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The extensive crosstalk between the developing heart and lung is critical to their proper morphogenesis and maturation. However, there remains a lack of models that investigate the critical cardio-pulmonary mutual interaction during human embryogenesis. Here, we reported a novel stepwise strategy for directing the simultaneous induction of both mesoderm-derived cardiac and endoderm-derived lung epithelial lineages within a single differentiation of human-induced pluripotent stem cells (hiPSCs) via temporal specific tuning of WNT and nodal signaling in the absence of exogenous growth factors. Using 3D suspension culture, we established concentric cardio-pulmonary micro-Tissues (μTs), and expedited alveolar maturation in the presence of cardiac accompaniment. Upon withdrawal of WNT agonist, the cardiac and pulmonary components within each dual-lineage μT effectively segregated from each other with concurrent initiation of cardiac contraction. We expect that our multilineage differentiation model will offer an experimentally tractable system for investigating human cardio-pulmonary interaction and tissue boundary formation during embryogenesis.

Mitochondrial complex I deficiency is associated with a diverse range of clinical phenotypes and can arise due to either mitochondrial DNA (mtDNA) or nuclear gene defects. We investigated two adult patients who exhibited non-syndromic neurological features and evidence of isolated mitochondrial complex I deficiency in skeletal muscle biopsies. The first presented with indolent myopathy, progressive since age 17, while the second developed deafness around age 20 and other relapsing-remitting neurological symptoms since. A novel, likely de novo, frameshift variant in MT-ND6 (m.14512_14513del) and a novel maternally-inherited transversion mutation in MT-ND1 were identified, respectively. Skewed tissue segregation of mutant heteroplasmy level was observed; the mutant heteroplasmy levels of both variants were greater than 70% in muscle homogenate, however, in blood the MT-ND6 variant was undetectable while the mutant heteroplasmy level of the MT-ND1 variant was low (12%). Assessment of complex I assembly by Blue-Native PAGE demonstrated a decrease in fully assembled complex I in the muscle of both cases. SDS-PAGE and immunoblotting showed decreased levels of mtDNA-encoded ND1 and several nuclear encoded complex I subunits in both cases, consistent with functional pathogenic consequences of the identified variants. Pathogenicity of the m.14512_14513del was further corroborated by single-fiber segregation studies.

Mitochondrial complex I deficiency is associated with a diverse range of clinical phenotypes and can arise due to either mitochondrial DNA (mtDNA) or nuclear gene defects. We investigated two adult patients who exhibited non-syndromic neurological features and evidence of isolated mitochondrial complex I deficiency in skeletal muscle biopsies. The first presented with indolent myopathy, progressive since age 17, while the second developed deafness around age 20 and other relapsing-remitting neurological symptoms since. A novel, likely de novo, frameshift variant in MT-ND6 (m.14512_14513del) and a novel maternally-inherited transversion mutation in MT-ND1 were identified, respectively. Skewed tissue segregation of mutant heteroplasmy level was observed; the mutant heteroplasmy levels of both variants were greater than 70% in muscle homogenate, however, in blood the MT-ND6 variant was undetectable while the mutant heteroplasmy level of the MT-ND1 variant was low (12%). Assessment of complex I assembly by Blue-Native PAGE demonstrated a decrease in fully assembled complex I in the muscle of both cases. SDS-PAGE and immunoblotting showed decreased levels of mtDNA-encoded ND1 and several nuclear encoded complex I subunits in both cases, consistent with functional pathogenic consequences of the identified variants. Pathogenicity of the m.14512_14513del was further corroborated by single-fiber segregation studies. [ABSTRACT FROM AUTHOR]

A diffusion molecular hypothesis from the dura and/or the leptomeninges below that would control the induction of calvarial membranous bone formation by the recombinant human transforming growth factor-β 3 (hTGF-β 3 ) was investigated. Coral-derived calcium carbonate-based macroporous constructs (25 mm diameter; 3.5/4 mm thickness) with limited hydrothermal conversion to hydroxyapatite (7% HA/CC) were inserted into forty calvarial defects created in 10 adult Chacma baboons Papio ursinus . In 20 defects, an impermeable nylon foil membrane (SupraFOIL ® ) was inserted between the cut endocranial bone and the underlying dura mater . Twenty of the macroporous constructs were preloaded with hTGF-β 3 (125 μg in 1000 μl 20 mM sodium succinate, 4% mannitol pH4.0), 10 of which were implanted into defects segregated by the SupraFOIL ® membrane, and 10 into non-segregated defects. Tissues were harvested on day 90, processed for decalcified and undecalcified histology and quantitative real-time polymerase chain reaction (qRT-PCR). Segregated untreated macroporous specimens showed a reduction of bone formation across the macroporous spaces compared to non-segregated constructs. qRT-PCR of segregated untreated specimens showed down regulation of osteogenic protein-1 (OP-1) , osteocalcin (OC) , bone morphogenetic protein-2 ( BMP-2 ), RUNX-2 and inhibitor of DNA binding-2 and -3 ( ID2 , ID3 ) and up regulation of TGF-β 3 , a molecular signalling pathway inhibiting the induction of membranous bone formation. Non-segregated hTGF-β 3 /treated constructs also showed non-osteogenic expression profiles when compared to non-segregated untreated specimens. Segregated hTGF-β 3 /treated 7% HA/CC constructs showed significantly greater induction of bone formation across the macroporous spaces and, compared to non-segregated hTGF-β 3 /treated constructs, showed up regulation of OP-1 , OC , BMP-2 , RUNX-2 , ID2 and ID3 . Similar up-regulated expression profiles were seen for untreated non-segregated constructs. TGF-β signalling via ID genes creates permissive or refractory micro-environments that regulate the induction of calvarial bone formation which is controlled by the exogenous hTGF-β 3 upon segregation of the calvarial defects. The dura is the common regulator of the induction of calvarial bone formation modulated by the presence or absence of the SupraFOIL ® membrane with or without hTGF-β 3 . [ABSTRACT FROM AUTHOR]

The issue of mitochondrial heteroplasmy has been cited as a theoretical problem for DNA barcoding but is only beginning to be examined in natural systems. We sequenced multiple DNA extractions from 20 individuals of four Hawaiian Hylaeus bee species known to be heteroplasmic. All species showed strong differences at polymorphic sites between abdominal and muscle tissue in most individuals, and only two individuals had no obvious segregation. Two specimens produced completely clean sequences from abdominal DNA. The fact that these differences are clearly visible by direct sequencing indicates that substantial intra-individual mtDNA diversity may be overlooked when DNA is taken from small tissue fragments. At the same time, differences in haplotype distribution among individuals may result in incorrect recognition of cryptic species. Because DNA barcoding studies typically use only a small fragment of an organism, they are particularly vulnerable to sequencing bias where heteroplasmy and haplotype segregation are present. It is important to anticipate this possibility prior to undertaking large-scale barcoding projects to reduce the likelihood of haplotype segregation confounding the results. [ABSTRACT FROM AUTHOR]