Diminished transmission of drug resistant HIV-1 variants with reduced replication capacity in a human transmission model

Background

Different patterns of drug resistance are observed in treated and therapy naïve HIV-1 infected populations. Especially the NRTI-related M184I/V variants, which are among the most frequently encountered mutations in treated patients, are underrepresented in the antiretroviral naïve population. M184I/V mutations are known to have a profound effect on viral replication and tend to revert over time in the new host. However it is debated whether a diminished transmission efficacy of HIV variants with a reduced replication capacity can also contribute to the observed discrepancy in genotypic patterns.As dendritic cells (DCs) play a pivotal role in HIV-1 transmission, we used a model containing primary human Langerhans cells (LCs) and DCs to compare the transmission efficacy M184 variants (HIV-M184V/I/T) to HIV wild type (HIV-WT). As control, we used HIV harboring the NNRTI mutation K103N (HIV-K103N) which has a minor effect on replication and is found at a similar prevalence in treated and untreated individuals.

Results

In comparison to HIV-WT, the HIV-M184 variants were less efficiently transmitted to CCR5⁺ Jurkat T cells by both LCs and DCs. The transmission rate of HIV-K103N was slightly reduced to HIV-WT in LCs and even higher than HIV-WT in DCs. Replication experiments in CCR5⁺ Jurkat T cells revealed no apparent differences in replication capacity between the mutant viruses and HIV-WT. However, viral replication in LCs and DCs was in concordance with the transmission results; replication by the HIV-M184 variants was lower than replication by HIV-WT, and the level of replication of HIV-K103N was intermediate for LCs and higher than HIV-WT for DCs.

Conclusions

Our data demonstrate that drug resistant M184-variants display a reduced replication capacity in LCs and DCs which directly impairs their transmission efficacy. As such, diminished transmission efficacy may contribute to the lower prevalence of drug resistant variants in therapy naive individuals.

     

Hydrodynamic effects on BHK cells grown as suspended natural aggregates

Baby hamster kidney (BHK) cell aggregates grown in stirred vessels with different working volumes and impeller sizes were characterized. Using batch cultures, the range of agitation rates studied (25-100 rpm) led to aggregates with maximum sizes of 150 mum. Necrotic centers were not observed and cell specific productivity was independent of aggregate size. High cell viability was found for both single and adherent cells without an increase in cell death when agitation rate was increased. The increase in agitation rate affected aggregates by reducing their size and increasing their concentration and cell concentration in aggregates, while increasing the fraction of free cells in suspension. The experimental relationship between aggregate size and power dissipation rate per unit of mass was close to -1/4, suggesting a correlation with a critical turbulence microscale; this was independent of vessel scale and impeller geometry over the range investigated. Viscous stresses in the viscous dissipation subrange (below Kolmogoroff eddies) appear to be responsible for aggregate breakage. Under intense agitation BHK cells grown in the absence of microcarriers existed as aggregates without cell damage, whereas cells grown on the surface of microcarriers were largely reduced. This is a clear advantage for scaleup purposes if aggregates are used as a natural immobilization system in stirred vessels. (c) 1995 John Wiley & Sons, Inc.     

Competition between nitrate and nitrite reduction in denitrification by Pseudomonas fluorescens

A pure culture of Pseudomonas fluorescens was used as a model system to study the kinetics of denitrification. An exponentially growing culture was harvested and resuspended in an anoxic acetate solution buffered with K/Na phosphate at pH values of 6.6, 7.0, 7.4, and 7.8. The temperature was kept at 28 degrees C in all assays. Nitrate pulses of approximately 0.2 mg N/L caused nitrite to accumulate due to a faster rate of nitrate reduction over nitrite reduction. The rate of nitrate reduction was observed to depend on its concentration as predicted by the Michaelis-Menten equation. At nonlimiting nitrate concentrations, nitrite reduction was described by the same equation. Otherwise, nitrite reduction also depended on nitrate concentration. Consequently, nitrate and nitrite reductions compete with each other for the oxidation of common electron donors. A kinetic model for nitrate competitive inhibition of nitrite reduction is proposed. The model was used to interpret the nitrate and nitrite profiles observed at the four pH values: the optimum pH value was 7.0 in both cases; the affinity for nitrite was also not affected by the medium pH in the range of values 6.6 to 7.4 (K(mNO(3) ) = 0.04 mg N/L); the affinity for nitrite was also not affected by the medium pH in the range of values 6.6 to 7.4 (K(mNO(2) ) = 0.06 mg N/L), but it decreased sharply for the pH value of 7.8. Although the ratio between the two maximum reduction rates (V(max NO(2) )/V(max NO(3) )) is constant, nitrite accumulation depends on the medium pH value. Therefore, the regulation mechanism that shifts the electron flow between the two terminal reductases is readily reversible and does not change their relative maximum reduction rates. (c) 1995 John Wiley & Sons, Inc.     

A graphical method for detecting recombination in phylogenetic data sets

Current phylogenetic tree reconstruction methods assume that there is a single underlying tree topology for all sites along the sequence. The presence of mosaic sequences due to recombination violates this assumption and will cause phylogenetic methods to give misleading results due to the imposition of a single tree topology on all sites. The detection of mosaic sequences caused by recombination is therefore an important first step in phylogenetic analysis. A graphical method for the detection of recombination, based on the least squares method of phylogenetic estimation, is presented here. This method locates putative recombination breakpoints by moving a window along the sequence. The performance of the method is assessed by simulation and by its application to a real data set.      

bilbo, a non-LTR retrotransposon of Drosophila subobscura: a clue to the evolution of LINE-like elements in Drosophila

We used the repetitive character of transposable elements to isolate a non-LTR retrotransposon in Drosophila subobscura. bilbo, as we have called it, has homology to TRIM and LOA elements. Sequence analysis showed a 5' untranslated region (UTR), an open reading frame (ORF) with no RNA-binding domains, a downstream ORF that had structural homology to that of the I factor, and, finally, a 3' UTR which ended in several 5-nt repeats. The results of our phylogenetic and structural analyses shed light on the evolution of Drosophila non-LTR retrotransposons and support the hypothesis that an ancestor of these elements was structurally complex.      

Desulfoferrodoxin structure determined by MAD phasing and refinement to 1.9-Å resolution reveals a unique combination of a tetrahedral FeS4 centre with a square pyramidal FeSN4 centre

The structure of desulfoferrodoxin (DFX), a protein containing two mononuclear non-heme iron centres, has been solved by the MAD method using phases determined at 2.8?Å resolution. The iron atoms in the native protein were used as the anomalous scatterers. The model was built from an electron density map obtained after density modification and refined against data collected at 1.9?Å. Desulfoferrodoxin is a homodimer which can be described in terms of two domains, each with two crystallographically equivalent non-heme mononuclear iron centres. Domain I is similar to desulforedoxin with distorted rubredoxin-type centres, and domain II has iron centres with square pyramidal coordination to four nitrogens from histidines as the equatorial ligands and one sulfur from a cysteine as the axial ligand. Domain I in DFX shows a remarkable structural fit with the DX homodimer. Furthermore, three β-sheets extending from one monomer to another in DFX, two in domain I and one in domain II, strongly support the assumption of DFX as a functional dimer. A calcium ion, indispensable in the crystallisation process, was assumed at the dimer interface and appears to contribute to dimer stabilisation. The C-terminal domain in the monomer has a topology fold similar to that of fibronectin III.     

Variation of physico-chemical parameters along a river transect through the Okavango Delta,Botswana

The Okavango Delta depends on water quantity and quality to sustain its ecosystem services. Whereas many studies have been carried out on its hydrology, few have been done on water quality in the delta. Water pH, electrical conductivity (EC), dissolved oxygen (DO), turbidity, total suspended solids (TSS) and dissolved organic carbon (DOC) were monitored at 10 sites along the Okavango–Boro–Thamalakane–Lake Ngami system almost fortnightly from June 2008 to June 2010. Water quality in the delta was generally good, despite high evapotranspiration rates which would normally produce very saline waters. Electrical conductivity and water temperature increased with distance from Mohembo to Lake Ngami, the former most likely due to evapoconcentration. In contrast, pH, DO, turbidity and TSS decreased with distance from Mohembo to Boro at the lower end of the seasonal floodplain, before increasing again to Lake Ngami. Dissolved oxygen and TSS most likely declined due to biological uptake and particle sedimentation, respectively. Strong and significant relationships were observed between TSS and turbidity and between DOC and EC, indicating that turbidity and EC could be useful proxies for routine estimations of TSS and DOC, respectively, in the delta.     

Macroinvertebrates as bioindicators of water quality in the Mkondoa River,Tanzania, in an agricultural area

The suitability of using macroinvertebrates as bioindicators of stream water quality was tested in the Mkondoa River in an agricultural area at Kilosa, using the rapid bioassessment protocol. The family biotic index (FBI) showed marked variation in water quality along the stream from values ranging from 4.1 to 5.0 in the upstream reaches, indicating good water quality, 5.3 to 5.5 in the mid-reaches and 6.0 to 6.5 in the lower reaches. The water quality index (WQI) indicated that water quality was fair (77 ± 0.98) in the upstream reach of the Mkondoa, marginal (55 ± 0.86) in the midstream reach and poor (33 ± 0.45) in the downstream reach. There were significant relationships between biological oxygen demand and dissolved oxygen and the occurrence of specific taxa, mainly Chironomus and Caenis. Significant changes in macroinvertebrate abundance were mostly related to changes in water quality. As in other parts of the world, macroinvertebrate communities proved to be good biological indicators of water quality and they should be used as bioindicators in long-term monitoring of this river.     

Starter culture production in fluidized bed reactor with a flocculent strain ofL. plantarum

Summary A lactic starter culture of a flocculentLactobacillus plantarum was produced in a fluidized bed reactor with higher cell volumetric productivities than in a continuous stirred tank reactor. The fluidized bed reactor was operated at optimised parameters obtained in batch reactor performed with and without pH control.