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Hemoglobin and myoglobin are widely responsible for oxygen transport and storage (see the Perspective by Rezende ). The ability of diving mammals to obtain enough oxygen to support extended dives and foraging is largely dependent on muscle myoglobin (Mb) content. Mirceta et al. (p. 1234192 ) found that in mammalian lineages with an aquatic or semiaquatic lifestyle, Mb net charge increases, which may represent an adaptation to inhibit self-association of Mb at high intracellular concentrations. Epistasis results from nonadditive genetic interactions and can affect phenotypic evolution. Natarajan et al. (p. 1324 ) found that epistatic interactions were able to explain the increased hemoglobin oxygen-binding affinity observed in deer mice populations at high altitude. In mammals, the offloading of oxygen from hemoglobin is facilitated by a reduction in the blood's pH, driven by metabolically produced CO 2 . However, in fish, a reduction in blood pH reduces oxygen carrying capacity of hemoglobin. Rummer et al. (p. 1327 ) implanted fiber optic oxygen sensors within the muscles of rainbow trout and found that elevated CO 2 levels in the water led to acidosis and elevated oxygen tensions. Increasing the number of charged amino acids allows for higher myoglobin concentrations in the muscles of diving mammals. [Also see Perspective by Rezende ] Extended breath-hold endurance enables the exploitation of the aquatic niche by numerous mammalian lineages and is accomplished by elevated body oxygen stores and adaptations that promote their economical use. However, little is known regarding the molecular and evolutionary underpinnings of the high muscle myoglobin concentration phenotype of divers. We used ancestral sequence reconstruction to trace the evolution of this oxygen-storing protein across a 130-species mammalian phylogeny and reveal an adaptive molecular signature of elevated myoglobin net surface charge in diving species that is mechanistically linked with maximal myoglobin concentration. This observation provides insights into the tempo and routes to enhanced dive capacity evolution within the ancestors of each major mammalian aquatic lineage and infers amphibious ancestries of echidnas, moles, hyraxes, and elephants, offering a fresh perspective on the evolution of this iconic respiratory pigment.

Remarkable feats of breath-hold endurance are observed in diving mammals, with some species routinely diving for an hour. During most mammalian dives metabolism remains aerobic in nature, which is accomplished by restricting the blood flow to parts of the body through peripheral vaso-constriction and bradycardia. This mechanism preserves essential oxygen (02) for heart and brain function, but also means some parts of the body, including locomotory muscles, become isolated and have to rely on O2 stored within the tissues. Due to the isolation of skeletal muscles, mammalian divers must be able to buffer large quantities of H+ ions due to the production of CO2 during aerobic metabolism and acidic end products of anaerobic metabolism once muscle O2 stores have been consumed. The protein responsible for storing molecular 02 is myoglobin (Mb), a small 17 kDa monomeric globular haemoprotein with the primary function of reversible O2 binding and facilitated diffusion of O2 to the mitochondria. A hallmark of mammalian divers is increased Mb concentrations ([Mb]. with divers exhibiting concentrations up to thirty times those seen in non-diving species. Previous research has found that proteins at high concentrations are prone to form aggregations leading to non-functioning protein. This raises the question of Mb solubility at such high concentrations as observed in mammalian divers. The central hypothesis of this thesis is that mammalian myoglobin has undergone previously unrecognised, parallel and adaptive evolution in several lineages of mammalian divers that has profoundly increased their maximal physiological diving capacity. To test the hypothesis, Mb amino acid sequences of 124 mammals, including 24 newly determined sequences, are analysed for the content and individual buffering properties of their ionisable amino acids. This is used to calculate the specific Mb buffer value (~Mb) for each species, which is experimentally verified by acid-base titration of purified Mb. Together with known [Mb], the contribution of Mb to whole muscle buffer capacity WmuscleMb) is then quantified. Amino acid sequences are assessed for substitutions that increase modelled net Mb charge in mammalian divers compared to terrestrial species and predictions are confirmed by measuring electrophoretic mobility of purified Mb. Observed changes in Mb buffer properties and net surface charge are mapped on a composite mammalian phylogeny to test whether they are significantly linked to the evolution of diving behaviour. Observed molecular changes in Mb amino acid sequence are integrated with diving capacity, producing a model that allows prediction of maximal dive duration from Mb amino acid sequence and body mass. Using ancestral Mb sequence reconstructions and body mass estimates, the model is applied to infer the evolution of maximal diving capacity in the cetacean lineage. Results suggest a general trend towards increasing ~Mb due to increased Mb histidine content in mammalian divers.βmuscleMb is significantly higher in divers compared to terrestrial species, and can account for up to 45%, of the increase in whole muscle buffering observed in diving mammals. This study shows a remarkable trend in all diving species to significantly increase the net charge of the Mb protein, which would convey increased Mb solubility. This is supported by a significant correlation between Mb net charge and maximal [Mb]. Evolutionary analysis shows that high Mb net charge is significantly linked to the occurrence of diving. Contrary to previous findings, the model developed here finds that increases in [Mb] convey greater increases in dive duration than similar increases in body mass. This study provides novel in sights into how cumulative substitutions on the molecular surface of Mb can have profound adaptive effects on the physiological properties conveyed to the whole animal. It suggests that adaptation to diving in multiple lineages of mammals involved not only evolution of increased expression levels of Mb, but also substantial qualitative changes to the protein to avoid aggregation and increase solubility and buffering.

The ATRX ATP-dependent chromatin remodelling/helicase protein associates with the DAXX histone chaperone to deposit histone H3.3 over repetitive DNA regions. Because ATRX-protein interactions impart functions, such as histone deposition, we used proximity-dependent biotinylation (BioID) to identify proximal associations for ATRX. The proteomic screen captured known interactors, such as DAXX, NBS1, and PML, but also identified a range of new associating proteins. To gauge the scope of their roles, we examined three novel ATRX-associating proteins that likely differed in function, and for which little data were available. We found CCDC71 to associate with ATRX, but also HP1 and NAP1, suggesting a role in chromatin maintenance. Contrastingly, FAM207A associated with proteins involved in ribosome biosynthesis and localized to the nucleolus. ATRX proximal associations with the SLF2 DNA damage response factor help inhibit telomere exchanges. We further screened for the proteomic changes at telomeres when ATRX, SLF2, or both proteins were deleted. The loss caused important changes in the abundance of chromatin remodelling, DNA replication, and DNA repair factors at telomeres. Interestingly, several of these have previously been implicated in alternative lengthening of telomeres. Altogether, this study expands the repertoire of ATRX-associating proteins and functions.

Objective. This study measured the relationship between lesions suggestive of subclinical pig illness at harvest to carcass contamination and human foodborne risk. Methods. Over the course of eight visits (December 2005 to January 2006), we swabbed 280 randomly selected carcasses, during normal slaughter operations, at three points in the slaughter line: skin pre-scald; the bung or pelvic cavity following removal of the distal colon and rectum; and pleural cavity, immediately before the final carcass rinse. Each swab sponge was used on five carcasses in bung and pleural cavity sampling. Swab sponges were cultured quantitatively for Campylobacter spp., Enterococcus spp., and Enterobacteriaceae spp., and qualitatively for Salmonella spp. Data on health indicators were collected for all pigs in the study (2,625 pigs) by experienced plant quality assurance personnel. Results. Campylobacter spp. were recovered from the pleural cavity in 58.9% (33/56) of pools (five carcasses/pool), and in 44.6% (25/56) of pools from the bung cavity. Enterococcus spp. were recovered from 66.1% (37/56) and 35.7% (20/56) of pleural and bung pools, respectively. The most common lesion identified was the peel-out (pleuritis or adhesions), with a total of 7.1% (186/2,625 total head). Linear regression showed that for every percentage point increase in peel-outs, Enterococcus spp. contamination increased by 4.4% and Campylobacter spp. increased by 5.1% (p<0.05). Conclusions. This study showed a correlation between animal health and human health risk, as measured by carcass contamination. Therefore, animal management decisions on-farm, such as housing, antibiotic use, environment, and level of veterinary care, may directly impact public health.