Characterization of a Novel Mouse Model of Alzheimer’s Disease—Amyloid Pathology and Unique β-Amyloid Oligomer Profile

Amyloid plaques composed of β-amyloid (Aβ) protein are a pathological hallmark of Alzheimer’s disease. We here report the generation and characterization of a novel transgenic mouse model of Aβ toxicity. The rTg9191 mice harbor a transgene encoding the 695 amino-acid isoform of human amyloid precursor protein (APP) with the Swedish and London mutations (APP_NLI) linked to familial Alzheimer’s disease, under the control of a tetracycline-response element, as well as a transgene encoding the tetracycline transactivator, under the control of the promoter for calcium-calmodulin kinase IIα. In these mice, APP_NLI is expressed at a level four-fold that of endogenous mouse APP and its expression is restricted to forebrain regions. Transgene expression was suppressed by 87% after two months of doxycycline administration. Histologically, we showed that (1) Aβ plaques emerged in cerebral cortex and hippocampus as early as 8 and 10.5-12.5 months of age, respectively; (2) plaque deposition progressed in an age-dependent manner, occupying up to 19% of cortex at ~25 months of age; and (3) neuropathology—such as abnormal neuronal architecture, tau hyperphosphorylation and misfolding, and neuroinflammation—was observed in the vicinity of neuritic plaques. Biochemically, we determined total Aβ production at varied ages of mice, and we showed that mice produced primarily fibrillar Aβ assemblies recognized by conformation-selective OC antibodies, but few non-fibrillar oligomers (e.g., Aβ*56) detectable by A11 antibodies. Finally, we showed that expression of the tetracycline transactivator resulted in reduced brain weight and smaller dentate-gyrus size. Collectively, these data indicate that rTg9191 mice may serve as a model for studying the neurological effects of the fibrillar Aβ assemblies in situ.     

Men’s Sexual Faithfulness Judgments May Contain a Kernel of Truth

Mechanisms enabling men to identify women likely to engage in extra-pair copulations (EPCs) would be advantageous in avoiding cuckoldry. Men’s judgments of female sexual faithfulness often show high consensus, but accuracy appears poor. We examined whether accuracy of these judgments made to images of women could be improved through i) employing a forced choice task, in which men were asked to select the more faithful of two women and/or ii) providing men with full person images. In Experiment 1, men rated 34 women, for whom we had self-reported EPC behavior, on faithfulness, trustworthiness or attractiveness from either face or full person photographs. They then completed a forced choice task, selecting the more faithful of two woman from 17 pairs of images, each containing one woman who had reported no EPCs and one who had reported two or more EPCs. Men were unable to rate faithfulness with any accuracy, replicating previous findings. However, when asked to choose the more faithful of two women, they performed significantly above chance, although the ability to judge faithfulness at above-chance levels did not generalize to all pairs of women. Although there was no significant difference in accuracy for face and full person image pairs, only judgments from faces were significantly above chance. In Experiment 2, we showed that this accuracy for faces was repeatable in a new sample of men. We also showed that individual variation in accuracy was unrelated to variation in preferences for faithfulness in a long-term partner. Overall, these results show that men’s judgments of faithfulness made from faces of unfamiliar women may contain a kernel of truth.     

The Effector Domain of MARCKS Is a Nuclear Localization Signal that Regulates Cellular PIP2 Levels and Nuclear PIP2 Localization

Translocation to the nucleus of diacylglycerol kinase (DGK)– ζ is dependent on a sequence homologous to the effector domain of Myristoylated Alanine Rich C-Kinase Substrate (MARCKS). These data would suggest that MARCKS could also localize to the nucleus. A single report demonstrated immunofluorescence staining of MARCKS in the nucleus; however, further experimental evidence confirming the specific domain responsible for this localization has not been reported. Here, we report that MARCKS is present in the nucleus in GBM cell lines. We then over-expressed wild-type MARCKS (WT) and MARCKS with the effector domain deleted (ΔED), both tagged with V5-epitope in a GBM cell line with low endogenous MARCKS expression (U87). We found that MARCKS-WT localized to the nucleus, while the MARCKS construct without the effector domain remained in the cytoplasm. We also found that over-expression of MARCKS-WT resulted in a significant increase in total cellular phosphatidyl-inositol (4,5) bisphosphate (PIP₂) levels, consistent with prior evidence that MARCKS can regulate PIP₂ levels. We also found increased staining for PIP₂ in the nucleus with MARCKS-WT over-expression compared to MARCKS ΔED by immunofluorescence. Interestingly, we observed MARCKS and PIP₂ co-localization in the nucleus. Lastly, we found changes in gene expression when MARCKS was not present in the nucleus (MARCKS ΔED). These data indicate that the MARCKS effector domain can function as a nuclear localization signal and that this sequence is critical for the ability of MARCKS to regulate PIP₂ levels, nuclear localization, and gene expression. These data suggests a novel role for MARCKS in regulating nuclear functions such as gene expression.     

Prevalence and Trends of Staphylococcus aureus Bacteraemia in Hospitalized Patients in South Africa, 2010 to 2012: Laboratory-Based Surveillance Mapping of Antimicrobial Resistance and Molecular Epidemiology

Introduction

We aimed to obtain an in-depth understanding on recent antimicrobial resistance trends and molecular epidemiology trends of S. aureus bacteraemia (SAB).

Methods

Thirteen academic centres in South Africa were included from June 2010 until July 2012. S. aureus susceptibility testing was performed on the MicroScan Walkaway. Real-time PCR using the LightCycler 480 II was done for mecA and nuc. SCCmec and spa-typing were finalized with conventional PCR. We selected one isolate per common spa type per province for multilocus sequence typing (MLST).

Results

S. aureus from 2709 patients were included, and 1231 (46%) were resistant to methicillin, with a significant decline over the three-year period (p-value = 0.003). Geographical distribution of MRSA was significantly higher in Gauteng compared to the other provinces (P<0.001). Children <5 years were significantly associated with MRSA with higher rates compared to all other age groups (P = 0.01). The most prevalent SCCmec type was SCCmec type III (531 [41%]) followed by type IV (402 [31%]). Spa-typing discovered 47 different spa-types. The five (87%) most common spa-types were t037, t1257, t045, t064 and t012. Based on MLST, the commonest was ST612 clonal complex (CC8) (n = 7) followed by ST5 (CC5) (n = 4), ST36 (CC30) (n = 4) and ST239 (CC8) (n = 3).

Conclusions

MRSA rate is high in South Africa. Majority of the isolates were classified as SCCmec type III (41%) and type IV (31%), which are typically associated with hospital and community- acquired infections, respectively. Overall, this study reveals the presence of a variety of hospital-acquired MRSA clones in South Africa dominance of few clones, spa 037 and 1257. Monitoring trends in resistance and molecular typing is recommended to detect changing epidemiological trends in AMR patterns of SAB.     

Comparative Prevalence of Immune Evasion Complex Genes Associated with β-Hemolysin Converting Bacteriophages in MRSA ST5 Isolates from Swine,Swine Facilities,Humans with Swine Contact,and Humans with No Swine Contact

Livestock associated methicillin-resistant Staphylococcus aureus (LA-MRSA) draws concern from the public health community because in some countries these organisms may represent the largest reservoir of MRSA outside hospital settings. Recent studies indicate LA-MRSA strains from swine are more genetically diverse than the first reported sequence type ST398. In the US, a diverse population of LA-MRSA is found including organisms of the ST398, ST9, and ST5 lineages. Occurrence of ST5 MRSA in swine is of particular concern since ST5 is among the most prevalent lineages causing clinical infections in humans. The prominence of ST5 in clinical disease is believed to result from acquisition of bacteriophages containing virulence or host-adapted genes including the immune-evasion cluster (IEC) genes carried by β-hemolysin converting bacteriophages, whose absence in LA-MRSA ST398 is thought to contribute to reduced rates of human infection and transmission associated with this lineage. The goal of this study was to investigate the prevalence of IEC genes associated with β-hemolysin converting bacteriophages in MRSA ST5 isolates obtained from agricultural sources, including swine, swine facilities, and humans with short- or long-term swine exposure. To gain a broader perspective, the prevalence of these genes in LA-MRSA ST5 strains was compared to the prevalence in clinical MRSA ST5 strains from humans with no known exposure to swine. IEC genes were not present in any of the tested MRSA ST5 strains from agricultural sources and the β-hemolysin gene was intact in these strains, indicating the bacteriophage’s absence. In contrast, the prevalence of the β-hemolysin converting bacteriophage in MRSA ST5 strains from humans with no exposure to swine was 90.4%. The absence of β-hemolysin converting bacteriophage in LA-MRSA ST5 isolates is consistent with previous reports evaluating ST398 strains and provides genetic evidence indicating LA-MRSA ST5 isolates may harbor a reduced capacity to cause severe disease in immunocompetent humans.     

Haemophilus ducreyi Cutaneous Ulcer Strains Are Nearly Identical to Class I Genital Ulcer Strains

Background

Although cutaneous ulcers (CU) in the tropics is frequently attributed to Treponema pallidum subspecies pertenue, the causative agent of yaws, Haemophilus ducreyi has emerged as a major cause of CU in yaws-endemic regions of the South Pacific islands and Africa. H. ducreyi is generally susceptible to macrolides, but CU strains persist after mass drug administration of azithromycin for yaws or trachoma. H. ducreyi also causes genital ulcers (GU) and was thought to be exclusively transmitted by microabrasions that occur during sex. In human volunteers, the GU strain 35000HP does not infect intact skin; wounds are required to initiate infection. These data led to several questions: Are CU strains a new variant of H. ducreyi or did they evolve from GU strains? Do CU strains contain additional genes that could allow them to infect intact skin? Are CU strains susceptible to azithromycin?

Methodology/Principal Findings

To address these questions, we performed whole-genome sequencing and antibiotic susceptibility testing of 5 CU strains obtained from Samoa and Vanuatu and 9 archived class I and class II GU strains. Except for single nucleotide polymorphisms, the CU strains were genetically almost identical to the class I strain 35000HP and had no additional genetic content. Phylogenetic analysis showed that class I and class II strains formed two separate clusters and CU strains evolved from class I strains. Class I strains diverged from class II strains ~1.95 million years ago (mya) and CU strains diverged from the class I strain 35000HP ~0.18 mya. CU and GU strains evolved under similar selection pressures. Like 35000HP, the CU strains were highly susceptible to antibiotics, including azithromycin.

Conclusions/Significance

These data suggest that CU strains are derivatives of class I strains that were not recognized until recently. These findings require confirmation by analysis of CU strains from other regions.     

Lights out: the evolution of bacterial bioluminescence in Loliginidae

Representatives of several metazoan clades engage in symbiotic interactions with bioluminescent bacteria, but the evolution and maintenance of these interactions remain poorly understood. Uroteuthis is a genus of loliginid squid (Cephalopoda: Loliginidae) characterized by paired ventral photophores (light organs) housing bioluminescent bacteria. While previous phylogenetic studies have suggested that Uroteuthis is closely related to Loliolus, a genus of non-bioluminescent species, this relationship remains unresolved. To illuminate Uroteuthis and Loliolus phylogeny and its implications for the evolution of bioluminescence in Loliginidae, we generated sequences from two mitochondrial genes from Uroteuthis specimens sampled from several sites in the Indian and western Pacific Oceans. We combined these data with data from GenBank, analyzed the concatenated data set using maximum likelihood and Bayesian methods, and reconstructed the evolution of bacterial bioluminescence on the resulting phylogenies. Our analyses support the hypothesis that Uroteuthis is paraphyletic with respect to Loliolus. Furthermore, our reconstructions suggest that the symbiosis between loliginid squid and bioluminescent bacteria evolved once in the ancestor of Loliolini (the clade comprising Uroteuthis and Loliolus), but was subsequently lost in the ancestor of Loliolus. These findings could have profound implications for our understanding of the evolution of symbiotic bioluminescence in squid.     

Probing the Structure of the Mechanosensitive Channel of Small Conductance in Lipid Bilayers with Pulsed Electron-Electron Double Resonance

Mechanosensitive channel proteins are important safety valves against osmotic shock in bacteria, and are involved in sensing touch and sound waves in higher organisms. The mechanosensitive channel of small conductance (MscS) has been extensively studied. Pulsed electron-electron double resonance (PELDOR or DEER) of detergent-solubilized protein confirms that as seen in the crystal structure, the outer ring of transmembrane helices do not pack against the pore-forming helices, creating an apparent void. The relevance of this void to the functional form of MscS in the bilayer is the subject of debate. Here, we report PELDOR measurements of MscS reconstituted into two lipid bilayer systems: nanodiscs and bicelles. The distance measurements from multiple mutants derived from the PELDOR data are consistent with the detergent-solution arrangement of the protein. We conclude, therefore, that the relative positioning of the transmembrane helices is preserved in mimics of the cell bilayer, and that the apparent voids are not an artifact of detergent solution but a property of the protein that will have to be accounted for in any molecular mechanism of gating.     

Reduced creatine kinase as a central and peripheral biomarker in Huntington's disease

A major goal of current clinical research in Huntington's disease (HD) has been to identify preclinical and manifest disease biomarkers, as these may improve both diagnosis and the power for therapeutic trials. Although the underlying biochemical alterations and the mechanisms of neuronal degeneration remain unknown, energy metabolism defects in HD have been chronicled for many years. We report that the brain isoenzyme of creatine kinase (CK-BB), an enzyme important in buffering energy stores, was significantly reduced in presymptomatic and manifest disease in brain and blood buffy coat specimens in HD mice and HD patients. Brain CK-BB levels were significantly reduced in R6/2 mice by ～ 18% to ～ 68% from 21 to 91 days of age, while blood CK-BB levels were decreased by ～ 14% to ～ 44% during the same disease duration. Similar findings in CK-BB levels were observed in the 140 CAG mice from 4 to 12 months of age, but not at the earliest time point, 2 months of age. Consistent with the HD mice, there was a grade-dependent loss of brain CK-BB that worsened with disease severity in HD patients from ～ 28% to ～ 63%, as compared to non-diseased control patients. In addition, CK-BB blood buffy coat levels were significantly reduced in both premanifest and symptomatic HD patients by ～ 23% and ～ 39%, respectively. The correlation of CK-BB as a disease biomarker in both CNS and peripheral tissues from HD mice and HD patients may provide a powerful means to assess disease progression and to predict the potential magnitude of therapeutic benefit in this disorder.