Involvement of Histone Acetyltransferase 1 (HAT1) in the Spermatogenesis of Non-Condensed Nuclear Sperm in Chinese Mitten Crab, Eriocheir sinensis.

Chinese mitten crab, Eriocheir sinensis, is a decapod crustacean with a special, non-condensated nucleus in the sperm. Studies have shown that the nuclear compact state of male germ cells during the spermatogenesis is closely related to histone modification. To explore the possible role of histone acetyltransferase 1 (HAT1) in the chromatin organization during the E. sinensis spermatogenesis, we took the testis tissues of both adult and juvenile crabs as the materials of study and analyzed the biological functions of HAT1 by whole transcriptome sequencing and bioinformatics, then further analyzed the expression and distribution of HAT1 using the methods of RT-qRCR, western blotting, and immunofluorescence location. The results showed that HAT1 is an alkaline-unstable hydrophilic protein. It was predicted to interact with a variety of histones and chromosome assembly proteins, including Asf1b, Chaf1b, and Hist1h3f, and is involved in many biological functions pertaining to chromatin dynamics such as chromatin organization, DNA dependent nucleosome assembly, DNA conformational changes, and so on. HAT1 was up-regulated in the adult testes compared to the juvenile (n = 3, P < 0.05). HAT1 was mainly located in the nuclei of male germ cells of E. sinensis. As spermatogenesis proceeded, the expression of HAT1 decreased and even disappeared in the nuclei (n = 3, P < 0.05). HAT1 is an important player in histone acetylation, which facilitates chromatin alteration in a three-dimensional conformation. The expression of HAT1 in different male germ cells might indicate the chromatin dynamics at the diversity stages of spermatogenesis. The high expression of HAT1 at the early stages of E. sinensis spermatogenesis hints the active involvement in chromatin organization, while its progressively reduced expression accompanied by the progression of spermatogenesis suggests a relatively gradual stabilization and stereotyping of chromatin. As for the disappearance of HAT1 in mature sperm with non-condensed nuclei, the reduction in histones targeted by HAT1 or histone acetylation may be an important initiator.

miR-124-3p regulates the involvement of Ptpn1 in testicular development and spermatogenesis in mouse.

Testicular development and spermatogenesis in mouse are a complex process in which phosphorylation modifications and regulation of genes by non-coding RNAs play an important role. However, protein tyrosine phosphatase, non-receptor type 1 (Ptpn1) is widely expressed in mammalian tissues. In this study, we analyzed the expression of Ptpn1 mRNA and its encoded proteins in testicular tissues of juvenile and adult mice by using experimental techniques such as biological information, real-time fluorescence quantitative PCR (RT-qPCR), western blot (WB), immunofluorescence (IF) and transfection, and further analyzed the possible target-regulatory relationship and regulatory mechanisms of miR-124-3p and Ptpn1. We found that Ptpn1 mRNA and its encoded protein were up-regulated in adult mouse testis compared to juvenile mouse testis. The expression trend of miR-124-3p was opposite to that of Ptpn1. In other cell types, Ptpn1 protein is localized in cell membrane, cytoplasm, endoplasmic reticulum and cytoplasmic vesicles. Immunofluorescence showed that Ptpn1 protein was mainly localized in the cytoplasm of male germ cells and was expressed at a high level in early-stage cells (spermatogonia) and at a low level in late-stage cells (sperm). Transfection results showed that the expression levels of Ptpn1 mRNA and its protein were significantly down-regulated after miR-124-3p overexpression in mouse spermatogonia. Bioinformatics analysis showed that Ptpn1 can involved in biological processes such as protein kinase inactivation through peptidyl tyrosine dephosphorylation. The reduction of miR-124-3p may be a key factor in promoting the high expression of Ptpn1 in testicular tissues of adult mice. Increased miR-124-3p may be a key factor in suppressing Ptpn1 expression in the mouse spermatogonia mimics group. The differential expression results from the negative regulation of miR-124-3p.

Expression, localization, and function of P4HB in the spermatogenesis of Chinese mitten crab (Eriocheir sinensis).

Background: The sperm of Chinese mitten crab (Eriocheir sinensis) have special noncondensed nuclei. The formation and stability of the special nuclei are closely related to the correct folding of proteins during spermatogenesis. P4HB plays a key role in protein folding, but its expression and role in the spermatogenesis of E. sinensis are unclear. Objective: To investigate the expression and distribution characteristics of P4HB in the spermatogenesis of E. sinensis as well as its possible role. Methods: The testis tissues of adult and juvenile E. sinensis were used as materials. We utilized a variety of techniques, including homology modeling, phylogenetic analysis, RT-qPCR, western blotting, and immunofluorescence staining to predict the protein structure and sequence homology of P4HB, analyze its expression in the testis tissues, and localize and semi-quantitatively assess its expression in different male germ cells. Results: The sequence of P4HB protein in E. sinensis shared a high similarity of 58.09% with the human protein disulfide isomerase, and the phylogenetic tree analysis indicated that the protein sequence was highly conserved among crustaceans, arthropods, and other animals species. P4HB was found to be expressed in both juvenile and adult E. sinensis testis tissues, with different localization patterns observed all over the developmental stages of male germ cells. It was higher expressed in the spermatogonia, spermatocytes, and stage I spermatids, followed by the mature sperm than in the stage II and III spermatids. The subcellular localization analysis revealed that P4HB was predominantly expressed in the cytoplasm, cell membrane, and extracellular matrix in the spermatogonia, spermatocytes, stage I and stage II spermatids, with some present in specific regions of the nuclei in the spermatogonia. In contrast, P4HB was mainly localized in the nuclei of stage III spermatids and sperm, with little expression observed in the cytoplasm. Conclusion: P4HB was expressed in the testis tissues of both adult and juvenile E. sinensis, but the expression and localization were different in male germ cells at various developmental stages. The observed differences in the expression and localization of P4HB may be an essential factor in maintaining the cell morphology and structure of diverse male germ cells in E. sinensis. Additionally, P4HB expressed in the nuclei of spermatogonia, late spermatids, and sperm may play an indispensable role in maintaining the stability of the noncondensed spermatozoal nuclei in E. sinensis.

Role of cytoskeleton-related proteins in the acrosome reaction of Eriocheir sinensis spermatozoa.

Cytoskeleton-related proteins are essential for cell shape maintenance and cytoskeleton remodeling. The spermatozoa of Eriocheir sinensis (Chinese mitten crab) have a unique cellular structure, and the mechanism of spermatozoal metamorphosis during the acrosome reaction is not well understood. In this study, the E. sinensis spermatozoa were induced using calcium ionophore A23187 to undergo the acrosome reaction in vitro, and the acrosome-reacting and fresh (non-reacting) spermatozoa were collected separately. The differential expression of cytoskeleton-related protein genes in acrosome-reacting and fresh spermatozoa of E. sinensis was analyzed by whole transcriptome sequencing and bioinformatics analysis, and PPI network and miRNA-mRNA regulation network were constructed to analyze their possible function and regulation mechanism. The results showed that numerous differentially expressed cytoskeleton-related protein genes, miRNAs and lncRNAs were found in acrosome-reacting and fresh spermatozoa of E. sinensis; 27 cytoskeleton-related protein genes were down regulated and 687 miRNAs were up regulated in acrosome-reacting spermatozoa; 147 miRNAs target these 27 cytoskeleton-related protein genes. In the PPI networks, RAC1, BCAR1, RDX, NCKAP1, EPS8, CDC42BPA, LIMK1, ELMO2, GNAI1 and OCRL were identified as hub proteins. These proteins are mainly involved in the regulation of cytoskeleton organization, actin cytoskeleton organization, microtubule skeleton organization and small GTPase-mediated signal transduction and other biological processes, and play roles in pathways such as actin cytoskeletal regulation and axon guidance. miR-9, miR-31 and two novel miRNAs in the miRNA-mRNA regulatory network are the core miRNAs targeting cytoskeleton-related protein genes. miR-9 targets and regulates OBSCN, CDC42BPA, ELMO2, BCAS3, TPR and OCRL; while miR-31 targets and regulates CDC42BPA and TPR. This study provides a theoretical basis for revealing the mechanism of acrosome reaction under the special spermatozoa morphology of E. sinensis.

Microenvironment Analysis of Prognosis and Molecular Signature of Immune-Related Genes in Lung Adenocarcinoma.

There is growing evidence on the clinical significance of tumor microenvironment (TME) cells in predicting prognosis and therapeutic effects. However, cell interactions in tumor microenvironments have not been thoroughly studied or systematically analyzed so far. In this study, 22 immune cell components in the lung adenocarcinoma (LUAD) TME were analyzed using gene expression profile from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). The TME-based molecular subtypes of LUAD were defined to evaluate further the relationship between molecular subtypes, prognosis, and clinical characteristics. A TME risk score model was constructed by using the differentially expressed genes (DEGs) of molecular subtypes. The relationship between the TME score and clinical characteristics and genomic mutations was compared to identify the genes that have significant associations with the TME. The comprehensive analysis of the TME characteristics may be helpful in revealing the response of LUAD patients to immunotherapy, providing a new strategy for immunotherapy.

Genes Induced by Panax Notoginseng in a Rodent Model of Ischemia-Reperfusion Injury.

Stroke is a cerebrovascular disease that results in decreased blood flow. Although Panax notoginseng (PN), a Chinese herbal medicine, has been proven to promote stroke recovery, its molecular mechanism remains unclear. In this study, middle cerebral artery occlusion (MCAO) was induced in rats with thrombi generated by thread and subsequently treated with PN. After that, staining with 2,3,5-triphenyltetrazolium chloride was employed to evaluate the infarcted area, and electron microscopy was used to assess ultrastructural changes of the neurovascular unit. RNA-Seq was performed to determine the differential expressed genes (DEGs) which were then verified by qPCR. In total, 817 DEGs were identified to be related to the therapeutic effect of PN on stroke recovery. Further analysis by Gene Oncology analysis and Kyoto Encyclopedia of Genes and Genomes revealed that most of these genes were involved in the biological function of nerves and blood vessels through the regulation of neuroactive live receptor interactions of PI3K-Akt, Rap1, cAMP, and cGMP-PKG signaling, which included in the 18 pathways identified in our research, of which, 9 were reported firstly that related to PN's neuroprotective effect. This research sheds light on the potential molecular mechanisms underlying the effects of PN on stroke recovery.

RNA binding proteins involved in regulation of protein synthesis to initiate biogenesis of secondary tumor in hepatocellular carcinoma in mice.

BACKGROUND: The tumor microenvironment (TM) in close contact with cancer cells is highly related to tumor growth and cancer metastasis. This study is to explore the biogenesis mechanism of a secondary hepatocellular carcinoma (HCC) based on the function of RNA binding proteins (RBPs)-encoding genes in the physiological microenvironment (PM). METHODS: The healthy and HCC mice were used to isolate the PM, pre-tumor microenvironment (PTM), and TM. The samples were analyzed using the technology of RNA-seq and bioinformatics. The differentially expressed RBPs-encoding genes (DERs) and differentially expressed DERs-associated genes (DEDs) were screened to undergo GO and KEGG analysis. RESULTS: 18 DERs and DEDs were identified in the PTM vs. PM, 87 in the TM vs. PTM, and 87 in the TM vs. PM. Those DERs and DEDs participated in the regulation of gene expression at the levels of chromatin conformation, gene activation and silencing, splicing and degradation of mRNA, biogenesis of piRNA and miRNA, ribosome assemble, and translation of proteins. CONCLUSION: The genes encoding RBPs and the relevant genes are involved in the transformation from PM to PTM, then constructing the TM by regulating protein synthesis. This regulation included whole process of biological genetic information transmission from chromatin conformation to gene activation and silencing to mRNA splicing to ribosome assemble to translation of proteins and degradation of mRNA. The abnormality of those functions in the organic microenvironments promoted the metastasis of HCC and initiated the biogenesis of a secondary HCC in a PM when the PM encountered the invasion of cancer cells.

Pretumor microenvironment of hepatocellular carcinoma: Cancerization or anticancerization?

BACKGROUND: Tumor microenvironment (TM) has been deeply concerned. However, the pretumor microenvironment (PTM) was poorly understood. The purpose in this study was to explore the possible pathophysiological features of PTM before hepatocellular carcinoma (HCC) appearance. METHODS: Mouse livers with no swelling but with tumors present elsewhere in the body after subcutaneous injection of H22 in the fore underarm were considered a PTM, HCC tumors presenting far away from the PTM were regarded as a TM, and the healthy livers of mice without injection of H22 were regarded as a physiological microenvironment (PM). The transcriptomes of samples were generated using RNA-seq and validated using RT-qPCR. RESULTS: Overall, 4483 differentially expressed genes (DEGs) were found in the TM compared with the PTM (TM/PTM), but only 194 were altered in the PTM compared with the PM (PTM/PM). Among those 194 DEGs, 104 displayed upregulation and 90 downregulation. Some of these DEGs could promote the ability to resist cancerization or facilitate cancer metastasis, while others indicated liver impairment. The DEGs were involved in 16 relevant pathways. Additionally, the frequency of alternative splicing (AS) in the DEGs in various samples was positively related to the expression of those DEGs. CONCLUSIONS: The PTM initiatively armed itself to combat cancerization when its indications appeared although the PTM did not manifest any tissue swelling. However, the cancer cells were negatively influencing immunity to prevent clearance and positively promoting transformation to construct a suitable environment. During transformation by cancer cells, some genes with acquired AS participated in the construction of the PTM. This alteration created an invadable space and an appropriate environment for cancer cells.

Extraction and characterization of dextran from Leuconostoc pseudomesenteroides YB-2 isolated from mango juice.

An exopolysaccharide (EPS)-producing strain of YB-2 isolated from mango juice was identified as Leuconostoc pseudomesenteroides. The molecular weight (Mw) of this EPS was 7.67×105 Da. Gas chromatography (GC) analysis confirmed the presence of only glucose monomers. Fourier transform infrared (FT-IR) spectroscopy and nuclear magnetic resonance (NMR) spectra displayed the glucan nature of the EPS with 96.8% α-(1→6) and 3.2% branching α-(1→3) linkages. Scanning electron microscopy (SEM) showed smooth surfaces and compact structure. The water solubility index (WSI) and water-holding capacity (WHC) of dextran were 97.48±2.46% and 287.51±7.93%, respectively. The rheological analysis of dextran elucidated a non-Newtonian pseudoplastic behavior. The dextran revealed an inhibitory activity against Escherichia coli and Staphylococcus aureus with minimal inhibitory concentrations (MIC) of 2.0 mg/mL and 3.0 mg/mL, respectively.

Identification of a Novel ACTN4 Gene Mutation Which Is Resistant to Primary Nephrotic Syndrome Therapy.

ACTN4, a gene which codes for the protein α-actinin-4, is critical for the maintenance of the renal filtration barrier. It is well known that ACTN4 mutations can lead to kidney dysfunction, such as familial focal segmental glomerulosclerosis (FSGS), a common cause of primary nephrotic syndrome (PNS). To elucidate whether other mutations of ACTN4 exist in PNS patients, we sequenced the ACTN4 gene in biopsies collected from 155 young PNS patients (≤16 years old). The patients were classified into five groups: FSGS, minimal change nephropathy, IgA nephropathy, membranous nephropathy, and those without renal puncture. Ninety-eight healthy people served as controls. Samples were subjected to Illumina's next generation sequencing protocols using FastTarget target gene capture method. We identified 5 ACTN4 mutations which occurred only in PNS patients: c.1516G > A (p.G506S) on exon 13 identified in two PNS patients, one with minimal change nephropathy and another without renal puncture; c.1442 + 10G > A at the splice site in a minimal change nephropathy patient; c.2191-4G > A at the cleavage site, identified from two FSGS patients; and c.1649A > G (p.D550G) on exon 14 together with c.2191-4G > A at the cleavage sites, identified from two FSGS patients. Among these, c.1649A > G (p.D550G) is a novel ACTN4 mutation. Patients bearing the last two mutations exhibited resistance to clinical therapies.

Hydroxymethylation of protein-encoding genes in the testes involved in precocious puberty of Eriocheir sinensis.

To investigate the possible effects of epigenetic modification of testis protein-encoding genes on precocious puberty of Eriocheir sinensis, we used MeDIP-seq and hMeDIP-seq techniques to compare the methylation and hydroxymethylation of 263 E. sinensis protein-encoding genes known in the NCBI database in precocious testes with those in normally developing testes. The results showed that total methylation level of those genes was lower than their total hydroxymethylation level. Moreover, their total hydroxymethylation level in precocious testes was significantly lower than that in normal testes. In addition, no methylated genes had significant difference, but there were 37 different hydroxymethylated genes (DhMGs) in the precocious testes compared to the normal ones. Among the DhMGs, 21 were hypo-hydroxymethylated and 16 were hyper-hydroxymethylated. The hypo-hydroxymethylated DhMGs were associated with development, cell structural and cytoskeletal proteins, and response to stress. However, the hyper-hydroxymethylated DhMGs included immune-related genes, free radicals removement-related genes, protein folding-related genes, and so on. In addition, some DhMGs were hyper-hydroxymethylated while their homologous DhMGs were hypo-hydroxymethylated. The results of a qRT-PCR assay showed that the expression levels of 5 DhMGs randomly chosen presented a positive correlation with their hydroxymethylation levels. It can be seen that hydroxymethylation might regulate the expression of genes and be involved in precocious puberty to cause high mortality of crabs. Therefore, the hydroxymethylation level of DhMGs may be used as an evaluation index with economically meaningful growth and breeding traits.

MeDIP-seq reveals the features of mitochondrial genomic methylation in immature testis of Chinese mitten crab Eriocheir sinensis.

In this study, the methylation of mitochondrial genome in the immature testis of Chinese mitten crab Eriocheir sinensis of the Yangtze River system was determined for the first time using MeDIP-seq. Our methylated DNA fragments covered more than 99% of the mitochondrial genome in E. sinensis loaded from GenBank. There were 8 mutated bases and 42 SNPs in the crab mitochondrial genome. The methylation presented in all genes as well as in an A + T region, but less in intergenic regions in the mitochondrial genome. However, the level of methylation of most genes coding proteins and the A + T region were high. But, the majority of genes encoding tRNAs were hypomethylated, and both the rRNA genes also showed methylation of low or median frequency. Especially, the level of methylation of the intergenic regions is the lowest. Those features indicated that the methylation of DNA may play an important role in gene expressing regulation in the mitochondrial genome of immature testis in E. sinensis.

Hypo-hydroxymethylation of rRNA genes in the precocious Eriocheir sinensis testes revealed using hMeDIP-seq.

Precocious puberty is a common phenomenon in crab breeding that seriously reduces the economic benefits for crab farmers. To address this problem, this study aimed to explore the potential functions of both methylation and hydroxymethylation of testis rRNA genes with respect to precocious puberty in Eriocheir sinensis. The results showed that the rRNA genes in normally developing testes of E. sinensis had low levels of methylation and high levels of hydroxymethylation; however, although methylation levels were similar, the level of hydroxymethylation in precocious testes was lower than normal. Highly significant differences (P < 0.01) in the hydroxymethylation of the 18S and 28S rRNA genes were found between precocious and normal testes. Our results suggested that both the 18S and 28S rRNA genes, which are normally downregulated by hypo-hydroxymethylation, might be involved in the process of precocious puberty. Our results also implied that hydroxymethylation of the 18S and 28S rRNA genes might be used as an important epigenetic molecular marker to evaluate economically significant potential for growth and breeding in this species.

Transcriptome using Illumina sequencing reveals the traits of spermatogenesis and developing testes in Eriocheir sinensis.

Chinese mitten crab (Eriocheir sinensis) has the spermatozoa with typical aflagellate, decondensed chromatin, cup-shaped nuclei, and radial arms. However, the mechanism of spermatogenesis during which the specific spermatozoa are generated in this species is yet unclear. Here, the transcriptome of developing testis in E. sinensis was analyzed using the ways of RNA-seq and bioinformatics analysis to identify candidate genes potentially involved in development of testis and spermatogenesis. The Illumina HiSeq2500 sequencing of three replicons of samples produced a total of 145.19 M clean reads representing with a total of 21.34 Gb bases and 45.48% GC content. 56.30% clean reads were mapped to the draft genome of E. sinensis. The assembly of the transcriptome yielded contigs of 5691802 sequences and unigenes of 406527 sequences. Total 24246 and 40793 transcripts were annotated using Swissprot and Nr database, respectively. There were 48213 (70.31%) and 7858 (46.25%) transcripts with identity of more than 99 matching to mature testis unigenes in the databases of Nr and EST, respectively. The analytic results of KOG, GO and KEGG showed wide potential molecular functions of transcripts in the developing testes. KEGG analysis of unigenes yielded total 9422 predicted genes. Those predicted genes were involved in total 216 KEGG pathways related to the physiological activities of developing testis. 1975 predicted genes were involved in cellular and subcellular structural alteration of male germ cells. There were important roles of some pathways in the processes of morphological and structural biogenesis pertaining to testis development and spermatogenesis. Other 583 unigenes encoding the genetic and epigenetic factors also be found, which might contribute to the decondensation and stability of decondensed nuclei in the spermatozoa. These predicted events provide a view of the potential molecular mechanisms of development of testis and spermatogenesis in E. sinensis.

H3K9ac involved in the decondensation of spermatozoal nuclei during spermatogenesis in Chinese mitten crab Eriocheir sinensis.

As a well-known crustacean model species, the Chinese mitten crab Eriocheir sinensis presents spermatozoa with decondensed DNA. Our aim was to analyze structural distribution of the histone H3 and its acetylated lysine 9 (H3K9ac) during spermatogenesis for the mechanistic understanding of the nuclear decondensation of the spermatozoa in E. sinensis. Using specific antibodies, we followed the structural distribution and acetylated lysine 9 of the histone H3 during spermatogenesis, especially spermiogenesis, of E. sinensis. Various spermary samples at different developmental stages were used for histological immunofluorescence and ultrastructural immunocytochemistry. Our results demonstrate a wide distribution of the histone H3 and H3K9ac during spermatogenesis, including spermatogonia, spermatocytes, spermatids, and immature and mature spermatozoa except for absence of H3K9ac in the secondary spermatocytes. Especially during the initial stage of nuclear decondensation, histone H3 lysine 9 was acetylated and then an amount of H3K9ac was removed from within to outside of the nuclei of late spermatids. The portion of remaining H3K9ac was gradually transferred from the nuclei during the stages of spermatozoa maturation. Our findings suggest both the acetylation of histone H3 lysine 9 and the remain of H3K9ac to contribute to the nuclear decondensation in spermatozoa of E. sinensis.

Spautin-1 Ameliorates Acute Pancreatitis via Inhibiting Impaired Autophagy and Alleviating Calcium Overload.

Acute pancreatitis is characterized by zymogen pre-activation. Severe inflammation caused by zymogen activation can eventually lead to multiple organ dysfunctions, which contributes to the high mortality rate of severe acute pancreatitis. However, there is no specific treatment available for acute pancreatitis therapy. Here, we show that spautin-1, which effectively inhibits autophagy flux, ameliorated the pathogenesis of acute pancreatitis induced by cerulein or L-Arginine. CaMKII phosphorylation due to cytosolic calcium oeverload was revealed in this paper. It was also demonstrated that autophagic protein aggregates degradation blockade accompanying with impaired autophagy correlated positively to intra acinar cells digestive aymogen activation sitimulated by cerulein or L-Arginine. The role of spautin-1 in ameliorating acute pancreatitis was shown here to be associated with impaired autophagy inhibition and Ca2+ overload alleviation. We provided a promising therapy for acute pancreatitis here through targeting both impaired autophagy and increased cytosolic calcium.

Induction of acrosome reaction of spermatozoa in Eriocheir sinensis by low temperature.

The effects of temperatures, durations of treatment, and derivations from spermatophores or spermaries on in vitro acrosome reaction of the spermatozoa in the Chinese mitten crab Eriocheir sinensis were investigated. The results showed that the different temperatures resulted in extremely significant differences (p < 0.01) in the time of beginning acrosome reaction, the time of the maximum percentage of acrosome reaction, and the maximum percentage of acrosome reaction of the spermatozoa from spermatophores; and the low temperature (-20, -80 degrees C and liquid nitrogen) induced acrosome reaction of more than 90% spermatozoa while 15 and 4 degrees C didn't. Similar results occur in the spermatozoa, treated with -80 degrees C for 15 min, from spermaries but the time of beginning acrosome reaction and the time of the maximum percentage of acrosome reaction were obviously longer than those from spermatophores. In conclusion, low temperature can induce acrosome reaction, which is a novel and efficient operating method of inducing acrosome reaction; the spermatozoa might be affected physiologically to capacitate with chilling. The study may be beneficial to new understandings of mechanism of acrosome reaction and provide the foundational material for artificial fertilization and breeding of this crab and other commercial aquatic crustaceans.