Design and synthesis of orally bioavailable serum and glucocorticoid-regulated kinase 1 (SGK1) inhibitors

The lead serum and glucocorticoid-related kinase 1 (SGK1) inhibitors 4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid (1) and {4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid (2) suffer from low DNAUC values in rat, due in part to formation and excretion of glucuronic acid conjugates. These PK/glucuronidation issues were addressed either by incorporating a substituent on the 3-phenyl ring ortho to the key carboxylate functionality of 1 or by substituting on the group in between the carboxylate and phenyl ring of 2. Three of these analogs have been identified as having good SGK1 inhibition potency and have DNAUC values suitable for in vivo testing.     

Effect of natural or vaccine-induced porcine circovirus type 2 (PCV2) immunity on fetal infection after artificial insemination with PCV2 spiked semen

The objectives of this study were to determine if vaccination against porcine circovirus type 2 (PCV2) or previous PCV2 infection of the dam are sufficient to prevent fetal infection when dams are artificially inseminated with PCV2-spiked semen. Nine sows (Sus domestica) were allocated into three groups of three dams each: The PCV2 naïve negative control Group 1 was artificially inseminated with extended PCV2 DNA negative semen during estrus, whereas the extended semen used in the vaccinated Group 2 (PCV2 vaccine was given 8 wk before insemination) and PCV2-exposed Group 3 (infected with PCV2 12 wk before insemination) was spiked with 5 mL of PCV2 inoculum with a titer of 10^4.2 tissue culture infectious dose (TCID₅₀) per milliliter at each breeding. The dams in the vaccinated and PCV2-exposed groups were positive for PCV2 antibody but negative for PCV2 DNA in serum at the time of insemination. Three negative control dams, two vaccinated dams, and three dams with previous PCV2 exposure became pregnant and maintained pregnancy to term. After artificial insemination, viremia was detected in one of three vaccinated dams and in two of three dams with previous PCV2 exposure. At farrowing, PCV2 infection was not detected in any piglets or fetuses expelled from the negative control dams or from dams with previous PCV2 exposure. In litters of the vaccinated dams, 15 of 24 live-born piglets were PCV2 viremic at birth, with 6 of 26 fetuses having detectable PCV2 antigen in tissues. In conclusion, vaccine-induced immunity did not prevent fetal infection in this sow model using semen spiked with PCV2.     

Survey of wastewater indicators and human pathogen genomes in biosolids produced by class a and class B stabilization treatments

Accurate modeling of the infectious aerosol risk associated with the land application of biosolids requires an in-depth knowledge of the magnitudes and changes in pathogen concentrations for a variety of class A and class B stabilization methods. The following survey used quantitative PCR (qPCR) and culture assays to detect environmentally resistant bacterial and viral pathogens and biosolid indicator organisms for 36 biosolid grab samples. Biosolids were collected from 14 U.S. states and included 16 class B mesophilic anaerobic digestion (MAD) samples and 20 class A biosolid samples from temperature-phased anaerobic digestion (TPAD), MAD plus composting (COM), and MAD plus heat pelletization processes. The indicator concentrations of fecal coliforms and male-specific coliphages as well as pathogen genome concentrations for human adenovirus species, Legionella pneumophila, Staphylococcus aureus, and Clostridium difficile were significantly lower in the class A samples, and a multivariate analysis of variance ranked the stabilization processes from the lowest pathogen/indicator load to the highest as (i) class A COM, (ii) class A TPAD, and (iii) class B MAD. Human adenovirus genomes were found in 88% of the class B samples and 70 to 100% of the class A samples. L. pneumophila, S. aureus, and C. difficile genomes were detected at the qPCR assay detection limits in 19 to 50% of the class B and class A anaerobic digestion samples, while L. pneumophila was detected in 50% of the class A compost samples. When considering all the stabilization methods, both the fecal coliform and the male-specific coliphage concentrations show a significant linear correlation with the pathogen genome concentrations. This survey provides the necessary pathogen concentrations to add to biosolid aerosol risk and pathogen exposure analyses and clarifies the effectiveness of class A stabilization methods with the pathogen and indicator loads in biosolids.     

Derivation and Characterization of a Simian Immunodeficiency Virus SIVmac239 Variant with Tropism for CXCR4

Like human immunodeficiency virus type 1 (HIV-1), most simian immunodeficiency virus (SIV) strains use CCR5 to establish infection. However, while HIV-1 can acquire the ability to use CXCR4, SIVs that utilize CXCR4 have rarely been reported. To explore possible barriers against SIV coreceptor switching, we derived an R5X4 variant, termed 239-ST1, from the R5 clone SIVmac239 by serially passaging virus in CD4⁺ CXCR4⁺ CCR5⁻ SupT1 cells. A 239-ST1 env clone, designated 239-ST1.2-32, used CXCR4 and CCR5 in cell-cell fusion and reporter virus infection assays and conferred the ability for rapid, cytopathic infection of SupT1 cells to SIVmac239. Viral replication was inhibitable by the CXCR4-specific antagonist AMD3100, and replication was abrogated in a novel CXCR4⁻ SupT1 line. Surprisingly, parental SIVmac239 exhibited low-level replication in SupT1 cells that was not observed in CXCR4⁻ SupT1 cells. Only two mutations in the 239-ST1.2-32 Env, K47E in the C1 domain and L328W in the V3 loop, were required for CXCR4 use in cell-cell fusion assays, although two other V3 changes, N316K and I324M, improved CXCR4 use in infection assays. An Env cytoplasmic tail truncation, acquired during propagation of 239-ST1 in SupT1 cells, was not required. Compared with SIVmac239, 239-ST1.2-32 was more sensitive to neutralization by five of seven serum and plasma samples from SIVmac239-infected rhesus macaques and was approximately 50-fold more sensitive to soluble CD4. Thus, SIVmac239 can acquire the ability to use CXCR4 with high efficiency, but the changes required for this phenotype may be distinct from those for HIV-1 CXCR4 use. This finding, along with the increased neutralization sensitivity of this CXCR4-using SIV, suggests a mechanism that could select strongly against this phenotype in vivo.Simian immunodeficiency viruses (SIVs) share many structural and biological features with human immunodeficiency virus (HIV), including target cell entry via interactions of the viral envelope glycoprotein (Env) with CD4 and a chemokine coreceptor. For HIV, the most important coreceptors in vivo are CCR5 (2, 13, 19, 21, 22) and CXCR4 (30). HIV type 1 (HIV-1) strains that use only CCR5 (R5 viruses) predominate during the early stages of infection and are critical for transmission (84, 90), as evidenced by the finding that individuals lacking a functional CCR5 protein due to a homozygous 32-bp deletion in the CCR5 gene (ccr5-Δ32) are largely resistant to HIV-1 infection (16, 54, 82). Although R5 viruses generally persist in late-stage disease, viruses that can use CXCR4, either exclusively (X4 viruses) or in addition to CCR5 (R5X4 viruses), emerge in approximately 50% of subtype B-infected individuals (15, 43). This coreceptor switch is associated with a more rapid decline in peripheral blood CD4⁺ T cells and a faster progression to AIDS (15, 43, 77), although it is unclear if CXCR4-using viruses are a cause or a consequence of progressing immunodeficiency. Like HIV, the vast majority of SIVs use CCR5 to establish infection (11, 12, 45). However, although CXCR4-using SIVs have been reported (47, 52, 65, 68, 69), their occurrence is rare, especially in models of pathogenic infection, where only one CXCR4-using SIV has been identified (17, 60, 71).This paucity of CXCR4-using SIVs is surprising for several reasons. First, SIV Envs tend to be more promiscuous than HIV-1 Envs and frequently use alternative coreceptors in addition to CCR5, including GPR1, GPR15, CXCR6, and CCR8 (20, 27, 29, 80, 81, 92) but not CXCR4. Second, HIV-2, which is more closely related to SIVmac than to HIV-1 (56, 57), commonly uses CXCR4 in vitro and in vivo (3, 28, 33, 58, 59, 67). Third, rhesus CXCR4 is ∼98% identical to human CXCR4 in amino acid sequence and can function as a coreceptor for HIV-1 in vitro (12). Finally, chimeric simian-human immunodeficiency viruses (SHIVs) that contain X4 HIV Envs on an SIV core can replicate to high levels in vivo and cause disease in rhesus macaques (39, 86). Moreover, it was recently shown that coreceptor switching can occur in rhesus macaques infected with an R5 SHIV (35). Thus, there does not appear to be any block per se against the use of rhesus CXCR4 as an entry coreceptor either in vitro or in vivo, suggesting that SIV is less capable of adapting to use CXCR4 and/or that mutations required for CXCR4 utilization may lead to a virus that is less fit and/or more susceptible to immune control in this host.For HIV-1, the Env determinants for CXCR4 use have been well documented and often involve the acquisition of positively charged amino acids in the V3 loop (18, 32, 87), particularly at positions 11, 24, and 25 (6, 18, 31, 32, 38, 75). Although the SIVmac239 V3 loop is a critical determinant for Env-coreceptor interactions (44, 63, 72), attempts to create an X4 SIVmac239 by introducing positively charged residues into the V3 loop (63) or by inserting a V3 loop from X4 HIV-1 (44) have been unsuccessful. SIVmac155T3, the only CXCR4-using variant of SIVmac that has been identified to date, was isolated from a rhesus macaque with advanced disease and contains additional positively charged residues in V3, although the determinants for CXCR4 use have not been determined (60, 71).Given questions concerning the possible determinants for and/or barriers to coreceptor switching in SIV, we sought to derive a CXCR4-using variant of the well-characterized pathogenic R5 SIV clone SIVmac239. Here we show that SIVmac239 could indeed acquire CXCR4 utilization when it was adapted in vitro for high-efficiency replication in the CXCR4⁺ CCR5⁻ human SupT1 cell line. An env clone from this virus could use CXCR4 in cell-cell fusion and reporter virus infection assays and conferred CXCR4 tropism to a replication-competent SIV. Although V3 mutations were important for CXCR4 use, an L328W change at the V3 crown rather than the acquisition of positively charged residues was required, as was an unusual K47E mutation in the conserved C1 domain of gp120. These changes also caused the highly neutralization-resistant SIVmac239 strain to become more neutralization sensitive to sera and plasmas from SIVmac239-infected animals, and particularly to soluble CD4. These results indicate that mutations distinct from those typically seen for HIV-1 may be required for SIVmac to gain CXCR4 utilization and suggest that these changes render this virus more susceptible to humoral immune control. Collectively, our findings indicate that there are likely to be strong viral and host selection pressures against CXCR4 use that may contribute to the paucity of X4 coreceptor switching for SIVmac in vivo.     

Assessing the in vitro toxicity of the lunar dust environment using respiratory cells exposed to Al<Subscript>2</Subscript>O<Subscript>3</Subscript> or SiO<Subscript>2</Subscript> fine dust particles

Prior chemical and physical analysis of lunar soil suggests a composition of dust particles that may contribute to the development of acute and chronic respiratory disorders. In this study, fine Al₂O₃ (0.7 μm) and fine SiO₂ (mean 1.6 μm) were used to assess the cellular uptake and cellular toxicity of lunar dust particle analogs. Respiratory cells, murine alveolar macrophages (RAW 264.7) and human type II epithelial (A549), were cultured as the in vitro model system. The phagocytic activity of both cell types using ultrafine (0.1 μm) and fine (0.5 μm) fluorescent polystyrene beads was determined. Following a 6-h exposure, RAW 264.7 cells had extended pseudopods with beads localized in the cytoplasmic region of cells. After 24 h, the macrophage cells were rounded and clumped and lacked pseudopods, which suggest impairment of phagocytosis. A549 cells did not contain beads, and after 24 h, the majority of the beads appeared to primarily coat the surface of the cells. Next, we investigated the cellular response to fine SiO₂ and Al₂O₃ (up to 5 mg/ml). RAW 264.7 cells exposed to 1.0 mg/ml of fine SiO₂ for 6 h demonstrated pseudopods, cellular damage, apoptosis, and necrosis. A549 cells showed slight toxicity when exposed to fine SiO₂ for the same time and dose. A549 cells had particles clustered on the surface of the cells. Only a higher dose (5.0 mg/ml) of fine SiO₂ resulted in a significant cytotoxicity to A549 cells. Most importantly, both cell types showed minimal cytotoxicity following exposure to fine Al₂O₃. Overall, this study suggests differential cellular toxicity associated with exposure to fine mineral dust particles.     

Not just black and white: Pigment pattern development and evolution in vertebrates

Animals display diverse colors and patterns that vary within and between species. Similar phenotypes appear in both closely related and widely divergent taxa. Pigment patterns thus provide an opportunity to explore how development is altered to produce differences in form and whether similar phenotypes share a common genetic basis. Understanding the development and evolution of pigment patterns requires knowledge of the cellular interactions and signaling pathways that produce those patterns. These complex traits provide unparalleled opportunities for integrating studies from ecology and behavior to molecular biology and biophysics.     

Sulfide persistence in oil field waters amended with nitrate and acetate

Nitrate amendment is normally an effective method for sulfide control in oil field-produced waters. However, this approach has occasionally failed to prevent sulfide accumulation, despite the presence of active nitrate-reducing bacterial populations. Here, we report our study of bulk chemical transformations in microcosms of oil field waters containing nitrate-reducing, sulfide-oxidizing bacteria, but lacking denitrifying heterotrophs. Amendment with combinations of nitrate, acetate, and phosphate altered the microbial sulfur and nitrogen transformations. Elemental sulfur produced by chemotrophic nitrate-reducing bacteria was re-reduced heterotrophically to sulfide. Ammonification, rather than denitrification, was the predominant pathway for nitrate reduction. The application of nitrite led to transient sulfide depletion, possibly due to higher rates of nitrite reduction. The addition of molybdate suppressed both the accumulation of sulfide and the heterotrophic reduction of nitrate. Therefore, sulfidogenesis was likely due to elemental sulfur-reducing heterotrophic bacteria, and the nitrate-reducing microbial community consisted mainly of facultatively chemotrophic microbes. This study describes one set of conditions for continued sulfidogenesis during nitrate reduction, with important implications for nitrate control of sulfide production in oil fields.