Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition,bi-directionally from the primed protospacer

Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems. To generate immunity, these systems acquire short sequences of nucleic acids from foreign invaders and incorporate these into their CRISPR arrays as spacers. This adaptation process is the least characterized step in CRISPR-Cas immunity. Here, we used Pectobacterium atrosepticum to investigate adaptation in Type I-F CRISPR-Cas systems. Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays. Endogenous expression of the cas genes was sufficient, yet required, for priming. The new spacers inhibited conjugation and transformation, and interference was enhanced with increasing numbers of new spacers. We analyzed ∼350 new spacers acquired in priming events and identified a 5′-protospacer-GG-3′ protospacer adjacent motif. In contrast to priming in Type I-E systems, new spacers matched either plasmid strand and a biased distribution, including clustering near the primed protospacer, suggested a bi-directional translocation model for the Cas1:Cas2–3 adaptation machinery. Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary.     

Transmitted antiretroviral drug resistance in new york state, 2006-2008: results from a new surveillance system

Background

HIV transmitted drug resistance (TDR) is a public health concern because it has the potential to compromise antiretroviral therapy (ART) at the population level. In New York State, high prevalence of TDR in a local cohort and a multiclass resistant case cluster led to the development and implementation of a statewide resistance surveillance system.

Methodology

We conducted a cross-sectional analysis of the 13,109 cases of HIV infection that were newly diagnosed and reported in New York State between 2006 and 2008, including 4,155 with HIV genotypes drawn within 3 months of initial diagnosis and electronically reported to the new resistance surveillance system. We assessed compliance with DHHS recommendations for genotypic resistance testing and estimated TDR among new HIV diagnoses.

Principal Findings

Of 13,109 new HIV diagnoses, 9,785 (75%) had laboratory evidence of utilization of HIV-related medical care, and 4,155 (43%) had a genotype performed within 3 months of initial diagnosis. Of these, 11.2% (95% confidence interval [CI], 10.2%–12.1%) had any evidence of TDR. The proportion with mutations associated with any antiretroviral agent in the NNRTI, NRTI or PI class was 6.3% (5.5%–7.0%), 4.3% (3.6%–4.9%) and 2.9% (2.4%–3.4%), respectively. Multiclass resistance was observed in <1%. TDR did not increase significantly over time (p for trend = 0.204). Men who have sex with men were not more likely to have TDR than persons with heterosexual risk factor (OR 1.0 (0.77–1.30)). TDR to EFV+TDF+FTC and LPV/r+TDF+FTC regimens was 7.1% (6.3%–7.9%) and 1.4% (1.0%–1.8%), respectively.

Conclusions/Significance

TDR appears to be evenly distributed and stable among new HIV diagnoses in New York State; multiclass TDR is rare. Less than half of new diagnoses initiating care received a genotype per DHHS guidelines.     

Effects of post-breeding moult and energetic condition on timing of songbird migration into the tropics

Each autumn billions of songbirds migrate between the temperate zone and tropics, but little is known about how events on the breeding grounds affect migration to the tropics. Here, we use light level geolocators to track the autumn migration of wood thrushes Hylocichla mustelina and test for the first time if late moult and poor physiological condition prior to migration delays arrival on the winter territory. Late nesting thrushes postponed feather moult, and birds with less advanced moult in August were significantly farther north on 10 October while en route to the tropics. Individuals in relatively poor energetic condition in August (high β-Hydroxybutyrate, low triglyceride, narrow feather growth bars) passed into the tropics significantly later in October. However, late moult and poor pre-migratory condition did not result in late arrival on the winter territory because stopover duration was highly variable late in migration. Although carry-over effects from the winter territory to spring migration may be strong in migratory songbirds, our study suggests that high reproductive effort late in the season does not impose time constraints that delay winter territory acquisition.     

Neural tube defects and maternal biomarkers of folate, homocysteine, and glutathione metabolism

BACKGROUND: Alterations in maternal folate and homocysteine metabolism are associated with neural tube defects (NTDs). The role played by specific micronutrients and metabolites in the causal pathway leading to NTDs is not fully understood. METHODS: We conducted a case-control study to investigate the association between NTDs and maternal alterations in plasma micronutrients and metabolites in two metabolic pathways: methionine remethylation and glutathione transsulfuration. Biomarkers were measured in a population-based sample of women who had NTD-affected pregnancies (n = 43) and a control group of women who had a pregnancy unaffected by a birth defect (n = 160). We compared plasma concentrations of folate, vitamin B(12), vitamin B(6), methionine, S-adenosylmethionine (SAM), s-adenosylhomocysteine (SAH), adenosine, homocysteine, cysteine, and reduced and oxidized glutathione between cases and controls after adjusting for lifestyle and sociodemographic factors. RESULTS: Women with NTD-affected pregnancies had significantly higher plasma concentrations of SAH (29.12 vs. 23.13 nmol/liter, P = .0011), adenosine (0.323 vs. 0.255 mumol/liter; P = .0269), homocysteine (9.40 vs. 7.56 micromol/liter; P < .001), and oxidized glutathione (0.379 vs. 0.262 micromol/liter; P = .0001), but lower plasma SAM concentrations (78.99 vs. 83.16 nmol/liter; P = .0172) than controls. This metabolic profile is consistent with reduced methylation capacity and increased oxidative stress in women with affected pregnancies. CONCLUSIONS: Increased maternal oxidative stress and decreased methylation capacity may contribute to the occurrence of NTDs. Further analysis of relevant genetic and environmental factors is required to define the basis for these observed alterations.     

Rapid Accumulation of Endogenous Tau Oligomers in a Rat Model of Traumatic Brain Injury: POSSIBLE LINK BETWEEN TRAUMATIC BRAIN INJURY AND SPORADIC TAUOPATHIES*

Traumatic brain injury (TBI) is a serious problem that affects millions of people in the United States alone. Multiple concussions or even a single moderate to severe TBI can also predispose individuals to develop a pathologically distinct form of tauopathy-related dementia at an early age. No effective treatments are currently available for TBI or TBI-related dementia; moreover, only recently has insight been gained regarding the mechanisms behind their connection. Here, we used antibodies to detect oligomeric and phosphorylated Tau proteins in a non-transgenic rodent model of parasagittal fluid percussion injury. Oligomeric and phosphorylated Tau proteins were detected 4 and 24 h and 2 weeks post-TBI in injured, but not sham control rats. These findings suggest that diagnostic tools and therapeutics that target only toxic forms of Tau may provide earlier detection and safe, more effective treatments for tauopathies associated with repetitive neurotrauma.     

Tetracyclines modulate cytosolic Ca2+ responses in the osteoclast associated with “Ca2+ receptor” activation

We report the effects of tetracycline analogues on cytosolic Ca²⁺ transients resulting from application of ionic nickel (Ni²⁺), a potent surrogate agonist of the osteoclast Ca²⁺ receptor. Preincubation with minocycline (1 mg/l) or a chemically modified tetracycline, 4-dedimethyl-aminotetracycline (CMT-1) (1 or 10 mg/l), resulted in a significant attenuation of the magnitude of the cytosolic [Ca²⁺] response to an application of 5 mM-[Ni²⁺]. Preincubation with doxycycline (1 or 10 mg/l) failed to produce similar results. In addition, application of minocycline alone (0.1–100 mg/l) resulted in a 3.5-fold elevation of cytosolic [Ca²⁺]. The results suggest a novel action of tetracyclines on the osteoclast Ca²⁺ receptor.     

Sex differences in circulating and renal angiotensins of hypertensive mRen(2). Lewis but not normotensive Lewis rats

Sex differences in blood pressure are evident in experimental models and human subjects, yet the mechanisms underlying this disparity remain equivocal. The current study sought to define the extent of male-female differences in the circulating and tissue renin-angiotensin aldosterone systems (RAASs) of congenic mRen(2). Lewis and control Lewis rats. Male congenics exhibited higher systolic blood pressure than females [200 +/- 4 vs. 146 +/- 7 mmHg, P < 0.01] or Lewis males and females [113 +/- 2 vs. 112 +/- 2 mmHg, P > 0.05]. Plasma ANG II levels were twofold higher in male congenics [47 +/- 3 vs. 19 +/- 3 pM, P < 0.01] and fivefold higher than in male or female Lewis rats [6 +/- 1 vs. 6 +/- 1 pM]. ANG I levels were also highest in the males; however, plasma ANG-(1-7) was higher in female congenics. Male congenics exhibited greater circulating renin and angiotensin-converting enzyme (ACE) activities, as well as angiotensinogen, than female littermates. Renal cortical and medullary ANG II levels were also higher in the male congenics versus all the other groups; ANG I was lower in the males. Cortical ACE2 activity was higher in male congenics, yet neprilysin activity and protein were greater in the females, which may contribute to reduced renal levels of ANG II. These data reveal that sex differences in both the circulating and renal RAAS are apparent primarily in the hypertensive group. The enhanced activity of the RAAS in male congenics may contribute to the higher pressure and tissue injury evident in the strain.     

Investigation of Genetic Structure between Deep and Shallow Populations of the Southern Rock Lobster,Jasus edwardsii in Tasmania,Australia

The southern rock lobster, Jasus edwardsii, shows clear phenotypic differences between shallow water (red coloured) and deeper water (pale coloured) individuals. Translocations of individuals from deeper water to shallower waters are currently being trialled as a management strategy to facilitate a phenotypic change from lower value pale colouration, common in deeper waters, to the higher value red colouration found in shallow waters. Although panmixia across the J. edwardsii range has been long assumed, it is critical to assess the genetic variability of the species to ensure that the level of population connectivity is appropriately understood and translocations do not have unintended consequences. Eight microsatellite loci were used to investigate genetic differentiation between six sites (three shallow, three deep) across southern Tasmania, Australia, and one from New Zealand. Based on analyses the assumption of panmixia was rejected, revealing small levels of genetic differentiation across southern Tasmania, significant levels of differentiation between Tasmania and New Zealand, and high levels of asymmetric gene flow in an easterly direction from Tasmania into New Zealand. These results suggest that translocation among Tasmanian populations are not likely to be problematic, however, a re-consideration of panmictic stock structure for this species is necessary.     

Equine Infectious Anemia Virus Gag Polyprotein Late Domain Specifically Recruits Cellular AP-2 Adapter Protein Complexes during Virion Assembly

We have identified an interaction between the equine infectious anemia virus (EIAV) late assembly domain and the cellular AP-2 clathrin-associated adapter protein complex. A YXXL motif within the EIAV Gag late assembly domain was previously characterized as a sequence critical for release of assembling virions. We now show that this YXXL sequence interacts in vitro with the AP-50 subunit of the AP-2 complex, while the functionally interchangeable late assembly domains carried by the Rous sarcoma virus p2b protein and human immunodeficiency virus type 1 p6 protein, which utilize PPPY and PTAPP L domains, respectively, do not bind AP-50 in vitro. In addition, EIAV late domain mutants containing mutations that have previously been shown to abrogate budding also exhibit marked decreases in AP-50 binding efficiencies. A role for AP-2 complex in viral assembly is supported by immunofluorescence analysis of EIAV-infected equine dermal cells demonstrating specific colocalization of the α adaptin subunit of AP-2 with the EIAV p9 protein at sites of virus budding on the plasma membrane. These data provide strong evidence that EIAV utilizes the cellular AP-2 complex to accomplish virion assembly and release.