Publicaciones por año
Publicaciones del Departamento de Histología y Embriología
Müller glia in short-term dark adaptation of the Austrolebias charrua retina: Cell proliferation and cytoarchitecture
Exp Cell Res 2025 Jan 15;444(2):114394
Laura Herrera-Astorga 1 , Stephanie Silva 2 , Inés Berrosteguieta 3 , Juan Carlos Rosillo 4 , Anabel Sonia Fernández 5
1 Departamento de Neurociencias Integrativas y Computacionales, Lab. Neurobiología Comparada, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Avenida. Italia 3318, 11600, Montevideo, Uruguay; Sección Fisiología y Nutrición, Facultad de Ciencias, Universidad de la República, Iguá 4225, 11400, Montevideo, Uruguay. Electronic address: lherrera@iibce.edu.uy. 2 Departamento de Neurociencias Integrativas y Computacionales, Lab. Neurobiología Comparada, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Avenida. Italia 3318, 11600, Montevideo, Uruguay. Electronic address: ssilva@fcien.edu.uy. 3 Departamento de Neurociencias Integrativas y Computacionales, Lab. Neurobiología Comparada, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Avenida. Italia 3318, 11600, Montevideo, Uruguay. Electronic address: iberrosteguieta@iibce.edu.uy. 4 Departamento de Neurociencias Integrativas y Computacionales, Lab. Neurobiología Comparada, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Avenida. Italia 3318, 11600, Montevideo, Uruguay; Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Avda. General Flores 2125, 11800, Montevideo, Uruguay. Electronic address: jrosillo@iibce.edu.uy. 5 Departamento de Neurociencias Integrativas y Computacionales, Lab. Neurobiología Comparada, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Avenida. Italia 3318, 11600, Montevideo, Uruguay; Laboratorio de Neurociencias, Facultad de Ciencias, Universidad de la República, Iguá 4225, 11400, Montevideo, Uruguay. Electronic address: afernandez@iibce.edu.uy.
DOI: 10.1016/j.yexcr.2024.114394
PMID: 39722301
Pubmed: https://pubmed.ncbi.nlm.nih.gov/39722301
Texto completo: https://linkinghub.elsevier.com/retrieve/pii/S0014-4827(24)00485-3
Abstract:
Fish with unique life cycles offer valuable insights into retinal plasticity, revealing mechanisms of environmental adaptation, cell proliferation, and thus, potentially regeneration. The variability of the environmental factors to which Austrolebias annual fishes are exposed has acted as a strong selective pressure shaping traits such as nervous system plasticity. This has contributed to adaptation to their extreme conditions including the decreased luminosity as ponds dry out. In particular, the retina of A. charrua has been shown to respond to 30 days of decreased luminosity by exacerbating cell proliferation Now, we aimed to determine the cellular component of the retina involved in shorter-term responses. To this end, we performed 5-bromo-2'-deoxyuridine (BrdU) experiments, exposing adult fish to a short period (11 days) of constant darkness. Strikingly, in control conditions, neurogenesis in the inner nuclear and ganglion cell layer in the differentiated retina was detected. In constant darkness, we observed an effect on inner nuclear layer cell proliferation and changes in retinal cytoarchitecture of the retina with cell clusters located in the inner plexiform layer. Additionally, increased BLBP (brain lipid-binding protein) presence was detected in darkness, which has been previously associated with immature and reactivated Müller glia. Thus, our results suggest that the A. charrua retina can respond to environmental changes via rapid activation of progenitor cells in the INL, namely the Müller glia This leads us to hypothesize, that cell proliferation and neurogenesis might contribute to the responses to the functional needs of organisms, potentially playing an adaptive role.
Evaluation of the efficacy and safety of gravitational therapy in a cohort of patients with systemic sclerosis
Reumatol Clin 2024 Nov;20(9):463-469
Luisa Fernanda Servioli 1 , Eugenia Isasi 2 , Alejandra Pérez 3 , Silvia Pouquette 3 , María Eloísa Isasi 3
1 Unidad de Enfermedades Autoinmunes Sistémicas, Hospital Militar, Montevideo, Uruguay. Electronic address: lservioli01@gmail.com. 2 Unidad Académica de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; Centro de Terapia Gravitacional, Montevideo, Uruguay. 3 Centro de Terapia Gravitacional, Montevideo, Uruguay.
DOI: 10.1016/j.reumae.2024.10.004
PMID: 39487058
Pubmed: https://pubmed.ncbi.nlm.nih.gov/39487058
Texto completo: http://www.elsevier.es/en/linksolver/pdf/pii/S2173-5743(24)00137-0
Abstract:
Background: The exposure to artificial gravity (AG) through human centrifugation is the basis of the treatment called gravity therapy (GT), in which the mechanical stimulation over the vessel wall, induces the synthesis and release of prostacyclin. It has been used for more than four decades in Uruguay in the treatment of different vascular-based pathologies. In patients with systemic sclerosis (SSc) it has shown good benefits and excellent safety profile over the years. However, there is a lack of knowledge in the scientific community about GT and its results.
Brain atlas of the annual Garcialebias charrua fish
Anat Rec (Hoboken) 2024 Oct;307(10):3384-3397.
Maximiliano Torres-Pérez 1 2 , María Laura Herrera 1 , Juan Carlos Rosillo 1 3 , Inés Berrosteguieta 1 , Gabriela Casanova 4 , Silvia Olivera-Bravo 2 , Anabel Sonia Fernández 1 5
1 División Neurociencias, Departamento de Neurociencias Integrativas y Computacionales, Laboratorio de Neurobiología Comparada, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay. 2 División Neurociencias, Departamento de Neurobiología y Neuropatología, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Montevideo, Uruguay. 3 Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República (UdelaR), Montevideo, Uruguay. 4 Unidad de Microscopía Electrónica, Facultad de Ciencias, Universidad de la República (UdelaR), Montevideo, Uruguay. 5 Laboratorio de Neurociencias, Facultad de Ciencias, Universidad de la República (UdelaR), Montevideo, Uruguay.
DOI: 10.1002/ar.25432
PMID:
Pubmed: https://pubmed.ncbi.nlm.nih.gov/
Texto completo: https://doi.org/10.1002/ar.25432
Abstract:
Annual fish have become attractive study models for a wide range of disciplines, including neurobiology. These fish have developed different survival strategies. As a result, their nervous system is under considerable selective pressure when facing extreme environmental situations. Fish from the Austrolebias group exhibit rapid neurogenesis in different brain regions, possibly as a result of the demanding conditions of a changing habitat. Knowledge of cerebral histology is essential for detecting ontogenic, anatomical, or cytoarchitectonic changes in the brain during the short lifespan of these fish, such as those reflecting functional adaptive plasticity in different systems, including sensory structures. The generation of an atlas of Garcialebias charrua (previously known as Austrolebias charrua) establishes its anatomical basis as a representative of a large group of fish that share similarities in their way of life. In this work, we present a detailed study of both gross anatomy and microscopic anatomy obtained through serial sections stained with the Nissl technique in three orientations: transverse, horizontal, and parasagittal planes. This atlas includes accurate drawings of the entire adult brain of the male fish Garcialebias charrua, showing dorsal, ventral, and lateral views, including where emergence and origin of cranial nerves. This brain atlas allows us to understand histoarchitecture as well as the location of neural structures that change during adult neurogenesis, enabling comparisons within the genus. Simultaneously, this atlas constitutes a valuable tool for comparing the brains of other fish species with different behaviors and neuroecologies.
Cerebral White Matter Alterations Associated With Oligodendrocyte Vulnerability in Organic Acidurias: Insights in Glutaric Aciduria Type I
Neurotox Res 2024 Jul 4;42(4):33
Eugenia Isasi 1 2 , Moacir Wajner 3 4 , Juliana Avila Duarte 5 , Silvia Olivera-Bravo 6
1 Laboratorio de Neurobiología Celular y Molecular, Unidad Académica de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay. 2 Departamento de Neurobiología y Neuropatología, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Montevideo, Uruguay. 3 Department of Biochemistry, Instituto de Ciencias Básicas da Saude, Universidade Federal de Río Grande do Sul, Porto Alegre, Brazil. 4 Medical Genetics Service, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil. 5 Departamento de Medicina Interna, Serviço de Radiología, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil. 6 Departamento de Neurobiología y Neuropatología, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Montevideo, Uruguay. solivera@iibce.edu.uy.
DOI: 10.1007/s12640-024-00710-6
PMID: 38963434
Pubmed: https://pubmed.ncbi.nlm.nih.gov/38963434
Texto completo: https://dx.doi.org/10.1007/s12640-024-00710-6
Abstract:
The white matter is an important constituent of the central nervous system, containing axons, oligodendrocytes, and its progenitor cells, astrocytes, and microglial cells. Oligodendrocytes are central for myelin synthesis, the insulating envelope that protects axons and allows normal neural conduction. Both, oligodendrocytes and myelin, are highly vulnerable to toxic factors in many neurodevelopmental and neurodegenerative disorders associated with disturbances of myelination. Here we review the main alterations in oligodendrocytes and myelin observed in some organic acidurias/acidemias, which correspond to inherited neurometabolic disorders biochemically characterized by accumulation of potentially neurotoxic organic acids and their derivatives. The yet incompletely understood mechanisms underlying the high vulnerability of OLs and/or myelin in glutaric acidemia type I, the most prototypical cerebral organic aciduria, are particularly discussed.
The complement system in neurodegenerative and inflammatory diseases of the central nervous system
Front Neurol 2024 Jul 3:15:1396520
Luciana Negro-Demontel # 1 2 3 , Adam F Maleki # 1 4 , Daniel S Reich 4 , Claudia Kemper 1
1 National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH), Complement and Inflammation Research Section (CIRS), Bethesda, MD, United States. 2 Department of Histology and Embryology, Faculty of Medicine, UDELAR, Montevideo, Uruguay. 3 Neuroinflammation and Gene Therapy Laboratory, Institut Pasteur de Montevideo, Montevideo, Uruguay. 4 Translational Neuroradiology Section, National Institute of Neurological Disorders and Stroke (NINDS), NIH, Bethesda, MD, United States. # Contributed equally.
DOI: 10.3389/fneur.2024.1396520
PMID: 39022733
Pubmed: https://pubmed.ncbi.nlm.nih.gov/39022733
Texto completo: https://doi.org/10.3389/fneur.2024.1396520
Abstract:
Neurodegenerative and neuroinflammatory diseases, including Alzheimer's disease, Parkinson's disease, and multiple sclerosis, affect millions of people globally. As aging is a major risk factor for neurodegenerative diseases, the continuous increase in the elderly population across Western societies is also associated with a rising prevalence of these debilitating conditions. The complement system, a crucial component of the innate immune response, has gained increasing attention for its multifaceted involvement in the normal development of the central nervous system (CNS) and the brain but also as a pathogenic driver in several neuroinflammatory disease states. Although complement is generally understood as a liver-derived and blood or interstitial fluid operative system protecting against bloodborne pathogens or threats, recent research, particularly on the role of complement in the healthy and diseased CNS, has demonstrated the importance of locally produced and activated complement components. Here, we provide a succinct overview over the known beneficial and pathological roles of complement in the CNS with focus on local sources of complement, including a discussion on the potential importance of the recently discovered intracellularly active complement system for CNS biology and on infection-triggered neurodegeneration.
Pyruvate dehydrogenase kinase 2 knockdown restores the ability of amyotrophic lateral sclerosis-linked SOD1G93A rat astrocytes to support motor neuron survival by increasing mitochondrial respiration
Glia 2024 May;72(5):999-1011
Ernesto Miquel 1 , Rosalía Villarino 1 , Laura Martínez-Palma 1 , Adriana Cassina 2 , Patricia Cassina 1
1 Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay. 2 Departamento de Bioquímica, Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Montevideo, Uruguay.
DOI: 10.1002/glia.24516
PMID: 38372421
Pubmed: https://pubmed.ncbi.nlm.nih.gov/38372421
Texto completo: https://doi.org/10.1002/glia.24516
Abstract:
Amyotrophic lateral sclerosis (ALS) is characterized by progressive motor neuron (MN) degeneration. Various studies using cellular and animal models of ALS indicate that there is a complex interplay between MN and neighboring non-neuronal cells, such as astrocytes, resulting in noncell autonomous neurodegeneration. Astrocytes in ALS exhibit a lower ability to support MN survival than nondisease-associated ones, which is strongly correlated with low-mitochondrial respiratory activity. Indeed, pharmacological inhibition of pyruvate dehydrogenase kinase (PDK) led to an increase in the mitochondrial oxidative phosphorylation pathway as the primary source of cell energy in SOD1G93A astrocytes and restored the survival of MN. Among the four PDK isoforms, PDK2 is ubiquitously expressed in astrocytes and presents low expression levels in neurons. Herein, we hypothesize whether selective knockdown of PDK2 in astrocytes may increase mitochondrial activity and, in turn, reduce SOD1G93A-associated toxicity. To assess this, cultured neonatal SOD1G93A rat astrocytes were incubated with specific PDK2 siRNA. This treatment resulted in a reduction of the enzyme expression with a concomitant decrease in the phosphorylation rate of the pyruvate dehydrogenase complex. In addition, PDK2-silenced SOD1G93A astrocytes exhibited restored mitochondrial bioenergetics parameters, adopting a more complex mitochondrial network. This treatment also decreased lipid droplet content in SOD1G93A astrocytes, suggesting a switch in energetic metabolism. Significantly, PDK2 knockdown increased the ability of SOD1G93A astrocytes to support MN survival, further supporting the major role of astrocyte mitochondrial respiratory activity in astrocyte-MN interactions. These results suggest that PDK2 silencing could be a cell-specific therapeutic tool to slow the progression of ALS.
Miquel_2024_Pyruvate dehydrogenase kinase 2 knockdown astrocytes ALS.pdf
Adult aberrant astrocytes submitted to late passage cultivation lost differentiation markers and decreased their pro-inflammatory profile
Heliyon 2024 Apr 26;10(9):e30360
Gabriel Otero 1 , Carmen Bolatto 2 1 , Eugenia Isasi 1 2 , Sofía Cerri 1 , Paola Rodríguez 1 , Daniela Boragno 1 , Marta Marco 1 3 , Cristina Parada 2 , Matías Stancov 1 , María Noel Cuitinho 1 , Silvia Olivera-Bravo 1
1 Department of Neurobiology and Neuropathology (NBNP), Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Montevideo, Uruguay. 2 Department of Histology and Embryology, Facultad de Medicina, Universidad de la República (UdelaR), Montevideo, Uruguay. 3 Department of Clinical Biochemistry, School of Chemistry (UdelaR), Montevideo, Uruguay.
DOI: 10.1016/j.heliyon.2024.e30360
PMID: 38711658
Pubmed: https://pubmed.ncbi.nlm.nih.gov/38711658
Texto completo: https://linkinghub.elsevier.com/retrieve/pii/S2405-8440(24)06391-6
Abstract:
In amyotrophic lateral sclerosis (ALS), astrocytes are considered key players in some non-cell non-neuronal autonomous mechanisms that underlie motor neuron death. However, it is unknown how much of these deleterious features were permanently acquired. To assess this point, we evaluated if the most remarkable features of neurotoxic aberrant glial phenotypes (AbAs) isolated from paralytic rats of the ALS model G93A Cu/Zn superoxide dismutase 1 (SOD1) could remain upon long lasting cultivation. Real time PCR, immunolabelling and zymography analysis showed that upon many passages, AbAs preserved the cell proliferation capacity, mitochondrial function and response to different compounds that inhibit some key astrocyte functions but decreased the expression of parameters associated to cell lineage, homeostasis and inflammation. As these results are contrary to the sustained inflammatory status observed along disease progression in SOD1G93A rats, we propose that the most AbAs remarkable features related to homeostasis and neurotoxicity were not permanently acquired and might depend on the signaling coming from the injuring microenvironment present in the degenerating spinal cord of terminal rats.
Mitochondrial function and reactive oxygen species production during human sperm capacitation: Unraveling key players
FASEB J 2024 Feb 29;38(4):e23486
Pilar Irigoyen 1 2 , Santiago Mansilla 2 3 , Laura Castro 2 4 , Adriana Cassina 2 4 , Rossana Sapiro 1 2
1 Unidad Académica Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay. 2 Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Montevideo, Uruguay. 3 Departamento de Métodos Cuantitativos, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay. 4 Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay.
DOI: 10.1096/fj.202301957RR
PMID: 38407497
Pubmed: https://pubmed.ncbi.nlm.nih.gov/38407497
Texto completo: https://doi.org/10.1096/fj.202301957RR
Abstract:
Sperm capacitation is a critical process for male fertility. It involves a series of biochemical and physiological changes that occur in the female reproductive tract, rendering the sperm competent for successful fertilization. The precise mechanisms and, specifically, the role of mitochondria, in sperm capacitation remain incompletely understood. Previously, we revealed that in mouse sperm mitochondrial activity (e.g., oxygen consumption, membrane potential, ATP/ADP exchange, and mitochondrial Ca2+ ) increases during capacitation. Herein, we studied mitochondrial function by high-resolution respirometry (HRR) and reactive oxygen species production in capacitated (CAP) and non-capacitated (NC) human spermatozoa. We found that in capacitated sperm from normozoospermic donors, the respiratory control ratio increased by 36%, accompanied by a double oxygen consumption rate (OCR) in the presence of antimycin A. Extracellular hydrogen peroxide (H2 O2 ) detection was three times higher in CAP than in NC sperm cells. To confirm that H2 O2 production depends on mitochondrial superoxide (
Vasotocin but not isotocin is involved in the emergence of the dominant-subordinate status in males of the weakly electric fish, Gymnotus omarorum
Horm Behav 2024 Feb:158:105446
Paula Pouso 1 , Álvaro Cabana 2 , Virginia Francia 3 , Ana Silva 4
1 Depto Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo 11800, Uruguay; Unidad Bases Neurales de la Conducta, Departamento de Neurofisiología Celular y Molecular, IIBCE, Montevideo 11600, Uruguay. 2 Instituto de Fundamentos y Métodos, Facultad de Psicología, Universidad de la República, Montevideo 11800, Uruguay. 3 Depto Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo 11800, Uruguay. 4 Unidad Bases Neurales de la Conducta, Departamento de Neurofisiología Celular y Molecular, IIBCE, Montevideo 11600, Uruguay; Laboratorio de Neurociencias, Facultad de Ciencias, Universidad de la República, Montevideo 11400, Uruguay. Electronic address: asilva@fcien.edu.uy.
DOI: 10.1016/j.yhbeh.2023.105446
PMID: 37945472
Pubmed: https://pubmed.ncbi.nlm.nih.gov/37945472
Texto completo: https://linkinghub.elsevier.com/retrieve/pii/S0018-506X(23)00144-7
Abstract:
The establishment of the dominant-subordinate status implies a clear behavioral asymmetry between contenders that arises immediately after the resolution of the agonistic encounter and persists during the maintenance of stable dominance hierarchies. Changes in the activity of the brain social behavior network (SBN) are postulated to be responsible for the establishment and maintenance of the dominant-subordinate status. The hypothalamic nonapeptides of the vasopressin (AVP) and oxytocin (OT) families are known to modulate the activity of the SBN in a context-dependent manner across vertebrates, including status-dependent modulations. We searched for status-dependent asymmetries in AVP-like (vasotocin, AVT) and OT-like (isotocin, IT) cell number and activation immediately after the establishment of dominance in males of the weakly electric fish, Gymnotus omarorum, which displays the best understood example of non-breeding territorial aggression among teleosts. We used immunolabeling (FOS, AVT, and IT) of preoptic area (POA) neurons after dyadic agonistic encounters. This study is among the first to show in teleosts that AVT, but not IT, is involved in the establishment of the dominant-subordinate status. We also found status-dependent subregion-specific changes of AVT cell number and activation. These results confirm the involvement of AVT in the establishment of dominance and support the speculation that AVT is released from dominants' AVT neurons.
Insights into the Y chromosome human diversity in Uruguay
Am J Hum Biol 2023 35(12):e23963
Patricia Mut 1 , Bernardo Bertoni 2 , Rossana Sapiro 3 , Pedro C Hidalgo 4 , Alejandra Torres 5 , Carlos Azambuja 5 , Mónica Sans 1
1 Departamento de Antropología Biológica, Facultad de Humanidades y Ciencias de la Educación, UdelaR, Montevideo, Uruguay. 2 Departamento de Genética, Facultad de Medicina, UdelaR, Montevideo, Uruguay. 3 Departamento de Histología y Embriología, Facultad de Medicina, UdelaR, Montevideo, Uruguay. 4 Polo de Desarrollo Universitario Diversidad Genética Humana, Centro Universitario Noreste, Tacuarembó, Uruguay. 5 Genia-Genetics Molecular Laboratory, Montevideo, Uruguay.
DOI: 10.1002/ajhb.23963
PMID: 37493343
Pubmed: https://pubmed.ncbi.nlm.nih.gov/37493343
Texto completo: https://doi.org/10.1002/ajhb.23963
Abstract:
Background: With regard to the origin of its population and microevolutionary processes, Uruguay exhibits distinctive features that distinguish it from other countries in Latin America, while at the same time sharing several similarities. In this article, we will focus on the variability of paternal genetic lineages in two geographical regions with different histories that can be considered as examples of distinct populations for the continent. In general terms, the genetic diversity is a result of different demographic processes related to the American conquest and colonisation. These resulted in distinct ancestral components which vary geographical and depend on the distribution by sex within these components. In Uruguay, native maternal haplogroups are significantly more frequent in the North. Although there are several studies about the geneticvariability of Uruguay, little is known about male genetic lineages.
Aims: The aim of this work is to present an updated study of the male genetic variability of the Uruguayan population.
Methods: We analyzed 13 biallelic markers and 27 STRs located in the male-specific region of the Y chromosome for 157 males: 98 from the capital, Montevideo, and 59 from Tacuarembó.
Results: Almost all haplogroups found in both locations are European (99% and 93.2% respectively). One Sub-Saharan African haplogroup was found in Montevideo (1%) and 2 in Tacuarembó (3%), while Native haplogroups were found only in Tacuarembó, evidencing a strong sex-biased admixture. By crossing genetic and genealogical information we could relate European haplogroups with different waves and times of migrations.
Discussion: Network analysis indicated a very diverse male population, suggesting that European migrants came from heterogeneous geographic locations and in different waves. Tacuarembó has closer population affinities with Iberian populations while Montevideo is more diverse. Male population expansion expansion, can be explained by the large number of migrants that arrived during the XIX century and the first half of the XX century.
Conclusions: The Uruguayan male gene pool is the result of several migration waves with diverse origins, with strong sex-biased admixture that can be explained by the European migration, the violence against the indigenous males, and the segregation of the Africansadmixture that can be explained due to European migration, violence against Natives, and segregation against African males.admixture that can be explained due to European migration, violence against Natives, and segregation against African males.admixture that can be explained due to European migration, violence against Natives, and segregation against African males.admixture that can be explained due to European migration, violence against Natives, and segregation of hte Africans.