High affinity human antibody fragments to dengue virus non-structural protein 3

Background

The enzyme activities catalysed by flavivirus non-structural protein 3 (NS3) are essential for virus replication. They are distributed between the N-terminal protease domain in the first one-third and the C-terminal ATPase/helicase and nucleoside 5′ triphosphatase domain which forms the remainder of the 618-aa long protein.

Methodology/Principal Findings

In this study, dengue full-length NS3 protein with residues 49 to 66 of NS2B covalently attached via a flexible linker, was used as bait in biopanning with a naïve human Fab phage-display library. Using a range of truncated constructs spanning the NS2B cofactor region and the full-length NS3, 10 unique Fab were identified and characterized. Of these, monoclonal Fab 3F8 was shown to bind α3″ (residues 526 through 531) within subdomain III of the helicase domain. The antibody inhibits the ATPase and helicase activites of NS3 in biochemical assays and reduces DENV replication in HEK293 cells that were previously transfected with Fab 3F8 compared with mock transfected cells.

Conclusions/Significance

Antibodies such as 3F8 are valuable tools for studying the molecular mechanisms of flaviviral replication and for the monospecific detection of replicating dengue virus in vivo.     

Hepatitis C Virus Induces the Cannabinoid Receptor 1

Background

Activation of hepatic CB₁ receptors (CB₁) is associated with steatosis and fibrosis in experimental forms of liver disease. However, CB₁ expression has not been assessed in patients with chronic hepatitis C (CHC), a disease associated with insulin resistance, steatosis and metabolic disturbance. We aimed to determine the importance and explore the associations of CB₁ expression in CHC.

Methods

CB₁ receptor mRNA was measured by real time quantitative PCR on extracted liver tissue from 88 patients with CHC (genotypes 1 and 3), 12 controls and 10 patients with chronic hepatitis B (CHB). The Huh7/JFH1 Hepatitis C virus (HCV) cell culture model was used to validate results.

Principal Findings

CB₁ was expressed in all patients with CHC and levels were 6-fold higher than in controls (P<0.001). CB₁ expression increased with fibrosis stage, with cirrhotics having up to a 2 fold up-regulation compared to those with low fibrosis stage (p<0.05). Even in mild CHC with no steatosis (F0-1), CB₁ levels remained substantially greater than in controls (p<0.001) and in those with mild CHB (F0-1; p<0.001). Huh7 cells infected with JFH-1 HCV showed an 8-fold upregulation of CB₁, and CB₁ expression directly correlated with the percentage of cells infected over time, suggesting that CB₁ is an HCV inducible gene. While HCV structural proteins appear essential for CB₁ induction, there was no core genotype specific difference in CB₁ expression. CB₁ significantly increased with steatosis grade, primarily driven by patients with genotype 3 CHC. In genotype 3 patients, CB₁ correlated with SREBP-1c and its downstream target FASN (SREBP-1c; R = 0.37, FASN; R = 0.39, p<0.05 for both).

Conclusions/Significance

CB₁ is up-regulated in CHC and is associated with increased steatosis in genotype 3. It is induced by the hepatitis C virus.     

Behavioral testing and preliminary analysis of the hamster visual system

The dependence of visual orienting ability in hamsters on the axonal projections from retina to midbrain tectum provides experimenters with a good model for assessing the functional regeneration of this central nervous system axonal pathway. For reliable testing of this behavior, male animals at least 10-12 weeks old are prepared by regular pretesting, with all procedures carried out during the less active portion of the daily activity cycle. Using a sunflower seed attached to a small black ball held at the end of a stiff wire, and avoiding whisker contact, turning movements toward visual stimuli are video recorded from above. Because at the eye level, the nasal-most 30 degrees of the visual field can be seen by both the eyes, this part of the field is avoided in assessments of a single side. Daily sessions consist of ten presentations per side. Measures are frequency of responding and detailed turning trajectories. Complete assessment of the functional return of behavior in this testing paradigm takes 3-6 months to complete.     

Fcgamma RI-triggered generation of arachidonic acid and eicosanoids requires iPLA2 but not cPLA2 in human monocytic cells

Aggregation of receptors for immunoglobulin G (FcgammaRs) on myeloid cells activates a series of events that are key in the inflammatory response and that can ultimately lead to targeted cell killing by antibody-directed cellular cytotoxicity. Generation of lipid-derived proinflammatory mediators is an important component of the integrated cellular response mediated by receptors for the constant region of immunoglobulins (Fc). We have demonstrated previously that, in interferon-gamma-primed U937 cells, the high affinity receptor for IgG, FcgammaRI, is coupled to a novel intracellular signaling pathway that involves the sequential activation of phospholipase D, sphingosine kinase, calcium transients, and protein kinase C isoforms, leading to the activation of the NADPH-oxidative burst. Here, we investigate the nature of the phospholipase that regulates arachidonic acid and eicosanoid production. Our data show that FcgammaRI couples to iPLA(2)beta for the release of arachidonic acid and the generation of leukotriene B(4) and prostaglandin E(2). Activation of iPLA(2)beta was protein kinase C-dependent; on the other hand, platelet-activating factor triggered cPLA(2)alpha by means of the mitogen-activated protein kinase pathway. These studies demonstrate that intracellular PLA(2)s can be selectively regulated by different stimuli and suggest a critical role for iPLA(2)beta in the intracellular signaling cascades initiated by FcgammaRI and its functional role in the generation of key inflammatory mediators.     

The Role of Cell Hydrophobicity in the Formation of Aerobic Granules

Cell hydrophobicity is an important affinity force in cell self-immobilization and attachment processes. The role of cell hydrophobicity in the formation of aerobic granules has not been clear. Therefore, two series of experiments were conducted to investigate the role of cell hydrophobicity in the formation of aerobic heterotrophic and nitrifying granules in sequencing batch reactors, while the effects of shear strength, hydraulic selection pressure, and organic loading rate on the cell hydrophobicity were also studied. Results showed that the formations of heterotrophic and nitrifying granules were associated very closely with the cell hydrophobicity. The hydrophobicity of granular sludge was nearly twofold higher than that of conventional bioflocs. A high shear force or hydraulic selection pressure imposed on microorganisms resulted in a significant increase in the cell hydrophobicity, while the cell hydrophobicity seemed not to be sensitive to the changes in the organic loading rates in the range studied. In conclusion, the cell hydrophobicity could induce and further strengthen cell–cell interaction, and might be a main triggering force to initiate the granulation of heterotrophic and nitrifying bacteria. Received: 21 May 2002 / Accepted: 21 June 2002     

Development and characteristics of phosphorus-accumulating microbial granules in sequencing batch reactors

Phosphorus (P)-accumulating microbial granules were developed at different substrate P/chemical oxygen demand (COD) ratios in the range of 1/100 to 10/100 by weight in sequencing batch reactors. The soluble COD and PO4-P profiles showed that the granules had typical P-accumulating characteristics, with concomitant uptake of soluble organic carbon and the release of phosphate in the anaerobic stage, followed by rapid phosphate uptake in the aerobic stage. The size of P-accumulating granules exhibited a decreasing trend with the increase in substrate P/COD ratio, while the structure of the granules became more compact and denser as the substrate P/COD ratio increased. The P uptake by granules fell within the range of 1.9% to 9.3% by weight, which is comparable with uptake obtained in conventional enhanced biological phosphorus removal (EBPR) processes. It was further found that low aerobic respirometric activity of granules in terms of specific oxygen utilization rate favors P uptake by granules. The results presented would be useful for the further development of a novel granule-based EBPR technology.     

A general model for biosorption of Cd2+, Cu2+ and Zn2+ by aerobic granules

Aerobic granules are microbial aggregates with a strong and compact structure. This study looked into the feasibility of aerobic granules as a novel type of biosorbent for the removal of individual Cd(2+), Cu(2+) and Zn(2+) from aqueous solution. Based on the thermodynamics of biosorption reaction, a general model was developed to describe the equilibrium biosorption of individual Cd(2+), Cu(2+) and Zn(2+) by aerobic granules. This model provides good insights into the thermodynamic mechanisms of biosorption of heavy metals. The model prediction was in good agreement with the experimental data obtained. It was further demonstrated that the Langmuir, Freundlich and Sips or Hill equations were particular cases of the proposed model. The biosorption capacity of individual Cd(2+), Cu(2+) and Zn(2+) on aerobic granules was 172.7, 59.6 and 164.5 mgg(-1), respectively. These values may imply that aerobic granules are effective biosorbent for the removal of Cd(2+), Cu(2+) and Zn(2+) from industrial wastewater.     

Thermotolerant guard cell protoplasts of tree tobacco do not require exogenous hormones to survive in culture and are blocked from reentering the cell cycle at the G1-to-S transition

When guard cell protoplasts (GCPs) of tree tobacco [Nicotiana glauca (Graham)] are cultured at 32 degrees C with an auxin (1-napthaleneacetic acid) and a cytokinin (6-benzylaminopurine), they reenter the cell cycle, dedifferentiate, and divide. GCPs cultured similarly but at 38 degrees C and with 0.1 micro M +/- -cis,trans-abscisic acid (ABA) remain differentiated. GCPs cultured at 38 degrees C without ABA dedifferentiate partially but do not divide. Cell survival after 1 week is 70% to 80% under all of these conditions. In this study, we show that GCPs cultured for 12 to 24 h at 38 degrees C accumulate heat shock protein 70 and develop a thermotolerance that, upon transfer of cells to 32 degrees C, enhances cell survival but inhibits cell cycle reentry, dedifferentiation, and division. GCPs dedifferentiating at 32 degrees C require both 1-napthaleneacetic acid and 6-benzylaminopurine to survive, but thermotolerant GCPs cultured at 38 degrees C +/- ABA do not require either hormone for survival. Pulse-labeling experiments using 5-bromo-2-deoxyuridine indicate that culture at 38 degrees C +/- ABA prevents dedifferentiation of GCPs by blocking cell cycle reentry at G1/S. Cell cycle reentry at 32 degrees C is accompanied by loss of a 41-kD polypeptide that cross-reacts with antibodies to rat (Rattus norvegicus) extracellular signal-regulated kinase 1; thermotolerant GCPs retain this polypeptide. A number of polypeptides unique to thermotolerant cells have been uncovered by Boolean analysis of two-dimensional gels and are targets for further analysis. GCPs of tree tobacco can be isolated in sufficient numbers and with the purity required to study plant cell thermotolerance and its relationship to plant cell survival, growth, dedifferentiation, and division in vitro.     

Effect of iron hydroxide on the phosphate elimination during anaerobic digestion of active sludge

Addition of iron (III) hydroxide during methanogenic digestion of active sludge by anaerobic sludge displaying an iron-reducing activity resulted in a microbial reduction of iron (III) with formation of iron (II), capable of precipitating phosphates. Feasibility of eliminating 66.6 to 99.6% of dissolved phosphate at initial concentrations of 1000 to 3500 mg PO4(3-)/l by adding 6420 mg/l iron (III) hydroxide into a reactor for anaerobic fermentation of active sludge. The optimal ratio of iron (III) added to dissolved phosphate eliminated (mg) providing a 95% elimination amounted to 2:1. These results may be used in a new technology for anaerobic wastewater treatment with phosphate elimination.     

Comparison of generalized and gender-specific transfer functions for the derivation of aortic waveforms

Arterial transfer functions have been promoted for the derivation of central aortic waveform characteristics not usually accessible noninvasively, but possibly of prognostic significance. The utility of generalized rather than gender-specific transfer functions has not been assessed. Invasive central aortic and noninvasive radial (Millar Mikro-tip tonometer) blood pressure waveforms were recorded simultaneously in 78 subjects (61 male and 17 female). Average transfer functions were obtained for the whole group and for each gender by two methods. Reverse transformation was performed with the use of each transfer function. Measured aortic waveform parameters were compared with those derived using average, gender-appropriate, and gender-inappropriate transfer functions. Differences in central waveform characteristics were demonstrated between men and women. Derived waveform parameters were significantly different from measured values [e.g., subendocardial viability index and augmentation index (P < 0.001)]. A gender-appropriate transfer function significantly improved the derivation of some parameters, including systolic pressure and systolic and diastolic pressure time integrals (P < 0.05). Generalized arterial transfer functions may not be universally applicable across all waveform parameters of potential interest, and gender-specific transfer functions may be more appropriate.