Cytochrome b5 reductase 3 overexpression and dietary nicotinamide riboside supplementation promote distinctive mitochondrial alterations in distal convoluted tubules of mouse kidneys during aging.

The kidney undergoes structural and physiological changes with age, predominantly studied in glomeruli and proximal tubules. However, limited knowledge is available about the impact of aging and anti-aging interventions on distal tubules. In this study, we investigated the effects of cytochrome b5 reductase 3 (CYB5R3) overexpression and/or dietary nicotinamide riboside (NR) supplementation on distal tubule mitochondria. Initially, transcriptomic data were analyzed to evaluate key genes related with distal tubules, CYB5R3, and NAD+ metabolism, showing significant differences between males and females in adult and old mice. Subsequently, our emphasis focused on assessing how these interventions, that have demonstrated the anti-aging potential, influenced structural parameters of distal tubule mitochondria, such as morphology and mass, as well as abundance, distance, and length of mitochondria-endoplasmic reticulum contact sites, employing an electron microscopy approach. Our findings indicate that both interventions have differential effects depending on the age and sex of the mice. Aging resulted in an increase in mitochondrial size and a decrease in mitochondrial abundance in males, while a reduction in abundance, size, and mitochondrial mass was observed in old females when compared with their adult counterparts. Combining both the interventions, CYB5R3 overexpression and dietary NR supplementation mitigated age-related changes; however, these effects were mainly accounted by NR in males and by transgenesis in females. In conclusion, the influence of CYB5R3 overexpression and dietary NR supplementation on distal tubule mitochondria depends on sex, genotype, and diet. This underscores the importance of incorporating these variables in subsequent studies to comprehensively address the multifaceted aspects of aging.

Prótesis mitral mecánica disfuncionante: cuando el trombo sobre pannus dificulta el diagnóstico / Dysfunctioning mechanical mitral valve prosthesis: When thrombus over pannus makes hinders diagnosis

La obstrucción de una válvula protésica es una complicación rara pero que puede ser letal. Las causas más frecuentes son la formación de trombos y pannus, en ausencia de datos infecciosos. El diagnóstico no siempre es sencillo recurriendo a la realización de tomografía computarizada (TC) cardiaca, y en el 46-85% de los casos coexisten trombo y pannus, por lo que el diagnóstico se complica. Un diagnóstico rápido es esencial para evitar un desenlace fatal de esta patología, cuya mortalidad, a pesar de un tratamiento correcto, es elevada.(AU)

Prosthetic valve obstruction is a rare but potentially lethal complication. The most frequent causes are thrombus and pannus formation, in the absence of infectious data. Diagnosis is not always easy using cardiac CT scanning and in 46-85% of cases thrombus and pannus coexist, complicating the diagnosis. A rapid diagnosis is essential to avoid a fatal outcome of this pathology whose mortality, despite correct treatment, is high.(AU)

Dysfunctioning mechanical mitral valve prosthesis: When thrombus over pannus makes hinders diagnosis.

Prosthetic valve obstruction is a rare but potentially lethal complication. The most frequent causes are thrombus and pannus formation, in the absence of infectious data. Diagnosis is not always easy using cardiac CT scanning and in 46%-85% of cases thrombus and pannus coexist, complicating the diagnosis. A rapid diagnosis is essential to avoid a fatal outcome of this pathology whose mortality, despite correct treatment, is high.

Identifying Biomarkers for Biological Age: Geroscience and the ICFSR Task Force.

The International Conference on Frailty and Sarcopenia Research Task Force met in March 2020, in the shadow of the COVID-19 pandemic, to discuss strategies for advancing the interdisciplinary field of geroscience. Geroscience explores biological mechanisms of aging as targets for intervention that may delay the physiological consequences of aging, maintain function, and prevent frailty and disability. Priorities for clinical practice and research include identifying and validating a range of biomarkers of the hallmarks of aging. Potential biomarkers discussed included markers of mitochondrial dysfunction, proteostasis, stem cell dysfunction, nutrient sensing, genomic instability, telomere dysfunction, cellular senescence, and epigenetic changes. The FRAILOMICS initiative is exploring many of these through various omics studies. Translating this knowledge into new therapies is being addressed by the U.S. National Institute on Aging Translational Gerontology Branch. Research gaps identified by the Task Force include the need for improved cellular and animal models as well as more reliable and sensitive measures.

Restoration of energy homeostasis by SIRT6 extends healthy lifespan.

Aging leads to a gradual decline in physical activity and disrupted energy homeostasis. The NAD+-dependent SIRT6 deacylase regulates aging and metabolism through mechanisms that largely remain unknown. Here, we show that SIRT6 overexpression leads to a reduction in frailty and lifespan extension in both male and female B6 mice. A combination of physiological assays, in vivo multi-omics analyses and 13C lactate tracing identified an age-dependent decline in glucose homeostasis and hepatic glucose output in wild type mice. In contrast, aged SIRT6-transgenic mice preserve hepatic glucose output and glucose homeostasis through an improvement in the utilization of two major gluconeogenic precursors, lactate and glycerol. To mediate these changes, mechanistically, SIRT6 increases hepatic gluconeogenic gene expression, de novo NAD+ synthesis, and systemically enhances glycerol release from adipose tissue. These findings show that SIRT6 optimizes energy homeostasis in old age to delay frailty and preserve healthy aging.

A roadmap to build a phenotypic metric of ageing: insights from the Baltimore Longitudinal Study of Aging.

Over the past three decades, considerable effort has been dedicated to quantifying the pace of ageing yet identifying the most essential metrics of ageing remains challenging due to lack of comprehensive measurements and heterogeneity of the ageing processes. Most of the previously proposed metrics of ageing have been emerged from cross-sectional associations with chronological age and predictive accuracy of mortality, thus lacking a conceptual model of functional or phenotypic domains. Further, such models may be biased by selective attrition and are unable to address underlying biological constructs contributing to functional markers of age-related decline. Using longitudinal data from the Baltimore Longitudinal Study of Aging (BLSA), we propose a conceptual framework to identify metrics of ageing that may capture the hierarchical and temporal relationships between functional ageing, phenotypic ageing and biological ageing based on four hypothesized domains: body composition, energy regulation, homeostatic mechanisms and neurodegeneration/neuroplasticity. We explored the longitudinal trajectories of key variables within these phenotypes using linear mixed-effects models and more than 10 years of data. Understanding the longitudinal trajectories across these domains in the BLSA provides a reference for researchers, informs future refinement of the phenotypic ageing framework and establishes a solid foundation for future models of biological ageing.

A toolbox for the longitudinal assessment of healthspan in aging mice.

The number of people aged over 65 is expected to double in the next 30 years. For many, living longer will mean spending more years with the burdens of chronic diseases such as Alzheimer's disease, cardiovascular disease, and diabetes. Although researchers have made rapid progress in developing geroprotective interventions that target mechanisms of aging and delay or prevent the onset of multiple concurrent age-related diseases, a lack of standardized techniques to assess healthspan in preclinical murine studies has resulted in reduced reproducibility and slow progress. To overcome this, major centers in Europe and the United States skilled in healthspan analysis came together to agree on a toolbox of techniques that can be used to consistently assess the healthspan of mice. Here, we describe the agreed toolbox, which contains protocols for echocardiography, novel object recognition, grip strength, rotarod, glucose tolerance test (GTT) and insulin tolerance test (ITT), body composition, and energy expenditure. The protocols can be performed longitudinally in the same mouse over a period of 4-6 weeks to test how candidate geroprotectors affect cardiac, cognitive, neuromuscular, and metabolic health.

The development of human digital Meissner's and Pacinian corpuscles.

Meissner's and Pacinian corpuscles are cutaneous mechanoreceptors responsible for different modalities of touch. The development of these sensory formations in humans is poorly known, especially regarding the acquisition of the typical immunohistochemical profile related to their full functional maturity. Here we used a panel of antibodies (to specifically label the main corpuscular components: axon, Schwann-related cells and endoneurial-perineurial-related cells) to investigate the development of digital Meissner's and Pacinian corpuscles in a representative sample covering from 11 weeks of estimated gestational age (wega) to adulthood. Development of Pacinian corpuscles starts at 13 wega, and it is completed at 4 months of life, although their basic structure and immunohistochemical characteristics are reached at 36 wega. During development, around the axon, a complex network of S100 positive Schwann-related processes is progressively compacted to form the inner core, while the surrounding mesenchyme is organized and forms the outer core and the capsule. Meissner's corpuscles start to develop at 22 wega and complete their typical morphology and immunohistochemical profile at 8 months of life. In developing Meissner's corpuscles, the axons establish complex relationships with the epidermis and are progressively covered by Schwann-like cells until they complete the mature arrangement late in postnatal life. The present results demonstrate an asynchronous development of the Meissner's and Pacini's corpuscles and show that there is not a total correlation between morphological and immunohistochemical maturation. The correlation of the present results with touch-induced cortical activity in developing humans is discussed.

Merkel cells and Meissner's corpuscles in human digital skin display Piezo2 immunoreactivity.

The transformation of mechanical energy into electrical signals is the first step in mechanotransduction in the peripheral sensory nervous system and relies on the presence of mechanically gated ion channels within specialized sensory organs called mechanoreceptors. Piezo2 is a vertebrate stretch-gated ion channel necessary for mechanosensitive channels in mammalian cells. Functionally, it is related to light touch, which has been detected in murine cutaneous Merkel cell-neurite complexes, Meissner-like corpuscles and lanceolate nerve endings. To the best of our knowledge, the occurrence of Piezo2 in human cutaneous mechanoreceptors has never been investigated. Here, we used simple and double immunohistochemistry to investigate the occurrence of Piezo2 in human digital glabrous skin. Piezo2 immunoreactivity was detected in approximately 80% of morphologically and immunohistochemically characterized (cytokeratin 20+ , chromogranin A+ and synaptophisin+ ) Merkel cells. Most of them were in close contact with Piezo2- nerve fibre profiles. Moreover, the axon, but not the lamellar cells, of Meissner's corpuscles was also Piezo2+ , but other mechanoreceptors, i.e. Pacinian or Ruffini's corpuscles, were devoid of immunoreactivity. Piezo2 was also observed in non-nervous tissue, especially the basal keratinocytes, endothelial cells and sweat glands. The present results demonstrate the occurrence of Piezo2 in cutaneous sensory nerve formations that functionally work as slowly adapting (Merkel cells) and rapidly adapting (Meissner's corpuscles) low-threshold mechanoreceptors and are related to fine and discriminative touch but not to vibration or hard touch. These data offer additional insight into the molecular basis of mechanosensing in humans.

Ultrastructure of Lingual Papillae in Common Chimpanzee (Pan troglodytes) Foetus, Newborn and Adult Specimens.

Among primates, the two recognized species of chimpanzees (common chimpanzee, Pan troglodytes; pygmy chimpanzee, Pan paniscus) are considered to be the most similar to humans. Importantly, in mammals, the food intake behaviour largely determines the tongue morphology, including the type, proportion and distribution of gustatory and non-gustatory tongue papillae. The lingual papillae form during its development and mature in post-natal life depending on the different feeding. In this study, we have used scanning electron microscopy to analyse the age-related changes in the lingual papillae of foetal, newborn and adult P. troglodytes. Four main types of lingual papillae, denominated filiform, fungiform, foliate and vallate, and one subtype of filiform papillae called conical papillae, were found. The main age-related changes observed in all kinds of papillae were a progressive keratinization and morphological complexity along the lifespan. During the foetal period, there was scarce keratinization, which progressively increases in young animals to adulthood. The number of filiform increased with ageing, and both filiform and fungiform papillae in adult tongues are divided into pseudopapillae. On the other hand, the vallate papillae vary from smooth simple surfaces in foetal tongues to irregular surfaces with grooves and pseudopapillae (microscopic papilla-shaped formations within the papilla itself) in adults. These results describe for the first time the age-related variations in the three-dimensional aspect of lingual papillae of the chimpanzee tongue and provide new data to characterize more precisely these structures in the human closest specie.

Endoneurial-CD34 positive cells define an intermediate layer in human digital Pacinian corpuscles.

The endoneurial and/or perineurial origin of the outer core; i.e. the concentric and continuous lamellae located outside the complex formed by the axon and the Schwann-related cells, in human Pacinian corpuscles is still debated. Here we used immunohistochemistry coupled with a battery of antibodies to investigate the expression of perineurial (Glucose transporter 1 and epithelial membrane antigen) or endoneurial (CD34 antigen) markers in human digital Pacinian corpuscles. CD34 immunoreactivity was restricted to one layer immediately outside the inner core, whereas the proper outer core displayed antigens typical of the perineurial cells. These results demonstrate an intermediate endoneurial layer that divides the Pacinian corpuscles into two distinct compartments: the avascular inner neural compartment (formed by the axon and the Schwann-related cells that form the inner core), and the outer non-neural compartment (formed by the outer core). The functional relevance of these findings, if any, remains to be clarified.

Immunohistochemical localization of periostin in human gingiva.

The periostin is a matricellular protein expressed in collagen-rich tissues including some dental and periodontal tissues where it is regulated by mechanical forces, growth factors and cytokines. Interestingly the expression of this protein has been found modified in different gingival pathologies although the expression of periostin in normal human gingiva was never investigated. Here we used Western blot and double immunofluorescence coupled to laser-confocal microscopy to investigated the occurrence and distribution of periostin in different segments of the human gingival in healthy subjects. By Western blot a protein band with an estimated molecular mass of 94 kDa was observed. Periostin was localized at the epithelial-connective tissue junction, or among the fibers of the periodontal ligament, and never co-localized with cytokeratin or vimentin thus suggesting it is an extracellular protein. These results demonstrate the occurrence of periostin in adult human gingiva; its localization suggests a role in the bidirectional interactions between the connective tissue and the epithelial cells, and therefore in the physiopathological conditions in which these interactions are altered.

Acid-sensing ion channels (ASICs) 2 and 4.2 are expressed in the retina of the adult zebrafish.

Acid-sensing ion channels (ASICs) are H(+)-gated, voltage-insensitive cation channels involved in synaptic transmission, mechanosensation and nociception. Different ASICs have been detected in the retina of mammals but it is not known whether they are expressed in adult zebrafish, a commonly used animal model to study the retina in both normal and pathological conditions. We study the expression and distribution of ASIC2 and ASIC4 in the retina of adult zebrafish and its regulation by light using PCR, in situ hybridization, western blot and immunohistochemistry. We detected mRNA encoding zASIC2 and zASIC4.2 but not zASIC4.1. ASIC2, at the mRNA or protein level, was detected in the outer nuclear layer, the outer plexiform layer, the inner plexiform layer, the retinal ganglion cell layer and the optic nerve. ASIC4 was expressed in the photoreceptors layer and to a lesser extent in the retinal ganglion cell layer. Furthermore, the expression of both ASIC2 and ASIC4.2 was down-regulated by light and darkness. These results are the first demonstration that ASIC2 and ASIC4 are expressed in the adult zebrafish retina and suggest that zebrafish could be used as a model organism for studying retinal pathologies involving ASICs.

Acid-sensing ion channel 2 (ASIC2) is selectively localized in the cilia of the non-sensory olfactory epithelium of adult zebrafish.

Ionic channels play key roles in the sensory cells, such as transducing specific stimuli into electrical signals. The acid-sensing ion channel (ASIC) family is voltage-insensitive, amiloride-sensitive, proton-gated cation channels involved in several sensory functions. ASIC2, in particular, has a dual function as mechano- and chemo-sensor. In this study, we explored the possible role of zebrafish ASIC2 in olfaction. RT-PCR, Western blot, chromogenic in situ hybridization and immunohistochemistry, as well as ultrastructural analysis, were performed on the olfactory rosette of adult zebrafish. ASIC2 mRNA and protein were detected in homogenates of olfactory rosettes. Specific ASIC2 hybridization was observed in the luminal pole of the non-sensory epithelium, especially in the cilia basal bodies, and immunoreactivity for ASIC2 was restricted to the cilia of the non-sensory cells where it was co-localized with the cilia marker tubulin. ASIC2 expression was always absent in the olfactory cells. These findings demonstrate for the first time the expression of ASIC2 in the olfactory epithelium of adult zebrafish and suggest that it is not involved in olfaction. Since the cilium sense and transduce mechanical and chemical stimuli, ASIC2 expression in this location might be related to detection of aquatic environment pH variations or to detection of water movement through the nasal cavity.

Brain-derived neurotrofic factor and its receptor TrkB are present, but segregated, within mature cutaneous Pacinian corpuscles of Macaca fascicularis.

Some mechanoreceptors in mammals depend totally or in part on the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4), and their receptor TrkB, for development and maintenance. These actions are presumably exerced regulating the survival of discrete sensory neurons in the dorsal root ganglia which form mechanoreceptors at the periphery. In addition, the cells forming the mechanoreceptors also express both neurotrophins and their receptors although large differences have been described among species. Pacinian corpuscles are rapidly adapting low-threshold mechanoreceptors whose dependence from neurotrophins is not known. In the present study, we analyzed expression of TrkB and their ligands BDNF and NT-4 in the cutaneous Pacinian corpuscles of Macaca fascicularis using immunohistochemistry and fluorescent microscopy. TrkB immunoreactivity was found in Pacinian corpuscles where it co-localized with neuron-specific enolase, and occasionally with S100 protein, thus suggesting that TrkB expression is primarily into axons but also in the lamellar cells and even in the outer core. On the other hand, BDNF immunoreactivity was found the inner core cells where it co-localized with S100 protein but also in the innermost layers of the outer core; NT-4 immunostaining was not detected. These results describe for the first time the expression and distribution of a full neurotrophin system in the axon-inner core complex of mature Pacinian corpuscles. The data support previous findings demonstrating large differences in the expression of BDNF-TrkB in mammalian mechanoreceptors, and also suggest the existence of a retrograde trophic signaling mechanism to maintain morphological and functional integrity of sensory neurons supplying Pacinian corpuscles.

ASIC2 is present in human mechanosensory neurons of the dorsal root ganglia and in mechanoreceptors of the glabrous skin.

Mechanosensory neurons lead to the central nervous system touch, vibration and pressure sensation. They project to the periphery and form different kinds of mechanoreceptors. The manner in which they sense mechanical signals is still not fully understood, but electrophysiological experiments have suggested that this may occur through the activation of ion channels that gate in response to mechanical stimuli. The acid-sensing ion channels (ASICs), especially ASIC2, may function as mechanosensors or are required for mechanosensation, and they are expressed in both mechanosensory neurons and mechanoreceptors. Here, we have used double immunohistochemistry for ASIC2 together with neuronal and glial markers associated with laser confocal microscopy and image analysis, to investigate the distribution of ASIC2 in human lumbar dorsal root ganglia, as well as in mechanoreceptors of the hand and foot glabrous skin. In lumbar dorsal root ganglia, ASIC2 immunoreactive neurons were almost all intermediate or large sized (mean diameter ≥20-70 µm), and no ASIC2 was detected in the satellite glial. ASIC2-positive axons were observed in Merkel cell-neurite complexes, Meissner and Pacinian corpuscles, all of them regarded as low-threshold mechanoreceptors. Moreover, a variable percent of Meissner (8 %) and Pacinian corpuscles (27 %) also displayed ASIC2 immunoreactivity in the Schwann-related cells. These results demonstrate the distribution of ASIC2 in the human cutaneous mechanosensory system and suggest the involvement of ASIC2 in mechanosensation.

Calcium-activated potassium channel SK1 is widely expressed in the peripheral nervous system and sensory organs of adult zebrafish.

Sensory cells contain ion channels involved in the organ-specific transduction mechanisms that convert different types of stimuli into electric energy. Here we focus on small-conductance calcium-activated potassium channel 1 (SK1) which plays an important role in all excitable cells acting as feedback regulators in after-hyperpolarization. This study was undertaken to analyze the pattern of expression of SK1 in the zebrafish peripheral nervous system and sensory organs using RT-PRC, Westernblot and immunohistochemistry. Expression of SK1 mRNA was observed at all developmental stages analyzed (from 10 to 100 days post fertilization, dpf), and the antibody used identified a protein with a molecular weight of 70kDa, at 100dpf (regarded to be adult). Cell expressing SK1 in adult animals were neurons of dorsal root and cranial nerve sensory ganglia, sympathetic neurons, sensory cells in neuromasts of the lateral line system and taste buds, crypt olfactory neurons and photoreceptors. Present results report for the first time the expression and the distribution of SK1 in the peripheral nervous system and sensory organs of adult zebrafish, and may contribute to set zebrafish as an interesting experimental model for calcium-activated potassium channels research. Moreover these findings are of potential interest because the potential role of SK as targets for the treatment of neurological diseases and sensory disorders.

Acid-sensing ion channels (ASICs) in the taste buds of adult zebrafish.

In detecting chemical properties of food, different molecules and ion channels are involved including members of the acid-sensing ion channels (ASICs) family. Consistently ASICs are present in sensory cells of taste buds of mammals. In the present study the presence of ASICs (ASIC1, ASIC2, ASIC3 and ASIC4) was investigated in the taste buds of adult zebrafish (zASICs) using Western blot and immunohistochemistry. zASIC1 and zASIC3 were regularly absent from taste buds, whereas faint zASIC2 and robust zASIC4 immunoreactivities were detected in sensory cells. Moreover, zASIC2 also immunolabelled nerves supplying taste buds. The present results demonstrate for the first time the presence of zASICs in taste buds of teleosts, with different patterns to that occurring in mammals, probably due to the function of taste buds in aquatic environment and feeding. Nevertheless, the role of zASICs in taste remains to be demonstrated.

Immunohistochemical detection of the putative mechanoproteins ASIC2 and TRPV4 in avian herbst sensory corpuscles.

The avian Herbst corpuscles are the equivalent of the Pacinian corpuscles in mammals, and detect vibration and the movement of joints and feathers. Therefore, they can be regarded as rapidly adapting low-threshold mechanoreceptors. In recent years, it has been establish that some ion channels are involved in mechanosensation and are present in both mechanosensory neurons and mechanoreceptors. Here we have used immunohistochemistry to localize some putative mechanoproteins in the Herbst corpuscles from the rictus of Columba livia. The proteins investigated were the subunits of the epithelial Na(+) channel (ENaC), the transient-receptor potential vanilloid 4 (TRPV4), and the acid-sensing ion channel 2 (ASIC2). Immunoreactivity for ENaC subunits was never found in Herbst corpuscles, while the axon expressed ASIC2 and TRPV4 immunoreactivity. Moreover, TRPV4 was also detected in the cell forming the inner core. The present results demonstrate for the first time the occurrence of mechanoproteins in avian low-threshold mechanoreceptors and provide further evidence for a possible role of the ion channels in mechanosensation.

Immunohistochemical localization of acid-sensing ion channel 2 (ASIC2) in cutaneous Meissner and Pacinian corpuscles of Macaca fascicularis.

Acid-sensing ion channel 2 (ASIC2) is a member of the degenerin/epithelial sodium channel superfamily, presumably involved mechanosensation. Expression of ASIC2 has been detected in mechanosensory neurons as well as in both axons and Schwann-like cells of cutaneous mechanoreceptors. In these studies we analysed expression of ASIC2 in the cutaneous sensory corpuscles of Macaca fascicularis using immunohistochemistry and laser confocal-scanner microscopy. ASIC2 immunoreactivity was detected in both Meissner and Pacinian corpuscles. It was found to co-localize with neuron-specific enolase and RT-97, but not with S100 protein, demonstrating that ASIC2 expression is restricted to axons supplying mechanoreceptors. These results demonstrate for the first time the presence of the protein ASIC2 in cutaneous rapidly adapting low-threshold mechanoreceptors of monkey, suggesting a role of this ion channel in touch sense.