: We implemented a 2-μg/L limit of quantification OF LC-MS/MS assay and compiled and reviewed all findings from a 22-month collection period for COC, benzoylecgonine (BZE), codeine (COD), 6-acetylmorphine (MAM), and morphine (MOR). We also compared the results of our clinical samples at different OF cutoffs and analytes specified in the new 2015 SAMHSA OF guidelines., Results: Of 3608 OF samples, COC and BZE were positive in 593 and 508, respectively. COC or BZE was positive in 662 samples. Importantly and unexpectedly, 154 samples were COC positive and BZE negative, with 125 having COC 2.0-7.9 μg/L. A simulation with the new guideline cutoffs confirmed 65% (430 of 662) of all COC- or BZE-positive data set samples. Similarly, the new guidelines confirmed 44% (263 of 603) of data set samples positive for MOR or COD. Simulation found that the new, lower MAM guideline cutoffs detected 89% of the 382 MAM-positive samples in the data set, 104 of which the new guidelines had identified as negative for MOR and COD., Conclusions: COC (not BZE) is the dominant low-concentration OF analyte in an addiction medicine setting. This information will aid OF test interpretation. It also illustrates the importance of the 2015 guideline's new immunoassay cross-reactivity requirements and the likely improvement in detection of heroin use stemming from the new, lower MAM cutoffs., (© 2016 American Association for Clinical Chemistry.)

Rising atmospheric [CO2] is a uniform, global change that increases C3 photosynthesis and could offset some of the negative effects of global climate change on crop yields. Genetic variation in yield responsiveness to rising [CO2] would provide an opportunity to breed more responsive crop genotypes. A multi-year study of 18 soybean (Glycine max Merr.) genotypes was carried out to identify variation in responsiveness to season-long elevated [CO2] (550 ppm) under fully open-air replicated field conditions. On average across 18 genotypes, elevated [CO2] stimulated total above-ground biomass by 22%, but seed yield by only 9%, in part because most genotypes showed a reduction in partitioning of energy to seeds. Over four years of study, there was consistency from year to year in the genotypes that were most and least responsive to elevated [CO2], suggesting heritability of CO2 response. Further analysis of six genotypes did not reveal a photosynthetic basis for the variation in yield response. Although partitioning to seed was decreased, cultivars with the highest partitioning coefficient in current [CO2 ] also had the highest partitioning coefficient in elevated [CO2]. The results show the existence of genetic variation in soybean response to elevated [CO2], which is needed to breed soybean to the future atmospheric environment., (© 2014 John Wiley & Sons Ltd.)

Receptor activator of NF-κB ligand (RANKL) is a TNFα-like cytokine that is produced by a diverse set of lineage-specific cells and is involved in a wide variety of physiological processes that include skeletal remodeling, lymph node organogenesis, mammary gland development, and thermal regulation. Consistent with these diverse functions, control of RANKL expression is accomplished in a cell-specific fashion via a set of at least 10 regulatory enhancers that are located up to 170 kb upstream of the gene's transcriptional start site. Here we examined the in vivo consequence of introducing a contiguous DNA segment containing these components into a genetically deleted RANKL null mouse strain. In contrast to RANKL null littermates, null mice containing the transgene exhibited normalized body size, skeletal development, and bone mass as well as normal bone marrow cavities, normalized spleen weights, and the presence of developed lymph nodes. These mice also manifested normalized reproductive capacity, including the ability to lactate and to produce normal healthy litters. Consistent with this, the transgene restored endogenous-like RANKL transcript levels in several RANKL-expressing tissues. Most importantly, restoration of RANKL expression from this segment of DNA was fully capable of rescuing the complex aberrant skeletal and immune phenotype of the RANKL null mouse. RANKL also restored appropriate levels of B220+ IgM+ and B220+ IgD+ B cells in spleen. Finally, we found that RANKL expression from this transgene was regulated by exogenously administered 1,25(OH)2 D3 , parathyroid hormone (PTH), and lipopolysaccharide (LPS), thus recapitulating the ability of these same factors to regulate the endogenous gene. These findings fully highlight the properties of the Tnfsf11 gene locus predicted through previous in vitro dissection. We conclude that the mouse Tnfsf11 gene locus identified originally through unbiased chromatin immunoprecipitation with DNA microarray (ChIP-chip) analysis contains the necessary genetic information to direct appropriate tissue-specific and factor-regulated RANKL expression in vivo., Competing Interests: All authors state that they have no conflicts of interest., (© 2014 American Society for Bone and Mineral Research.)

In addition to osteoblast lineage cells, the TNF-like factor receptor activator of NF-κB ligand (RANKL) is expressed in both B and T cells and may play a role in bone resorption. Rankl gene (Tnfsf11) expression in mouse T cells is mediated through multiple distal elements marked by increased transcription factor occupancy, histone tail acetylation, and RNA polymerase II recruitment. Little is known, however, of the regulation of human TNFSF11 in T cells. Accordingly, we examined the consequence of T cell activation on the expression of this factor both in Jurkat cells and in primary human T cells. We then explored the mechanism of this regulation by scanning over 400 kb of DNA surrounding the TNFSF11 locus for regulatory enhancers using ChIP-chip analysis. Histone H3/H4 acetylation enrichment identified putative regulatory regions located between -170 and -220 kb upstream of the human TNFSF11 TSS that we designated the human T cell control region (hTCCR). This region showed high sequence conservation with the mouse TCCR. Inhibition of MEK1/2 by U0126 resulted in decreased RANKL expression suggesting that stimulation through MEK1/2 was a prerequisite. ChIP-chip analysis also revealed that c-FOS was recruited to the hTCCR as well. Importantly, both the human TNFSF11 D5a/b (RLD5a/b) enhancer and segments of the hTCCR mediated robust inducible reporter activity following TCR activation. Finally, SNPs implicated in diseases characterized by dysregulated BMD co-localized to the hTCCR region. We conclude that the hTCCR region contains a cell-selective set of enhancers that plays an integral role in the transcriptional regulation of the TNFSF11 gene in human T cells., (© 2014 Wiley Periodicals, Inc.)

Osteocytes are derived from osteoblast lineage cells that become progressively embedded in mineralized bone. Development of the osteocytogenic cell line IDG-SW3 has enabled a temporal and mechanistic investigation of this process. Through RNA-sequencing analyses, we show that although substantial changes in gene expression occur during the osteoblast to osteocyte transition, the majority of the transcriptome remains qualitatively osteoblast like. Genes either up-regulated or expressed uniquely in the osteocyte include local and systemic factors such as Sost and Fgf23 as well as genes implicated in neuronal, muscle, vascular, or regulatory function. As assessed by chromatin immunoprecipitation coupled to high-throughput sequencing, numerous changes in epigenetic histone modifications also occur during osteocytogenesis; these are largely qualitative rather than quantitative. Specific epigenetic changes correlate with altered gene expression patterns that are observed during the transition. These genomic changes likely influence the highly restricted transcriptomic response to 1,25(OH)(2)D(3) that occurs during differentiation. VDR binding in osteocytes revealed an extensive cistrome co-occupied by retinoid X receptor and located predominantly at sites distal to regulated genes. Although sites of VDR binding were apparent near many 1,25(OH)(2)D(3)-regulated genes, the expression of others adjacent to VDR-binding sites were unaffected; lack of VDR binding was particularly prevalent at down-regulated genes. Interestingly, 1,25(OH)(2)D(3) was found to induce the Boc and Cdon coreceptors that are active in hedgehog signaling in osteocytes. We conclude that osteocytogenesis is accompanied by changes in gene expression that may be driven by both genetic and epigenetic components. These changes are likely responsible for the osteocyte phenotype and may contribute to reduced sensitivity to 1,25(OH)(2)D(3).

The biological actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are mediated by the vitamin D receptor (VDR), which is expressed in numerous target tissues in a cell type-selective manner. Recent studies using genomic analyses and recombineered bacterial artificial chromosomes (BACs) have defined the specific features of mouse and human VDR gene loci in vitro. In the current study, we introduced recombineered mouse and human VDR BACs as transgenes into mice and explored their expression capabilities in vivo. Individual transgenic mouse strains selectively expressed BAC-derived mouse or human VDR proteins in appropriate vitamin D target tissues, thereby recapitulating the tissue-specific expression of endogenous mouse VDR. The mouse VDR transgene was also regulated by 1,25(OH)2D3 and dibutyryl-cAMP. When crossed into a VDR-null mouse background, both transgenes restored wild-type basal as well as 1,25(OH)2D3-inducible gene expression patterns in the appropriate tissues. This maneuver resulted in the complete rescue of the aberrant phenotype noted in the VDR-null mouse, including systemic features associated with altered calcium and phosphorus homeostasis and disrupted production of parathyroid hormone and fibroblast growth factor 23, and abnormalities associated with the skeleton, kidney, parathyroid gland, and the skin. This study suggests that both mouse and human VDR transgenes are capable of recapitulating basal and regulated expression of the VDR in the appropriate mouse tissues and restore 1,25(OH)2D3 function. These results provide a baseline for further dissection of mechanisms integral to mouse and human VDR gene expression and offer the potential to explore the consequence of selective mutations in VDR proteins in vivo.

Progesterone (P4) stimulates proliferation of the mammary epithelium by a mechanism that involves paracrine signaling mediated from progesterone receptor (PR)-positive to neighboring PR-negative cells. Here we used a primary mouse mammary epithelial cell (MEC) culture system to define the molecular mechanism by which P4 regulates the expression of target gene effectors of proliferation including the paracrine factor receptor and activator of nuclear factor κB ligand (RANKL). MECs from adult virgin mice grown and embedded in three-dimensional basement-membrane medium resemble mammary ducts in vivo structurally and with respect to other properties including a heterogeneous pattern of PR expression, P4 induction of RANKL and other target genes in a PR-dependent manner, and a proliferative response to progestin. RANKL was demonstrated to have multiple functional P4-responsive enhancers that bind PR in a hormone-dependent manner as detected by chromatin immunoprecipitation assay. P4 also stimulated recruitment of signal transducer and activator of transcription (Stat)5a to RANKL enhancers through an apparent tethering with PR. Analysis of primary MECs from Stat5a knockout mice revealed that P4 induction of RANKL and a broad range of other PR target genes required Stat5a, as did P4-stimulated cell proliferation. In the absence of Stat5a, PR binding was lost at selective RANKL enhancers but was retained with others, suggesting that Stat5a acts to facilitate PR DNA binding at selective sites and to function as a coactivator with DNA-bound PR at others. These results show that RANKL is a direct PR target gene and that Stat5a has a novel role as a cofactor in PR-mediated transcriptional signaling in the mammary gland.

Fat mass may be modulated by the number of brown-like adipocytes in white adipose tissue (WAT) in humans and rodents. Bone remodeling is dependent on systemic energy metabolism and, with age, bone remodeling becomes uncoupled and brown adipose tissue (BAT) function declines. To test the interaction between BAT and bone, we employed Misty (m/m) mice, which were reported be deficient in BAT. We found that Misty mice have accelerated age-related trabecular bone loss and impaired brown fat function (including reduced temperature, lower expression of Pgc1a, and less sympathetic innervation compared to wild-type (+/ +)). Despite reduced BAT function, Misty mice had normal core body temperature, suggesting heat is produced from other sources. Indeed, upon acute cold exposure (4°C for 6 hours), inguinal WAT from Misty mice compensated for BAT dysfunction by increasing expression of Acadl, Pgc1a, Dio2, and other thermogenic genes. Interestingly, acute cold exposure also decreased Runx2 and increased Rankl expression in Misty bone, but only Runx2 was decreased in wild-type. Browning of WAT is under the control of the sympathetic nervous system (SNS) and, if present at room temperature, could impact bone metabolism. To test whether SNS activity could be responsible for accelerated trabecular bone loss, we treated wild-type and Misty mice with the β-blocker, propranolol. As predicted, propranolol slowed trabecular bone volume/total volume (BV/TV) loss in the distal femur of Misty mice without affecting wild-type. Finally, the Misty mutation (a truncation of DOCK7) also has a significant cell-autonomous role. We found DOCK7 expression in whole bone and osteoblasts. Primary osteoblast differentiation from Misty calvaria was impaired, demonstrating a novel role for DOCK7 in bone remodeling. Despite the multifaceted effects of the Misty mutation, we have shown that impaired brown fat function leads to altered SNS activity and bone loss, and for the first time that cold exposure negatively affects bone remodeling., (Copyright © 2013 American Society for Bone and Mineral Research.)

A key finding from elevated [CO(2)] field experiments is that the impact of elevated [CO(2)] on plant and ecosystem function is highly dependent upon other environmental conditions, namely temperature and the availability of nutrients and soil moisture. In addition, there is significant variation in the response to elevated [CO(2)] among plant functional types, species and crop varieties. However, experimental data on plant and ecosystem responses to elevated [CO(2)] are strongly biased to economically and ecologically important systems in the temperate zone. There is a multi-biome gap in experimental data that is most severe in the tropics and subtropics, but also includes high latitudes. Physiological understanding of the environmental conditions and species found at high and low latitudes suggest they may respond differently to elevated [CO(2)] than well-studied temperate systems. Addressing this knowledge gap should be a high priority as it is vital to understanding 21st century food supply and ecosystem feedbacks on climate change., (Published by Elsevier Ltd.)

Invariant NKT cells (iNKT cells) are innate T lymphocytes that are thought to play an important role in producing an early burst of IFN-γ that promotes successful tumor immunosurveillance and antimicrobial immunity. The cellular activation processes underlying innate IFN-γ production remain poorly understood. We show here that weak T cell receptor (TCR) stimulation that does not directly activate iNKT cell IFN-γ messenger RNA transcription nevertheless induces histone H4 acetylation at specific regions near the IFNG gene locus. This renders the iNKT cells able to produce IFN-γ in an innate manner (i.e., not requiring concurrent TCR stimulation) upon exposure to IL-12 and IL-18. The iNKT cells retain the capacity for innate activation for hours to days after the initial weak TCR stimulation, although their innate responsiveness gradually declines as a function of histone deacetylation. These results explain how iNKT cells are able to mediate rapid innate IFN-γ secretion in a manner that does not require them to undergo permanent T(H1) differentiation. Moreover, our results also indicate that iNKT cell motility is maintained during activation by IL-12 and IL-18. Therefore, iNKT cells activated through this pathway can continue to migrate and may thus disseminate the IFN-γ that they produce, which may amplify its impact.

We have studied the subtleties of fragment docking and binding using data generated in a Pim-1 kinase inhibitor program. Crystallographic and docking data analyses have been undertaken using inhibitor complexes derived from an in-house surface plasmon resonance (SPR) fragment screen, a virtual needle screen, and a de novo designed fragment inhibitor hybrid. These investigations highlight that fragments that do not fill their binding pocket can exhibit promiscuous hydrophobic interactions due to the lack of steric constraints imposed on them by the boundaries of said pocket. As a result, docking modes that disagree with an observed crystal structure but maintain key crystallographically observed hydrogen bonds still have potential value in ligand design and optimization. This observation runs counter to the lore in fragment-based drug design that all fragment elaboration must be based on the parent crystal structure alone.

Virtually all of the known biological actions of the hormonal ligand 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) are mediated by the vitamin D receptor (VDR). Following binding and activation by the ligand, the VDR localizes in the nucleus to the regulatory regions of target genes and recruits chromatin-active coregulatory complexes which, in turn, modulate transcriptional output. The failure of the VDR to function due to crippling mutations results in total hereditary resistance to 1,25(OH)(2)D(3) in both mice and humans. In this review, we summarize the structural and functional properties of the VDR and the role of 1,25(OH)(2)D(3) in receptor activation, and then describe the results of recent studies using genome-wide analyses that define the overarching principles through which the VDR modulates genes expression. We also focus on the recent analysis of a specific 1,25(OH)(2)D(3) regulated gene that provides confirmation of the principles identified through these genome-wide methodologies. Taken together, these studies suggest an unanticipated increase in the complexity of the molecular processes that govern gene regulation by hormones and other factors.

Receptor activator of NF-κB ligand (Rankl) is a TNF-like factor that induces the formation of osteoclasts responsible for bone resorption. Although T cell activation up-regulates this gene, the molecular mechanism of its transcriptional control remains unknown. We used ChIP-chip analysis in mouse primary T cells and a T cell hybridoma to define the regulatory enhancers responsible for this up-regulation and to characterize their properties. Elevated H3/H4 acetylation and increased RNA polymerase II density were evident at mRL-D5, a known enhancer located 76 kb upstream of the TSS, as well as at a cluster of regulatory sites located even further upstream between -123 to -156 kb, termed the T cell control region (TCCR). Based upon the ability of calcium signaling and MAPK inhibitors to block Rankl expression, we conducted further ChIP-chip analysis of the transcriptional mediators c-Fos, NF-κB, and Nfat. T cell activation induced c-Fos binding at the mRL-D5 enhancer and within the TCCR. The interaction of NF-κB was observed at the transcriptional start site and at mRL-D5. Both mRL-D5 and segments of the TCCR exhibited robust transcriptional activity in reporter assays, and site-specific mutagenesis of c-Fos and Nfat elements abrogated reporter activity, suggesting a role for both factors in the control of enhancer-mediated Rankl transcription. Finally, chromosome conformation capture analysis confirmed that mRL-D5 and segments of the TCCR were located in proximity to the Rankl gene promoter and thus potentially able to influence directly Rankl gene promoter activity. We conclude that both mRL-D5 and the TCCR represent control segments that play an integral role in Rankl expression in T cells.

Novel benzofuran-2-carboxylic acids, exemplified by 29, 38 and 39, have been discovered as potent Pim-1 inhibitors using fragment based screening followed by X-ray structure guided medicinal chemistry optimization. The compounds demonstrate potent inhibition against Pim-1 and Pim-2 in enzyme assays. Compound 29 has been tested in the Ambit 442 kinase panel and demonstrates good selectivity for the Pim kinase family. X-ray structures of the inhibitor/Pim-1 binding complex reveal important salt-bridge and hydrogen bond interactions mediated by the compound's carboxylic acid and amino groups., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of 1,4-dienes to produce conjugated diene hydroperoxides. The best substrates are anions of fatty acids; for example, linoleate is converted to 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate. The manner in which SBLO-1 binds substrates is uncertain. In the present work, it was found that SBLO-1 will oxygenate linoleyltrimethylammonium ion (LTMA) to give primarily13(S)-hydroperoxy-9(Z),11(E)-octadecadienyltrimethylammonium ion. The rate of this process is about the same at pH 7 and pH 9 and is about 30% of the rate observed with linoleate at pH 9. At pH 7, SBLO-1 oxygenates linoleyldimethylamine (LDMA) to give primarily 13(S)-hydroperoxy-9(Z),11(E)-octadecadienyldimethylamine. The oxygenation of LDMA occurs at about the same rate as LTMA at pH 7, but more slowly at pH 9. The results demonstrate that SBLO-1 will readily oxygenate substrates in which the carboxylate of linoleate is replaced with a cationic group, and the products of these reactions have the same stereo- and regiochemistry as the products obtained from fatty acid substrates., (Copyright © 2011 Elsevier Inc. All rights reserved.)

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) functions as a steroid hormone to modulate the expression of genes. Its actions are mediated by the vitamin D receptor (VDR) which binds to target genes and functions to recruit coregulatory complexes that are essential for transcriptional modulation. ChIP analysis coupled to tiled DNA microarray hybridization (ChIP-chip) or massively parallel DNA sequencing (ChIP-seq) is now providing critical new insight into how genes are regulated. In studies herein, we utilized these techniques as well as gene expression analysis to explore the actions of 1,25(OH)2D3 at the genome-wide and individual target gene levels in cells. We identify a series of overarching principles that likely define the actions of 1,25(OH)2D3 at most target genes. We discover that while VDR binding to target sites is ligand-dependent, RXR binding is ligand-independent. We also show that while VDR/RXR binding can localize to promoters, it occurs more frequently at multiple sites many kilobases from target gene promoters. We then describe a new method whereby the regulatory regions of complex genes can be evaluated using large recombineered bacterial artificial chromosomes. We conclude that these new approaches are likely to replace many of the traditional methods used to explore the regulation of transcription., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Chronic inflammatory states are associated with increased bone loss. This increase is often linked to an elevation in receptor activator of nuclear factor-kappaB ligand (RANKL), a TNFalpha-like factor essential to osteoclast formation. In this study, we document the ability of IL-6 in combination with IL-6 soluble receptor (IL-6/IL-6sR) and oncostatin M to induce Rankl expression in stromal cells via signal transducer and activator of transcription 3 (STAT3). We used chromatin immunoprecipitation-tiled DNA microarray analysis to determine sites of action of STAT3 at the Rankl locus and to assess the consequences of binding on histone H4 acetylation and RNA polymerase II recruitment. Both IL-6/IL-6 soluble receptor and oncostatin M stimulated STAT3 binding upstream of the Rankl transcriptional start site. Although previously identified enhancers bound STAT3, a more distal enhancer termed mRLD6 was a particular focus of STAT3 binding. When fused to a heterologous promoter, this enhancer was highly active, containing two functionally active STAT response elements. Importantly, small interfering RNA knockdown of Stat3 mRNA and protein, but not that of Stat1 or Stat5a, was effective in limiting Rankl mRNA up-regulation. Interestingly, although RNA polymerase II and histone H4 acetylation marked many of the enhancers under basal conditions, the levels of both were strongly increased after cytokine treatment, particularly at mRLD6. Finally, mRLD6 was also a target for forskolin-induced cellular response element-binding protein (CREB) recruitment, which potentiated cytokine activity. Our studies provide new insight into mechanisms by which glycoprotein 130 activating cytokines induce RANKL expression.

Hendra virus (HeV) is a member of the broadly tropic and highly pathogenic paramyxovirus genus Henipavirus. HeV is enveloped and infects cells by using membrane-anchored attachment (G) and fusion (F) glycoproteins. G possesses an N-terminal cytoplasmic tail, an external membrane-proximal stalk domain, and a C-terminal globular head that binds the recently identified receptors ephrinB2 and ephrinB3. Receptor binding is presumed to induce conformational changes in G that subsequently trigger F-mediated fusion. The stalk domains of other attachment glycoproteins appear important for oligomerization and F interaction and specificity. However, this region of G has not been functionally characterized. Here we performed a mutagenesis analysis of the HeV G stalk, targeting a series of isoleucine residues within a hydrophobic alpha-helical domain that is well conserved across several attachment glycoproteins. Nine of 12 individual HeV G alanine substitution mutants possessed a complete defect in fusion-promotion activity yet were cell surface expressed and recognized by a panel of conformation-dependent monoclonal antibodies (MAbs) and maintained their oligomeric structure. Interestingly, these G mutations also resulted in the appearance of an additional electrophoretic species corresponding to a slightly altered glycosylated form. Analysis revealed that these G mutants appeared to adopt a receptor-bound conformation in the absence of receptor, as measured with a panel of MAbs that preferentially recognize G in a receptor-bound state. Further, this phenotype also correlated with an inability to associate with F and in triggering fusion even after receptor engagement. Together, these data suggest the stalk domain of G plays an important role in the conformational stability and receptor binding-triggered changes leading to productive fusion, such as the dissociation of G and F.

We report that use of the popular analgesic tramadol can cause false-positive urine buprenorphine results. We examined the extent of tramadol cross-reactivity in three point-of-care urine buprenorphine immunoassays (ACON, QuikStrip, and ABMC) and an instrument-based one (Cedia). We tested 29 urine samples from patients known to be taking tramadol. Ten different samples tested positive for urine buprenorphine by at least one immunoassay. Samples with positive buprenorphine screens by immunoassay were tested for total buprenorphine and total norbuprenorphine content by liquid chromatography-tandem mass spectrometry (LC-MS-MS), which confirmed that seven of the 10 positive samples were false-positives. The remaining three positive immunoassay samples had insufficient quantity for LC-MS-MS testing. No false-positives were detected with the ACON (10 ng/mL calibration cutoff) or the Cedia assay (using a 20 ng/mL calibration cutoff). All four false-positive Cedia results (using a 5 ng/mL cutoff) in this study tested negative using the ACON device. Our data suggest that tramadol use can cause false-positive urine buprenorphine immunoassays, and this effect appears to be assay-dependent. Tramadol interference with the Cedia assay is clinically relevant, especially if the 5 ng/mL calibration cutoff is used.

We have previously identified neutralizing human monoclonal antibodies against Nipah virus (NiV) and Hendra virus (HeV) by panning a large nonimmune antibody library against a soluble form of the HeV attachment-envelope glycoprotein G (sG HeV). One of these antibodies, m102, which exhibited the highest level of cross-reactive neutralization of both NiV and HeV G, was affinity maturated by light-chain shuffling combined with random mutagenesis of its heavy-chain variable domain and panning against sGHeV. One of the selected antibody Fab clones, m102.4, had affinity of binding to sGHeV that was equal to or higher than that of the other Fabs; it was converted to IgG1 and tested against infectious NiV and HeV. It exhibited exceptionally potent and cross-reactive inhibitory activity with 50% inhibitory concentrations below 0.04 and 0.6 microg/mL, respectively. The virus-neutralizing activity correlated with the binding affinity of the antibody to sG HeV and sG NiV. m102.4 bound a soluble form of NiV G (sG NiV) better than it bound sG HeV, and it neutralized NiV better than HeV, despite being originally selected against sG HeV. These results suggest that m102.4 has potential as a therapeutic agent for the treatment of diseases caused by henipaviruses. It could be also used for prophylaxis and diagnosis, and as a research reagent.

Hendra virus (HeV) is an emerging paramyxovirus capable of infecting and causing disease in a variety of mammalian species, including humans. The virus infects its host cells through the coordinated functions of its fusion (F) and attachment (G) glycoproteins, the latter of which is responsible for binding the virus receptors ephrinB2 and ephrinB3. In order to identify the receptor binding site, a panel of G glycoprotein constructs containing mutations was generated using an alanine-scanning mutagenesis strategy. Based on a predicted G structure, charged amino acids residing in regions that could be homologous to those in the measles virus H attachment glycoprotein known to be involved in its protein receptor interaction were targeted. Using a coprecipitation-based assay, seven single-amino-acid substitutions in HeV G were identified as having significantly impaired binding to both the ephrinB2 and ephrinB3 viral receptors: D257A, D260A, G439A, K443A, G449A, K465A, and D468A. The impairment of receptor interaction conferred a concomitant diminution in their abilities to promote membrane fusion when coexpressed with F. The G glycoprotein mutants were also recognized by three or more conformation-dependent monoclonal antibodies of a panel of five, were expressed on the cell surface, and retained their abilities to bind and coprecipitate F. Interestingly, some of these mutant G glycoproteins coprecipitated with F more efficiently than wild-type G. Taken together, these data provide strong biochemical and functional evidence that some of these residues could be part of a conformation-dependent, discontinuous, and overlapping ephrinB2 and -B3 binding domain within the HeV G glycoprotein.

Hendra virus (HeV) and Nipah virus (NiV) are closely related emerging viruses comprising the Henipavirus genus of the Paramyxovirinae. Each has a broad species tropism and can cause disease with high mortality in both animal and human hosts. These viruses infect cells by a pH-independent membrane fusion event mediated by their attachment (G) and fusion (F) envelope glycoproteins (Envs). Seven Fabs, m101 to -7, were selected for their significant binding to a soluble form of Hendra G (sG) which was used as the antigen for panning of a large naïve human antibody library. The selected Fabs inhibited, to various degrees, cell fusion mediated by the HeV or NiV Envs and virus infection. The conversion of the most potent neutralizer of infectious HeV, Fab m101, to immunoglobulin G1 (IgG1) significantly increased its cell fusion inhibitory activity: the 50% inhibitory concentration was decreased more than 10-fold to approximately 1 microg/ml. The IgG1 m101 was also exceptionally potent in neutralizing infectious HeV; complete (100%) neutralization was achieved with 12.5 microg/ml, and 98% neutralization required only 1.6 microg/ml. The inhibition of fusion and infection correlated with binding of the Fabs to full-length G as measured by immunoprecipitation and less with binding to sG as measured by enzyme-linked immunosorbent assay and Biacore. m101 and m102 competed with the ephrin-B2, which we recently identified as a functional receptor for both HeV and NiV, indicating a possible mechanism of neutralization by these antibodies. The m101, m102, and m103 antibodies competed with each other, suggesting that they bind to overlapping epitopes which are distinct from the epitopes of m106 and m107. In an initial attempt to localize the epitopes of m101 and m102, we measured their binding to a panel of 11 G alanine-scanning mutants and identified two mutants, P185A and Q191 K192A, which significantly decreased binding to m101 and one, G183, which decreased binding of m102 to G. These results suggest that m101 to -7 are specific for HeV or NiV or both and exhibit various neutralizing activities; they are the first human monoclonal antibodies identified against these viruses and could be used for treatment, prophylaxis, and diagnosis and as research reagents and could aid in the development of vaccines.

Hendra virus (HeV) and Nipah virus (NiV) belong to the genus Henipavirus of the family Paramyxoviridae and are unique in that they exhibit a broad species tropism and cause fatal disease in both animals and humans. They infect cells through a pH-independent membrane fusion process mediated by their fusion and attachment glycoproteins. Previously, we demonstrated identical cell fusion tropisms for HeV and NiV and the protease-sensitive nature of their unknown cell receptor and identified a human cell line (HeLa-USU) that was nonpermissive for fusion and virus infection. Here, a microarray analysis was performed on the HeLa-USU cells, permissive HeLa-CCL2 cells, and two other permissive human cell lines. From this analysis, we identified a list of genes encoding known and predicted plasma membrane surface-expressed proteins that were highly expressed in all permissive cells and absent from the HeLa-USU cells and rank-ordered them based on their relative levels. Available expression vectors containing the first 10 genes were obtained and individually transfected into HeLa-USU cells. One clone, encoding human ephrin-B2 (EFNB2), was found capable of rendering HeLa-USU cells permissive for HeV- and NiV-mediated cell fusion as well as infection by live virus. A soluble recombinant EFNB2 could potently block fusion and infection and bind soluble recombinant HeV and NiV attachment glycoproteins with high affinity. Together, these data indicate that EFNB2 serves as a functional receptor for both HeV and NiV. The highly conserved nature of EFNB2 in humans and animals is consistent with the broad tropism exhibited by these emerging zoonotic viruses.

The Australian climate is highly variable and many Australian estuaries lack a seasonal pattern of freshwater flow. During periods of low freshwater inflow, saline waters enter from the ocean through the estuary mouth. These saline waters enter as density currents or as a result of tidal mixing. During periods of high freshwater inflow from the estuary catchment, salt water is flushed towards the estuary mouth. As a consequence, the saline structure of Australian estuaries can be highly variable, depending on the antecedent rainfall. The Richmond River in northern New South Wales in such an estuary. The biota inhabiting estuaries have varying levels of freshwater and saltwater tolerance and reducing the freshwater flow into and along an estuary will favour saltwater species. However, if extractions of freshwater from an estuary are sufficiently high, freshwater habitat could be eliminated entirely for short periods (-one month) but with consequent, and perhaps long-standing, damage to the environment. This contribution describes a new approach to quantify the impact of changes to freshwater flows to the Richmond River estuary. This approach includes a review of hydrological data gathered over the past century and includes a detailed assessment of: changes to the highly variable freshwater inflows and freshwater extraction below the tidal limit; consequent changes to the highly variable saline structure; and the risk to aquatic biota. The crucial factors of magnitude, frequency and duration of short-term intrusions of saline water into freshwater habitat and their impact are quantified by the methods developed.

This article describes the restorative management of a case of advanced, localised tooth-surface loss using modern materials and techniques. Particular emphasis is placed on the conservation of tooth structure while providing restorations with optimum physical and aesthetic characteristics.

The primary objective of the biological monitoring program of the ARRRI is to develop techniques and procedures to monitor and assess effects of the mining and processing of ores on aquatic ecosystems of the Region. Studies have been made in a seasonal tributary (Magela Creek) of the East Alligator River near the Ranger Uranium Mine, and in the upper South Alligator River (SAR) near the Coronation Hill gold, platinum and palladium prospect. Ranger and Coronation Hill are enclosed by Kakadu National Park, environmental safeguards for which require the minimization of impairment of water quality in the aquatic ecosystems. Present studies on Magela Creek are designed to verify the adequacy of release standards, based on biological tests, in protecting the aquatic environment during and after releases of waste-water from Ranger during the Wet season. Detection of short-term effects will be sought by either creekside orin situ monitoring methods. The detection and assessment of any longer-term impacts, however, will rely chiefly on comparisons of post-release data with those of historical baselines. Such baseline information is provided by studies on the structure of macroinvertebrate and fish communities and/or on concentrations of elements in organisms in those communities, in Magela Creek and SAR catchments.

Collagen of types I, III and V has been identified and localised in adult human aorta, coronary arteries and atherosclerotic plaques using an indirect immunofluorescent method with specific antibodies to human collagen antigens. The distribution of the three types of collagen was distinct. Type I collagen was found around smooth muscle cells in the aortic media and in large amounts in the atherosclerotic plaque. Type III collagen was found in dense deposits alongside the elastic laminae in the aortic media and in diffuse intimal thickening. In the aortic media, there appeared to be more type III collagen than type I. The reverse was true in advanced atherosclerotic plaques. Type V collagen was distributed throughout the extracellular matrix in the aortic media and in the subendothelial region of plaques. These findings confirm earlier biochemical studies and suggest that a major shift in the nature of collagen synthesis occurs within advanced atherosclerotic plaques.

Chronic pyelonephritis was induced in young adult cats by the intravenous injection of a human or a feline strain of Escherichia coli after ligation of one ureter for 24 or 48 h. In the 3 cats infected with the feline strain, scarred kidneys from the obstructed side were removed at necropsy 3, 4 and 5 months later. Collagen was extracted from pyelonephritic and normal kidney tissue with dilute acetic acid and limited proteolysis with pepsin. Scarred kidneys gave higher yields of both acid-soluble collagen (normal = 0.57 +/- 0.12 mg per g tissue; scarred = 0.88 +/- 0.10 mg per g tissue) and pepsin-solubilized collagen (normal = 9.69 +/- 1.79 mg per g tissue; scarred = 20.02 +/- 2.84 mg per g tissue). There was no significant increase in the collagen yield from the kidneys of the 2 cats in which mild focal lesions were found 14 and 16 months after infection with the human strain of E. coli. Pepsin released collagens were separated by fractional salt precipitation and identified by agarose gel chromatography and polyacrylamide gel electrophoresis. Normal kidney was shown to contain collagen of Types I, IV and V (AB). The Type IV collagen extracted consisted of a mixture of 4 major pepsin-resistant chains of apparent molecular weights of 150 000, 115 000, 85 000 and 60 000. The collagen extracted from scarred kidneys was predominantly Type I, only trace amounts of Type IV and V components being present. These findings suggest that basement membrane collagens of the kidney are selectively degraded during the atrophy and scarring of chronic feline pyelonephritis and are preferentially replaced by interstitial Type I collagen.