Phosphoregulation is ubiquitous in biology. Defining the functional roles of individual phosphorylation sites within a multivalent system remains particularly challenging. We have therefore applied a chemical biology approach to light-control the state of single candidate phosphoserines in the canonical anion channel CFTR while simultaneously measuring channel activity. The data show striking non-equivalency among protein kinase A consensus sites, which vary from <10% to >1,000% changes in channel activity upon phosphorylation. Of note, slow phosphorylation of S813 suggests that this site is rate-limiting to the full activation of CFTR. Further, this approach reveals an unexpected coupling between the phosphorylation of S813 and a nearby site, S795. Overall, these data establish an experimental route to understanding roles of specific phosphoserines within complex phosphoregulatory domains. This strategy may be employed in the study of phosphoregulation of other eukaryotic proteins.

Sodium appetite is a complex, motivated behavioral state. It requires low-sodium status detection and a coordinated drive to seek and ingest salty foods. Interestingly, this elaborate behavior can be elicited by administering a singular hormone, aldosterone (Formenti et al., 2013; Gasparini et al., 2018; Geerling & Loewy, 2008). Aldosterone is a mineralocorticoid produced in the zona glomerulosa of the adrenal cortex. It affects cells that express the mineralocorticoid receptor (MR) by translocating this receptor into the cell nucleus to promote genomic activity (Robertson et al., 1993). Aldosterone-sensitive cells also require the enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2), which protects the MR from glucocorticoids, leaving it more accessible to aldosterone (Funder et al., 1988; Naray-Fejes-Toth et al.,

Quantifying complexity from heart rate variability (HRV) series is a challenging task, and multiscale entropy (MSE), along with its variants, has been demonstrated to be one of the most robust approaches to achieve this goal. Although physical training is known to be beneficial, there is little information about the long-term complexity changes induced by the physical conditioning. The present study aimed to quantify the changes in physiological complexity elicited by physical training through multiscale entropy-based complexity measurements. Rats were subject to a protocol of medium intensity training (n = 13) or a sedentary protocol (n = 12). One-hour HRV series were obtained from all conscious rats five days after the experimental protocol. We estimated MSE, multiscale dispersion entropy (MDE) and multiscale SDiff q from HRV series. Multiscale SDiff q is a recent approach that accounts for entropy differences between a given time series and its shuffled dynamics. From SDiff q , three attributes (q-attributes) were derived, namely SDiff q max , q max and q zero. MSE, MDE and multiscale q-attributes presented similar profiles, except for SDiff q max. q max showed significant differences between trained and sedentary groups on Time Scales 6 to 20. Results suggest that physical training increases the system complexity and that multiscale q-attributes provide valuable information about the physiological complexity.

The literature is extensive on how hypertension affects the morphology and function of the central nervous system (CNS) and is being focused on multiple organ damage involving the kidneys, heart, endothelium and retina. Hypertension damage to the peripheral nervous system is less explored in the literature. We have previously shown morphometric alterations in large and small caliber myelinated fibers of nerves in the adult spontaneously hypertensive rat (SHR). However, the functional correlation of these findings has not been explored. We performed an electrophysiological investigation of hind limb nerves in SHR of both genders in different ages. Normotensive Wistar-Kyoto (WKY) rats were used as controls. Electrophysiological recordings and determination of motor (MCV) and sensory (SCV) nerve conduction velocity were performed in the same animals at four different ages: 5, 8, 20 and 40 weeks after birth. Comparisons were made between ages, genders and animal strain. We showed a continuous body weight increase in adult life in all animals studied. MCV got stable at 20-week old hypertensive animals and continued to increase in normotensive ones. The SCV was constant between the ages of 20 and 40 weeks old in female SHR and decreased in male SHR while it continued to increase in WKY animals. The electrophysiological investigation of the nerves in WKY and SHR from both genders and different ages, associated with morphological and morphometric data from the literature suggest that hypertension affects the nerve function and might corroborate the development of a peripheral neuropathy.

The aromatic side-chains of phenylalanine, tyrosine, and tryptophan interact with their environments via both hydrophobic and electrostatic interactions. Determining the extent to which these contribute to protein function and stability is not possible with conventional mutagenesis. Serial fluorination of a given aromatic is a validated method in vitro and in silico to specifically alter electrostatic characteristics, but this approach is restricted to a select few experimental systems. Here, we report a group of pyrrolysine-based aminoacyl-tRNA synthetase/tRNA pairs (tRNA/RS pairs) that enable the site-specific encoding of a varied spectrum of fluorinated phenylalanine amino acids in E. coli and mammalian (HEK 293T) cells. By allowing the cross-kingdom expression of proteins bearing these unnatural amino acids at biochemical scale, these tools may potentially enable the study of biological mechanisms which utilize aromatic interactions in structural and cellular contexts.

In addition to its renal and cardiovascular functions, angiotensin signalling is thought to be responsible for the increases in salt and water intake caused by hypovolaemia. However, it remains unclear whether these behaviours require angiotensin production in the brain or liver. Here, we use in situ hybridization to identify tissue‐specific expression of the genes required for producing angiotensin peptides, and then use conditional genetic deletion of the angiotensinogen gene (Agt) to test whether production in the brain or liver is necessary for sodium appetite and thirst. In the mouse brain, we identified expression of Agt (the precursor for all angiotensin peptides) in a large subset of astrocytes. We also identified Ren1 and Ace (encoding enzymes required to produce angiotensin II) expression in the choroid plexus, and Ren1 expression in neurons within the nucleus ambiguus compact formation. In the liver, we confirmed that Agt is widely expressed in hepatocytes. We next tested whether thirst and sodium appetite require angiotensinogen production in astrocytes or hepatocytes. Despite virtually eliminating expression in the brain, deleting astrocytic Agt did not reduce thirst or sodium appetite. Despite markedly reducing angiotensinogen in the blood, eliminating Agt from hepatocytes did not reduce thirst or sodium appetite, and in fact, these mice consumed the largest amounts of salt and water after sodium deprivation. Deleting Agt from both astrocytes and hepatocytes also did not prevent thirst or sodium appetite. Our findings suggest that angiotensin signalling is not required for sodium appetite or thirst and highlight the need to identify alternative signalling mechanisms. imageKey points Angiotensin signalling is thought to be responsible for the increased thirst and sodium appetite caused by hypovolaemia, producing elevated water and sodium intake. Specific cells in separate brain regions express the three genes needed to produce angiotensin peptides, but brain‐specific deletion of the angiotensinogen gene (Agt), which encodes the lone precursor for all angiotensin peptides, did not reduce thirst or sodium appetite. Double‐deletion of Agt from brain and liver also did not reduce thirst or sodium appetite. Liver‐specific deletion of Agt reduced circulating angiotensinogen levels without reducing thirst or sodium appetite. Instead, these angiotensin‐deficient mice exhibited an enhanced sodium appetite. Because the physiological mechanisms controlling thirst and sodium appetite continued functioning without angiotensin production in the brain and liver, understanding these mechanisms requires a renewed search for the hypovolaemic signals necessary for activating each behaviour.

A variety of animal models have been proposed to study hyperkalaemia, but most of them have meaningful limitations when the goal is to study the effect of potassium overload on healthy kidneys. In this study, we aimed to introduce a new approach for induction of hyperkalaemia in a reliable and reproducible animal model. We used intragastric administration of potassium chloride [KCl 2.3 M, 10 ml/(kg body weight)] to male Holtzman rats (300-350 g) to induce hyperkalaemia. The results showed that this potassium load can temporarily overwhelm the renal and extrarenal handling of this ion, causing an acute and severe hyperkalaemia that can be useful to study the effect of potassium imbalance in a variety of scenarios. Severe hyperkalaemia (>8 meqiv/l) and very profound ECG alterations, characterized by lengthening waves and intervals, were seen as early as 30 min after intragastric administration of KCl in rats. In addition, a transient increase in arterial blood pressure and time-dependent bradycardia were also seen after the KCl administration. No metabolic acidosis was present in the animals, and the potassium ion did not increase proportionally to chloride ion in the blood, leading to an increased anion gap. In conclusion, the results suggest that intragastric KCl loading is a reliable model to promote rapid and severe hyperkalaemia that can be used for further research on this topic.

Coronary reperfusion is an important tool to limit infarct size and preserve cardiac function after acute myocardial infarction. However, the reperfusion of acutely ischemic myocardium can induce arrhythmias and cardiomyocyte death, a phenomenon known as myocardial reperfusion injury. The study of the mechanisms and the search for therapies to protect the heart from reperfusion injury is an important field of research. The aim of this study was to evaluate mophofunctional and molecular aspects involved in a rat model of acute myocardial ischemia followed by reperfusion (IR). Male Wistar rats (N=47) were anesthetized with ketamine and xylazine (50 and 10 mg/kg, ip) and subjected to myocardial IR by temporary (30 min) ligation of left descending coronary artery. Control rats (N=7) underwent the same surgery without coronary ligation. After 4 weeks 45% of the rats subjected to IR showed no cardiac lesion (NL) while 38% presented mild (ML) and 17% large (LL) lesion of the heart. Cardiac function, evaluated by echocardiography, was impaired in all rats subjected to IR, independently of the degree of cardiac lesion. Interstitial fibrosis, quantified in non‐infarcted myocardium, was 11±0.8% in control rats and was found increased in rats with IR (19.7±1.4, 19.4±1.2 and 21.6±1.2% in rats with NL, ML and LL, respectively). Activity of matrix‐metalloproteinase‐2 was higher in the myocardium of rats subjected to IR (0,44 to 0.48 AU) as compared to their control counterparts (0.22 AU). Moreover, concentration of superoxide anion in the myocardium of rats subjected to IR was higher (176±44, 127±13 and 108±30 RLU/mg of protein in rats with NL, ML and LL, respectively) as compared to their controls (50±5 RLU/mg of protein). Finally, hydrogen peroxide was measured 86±29 nmol/mg of protein in myocardium of control rats and was found markedly higher (1077±192, 1194±238 and 639±117 nmol/mg of protein in IR rats with NL, ML and LL, respectively) in the animals subjected to IR. These results show that IR leads to distinct degrees of myocardial injury in a rat model, which contributes to cardiac dysfunction over time. Reactive oxygen species (O2− and H2O2) and MMP‐2 seems to be involved in cardiac dysfunction and heart failure development after reperfusion.Support or Funding InformationFinancial support: CNPq (870308/1997‐1) e FAPESP (2013/20549‐7).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.