Predicting the effects of nectar robbing on plant reproduction: implications of pollen limitation and plant mating system

The outcome of species interactions is often difficult to predict, depending on the organisms involved and the ecological context. Nectar robbers remove nectar from flowers, often without providing pollination service, and their effects on plant reproduction vary in strength and direction. In two case studies and a meta-analysis, we tested the importance of pollen limitation and plant mating system in predicting the impacts of nectar robbing on female plant reproduction. We predicted that nectar robbing would have the strongest effects on species requiring pollinators to set seed and pollen limited for seed production. Our predictions were partially supported. In the first study, natural nectar robbing was associated with lower seed production in Delphinium nuttallianum, a self-compatible but non-autogamously selfing, pollen-limited perennial, and experimental nectar robbing reduced seed set relative to unrobbed plants. The second study involved Linaria vulgaris, a self-incompatible perennial that is generally not pollen limited. Natural levels of nectar robbing generally had little effect on estimates of female reproduction in L. vulgaris, while experimental nectar robbing reduced seed set per fruit but not percentage of fruit set. A meta-analysis revealed that nectar robbing had strong negative effects on pollen-limited and self-incompatible plants, as predicted. Our results suggest that pollination biology and plant mating system must be considered to understand and predict the ecological outcome of both mutualistic and antagonistic plant-animal interactions.     

Mitochondrial Protein Acylation and Intermediary Metabolism: Regulation by Sirtuins and Implications for Metabolic Disease

The sirtuins are a family of NAD⁺-dependent protein deacetylases that regulate cell survival, metabolism, and longevity. Three sirtuins, SIRT3–5, localize to mitochondria. Expression of SIRT3 is selectively activated during fasting and calorie restriction. SIRT3 regulates the acetylation level and enzymatic activity of key metabolic enzymes, such as acetyl-CoA synthetase, long-chain acyl-CoA dehydrogenase, and 3-hydroxy-3-methylglutaryl-CoA synthase 2, and enhances fat metabolism during fasting. SIRT5 exhibits demalonylase/desuccinylase activity, and lysine succinylation and malonylation are abundant mitochondrial protein modifications. No convincing enzymatic activity has been reported for SIRT4. Here, we review the emerging role of mitochondrial sirtuins as metabolic sensors that respond to changes in the energy status of the cell and modulate the activities of key metabolic enzymes via protein deacylation.     

Evaluation of the effect of closed areas on a unique and shallow water coral reef fish assemblage reveals complex responses

Areas closed to fishing are advocated as both fisheries management and biodiversity conservation tools. However, few studies investigate the responses of suites of both target and non-target fish species within an assemblage, which is an important consideration for ecosystem-based fisheries management approaches. Diver-operated stereo-video was used to assess the abundance and length of coral reef fish across multiple areas both open and closed to fishing at the Houtman Abrolhos Islands, Western Australia. After taking into consideration spatial differences in benthic habitat, the composition of fish assemblages was found to differ between open and closed areas. The target species, Plectropomus leopardus, was approximately two times more abundant in closed areas. Furthermore, 51 % of P. leopardus were larger than the minimum legal length (MLL) for retention in closed areas compared with only 1.8 % in areas open to fishing. Another target species, Choerodon rubescens was surveyed in greater abundance at sizes larger than the MLL in closed areas (64 % >400 mm) in comparison with areas open to fishing (36 %). A number of non-target species were also larger in closed areas (e.g., Kyphosus cornelii, Scarus schlegeli). In contrast, several non-targeted prey species were more abundant in open areas (e.g., Pomacentrus milleri was six times more abundant in open areas). Our results document complex responses of target and non-target species in closed areas at the Houtman Abrolhos Islands.     

Survey of the fragile X syndrome CGG repeat and the short-tandem-repeat and single-nucleotide-polymorphism haplotypes in an African American population

Previous studies have shown that specific short-tandem-repeat (STR) and single-nucleotide-polymorphism (SNP)-based haplotypes within and among unaffected and fragile X white populations are found to be associated with specific CGG-repeat patterns. It has been hypothesized that these associations result from different mutational mechanisms, possibly influenced by the CGG structure and/or cis-acting factors. Alternatively, haplotype associations may result from the long mutational history of increasing instability. To understand the basis of the mutational process, we examined the CGG-repeat size, three flanking STR markers (DXS548-FRAXAC1-FRAXAC2), and one SNP (ATL1) spanning 150 kb around the CGG repeat in unaffected (n=637) and fragile X (n=63) African American populations and compared them with unaffected (n=721) and fragile X (n=102) white populations. Several important differences were found between the two ethnic groups. First, in contrast to that seen in the white population, no associations were observed among the African American intermediate or "predisposed" alleles (41-60 repeats). Second, two previously undescribed haplotypes accounted for the majority of the African American fragile X population. Third, a putative "protective" haplotype was not found among African Americans, whereas it was found among whites. Fourth, in contrast to that seen in whites, the SNP ATL1 was in linkage equilibrium among African Americans, and it did not add new information to the STR haplotypes. These data indicate that the STR- and SNP-based haplotype associations identified in whites probably reflect the mutational history of the expansion, rather than a mutational mechanism or pathway.     

Human macrophages do not require phagosome acidification to mediate fungistatic/fungicidal activity against Histoplasma capsulatum

Histoplasma capsulatum (Hc) is a facultative intracellular fungus that modulates the intraphagosomal environment to survive within macrophages (Mphi). In the present study, we sought to quantify the intraphagosomal pH under conditions in which Hc yeasts replicated or were killed. Human Mphi that had ingested both viable and heat-killed or fixed yeasts maintained an intraphagosomal pH of approximately 6.4-6.5 over a period of several hours. These results were obtained using a fluorescent ratio technique and by electron microscopy using the 3-(2,4-dinitroanilo)-3'-amino-N-methyldipropylamine reagent. Mphi that had ingested Saccharomyces cerevisae, a nonpathogenic yeast that is rapidly killed and degraded by Mphi, also maintained an intraphagosomal pH of approximately 6.5 over a period of several hours. Stimulation of human Mphi fungicidal activity by coculture with chloroquine or by adherence to type 1 collagen matrices was not reversed by bafilomycin, an inhibitor of the vacuolar ATPase. Human Mphi cultured in the presence of bafilomycin also completely degraded heat-killed Hc yeasts, whereas mouse peritoneal Mphi digestion of yeasts was completely reversed in the presence of bafilomycin. However, bafilomycin did not inhibit mouse Mphi fungistatic activity induced by IFN-gamma. Thus, human Mphi do not require phagosomal acidification to kill and degrade Hc yeasts, whereas mouse Mphi do require acidification for fungicidal but not fungistatic activity.     

Global Estimates of the Prevalence and Incidence of Four Curable Sexually Transmitted Infections in 2012 Based on Systematic Review and Global Reporting

Background

Quantifying sexually transmitted infection (STI) prevalence and incidence is important for planning interventions and advocating for resources. The World Health Organization (WHO) periodically estimates global and regional prevalence and incidence of four curable STIs: chlamydia, gonorrhoea, trichomoniasis and syphilis.

Methods and Findings

WHO’s 2012 estimates were based upon literature reviews of prevalence data from 2005 through 2012 among general populations for genitourinary infection with chlamydia, gonorrhoea, and trichomoniasis, and nationally reported data on syphilis seroprevalence among antenatal care attendees. Data were standardized for laboratory test type, geography, age, and high risk subpopulations, and combined using a Bayesian meta-analytic approach. Regional incidence estimates were generated from prevalence estimates by adjusting for average duration of infection. In 2012, among women aged 15–49 years, the estimated global prevalence of chlamydia was 4.2% (95% uncertainty interval (UI): 3.7–4.7%), gonorrhoea 0.8% (0.6–1.0%), trichomoniasis 5.0% (4.0–6.4%), and syphilis 0.5% (0.4–0.6%); among men, estimated chlamydia prevalence was 2.7% (2.0–3.6%), gonorrhoea 0.6% (0.4–0.9%), trichomoniasis 0.6% (0.4–0.8%), and syphilis 0.48% (0.3–0.7%). These figures correspond to an estimated 131 million new cases of chlamydia (100–166 million), 78 million of gonorrhoea (53–110 million), 143 million of trichomoniasis (98–202 million), and 6 million of syphilis (4–8 million). Prevalence and incidence estimates varied by region and sex.

Conclusions

Estimates of the global prevalence and incidence of chlamydia, gonorrhoea, trichomoniasis, and syphilis in adult women and men remain high, with nearly one million new infections with curable STI each day. The estimates highlight the urgent need for the public health community to ensure that well-recognized effective interventions for STI prevention, screening, diagnosis, and treatment are made more widely available. Improved estimation methods are needed to allow use of more varied data and generation of estimates at the national level.     

Testing an Electrostatic Interaction Hypothesis of Hepatitis B Virus Capsid Stability by Using an In Vitro Capsid Disassembly/Reassembly System

To test a previously coined “charge balance hypothesis” of human hepatitis B virus (HBV) capsid stability, we established an in vitro disassembly and reassembly system using bacterially expressed HBV capsids. Capsid disassembly can be induced by micrococcal nuclease digestion of encapsidated RNA. HBV core protein (HBc) mutants containing various amounts of arginine were constructed by serial truncations at the C terminus. Capsids containing smaller amounts of arginine (HBc 149, 154, and 157) remained intact after micrococcal nuclease digestion by native gel electrophoresis. Capsids containing larger amounts of arginine (HBc 159, 164, 169, and 171) exhibited reduced and more diffuse banding intensity and slightly upshifted mobility (HBc 159 and 164). Capsids containing the largest amounts of arginine (HBc 173, 175, and 183), as well as HBc 167, exhibited no detectable banding signal, indicating loss of capsid integrity or stability. Interestingly, capsid reassembly can be induced by polyanions, including oligonucleotides, poly-glutamic acid, and nonbiological polymer (polyacrylic acid). In contrast, polycations (polylysine and polyethylenimine) and low-molecular-weight anions (inositol triphosphate) induced no capsid reassembly. Results obtained by gel assay were confirmed by electron microscopy. Reassembled capsids comigrated with undigested parental capsids on agarose gels and cosedimented with undigested capsids by sucrose gradient ultracentrifugation. Taken together, the results indicate that HBV capsid assembly and integrity depend on polyanions, which probably can help minimize intersubunit charge repulsion caused mainly by arginine-rich domain III or IV in close contact. The exact structure of polyanions is not important for in vitro capsid reassembly. A large amount of independent experimental evidence for this newly coined “electrostatic interaction hypothesis” is discussed.Chronic infection with hepatitis B virus (HBV) leads to the development of cirrhosis and hepatocellular carcinoma (6, 31, 36). HBV core protein (HBc) consists of the assembly domain (HBc amino acids 1 to 149) at the N terminus and the arginine-rich domain (ARD) at the C terminus (HBc amino acids 150 to 183) (33, 34). Escherichia coli-expressed HBc can spontaneously self-assemble into 28-nm capsid particles with a spherical appearance indistinguishable from that of human liver-derived capsid particles (7). Such capsid particles have been shown to package RNAs transcribed in E. coli (5, 8, 11, 28, 37). The four-helix bundle structure of HBV capsid particles is based on cryo-electron microscopy and X-ray crystallography using C-terminally truncated HBc (34, 38). At present, there is no known structure at the C terminus of HBc capsids (34, 41). HBc amino acids 150 to 183 contains four stretches of clustering arginine residues (ARD-I, -II, -III, and -IV) (Fig. ​(Fig.1).1). When the C-terminal domain of hepadnaviral core protein was serially truncated, a viral replication defect was observed (4, 19, 22, 27, 39). To date, it remains to be elucidated why the C terminus is so important for diverse biological activities, including RNA encapsidation and DNA replication. To address this issue, we proposed previously a so-called “charge balance hypothesis” (22), which highlights the importance of adequate electrostatic interactions between positive charge (basic residues) from HBc and negative charge from encapsidated RNA or DNA.Open in a separate windowFIG. 1.Effects of micrococcal nuclease treatment on HBV nucleocapsids with serially truncated C termini of core proteins. A series of HBV core expression vectors with different lengths and arginine contents were constructed in pET-Blue-1. The truncations are illustrated in the top panel, and the four ARDs (ARD-I, -II, -III, and -IV) are underlined. All constructs self-assembled into capsids when expressed in E. coli. The capsids run as a distinct band on 1% native agarose gels and can be stained with EtBr (upper panel) and Coomassie blue (middle panel). Untreated controls without micrococcal nuclease were incubated with the same buffer and conditions as for micrococcal nuclease-digested samples. In the upper panel, note the loss of the EtBr signal when the encapsidated RNAs were digested by micrococcal nuclease. In the middle panel, we observed three different groups of mutants with three different CBS patterns. Group 1 mutants, including HBc mutants 149, 154, and 157, exhibited no significant change in CBS banding pattern before or after micrococcal nuclease digestion. In group 2 capsids 159, 164, 169, and 171 (#), the CBS banding pattern became more diffuse, less intense, and slightly (yet reproducibly) upshifted. * indicates the near-complete loss of CBS banding in group 3 capsids 167, 173, 175, and 183. To confirm that this loss of CBS probably results from a loss of structural integrity of the capsid particle, rather than from a loss of the core protein per se, aliquots of samples were also run on denaturing SDS-PAGE (bottom panel).The first clue that such an electrostatic interaction could play an important role in RNA encapsidation is from the study of an engineered mutant, HBc 164. Despite its reduced arginine content relative to the wild-type (WT) full-length HBc 183 (Fig. ​(Fig.1),1), HBc mutant 164 can encapsidate, as efficiently as WT HBV, both 3.5-kb pregenomic RNA (pgRNA) and a spliced 2.2-kb subgenomic RNA (sgRNA) (19, 22). When WT HBV nucleocapsids (capsids) were treated with micrococcal nuclease, the encapsidated 3.5-kb pgRNA and its reverse-transcribing RNA template were resistant to micrococcal nuclease treatment. In contrast, when mutant 164 capsids were treated with micrococcal nuclease, the encapsidated 3.5-kb RNA was highly nuclease sensitive, while the encapsidated 2.2-kb sgRNA appeared to be nuclease resistant (19, 22).To further elucidate the mechanism behind this phenomenon, we hypothesized that this result could be due to an abnormal or less stable capsid structure generated by a charge imbalance (insufficient positive charge or excessive negative charge) when arginine-deficient mutant 164 encapsidates the full-length 3.5-kb pgRNA. In contrast, a more normal or stable capsid structure can be generated when arginine-deficient mutant 164 encapsidates the 2.2-kb sgRNA (with a reduced negative charge content relative to the 3.5-kb pgRNA) (22). In addition to RNA encapsidation and capsid stability, electrostatic interaction could also play a role in HBV DNA synthesis and genome maturation. For example, when the truncated C terminus of HBc 164 was progressively restored, the core-associated viral DNA gradually increased in both size and signal intensity (22).In this study, we designed an in vitro capsid disassembly/reassembly experiment which is complementary to the in vivo approach in tissue culture (22; P. K. Chua, F. M. Tang, J. Y. Huang, C. S. Suen, and C. Shih, submitted for publication). We investigated the relationship between the arginine content of HBV capsids and the encapsidated nucleic acid in maintaining capsid stability in vitro. In addition, we demonstrated that HBV capsids that are disassembled by depletion of encapsidated RNA can be efficiently reassembled in the presence of exogenous nucleic acid and non-nucleic acid polyanions but not in the presence of polycations or low-molecular-weight (low-MW) anions. Capsid disassembly induced by RNA digestion appears to be related to intersubunit charge repulsion between ARD-III or ARD-IV in close contact.