Investigation of a functional requirement for isoprenylation by the human prostacyclin receptor.

In the current study, we have established that the human (h) prostacyclin receptor (IP) is isoprenylated in whole cells. Through site directed mutagenesis and generation of the isoprenylation defective hIPSSLC, it was established that while isoprenylation of hIP does not influence ligand binding, it is obligatory for agonist activation of adenylyl cyclase and cAMP generation. Overexpression of GalphaS significantly augmented cAMP generation by the hIP but not by the hIPSSLC. Moreover, GalphaS co-immunoprecipitated with hIP following agonist activation but did not co-immunoprecipitate with hIPSSLC. Whereas hIP mediated concentration-dependent activation of phospholipase C (PLC); the extent of PLC activation by hIPSSLC was impaired compared to hIP. Co-expression of Galphaq significantly augmentated intracellular calcium mobilization by the hIP but not by hIPSSLC. Moreover, whereas Galphaq co-immunoprecipitated with hIP, it failed to co-immunoprecipitate with hIPSSLC. While both the hIP and hIPSSLC underwent agonist-induced internalization, the kinetics and extent of hIPSSLC internalization was impaired compared to hIP. Altering the CAAX motif of the hIP from a farnesyl (-CSLC) to a geranylgeranyl (-CSLL) isoprene acceptor, to generate hIPCSLL, did not affect ligand binding and yielded a receptor that exhibited identical signalling through both Gs- and Gq-coupled effectors to that of hIP. Thus, whereas isoprenylation of hIP does not influence ligand binding, it is functionally imperative in regulating post-receptor events including agonist-activation of adenylyl cyclase, for efficient activation of PLC and for receptor internalization. Though the nature of the isoprenoid attached to hIP does not act as a major determinant, the presence of an isoprenoid group, for example farnesyl or geranylgeranyl, is required for functional receptor-G protein interaction and coupling and for efficient agonist- induced receptor internalization.     

Molecular characterization of ribosomal gene variation within and among NORs segregating in specialized populations of chicken.

The molecular organization of the 18S, 5.8S, and 28S ribosomal RNA gene repeat units, located at the single nucleolus organizer region (NOR) locus in the chicken, was investigated in genetically distinct populations of research and commercial chickens. Substantial gene repeat variation within and among NORs was documented. Intact ribosomal gene repeat size ranged from 11 kb to over 50 kb. Unique combinations of ribosomal genes, of different size, were specific to particular populations. It was determined that the basis for the ribosomal gene repeat size variation was intergenic spacer (IGS) length heterogeneity. Interestingly, in different populations, the location of the variation that contributes to length heterogeneity was specific to particular IGS subregions. In addition to IGS variation, an inbred line of Red Jungle Fowl exhibited coding region variation. Ribosomal gene copy number variation was also studied, and line averages ranged from 279 to 368. Average rDNA array size (a function of copy number and gene repeat length) was calculated for each of the populations and found to vary over a range of two megabases, from 5 to 7 Mb.     

A subset of CD16-natural killer cells without antibody-dependent cellular cytotoxicity function

The low affinity IgG receptor, CD16 (Fc gamma RIII), is expressed on almost all peripheral blood natural killer (NK) cells. A small subset of CD3- CD16- CD56+ NK cells, representing less than 1% of peripheral blood lymphocytes, expands during in vivo IL-2 treatment. To analyze this CD16- NK cell subset in more detail, NK clones have been generated. One of them (TNK2) has been used to study the function of these cells in more detail. It is demonstrated that TNK2 exerts normal NK activity and displays large granular lymphocyte morphology. Since this clone lacks CD16 expression, antibody-dependent cellular cytotoxicity cannot be exerted. CD16 monoclonal antibodies fail to induce cytotoxic activity against NK-resistant target cells. These studies reveal that the lack of CD16 detection is not due to the modulation or the stage of activation of these NK cells. TNK2 is representative of this small subset of peripheral blood NK cells, expanded during IL-2 treatment, which does not express Fc gamma RIII and therefore cannot perform antibody-dependent cellular cytotoxicity.     

The population dynamics of small rodents in a tropical African grassland

Monthly estimates were obtained of the density of small rodents in tropical grassland in Rwenzori Park, Uganda between April 1972 and September 1973. Of the 12 species of rodents present, the most numerous were Lemniscomys striatus, Lophuromys sikapusi, Mus triton, Mylomys dybowskii and Praomys natalensis . In a live trapping grid estimates were obtained using direct enumeration, numbers caught, Hayne's Lincoln Index and Jolly's method. Monthly estimates ranged between 16·67 and 63·32 animals per hectare. Intensive removal trappings were also undertaken and these gave estimates of 6·45 to 27·24 per hectare. The inconsistency of the two estimates may be accounted for by small microhabitat and vegetational differences.
Breeding occurs in Lemniscomys and Praomys during the wet season whilen Lophuromys it is more extended but nevertheless seasonal. There is considerable evidence of rapid population turnover as the five species examined in detail had a mean duration of residence between two and three months. Few animals were resident for more than eight months.
The monthly standing crop biomass ranged from 672 to 2221 g/ha on the live trapping grid and from 348 to 1126 g/ha on the intensive removal grids. Estimates of net annual production on the live trapping grid rely on a number of assumptions but are nevertheless relatively high ranging from 5897 to 7072 g/ha.
Fires had a significant indirect effect on the composition of the fauna. Mus triton and Lemniscomys increased their numbers in the five months following the burn to levels not previously attained whereas Mylomys and Lophuromys were less frequent during this period. Several species of avian, reptilian and mammalian predators are recorded.     

Teratogenic development in chicken embryos associated with a major deletion in the rRNA gene cluster

A new strain of chickens (mPNU) that segregates a severely deleted rDNA cluster was studied. Individuals heterozygous (+/p²) and homozygous (p²/p²) for the deletion were found to have 56 and 27%, respectively, of the normal complement of rRNA genes (290 copies/cell). Morphogenesis, cellular rRNA levels, and nucleolar sizes, were investigated and compared in normal +/+, +/p², and p²/p² embryos. Cellular rRNA contents were similar among the three genotypes at stage X, but subsequently during gastrulation, p²/p² levels were reduced to 56% and eventually to 43% of +/+. Viability and morphogenesis were normal in p²/p² embryos until the initial primitive streak stage of gastrulation. However, further development was abnormal and characterized by disrupted axis formation. In +/+ and +/p² embryos, rRNA levels and nucleolar sizes increased during early development; however, the profile of these increases differed temporally and quantitatively between the genotypes. The +/p² embryos, at the full streak stage of gastrulation, exhibited reduced rRNA levels and nucleolar sizes (80% of +/+), yet the +/p² embryos developed normally. These studies establish a minimum copy number requirement lower than previously demonstrated, that is, a rDNA genotype with only 56% of the normal gene complement (～160 genes) is compatible with early embryonic viability. Also, a rRNA threshold was detected: rRNA levels that were 56% of +/+ failed to support normal gastrulation; however, even under the circumstance of reduced rRNA levels (43% of control), some aspects of gastrulation apparently continue (cell migration and invagination). The teratogenic development of p²/p² embryos is a biological consequence unique from that found in other metazoan models of rDNA-deficiency, and will be useful as a model to investigate mechanisms of vertebrate gastrulation and axis formation.     

Space-for-time substitutions in climate change ecology and evolution

In an epoch of rapid environmental change, understanding and predicting how biodiversity will respond to a changing climate is an urgent challenge. Since we seldom have sufficient long-term biological data to use the past to anticipate the future, spatial climate–biotic relationships are often used as a proxy for predicting biotic responses to climate change over time. These ‘space-for-time substitutions’ (SFTS) have become near ubiquitous in global change biology, but with different subfields largely developing methods in isolation. We review how climate-focussed SFTS are used in four subfields of ecology and evolution, each focussed on a different type of biotic variable – population phenotypes, population genotypes, species' distributions, and ecological communities. We then examine the similarities and differences between subfields in terms of methods, limitations and opportunities. While SFTS are used for a wide range of applications, two main approaches are applied across the four subfields: spatial in situ gradient methods and transplant experiments. We find that SFTS methods share common limitations relating to (i) the causality of identified spatial climate–biotic relationships and (ii) the transferability of these relationships, i.e. whether climate–biotic relationships observed over space are equivalent to those occurring over time. Moreover, despite widespread application of SFTS in climate change research, key assumptions remain largely untested. We highlight opportunities to enhance the robustness of SFTS by addressing key assumptions and limitations, with a particular emphasis on where approaches could be shared between the four subfields.     

Comparison of miRNA expression patterns using total RNA extracted from matched samples of formalin-fixed paraffin-embedded (FFPE) cells and snap frozen cells

Background  

Archival formalin-fixed paraffin-embedded (FFPE) tissues have limited utility in applications involving analysis of gene expression due to mRNA degradation and modification during fixation and processing. This study analyzed 160 miRNAs in paired snap frozen and FFPE cells to investigate if miRNAs may be successfully detected in archival specimens.     

Diagnostic accuracy of cervical cancer screening and screening–triage strategies among women living with HIV-1 in Burkina Faso and South Africa: A cohort study

BackgroundCervical cancer screening strategies using visual inspection or cytology may have suboptimal diagnostic accuracy for detection of precancer in women living with HIV (WLHIV). The optimal screen and screen–triage strategy, age to initiate, and frequency of screening for WLHIV remain unclear. This study evaluated the sensitivity, specificity, and positive predictive value of different cervical cancer strategies in WLHIV in Africa.Methods and findingsWLHIV aged 25–50 years attending HIV treatment centres in Burkina Faso (BF) and South Africa (SA) from 5 December 2011 to 30 October 2012 were enrolled in a prospective evaluation study of visual inspection using acetic acid (VIA) or visual inspection using Lugol’s iodine (VILI), high-risk human papillomavirus DNA test (Hybrid Capture 2 [HC2] or careHPV), and cytology for histology-verified high-grade cervical intraepithelial neoplasia (CIN2+/CIN3+) at baseline and endline, a median 16 months later. Among 1,238 women (BF: 615; SA: 623), median age was 36 and 34 years (p < 0.001), 28.6% and 49.6% ever had prior cervical cancer screening (p < 0.001), and 69.9% and 64.2% were taking ART at enrolment (p = 0.045) in BF and SA, respectively. CIN2+ prevalence was 5.8% and 22.4% in BF and SA (p < 0.001), respectively. VIA had low sensitivity for CIN2+ (44.7%, 95% confidence interval [CI] 36.9%–52.7%) and CIN3+ (56.1%, 95% CI 43.3%–68.3%) in both countries, with specificity for ≤CIN1 of 78.7% (95% CI 76.0%–81.3%). HC2 had sensitivity of 88.8% (95% CI 82.9%–93.2%) for CIN2+ and 86.4% (95% CI 75.7%–93.6%) for CIN3+. Specificity for ≤CIN1 was 55.4% (95% CI 52.2%–58.6%), and screen positivity was 51.3%. Specificity was higher with a restricted genotype (HPV16/18/31/33/35/45/52/58) approach (73.5%, 95% CI 70.6%–76.2%), with lower screen positivity (33.7%), although there was lower sensitivity for CIN3+ (77.3%, 95% CI 65.3%–86.7%). In BF, HC2 was more sensitive for CIN2+/CIN3+ compared to VIA/VILI (relative sensitivity for CIN2+ = 1.72, 95% CI 1.28–2.32; CIN3+: 1.18, 95% CI 0.94–1.49). Triage of HC2-positive women with VIA/VILI reduced the number of colposcopy referrals, but with loss in sensitivity for CIN2+ (58.1%) but not for CIN3+ (84.6%). In SA, cytology high-grade squamous intraepithelial lesion or greater (HSIL+) had best combination of sensitivity (CIN2+: 70.1%, 95% CI 61.3%–77.9%; CIN3+: 80.8%, 95% CI 67.5%–90.4%) and specificity (81.6%, 95% CI 77.6%–85.1%). HC2 had similar sensitivity for CIN3+ (83.0%, 95% CI 70.2%–91.9%) but lower specificity compared to HSIL+ (42.7%, 95% CI 38.4%–47.1%; relative specificity = 0.57, 95% CI 0.52–0.63), resulting in almost twice as many referrals. Compared to HC2, triage of HC2-positive women with HSIL+ resulted in a 40% reduction in colposcopy referrals but was associated with some loss in sensitivity. CIN2+ incidence over a median 16 months was highest among VIA baseline screen-negative women (2.2%, 95% CI 1.3%–3.7%) and women who were baseline double-negative with HC2 and VIA (2.1%, 95% CI 1.3%–3.5%) and lowest among HC2 baseline screen-negative women (0.5%, 95% CI 0.1%–1.8%). Limitations of our study are that WLHIV included in the study may not reflect a contemporary cohort of WLHIV initiating ART in the universal ART era and that we did not evaluate HPV tests available in study settings today.ConclusionsIn this cohort study among WLHIV in Africa, a human papillomavirus (HPV) test targeting 14 high-risk (HR) types had higher sensitivity to detect CIN2+ compared to visual inspection but had low specificity, although a restricted genotype approach targeting 8 HR types decreased the number of unnecessary colposcopy referrals. Cytology HSIL+ had optimal performance for CIN2+/CIN3+ detection in SA. Triage of HPV-positive women with HSIL+ maintained high specificity but with some loss in sensitivity compared to HC2 alone.

In this cohort study, Helen Kelly and colleagues explore cervical cancer screening strategies for women living with HIV.