Bacterial genome replication at subzero temperatures in permafrost

Microbial metabolic activity occurs at subzero temperatures in permafrost, an environment representing ∼25% of the global soil organic matter. Although much of the observed subzero microbial activity may be due to basal metabolism or macromolecular repair, there is also ample evidence for cellular growth. Unfortunately, most metabolic measurements or culture-based laboratory experiments cannot elucidate the specific microorganisms responsible for metabolic activities in native permafrost, nor, can bulk approaches determine whether different members of the microbial community modulate their responses as a function of changing subzero temperatures. Here, we report on the use of stable isotope probing with ¹³C-acetate to demonstrate bacterial genome replication in Alaskan permafrost at temperatures of 0 to −20 °C. We found that the majority (80%) of operational taxonomic units detected in permafrost microcosms were active and could synthesize ¹³C-labeled DNA when supplemented with ¹³C-acetate at temperatures of 0 to −20 °C during a 6-month incubation. The data indicated that some members of the bacterial community were active across all of the experimental temperatures, whereas many others only synthesized DNA within a narrow subzero temperature range. Phylogenetic analysis of ¹³C-labeled 16S rRNA genes revealed that the subzero active bacteria were members of the Acidobacteria, Actinobacteria, Chloroflexi, Gemmatimonadetes and Proteobacteria phyla and were distantly related to currently cultivated psychrophiles. These results imply that small subzero temperature changes may lead to changes in the active microbial community, which could have consequences for biogeochemical cycling in permanently frozen systems.     

M234Glu is a component of the proton sponge in the reaction center from photosynthetic bacteria

Bacterial reaction centers use light energy to couple the uptake of protons to the successive semi-reduction of two quinones, namely Q_A and Q_B. These molecules are situated symmetrically in regard to a non-heme iron atom. Four histidines and one glutamic acid, M234Glu, constitute the five ligands of this atom. By flash-induced absorption spectroscopy and delayed fluorescence we have studied in the M234EH and M234EL variants the role played by this acidic residue on the energetic balance between the two quinones as well as in proton uptake. Delayed fluorescence from the P⁺Q_A^? state (P is the primary electron donor) and temperature dependence of the rate of P⁺Q_A^? charge recombination that are in good agreement show that in the two RC variants, both Q_A^? and Q_B^? are destabilized by about the same free energy amount: respectively ～ 100 ± 5 meV and 90 ± 5 meV for the M234EH and M234EL variants, as compared to the WT. Importantly, in the M234EH and M234EL variants we observe a collapse of the high pH band (present in the wild-type reaction center) of the proton uptake amplitudes associated with formation of Q_A^? and Q_B^?. This band has recently been shown to be a signature of a collective behaviour of an extended, multi-entry, proton uptake network. M234Glu seems to play a central role in the proton sponge-like system formed by the RC protein.     

PYPAF7, a novel PYRIN-containing Apaf1-like protein that regulates activation of NF-kappa B and caspase-1-dependent cytokine processing

PYRIN-containing Apaf1-like proteins (PYPAFs) are members of the nucleotide-binding site/leucine-rich repeat (NBS/LRR) family of signal transduction proteins. We report here that PYPAF7 is a novel PYPAF protein that activates inflammatory signaling pathways. The expression of PYPAF7 is highly restricted to immune cells, and its gene maps to chromosome 19q13.4, a locus that contains a cluster of genes encoding numerous PYPAF family members. Co-expression of PYPAF7 with ASC results in the recruitment of PYPAF7 to distinct cytoplasmic loci and a potent synergistic activation of NF-kappa B. To identify other proteins involved in PYPAF7 and ASC signaling pathways, we performed a mammalian two-hybrid screen and identified pro-caspase-1 as a binding partner of ASC. Co-expression of PYPAF7 and ASC results in the synergistic activation of caspase-1 and a corresponding increase in secretion of interleukin-1 beta. In addition, PYPAF1 induces caspase-1-dependent cytokine processing when co-expressed with ASC. These findings indicate that PYPAF family members participate in inflammatory signaling by regulating the activation of NF-kappa B and cytokine processing.     

Isolation of a putative progenitor subpopulation of alveolar epithelial type 2 cells

Alveolar epithelial type 2 cells (AEC2) isolated from hyperoxia-treated animals exhibit increases in both proliferation and DNA damage in response to culture. AEC2 express the zonula adherens proteins E-cadherin, -, - and -catenin, desmoglein, and pp120, as demonstrated by Western blotting. Immunohistochemical analysis of cultured AEC2 showed expression of E-cadherin on cytoplasmic membranes varying from strongly to weakly staining. When cultured AEC2 placed in suspension were labeled with fluorescent-tagged antibodies to E-cadherin, cells could be sorted into at least two subpopulations, either dim or brightly staining for this marker. With the use of antibody to E-cadherin bound to magnetic beads, cells were physically separated into E-cadherin-positive and -negative subpopulations, which were then analyzed for differences in proliferation and DNA damage. The E-cadherin-positive subpopulation contained the majority of damaged cells, was quiescent, and expressed low levels of telomerase activity, whereas the E-cadherin-negative subpopulation was undamaged, proliferative, and expressed high levels of telomerase activity.     

Cas9 Cuts and Consequences; Detecting,Predicting, and Mitigating CRISPR/Cas9 On- and Off-Target Damage

Large deletions and genomic re-arrangements are increasingly recognized as common products of double-strand break repair at Clustered Regularly Interspaced, Short Palindromic Repeats - CRISPR associated protein 9 (CRISPR/Cas9) on-target sites. Together with well-known off-target editing products from Cas9 target misrecognition, these are important limitations, that need to be addressed. Rigorous assessment of Cas9-editing is necessary to ensure validity of observed phenotypes in Cas9-edited cell-lines and model organisms. Here the mechanisms of Cas9 specificity, and strategies to assess and mitigate unwanted effects of Cas9 editing are reviewed; covering guide-RNA design, RNA modifications, Cas9 modifications, control of Cas9 activity; computational prediction for off-targets, and experimental methods for detecting Cas9 cleavage. Although recognition of the prevalence of on- and off-target effects of Cas9 editing has increased in recent years, broader uptake across the gene editing community will be important in determining the specificity of Cas9 across diverse applications and organisms.     

A rare insertional translocation of proximal segment with heterochromatic region of 1q into 7p in monozygotic twins and spontaneous abortions

Summary We present a family identified through a healthy 20-year-old female with a history of multiple successive spontaneous abortions. Her karyotype demonstrates a rare balanced insertional translocation between chromosomes 1 and 7, 46,XX,dir ins(7;1)(p15.3;q12q21.3). This is the first reported case of a 7;1 insertional translocation involving the proximal segment of chromosome 1 and may well be the cause of the multiple spontaneous abortions in our proband.     

Neuroprotective Efficacy of Methylene Blue in Ischemic Stroke: An MRI Study

Methylene blue (MB) has unique energy-enhancing and antioxidant properties and is FDA-approved drug to treat methemoglobinemia and cyanide poisoning. This study evaluated the efficacy of MB to treat ischemic stroke in rats using longitudinal MRI and behavioral measures. Rats were subjected to 60-minute middle-cerebral-artery occlusion. In a randomized double-blinded design, vehicle or MB was administered after reperfusion. The initial lesion volumes at 30 minutes post-ischemia were not significantly different between the two groups (P = 0.92). The final infarct volumes two days after stroke increased in the vehicle group but decreased in the MB group, yielding a 30% difference in infarct volume (P = 0.03). Tracking tissue fate on a pixel-by-pixel basis showed that MB salvaged more initial core pixels compared to controls (22±3% versus 11±3%, P = 0.03), and more mismatch pixels compared to controls (83±3% versus 61±8%, P = 0.02). This study demonstrates MB treatment minimizes ischemic brain injury and improves functional outcomes.     

Dysbiosis-Associated Change in Host Metabolism Generates Lactate to Support Salmonella Growth

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