Optimizing clinical dosing of combination broadly neutralizing antibodies for HIV prevention

Broadly neutralizing antibodies (bNAbs) are promising agents to prevent HIV infection and achieve HIV remission without antiretroviral therapy (ART). As with ART, bNAb combinations are likely needed to cover HIV’s extensive diversity. Not all bNAbs are identical in terms of their breadth, potency, and in vivo longevity (half-life). Given these differences, it is important to optimally select the composition, or dose ratio, of combination bNAb therapies for future clinical studies. We developed a model that synthesizes 1) pharmacokinetics, 2) potency against a wide HIV diversity, 3) interaction models for how drugs work together, and 4) correlates that translate in vitro potency to clinical protection. We found optimization requires drug-specific balances between potency, longevity, and interaction type. As an example, tradeoffs between longevity and potency are shown by comparing a combination therapy to a bi-specific antibody (a single protein merging both bNAbs) that takes the better potency but the worse longevity of the two components. Then, we illustrate a realistic dose ratio optimization of a triple combination of VRC07, 3BNC117, and 10–1074 bNAbs. We apply protection estimates derived from both a non-human primate (NHP) challenge study meta-analysis and the human antibody mediated prevention (AMP) trials. In both cases, we find a 2:1:1 dose emphasizing VRC07 is nearly optimal. Our approach can be immediately applied to optimize the next generation of combination antibody prevention and cure studies.     

Cost-effective and concurrent production of industrially valuable xylano-pectinolytic enzymes by a bacterial isolate Bacillus pumilus AJK

Simultaneous production of xylanase and pectinase by Bacillus pumilus AJK under submerged fermentation was investigated in this study. Under optimized conditions, it produced 315?±?16 IU/mL acidic xylanase, 290?±?20 IU/mL alkaline xylanase, and 88?±?9 IU/mL pectinase. The production of xylano-pectinolytic enzymes was the highest after inoculating media (containing 2% each of wheat bran and Citrus limetta peel, 0.5% peptone, 10?mM MgSO₄, pH 7.0) with 2% of 21-hr-old culture and incubated at 37°C for 60?hr at 200?rpm. Xylanase retained 100% activity from pH 6.0 to10.0 after 3?hr of incubation, while pectinase showed 100% stability from pH 6.0 to 9.0 even after 6?hr of incubation. Cost-effective and concurrent production of xylanase and pectinase by a bacterial isolate in the same production media suggests its potential for various biotechnological applications. This is the first report of simultaneous production of industrially important extracellular xylano-pectinolytic enzymes by B. pumilus.     

Mechanistic insights into the inhibition of prostate specific antigen by beta-lactam class compounds

Prostate Specific Antigen (PSA) is a biomarker used in the diagnosis of prostate cancer and to monitor therapeutic response. However, its precise role in prostate carcinogenesis and metastasis remains largely unknown. A number of studies arguing in the favor of an active role of PSA in prostate cancer development and progression have implicated this serine protease in the release and activation of growth factors such as insulin-like growth factor 1 (IGF1) through cleavage of insulin like growth factor binding protein 3 and Transforming Growth Factor beta (TGF-beta) through cleavage of Latent TGF-beta. In contrast, other studies suggest that PSA activity might hinder tumor development and progression. In light of these contradictory findings, efficient inhibitors of PSA are needed for exploring its biological role in tumor development and metastasis. Towards the goal of developing potent inhibitors of PSA, we have explored the molecular mechanism of a series of beta-lactam based compounds on binding to PSA using activity assays, matrix assisted laser desorption ionization with a time-of-flight mass spectrometry, and GOLD docking methodology. The mass spectrometry experiments and the activity assays confirmed the time-dependent and covalent nature of beta-lactam binding. To gain insights on the reaction intermediates at the molecular level, we docked beta-lactam inhibitors to a homology modeled PSA using the GOLD docking program in noncovalent and covalent binding modes. The docking studies elucidated the molecular details of the early noncovalent Michaelis complex, the acyl-enzyme covalent complex, and the nature of conformational reorganization required for the long term stability of the covalent complex. Additionally, the molecular basis for the effect of stereochemistry of the lactam ring on the inhibitory potency was elucidated through docking of beta-lactam enantiomers. As a validation of our docking methodology, two novel enantiomers were synthesized and evaluated for their inhibitory potency using fluorogenic substrate based activity assays. Additionally, cis enantiomers of eight beta-lactam compounds reported in a previous study were docked and their GOLD scores and binding modes were analyzed in order to assess the general applicability of our docking results. The close agreement of our docking results with the experimental data validates the mechanistic insights revealed through the docking studies and paves the way for the design and development of potent and specific inhibitors of PSA.     

Grassland birds and rotational-grazing in the northeast: Breeding ecology,survival and management opportunities

We studied long-term (2002–2009) demographics of bobolinks (Dolichonyx oryzivorus) and savannah sparrows (Passerculus sandwichensis) breeding in rotationally grazed systems in the northeastern United States. Both nest success (6–44% bobolink; 7–48% savannah sparrow) and annual productivity (0.43–2.83 bobolink; 0.70–2.35 savannah sparrow) varied annually. Predation (48%) and trampling by cows (32%) accounted for most failed nests. Annual adult apparent survival ranged from 23% to 85% and showed substantial variation by species and sex. Although grazing resulted in substantial loss of vegetative cover, savannah sparrows responded similarly to predation and grazing-induced nest failure (time to renest, distance moved between nests) and generally remained in the same paddock to renest. Minimum paddock size to accommodate one female within this rotation was 70 m × 70 m (0.49 ha), allowing a female to stay within a paddock and move (approx. 50 m) between nesting attempts. To balance pasturing objectives and birds' needs in paddocks first grazed before 25 June, managers in the Northeast should rest that paddock 42–50 days before the second grazing. © 2011 The Wildlife Society.     

Phytotoxic effects of β-pinene on early growth and associated biochemical changes in rice

β-Pinene, an oxygenated monoterpene, is one of the major monoterpenes emitted into the atmosphere from forest areas and trees. Besides, it is a principal component of essential oils of a number of aromatic plants, which are involved in a variety of ecological interactions, including allelopathy, in the natural environment. However, studies pertaining to phytotoxicity and biochemical effect(s) of β-pinene are largely lacking. We investigated the effect of β-pinene (0.02, 0.04, 0.08, 0.20, 0.40 and 0.80 mg/ml) in a dose- and time-dependent manner on early seedling growth, dry weight accumulation, photosynthetic pigments and changes in macromolecule (protein and carbohydrate) content and activities of enzymes—proteases, α- and β-amylases, polyphenol oxidases and peroxidases- in rice (Oryza sativa) after 3rd, 5th and 7th day of exposure. β-pinene (≥0.04 mg/ml) significantly reduced the root (by 13–87%) and coleoptile (by 5–80%) length of rice. Exposure to β-pinene reduced total chlorophyll content in rice coleoptiles suggesting a negative impact on photosynthesis. The content of macromolecules (proteins and carbohydrates) enhanced significantly in response to β-pinene, whereas the activities of hydrolyzing enzymes—proteases, α-amylases, and β-amylases—declined (by 30–85, 26–84, 27–74%, respectively) in β-pinene-exposed seedlings. In contrast, the activities of peroxidases (POX) and polyphenol oxidases (PPO) enhanced significantly (by 16–152 and 53–290%, respectively) in rice roots in response to β-pinene in a dose- and time-dependent manner. Increased activities of POX and PPO indicate their involvement in providing protection and/or conferring resistance against β-pinene-induced stress. The study concludes that β-pinene inhibits the early growth of rice by altering the plant biochemical status and enhancing activities of POXs and PPOs involved in general plant defense.     

Anti-hemolytic and Peroxyl Radical Scavenging Activity of Organoselenium Compounds: An In Vitro Study

Selenium-containing amino acids, selenocystine (CysSeSeCys), methylselenocysteine (MeSeCys), and selenomethionine (SeMet) have been examined for anti-hemolytic and peroxyl radical scavenging ability. Effect of these compounds on membrane lipid peroxidation, release of hemoglobin, and loss of intracellular K⁺ ion as a consequence of peroxyl radicals-induced oxidation of human red blood cells were used to evaluate their anti-hemolytic ability. The peroxyl radicals were generated from thermal degradation of 2,2′-azobis(2-methylpropionamidine) dihydrochloride. Significant delay (t _eff) was observed in oxidative damage in the presence of the selenium compounds. From the IC₅₀ values for the inhibition of hemolysis, lipid peroxidation, and K⁺ ion leakage, the relative anti-hemolytic ability of the compounds were found to be in the order of CysSeSeCys > MeSeCys > SeMet. The anti-hemolytic abilities of the compounds, when compared with sodium selenite (Na₂SeO₃) under identical experimental conditions, were found to be better than Na₂SeO₃. Relative rate constants estimated for the reaction of MeSeCys and SeMet with peroxyl radicals by competition kinetics using ABTS²⁻ as a reference confirmed that all the compounds are efficient peroxyl radical scavengers. Comparison of the GPx-like activity of these compounds, by NADPH–GSH reductase coupled assay, indicated that CysSeSeCys exhibits the highest activity. Based on these results, it is concluded that among the compounds examined, CysSeSeCys, possessing the ability to reduce peroxyl radicals and hydroperoxides showed efficient anti-hemolytic activity.     

Psychosine-induced alterations in peroxisomes of twitcher mouse liver

Krabbe disease is a neuroinflammatory disorder in which galactosylsphingosine (psychosine) accumulates in nervous tissue. To gain insight into whether the psychosine-induced effects in nervous tissue extend to peripheral organs, we investigated the expression of cytokines and their effects on peroxisomal structure/functions in twitcher mouse liver (animal model of Krabbe disease). Immunofluorescence analysis demonstrated TNF-α and IL-6 expression, which was confirmed by mRNAs quantitation. Despite the presence of TNF-α, lipidomic analysis did not indicate a significant decrease in sphingomyelin or an increase in ceramide fractions. Ultrastructural analysis of catalase-dependent staining of liver sections showed reduced reactivity without significant changes in peroxisomal contents. This observation was confirmed by assaying catalase activity and quantitation of its mRNA, both of which were found significantly decreased in twitcher mouse liver. Western blot analysis demonstrated a generalized reduction of peroxisomal matrix and membrane proteins. These observations indicate that twitcher mouse pathobiology extends to the liver, where psychosine-induced TNF-α and IL-6 compromise peroxisomal structure and functions.     

Matrix-Assisted Refolding,Purification and Activity Assessment Using a ‘Form Invariant’ Assay for Matrix Metalloproteinase 2 (MMP2)

Matrix metalloproteinases expression is used as biomarker for various cancers and associated malignancies. Since these proteinases can cleave many intracellular proteins, overexpression tends to be toxic; hence, a challenge to purify them. To overcome these limitations, we designed a protocol where full length pro-MMP2 enzyme was overexpressed in E. coli as inclusion bodies and purified using 6xHis affinity chromatography under denaturing conditions. In one step, the enzyme was purified and refolded directly on the affinity matrix under redox conditions to obtain a bioactive protein. The pro-MMP2 protein was characterized by mass spectrometry, CD spectroscopy, zymography and activity analysis using a simple in-house developed ‘form invariant’ assay, which reports the total MMP2 activity independent of its various forms. The methodology yielded higher yields of bioactive protein compared to other strategies reported till date, and we anticipate that using the protocol, other toxic proteins can also be overexpressed and purified from E. coli and subsequently refolded into active form using a one step renaturation protocol.