Advancing cervical cancer prevention initiatives in resource-constrained settings: insights from the Cervical Cancer Prevention Program in Zambia

Groesbeck Parham and colleagues describe their Cervical Cancer Prevention Program in Zambia, which has provided services to over 58,000 women over the past five years, and share lessons learned from the program's implementation and integration with existing HIV/AIDS programs.     

Fine root foraging strategies in Norway spruce forests across a European climate gradient

Fine root acclimation to different environmental conditions is crucial for growth and sustainability of forest trees. Relatively small changes in fine root standing biomass (FRB), morphology or mycorrhizal symbiosis may result in a large change in forest carbon, nutrient and water cycles. We elucidated the changes in fine root traits and associated ectomycorrhizal (EcM) fungi in 12 Norway spruce stands across a climatic and N deposition gradient from subarctic‐boreal to temperate regions in Europe (68°N–48°N). We analysed the standing FRB and the ectomycorrhizal root tip biomass (EcMB, g m^?2) simultaneously with measurements of the EcM root morphological traits (e.g. mean root length, root tissue density (RTD), N% in EcM roots) and frequency of dominating EcM fungi in different stands in relation to climate, soil and site characteristics. Latitude and N deposition explained the greatest proportion of variation in fine root traits. EcMB per stand basal area (BA) increased exponentially with latitude: by about 12.7 kg m^?2 with an increase of 10° latitude from southern Germany to Estonia and southern Finland and by about 44.7 kg m^?2 with next latitudinal 10° from southern to northern Finland. Boreal Norway spruce forests had 4.5 to 11 times more EcM root tips per stand BA, and the tips were 2.1 times longer, with 1.5 times higher RTD and about 1/3 lower N concentration. There was 19% higher proportion of root tips colonized by long‐distance exploration type forming EcM fungi in the southern forests indicating importance of EcM symbiont foraging strategy in fine root nutrient acquisition. In the boreal zone, we predict ca. 50% decrease in EcMB per stand BA with an increase of 2 °C annual mean temperature. Different fine root foraging strategies in boreal and temperate forests highlight the importance of complex studies on respective regulatory mechanisms in changing climate.     

Protease-activated receptor 2 activation of myeloid dendritic cells regulates allergic airway inflammation

Background

A common characteristic of allergens is that they contain proteases that can activate protease-activated receptor (PAR-2); however the mechanism by which PAR-2 regulates allergic airway inflammation is unclear.

Methods

Mice (wild type and PAR-2-deficient) were sensitized using German cockroach (GC) feces (frass), the isolated protease from GC frass, or through adoptive transfer of GC frass-treated bone marrow-derived dendritic cells (BMDC) and measurements of airway inflammation (cellular infiltration, cytokine expression, and mucin production), serum IgE levels and airway hyperresponsiveness (AHR) were assessed. BMDC were cultured, treated with GC frass and assessed for cytokine production. PAR-2 expression on pulmonary mDCs was determined by flow cytometry.

Results

Exposure to GC frass induced AHR and airway inflammation in wild type mice; however PAR-2-deficient mice had significantly attenuated responses. To directly investigate the role of the protease, we isolated the protease from GC frass and administered the endotoxin-free protease into the airways of mice in the presence of OVA. GC frass proteases were sufficient to promote the development of AHR, serum IgE, and Th2 cytokine production. PAR-2 expression on mDC was upregulated following GC frass exposure, but the presence of a functional PAR-2 did not alter antigen uptake. To determine if PAR-2 activation led to differential cytokine production, we cultured BMDC in the presence of GM-CSF and treated these cells ex vivo with GC frass. PAR-2-deficient BMDC released significantly less IL-6, IL-23 and TNFα compared to BMDC from wild type mice, suggesting PAR-2 activation was important in Th2/Th17 skewing cytokine production. To determine the role for PAR-2 on mDCs on the initiation of allergic airway inflammation, BMDCs from wild type and PAR-2-deficient mice were treated in the presence or absence of GC frass and then adoptively transferred into the airway of wild type mice. Importantly, GC frass-stimulated wild type BMDCs were sufficient to induce AHR and allergic airway inflammation, while GC frass-stimulated PAR-2-deficient BMDC had attenuated responses.

Conclusions

Together these data suggest an important role for allergen activation of PAR-2 on mDCs in mediating Th2/Th17 cytokine production and allergic airway responses.     

Genomic DNA methylation changes in response to folic acid supplementation in a population-based intervention study among women of reproductive age

Folate is a source of one-carbons necessary for DNA methylation, a critical epigenetic modification necessary for genomic structure and function. The use of supplemental folic acid is widespread however; the potential influence on DNA methylation is unclear. We measured global DNA methylation using DNA extracted from samples from a population-based, double-blind randomized trial of folic acid supplementation (100, 400, 4000 μg per day) taken for 6 months; including a 3 month post-supplementation sample. We observed no changes in global DNA methylation in response to up to 4,000 μg/day for 6 months supplementation in DNA extracted from uncoagulated blood (approximates circulating blood). However, when DNA methylation was determined in coagulated samples from the same individuals at the same time, significant time, dose, and MTHFR genotype-dependent changes were observed. The baseline level of DNA methylation was the same for uncoagulated and coagulated samples; marked differences between sample types were observed only after intervention. In DNA from coagulated blood, DNA methylation decreased (-14%; P<0.001) after 1 month of supplementation and 3 months after supplement withdrawal, methylation decreased an additional 23% (P<0.001) with significant variation among individuals (max+17%; min-94%). Decreases in methylation of ≥25% (vs. <25%) after discontinuation of supplementation were strongly associated with genotype: MTHFR CC vs. TT (adjusted odds ratio [aOR] 12.9, 95%CI 6.4, 26.0). The unexpected difference in DNA methylation between DNA extracted from coagulated and uncoagulated samples in response to folic acid supplementation is an important finding for evaluating use of folic acid and investigating the potential effects of folic acid supplementation on coagulation.     

Bioenergetic profiling of zebrafish embryonic development

Many debilitating conditions are linked to bioenergetic defects. Developing screens to probe the genetic and/or chemical basis for such links has proved intractable. Furthermore, there is a need for a physiologically relevant assay of bioenergetics in whole organisms, especially for early stages in life where perturbations could increase disease susceptibility with aging. Thus, we asked whether we could screen bioenergetics and mitochondrial function in the developing zebrafish embryo. We present a multiplexed method to assay bioenergetics in zebrafish embryos from the blastula period (3 hours post-fertilization, hpf) through to hatching (48 hpf). In proof of principle experiments, we measured respiration and acid extrusion of developing zebrafish embryos. We quantified respiratory coupling to various bioenergetic functions by using specific pharmacological inhibitors of bioenergetic pathways. We demonstrate that changes in the coupling to ATP turnover and proton leak are correlated with developmental stage. The multiwell format of this assay enables the user to screen for the effects of drugs and environmental agents on bioenergetics in the zebrafish embryo with high sensitivity and reproducibility.     

Anchor profiles of HLA-specific peptides: analysis by a novel affinity scoring method and experimental validation

The study of intermolecular interactions is a fundamental research subject in biology. Here we report on the development of a quantitative structure-based affinity scoring method for peptide-protein complexes, named PepScope. The method operates on the basis of a highly specific force field function (CHARMM) that is applied to all-atom structural representations of peptide-receptor complexes. Peptide side-chain contributions to total affinity are scored after detailed rotameric sampling followed by controlled energy refinement. A de novo approach to estimate dehydration energies was developed, based on the simulation of individual amino acids in a solvent box filled with explicit water molecules. Transferability of the method was demonstrated by its application to the hydrophobic HLA-A2 and -A24 receptors, the polar HLA-A1, and the sterically ruled HLA-B7 receptor. A combined theoretical and experimental study on 39 anchor substitutions in FxSKQYMTx/HLA-A2 and -A24 complexes indicated a prediction accuracy of about two thirds of a log-unit in Kd. Analysis of free energy contributions identified a great role of desolvation and conformational strain effects in establishing a given specificity profile. Interestingly, the method rightly predicted that most anchor profiles are less specific than so far assumed. This suggests that many potential T-cell epitopes could be missed with current prediction methods. The results presented in this work may therefore significantly affect T-cell epitope discovery programs applied in the field of peptide vaccine development.     

Iron metabolism mutant hbd mice have a deletion in Sec15l1, which has homology to a yeast gene for vesicle docking

Defects in iron absorption and utilization lead to iron deficiency and anemia. While iron transport by transferrin receptor-mediated endocytosis is well understood, it is not completely clear how iron is transported from the endosome to the mitochondria where heme is synthesized. We undertook a positional cloning project to identify the causative mutation for the hemoglobin-deficit (hbd) mouse mutant, which suffers from a microcytic, hypochromic anemia apparently due to defective iron transport in the endocytosis cycle. As shown by previous studies, reticulocyte iron accumulation in homozygous hbd/hbd mice is deficient despite normal binding of transferrin to its receptor and normal transferrin uptake in the cell. We have identified a strong candidate gene for hbd, Sec15l1, a homologue to yeast SEC15, which encodes a key protein in vesicle docking. The hbd mice have an exon deletion in Sec15l1, which is the first known mutation of a SEC gene homologue in mammals.     

Effects of acid sphingomyelinase deficiency on male germ cell development and programmed cell death

Deficiency of acid sphingomyelinase (ASM), an enzyme responsible for producing a pro-apoptotic second messenger ceramide, has previously been shown to promote the survival of fetal mouse oocytes in vivo and to protect oocytes from chemotherapy-induced apoptosis in vitro. Here we investigated the effects of ASM deficiency on testicular germ cell development and on the ability of germ cells to undergo apoptosis. At the age of 20 weeks, ASM knock-out (ASMKO) sperm concentrations were comparable with wild-type (WT) sperm concentrations, whereas sperm motility was seriously affected. ASMKO testes contained significantly elevated levels of sphingomyelin at the age of 8 weeks as detected by high-performance, thin-layer chromatography. Electron microscopy revealed that the testes started to accumulate pathological vesicles in Sertoli cells and in the interstitium at the age of 21 days. Irradiation of WT and ASMKO mice did not elevate intratesticular ceramide levels at 16 h after irradiation. In situ end labeling of apoptotic cells also showed a similar degree of cell death in both groups. After a 21-day recovery period, the numbers of primary spermatocytes and spermatogonia at G2 as well as spermatids were essentially the same in the WT and ASMKO testes, as detected by flow cytometry. In serum-free cultures both ASMKO and WT germ cells showed a significant increase in the level of ceramide, as well as massive apoptosis. In conclusion, ASM is required for maintenance of normal sphingomyelin levels in the testis and for normal sperm motility, but not for testicular ceramide production or for the ability of the germ cells to undergo apoptosis.     

Receptor for retrograde transport in the apicomplexan parasite Toxoplasma gondii

Toxoplasma gondii and its apicomplexan relatives (such as Plasmodium falciparum, which causes malaria) are obligate intracellular parasites that rely on sequential protein release from specialized secretory organelles for invasion and multiplication within host cells. Because of the importance of these unusual membrane trafficking pathways for drug development and comparative cell biology, characterizing them is essential. In particular, it is unclear what role retrieval mechanisms play in parasite membrane trafficking or where they operate. Previously, we showed that T. gondii's beta-COP (TgBetaCOP; a subunit of coatomer protein complex I, COPI) and retrieval reporters localize exclusively to the zone between the parasite endoplasmic reticulum (ER) and Golgi apparatus. This suggested the existence of an HDEL receptor in T. gondii. We have now identified, cloned, and sequenced this receptor, TgERD2. TgERD2 localizes in a Golgi or ER pattern suggestive of the HDEL retrieval reporter (K. M. Hager, B. Striepen, L. G. Tilney, and D. S. Roos, J. Cell Sci. 112:2631-2638, 1999). A functional assay reveals that TgERD2 is able to complement the Saccharomyces cerevisiae ERD2 null mutant. Retrieval studies reveal that stable expression of a fluorescent exogenous retrieval ligand results in a dispersal of betaCOP signal throughout the cytoplasm and, surprisingly, results in betaCOP staining of the vacuolar space of the parasite. In contrast, stable expression of TgERD2GFP does not appear to disturb betaCOP staining. In addition to TgERD2, Toxoplasma contains two more divergent ERD2 relatives. Phylogenetic analysis reveals that these proteins belong to a previously unrecognized ERD2 subfamily common to plants and alveolate organisms and as such could represent mediators of parasite-specific retrieval functions. No evidence of class 2 ERD2 proteins was found in metazoan organisms or fungi.