Natural underlying mtDNA heteroplasmy as a potential source of intra‐person hiPSC variability

Functional variability among human clones of induced pluripotent stem cells (hiPSCs) remains a limitation in assembling high‐quality biorepositories. Beyond inter‐person variability, the root cause of intra‐person variability remains unknown. Mitochondria guide the required transition from oxidative to glycolytic metabolism in nuclear reprogramming. Moreover, mitochondria have their own genome (mitochondrial DNA [mtDNA]). Herein, we performed mtDNA next‐generation sequencing (NGS) on 84 hiPSC clones derived from a cohort of 19 individuals, including mitochondrial and non‐mitochondrial patients. The analysis of mtDNA variants showed that low levels of potentially pathogenic mutations in the original fibroblasts are revealed through nuclear reprogramming, generating mutant hiPSCs with a detrimental effect in their differentiated progeny. Specifically, hiPSC‐derived cardiomyocytes with expanded mtDNA mutations non‐related with any described human disease, showed impaired mitochondrial respiration, being a potential cause of intra‐person hiPSC variability. We propose mtDNA NGS as a new selection criterion to ensure hiPSC quality for drug discovery and regenerative medicine.     

Designing healthy,climate friendly and affordable school lunches

Purpose

This study aims to develop a model with which to build diets taking into account nutritional, climate change and economic aspects. A case study is used to test the proposed model, consisting of finding the optimal menus for school children in Spain from combinations of 20 starters, 20 main dishes and 7 desserts for a 20-day planning period.

Methods

An optimizing technique, specifically integer goal programming, is used as a means of designing diets which take into account the aforementioned aspects. Goal programming (GP) is used to design those menus that meet, or nearly meet, all the requirements with respect to caloric content, caloric share among macronutrients, nutrients to encourage and nutrients to limit, while reducing the carbon footprint (CFP) and the lunch budget. In order to have real, acceptable dishes, a school catering company provided information about the typical dishes they serve. The CFP of each dish was assessed, based on literature about life cycle assessment and CFP studies on food products. The nutritional value of each dish was obtained from databases, whereas prices were gathered from a wholesaler.

Results and discussion

After solving the goal programming model for several CFP and budget goals, the results show reductions with respect to the average CFP of between ?13 and ?24 %, and reductions with respect to the average budget between ?10 and ?15 % while maintaining the nutritional aspects similar to the average of the proposed menus. The results show that a wide range of budget is available, maintaining an almost constant CFP and meeting nutritional requirements to a similar degree; therefore, it is possible to avoid trade-offs between the CFP and the budget. The analysis of the dishes selected shows how the optimization model, in general, avoids the dishes which have a high CFP and high price and which are low in iron content, but high in protein and cholesterol.

Conclusions

Goal programming constitutes a suitable tool for designing diets which are economically, environmentally and nutritionally sustainable. Its flexibility enables specific issues to be studied, such as the existence of possible trade-offs between budget and CFP, attained by changing the budget and the CFP goals. By means of an iterative process, new dishes could be introduced or the existing ones could be improved, thus providing catering companies with useful information.

     

Cryopreserved CD90+ cells obtained from mobilized peripheral blood in sheep: a new source of mesenchymal stem cells for preclinical applications

Mobilized peripheral blood (MPB) bone marrow cells possess the potential to differentiate into a variety of mesenchymal tissue types and offer a source of easy access for obtaining stem cells for the development of experimental models with applications in tissue engineering. In the present work, we aimed to isolate by magnetic activated cell sorting CD90+ cells from MPB by means of the administration of Granulocyte-Colony Stimulating Factor and to evaluate cell proliferation capacity, after thawing of the in vitro culture of this population of mesenchymal stem cells (MSCs) in sheep. We obtained a median of 8.2 ± 0.6 million of CD90+ cells from the 20-mL MPB sample. After thawing, at day 15 under in vitro culture, the mean CD90+ cells determined by flow cytometry was 92.92 ± 1.29 % and cell duplication time determined by crystal violet staining was 47.59 h. This study describes for the first time the isolation, characterization, and post-in vitro culture thawing of CD90+ MSCs from mobilized peripheral blood in sheep. This population can be considered as a source of MSCs for experimental models in tissue engineering research.     

Crystal structure of the bovine α-chymotrypsin:kunitz inhibitor complex. An example of multiple protein:protein recognition sites

The crystal structure of bovine α-chymotrypsin (α-CHT) in complex with the bovine basic pancreatic trypsin inhibitor (BPTI) has been solved and refined at 2.8 Å resolution (R-factor=0.18). The proteinase:inhibitor complex forms a compact dimer (two α-CHT and two BPTI molecules), which may be stabilized by surface-bound sulphate ions, in the crystalline state. Each BPTI molecule, at opposite ends, is contacting both proteinase molecules in the dimer, through the reactive site loop and through residues next to the inhibitor's C-terminal region. Specific recognition between α-CHT and BPTI occurs at the (re)active site interface according to structural rules inferred from the analysis of homologous serine proteinase:inhibitor complexes. Lys15, the P₁ residue of BPTI, however, does not occupy the α-CHT S₁ specificity pocket, being hydrogen bonded to backbone atoms of the enzyme surface residues Gly216 and Ser217. © 1997 John Wiley & Sons, Ltd.     

Burkholderia pseudomallei γ-carbonic anhydrase is strongly activated by amino acids and amines

Activation of the γ-class carbonic anhydrase (CAs, EC 4.2.1.1) from the pathogenic bacterium Burkholderia pseudomallei (BpsγCA) with a series of natural and non-natural amino acids and aromatic/heterocyclic amines has been investigated. The best BpsγCA activators were d-His, l-DOPA, d-Trp, 4-amino-l-Phe, dopamine, 2-(2-aminoethyl)pyridine, 2-aminoethyl-piparazine/morpholine and l-adrenaline, which showed activation constants ranging between 9 and 86 nM. The least effective activators were l-His, l-Phe and 2-pyridyl-methylamine, with K_As in the range of 1.73–24.7 μM. As little is known about the role of γ-CAs in the lifecycle and virulence of this saprophytic bacterium, this study may shed some light on such phenomena. This is the first CA activation study of a γ-CA from a pathogenic bacterium, the only other such study being on the enzyme discovered in the archaeon Methanosarcina thermophila, Cam.     

Archipelagos of the Anthropocene: rapid and extensive differentiation of native terrestrial vertebrates in a single metropolis

Some of the best evidence for rapid evolutionary change comes from studies of archipelagos and oceanic islands. City parks are analogous systems as they create geographically isolated green spaces that differ in size, structure and complexity. Very little, however, is known about whether city parks within a single urban centre drive selection and result in the diversification of native species. Here, we provide evidence for the rapid genetic and morphological differentiation of a native lizard (Intellagama lesueurii) at four geographically close yet unconnected parks within one city. Year of establishment of each city park varied from 1855 (oldest) to 2001 (youngest) equating to a generation time range of 32 to three generations. Genetic divergence among city park populations was large despite the small pairwise geographic distances (<5 km) and found to be two to three times higher for microsatellites and three to 33 times higher for mtDNA relative to nonurban populations. Patterns of morphological differentiation were also found to be most extensive among the four city park populations. In contrast to nonurban populations, city park populations showed significant differentiation in relative body size, relative head and limb morphology and relative forelimb and hindlimb length. Crucially, we show that these patterns of differentiation are unlikely to have been caused by founder events and/or drift alone. Our results suggest that city park ‘archipelagos’ could represent theatres for rapid evolution that may, in time, favour adaptive diversification.     

Cell Entry of Borna Disease Virus Follows a Clathrin-Mediated Endocytosis Pathway That Requires Rab5 and Microtubules

Borna disease virus (BDV), the prototypic member of the Bornaviridae family within the order Mononegavirales, exhibits high neurotropism and provides an important and unique experimental model system for studying virus-cell interactions within the central nervous system. BDV surface glycoprotein (G) plays a critical role in virus cell entry via receptor-mediated endocytosis, and therefore, G is a critical determinant of virus tissue and cell tropism. However, the specific cell pathways involved in BDV cell entry have not been determined. Here, we provide evidence that BDV uses a clathrin-mediated, caveola-independent cell entry pathway. We also show that BDV G-mediated fusion takes place at an optimal pH of 6.0 to 6.2, corresponding to an early-endosome compartment. Consistent with this finding, BDV cell entry was Rab5 dependent but Rab7 independent and exhibited rapid fusion kinetics. Our results also uncovered a key role for microtubules in BDV cell entry, whereas the integrity and dynamics of actin cytoskeleton were not required for efficient cell entry of BDV.Borna disease virus (BDV) causes central nervous system disease in a variety of vertebrate species that is frequently manifested by behavioral abnormalities (27, 59). BDV is the causative agent of Borna disease, an often fatal immune-mediated neurological disease naturally occurring mainly in horses and sheep (21, 26, 47). However, current evidence indicates that the natural host range, prevalence, and geographic distribution of BDV are wider than originally thought (25, 31). Experimentally, BDV has a wide host range, and both host and viral factors contribute to a variable period of incubation and heterogeneity in the symptoms and pathology associated with BDV infection (20, 23, 34, 50). Notably, cases of proventricular dilatation disease affecting different species of psittacine birds have recently been linked to infection with avian bornaviruses (24, 29), a finding that expands the natural host range of bornavirus infections associated with clinical manifestations.BDV is an enveloped virus with a nonsegmented negative-strand RNA genome (11, 33, 53, 55) whose gene organization [3′-N-P-p10 (X)-M-G-L-5′] is characteristic of mononegaviruses. However, on the basis of its unique genetic and biological features, BDV is considered to be the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales.The BDV surface glycoprotein G plays a key role in receptor recognition and cell entry (20, 46). The G gene directs the synthesis of a precursor, GPC, with a predicted M_r of ca. 56 kDa, but due to its extensive glycosylation, GPC migrates with an M_r of 84 to 94 kDa. GPC is posttranslationally cleaved by the cellular protease furin into GP-1 and GP-2, corresponding to the N-and C-terminal regions, respectively, of G (2, 8, 19, 49). GP-1 has been shown to be sufficient for virus cell entry via receptor-mediated endocytosis (46), whereas GP-2 likely mediates the pH-dependent fusion event between BDV and cell membranes required for a BDV productive infection (19). In vivo, neurons are the initial target of BDV, suggesting a restricted expression pattern of a yet-unidentified virus receptor. Late in infection, BDV is detected in many tissues and organs as a consequence of its centrifugal spread via the axoplasm of peripheral nerve tissues. Receptor-independent mechanisms also contribute to cell-to-cell propagation of BDV (8).The paucity of cell-free virus associated with BDV infection has hindered studies aimed at the elucidation of the mechanisms involved in BDV cell entry. To overcome this problem, we generated a replication-competent recombinant vesicular stomatitis virus expressing BDV G (rVSVΔG*/BDVG) (45). Cells infected with rVSVΔG*/BDVG produced high titers (10⁷ PFU/ml) of cell-free virus progeny. Notably, rVSVΔG*/BDVG recreated the cell tropism and entry pathway of bona fide BDV, thus providing a unique tool for the investigation of BDV G-mediated cell entry.Viruses that enter cells via receptor-mediated endocytosis mainly use trafficking pathways mediated by either clathrin or caveola, although alternative entry pathways have been also reported (36). Nevertheless, clathrin-mediated endocytosis (CME) is the route most commonly used by enveloped viruses for cell internalization (35). The initial virus-cell surface receptor interaction results in the activation of different signaling pathways leading to the accumulation of clathrin coated-pits and subsequent formation of endocytotic vesicles (43). Another major endocytotic pathway used by several viruses, including Ebola virus (16) and SV40 (44), uses caveolae for viral internalization into the cell. This endocytotic pathway is strictly dependent on recruitment of lipid rafts to the cell surface, an event mediated by cholesterol. In this regard, we have recently documented the requirements of cholesterol and structural integrity of cell surface lipid rafts for efficient cell entry of BDV (9).In this work, we provide evidence for the first time that BDV cell entry follows a CME-dependent, caveola-independent pathway. Moreover, we show that BDV entry is Rab5 dependent but Rab7 independent and that BDV G-mediated fusion has a rapid kinetics and an optimal pH between 6.0 and 6.2. These findings indicate that BDV G-mediated fusion occurs within the early-endosome compartment. We also provide evidence that microtubules, but not actin dynamics, play a role in BDV cell entry likely by mediating trafficking of BDV-containing endosomes to the subcellular location where viral and endosomal membranes fuse.     

SHP-2 regulates myogenesis by coupling to FAK signaling pathway

Transient dephosphorylation of FAK at Tyr-397 is required for cell cycle withdrawal early on during myogenesis. Here, we show that upon serum starvation of C2C12 myoblasts, FAK is transiently dephosphorylated in parallel with SHP-2 activation and association with FAK. SHP-2 knockdown by RNA interference suppressed the transient upregulation of SHP-2 and dephosphorylation of FAK during myogenesis. Furthermore, depletion of SHP-2 retarded the cell cycle withdrawal and the differentiation of serum-starved myoblasts into myotubes. These data provide a mechanistic basis for the reduction in FAK activity in differentiating myoblasts, indicating that myogenesis is critically triggered by FAK/SHP-2 complex.

Structured summary

MINT-7258938: Fak1 (uniprotkb:P34152) physically interacts (MI:0915) with shp2 (uniprotkb:P35235) by anti bait coimmunoprecipitation (MI:0006)     

Co-culture of dedifferentiated and primary human chondrocytes obtained from cadaveric donor enhance the histological quality of repair tissue: an in-vivo animal study

To compare the quality of the repair tissue in three-dimensional co-culture of human chondrocytes implanted in an in vivo model. Six cadaveric and five live human donors were included. Osteochondral biopsies from the donor knees were harvested for chondrocyte isolation. Fifty percent of cadaveric chondrocytes were expanded until passage-2 (P2) while the remaining cells were cryopreserved in passage-0 (P0). Fresh primary chondrocytes (P0f) obtained from live human donors were co-cultured. Three-dimensional constructs were prepared with a monolayer of passage-2 chondrocytes, collagen membrane (Geistlich Bio-Gide^®), and pellet of non-co-cultured (P2) or co-cultured chondrocytes (P2 + P0c, P2 + P0f). Constructs were implanted in the subcutaneous tissue of athymic mice and left for 3 months growth. Safranin-O and Alcian blue staining were used to glycosaminoglycan content assessment. Aggrecan and type-II collagen were evaluated by immunohistochemistry. New-formed tissue quality was evaluated with an adaptation of the modified O’Driscoll score. Histological quality of non-co-cultured group was 4.37 (SD ±4.71), while co-cultured groups had a mean score of 8.71 (SD ±3.98) for the fresh primary chondrocytes and 9.57 (SD ±1.27) in the cryopreserved chondrocytes. In immunohistochemistry, Co-culture groups were strongly stained for type-II and aggrecan not seen in the non-co-cultured group. It is possible to isolate viable chondrocytes from cadaveric human donors in samples processed in the first 48-h of dead. There is non-significant difference between the numbers of chondrocytes isolated from live or cadaveric donors. Cryopreservation of cadaveric primary chondrocytes does not alter the capability to form cartilage like tissue. Co-culture of primary and passaged chondrocytes enhances the histological quality of new-formed tissue compared to non-co-cultured cells.