Accessing naïve human pluripotency

Pluripotency manifests during mammalian development through formation of the epiblast, founder tissue of the embryo proper. Rodent pluripotent stem cells can be considered as two distinct states: na?ve and primed. Na?ve pluripotent stem cell lines are distinguished from primed cells by self-renewal in response to LIF signaling and MEK/GSK3 inhibition (LIF/2i conditions) and two active X chromosomes in female cells. In rodent cells, the na?ve pluripotent state may be accessed through at least three routes: explantation of the inner cell mass, somatic cell reprogramming by ectopic Oct4, Sox2, Klf4, and C-myc, and direct reversion of primed post-implantation-associated epiblast stem cells (EpiSCs). In contrast to their rodent counterparts, human embryonic stem cells and induced pluripotent stem cells more closely resemble rodent primed EpiSCs. A critical question is whether na?ve human pluripotent stem cells with bona fide features of both a pluripotent state and na?ve-specific features can be obtained. In this review, we outline current understanding of the differences between these pluripotent states in mice, new perspectives on the origins of na?ve pluripotency in rodents, and recent attempts to apply the rodent paradigm to capture na?ve pluripotency in human cells. Unraveling how to stably induce na?ve pluripotency in human cells will influence the full realization of human pluripotent stem cell biology and medicine.     

Engineering domain-swapped binding interfaces by mutually exclusive folding

Domain swapping is a mechanism for forming protein dimers and oligomers with high specificity. It is distinct from other forms of oligomerization in that the binding interface is formed by reciprocal exchange of polypeptide segments. Swapping plays a physiological role in protein–protein recognition, and it can also potentially be exploited as a mechanism for controlled self-assembly. Here, we demonstrate that domain-swapped interfaces can be engineered by inserting one protein into a surface loop of another protein. The key to facilitating a domain swap is to destabilize the protein when it is monomeric but not when it is oligomeric. We achieve this condition by employing the “mutually exclusive folding” design to apply conformational stress to the monomeric state. Ubiquitin (Ub) is inserted into one of six surface loops of barnase (Bn). The 38-Å amino-to-carboxy-terminal distance of Ub stresses the Bn monomer, causing it to split at the point of insertion. The 2.2-Å X-ray structure of one insertion variant reveals that strain is relieved by intermolecular folding with an identically unfolded Bn domain, resulting in a domain-swapped polymer. All six constructs oligomerize, suggesting that inserting Ub into each surface loop of Bn results in a similar domain-swapping event. Binding affinity can be tuned by varying the length of the peptide linkers used to join the two proteins, which modulates the extent of stress. Engineered, swapped proteins have the potential to be used to fabricate “smart” biomaterials, or as binding modules from which to assemble heterologous, multi-subunit protein complexes.     

Diminution of hypertriglyceridemia-induced pressor effect under hyperinsulinemic condition in normal and fructose-induced insulin resistant rats

This study aimed to evaluate the effect of hyperinsulinemia on hypertriglyceridemia-induced pressor response in normal and fructose-induced insulin resistant rats. The rats were divided into six groups of eight rats and were fed a fructose-enriched diet (FINs, F(INS+TG)) or a regular chow diet (C, C(TG), C(INS), C(INS+TG)) for 8 wks. The acute experiment was conducted at the end of wk 8 and consisted of a 30-min basal period and followed by a 120-min test period. After the basal period, somatostatin (1.3 microg/kg/ min) combined with regular insulin (0.6 or 4 mU/kg/min) and variable glucose infusion were given to clamp euglycemia and euinsulinemia in C and C(TG) or euglycemia and hyperinsulinemia in CINs, C(INS+TG), F(INS) and F(INS+TG). During test period, lipofundin (a triglyceride emulsion) was infused into CTG, C(INS+TG), F(INS+TG) and saline instead was infused into C, C(INS), FINS. Plasma insulin and triglyceride levels were significantly higher in fructose-fed rats than in normal rats. During the test period, the lipofundin infusion (1.2 ml/kg/hr) increased plasma triglyceride levels by 368 +/- 39, 351 +/- 71 and 489 +/- 38 mg/dl compared with their baseline levels in lipid-infused groups. During the test period, low-dose insulin infusion kept plasma insulin at basal levels in C and C(TG) and high-dose insulin infusion increased plasma insulin levels about 6 times the baseline insulin level in C. Glucose infusion rate (GIR) was significantly higher in rats with high insulin infusion than those with low insulin infusion. The increase in GIR was lower in fructose-fed groups than in control groups under similar hyperinsulinemia. Rats with or without lipofundin infusion did not alter GIR during the test period. The present results demonstrated that hypertriglyceridemia-induced pressor response was diminished under hyperinsulinemic condition in both normal and fructose-induced insulin resistant rats.     

The isotopic biosignatures of photo‐ vs. thiotrophic bivalves: are they preserved in fossil shells?

Symbiont‐bearing and non‐symbiotic marine bivalves were used as model organisms to establish biosignatures for the detection of distinctive symbioses in ancient bivalves. For this purpose, the isotopic composition of lipids (δ¹³C) and bulk organic shell matrix (δ¹³C, δ³⁴S, δ¹⁵N) from shells of several thiotrophic, phototrophic, or non‐symbiotic bivalves were compared (phototrophic: Fragum fragum, Fragum unedo, Tridacna maxima; thiotrophic: Codakia tigerina, Fimbria fimbriata, Anodontia sp.; non‐symbiotic: Tapes dorsatus, Vasticardium vertebratum, Scutarcopagia sp.). ?¹³C values of bulk organic shell matrices, most likely representing mainly original shell protein/chitin biomass, were depleted in thio‐ and phototrophic bivalves compared to non‐symbiotic bivalves. As the bulk organic shell matrix also showed a major depletion of δ¹⁵N (down to –2.2 ‰) for thiotrophic bivalves, combined δ¹³C and δ¹⁵N values are useful to differentiate between thio‐, phototrophic, and non‐symbiotic lifestyles. However, the use of these isotopic signatures for the study of ancient bivalves is limited by the preservation of the bulk organic shell matrix in fossils. Substantial alteration was clearly shown by detailed microscopic analyses of fossil (late Pleistocene) T. maxima and Trachycardium lacunosum shell, demonstrating a severe loss of quantity and quality of bulk organic shell matrix with time. Likewise, the composition and δ¹³C‐values of lipids from empty shells indicated that a large part of these compounds derived from prokaryotic decomposers. The use of lipids from ancient shells for the reconstruction of the bivalve's life style therefore appears to be restricted.     

High-fat diet alters gut microbiota physiology in mice

The intestinal microbiota is known to regulate host energy homeostasis and can be influenced by high-calorie diets. However, changes affecting the ecosystem at the functional level are still not well characterized. We measured shifts in cecal bacterial communities in mice fed a carbohydrate or high-fat (HF) diet for 12 weeks at the level of the following: (i) diversity and taxa distribution by high-throughput 16S ribosomal RNA gene sequencing; (ii) bulk and single-cell chemical composition by Fourier-transform infrared- (FT-IR) and Raman micro-spectroscopy and (iii) metaproteome and metabolome via high-resolution mass spectrometry. High-fat diet caused shifts in the diversity of dominant gut bacteria and altered the proportion of Ruminococcaceae (decrease) and Rikenellaceae (increase). FT-IR spectroscopy revealed that the impact of the diet on cecal chemical fingerprints is greater than the impact of microbiota composition. Diet-driven changes in biochemical fingerprints of members of the Bacteroidales and Lachnospiraceae were also observed at the level of single cells, indicating that there were distinct differences in cellular composition of dominant phylotypes under different diets. Metaproteome and metabolome analyses based on the occurrence of 1760 bacterial proteins and 86 annotated metabolites revealed distinct HF diet-specific profiles. Alteration of hormonal and anti-microbial networks, bile acid and bilirubin metabolism and shifts towards amino acid and simple sugars metabolism were observed. We conclude that a HF diet markedly affects the gut bacterial ecosystem at the functional level.