Naked mole-rat brown fat thermogenesis is diminished during hypoxia through a rapid decrease in UCP1

Naked mole-rats are among the most hypoxia-tolerant mammals. During hypoxia, their body temperature (Tb) decreases via unknown mechanisms to conserve energy. In small mammals, non-shivering thermogenesis in brown adipose tissue (BAT) is critical to Tb regulation; therefore, we hypothesize that hypoxia decreases naked mole-rat BAT thermogenesis. To test this, we measure changes in Tb during normoxia and hypoxia (7% O2; 1–3 h). We report that interscapular thermogenesis is high in normoxia but ceases during hypoxia, and Tb decreases. Furthermore, in BAT from animals treated in hypoxia, UCP1 and mitochondrial complexes I-V protein expression rapidly decrease, while mitochondria undergo fission, and apoptosis and mitophagy are inhibited. Finally, UCP1 expression decreases in hypoxia in three other social African mole-rat species, but not a solitary species. These findings suggest that the ability to rapidly down-regulate thermogenesis to conserve oxygen in hypoxia may have evolved preferentially in social species.

CENP-V is required for proper chromosome segregation through interaction with spindle microtubules in mouse oocytes

Proper chromosome segregation is essential to avoid aneuploidy, yet this process fails with increasing age in mammalian oocytes. Here we report a role for the scarcely described protein CENP-V in oocyte spindle formation and chromosome segregation. We show that depending on the oocyte maturation state, CENP-V localizes to centromeres, to microtubule organizing centers, and to spindle microtubules. We find that Cenp-V−/− oocytes feature severe deficiencies, including metaphase I arrest, strongly reduced polar body extrusion, increased numbers of mis-aligned chromosomes and aneuploidy, multipolar spindles, unfocused spindle poles and loss of kinetochore spindle fibres. We also show that CENP-V protein binds, diffuses along, and bundles microtubules in vitro. The spindle assembly checkpoint arrests about half of metaphase I Cenp-V−/− oocytes from young adults only. This finding suggests checkpoint weakening in ageing oocytes, which mature despite carrying mis-aligned chromosomes. Thus, CENP-V is a microtubule bundling protein crucial to faithful oocyte meiosis, and Cenp-V−/− oocytes reveal age-dependent weakening of the spindle assembly checkpoint.

Nannochloropsis oceanica as a Sustainable Source of n-3 Polyunsaturated Fatty Acids for Enrichment of Hen Eggs

This study aimed to evaluate the potential of the marine microalgae Nannochloropsis oceanica as a sustainable source of n-3 polyunsaturated fatty acids (n-3 PUFA) for hen eggs enrichment. During 4 weeks, hens were fed with 3% (w/w) of Nannochloropsis oceanica supplemented diet. Throughout the assay, eggs were analyzed according to several nutritional and physical parameters, namely: (i) protein, fat, and ash content; (ii) fatty acid profile; (iii) thickness and colour of the shell; (iv) total egg weight; (v) protein quality (HU) and (vi) yolk colour. A remarkable increase in eicosapentaenoic (EPA), from 2.1 ± 0.1 to 5.2 ± 1.2 mg/100 g, and docosahexaenoic (DHA), from 50.3 ± 4.0 to 105 ± 18 mg/100 g, fatty acids was observed. Yolk colour also changed significantly according to the La Roche scale, from 9.6 ± 0.8 to 11.4 ± 0.8 (more orange). Feed supplementation did not lead to changes in the remaining analyzed parameters. A shelf life study, carried out for 28 days at room temperature, showed a decrease in eggs protein quality. In conclusion, eggs from hens fed with Nannochloropsis oceanica had a yolk colour more appealing to consumers and higher levels of EPA and DHA, allowing its classification as high in n-3 PUFA (CE nº 1924/2006).

Identification of a Region in the Common Amino-Terminal Domain of Hendra Virus P, V, and W Proteins Responsible for Phase Transition and Amyloid Formation

Henipaviruses are BSL-4 zoonotic pathogens responsible in humans for severe encephalitis. Their V protein is a key player in the evasion of the host innate immune response. We previously showed that the Henipavirus V proteins consist of a long intrinsically disordered N-terminal domain (NTD) and a β-enriched C-terminal domain (CTD). These terminals are critical for V binding to DDB1, which is a cellular protein that is a component of the ubiquitin ligase E3 complex, as well as binding to MDA5 and LGP2, which are two host sensors of viral RNA. Here, we serendipitously discovered that the Hendra virus V protein undergoes a liquid-to-hydrogel phase transition and identified the V region responsible for this phenomenon. This region, referred to as PNT3 and encompassing residues 200–310, was further investigated using a combination of biophysical and structural approaches. Congo red binding assays, together with negative-staining transmisison electron microscopy (TEM) studies, show that PNT3 forms amyloid-like fibrils. Fibrillation abilities are dramatically reduced in a rationally designed PNT3 variant in which a stretch of three contiguous tyrosines, falling within an amyloidogenic motif, were replaced by three alanines. Worthy to note, Congo red staining experiments provided hints that these amyloid-like fibrils form not only in vitro but also in cellula after transfection or infection. The present results set the stage for further investigations aimed at assessing the functional role of phase separation and fibrillation by the Henipavirus V proteins.

Reduced pathogenicity of velogenic NDV strain AF22420-I via site-directed mutagenesis of V gene

Newcastle disease virus (NDV), an avian paramyxovirus, has the potential to be used as an anti-cancer therapeutic vaccine due to its oncolytic and immunostimulatory activities. The virus can be categorised into three pathotypes: lentogenic, mesogenic, and velogenic; of the three pathotypes, the lentogenic strains such as the La Sota are the preferred pathotype for vaccine development due to their low virulence to birds. On the other hand, the translation of the virus to clinic of the velogenic strain AF2240-I is hindered by its virulence towards birds although it exhibits strong oncolysis with significant outcomes both in vitro and in vivo. This study aims to reduce the pathogenicity of AF2240-I yet retaining the anti-cancer properties of the virus. To achieve this, the V protein that acts as an interferon antagonist was chosen to be mutated. It is a non-structural protein that does not interfere with the binding and infection of the virus; hence, mutation of this virulence factor was deducted to be able to reduce harm to the avian species but retain its anti-cancer properties as much as possible. The V protein, which was produced from the insertion of an additional G into a conserved editing site of the P gene, was mutated by substituting the G nucleotide at position 411 from the start of P gene to a T nucleotide. This mutation will produce a premature stop codon from the V mRNA, resulting in a truncated V protein; but only causes a silent mutation in the P protein. The recombinant virus was recovered by the use of BHK cells stably expressing the phage T7 RNA polymerase. The pathogenicity of the mutated virus was determined in 9- to 11-day-old embryonated SPF chicken eggs. The mean death time (MDT) was determined to be 73.6 hours at the minimal lethal dose of 10-7, resembling to that of a mesogenic strain. The virulence of the mutated virus has been successfully reduced where it could be potentially used as the vector for the development of recombinant oncolytic virus for cancer treatment.

Characterisation of the Myocardial Mitochondria Structural and Functional Phenotype in a Murine Model of Diabetic Cardiomyopathy

People affected by diabetes are at an increased risk of developing heart failure than their non-diabetic counterparts, attributed in part to a distinct cardiac pathology termed diabetic cardiomyopathy. Mitochondrial dysfunction and excess reactive oxygen species (ROS) have been implicated in a range of diabetic complications and are a common feature of the diabetic heart. In this study, we sought to characterise impairments in mitochondrial structure and function in a recently described experimental mouse model of diabetic cardiomyopathy. Diabetes was induced in 6-week-old male FVB/N mice by the combination of three consecutive-daily injections of low-dose streptozotocin (STZ, each 55 mg/kg i.p.) and high-fat diet (42% fat from lipids) for 26 weeks. At study end, diabetic mice exhibited elevated blood glucose levels and impaired glucose tolerance, together with increases in both body weight gain and fat mass, replicating several aspects of human type 2 diabetes. The myocardial phenotype of diabetic mice included increased myocardial fibrosis and left ventricular (LV) diastolic dysfunction. Elevated LV superoxide levels were also evident. Diabetic mice exhibited a spectrum of LV mitochondrial changes, including decreased mitochondria area, increased levels of mitochondrial complex-III and complex-V protein abundance, and reduced complex-II oxygen consumption. In conclusion, these data suggest that the low-dose STZ-high fat experimental model replicates some of the mitochondrial changes seen in diabetes, and as such, this model may be useful to study treatments that target the mitochondria in diabetes.

Effects of Protein, Calcium, and pH on Gene Transcription, Cell-Envelope Peptidase Activity of Lactococcus lactis Strains, and the Formation of Bitter Peptides

Calcium- and protein-rich fermented milk products, such as concentrated yoghurts and fresh cheeses, may contain undesired bitter peptides, which are generated by the proteolytic cleavage of casein. Up to now, it is not clear whether this process is caused by endogenous milk enzymes, such as plasmin and cathepsin D, or whether proteolytic enzymes from applied starter cultures, such as the lactococcal cell-envelope peptidase PrtP, are involved. A sensory analysis of fresh cheese products made from milk concentrates fermented with prtP-negative and -positive Lactococcus lactis strains revealed bitterness in the products fermented with prtP-positive L. lactis strains. Two prtP-positive strains, LTH 7122 and LTH 7123, were selected to investigate the effect of increased calcium concentrations (additional 5 mM and 50 mM CaCl2) at neutral (pH 6.6) and acidic (pH 5.5) pH-values on the transcription of the prtP gene and its corresponding PrtP peptidase activity in milk citrate broth (MCB). For both strains, it was shown that prtP transcription was upregulated only under slightly elevated calcium conditions (5 mM CaCl2) after 5 h of growth. In concordance with these findings, PrtP peptidase activity also increased. When higher concentrations of calcium were used (50 mM), prtP expression of both strains decreased strongly by more than 50%. Moreover, PrtP peptidase activity of strain LTH 7123 decreased by 15%, but enzymatic activity of strain LTH 7122 increased slightly during growth under elevated calcium concentrations (50 mM CaCl2). Fermentations of reconstituted casein medium with 3.4% (w/v) and 8.5% (w/v) protein and different calcium concentrations using strain LTH 7122 revealed no clear relationship between prtP transcription and calcium or protein concentration. However, an increase in PrtP peptidase activity under elevated protein and calcium conditions was observed. The activity increase was accompanied by increased levels of bitter peptides derived from different casein fractions. These findings could be a possible explanation for the bitterness in fermented milk concentrates that was detected by a trained bitter panel.