Translational enhancement by base editing of the Kozak sequence rescues haploinsufficiency

A variety of single-gene human diseases are caused by haploinsufficiency, a genetic condition by which mutational inactivation of one allele leads to reduced protein levels and functional impairment. Translational enhancement of the spare allele could exert a therapeutic effect. Here we developed BOOST, a novel gene-editing approach to rescue haploinsufficiency loci by the change of specific single nucleotides in the Kozak sequence, which controls translation by regulating start codon recognition. We evaluated for translational strength 230 Kozak sequences of annotated human haploinsufficient genes and 4621 derived variants, which can be installed by base editing, by a high-throughput reporter assay. Of these variants, 149 increased the translation of 47 Kozak sequences, demonstrating that a substantial proportion of haploinsufficient genes are controlled by suboptimal Kozak sequences. Validation of 18 variants for 8 genes produced an average enhancement in an expression window compatible with the rescue of the genetic imbalance. Base editing of the NCF1 gene, whose monoallelic loss causes chronic granulomatous disease, resulted in the desired increase of NCF1 (p47^phox) protein levels in a relevant cell model. We propose BOOST as a fine-tuned approach to modulate translation, applicable to the correction of dozens of haploinsufficient monogenic disorders independently of the causing mutation.     

Reproductive performance of Eurasian Kestrel Falco tinnunculus in an agricultural landscape with a mosaic of land uses

There are serious concerns about the environmental and ecological degradation caused by modern agriculture and its impact on animal populations. There is therefore a need to assess the reproductive performance of free‐living animals in agricultural landscapes. We undertook a 4‐year study on the reproductive biology of Eurasian Kestrel Falco tinnunculus in the Parma district of Italy. Kestrels breeding in areas of intensive agriculture delayed egg‐laying and their offspring were in poorer condition compared with those breeding in areas of temporary or permanent grasslands. However, egg volume and fledging success were similar among sites. The availability of temporary and permanent grasslands may limit the negative impacts of intensive agriculture on the breeding activity of Kestrels.     

Ecology of Staphylococcus aureus: characterization of strains from chicken

The majority of S. aureus strains isolated from beak-swabs and pathological processes in chicken shows coagulation of human plasma (not of bovine plasma), crystal violet-type A, hemolysine-type A, formation of fibrinolysin, not formation of DNase and reactions with the experimental phage A1591. Because of the absence of DNase-formation and the reaction-specificity for phage A1591 we propose to designate these strains as host-specific variety gallinae of S. aureus. The strains from chicken are compared with strains of human, bovine, and ovine origin. An ecological study in a chicken farm has shown that S. aureus strains from chicken are not found in man and vice versa.     

Postnatal Environmental Tobacco Smoke Exposure Related to Behavioral Problems in Children

ObjectiveThe purpose of this study was to examine the association between pre and post environmental tobacco smoke (ETS) exposure and behavioral problems in schoolchildren.MethodsIn the cross-sectional 6 cities Study conducted in France, 5221 primary school children were investigated. Pre- and postnatal exposure to secondhand tobacco smoke at home was assessed using a parent questionnaire. Child’s behavioral outcomes (emotional symptoms and conduct problems) were evaluated by the Strengths and Difficulties Questionnaire (SDQ) completed by the parents.ResultsETS exposure during the postnatal period and during both pre- and postnatal periods was associated with behavioral problems in children. Abnormal emotional symptoms (internalizing problems) were related to ETS exposure in children who were exposed during the pre- and postnatal periods with an OR of 1.72 (95% Confidence Interval (CI)= 1.36-2.17), whereas the OR was estimated to be 1.38 (95% CI= 1.12-1.69) in the case of postnatal exposure only. Abnormal conduct problems (externalizing problems) were related to ETS exposure in children who were exposed during the pre- and postnatal periods with an OR of 1.94 (95% CI= 1.51-2.50), whereas the OR was estimated to be 1.47 (95% CI=1.17-1.84) in the case of postnatal exposure only. Effect estimates were adjusted for gender, study center, ethnic origin, child age, low parental education, current physician diagnosed asthma, siblings, preterm birth and single parenthood.ConclusionPostnatal ETS exposure, alone or in association with prenatal exposure, increases the risk of behavioral problems in school-age children.     

Pathway-Focused PCR Array Profiling of Enriched Populations of Laser Capture Microdissected Hippocampal Cells after Traumatic Brain Injury

Cognitive deficits in survivors of traumatic brain injury (TBI) are associated with irreversible neurodegeneration in brain regions such as the hippocampus. Comparative gene expression analysis of dying and surviving neurons could provide insight into potential therapeutic targets. We used two pathway-specific PCR arrays (RT2 Profiler Apoptosis and Neurotrophins & Receptors PCR arrays) to identify and validate TBI-induced gene expression in dying (Fluoro-Jade-positive) or surviving (Fluoro-Jade- negative) pyramidal neurons obtained by laser capture microdissection (LCM). In the Apoptosis PCR array, dying neurons showed significant increases in expression of genes associated with cell death, inflammation, and endoplasmic reticulum (ER) stress compared with adjacent, surviving neurons. Pro-survival genes with pleiotropic functions were also significantly increased in dying neurons compared to surviving neurons, suggesting that even irreversibly injured neurons are able to mount a protective response. In the Neurotrophins & Receptors PCR array, which consists of genes that are normally expected to be expressed in both groups of hippocampal neurons, only a few genes were expressed at significantly different levels between dying and surviving neurons. Immunohistochemical analysis of selected, differentially expressed proteins supported the gene expression data. This is the first demonstration of pathway-focused PCR array profiling of identified populations of dying and surviving neurons in the brain after TBI. Combining precise laser microdissection of identifiable cells with pathway-focused PCR array analysis is a practical, low-cost alternative to microarrays that provided insight into neuroprotective signals that could be therapeutically targeted to ameliorate TBI-induced neurodegeneration.     

Anaphylaxis Imaging: Non-Invasive Measurement of Surface Body Temperature and Physical Activity in Small Animals

In highly sensitized patients, the encounter with a specific allergen from food, insect stings or medications may rapidly induce systemic anaphylaxis with potentially lethal symptoms. Countless animal models of anaphylaxis, most often in BALB/c mice, were established to understand the pathophysiology and to prove the safety of different treatments. The most common symptoms during anaphylactic shock are drop of body temperature and reduced physical activity. To refine, improve and objectify the currently applied manual monitoring methods, we developed an imaging method for the automated, non-invasive measurement of the whole-body surface temperature and, at the same time, of the horizontal and vertical movement activity of small animals. We tested the anaphylaxis imaging in three in vivo allergy mouse models for i) milk allergy, ii) peanut allergy and iii) egg allergy. These proof-of-principle experiments suggest that the imaging technology represents a reliable non-invasive method for the objective monitoring of small animals during anaphylaxis over time. We propose that the method will be useful for monitoring diseases associated with both, changes in body temperature and in physical behaviour.     

The anatomy of Neotenophycus ichthyosteus gen. et sp. nov. (Rhodomelaceae,Ceramiales), a bizarre red algal parasite from the central Pacific

A minute parasite of Neosiphonia poko (Hollenberg) Abbott from a shallow lagoon on the central-Pacific Johnston Atoll is described as Neotenophycus ichthyosteus Kraft et Abbott, gen. et sp. nov. The infective parasite cell first connects to a central-axial cell of the host, then emerges from between host pericentral cells at a node before dividing into a three- or four-celled primary axis. Epibasal cells of the parasite divide to form three pericentral cells whose derivatives produce a globular head on the basal cell and on which reproductive structures differentiate almost immediately. Trichoblasts on any life-history stage are completely lacking. Spermatangia are borne on mother cells across the whole thallus surface. Procarps consist of four pericentral cells that encircle a subapical fertile-axial cell in an ampullar configuration, one of the pericentral cells serving as the supporting cell and bearing a four-celled carpogonial branch and a single sterile cell. Diploidization results in a longitudinal/concave division of the auxiliary cell and formation of an arching linear series of inner gonimoblast cells, each dividing toward the thallus surface into gonimoblast filaments of very narrow, horizontally aligned cells terminated by initially monopodial, later by sympodial, carposporangia, the whole of the mature female gametophyte consisting of an amalgam of several cystocarps within a lax jacket of sterile gametophytic tissue. Tetrasporophytes are composed of lobes of pericentral-cell-derived filaments, each axial cell of which is ringed by three pericentral cells producing tetrahedral tetrasporangia enclosed by two pre-sporangial cover cells. Affinities of the new genus are discussed and comparison is made particularly to the enigmatic parasite Episporium centroceratis Möbius. It is concluded that relationships with any previously described tribe are so remote or obscure that the new tribe Neotenophyceae should be proposed for it.