Efficient target cleavage by Type V Cas12a effectors programmed with split CRISPR RNA

CRISPR RNAs (crRNAs) that direct target DNA cleavage by Type V Cas12a nucleases consist of constant repeat-derived 5′-scaffold moiety and variable 3′-spacer moieties. Here, we demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by a Cas12a ortholog from Acidaminococcus sp. (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer moiety only. crRNAs split into separate scaffold and spacer RNAs catalyzed highly specific and efficient cleavage of target DNA by AsCas12a in vitro and in lysates of human cells. In addition to dsDNA target cleavage, AsCas12a programmed with split crRNAs also catalyzed specific ssDNA target cleavage and non-specific ssDNA degradation (collateral activity). V-A effector nucleases from Francisella novicida (FnCas12a) and Lachnospiraceae bacterium (LbCas12a) were also functional with split crRNAs. Thus, the ability of V-A effectors to use split crRNAs appears to be a general property. Though higher concentrations of split crRNA components are needed to achieve efficient target cleavage, split crRNAs open new lines of inquiry into the mechanisms of target recognition and cleavage and may stimulate further development of single-tube multiplex and/or parallel diagnostic tests based on Cas12a nucleases.     

Theoretical study of the electron affinities of the alkaline-earth tetramers possessing T d symmetry: Be4 and Mg4

The electron affinities of beryllium and magnesium tetramers are calculated at the ROMP2 level employing the Dunning-type aug-cc-pVQZ basis set. The vertical electron detachment energy (VEDE) amounts to 1.685 eV for Be₄^– and 0.943 eV for Mg₄ . The decomposition of the VEDE into physical components and an atomic orbital population analysis are used to elucidate the nature of the outer electron binding in these anions.Figure The lowest unoccupied molecular orbitals in the ground state of Mg4 : a LUMO, symmetry A₁, b LUMO + 1, symmetry T₂; c the highest occupied molecular orbital (HOMO), symmetry A1 in the ground state of Mg₄^–.      

Environmental factors function as constraints on soil nitrous oxide fluxes in bioenergy feedstock cropping systems

Nitrous oxide (N₂O) is a potent greenhouse gas and major component of the net global warming potential of bioenergy feedstock cropping systems. Numerous environmental factors influence soil N₂O production, making direct correlation difficult to any one factor of N₂O fluxes under field conditions. We instead employed quantile regression to evaluate whether soil temperature, water‐filled pore space (WFPS), and concentrations of soil nitrate () and ammonium () determined upper bounds for soil N₂O flux magnitudes. We collected data over 6 years from a range of bioenergy feedstock cropping systems including no‐till grain crops, perennial warm‐season grasses, hybrid poplar, and polycultures of tallgrass prairie species each with and without nitrogen (N) addition grown at two sites. The upper bounds for soil N₂O fluxes had a significant and positive correlation with all four environmental factors, although relatively large fluxes were still possible at minimal values for nearly all factors. The correlation with was generally weaker, suggesting it is less important than in driving large fluxes. Quantile regression slopes were generally lower for unfertilized perennials than for other systems, but this may have resulted from a perpetual state of nitrogen limitation, which prevented other factors from being clear constraints. This framework suggests efforts to reduce concentrations of in the soil may be effective at reducing high‐intensity periods—”hot moments”—of N₂O production.     

High Rate of Subclinical Chikungunya Virus Infection and Association of Neutralizing Antibody with Protection in a Prospective Cohort in The Philippines

Background

Chikungunya virus (CHIKV) is a globally re-emerging arbovirus for which previous studies have indicated the majority of infections result in symptomatic febrile illness. We sought to characterize the proportion of subclinical and symptomatic CHIKV infections in a prospective cohort study in a country with known CHIKV circulation.

Methods/Findings

A prospective longitudinal cohort of subjects ≥6 months old underwent community-based active surveillance for acute febrile illness in Cebu City, Philippines from 2012-13. Subjects with fever history were clinically evaluated at acute, 2, 5, and 8 day visits, and at a 3-week convalescent visit. Blood was collected at the acute and 3-week convalescent visits. Symptomatic CHIKV infections were identified by positive CHIKV PCR in acute blood samples and/or CHIKV IgM/IgG ELISA seroconversion in paired acute/convalescent samples. Enrollment and 12-month blood samples underwent plaque reduction neutralization test (PRNT) using CHIKV attenuated strain 181/clone25. Subclinical CHIKV infections were identified by ≥8-fold rise from a baseline enrollment PRNT titer <10 without symptomatic infection detected during the intervening surveillance period. Selected CHIKV PCR-positive samples underwent viral isolation and envelope protein-1 gene sequencing. Of 853 subjects who completed all study procedures at 12 months, 19 symptomatic infections (2.19 per 100 person-years) and 87 subclinical infections (10.03 per 100 person-years) occurred. The ratio of subclinical-to-symptomatic infections was 4.6:1 varying with age from 2:1 in 6 month-5 year olds to 12:1 in those >50 years old. Baseline CHIKV PRNT titer ≥10 was associated with 100% (95%CI: 46.1, 100.0) protection from symptomatic CHIKV infection. Phylogenetic analysis demonstrated Asian genotype closely related to strains from Asia and the Caribbean.

Conclusions

Subclinical infections accounted for a majority of total CHIKV infections. A positive baseline CHIKV PRNT titer was associated with protection from symptomatic CHIKV infection. These findings have implications for assessing disease burden, understanding virus transmission, and supporting vaccine development.     

<Emphasis Type="Italic">Helicobacter pylori</Emphasis> genome variability in a framework of familial transmission

Background  

Helicobacter pylori infection is exceptionally prevalent and is considered to be acquired primarily early in life through person-to-person transmission within the family. H. pylori is a genetically diverse bacterial species, which may facilitate adaptation to new hosts and persistence for decades. The present study aimed to explore the genetic diversity of clonal isolates from a mother and her three children in order to shed light on H. pylori transmission and host adaptation.     

Disease modifying treatment of spinal cord injury with directly reprogrammed neural precursor cells in non-human primates

BACKGROUNDThe development of regenerative therapy for human spinal cord injury (SCI) is dramatically restricted by two main challenges: the need for a safe source of functionally active and reproducible neural stem cells and the need of adequate animal models for preclinical testing. Direct reprogramming of somatic cells into neuronal and glial precursors might be a promising solution to the first challenge. The use of non-human primates for preclinical studies exploring new treatment paradigms in SCI results in data with more translational relevance to human SCI.AIMTo investigate the safety and efficacy of intraspinal transplantation of directly reprogrammed neural precursor cells (drNPCs).METHODSSeven non-human primates with verified complete thoracic SCI were divided into two groups: drNPC group (n = 4) was subjected to intraspinal transplantation of 5 million drNPCs rostral and caudal to the lesion site 2 wk post injury, and lesion control (n = 3) was injected identically with the equivalent volume of vehicle.RESULTSFollow-up for 12 wk revealed that animals in the drNPC group demonstrated a significant recovery of the paralyzed hindlimb as well as recovery of somatosensory evoked potential and motor evoked potential of injured pathways. Magnetic resonance diffusion tensor imaging data confirmed the intraspinal transplantation of drNPCs did not adversely affect the morphology of the central nervous system or cerebrospinal fluid circulation. Subsequent immunohistochemical analysis showed that drNPCs maintained SOX2 expression characteristic of multipotency in the transplanted spinal cord for at least 12 wk, migrating to areas of axon growth cones.CONCLUSIONOur data demonstrated that drNPC transplantation was safe and contributed to improvement of spinal cord function after acute SCI, based on neurological status assessment and neurophysiological recovery within 12 wk after transplantation. The functional improvement described was not associated with neuronal differentiation of the allogeneic drNPCs. Instead, directed drNPCs migration to the areas of active growth cone formation may provide exosome and paracrine trophic support, thereby further supporting the regeneration processes.     

Identification of Extracellular Domain Residues Required for Epithelial Na+ Channel Activation by Acidic pH

A growing body of evidence suggests that the extracellular domain of the epithelial Na⁺ channel (ENaC) functions as a sensor that fine tunes channel activity in response to changes in the extracellular environment. We previously found that acidic pH increases the activity of human ENaC, which results from a decrease in Na⁺ self-inhibition. In the current work, we identified extracellular domain residues responsible for this regulation. We found that rat ENaC is less sensitive to pH than human ENaC, an effect mediated in part by the γ subunit. We identified a group of seven residues in the extracellular domain of γENaC (Asp-164, Gln-165, Asp-166, Glu-292, Asp-335, His-439, and Glu-455) that, when individually mutated to Ala, decreased proton activation of ENaC. γ_E455 is conserved in βENaC (Glu-446); mutation of this residue to neutral amino acids (Ala, Cys) reduced ENaC stimulation by acidic pH, whereas reintroduction of a negative charge (by MTSES modification of Cys) restored pH regulation. Combination of the seven γENaC mutations with β_E446A generated a channel that was not activated by acidic pH, but inhibition by alkaline pH was intact. Moreover, these mutations reduced the effect of pH on Na⁺ self-inhibition. Together, the data identify eight extracellular domain residues in human β- and γENaC that are required for regulation by acidic pH.     

Suppression of TRPC3 leads to disappearance of store-operated channels and formation of a new type of store-independent channels in A431 cells

In most non-excitable cells, calcium (Ca(2+)) release from the inositol 1,4,5-trisphosphate (InsP(3))-sensitive intracellular Ca(2+) stores is coupled to Ca(2+) influx through the plasma membrane Ca(2+) channels whose molecular composition is poorly understood. Several members of mammalian TRP-related protein family have been implicated to both receptor- and store-operated Ca(2+) influx. Here we investigated the role of the native transient receptor potential 3 (TRPC3) homologue in mediating the store- and receptor-operated calcium entry in A431 cells. We show that suppression of TRPC3 protein levels by small interfering RNA (siRNA) leads to a significant reduction in store-operated calcium influx without affecting the receptor-operated calcium influx. With single-channel analysis, we further demonstrate that reduction of TRPC3 levels results in suppression of specific subtype of store-operated calcium channels and activation of store-independent channels. Our data suggest that TRPC3 is required for the formation of functional store-operated channels in A431 cells.