Comparative Population Genomics of the Borrelia burgdorferi Species Complex Reveals High Degree of Genetic Isolation among Species and Underscores Benefits and Constraints to Studying Intra-Specific Epidemiological Processes

Lyme borreliosis, one of the most frequently contracted zoonotic diseases in the Northern Hemisphere, is caused by bacteria belonging to different genetic groups within the Borrelia burgdorferi species complex, which are transmitted by ticks among various wildlife reservoirs, such as small mammals and birds. These features make the Borrelia burgdorferi species complex an attractive biological model that can be used to study the diversification and the epidemiology of endemic bacterial pathogens. We investigated the potential of population genomic approaches to study these processes. Sixty-three strains belonging to three species within the Borrelia burgdorferi complex were isolated from questing ticks in Alsace (France), a region where Lyme disease is highly endemic. We first aimed to characterize the degree of genetic isolation among the species sampled. Phylogenetic and coalescent-based analyses revealed clear delineations: there was a ∼50 fold difference between intra-specific and inter-specific recombination rates. We then investigated whether the population genomic data contained information of epidemiological relevance. In phylogenies inferred using most of the genome, conspecific strains did not cluster in clades. These results raise questions about the relevance of different strategies when investigating pathogen epidemiology. For instance, here, both classical analytic approaches and phylodynamic simulations suggested that population sizes and migration rates were higher in B. garinii populations, which are normally associated with birds, than in B. burgdorferi s.s. populations. The phylogenetic analyses of the infection-related ospC gene and its flanking region provided additional support for this finding. Traces of recombination among the B. burgdorferi s.s. lineages and lineages associated with small mammals were found, suggesting that they shared the same hosts. Altogether, these results provide baseline evidence that can be used to formulate hypotheses regarding the host range of B. burgdorferi lineages based on population genomic data.     

Uroguanylin regulates net fluid secretion via the NHE2 isoform of the Na/H+ exchanger in an intestinal cellular model

Uroguanylin (UGN) has been proposed as a key regulator of salt and water intestinal transport. Uroguanylin activates cell-surface guanylate cyclase C receptor (GC-C) and modulates cellular function via cyclic GMP (cGMP), thus increasing electrolyte and net water secretion. It has been suggested that the action of UGN could involve the Na(+)/H(+) exchanger, but the actual contribution of this transporter still remains unclear. The objective of our study was to investigate the putative effects of UGN on some members of the Na(+)/H(+) exchanger family (NHEs), as well as to clarify its consequences on transepithelial fluid flow in T84 cells. In order to do so, transepithelial fluid flow (J(v)) was studied by optic techniques and intracellular pH (pH(i)) was measured with a fluorescence method. Results showed that NHE2 is found at the apical membrane and has a major role in Na(+) absorption; NHE1 and NHE4 are localized at the basolateral membrane with a house-keeping role in steady state pH(i). In the assayed conditions, cell exposure to apical UGN increases net secretory J(v), without changing short-circuit currents nor transepithelial resistance, and reduces NHE2 activity. Therefore, at physiological pH, the effect on net J(v) was produced mainly by a reduction in normal Na(+) absorption through NHE2, rather than by the stimulation of electrolyte secretion. Our study shows that the effect of UGN on pH(i) is GC-C/cGMP-mediated and enhanced by sildenafil, thus involving PDE5 enzyme. Additionally, cell exposure to apical UGN results in intracellular alkalinization, probably due to indirect effects on basolateral NHE1 and NHE4, which have a major role in pH(i) regulation.     

Notes on Scleroderma,Puff balls and Geastrum of Azad Jammu and Kashmir,Pakistan

Six species of mushrooms allied to the Family Sclerodermataceae, Lycoperdaceae and Geastraceae have been described for the first time from Azad Jammu and Kashmir. These are Scleroderma aurantium, Calvatia verrucosia sp. nov., Lycoperdon pedicellaton sp. nov. L. sphaericon sp. nov., L. echinulaton sp. nov., and Geastrum heptaplex sp. nov.     

Identification of PDZ ligands by docking-based virtual screening for the development of novel analgesic agents

Disrupting the interaction between the PDZ protein, PSD-95, and its target ligands (such as the glutamate NMDA receptor or the serotonin 5-HT_2A receptor) was found to reduce hyperalgesia in various models of neuropathic pain. Here, we set out to identify lead molecules which would interact with PSD-95, and hence, would potentially display analgesic activity. We describe the virtual screening of the Asinex and Cambridge databases which together contain almost one million molecules. Using three successive docking filters and visual inspection, we identified three structural classes of molecules and synthesized a potential lead compound from each class. The binding of the molecules with the PDZ domains of PSD-95 was assessed by ¹H–¹⁵N HSQC NMR experiments. The analgesic activity of the best ligand, quinoline 2, was evaluated in vivo in a model of neuropathic pain and showed promising results.     

Triterpene derivatives that inhibit human immunodeficiency virus type 1 replication

Triterpene derivatives were analyzed for anti-HIV-1 activity and for cellular toxicity. Betulinic aldehyde, betulinic nitrile, and morolic acid derivatives were identified to have anti-HIV-1 activity. These derivatives inhibit a late step in virus replication, likely virus maturation.     

Reduced maximal inhibition in phenotypic susceptibility assays indicates that viral strains resistant to the CCR5 antagonist maraviroc utilize inhibitor-bound receptor for entry

Maraviroc is a CCR5 antagonist in clinical development as one of a new class of antiretrovirals targeting human immunodeficiency virus type 1 (HIV-1) coreceptor binding. We investigated the mechanism of HIV resistance to maraviroc by using in vitro sequential passage and site-directed mutagenesis. Serial passage through increasing maraviroc concentrations failed to select maraviroc-resistant variants from some laboratory-adapted and clinical isolates of HIV-1. However, high-level resistance to maraviroc was selected from three of six primary isolates passaged in peripheral blood lymphocytes (PBL). The SF162 strain acquired resistance to maraviroc in both treated and control cultures; all resistant variants were able to use CXCR4 as a coreceptor. In contrast, maraviroc-resistant virus derived from isolates CC1/85 and RU570 remained CCR5 tropic, as evidenced by susceptibility to the CCR5 antagonist SCH-C, resistance to the CXCR4 antagonist AMD3100, and an inability to replicate in CCR5 Delta32/Delta32 PBL. Strain-specific mutations were identified in the V3 loop of maraviroc-resistant CC1/85 and RU570. The envelope-encoding region of maraviroc-resistant CC1/85 was inserted into an NL4-3 background. This recombinant virus was completely resistant to maraviroc but retained susceptibility to aplaviroc. Reverse mutation of gp120 residues 316 and 323 in the V3 loop (numbering from HXB2) to their original sequence restored wild-type susceptibility to maraviroc, while reversion of either mutation resulted in a partially sensitive virus with reduced maximal inhibition (plateau). The plateaus are consistent with the virus having acquired the ability to utilize maraviroc-bound receptor for entry. This hypothesis was further corroborated by the observation that a high concentration of maraviroc blocks the activity of aplaviroc against maraviroc-resistant virus.     

Resequencing the susceptibility gene,ITGAM, identifies two functionally deleterious rare variants in systemic lupus erythematosus cases

Introduction

The majority of the genetic variance of systemic lupus erythematosus (SLE) remains unexplained by the common disease-common variant hypothesis. Rare variants, which are not detectable by genome-wide association studies because of their low frequencies, are predicted to explain part of this ”missing heritability.” However, recent studies identifying rare variants within known disease-susceptibility loci have failed to show genetic associations because of their extremely low frequencies, leading to the questioning of the contribution of rare variants to disease susceptibility. A common (minor allele frequency = 17.4% in cases) nonsynonymous coding variant rs1143679 (R77H) in ITGAM (CD11b), which forms half of the heterodimeric integrin receptor, complement receptor 3 (CR3), is robustly associated with SLE and has been shown to impair CR3-mediated phagocytosis.

Methods

We resequenced ITGAM in 73 SLE cases and identified two previously unidentified, case-specific nonsynonymous variants, F941V and G1145S. Both variants were genotyped in 2,107 and 949 additional SLE cases, respectively, to estimate their frequencies in a disease population. An in vitro model was used to assess the impact of F941V and G1145S, together with two nonsynonymous ITGAM polymorphisms, A858V (rs1143683) and M441T (rs11861251), on CR3-mediated phagocytosis. A paired two-tailed t test was used to compare the phagocytic capabilities of each variant with that of wild-type CR3.

Results

Both rare variants, F941V and G1145S, significantly impair CR3-mediated phagocytosis in an in vitro model (61% reduction, P = 0.006; 26% reduction, P = 0.0232). However, neither of the common variants, M441T and A858V, had an effect on phagocytosis. Neither rare variant was observed again in the genotyping of additional SLE cases, suggesting that there frequencies are extremely low.

Conclusions

Our results add further evidence to the functional importance of ITGAM in SLE pathogenesis through impaired phagocytosis. Additionally, this study provides a new example of the identification of rare variants in common-allele-associated loci, which, because of their extremely low frequencies, are not statistically associated. However, the demonstration of their functional effects adds support to their contribution to disease risk, and questions the current notion of dismissing the contribution of very rare variants on purely statistical analyses.     

<Emphasis Type="Italic">Apeiba trombetensis</Emphasis> (Malvaceae: Grewioideae), a new species from northern Brazil

Apeiba trombetensis is described from northern Brazil, illustrated, and distinguished from related species. It is remarkable for its tetramerous calyx, sepal pubescence, and its large, globose capsules with stout spines.