Mode of inhibition of HIV-1 Integrase by a C-terminal domain-specific monoclonal antibody*

BACKGROUND: To further our understanding of the structure and function of HIV-1 integrase (IN) we developed and characterized a library of monoclonal antibodies (mAbs) directed against this protein. One of these antibodies, mAb33, which is specific for the C-terminal domain, was found to inhibit HIV-1 IN processing activity in vitro; a corresponding Fv fragment was able to inhibit HIV-1 integration in vivo. Our subsequent studies, using heteronuclear nuclear magnetic resonance spectroscopy, identified six solvent accessible residues on the surface of the C-terminal domain that were immobilized upon binding of the antibody, which were proposed to comprise the epitope. Here we test this hypothesis by measuring the affinity of mAb33 to HIV-1 proteins that contain Ala substitutions in each of these positions. To gain additional insight into the mode of inhibition we also measured the DNA binding capacity and enzymatic activities of the Ala substituted proteins. RESULTS: We found that Ala substitution of any one of five of the putative epitope residues, F223, R224, Y226, I267, and I268, caused a decrease in the affinity of the mAb33 for HIV-1 IN, confirming the prediction from NMR data. Although IN derivatives with Ala substitutions in or near the mAb33 epitope exhibited decreased enzymatic activity, none of the epitope substitutions compromised DNA binding to full length HIV-1 IN, as measured by surface plasmon resonance spectroscopy. Two of these derivatives, IN (I276A) and IN (I267A/I268A), exhibited both increased DNA binding affinity and uncharacteristic dissociation kinetics; these proteins also exhibited non-specific nuclease activity. Results from these investigations are discussed in the context of current models for how the C-terminal domain interacts with substrate DNA. CONCLUSION: It is unlikely that inhibition of HIV-1 IN activity by mAb33 is caused by direct interaction with residues that are essential for substrate binding. Rather our findings are most consistent with a model whereby mAb33 binding distorts or constrains the structure of the C-terminal domain and/or blocks substrate binding indirectly. The DNA binding properties and non-specific nuclease activity of the I267A derivatives suggest that the C-terminal domain of IN normally plays an important role in aligning the viral DNA end for proper processing

Brief Report: The Value of a Patient Global Assessment of Disease Activity in Granulomatosis With Polyangiitis (Wegener's)

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102695/1/art38248.pd

Distribution and Activity of Bacteria in the Headwaters of the Rhode River Estuary, Maryland, USA

Abstract: A transect along the axis of the headwaters of a tidal estuary was sampled for microbial, nutrient, and physical parameters. Chlorophyll a averaged 42 μg 1 -1 and phytoplankton comprised an estimated 80% of the total microbial biomass as determined by adenosine triphosphate (ATP). Bacterial concentrations ranged from 0.3-53.9 × 10 6 cells ml -1 and comprised about 4% of the total living microbial biomass. Bacterial production, determined by 3H-methyl-thymidine incorporation was about 0.05-2.09 × 10 9 cells 1 -1 h -1 , with specific growth rates of 0.26-1.69 d -1 . Most bacterial production was retained on 0.2 μm pore size filters, but passed through 1.0 μm filters. Significant positive correlations were found between all biomass measures and most nutrient measures with the exception of dissolved inorganic nitrogen nutrients where correlations were negative. Seasonal variability was evident in all parameters and variability among the stations was evident in most. The results suggest that bacterial production requires a significant carbon input, likely derived from autotrophic production, and that microbial trophic interactions are important. Article: Introduction Coastal and estuarine systems are sites where competition for nutrients by microorganisms and remineralization of photosynthetically fixed carbon are important processes. High rates of bacterial activity have been found in such systems for both inorganic nutrients and organic compounds Utilization of carbon fixed by primary production is a primary role of heterotrophic microorganisms, and recent studies suggest that the magnitude of this role has been underestimated Estuarine systems may exhibit even greater dependence upon trophic links through microheterotrophs tha

Effect of Aspect Ratio and Deformability on Nanoparticle Extravasation through Nanopores

We describe the fabrication of filamentous hydrogel nanoparticles using a unique soft lithography based particle molding process referred to as PRINT (Particle Replication in Non-wetting Templates). The nanoparticles possess a constant width of 80 nm, and we varied their lengths ranging from 180 nm to 5000 nm. In addition to varying the aspect ratio of the particles, the deformability of the particles was tuned by varying the cross-link density within the particle matrix. Size characteristics such as hydrodynamic diameter and persistence length of the particles were analyzed using dynamic light scattering and electron microscopy techniques, respectively, while particle deformability was assessed by atomic force microscopy. Additionally, the ability of the particles to pass through membranes containing 0.2 μm pores was assessed by means of a simple filtration technique, and particle recovery was determined using fluorescence spectroscopy. The results show that particle recovery is mostly independent of aspect ratio at all cross-linker concentrations utilized, with the exception of 96 wt% PEG diacrylate 80 × 5000 nm particles, which showed the lowest percent recovery

Seventy-Five Years (1940-2015) of Lehigh University\u27s Chemistry Department

The 75-years 1940 to 2015 have been exciting ones for the Department of Chemistry; new buildings, new programs, energetic young faculty, enhanced research image, and a far broader coverage of Chemistry than our ancestors ever presumed. Five chairs guided the department through its first 75-years but it took 11 chairs (with two of them serving twice) to manage the second 75-years. As one of the Lehigh founding departments in 1865 our first 75-years have already been covered. The reader is directed to a history written by Robert D. Billinger, A History of the Department of Chemistry and Chemical Engineering of Lehigh University, Bethlehem, Pennsylvania (1866-1941) which is available in original in the Lehigh Archives and as an on-line document. This sesquicentennial volume is also available in hardcopy with original illustrations in the archives or on-line

Assessment of the item selection and weighting in the Birmingham Vasculitis Activity Score for Wegener's Granulomatosis

Objective To assess the Birmingham Vasculitis Activity Score for Wegener's Granulomatosis (BVAS/WG) with respect to its selection and weighting of items. Methods This study used the BVAS/WG data from the Wegener's Granulomatosis Etanercept Trial. The scoring frequencies of the 34 predefined items and any “other” items added by clinicians were calculated. Using linear regression with generalized estimating equations in which the physician global assessment (PGA) of disease activity was the dependent variable, we computed weights for all predefined items. We also created variables for clinical manifestations frequently added as other items, and computed weights for these as well. We searched for the model that included the items and their generated weights yielding an activity score with the highest R 2 to predict the PGA. Results We analyzed 2,044 BVAS/WG assessments from 180 patients; 734 assessments were scored during active disease. The highest R 2 with the PGA was obtained by scoring WG activity based on the following items: the 25 predefined items rated on ≥5 visits, the 2 newly created fatigue and weight loss variables, the remaining minor other and major other items, and a variable that signified whether new or worse items were present at a specific visit. The weights assigned to the items ranged from 1 to 21. Compared with the original BVAS/WG, this modified score correlated significantly more strongly with the PGA. Conclusion This study suggests possibilities to enhance the item selection and weighting of the BVAS/WG. These changes may increase this instrument's ability to capture the continuum of disease activity in WG.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/60211/1/23707_ftp.pd

Scalable, Shape-Specific, Top-Down Fabrication Methods for the Synthesis of Engineered Colloidal Particles

The search for a method to fabricate non-spherical colloidal particles from a variety of materials is of growing interest. As the commercialization of nanotechnology continues to expand, the ability to translate particle fabrication methods from a laboratory to an industrial scale is of increasing significance. In this article, we examine several of the most readily scalable top-down methods for the fabrication of such shape specific particles and compare their capabilities with respect to particle composition, size, shape and complexity as well as the scalability of the method. We offer an extensive examination of Particle Replication In Non-wetting Templates (PRINT®) with regards to the versatility and scalability of this technique. We also detail the specific methods used in PRINT particle fabrication, including harvesting, purification and surface modification techniques, with examination of both past and current methods

Bringing numerous methods for expression and promoter analysis to a public cloud computing service

Every year, a large number of novel algorithms are introduced to the scientific community for a myriad of applications, but using these across different research groups is often troublesome, due to suboptimal implementations and specific dependency requirements. This does not have to be the case, as public cloud computing services can easily house tractable implementations within self-contained dependency environments, making the methods easily accessible to a wider public. We have taken 14 popular methods, the majority related to expression data or promoter analysis, developed these up to a good implementation standard and housed the tools in isolated Docker containers which we integrated into the CyVerse Discovery Environment, making these easily usable for a wide community as part of the CyVerse UK project

Solid malignancies among etanercept‐treated patients with granulomatosis with polyangiitis (Wegener's): Long‐term followup of a multicenter longitudinal cohort

Objective An association between therapeutic inhibition of tumor necrosis factor (TNF) and solid malignancies was observed during the Wegener's Granulomatosis Etanercept Trial (WGET), which included 180 patients with granulomatosis with polyangiitis (Wegener's) (GPA). The present study was conducted to determine the malignancy risk beyond the time of exposure to study therapy. Methods The occurrence and type of solid malignancies were ascertained using a standardized data form. Data collected included vital status, histologic findings, and therapeutic interventions. The Surveillance, Epidemiology, and End‐Results database was used to estimate a standardized incidence rate (SIR) for solid malignancies. Results Post‐trial followup data were available for 153 patients (85% of the original cohort), with a median followup time of 43 months. Fifty percent of these patients had received etanercept. There were no differences in demographic characteristics between the etanercept and placebo groups. Thirteen new solid malignancies were detected, 8 in the etanercept group and 5 in the placebo group. Compared to the general population, the risk of solid malignancies in the etanercept group was increased (SIR 3.92 [95% confidence interval 1.69–7.72]), but was not different from the risk in the placebo group compared to the general population (SIR 2.89 [95% confidence interval 0.94–6.73]). All solid malignancies occurred in patients who had been exposed to cyclophosphamide. The overall duration of disease and a history of malignancy before trial enrollment were associated with the development of malignancy during post‐trial followup. Conclusion The incidence of solid malignancy remained increased during long‐term followup of the WGET cohort. However, this could not be attributed solely to etanercept exposure during the trial. Anti‐TNF therapy with etanercept appears to further increase the risk of malignancy observed in patients with GPA treated with cytotoxic agents and should be avoided in these patients.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/87143/1/30394_ftp.pd

Ovarian reserve diminished by oral cyclophosphamide therapy for granulomatosis with polyangiitis (Wegener's)

Objective Standard treatment for severe granulomatosis with polyangiitis (Wegener's) (GPA) is daily oral cyclophosphamide (CYC), a cytotoxic agent associated with ovarian failure. In this study, we assessed the rate of diminished ovarian reserve in women with GPA who received CYC versus methotrexate (MTX). Methods Patients in the Wegener's Granulomatosis Etanercept Trial received either daily CYC or weekly MTX and were randomized to etanercept or placebo. For all women ages <50 years, plasma samples taken at baseline or early in the study were evaluated against samples taken later in the study to compare levels of anti‐Müllerian hormone (AMH) and follicle‐stimulating hormone (FSH), endocrine markers of remaining egg supply. Diminished ovarian reserve was defined as an AMH level of <1.0 ng/ml. Results Of 42 women in this analysis (mean age 35 years), 24 had CYC exposure prior to enrollment and 28 received the drug during the study. At study entry, women with prior CYC exposure had significantly lower AMH, higher FSH, and a higher rate of early menstruation cessation. For women with normal baseline ovarian function, 6 of 8 who received CYC during the trial developed diminished ovarian reserve, compared to 0 of 4 who did not receive CYC ( P < 0.05). Changes in AMH correlated inversely with cumulative CYC dose ( P < 0.01), with a 0.74 ng/ml decline in AMH level for each 10 gm of CYC. Conclusion Daily oral CYC, even when administered for less than 6 months, causes diminished ovarian reserve, as indicated by low AMH levels. These data highlight the need for alternative treatments for GPA in women of childbearing age.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/88079/1/20605_ftp.pd