Global Biogeography and Quantitative Seasonal Dynamics of Gemmatimonadetes in Soil

Bacteria belonging to phylum Gemmatimonadetes comprise approximately 2% of soil bacterial communities. However, little is known of their ecology due to a lack of cultured representation. Here we present evidence from biogeographical analyses and seasonal quantification of Gemmatimonadetes in soils, which suggests an adaptation to low soil moisture.     

Epigenetic interactions between transposons and genes: lessons from plants

Transposons replicate, increase in copy number and persist in nature by moving, but insertion into genes is generally mutagenic. There is thus a strong selection for transposons that can achieve a balance between their own replication and minimal damage to their host. Epigenetic regulation proves to be a widespread way to achieve this balance, quieting transposition on the one hand, yet reversible on the other. As our understanding of epigenetics improves, the subtleties and the scope of how transposons can affect gene expression, both directly and indirectly, are becoming clearer.     

Give-and-take: interactions between DNA transposons and their host plant genomes

Recent genome sequencing efforts have revealed how extensively transposable elements (TEs) have contributed to the shaping of present day plant genomes. DNA transposons associate preferentially with the euchromatic or genic component of plant genomes and have had the opportunity to interact intimately with the genes of the plant host. These interactions have resulted in TEs acquiring host sequences, forming chimeric genes through exon shuffling, replacing regulatory sequences, mobilizing genes around the genome, and contributing genes to the host. The close interaction of transposons with genes has also led to the evolution of intricate cellular mechanisms for silencing transposon activity. Transposons have thus become important subjects of study in understanding epigenetic regulation and, in cases where transposons have amplified to high numbers, how to escape that regulation.     

Tiered Categorization of a Diverse Panel of HIV-1 Env Pseudoviruses for Assessment of Neutralizing Antibodies

The restricted neutralization breadth of vaccine-elicited antibodies is a major limitation of current human immunodeficiency virus-1 (HIV-1) candidate vaccines. In order to permit the efficient identification of vaccines with enhanced capacity for eliciting cross-reactive neutralizing antibodies (NAbs) and to assess the overall breadth and potency of vaccine-elicited NAb reactivity, we assembled a panel of 109 molecularly cloned HIV-1 Env pseudoviruses representing a broad range of genetic and geographic diversity. Viral isolates from all major circulating genetic subtypes were included, as were viruses derived shortly after transmission and during the early and chronic stages of infection. We assembled a panel of genetically diverse HIV-1-positive (HIV-1⁺) plasma pools to assess the neutralization sensitivities of the entire virus panel. When the viruses were rank ordered according to the average sensitivity to neutralization by the HIV-1⁺ plasmas, a continuum of average sensitivity was observed. Clustering analysis of the patterns of sensitivity defined four subgroups of viruses: those having very high (tier 1A), above-average (tier 1B), moderate (tier 2), or low (tier 3) sensitivity to antibody-mediated neutralization. We also investigated potential associations between characteristics of the viral isolates (clade, stage of infection, and source of virus) and sensitivity to NAb. In particular, higher levels of NAb activity were observed when the virus and plasma pool were matched in clade. These data provide the first systematic assessment of the overall neutralization sensitivities of a genetically and geographically diverse panel of circulating HIV-1 strains. These reference viruses can facilitate the systematic characterization of NAb responses elicited by candidate vaccine immunogens.The development of an HIV-1 vaccine that can elicit protective humoral and cellular immunity is one of the highest priorities in the global fight against HIV/AIDS (2, 44). Data from lentiviral animal models suggest that antibodies capable of neutralizing primary strains of HIV-1 may have the capacity to prevent HIV-1 infection (1, 28, 30, 35). However, the ability to design immunogens that can elicit such broadly reactive neutralizing antibodies (NAbs) has proven to be a formidable obstacle, due in part to the extensive genetic diversity of HIV-1 and the complex escape mechanisms employed by the envelope gp120 and gp41 glycoproteins that form the trimeric viral envelope spike (Env) (20, 34, 45). As improved vaccine immunogens enter the stage of detailed preclinical analysis, the in vitro assays used for evaluating vaccine sera will need to detect incremental advances in the magnitude, breadth, and durability of NAb responses (37). Such data can then be used to distinguish and prioritize among antibody-based vaccine immunogens. Furthermore, highly reproducible and quantitative data on vaccine-elicited NAbs can enhance our understanding of the relationship between Env immunogen design and the resulting antibody response generated.Current recommendations for evaluating candidate vaccine sera for NAb activity include the use of standard reference panels of molecularly cloned HIV-1 Env pseudoviruses and a tiered algorithm of testing (27). Reference virus panels should represent genetically and geographically diverse subsets of viruses with neutralization phenotypes that are generally representative of primary isolate strains that a vaccine would need to protect against. As such, standard reference panels for HIV-1 subtypes B and C have been described (22, 23), and efforts continue toward the creation of virus reference panels representing additional genetic subtypes. For tiered evaluation of NAb activity, vaccine sera are first tested against homologous Env pseudoviruses and/or a small number of isolates that are known to be highly sensitive to antibody-mediated neutralization (commonly referred to as tier 1 viruses). A more rigorous assessment of the potency and breadth of vaccine-induced NAbs entails testing against more resistant reference panel viruses (commonly referred to as tier 2 viruses) that are either matched or mismatched in genetic subtype to the vaccine immunogen (second and third tiers of testing, respectively). This tiered approach for testing candidate HIV-1 vaccine sera is advantageous in that it provides increasingly stringent levels for assessing the potency and breadth of NAbs, uses standardized panels of reference viruses for consistency and reproducibility, and allows for the generation of comparative data sets for evaluating different candidate vaccine regimens.While the tiered algorithm for evaluating vaccine sera has gained acceptance in the field, a major limitation has been the lack of objective data to characterize HIV-1 Env pseudoviruses according to their overall sensitivity or resistance to antibody-mediated neutralization. The category of sensitive, tier 1 viruses arose in part from the observation that HIV-1 isolates passaged through T-cell lines often become highly sensitive to antibody-mediated neutralization (33). Compared to these laboratory-adapted viruses, most primary isolate strains are moderately resistant to NAbs. Yet, even among recently isolated circulating viral Envs, there is a wide spectrum of neutralization sensitivity. Some HIV-1 isolates have a neutralization phenotype closer to that of tier 1 viruses, while others appear to be quite neutralization resistant (6, 19, 22, 23). Overall, there are few data from which to understand or categorize the viral neutralization phenotypes of HIV-1 strains. As a result, we have a limited ability to assess the potential potency of vaccine-elicited NAbs or to estimate the percentage of circulating HIV-1 isolates that would be neutralized. Further categorization of isolates into distinct subgroups based on sensitivity to NAbs may reveal patterns of neutralization that could provide a greater understanding of the NAb response generated by current and future vaccine immunogens. In addition, the structure-based design of novel immunogens may be facilitated by an ability to monitor the types of viruses neutralized and to specifically map the viral epitopes targeted by vaccine-elicited NAbs.In this study, we assembled a diverse panel of 109 HIV-1 Env pseudoviruses, including multiple representatives from clades A, B, and C and circulating recombinant forms (CRFs) CRF07_BC and CRF02_AG-related. These were tested for their sensitivities using HIV-1-positive (HIV-1⁺) plasma samples representative of clades A, B, and C and CRF01_AE and CRF02_AG. Clinical, demographic, and viral genetic sequence data were collected for each virus. The neutralization phenotype of each virus was assessed with a panel of seven clade-specific HIV-1⁺ plasma pools. Viruses were rank ordered according to average neutralization sensitivity, and k-means clustering was utilized to identify four subgroups of viruses with neutralization phenotypes ranging from highly sensitive to resistant. Together, these results will improve the ability to rigorously evaluate antibody-based HIV-1 vaccines and will facilitate the interpretation of assay results to identify immunogens with improved capacity to elicit broadly cross-reactive NAbs.     

Idaho forest growth response to post‐thinning energy biomass removal and complementary soil amendments

Utilization of woody biomass for biofuel can help meet the need for renewable energy production. However, there is a concern biomass removal will deplete soil nutrients, having short‐ and long‐term effects on tree growth. This study aimed to develop short‐term indicators to assess the impacts of the first three years after small‐diameter woody biomass removal on forest productivity to establish optimal biomass retention levels for mixed‐conifer forests in the Inland Northwest region, and to evaluate the ability of soil amendments to compensate for potential adverse effects from biomass removal. We examined impacts of four biomass retention‐level treatments at two study locations: full biomass removal (0x), full biomass retention (1x), double biomass retention (2x), and unthinned control. We combined biomass retention with four soil amendment treatments: biochar (B), fertilizer (F), fertilizer and biochar combined (FB), and an untreated control (C). We considered treatment effects on basal area and total stem volume growth for all trees per plot (plot trees) and for the six largest trees per plot (crop trees). Biomass removal had no effect on plot (P > 0.40) or crop tree growth (P > 0.65) compared to normal biomass retention. High biomass retention (2x) decreased plot tree growth as compared to normal biomass retention (1x) levels (P < 0.05) after three years. This growth difference was not explained by soil moisture, temperature, or nutrient uptake. While there were strong tree growth differences between study locations, patterns of biomass and amendment treatment responses did not differ. Fertilizer increased basal area growth and total volume growth (P < 0.10) as expected, because nitrogen is limiting in the region. Biochar had no effect on tree growth (P > 0.47). Initial findings after three years suggest removing small‐diameter biomass for biofuel feedstocks is feasible in the Inland Northwest without negative impacts on tree growth.     

A quantitative polymerase chain reaction assay for detecting and identifying fungal contamination in human allograft tissue

To complement donor selection and tissue processing, rapid and reliable detection, discrimination, and quantification of fungal pathogens are extremely important for tissues destined to be implanted into humans. The current detection method for fungal pathogens, in particular, is difficult and time-consuming. Quantitative polymerase chain reaction (qPCR) technology is considered one of the most sensitive methods to detect low levels of DNA. Here a qPCR method is described that can detect clinically relevant, pathogenic fungal organisms. The assay allowed the quantification of fungal organisms within a tissue implant and provides a means to identify the contaminating species. The primers for the qPCR assay were designed to amplify a conserved region of the L2 region of the large ribosomal subunit (LSU) gene. This set of primers was able to detect fewer than 10 colony forming units from Aspergillus and Candida species in spiked samples. Clinical samples were also evaluated using this method and the data compared positively to the existing accepted 28-day fungal culture method for fungal detection. The qPCR method described herein significantly reduced the time required to identify fungal contamination in tissue implants.     

A study of chromosomal changes associated with amplified dihydrofolate reductase genes in rat hepatoma cells and their dedifferentiated variants

We have examined the karyological consequences of dihydrofolate reductase gene amplification in a series of six rat hepatoma cell lines, all derived from the same clone. Cells of three of these lines express a series of liver-specific functions whereas those of three others fail to express these functions. Cells of each line have been subjected to stepwise selection for methotrexate resistance and, in most cases, resistance is associated with a 40-50-fold amplification of sequences hybridizing to a dihydrofolate reductase cDNA probe. In one line no modified chromosome is observed, whereas in two others the amplified genes are associated with an expanded chromosomal region. R- banding analysis of these karyotypes showed that few changes have occurred. These observations apply to two of the well-differentiated lines, and to a variant able to revert to the differentiated state. In contrast, in the two stably dedifferentiated hepatoma cell lines, amplified dihydrofolate reductase genes are found on large chromosomes of variable size, on ring chromosomes, and on chromosomes containing terminal, median, or multiple centromeres. We conclude that the nature of the chromosomal changes associated with dihydrofolate reductase gene amplification are the result of differences in cell lines rather than in the protocols employed for selection.     

Identification of a Cryptic Bacterial Promoter in Mouse (mdr1a) P-Glycoprotein cDNA

The efflux transporter P-glycoprotein (P-gp) is an important mediator of various pharmacokinetic parameters, being expressed at numerous physiological barriers and also in multidrug-resistant cancer cells. Molecular cloning of homologous cDNAs is an important tool for the characterization of functional differences in P-gp between species. However, plasmids containing mouse mdr1a cDNA display significant genetic instability during cloning in bacteria, indicating that mdr1a cDNA may be somehow toxic to bacteria, allowing only clones containing mutations that abrogate this toxicity to survive transformation. We demonstrate here the presence of a cryptic promoter in mouse mdr1a cDNA that causes mouse P-gp expression in bacteria. This expression may account for the observed toxicity of mdr1a DNA to bacteria. Sigma 70 binding site analysis and GFP reporter plasmids were used to identify sequences in the first 321 bps of mdr1a cDNA capable of initiating bacterial protein expression. An mdr1a M107L cDNA containing a single residue mutation at the proposed translational start site was shown to allow sub-cloning of mdr1a in E. coli while retaining transport properties similar to wild-type P-gp. This mutant mdr1a cDNA may prove useful for efficient cloning of mdr1a in E. coli.