Accelerated evolution of SARS-CoV-2 in free-ranging white-tailed deer

The zoonotic origin of the COVID-19 pandemic virus highlights the need to fill the vast gaps in our knowledge of SARS-CoV-2 ecology and evolution in non-human hosts. Here, we detected that SARS-CoV-2 was introduced from humans into white-tailed deer more than 30 times in Ohio, USA during November 2021-March 2022. Subsequently, deer-to-deer transmission persisted for 2–8 months, disseminating across hundreds of kilometers. Newly developed Bayesian phylogenetic methods quantified how SARS-CoV-2 evolution is not only three-times faster in white-tailed deer compared to the rate observed in humans but also driven by different mutational biases and selection pressures. The long-term effect of this accelerated evolutionary rate remains to be seen as no critical phenotypic changes were observed in our animal models using white-tailed deer origin viruses. Still, SARS-CoV-2 has transmitted in white-tailed deer populations for a relatively short duration, and the risk of future changes may have serious consequences for humans and livestock

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Ammonium chloride administration prior to exercise has muscle-specific effects on mitochondrial and myofibrillar protein synthesis in rats

Aim: Exercise is able to increase both muscle protein synthesis and mitochondrial biogenesis. However, acidosis, which can occur in pathological states as well as during high-intensity exercise, can decrease mitochondrial function, whilst its impact on muscle protein synthesis is disputed. Thus, the aim of this study was to determine the effect of a mild physiological decrease in pH, by administration of ammonium chloride, on myofibrillar and mitochondrial protein synthesis, as well as associated molecular signaling events. Methods: Male Wistar rats were given either a placebo or ammonium chloride prior to a short interval training session. Rats were killed before exercise, immediately after exercise, or 3 h after exercise. Results: Myofibrillar (p = 0.036) fractional protein synthesis rates was increased immediately after exercise in the soleus muscle of the placebo group, but this effect was absent in the ammonium chloride group. However, in the gastrocnemius muscle NH4Cl increased myofibrillar (p = 0.044) and mitochondrial protein synthesis (0 h after exercise p = 0.01; 3 h after exercise p = 0.003). This was accompanied by some small differences in protein phosphorylation and mRNA expression. Conclusion: This study found ammonium chloride administration immediately prior to a single session of exercise in rats had differing effects on mitochondrial and myofibrillar protein synthesis rates in soleus (type I) and gastrocnemius (type II) muscle in rats

Densitometric gel-like image (virtual gel image) of RNA samples extracted from the same muscle sample using different methods.

<p>The images were generated by a Bio-Rad Experion microfluidic gel electrophoresis system. The first lane is the molecular size marker (ladder), which indicates the approximate size of molecules on the gel. For RNA samples extracted using TRIzol (Lane 2) or an extraction kit containing a Tri-reagent lysis step and precipitating RNA using 2-propanol (Lane 3), 28S and 18S rRNA are clearly visible as two sharp bands. For RNA samples extracted using an extraction kit containing a Tri-reagent lysis step and precipitating RNA using ethanol (Lane 4), 28S and 18S rRNA appear as a smear (indicating the accumulation of low molecular weight components) rather than sharp bands. The location of 28S and 18S rRNA is indicated beside the gel image.</p

The sequential stages of the quantitative real-time PCR workflow.

<p>Skeletal muscle samples are cleaned quickly and snap-frozen in liquid nitrogen after the muscle biopsy, and then stored at -80°C until RNA extraction. RNA is extracted using TRIzol or another Tri-reagent, and assessed for concentration and quality. RNA that passes the quality test is used to synthesise cDNA, which is used as template in the final qPCR assay. The conditions of the final qPCR assay must be optimised for each experiment, and the data should be processed using the correct analysis methods.</p

RNA concentration, yield, and quality with different RNA extraction protocols.

<p>RNA concentration, yield, and quality with different RNA extraction protocols.</p

An example of an amplification efficiency test using <i>Cyclophilin</i> (see Table 4 for details) and <i>PPARG coactivator 1 alpha</i> (<i>PGC-1α</i>, see Experiment 4 for details) primers.

<p>A standard curve was generated using a 10-fold dilution of cDNA as template for qPCR reactions. The resulting C_q values are plotted against the Log of the cDNA input. The efficiency, as well as the R² value, are within the acceptable range. The efficiencies of <i>Cyclophilin</i> and <i>PGC-1α</i> are approximately equal, as the absolute value of the slope of ΔC_q against the Log of the cDNA input is < 0.1.</p

Evaluation of reference genes using RefFinder.

<p>Evaluation of reference genes using RefFinder.</p