Widespread impact of HLA restriction on immune control and escape pathways of HIV-1

The promiscuous presentation of epitopes by similar HLA class I alleles holds promise for a universal T-cell-based HIV-1 vaccine. However, in some instances, cytotoxic T lymphocytes (CTL) restricted by HLA alleles with similar or identical binding motifs are known to target epitopes at different frequencies, with different functional avidities and with different apparent clinical outcomes. Such differences may be illuminated by the association of similar HLA alleles with distinctive escape pathways. Using a novel computational method featuring phylogenetically corrected odds ratios, we systematically analyzed differential patterns of immune escape across all optimally defined epitopes in Gag, Pol, and Nef in 2,126 HIV-1 clade C-infected adults. Overall, we identified 301 polymorphisms in 90 epitopes associated with HLA alleles belonging to shared supertypes. We detected differential escape in 37 of 38 epitopes restricted by more than one allele, which included 278 instances of differential escape at the polymorphism level. The majority (66 to 97%) of these resulted from the selection of unique HLA-specific polymorphisms rather than differential epitope targeting rates, as confirmed by gamma interferon (IFN-γ) enzyme-linked immunosorbent spot assay (ELISPOT) data. Discordant associations between HLA alleles and viral load were frequently observed between allele pairs that selected for differential escape. Furthermore, the total number of associated polymorphisms strongly correlated with average viral load. These studies confirm that differential escape is a widespread phenomenon and may be the norm when two alleles present the same epitope. Given the clinical correlates of immune escape, such heterogeneity suggests that certain epitopes will lead to discordant outcomes if applied universally in a vaccine.     

Next-generation sequencing and syntenic integration of flow-sorted arms of wheat chromosome 4A exposes the chromosome structure and gene content

Wheat is the third most important crop for human nutrition in the world. The availability of high-resolution genetic and physical maps and ultimately a complete genome sequence holds great promise for breeding improved varieties to cope with increasing food demand under the conditions of changing global climate. However, the large size of the bread wheat (Triticum aestivum) genome (approximately 17 Gb/1C) and the triplication of genic sequence resulting from its hexaploid status have impeded genome sequencing of this important crop species. Here we describe the use of mitotic chromosome flow sorting to separately purify and then shotgun-sequence a pair of telocentric chromosomes that together form chromosome 4A (856 Mb/1C) of wheat. The isolation of this much reduced template and the consequent avoidance of the problem of sequence duplication, in conjunction with synteny-based comparisons with other grass genomes, have facilitated construction of an ordered gene map of chromosome 4A, embracing ≥85% of its total gene content, and have enabled precise localization of the various translocation and inversion breakpoints on chromosome 4A that differentiate it from its progenitor chromosome in the A genome diploid donor. The gene map of chromosome 4A, together with the emerging sequences of homoeologous wheat chromosome groups 4, 5 and 7, represent unique resources that will allow us to obtain new insights into the evolutionary dynamics between homoeologous chromosomes and syntenic chromosomal regions.     

Prairie plant phenology driven more by temperature than moisture in climate manipulations across a latitudinal gradient in the Pacific Northwest,USA

Plant phenology will likely shift with climate change, but how temperature and/or moisture regimes will control phenological responses is not well understood. This is particularly true in Mediterranean climate ecosystems where the warmest temperatures and greatest moisture availability are seasonally asynchronous. We examined plant phenological responses at both the population and community levels to four climate treatments (control, warming, drought, and warming plus additional precipitation) embedded within three prairies across a 520 km latitudinal Mediterranean climate gradient within the Pacific Northwest, USA. At the population level, we monitored flowering and abundances in spring 2017 of eight range‐restricted focal species planted both within and north of their current ranges. At the community level, we used normalized difference vegetation index (NDVI) measured from fall 2016 to summer 2018 to estimate peak live biomass, senescence, seasonal patterns, and growing season length. We found that warming exerted a stronger control than our moisture manipulations on phenology at both the population and community levels. Warming advanced flowering regardless of whether a species was within or beyond its current range. Importantly, many of our focal species had low abundances, particularly in the south, suggesting that establishment, in addition to phenological shifts, may be a strong constraint on their future viability. At the community level, warming advanced the date of peak biomass regardless of site or year. The date of senescence advanced regardless of year for the southern and central sites but only in 2018 for the northern site. Growing season length contracted due to warming at the southern and central sites (~3 weeks) but was unaffected at the northern site. Our results emphasize that future temperature changes may exert strong influence on the timing of a variety of plant phenological events, especially those events that occur when temperature is most limiting, even in seasonally water‐limited Mediterranean ecosystems.     

The use of PGF2α as ovulatory stimulus for timed artificial insemination in cattle

The objective of this study was to evaluate the effect of a PGF2α-analogue (PGF) on ovulation and pregnancy rates after timed artificial insemination (TAI) in cattle. In experiment 1, crossbred dual-purpose heifers, in a crossover design (3 × 3), were given an intravaginal progesterone-releasing insert (controlled internal drug release [CIDR]) plus 1 mg estradiol benzoate (EB) intramuscularly (im) and 250 μg of a PGF-analogue im on Day 0. The CIDR inserts were removed 5 days after follicular wave emergence, and the heifers were randomly divided into three treatment groups to receive the following treatments: (1) 1 mg of EB im (EB group, n = 13); (2) 500 μg of PGF im (PG group, n = 13); or (3) saline (control group, n = 13), 24 hours after CIDR removal. Ovulation occurred earlier in EB (69.81 ± 3.23 hours) and PG groups (73.09 ± 3.23 hours) compared with control (83.07 ± 4.6 hours; P = 0.01) after CIDR removal. In experiment 2, pubertal beef heifers (n = 444), 12 to 14 months of age were used. On Day 0, the heifers were given a CIDR insert plus 2 mg EB im. On Day 9, the CIDR was removed and the heifers were given 500 μg of PGF im. Heifers were randomly assigned into one of three treatment groups: (1) 1 mg of EB (EB group; n = 145); (2) 500 μg of PGF (PG group; n = 149), both 24 hours after CIDR removal; or (3) 600 μg of estradiol cypionate (ECP group; n = 150) at CIDR removal. Timed artificial insemination occurred 48 hours after CIDR removal in the ECP group and 54 hours in the PG and EB groups. The percentage of heifers ovulating was higher in the PG group compared with the other groups (P = 0.08). However, the pregnancy rates did not differ among groups (47.6%, 45%, and 46.6%, for EB, PG, and ECP, respectively; P = 0.9). In experiment 3, 224 lactating beef cows, 40 to 50 days postpartum with 2.5 to 3.5 of body condition score were treated similarly as described in experiment 2, except for the ECP group, which was excluded. The treatments were as follows: 1 mg EB (EB group; n = 117) or 500 μg PGF (PG group; n = 107), 24 hours after CIDR removal. The calves were temporarily separated from their dams from Days 9 to 11. No difference was detected on the pregnancy rate between the EB and PG groups (58.1% vs. 47.6%, respectively; P = 0.11). Taken together, the combined results suggested that PGF2α could be successfully used to induce and synchronize ovulation in cattle undergoing TAI, with similar pregnancy rates when compared with other ovulatory stimuli (ECP and EB).     

Metabolic flux analysis and pharmaceutical production

Rational engineering of biological systems is an inherently complex process due to their evolved nature. Metabolic engineering emerged and developed over the past 20 years as a field in which methodologies for the rational engineering of biological systems is now being applied to specific industrial, medical, or scientific problems. Of considerable interest is the determination of metabolic fluxes within the cell itself, called metabolic flux analysis. This special issue and this review have a particular interest in the application of metabolic flux analysis for improving the pharmaceutical production process (for both small and large molecules). Though metabolic flux analysis has been somewhat limited in application towards pharmaceutical production, the overall goal is to: (1) have a better understanding of the organism and/or process in question, and (2) provide a rational basis to further engineer (on both metabolic and process scales) improved pharmaceutical production in these organisms. The focus of this review article is to present how experimental and computational methods of metabolic flux analysis have matured, mirroring the maturation of the metabolic engineering field itself, while highlighting some of the successful applications towards both small- and large-molecule pharmaceuticals.     

Profilin 1 is required for abscission during late cytokinesis of chondrocytes

Profilins are key factors for dynamic rearrangements of the actin cytoskeleton. However, the functions of profilins in differentiated mammalian cells are uncertain because profilin deficiency is early embryonic lethal for higher eukaryotes. To examine profilin function in chondrocytes, we disrupted the profilin 1 gene in cartilage (Col2pfn1). Homozygous Col2pfn1 mice develop progressive chondrodysplasia caused by disorganization of the growth plate and defective chondrocyte cytokinesis, indicated by the appearance of binucleated cells. Surprisingly, Col2pfn1 chondrocytes assemble and contract actomyosin rings normally during cell division; however, they display defects during late cytokinesis as they frequently fail to complete abscission due to their inability to develop strong traction forces. This reduced force generation results from an impaired formation of lamellipodia, focal adhesions and stress fibres, which in part could be linked to an impaired mDia1‐mediated actin filament elongation. Neither an actin nor a poly‐proline binding‐deficient profilin 1 is able to rescue the defects. Taken together, our results demonstrate that profilin 1 is not required for actomyosin ring formation in dividing chondrocytes but necessary to generate sufficient force for abscission during late cytokinesis.     

Effects of low versus physiologic plasma progesterone concentrations on ovarian follicular development and fertility in beef cattle

The objective of this study was to determine the effects of low versus physiologic plasma progesterone concentrations during the ovulatory wave on fertility in cattle. Suckled beef cows (Bos taurus; n = 129) and pubertal heifers (Bos taurus; n = 150) at random stages of the estrous cycle were given a luteolytic dose of prostaglandin F_2α (500 μg cloprostenol; PGF) twice, 11 d apart. Ten days after the second PGF treatment, cattle were given estradiol benzoate im (1.5 and 1.0 mg for cows and heifers, respectively) and a progesterone-releasing intravaginal device (Cue-Mate) with a single pod containing 0.78 g progesterone (Day 0). Cattle in the low-progesterone group (n = 148) received a luteolytic dose of PGF on Day 0, whereas those in the high-progesterone (i.e., physiologic plasma concentrations) group (n = 131) were allowed to retain their corpora lutea. On Day 8, the Cue-Mate was removed, and PGF was given to both groups. Fifty-four hours to 56 h later, cattle received 12.5 mg of porcine LH (pLH) im and were concurrently artificially inseminated. The dominant follicle in the low-progesterone group was larger (P < 0.001) than that in the high-progesterone group on the day of insemination (14.9 ± 0.3 mm vs. 12.7 ± 0.3 mm, mean ± SEM). At 7 d after ovulation, the low-progesterone group had a larger corpus luteum (24.5 ± 0.54 mm vs. 21.9 ± 0.64 mm, P < 0.01) and higher plasma progesterone concentration (4.0 ± 0.3 vs. 3.1 ± 0.2, P < 0.01) than that of the high-progesterone group. However, pregnancy rates did not differ (79 of 148, 53.4%, and 70 of 131, 53.4%) for low- and high-progesterone groups, respectively). In summary, low circulating progesterone concentrations during the growing phase of the ovulatory follicle resulted in a larger dominant follicle and a larger CL that produced more progesterone, with no significant effect on pregnancy rate.     

Examination of the specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine

DNA cytosine-5 methylation is a well-studied epigenetic pathway implicated in gene expression control and disease pathogenesis. Different technologies have been developed to examine the distribution of 5-methylcytosine (5mC) in specific sequences of the genome. Recently, substantial amounts of 5-hydroxymethylcytosine (5hmC), most likely derived from enzymatic oxidation of 5mC by TET1, have been detected in certain mammalian tissues. Here, we have examined the ability of several commonly used DNA methylation profiling methods to distinguish between 5mC and 5hmC. We show that techniques based on sodium bisulfite treatment of DNA are incapable of distinguishing between the two modified bases. In contrast, techniques based on immunoprecipitation with anti-5mC antibody (methylated DNA immunoprecipitation, MeDIP) or those based on proteins that bind to methylated CpG sequences (e.g. methylated-CpG island recovery assay, MIRA) do not detect 5hmC and are specific for 5mC unless both modified bases occur in the same DNA fragment. We also report that several methyl-CpG binding proteins including MBD1, MBD2 and MBD4 do not bind to sequences containing 5hmC. Selective mapping of 5hmC will require the development of unique tools for the detection of this modified base.     

Structural model of the gas vesicle protein GvpA and analysis of GvpA mutants in vivo

Gas vesicles are gas-filled protein structures increasing the buoyancy of cells. The gas vesicle envelope is mainly constituted by the 8 kDa protein GvpA forming a wall with a water excluding inner surface. A structure of GvpA is not available; recent solid-state NMR results suggest a coil-α-β-β-α-coil fold. We obtained a first structural model of GvpA by high-performance de novo modelling. Attenuated total reflection (ATR)-Fourier transform infrared spectroscopy (FTIR) supported this structure. A dimer of GvpA was derived that could explain the formation of the protein monolayer in the gas vesicle wall. The hydrophobic inner surface is mainly constituted by anti-parallel β-strands. The proposed structure allows the pinpointing of contact sites that were mutated and tested for the ability to form gas vesicles in haloarchaea. Mutations in α-helix I and α-helix II, but also in the β-turn affected the gas vesicle formation, whereas other alterations had no effect. All mutants supported the structural features deduced from the model. The proposed GvpA dimers allow the formation of a monolayer protein wall, also consistent with protease treatments of isolated gas vesicles.