A mouse model of Proteus syndrome.

Proteus syndrome is a mosaic, progressive overgrowth disorder caused by a somatic activating variant c.49G > A p.(E17K) in AKT1. The presentation in affected individuals is variable, with a diversity of tissues demonstrating abnormalities. Common manifestations include skin and bony overgrowth, vascular malformations (VMs), cysts and benign tumors. We used two methods to create mouse models that had endogenously-regulated mosaic expression of the Proteus syndrome variant. Variant allele fractions (VAFs) ranged from 0% to 50% across numerous tissues in 44 Proteus syndrome mice. Mice were phenotypically heterogeneous with lesions rarely observed before 12 months of age. VMs were the most frequent finding with a total of 69 found in 29 of 44 Proteus syndrome mice. Twenty-eight cysts and ectasia, frequently biliary, were seen in 22 of 44 Proteus syndrome mice. Varying levels of mammary hyperplasia were seen in 10 of 16 female Proteus syndrome mice with other localized regions of hyperplasia and stromal expansion noted in several additional animals. Interestingly, 27 of 31 Proteus syndrome animals had non-zero blood VAF that is in contrast to the human disorder where it is rarely seen in peripheral blood. Identification of variant-positive cells by green fluorescent protein (GFP) staining in chimeric Proteus syndrome mice showed that in some lesions, hyperplastic cells were predominantly GFP/Akt1E17K-positive and showed increased pAKT signal compared to GFP-negative cells. However, hyperplastic mammary epithelium was a mixture of GFP/Akt1E17K-positive and negative cells with some GFP/Akt1E17K-negative cells also having increased pAKT signal suggesting that the variant-positive cells can induce lesion formation in a non-cell autonomous manner.

Bi-allelic TMEM94 Truncating Variants Are Associated with Neurodevelopmental Delay, Congenital Heart Defects, and Distinct Facial Dysmorphism.

Neurodevelopmental disorders (NDD) are genetically and phenotypically heterogeneous conditions due to defects in genes involved in development and function of the nervous system. Individuals with NDD, in addition to their primary neurodevelopmental phenotype, may also have accompanying syndromic features that can be very helpful diagnostically especially those with recognizable facial appearance. In this study, we describe ten similarly affected individuals from six unrelated families of different ethnic origins having bi-allelic truncating variants in TMEM94, which encodes for an uncharacterized transmembrane nuclear protein that is highly conserved across mammals. The affected individuals manifested with global developmental delay/intellectual disability, and dysmorphic facial features including triangular face, deep set eyes, broad nasal root and tip and anteverted nostrils, thick arched eye brows, hypertrichosis, pointed chin, and hypertelorism. Birthweight in the upper normal range was observed in most, and all but one had congenital heart defects (CHD). Gene expression analysis in available cells from affected individuals showed reduced expression of TMEM94. Global transcriptome profiling using microarray and RNA sequencing revealed several dysregulated genes essential for cell growth, proliferation and survival that are predicted to have an impact on cardiotoxicity hematological system and neurodevelopment. Loss of Tmem94 in mouse model generated by CRISPR/Cas9 was embryonic lethal and led to craniofacial and cardiac abnormalities and abnormal neuronal migration pattern, suggesting that this gene is important in craniofacial, cardiovascular, and nervous system development. Our study suggests the genetic etiology of a recognizable dysmorphic syndrome with NDD and CHD and highlights the role of TMEM94 in early development.

The Absence of Endothelial Sodium Channel α (αENaC) Reduces Renal Ischemia/Reperfusion Injury.

The epithelial sodium channel (ENaC) has a key role in modulating endothelial cell stiffness and this in turn regulates nitric oxide (NO) synthesis. The physiological relevance of endothelial ENaC in pathological conditions where reduced NO bioavailability plays an essential role remains largely unexplored. Renal ischemia/reperfusion (IR) injury is characterized by vasoconstriction and sustained decrease in renal perfusion that is partially explained by a reduction in NO bioavailability. Therefore, we aimed to explore if an endothelial ENaC deficiency has an impact on the severity of renal injury induced by IR. Male mice with a specific endothelial sodium channel α (αENaC) subunit gene inactivation in the endothelium (endo-αENaCKO) and control littermates were subjected to bilateral renal ischemia of 22 min and were studied after 24 h of reperfusion. In control littermates, renal ischemia induced an increase in plasma creatinine and urea, augmented the kidney injury molecule-1 (Kim-1) and neutrophil gelatinase associated lipocalin-2 (NGAL) mRNA levels, and produced severe tubular injury. The absence of endothelial αENaC expression prevented renal tubular injury and renal dysfunction. Moreover, endo-αENaCKO mice recovered faster from renal hypoxia after the ischemia episode as compared to littermates. In human endothelial cells, pharmacological ENaC inhibition promoted endothelial nitric oxide synthase (eNOS) coupling and activation. Altogether, these data suggest an important role for endothelial αENaC in kidney IR injury through improving eNOS activation and kidney perfusion, thus, preventing ischemic injury.

The C-terminus of CBFß-SMMHC is required to induce embryonic hematopoietic defects and leukemogenesis.

The C-terminus of CBFß-SMMHC, the fusion protein produced by a chromosome 16 inversion in acute myeloid leukemia subtype M4Eo, contains domains for self-multimerization and transcriptional repression, both of which have been proposed to be important for leukemogenesis by CBFß-SMMHC. To test the role of the fusion protein's C-terminus in vivo, we generated knock-in mice expressing a C-terminally truncated CBFß-SMMHC (CBFß-SMMHCΔC95). Embryos with a single copy of CBFß-SMMHCΔC95 were viable and showed no defects in hematopoiesis, whereas embryos homozygous for the CBFß-SMMHCΔC95 allele had hematopoietic defects and died in mid-gestation, similar to embryos with a single-copy of the full-length CBFß-SMMHC. Importantly, unlike mice expressing full-length CBFß-SMMHC, none of the mice expressing CBFß-SMMHCΔC95 developed leukemia, even after treatment with a mutagen, although some of the older mice developed a nontransplantable myeloproliferative disease. Our data indicate that the CBFß-SMMHC's C-terminus is essential to induce embryonic hematopoietic defects and leukemogenesis.

Funciones ejecutivas de jóvenes que han infringido la ley: una revisión sistemática / Executive functions of juvenile offenders: a systematic review / Funções executivas dos delinquentes juvenis: uma revisão sistemática

Se propone una revisión sistemática que indaga en profundidad sobre el desempeño de jóvenes que han infringido la ley en mediciones de funciones ejecutivas. Una vez revisadas más de 1500 fuentes bibliográficas, se extrajeron 64 publicaciones que cumplieron los criterios de inclusión establecidos. Se evaluó que la enorme mayoría de las investigaciones revisadas constatan déficits específicos en funcionamiento ejecutivo entre jóvenes que han infringido la ley, elemento fundamental en la comprensión del origen del comportamiento delictual y la reincidencia, así como antecedente importante sobre la capacidad de respuesta de los atendidos, en el momento de impulsar acciones interventivas en el marco de programas de medida y sanción en justicia juvenil. Junto con lo anterior, se vincularán los resultados de la síntesis cualitativa con elementos teóricos, al igual que con un abordaje práctico sobre las herramientas de medición que podrían utilizarse en países de habla hispana. Finalmente, los resultados dan cuenta de la urgencia de implementar prácticas más informadas sobre neurodiversidad, así como la necesidad de disponer de planteles profesionales efectivamente interdisciplinarios en los sistemas de justicia juvenil.

A systematic review is proposed that investigates in depth the performance of young people who have broken the law in executive function measurements. After reviewing more than 1500 bibliographic sources, 64 publications that met the established inclusion criteria were extracted. It was evaluated that the vast majority of the research reviewed showed specific deficits in executive functioning among young people who have broken the law, a fundamental element in the understanding of the origin of delinquent behavior and recidivism, as well as an important antecedent on the response capacity of those served, at the time of promoting intervention actions in the framework of juvenile justice programs of measures and sanctions. Together with the above, the results of the qualitative synthesis will be linked to theoretical elements, as well as to a practical approach to measurement tools that could be used in Spanish-speaking countries. Finally, the results point to the urgency of implementing more informed practices on neurodiversity, as well as the need for effective interdisciplinary professional staffs in juvenile justice systems.

É proposta uma revisão sistemática que investiga em profundidade o desempenho dos jovens infratores em medidas de funções executivas. Após a revisão de mais de 1500 fontes de literatura, foram extraídas 64 publicações que preenchiam os critérios de inclusão estabelecidos. Foi avaliado que a grande maioria das pesquisas analisadas encontrou déficits específicos no funcionamento executivo entre os jovens que infringiram a lei, um elemento fundamental para entender a origem do comportamento delinqüente e da reincidência, assim como um antecedente importante sobre a capacidade de resposta dos que estão sob cuidados, no momento de promover ações de intervenção no âmbito de programas de medidas e sanções na justiça juvenil. Juntamente com o acima exposto, os resultados da síntese qualitativa estarão ligados a elementos teóricos, bem como a uma abordagem prática das ferramentas de medição que poderiam ser utilizadas nos países de língua espanhola. Finalmente, os resultados apontam para a urgência de implementar mais práticas informadas sobre a neurodiversidade, bem como a necessidade de uma equipe profissional interdisciplinar eficaz nos sistemas de justiça juvenil.

Highly Efficient Cpf1-Mediated Gene Targeting in Mice Following High Concentration Pronuclear Injection.

Cpf1 has emerged as an alternative to the Cas9 RNA-guided nuclease. Here we show that gene targeting rates in mice using Cpf1 can meet, or even surpass, Cas9 targeting rates (approaching 100% targeting), but require higher concentrations of mRNA and guide. We also demonstrate that coinjecting two guides with close targeting sites can result in synergistic genomic cutting, even if one of the guides has minimal cutting activity.

Loci associated with skin pigmentation identified in African populations.

Despite the wide range of skin pigmentation in humans, little is known about its genetic basis in global populations. Examining ethnically diverse African genomes, we identify variants in or near SLC24A5, MFSD12, DDB1, TMEM138, OCA2, and HERC2 that are significantly associated with skin pigmentation. Genetic evidence indicates that the light pigmentation variant at SLC24A5 was introduced into East Africa by gene flow from non-Africans. At all other loci, variants associated with dark pigmentation in Africans are identical by descent in South Asian and Australo-Melanesian populations. Functional analyses indicate that MFSD12 encodes a lysosomal protein that affects melanogenesis in zebrafish and mice, and that mutations in melanocyte-specific regulatory regions near DDB1/TMEM138 correlate with expression of ultraviolet response genes under selection in Eurasians.

Fertilization efficiency of in vitro matured oocytes transferred to oviducts of inseminated goats: a model to assess in vivo fertilization performance of goat spermatozoa.

An alternative to conventional in vivo validation of sperm assays might be to assess the fertilization rate of multiple oocytes transferred to the oviducts of inseminated females. Increasing the number of oocytes increases the egg-sperm ratio in the oviduct under an unaltered endocrine milieu, setting the basis for picking up statistical differences between treatments in small populations. The study evaluated the model by transferring oocytes to females inseminated under conditions that are known to modify the fertilization rate in the field. The study then evaluated the use of cattle oocytes to replace goat oocytes for assessing sperm function under this model. In Experiment 1, 12 females were inseminated at estrus with either 100 or 300 million spermatozoa 20 h before transferring homologous oocytes into the oviduct ipsilateral to the ovulation point. In Experiment 2, 10 females were inseminated either once or twice; 10-20 h later, homologous oocytes were transferred into the oviduct ipsilateral to the ovulation point. In Experiment 3, 13 bilateral-ovulated females were inseminated and 20 h later goat and cattle oocytes were transferred to contralateral oviducts. Then, 16-20 h later, oocytes were flushed from the oviduct, cleaned of spermatozoa and stained to assess the fertilization rate. The fertilization rate was improved by increasing sperm numbers at insemination (P < 0.04) and by increasing the number of inseminations (P < 0.02). The results in Experiment 3 showed that fertilization rates were similar for goat and cattle oocyte (P > 0.05) and that fertilization values were highly correlated (r = 0.811, P < 0.001). Results suggest that the model can be used for in vivo validation of in vitro sperm assays by facilitating the expression of statistical differences in small number of animals. In addition, cattle oocytes can be used to replace goat oocytes to study in vivo sperm function in goats.

Differences in sperm migration through cervical mucus in vitro relates to sperm colonization of the oviduct and fertilizing ability in goats.

Sperm migration in estrous cervical mucus can be used to measure the ability of spermatozoa to migrate through the genital tract. The relationship of this test with the sperm colonization of the isthmus, and its impact on fertility has not been evaluated in goats. Our objectives were to determine the differences among spermatozoa of different bucks in their ability to penetrate homologous cervical mucus in vitro and to determine the relationship between sperm displacement through cervical mucus and the ability of spermatozoa to colonize the oviduct and penetrate IVM oocytes, in vivo. Sperm migration in cervical mucus was assessed in flat capillary tubes with a phase contrast microscope. In the first experiment, fresh semen was used to establish differences between males in the ability of their spermatozoa to migrate in cervical mucus. In the second experiment, goats in estrus were inseminated with fresh spermatozoa from males with significant differences in mucus migration ability, and sperm numbers were evaluated at the UTJ. In the third experiment, the fertilization efficiency of IVM oocytes transferred to the oviduct of estrous females inseminated with semen from the same males as earlier, was used to assess the relationship between the mucus migration test and the in vivo fertilization performance of their spermatozoa. Spermatozoa from different males varies significantly in sperm migration efficiency in cervical mucus (15.5a +/- 1.2; 14.9a +/- 1.4; 17.5ab +/- 1.2; 17.0ab +/- 1.5; 19.7b +/- 1.2; 20.1b +/- 1.4 mm; media +/- S.E.M. for males A-F, respectively, P < 0.05). Spermatozoa from males with different mucus migration efficiency values produced different sperm populations at the oviduct reservoir of inseminated females (1,233 +/- 92.3 versus 28.8 +/- 17.0 spermatozoa of males with high and low relative migration efficiency, respectively, P < 0.02). Spermatozoa from males with different mucus migration efficiency values have different fertilization rates of IVM oocytes transferred to oviduct (47/96 (49.0%) versus 25/91 (27.5%) for males with high and low relative migration efficiency, respectively, P < 0.05). Cumulative results suggest that sperm migration in cervical mucus is related to the ability of spermatozoa to colonize the oviduct and to fertilize matured oocytes in vivo.