Preserving avian blood and DNA sampled in the wild: A survey of personal experiences

Collecting and storing biological material from wild animals in a way that does not deteriorate DNA quality for subsequent analyses is instrumental for research in ecology and evolution. Our aims were to gather reports on the effectiveness of methods commonly used by researchers for the field collection and long‐term storage of blood samples and DNA extracts from wild birds. Personal experiences were collected with an online survey targeted specifically at researchers sampling wild birds. Many researchers experienced problems with blood sample storage but not with DNA extract storage. Storage issues generated problems with obtaining adequate DNA quality and sufficient DNA quantity for the targeted molecular analyses but were not related to season of blood sampling, access to equipment, transporting samples, temperature, and method of blood storage. Final DNA quality and quantity were also not affected by storage time before DNA extraction or the methods used to extract DNA. We discuss practical aspects of field collection and storage and provide some general recommendations, with a list of pros and cons of different preservation methods of avian blood samples and DNA extracts.     

Bacterial division ring stabilizing ZapA versus destabilizing SlmA modulate FtsZ switching between biomolecular condensates and polymers

Cytokinesis is a fundamental process for bacterial survival and proliferation, involving the formation of a ring by filaments of the GTPase FtsZ, spatio-temporally regulated through the coordinated action of several factors. The mechanisms of this regulation remain largely unsolved, but the inhibition of FtsZ polymerization by the nucleoid occlusion factor SlmA and filament stabilization by the widely conserved cross-linking protein ZapA are known to play key roles. It was recently described that FtsZ, SlmA and its target DNA sequences (SlmA-binding sequence (SBS)) form phase-separated biomolecular condensates, a type of structure associated with cellular compartmentalization and resistance to stress. Using biochemical reconstitution and orthogonal biophysical approaches, we show that FtsZ-SlmA-SBS condensates captured ZapA in crowding conditions and when encapsulated inside cell-like microfluidics microdroplets. We found that, through non-competitive binding, the nucleotide-dependent FtsZ condensate/polymer interconversion was regulated by the ZapA/SlmA ratio. This suggests a highly concentration-responsive tuning of the interconversion that favours FtsZ polymer stabilization by ZapA under conditions mimicking intracellular crowding. These results highlight the importance of biomolecular condensates as concentration hubs for bacterial division factors, which can provide clues to their role in cell function and bacterial survival of stress conditions, such as those generated by antibiotic treatment.     

Chronic inhibition of the mitochondrial ATP synthase in skeletal muscle triggers sarcoplasmic reticulum distress and tubular aggregates

Tubular aggregates (TA) are honeycomb-like arrays of sarcoplasmic-reticulum (SR) tubules affecting aged glycolytic fibers of male individuals and inducing severe sarcomere disorganization and muscular pain. TA develop in skeletal muscle from Tubular Aggregate Myopathy (TAM) patients as well as in other disorders including endocrine syndromes, diabetes, and ageing, being their primary cause unknown. Nowadays, there is no cure for TA. Intriguingly, both hypoxia and calcium dyshomeostasis prompt TA formation, pointing to a possible role for mitochondria in their setting. However, a functional link between mitochondrial dysfunctions and TA remains unknown. Herein, we investigate the alteration in muscle-proteome of TAM patients, the molecular mechanism of TA onset and a potential therapy in a preclinical mouse model of the disease. We show that in vivo chronic inhibition of the mitochondrial ATP synthase in muscle causes TA. Upon long-term restrained oxidative phosphorylation (OXPHOS), oxidative soleus experiments a metabolic and structural switch towards glycolytic fibers, increases mitochondrial fission, and activates mitophagy to recycle damaged mitochondria. TA result from the overresponse of the fission controller DRP1, that upregulates the Store-Operate-Calcium-Entry and increases the mitochondria-SR interaction in a futile attempt to buffer calcium overloads upon prolonged OXPHOS inhibition. Accordingly, hypoxic muscles cultured ex vivo show an increase in mitochondria/SR contact sites and autophagic/mitophagic zones, where TA clusters grow around defective mitochondria. Moreover, hypoxia triggered a stronger TA formation upon ATP synthase inhibition, and this effect was reduced by the DRP1 inhibitor mDIVI. Remarkably, the muscle proteome of TAM patients displays similar alterations in mitochondrial dynamics and in ATP synthase contents. In vivo edaravone treatment in mice with restrained OXPHOS restored a healthy phenotype by prompting mitogenesis and mitochondrial fusion. Altogether, our data provide a functional link between the ATP synthase/DRP1 axis and the setting of TA, and repurpose edaravone as a possible treatment for TA-associated disorders.Subject terms: Musculoskeletal abnormalities, Energy metabolism     

Anti‐inflammatory properties of lemon‐derived extracellular vesicles are achieved through the inhibition of ERK/NF‐κB signalling pathways

Chronic inflammation is associated with the occurrence of several diseases. However, the side effects of anti‐inflammatory drugs prompt the identification of new therapeutic strategies. Plant‐derived extracellular vesicles (PDEVs) are gaining increasing interest in the scientific community for their biological properties. We isolated PDEVs from the juice of Citrus limon L. (LEVs) and characterized their flavonoid, limonoid and lipid contents through reversed‐phase high‐performance liquid chromatography coupled to electrospray ionization quadrupole time‐of‐flight mass spectrometry (RP‐HPLC–ESI‐Q‐TOF‐MS). To investigate whether LEVs have a protective role on the inflammatory process, murine and primary human macrophages were pre‐treated with LEVs for 24 h and then were stimulated with lipopolysaccharide (LPS). We found that pre‐treatment with LEVs decreased gene and protein expression of pro‐inflammatory cytokines, such as IL‐6, IL1‐β and TNF‐α, and reduced the nuclear translocation and phosphorylation of NF‐κB in LPS‐stimulated murine macrophages. The inhibition of NF‐κB activation was associated with the reduction in ERK1‐2 phosphorylation. Furthermore, the ability of LEVs to decrease pro‐inflammatory cytokines and increase anti‐inflammatory molecules was confirmed ex vivo in human primary T lymphocytes. In conclusion, we demonstrated that LEVs exert anti‐inflammatory effects both in vitro and ex vivo by inhibiting the ERK1‐2/NF‐κB signalling pathway.     

RNA polymerase heterogeneity in Streptomyces aureofaciens: characterization by antibody-linked polymerase assay

The ability of Escherichia coli with different receptor specificities to interact with meconium was studied. E. coli strains expressing P-fimbriae, specific for Gal alpha 1-4Gal beta-containing receptors, were agglutinated by meconium at high titres. This reaction was inhibited by globotetraosylceramide. The attachment of P-fimbriated E. coli to human colonic epithelial cells of the HT-29 cell line was inhibited by meconium. Some type 1 fimbriated strains were agglutinated by meconium, but the agglutination was rarely blocked by methyl alpha-D-mannoside. The attachment by type 1 fimbriated strains to HT-29 cells was reduced by meconium only in some cases. These results suggest that meconium interacts with the P-fimbriae of E. coli, in a way that may influence bacterial colonization of the neonatal intestine.