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Ex vivo studies with first trimester placental tissues are crucial for understanding human placental development, and culturing placental villi in floating conditions is vital to mimic the natural setting. Moreover, being able to cryopreserve these scarce materials for ex vivo studies would open unprecedented avenues, as they are very limited to research and the need to culture freshly adds further hurdles while limiting efficient use. Here we showed that hanging drop method is a simple yet effective approach to culture placental floating villi. We also revealed that these functional units of the early-stage human placenta can be cryopreserved for culturing, and that frozen-thawed villi can regenerate the syncytial layer following denudation. We further illustrated the utility of frozen-thawed tissues to study syncytialization, while validating the importance of HtrA4 in the process which was shown previously in cell models. These represent significant new knowledge to the field of placental biology.

The outer layer of the human placenta comprises syncytiotrophoblast, which forms through fusion of cytotrophoblasts (syncytialization), and plays a critical role in maternal–fetal communication including nutrient/oxygen transportation and hormone secretion. Impairment in syncytialization inevitably affects pregnancy outcomes. High temperature requirement factor A 4 (HtrA4) is a placental-specific protease, expressed by various trophoblasts including syncytiotrophoblast, and significantly elevated in preeclampsia at disease presentation. However, it is unknown whether HtrA4 is important for syncytialization. Here we first examined HtrA4 expression in primary human cytotrophoblasts during syncytialization which occurs spontaneously in culture, and in BeWo cells which syncytialize upon forskolin stimulation. The success of syncytialization in each model was confirmed by significant up-regulation/secretion of β-hCG, and the concurrent down-regulation of E-cadherin. In both models, HtrA4 mRNA and protein increased concomitantly with syncytialization. Furthermore, the secreted levels of β-hCG and HtrA4 correlated significantly and positively in both models. We next knocked out HtrA4 in BeWo by CRISPR/Cas9. Upon forskolin treatment, control BeWo profoundly up-regulated β-hCG and syncytin-1, down-regulated E-cadherin, and at the same time increased the formation of multinucleated cells, whereas BeWo cells without HtrA4 did not alter any of these parameters. Our data thus suggest that HtrA4 plays an essential role in syncytialization.

Nonsyndromic cleft lip and palate (NS CLP) is a complex birth defect resulting from a combination of genetic and environmental factors. Several members of the FGF and FGFR families are expressed during craniofacial development and can rarely harbor mutations that result in human clefting syndromes. We hypothesized that disruptions in this pathway might also contribute to NS CLP. We sequenced the coding regions and performed association testing on 12 genes (FGFR1, FGFR2, FGFR3, FGF2, FGF3, FGF4, FGF7, FGF8, FGF9, FGF10, FGF18, and NUDT6) and used protein structure analyses to predict the function of amino acid variants. Seven likely disease-causing mutations were identified, including: one nonsense mutation (R609X) in FGFR1, a de novo missense mutation (D73H) in FGF8, and other missense variants in FGFR1, FGFR2, and FGFR3. Structural analysis of FGFR1, FGFR2, and FGF8 variants suggests that these mutations would impair the function of the proteins, albeit through different mechanisms. Genotyping of SNPs in the genes found associations between NS CLP and SNPs in FGF3, FGF7, FGF10, FGF18, and FGFR1. The data suggest that the FGF signaling pathway may contribute to as much as 3-5% of NS CLP and will be a consideration in the clinical management of CLP.

Based on the ability of apoptosis to induce immunological tolerance, liposomes were generated mimicking apoptotic cells, and they arrest autoimmunity in Type 1 diabetes. Our aim was to validate the immunotherapy in other autoimmune disease: multiple sclerosis. Phosphatidylserine-rich liposomes were loaded with disease-specific autoantigen. Therapeutic capability of liposomes was assessed and . Liposomes induced a tolerogenic phenotype in dendritic cells, and arrested autoimmunity, thus decreasing the incidence, delaying the onset and reducing the severity of experimental disease, correlating with an increase in a probably regulatory CD25 FoxP3 CD4 T-cell subset. This is the first work that confirms phosphatidylserine-liposomes as a powerful tool to arrest multiple sclerosis, demonstrating its relevance for clinical application.

The South American temperate and sub-Antarctic forests cover the longest latitudinal range in the Southern Hemisphere and include the world's southernmost forests. However, until now, this unique biome has been absent from global ecosystem research and monitoring networks. Moreover, the latitudinal range of between 40 degrees (°) south (S) and 60° S constitutes a conspicuous gap in the International Long-Term Ecological Research (ILTER) and other international networks. We first identify 10 globally salient attributes of biological and cultural diversity in southwestern South America. We then present the nascent Chilean Long-Term Socio-Ecological Research (LTSER) network, which will incorporate a new biome into ILTER. Finally, we introduce the field environmental philosophy methodology, developed by the Chilean LTSER network to integrate ecological sciences and environmental ethics into graduate education and biocultural conservation. This approach broadens the prevailing economic spectrum of social dimensions considered by LTSER programs and helps foster bioculturally diverse forms of Earth stewardship.

Objectives: Non-syndromic orofacial clefts, i.e. cleft lip (CL) and cleft palate (CP), are among the most common birth defects. The goal of this study was to identify genomic regions and genes for CL with or without CP (CL/P). Methods: We performed linkage analyses of a 10 cM genome scan in 820 multiplex CL/P families (6,565 individuals). Significant linkage results were followed by association analyses of 1,476 SNPs in candidate genes and regions, utilizing a weighted false discovery rate (wFDR) approach to control for multiple testing and incorporate the genome scan results. Results: Significant (multipoint HLOD ≥3.2) or genome-wide-significant (HLOD ≥4.02) linkage results were found for regions 1q32, 2p13, 3q27-28, 9q21, 12p11, 14q21-24 and 16q24. SNPs in IRF6 (1q32) and in or near FOXE1 (9q21) reached formal genome-wide wFDR-adjusted significance. Further, results were phenotype dependent in that the IRF6 region results were most significant for families in which affected individuals have CL alone, and the FOXE1 region results were most significant in families in which some or all of the affected individuals have CL with CP. Conclusions: These results highlight the importance of careful phenotypic delineation in large samples of families for genetic analyses of complex, heterogeneous traits such as CL/P.

Terri Beaty and colleagues report a genome-wide association study of cleft lip with/without cleft palate. They identified variants near MAFB and ABCA4 associated with risk of this birth defect in case-parent trios of European and Asian ancestry. Case-parent trios were used in a genome-wide association study of cleft lip with and without cleft palate. SNPs near two genes not previously associated with cleft lip with and without cleft palate ( MAFB , most significant SNP rs13041247, with odds ratio (OR) per minor allele = 0.704, 95% CI 0.635–0.778, P = 1.44 × 10 −11 ; and ABCA4 , most significant SNP rs560426, with OR = 1.432, 95% CI 1.292–1.587, P = 5.01 × 10 −12 ) and two previously identified regions (at chromosome 8q24 and IRF6 ) attained genome-wide significance. Stratifying trios into European and Asian ancestry groups revealed differences in statistical significance, although estimated effect sizes remained similar. Replication studies from several populations showed confirming evidence, with families of European ancestry giving stronger evidence for markers in 8q24, whereas Asian families showed stronger evidence for association with MAFB and ABCA4 . Expression studies support a role for MAFB in palatal development.

We present a versatile diagram to envisage the dominant relaxation mechanism of single-domain magnetic nanoparticles (MNPs) under alternating magnetic fields, as those used in magnetic fluid hyperthermia (MFH). The diagram allows estimating the heating efficiency, measured by the Specific Power Absorption (SPA), originated in the magnetic and viscous relaxation times of single-domain MNPs for a given frequency of the ac magnetic field (AFM). The diagram has been successfully applied to different colloids, covering a wide variety of MNPs with different magnetic anisotropy and particle size, and dispersed in different viscous liquid carriers. From the general diagram, we derived a specific chart based on the Linear Response Theory in order to easily estimate the experimental condition for the optimal SPA values of most colloids currently used in MFH.

Superparamagnetic iron oxide nanoparticles (SPIONs) have been identified as a promising material for biomedical applications. These include as contrast agents for medical imaging, drug delivery and/or cancer cell treatment. The nanotoxicological profile of SPIONs has been investigated in different studies and the distribution of SPIONs in the human body has not been fully characterized. The aim of this study was to develop a physiologically-based pharmacokinetic (PBPK) model to predict the pharmacokinetics of SPIONs. The distribution and accumulation of SPIONs in organs were simulated taking into consideration their penetration through capillary walls and their active uptake by specialized macrophages in the liver, spleen and lungs. To estimate the kinetics of SPION uptake, a novel experimental approach using primary macrophages was developed. The murine PBPK model was validated against in vivo pharmacokinetic data, and accurately described accumulation in liver, spleen and lungs. After validation of the murine model, a similar PBPK approach was developed to simulate the distribution of SPIONs in humans. These data demonstrate the utility of PBPK modeling for estimating biodistribution of inorganic nanoparticles and represents an initial platform to provide computational prediction of nanoparticle pharmacokinetics.

Objective To assess the association between nonsyndromic (NS) cleft lip with or without cleft palate (CL(P)) and single-nucleotide polymorphisms (SNPs) within the CRISPLD2 gene (cysteine-rich secretory protein LCCL domain containing 2). Design Four SNPs within the CRISPLD2 gene domain (rs1546124, rs8061351, rs2326398, rs4783099) were genotyped to test for association via family-based association methods. Participants A total of 5826 individuals from 1331 families in which one or more family member is affected with CL(P). Results Evidence of association was seen for SNP rs1546124 in U.S. (p = .02) and Brazilian (p = .04) Caucasian cohorts. We also found association of SNP rs1546124 with cleft palate alone (CP) in South Americans (Guatemala and ECLAMC) and combined Hispanics (Guatemala, ECLAMC, and Texas Hispanics; p = .03 for both comparisons) and with both cleft lip with cleft palate (CLP; p = .04) and CL(P) (p = .02) in North Americans. Strong evidence of association was found for SNP rs2326398 with CP in Asian populations (p = .003) and with CL(P) in Hispanics (p = .03) and also with bilateral CL(P) in Brazilians (p = .004). In Brazilians, SNP rs8061351 showed association with cleft subgroups incomplete CL(P) (p = .004) and unilateral incomplete CL(P) (p = .003). Prediction of SNP functionality revealed that the C allele in the C471T silent mutation (overrepresented in cases with CL(P) presents two putative exonic splicing enhancer motifs and creates a binding site AP-2 alpha, a transcription factor involved in craniofacial development. Conclusions Our results support the hypothesis that variants in the CRISPLD2 gene may be involved in the etiology of NS CL(P).