Molecular functions of Mrhl lncRNA in mouse spermatogenesis.

In brief: Mrhl lncRNA regulates the Wnt signaling pathway in mouse spermatogonial cells, resulting in the commitment of B-type spermatogonia to meiotic entry through Mrhl lncRNA-mediated regulation of Sox8. Mrhl lncRNA regulates chromatin dynamics of the Sox8 promoter/enhancer interaction through chromatin looping. Abstract: Proliferation and meiotic division of spermatogonial stem cells are highly regulated biological processes that occur during spermatogenesis. In addition to protein-coding genes, the mammalian genome encodes thousands of lncRNAs which are spatio-temporally expressed and play key role(s) in cellular differentiation and development. Mrhl lncRNA is one such mono-exonic polyadenylated non-coding RNA encoded within the 15th intron of the mouse Phkb gene. Mrhl lncRNA is expressed in mouse testis amongst other tissues. The RNA is nuclear localized and predominantly bound to chromatin in GC-1 spg cells (derived from B-type spermatogonia). It regulates multiple genes belonging to different biological processes including Wnt signaling. Wnt activation of GC-1 spg cells downregulates Mrhl lncRNA expression, in turn activating the expression of meiotic marker genes and downregulating stem cell markers. We have mapped the genomic loci bound by Mrhl lncRNA and identified 37 genes to be regulated by its physical association. Sox8 gene, one among these, is regulated through its interaction at its promoter through RNA:DNA:DNA triplex structure and chromatin looping mediated by the architectural proteins CTCF and YY1. Here, we summarize our major findings on this novel lncRNA starting from its discovery to biological function(s), particularly during meiotic commitment/initiation in mouse spermatogenesis.

Identification of PAX6 and NFAT4 as the Transcriptional Regulators of the Long Noncoding RNA Mrhl in Neuronal Progenitors.

The long noncoding RNA (lncRNA) Mrhl has been shown to be involved in coordinating meiotic commitment of mouse spermatogonial progenitors and differentiation events in mouse embryonic stem cells. Here, we characterized the interplay of Mrhl with lineage-specific transcription factors during mouse neuronal lineage development. Our results demonstrate that Mrhl is expressed in the neuronal progenitor populations in mouse embryonic brains and in retinoic acid-derived radial-glia-like neuronal progenitor cells. Depletion of Mrhl leads to early differentiation of neuronal progenitors to a more committed state. A master transcription factor, PAX6, directly binds to the Mrhl promoter at a major site in the distal promoter, located at 2.9 kb upstream of the transcription start site (TSS) of Mrhl. Furthermore, NFAT4 occupies the Mrhl-proximal promoter at two sites, at 437 base pairs (bp) and 143 bp upstream of the TSS. Independent knockdown studies for PAX6 and NFAT4 confirm that they regulate Mrhl expression in neuronal progenitors. We also show that PAX6 and NFAT4 associate with each other in the same chromatin complex. NFAT4 occupies the Mrhl promoter in PAX6-bound chromatin, implying possible coregulation of Mrhl. Our studies are crucial for understanding how lncRNAs are regulated by major lineage-specific transcription factors, in order to define specific development and differentiation events.

Regulation of Sox8 through lncRNA Mrhl-Mediated Chromatin Looping in Mouse Spermatogonia.

Sox8 is a developmentally important transcription factor that plays an important role in sex maintenance and fertility of adult mice. In the B-type spermatogonial cells, Sox8 is regulated by the long noncoding RNAs (lncRNA) Mrhl in a p68-dependant manner under the control of the Wnt signaling pathway. The downregulation of Mrhl leads to the meiotic commitment of the spermatogonial cells in a Sox8-dependant manner. While the molecular players involved in the regulation of transcription at the Sox8 promoter have been worked out, our current study points to the involvement of the architectural proteins CTCF and cohesin in mediating a chromatin loop that brings the Sox8 promoter in contact with a silencer element present within the gene body in the presence of lncRNA Mrhl concomitant with transcriptional repression. Further, lncRNA Mrhl interacts with the Sox8 locus through the formation of a DNA:DNA:RNA triplex, which is necessary for the recruitment of PRC2 to the locus. The downregulation of lncRNA Mrhl results in the promoter-silencer loop giving way to a promoter-enhancer loop. This active transcription-associated chromatin loop is mediated by YY1 and brings the promoter in contact with the enhancer present downstream of the gene.

LncRNA Mrhl orchestrates differentiation programs in mouse embryonic stem cells through chromatin mediated regulation.

Long non-coding RNAs (lncRNAs) have been well-established to act as regulators and mediators of development and cell fate specification programs. LncRNA Mrhl (meiotic recombination hotspot locus) has been shown to act in a negative feedback loop with WNT signaling to regulate male germ cell meiotic commitment. In our current study, we have addressed the role of Mrhl in development and differentiation using mouse embryonic stem cells (mESCs) as our model system of study. Mrhl is a nuclear-localized, chromatin-bound lncRNA with moderately stable expression in mESCs. Transcriptome analyses and loss-of-function phenotype studies revealed dysregulation of developmental processes, lineage-specific transcription factors and key networks along with aberrance in specification of early lineages during differentiation of mESCs. Genome-wide chromatin occupancy studies suggest regulation of chromatin architecture at key target loci through triplex formation. Our studies thus reveal a role for lncRNA Mrhl in regulating differentiation programs in mESCs in the context of appropriate cues through chromatin-mediated responses.

A novel enhancer RNA, Hmrhl, positively regulates its host gene, phkb, in chronic myelogenous leukemia.

Noncoding RNAs are increasingly being accredited with key roles in gene regulation during development and disease. Here we report the discovery and characterization of a novel long noncoding RNA, Hmrhl, which shares synteny and partial sequence similarity with the mouse lncRNA, Mrhl. The human homolog, Hmrhl, transcribed from intron 14 of phkb gene, is 5.5 kb in size, expressed in all tissues examined and is associated with chromatin. Analysis of Hmrhl locus using ENCODE database revealed that it exhibits hallmarks of enhancers like the open chromatin configuration, binding of transcription factors, enhancer specific histone signature etc. in the K562 Chronic Myelogenous Leukemia (CML) cells. We compared the expression of Hmrhl in the normal lymphoblast cell line, GM12878, with that of K562 cells and lymphoma samples and show that it is highly upregulated in leukemia as well as several cases of lymphoma. Further, we validated the enhancer properties of Hmrhl locus in K562 cells with the help of ChIP-qPCR and Luciferase assay. Moreover, siRNA mediated down-regulation of Hmrhl in K562 cells leads to a concomitant down regulation of its parent gene, phkb, showing that Hmrhl functions as an enhancer RNA and positively regulates its host gene, phkb, in chronic myelogenous leukemia. This study is significant in view of the fact that a better understanding of mechanism of gene regulation under normal conditions and its perturbation in cancer could in turn help in its therapeutic intervention through molecular medicine/RNA based drug discovery.

Long Noncoding RNAs in Pluripotency of Stem Cells and Cell Fate Specification.

Since the annotation of the mouse genome (FANTOM project) [Kawai J et al (2001) Functional annotation of a full-length mouse cDNA collection. Nature 409(6821):685-690] or the human genome [An integrated encyclopedia of DNA elements in the human genome. (2012) Nature 489(7414):57-74; Harrow J et al (2012) GENCODE: the reference human genome annotation for the ENCODE project. Genome Res 22(9):1760-1774], the roles of long noncoding RNAs in coordinating specific signaling pathways have been established in a wide variety of model systems. They have emerged as crucial and key regulators of stem cell maintenance and/or their differentiation into different lineages. In this chapter we have discussed the recently discovered lncRNAs that have been shown to be necessary for the maintenance of pluripotency of both mouse and human ES cells. We have also highlighted the different lncRNAs which are involved in directed differentiation of stem cells into any of the three germ layers. In recent years stem cell therapies including bone marrow transplantation are becoming an integral part of modern medicinal practices. However, there are still several challenges in making stem cell therapy more reproducible so that the success rate reaches a high percentage in the clinic. It is hoped that understanding the molecular mechanisms pertaining to the role of these newly discovered lncRNAs in the differentiation process of stem cells to specific lineages should pave the way to make stem cell therapy and regenerative medicine as a normal clinical practice in the near future.

Identification of Posttranslational Modifications of Endogenous Chromatin Proteins From Testicular Cells by Mass Spectrometry.

Chromatin architecture in mammalian spermatogenesis undergoes extensive structural and functional reorganization during which several testis-specific histone variants and other chromatin proteins are expressed in a stage-dependent manner. The most dramatic change in chromatin composition is observed during spermiogenesis where nucleosomal chromatin is transformed into nucleoprotamine fiber. Role of posttranslational modification (PTM) of somatic canonical histones and histone variants is well documented and effect several chromatin-templated events. PTM of testis-specific chromatin proteins is proposed to orchestrate chromatin-templated events during mammalian spermatogenesis and their identification and subsequent functional characterization is key to understand chromatin restructuring events and establishment of sperm epigenome. Here, we present protocols for the purification of endogenous testis chromatin proteins from different stages of spermatogenesis and identification of their PTM repertoire by mass spectrometry through examples of testis-specific histone variants (TH2B and HILS1), and transition proteins (TP1 and TP2).

Higher topoisomerase 2 alpha gene transcript levels predict better prognosis in GBM patients receiving temozolomide chemotherapy: identification of temozolomide as a TOP2A inhibitor.

The search for molecular markers which predict response to chemotherapy is an important aspect of current neuro-oncology research. MGMT promoter methylation is the only proved marker of glioblastoma. The purpose of this study was to assess the effect of topoisomerase expression on glioblastoma survival and study the mechanisms involved. The transcript levels of all isoforms of the topoisomerase family in all grades of diffuse astrocytoma were assessed. A prospective study of patients with glioblastoma treated by a uniform treatment procedure was performed with the objective of correlating outcome with gene expression. The ability of TOP2A enzyme to relax the super coiled plasmid DNA in the presence of temozolomide was evaluated to assess its effect on TOP2A. The temozolomide cyctotoxicity of TOP2A-silenced U251 cells was assessed. The transcript levels of TOP2A, TOP2B, and TOP3A are upregulated significantly in GBM in comparison with lower grades of astrocytoma and normal brain samples. mRNA levels of TOP2A correlated significantly with survival of the patients. Higher TOP2A transcript levels in GBM patients predicted better prognosis (P = 0.043; HR = 0.889). Interestingly, we noted that temozolomide inhibited TOP2A activity in in-vitro enzyme assays. We also noted that siRNA knock down of TOP2A rendered a glioma cell line resistant to temozolomide chemotherapy. We demonstrated for the first time that temozolomide is also a TOP2A inhibitor and established that TOP2A transcript levels determine the chemosensitivity of glioblastoma to temozolomide therapy. Very high levels of TOP2A are a good prognostic indicator in GBM patients receiving temozolomide chemotherapy.

A haplotype at the UCP1 gene locus contributes to genetic risk for type 2 diabetes in Asian Indians (CURES-72).

BACKGROUND: The gene encoding for uncoupling protein-1 (UCP1) is considered to be a candidate gene for type 2 diabetes because of its role in thermogenesis and energy expenditure. The objective of the study was to examine whether genetic variations in the UCP1 gene are associated with type 2 diabetes and its related traits in Asian Indians. METHODS: The study subjects, 810 type 2 diabetic subjects and 990 normal glucose tolerant (NGT) subjects, were chosen from the Chennai Urban Rural Epidemiological Study (CURES), an ongoing population-based study in southern India. The polymorphisms were genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Linkage disequilibrium (LD) was estimated from the estimates of haplotypic frequencies. RESULTS: The three polymorphisms, namely -3826A-->G, an A-->C transition in the 5'-untranslated region (UTR) and Met229Leu, were not associated with type 2 diabetes. However, the frequency of the A-C-Met (-3826A-->G-5'UTR A-->C-Met229Leu) haplotype was significantly higher among the type 2 diabetic subjects (2.67%) compared with the NGT subjects (1.45%, P < 0.01). The odds ratio for type 2 diabetes for the individuals carrying the haplotype A-C-Met was 1.82 (95% confidence interval, 1.29-2.78, P = 0.009). CONCLUSIONS: The haplotype, A-C-Met, in the UCP1 gene is significantly associated with the increased genetic risk for developing type 2 diabetes in Asian Indians.

WITHDRAWN: Association of uncoupling protein 2 and 3 gene polymorphisms with type 2 diabetes in Asian Indians.

Ahead of Print article withdrawn by publisher.

Identification of a novel nucleolin related protein (NRP) gene expressed during rat spermatogenesis.

BACKGROUND: Nucleolin is a major nucleolar phosphoprotein involved in various steps of ribosome biogenesis in eukaryotic cells. As nucleolin plays a significant role in ribosomal RNA transcription we were interested in examining in detail the expression of nucleolin across different stages of spermatogenesis and correlate with the transcription status of ribosomal DNA in germ cells. RESULTS: By RT PCR and western blot analysis we found that nucleolin is strongly down regulated in meiotic spermatocytes and haploid germ cells. We have identified a new nucleolin related protein (NRP) gene in the rat genome, which is over expressed in the testis and is up regulated several fold in meiotic spermatocytes and haploid germ cells. The NRP protein lacks the acidic stretches in its N terminal domain, and it is encoded in rat chromosome 15 having a different genomic organization as compared to nucleolin gene present on chromosome 9. We have also found NRP genes encoded in genomes of other mammalian species. We performed run-on transcription assay where we have observed that rDNA is transcribed at much lower level in meiotic spermatocytes and haploid spermatids as compared to diploid cells. By siRNA knock down experiments we could also demonstrate that NRP can support rDNA transcription in the absence of nucleolin. CONCLUSION: We have identified a new nucleolin variant over expressed in germ cells in rat and analyzed its domain structure. We attribute that the transcriptional activity of rDNA genes in the late spermatogenesis is due to the presence of this variant NRP. The expression of this variant in the germ cells in the absence of nucleolin, could have additional functions in the mammalian spermatogenesis which needs to be investigated further.

PBEF1/NAmPRTase/Visfatin: a potential malignant astrocytoma/glioblastoma serum marker with prognostic value.

Malignant astrocytomas comprise anaplastic astrocytoma (AA; grade III) and Glioblastoma (GBM; grade IV). GBM is the most malignant with a median survival of 10-12 months in patients. Using cDNA microarray based expression profiling of different grades of astrocytomas, we identified several fold increased levels of PBEF1 transcripts in GBM samples. Pre-B-cell colony enhancing factor 1 gene (PBEF1) encodes Nicotinamide phosphoribosyltransferase (NAmPRTase), which catalyses the rate limiting step in the salvage pathway of NAD metabolism in mammalian cells. Further validation using real time RT-qPCR on an independent set of tumor samples (n=91) and normal brain samples (n=9), GBM specific higher expression of PBEF1 was confirmed. Immunohistochemical staining for PBEF1 on a subset of the above samples largely reinforced our finding. We carried out ELISA analysis on serum samples of astrocytoma patients to determine whether this protein levels would correlate with the presence of tumor and tumor grade. PBEF1 serum levels were substantially elevated in many of the AA and GBM patients. Statistical analysis of these data indicates that in patients with astrocytoma, serum PBEF1 levels correlate with tumor grade and is highest in GBM. Immunohistochemical analysis of an independent set of 51 retrospective GBM cases with known survival data revealed that PBEF1 expression in the tumor tissue along with its co-expression with p53 was associated with poor survival. Thus, we have identified PBEF1 as a potential malignant astrocytoma serum marker and prognostic indicator among GBMs.

Involvement of importin-4 in the transport of transition protein 2 into the spermatid nucleus.

Mammalian spermiogenesis is characterized by a unique chromatin-remodeling process in which histones are replaced by transition protein 1 (TP1), TP2, and TP4, which are further replaced by protamines. We showed previously that the import of TP2 into the haploid spermatid nucleus requires the components of cytosol and ATP. We have now carried out a detailed analysis to characterize the molecular components underlying the nuclear translocation of TP2. Real-time PCR analysis of the expression of different importins in testicular germ cells revealed that importin-4 and importin-beta3 are significantly up-regulated in tetraploid and haploid germ cells. We carried out physical interaction studies as well as an in vitro nuclear transport assay using recombinant TP2 and the nuclear localization signal of TP2 (TP2(NLS)) fused to glutathione S-transferase in digitonin-permeabilized, haploid, round spermatids and identified importin-4 to be involved in the import of TP2. A three-dimensional model of the importin-4 protein was generated using the crystal structure of importin-beta1 as the template. Molecular docking simulations of TP2(NLS) with the importin-4 structure led to the identification of a TP2(NLS) binding pocket spanning the three helices (helices 21 to 23) of importin-4, which was experimentally confirmed by in vitro interaction and import studies with different deletion mutants of importin-4. In contrast to TP2, TP1 import was accomplished through a passive diffusion process.

Chromatin remodeling during mammalian spermatogenesis: role of testis specific histone variants and transition proteins.

The structure of chromatin undergoes extensive alteration during mammalian spermatogenesis. Several testis specific histone subtypes are synthesised and replace their somatic counterparts during pre-meiotic, meiotic and post-meiotic stages of germ cell differentiation. Early work from our laboratory showed that pachytene spermatocyte nuclei as well as nucleosome core particle are more accessible to DNasel than the interphase liver nuclei. The higher order structure of chromatin in pachytene spermatocytes is also loosely packed due to the poor DNA and chromatin condensing property of the testis specific linker histone H1t. A careful analysis of the amino acid sequence of histone H1t revealed the absence of the DNA condensing domain containing SPKK/TPKK motifs in the C-terminus of the histone H1t. The spermiogenesis process following the meiotic division is characterised by extensive remodeling of chromatin. Transition proteins, TP1 and TP2, unique to mammalian spermatogenesis play an important role in this spermiogenesis process. We have shown that TP1 is a DNA melting protein while TP2 is a DNA condensing protein. We have delineated the molecular anatomy of TP2 including the presence of two novel zinc finger modules, which are essential for the recognition of CpG islands in the genome. TP2 is also phosphorylated by sperm specific protein kinase A and the phosphorylation/dephosphorylation cycle plays an important role in the chromatin condensation process.

Lack of association between serum adiponectin levels and the Pro12Ala polymorphism in Asian Indians.

AIMS: The aim of the study was to investigate the association of serum adiponectin levels with the Pro12Ala polymorphism of the peroxisome proliferator activated receptor-gamma (PPARG) gene in Asian Indians. METHODS: We selected 400 diabetic subjects, 200 with the Pro12Pro genotype (100 male and 100 female) and 200 with the Pro12Ala genotype (100 male and 100 female) and 400 age- and sex-matched normal glucose tolerance subjects with similar genotype profiles from the Chennai Urban Rural Epidemiology Study. Fasting serum adiponection levels were measured using radioimmunoassay. The Pro12Ala polymorphism was genotyped by PCR-restriction fragment length polymorphism using BstUI. RESULTS: All clinical and biochemical parameters were similar in the subjects with the Pro12Pro and Pro12Ala genotypes. There was no significant difference in serum adiponectin values between subjects with the Pro12Pro and Pro12Ala genotypes (males 5.4 vs. 5.8 microg/ml, P = 0.546; females 6.9 vs. 7.2 microg/ml, P = 0.748). Adiponectin values did not differ among these two genotypes even when categorized based on their diabetes status (normal glucose tolerance Pro12Pro 7.9 vs. Pro12Ala 7.7 microg/ml, P = 0.994; diabetes Pro12Pro 4.7 vs. Pro12Ala 5.4 microg/ml, P = 0.622). CONCLUSION: The Pro12Ala polymorphism of the PPARG gene is not associated with serum adiponectin levels in Asian Indians.

Single nucleotide polymorphism analysis of the nucleotide excision repair genes XPC, XPA, and XPG in the Indian population.

We have analyzed single nucleotide polymorphisms (SNPs) in the XPC, XPA, and XPG genes of the nucleotide excision repair (NER) pathway in the Indian population. In the XPC gene we observed nine polymorphisms in the coding region, four polymorphisms in the intronic region, and two polymorphisms in the 5' untranslated region (UTR). In the XPA gene we observed one frequent SNP (allele frequency 0.48) within the 5' UTR at the 1665 position in a large proportion of the sample. In addition, we observed three novel heterozygous polymorphisms (a C to A transversion at position 1523 and a G to A transition at positions 1418 and 1458, with an allele frequency of 0.004) within the promoter region. In silico PCR analysis demonstrated that all three novel polymorphisms lie within a putative CpG island and that the variation at position 1418 falls within the potential GATA1/2/3 transcription factor(s) binding site and also within the negative control element. We performed a gel retardation assay with HeLa cell nuclear extract with an oligonucleotide encompassing this region. One of the alleles found at position 1458 of the XPA gene showed a significant change in protein-DNA interaction. In the XPG gene we found five polymorphisms in the coding region and one each in the 5' UTR of exon 1 and in intron 13.

The G1057D polymorphism of IRS-2 gene and its relationship with obesity in conferring susceptibility to type 2 diabetes in Asian Indians.

OBJECTIVE: To investigate the association of insulin receptor substrate-2 (IRS-2) G1057D polymorphism with type 2 diabetes and obesity in Asian Indians. METHODS: The study comprised of 1193 normal glucose tolerant (NGT) subjects and 1018 subjects with type 2 diabetes, aged >/=20 years with an average body mass index of 23.7+/-4.6 and 25.3+/-4.2 kg/m(2), respectively. The subjects were unrelated and randomly selected from the Chennai Urban Rural Epidemiology Study (CURES), a population-based study in Chennai in southern India. The G1057D polymorphism of the IRS-2 gene was genotyped using PCR-RFLP assay. RESULTS: The genotype frequency of the IRS-2 G1057D polymorphism was significantly different between the NGT and type 2 diabetic groups (P=0.0007) in the total study subjects and among the obese subjects (P=0.00007). Logistic regression analysis showed that the DD genotype showed an increased susceptibility to diabetes with an odds ratio (adjusted for age and sex) of 2.19 (95% CI: 1.34-3.57, P=0.002) when compared to the GG+GD genotype, among the obese subjects, but not in non obese subjects. In order to explore possible interaction with obesity, logistic regression analysis was performed and the coefficient corresponding to the interaction parameter (genotype x obesity) was significant (P=0.0001). CONCLUSION: In Asian Indians, the DD genotype increases susceptibility to type 2 diabetes by interacting with obesity.

Rage gene promoter polymorphisms and diabetic retinopathy in a clinic-based population from South India.

PURPOSE: The main objective of this study was to evaluate if the -429T/C, -374T/A and 63 bp deletion polymorphisms in the RAGE gene are associated with diabetic retinopathy (DR) among Type 2 diabetic subjects in a clinic-based population from South India. METHODS: We screened 149 normal glucose tolerant subjects (NGT), 189 Type 2 diabetes subjects without retinopathy (DM) and 190 subjects with DR for these polymorphisms using the PCR-RFLP method. DR was diagnosed by grading color fundus photography. Logistic regression models were used to evaluate the association of individual polymorphisms with DR. Expectation-maximization algorithms were implemented in haplotype tests of association to examine the combined effects of -429T/C and -374T/A polymorphisms on DR. RESULTS: The allelic frequencies of -429T are 0.83 in NGT, 0.84 in DM and 0.85 in DR subjects, and that of -374T are 0.93 in NGT, 0.92 in DM and 0.88 in DR subjects. The -374 polymorphism was found to be associated with non-proliferative retinopathy when this subgroup was compared to the DM group (OR=1.814, 95% CI=1.005-3.273). However, this association was not obvious when both the subphenotypes of DR (the nonproliferative and proliferative DR groups) were studied jointly. We found no evidence for associations between the -429T/C polymorphism and the DR phenotype. Finally, extension to a 2-SNP haplotype did not reveal any significant statistical difference between the groups (P=0.668). CONCLUSION: In this study, we found a modest association with the -374T/A polymorphism in the nonproliferative DR subgroup.

CREMOFAC--a database of chromatin remodeling factors.

MOTIVATION: Chromatin-remodeling is an important event in the eukaryotic nucleus rendering nucleosomal DNA accessible for various transaction processes. Remodeling Factors facilitate the dynamic nature of chromatin through participation of the collective action of (i) ATP and (ii) Non-ATP dependent factors. Considering the importance of these factors in eukaryotes, we have developed, CREMOFAC, a dedicated and frequently updated web-database for chromatin-remodeling factors. RESULTS: The database harbors factors from 49 different organisms reported in literature and facilitates a comprehensive search for them. In addition, it also provides in-depth information for the factors reported in the three widely studied mammals namely, human, mouse and rat. Further, information on literature, pathways and phylogenetic relationships has also been covered. The development of CREMOFAC as a central repository for chromatin-remodeling factors and the absence of such a pre-existing database heighten its utility thus making its presence indispensable. AVAILABILITY: http://www.jncasr.ac.in/cremofac/