Antispacer peptide nucleic acids for sequence-specific CRISPR-Cas9 modulation

Despite the rapid and broad implementation of CRISPR-Cas9-based technologies, convenient tools to modulate dose, timing, and precision remain limited. Building on methods using synthetic peptide nucleic acids (PNAs) to bind RNA with unusually high affinity, we describe guide RNA (gRNA) spacer-targeted, or ‘antispacer’, PNAs as a tool to modulate Cas9 binding and activity in cells in a sequence-specific manner. We demonstrate that PNAs rapidly and efficiently target complexed gRNA spacer sequences at low doses and without design restriction for sequence-selective Cas9 inhibition. We further show that short PAM-proximal antispacer PNAs achieve potent cleavage inhibition (over 2000-fold reduction) and that PAM-distal PNAs modify gRNA affinity to promote on-target specificity. Finally, we apply antispacer PNAs for temporal regulation of two dCas9-fusion systems. These results present a novel rational approach to nucleoprotein engineering and describe a rapidly implementable antisense platform for CRISPR-Cas9 modulation to improve spatiotemporal versatility and safety across applications.     

An integrative taxonomic revision of the Cape Verdean skinks (Squamata,Scincidae)

Miralles, A., Vasconcelos, R., Perera, A., Harris, D. J. & Carranza, S. (2010). An integrative taxonomic revision of the Cape Verdean skinks (Squamata, Scincidae). —Zoologica Scripta, 40, 16–44. A comprehensive taxonomic revision of the Cape Verdean skinks is proposed based on an integrative approach combining (i) a phylogenetic study pooling all the previously published molecular data, (ii) new population genetic analyses using mitochondrial and nuclear data resulting from additional sampling, together with (iii) a morphological study based on an extensive examination of the scalation and colour patterns of 516 live and museum specimens, including most of the types. All Cape Verdean species of skinks presently recognised, formerly regarded as members of the genera Mabuya Fitzinger, 1826 and Macroscincus Bocage, 1873 are considered as members of the Cape Verdean endemic genus Chioninia Gray, 1845. The new phylogeny and networks obtained are congruent with the previously published phylogenetic studies, although suggesting older colonization events (between 11.6 and 0.8 Myr old), and indicate the need for taxonomic changes. Intraspecific diversity has been analysed and points to a very recent expansion of Chioninia delalandii on the southern islands and its introduction on Maio, to a close connection between Chioninia stangeri island populations due to Pleistocene sea‐level falls and to a generally low haplotypic diversity due to the ecological and geological characteristics of the archipelago. Three new consistent morphological synapomorphies supporting two of the four main clades of the genus have been identified. The complex taxonomic status of Euprepes fogoensis O’Shaughnessy, 1874 has been resolved and a lectotype has been designated for this species; Chioninia fogoensis nicolauensis (Schleich, 1987) is elevated to species rank, whereas Chioninia fogoensis antaoensis (Schleich, 1987) is now regarded as a junior subjective synonym of C. fogoensis. Additionally, one new subspecies of Chioninia vaillanti and two of Chioninia spinalis are described (Chioninia vaillanti xanthotis ssp. n., Chioninia spinalis santiagoensis ssp. n. and Chioninia spinalis boavistensis ssp. n.) and a lectotype has been designated for Mabuia spinalis Boulenger, 1906. Finally, an identification key for the Chioninia species is presented.     

Computational study of the putative active form of protein Z (PZa): sequence design and structural modeling

Although protein Z (PZ) has a domain arrangement similar to the essential coagulation proteins FVII, FIX, FX, and protein C, its serine protease (SP)-like domain is incomplete and does not exhibit proteolytic activity. We have generated a trial sequence of putative activated protein Z (PZa) by identifying amino acid mutations in the SP-like domain that might reasonably resurrect the serine protease catalytic activity of PZ. The structure of the activated form was then modeled based on the proposed sequence using homology modeling and solvent-equilibrated molecular dynamics simulations. In silico docking of inhibitors of FVIIa and FXa to the putative active site of equilibrated PZa, along with structural comparison with its homologous proteins, suggest that the designed PZa can possibly act as a serine protease.     

In vitro and in vivo characterization of an interleukin‐15 antagonist peptide by metabolic stability, 99mTc‐labeling,and biological activity assays

Interleukin (IL)–15 is an inflammatory cytokine that constitutes a validated therapeutic target in some immunopathologies, including rheumatoid arthritis (RA). Previously, we identified an IL‐15 antagonist peptide named [K6T]P8, with potential therapeutic application in RA. In the current work, the metabolic stability of this peptide in synovial fluids from RA patients was studied. Moreover, [K6T]P8 peptide was labeled with ^99mTc to investigate its stability in human plasma and its biodistribution pattern in healthy rats. The biological activity of [K6T]P8 peptide and its dimer was evaluated in CTLL‐2 cells, using 3 different additives to improve the solubility of these peptides. The half‐life of [K6T]P8 in human synovial fluid was 5.88 ± 1.73 minutes, and the major chemical modifications included peptide dimerization, cysteinylation, and methionine oxidation. Radiolabeling of [K6T]P8 with ^99mTc showed a yield of approximately 99.8%. The ^99mTc‐labeled peptide was stable in a 30‐fold molar excess of cysteine and in human plasma, displaying a low affinity to plasma proteins. Preliminary biodistribution studies in healthy Wistar rats suggested a slow elimination of the peptide through the renal and hepatic pathways. Although citric acid, sucrose, and Tween 80 enhanced the solubility of [K6T]P8 peptide and its dimer, only the sucrose did not interfere with the in vitro proliferation assay used to assess their biological activity. The results here presented, reinforce nonclinical characterization of the [K6T]P8 peptide, a potential agent for the treatment of RA and other diseases associated with IL‐15 overexpression.     

The potential therapeutic use of stem cells in cartilage repair

As our population demographics change, osteoarthritis and cartilage defects are becoming more prevalent. The discovery of stems cells and their ability for indefinite regeneration has revolutionised the way cartilage problems are viewed. Tissue engineering has been shown to be the ideal way of repairing articular cartilage lesions, i.e. back to native tissue. Cartilage is an ideal tissue engineering target as it is avascular, aneural and alymphatic. The two main types of stem cells being investigated in chondrogenesis are embryological and mesenchymal stem cells. Research into embryological stem cells has been surrounded by controversy because of ethical, religious and social concerns. We discuss the use of embryological and mesenchymal stem cells in cartilage repair and the various factors involved in the differentiation into chondrocytes. We also discuss commonly used mesenchymal stem cell markers and their limitations.     

Peripheral genetic structure of Helicoverpa zea indicates asymmetrical panmixia

Seasonal climatic shifts create peripheral habitats that alternate between habitable and uninhabitable for migratory species. Such dynamic peripheral habitats are potential sites where migratory species could evolve high genetic diversity resulting from convergence of immigrants from multiple regionally distant areas. Migrant populations of Helicoverpa zea (Boddie) captured during two different seasons were assessed for genetic structure using microsatellite markers and for host plant type using stable carbon isotope analysis. Individuals (N = 568) were genotyped and divided into 13 putative populations based on collection site and time. Fixation indices (F‐statistics), analysis of molecular variance (AMOVA), and discriminant analysis of principal components (DAPC) were used to examine within and among population genetic variation. Mean number of alleles per locus was 10.25 (± 3.2 SD), and allelic richness ranged from 2.38 to 5.13 (± 3.2 SD). The observed and expected heterozygosity ranged from 0.07 to 0.48 and 0.08 to 0.62, respectively. Low F_ST (0.01 to 0.02) and high F_IS (0.08 to 0.33) values suggest captured migrants originated from breeding populations with different allele frequencies. We postulate that high genetic diversity within migrant populations and low genetic differentiation among migrant populations of H. zea are the result of asymmetrical immigration due to the high dispersal and reproductive behavior of H. zea, which may hinder the adaptation and establishment of H. zea to peripheral habitat. These findings highlight the importance of assessing peripheral population structure in relation to ecological and evolutionary dynamics of this and other highly reproductive and dispersive species.     

The evidence base for interventions delivered to children in primary care: an overview of cochrane systematic reviews

Background

As a first step in developing a framework to evaluate and improve the quality of care of children in primary care there is a need to identify the evidence base underpinning interventions relevant to child health. Our objective was to identify all Cochrane systematic reviews relevant to the management of childhood conditions in primary care and to assess the extent to which Cochrane reviews reflect the burden of childhood illness presenting in primary care.

Methodology/Principal Findings

We used the Cochrane Child Health Field register of child-relevant systematic reviews to complete an overview of Cochrane reviews related to the management of children in primary care. We compared the proportion of systematic reviews with the proportion of consultations in Australia, US, Dutch and UK general practice in children. We identified 396 relevant systematic reviews; 358 included primary studies on children while 251 undertook a meta-analysis. Most reviews (n = 218, 55%) focused on chronic conditions and over half (n = 216, 57%) evaluated drug interventions. Since 2000, the percentage of pediatric primary care relevant reviews only increased by 2% (7% to 9%) compared to 18% (10% to 28%) in all child relevant reviews. Almost a quarter of reviews (n = 78, 23%) were published on asthma treatments which only account for 3–5% of consultations. Conversely, 15–23% of consultations are due to skin conditions yet they represent only 7% (n = 23) of reviews.

Conclusions/Significance

Although Cochrane systematic reviews focus on clinical trials and do not provide a comprehensive picture of the evidence base underpinning the management of children in primary care, the mismatch between the focus of the published research and the focus of clinical activity is striking. Clinical trials are an important component of the evidence base and the lack of trial evidence to demonstrate intervention effectiveness in substantial areas of primary care for children should be addressed.     

Early inflorescence and floral development in Cocos nucifera L. (Arecaceae: Arecoideae)

Palms are generally characterized by a large structure with a massive crown that creates difficulties in anatomical studies. The flowering behaviour of palm species may be a useful indicator of phylogenetic relationships and therefore evolutionary events. This paper presents a detailed histological study of reproductive development in coconut (Cocos nucifera L.), from initiation up to maturation of staminate and pistillate flowers. Reproductive development in coconut consists of a sequence of individual events that span more than two years. Floral morphogenesis is the longest event, taking about one year, while sex determination is a rapid process that occurs within one month. The inflorescence consists of different ultimate floral structural components. Pistillate flowers are borne in floral triads that are flanked by two functional staminate flowers. The staminate flowers are born in floral diads towards the base of the rachilla followed by solitary flowers in the middle to top of the rachilla. Three primary phases were identified in reproductive development, namely, transition of axillary bud into inflorescence bud, formation of floral buds, and sexualisation of individual flower buds. All developmental events with respect to stage or time of occurrence were determined.     

Admixture in Mexico City: implications for admixture mapping of Type 2 diabetes genetic risk factors

Admixture mapping is a recently developed method for identifying genetic risk factors involved in complex traits or diseases showing prevalence differences between major continental groups. Type 2 diabetes (T2D) is at least twice as prevalent in Native American populations as in populations of European ancestry, so admixture mapping is well suited to study the genetic basis of this complex disease. We have characterized the admixture proportions in a sample of 286 unrelated T2D patients and 275 controls from Mexico City and we discuss the implications of the results for admixture mapping studies. Admixture proportions were estimated using 69 autosomal ancestry-informative markers (AIMs). Maternal and paternal contributions were estimated from geographically informative mtDNA and Y-specific polymorphisms. The average proportions of Native American, European and, West African admixture were estimated as 65, 30, and 5%, respectively. The contributions of Native American ancestors to maternal and paternal lineages were estimated as 90 and 40%, respectively. In a logistic model with higher educational status as dependent variable, the odds ratio for higher educational status associated with an increase from 0 to 1 in European admixture proportions was 9.4 (95%, credible interval 3.8–22.6). This association of socioeconomic status with individual admixture proportion shows that genetic stratification in this population is paralleled, and possibly maintained, by socioeconomic stratification. The effective number of generations back to unadmixed ancestors was 6.7 (95% CI 5.7–8.0), from which we can estimate that genome-wide admixture mapping will require typing about 1,400 evenly distributed AIMs to localize genes underlying disease risk between populations of European and Native American ancestry. Sample sizes of about 2,000 cases will be required to detect any locus that contributes an ancestry risk ratio of at least 1.5.