Proteomic Analysis of Adult Human Hippocampal Subfields Demonstrates Regional Heterogeneity in the Protein Expression.

Background: Distinct hippocampal subfields are known to get affected during aging, psychiatric disorders, and various neurological and neurodegenerative conditions. To understand the biological processes associated with each subfield, it is important to understand its heterogeneity at the molecular level. To address this lacuna, we investigated the proteomic analysis of hippocampal subfields─the cornu ammonis sectors (CA1, CA2, CA3, CA4) and dentate gyrus (DG) from healthy adult human cohorts. Findings: Microdissection of hippocampal subfields from archived formalin-fixed paraffin-embedded tissue sections followed by TMT-based multiplexed proteomic analysis resulted in the identification of 5,593 proteins. Out of these, 890 proteins were found to be differentially abundant among the subfields. Further bioinformatics analysis suggested proteins related to gene splicing, transportation, myelination, structural activity, and learning processes to be differentially abundant in DG, CA4, CA3, CA2, and CA1, respectively. A subset of proteins was selected for immunohistochemistry-based validation in an independent set of hippocampal samples. Conclusions: We believe that our findings will effectively pave the way for further analysis of the hippocampal subdivisions and provide awareness of its subfield-specific association to various neurofunctional anomalies in the future. The current mass spectrometry data is deposited and publicly made available through ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD029697.

Multi-Omics Driven Assembly and Annotation of the Sandalwood (Santalum album) Genome.

Indian sandalwood (Santalum album) is an important tropical evergreen tree known for its fragrant heartwood-derived essential oil and its valuable carving wood. Here, we applied an integrated genomic, transcriptomic, and proteomic approach to assemble and annotate the Indian sandalwood genome. Our genome sequencing resulted in the establishment of a draft map of the smallest genome for any woody tree species to date (221 Mb). The genome annotation predicted 38,119 protein-coding genes and 27.42% repetitive DNA elements. In-depth proteome analysis revealed the identities of 72,325 unique peptides, which confirmed 10,076 of the predicted genes. The addition of transcriptomic and proteogenomic approaches resulted in the identification of 53 novel proteins and 34 gene-correction events that were missed by genomic approaches. Proteogenomic analysis also helped in reassigning 1,348 potential noncoding RNAs as bona fide protein-coding messenger RNAs. Gene expression patterns at the RNA and protein levels indicated that peptide sequencing was useful in capturing proteins encoded by nuclear and organellar genomes alike. Mass spectrometry-based proteomic evidence provided an unbiased approach toward the identification of proteins encoded by organellar genomes. Such proteins are often missed in transcriptome data sets due to the enrichment of only messenger RNAs that contain poly(A) tails. Overall, the use of integrated omic approaches enhanced the quality of the assembly and annotation of this nonmodel plant genome. The availability of genomic, transcriptomic, and proteomic data will enhance genomics-assisted breeding, germplasm characterization, and conservation of sandalwood trees.

Role of indole curcumin in the epigenetic activation of apoptosis and cell cycle regulating genes.

Background: Head-and-neck squamous cell carcinoma is associated with the epigenetic silencing of various genes such as DAPK, ataxia telangiectasia mutated (ATM), BRCA1, p16INK4a, pVHL, p16, and RASSF1A. The most common epigenetic change observed in these genes is DNA methylation that directs the studies toward finding inhibitors for DNA methyltransferases (DNMTs), the protagonist in the action. The present study focuses on analyzing the possibility whether indole curcumin can reverse epigenetic changes of the various tumor suppressor genes, characteristically silenced by methylation, by inhibiting the major methylation enzyme DNA methyltransferase 1 or DNMT1. Materials and Methods: The cytotoxic effects of indole curcumin were studied through the MTT and lactate dehydrogenase assays. To determine the apoptosis-mediated death of HEp-2 cells, fluorescence imaging using different stains was done. Gene or mRNA expression analysis was done for p53, ATM, and DAPK genes. Results: The results obtained from this study clearly indicate that the indole analog of curcumin plays a remarkable role in activating genes involved in cell cycle regulation and apoptosis induction through epigenetic regulation. The influence that the drug has on the methylation status of gene promoter sequence of the ATM gene is also very significant. Conclusion: Indole curcumin, being an analog of curcumin, promises to be a very useful drug molecule having various potential targets. The target selected for this study was DNMT1 enzyme and the drug seems to actually show the effects; it was predicted to be having on the target molecule.

A molecular network map of orexin-orexin receptor signaling system.

Orexins are excitatory neuropeptides, which are predominantly associated with feeding behavior, sleep-wake cycle and energy homeostasis. The orexinergic system comprises of HCRTR1 and HCRTR2, G-protein-coupled receptors of rhodopsin family and the endogenous ligands processed from HCRT pro-hormone, Orexin A and Orexin B. These neuropeptides are biosynthesized by the orexin neurons present in the lateral hypothalamus area, with dense projections to other brain regions. The orexin-receptor signaling is implicated in various metabolic as well as neurological disorders, making it a promising target for pharmacological interventions. However, there is limited information available on the collective representation of the signal transduction pathways pertaining to the orexin-orexin receptor signaling system. Here, we depict a compendium of the Orexin A/B stimulated reactions in the form of a basic signaling pathway map. This map catalogs the reactions into five categories: molecular association, activation/inhibition, catalysis, transport, and gene regulation. A total of 318 downstream molecules were annotated adhering to the guidelines of NetPath curation. This pathway map can be utilized for further assessment of signaling events associated with orexin-mediated physiological functions and is freely available on WikiPathways, an open-source pathway database ( https://www.wikipathways.org/index.php/Pathway:WP5094 ).

Opioid receptors signaling network.

Opioid receptors belong to the class A G-protein-coupled receptors and are activated by alkaloid opiates such as morphine, and endogenous ligands such as endorphins and enkephalins. Opioid receptors are widely distributed in the human body and are involved in numerous physiological processes through three major classical opioid receptor subtypes; the mu, delta and kappa along with a lesser characterized subtype, opioid receptor-like (ORL1). Opioids are the most potent analgesics and have been extensively used as a therapeutic drug for the treatment of pain and related disorders. Chronic administration of clinically used opioids is associated with adverse effects such as drug tolerance, addiction and constipation. Several investigations attempted to identify the molecular signaling networks associated with endogenous as well as synthetic opiates, however, there is a paucity of a cumulative depiction of these signaling events. Here, we report a systemic collection of downstream molecules pertaining to four subtypes of opioid receptors (MOR, KOR, DOR and ORL1) in the form of a signaling pathway map. We manually curated reactions induced by the activation of opioid receptors from the literature into five categories- molecular association, activation/inhibition, catalysis, transport, and gene regulation. This led to a dataset of 180 molecules, which is collectively represented in the opioid receptor signaling network following NetPath criteria. We believe that the public availability of an opioid receptor signaling pathway map can accelerate biomedical research in this area because of its high therapeutic significance. The opioid receptors signaling pathway map is uploaded to a freely available web resource, WikiPathways enabling ease of access ( https://www.wikipathways.org/index.php/Pathway:WP5093 ).

Age-Associated Molecular Changes in Human Hippocampus Subfields as Determined by Quantitative Proteomics.

The hippocampus demonstrates age-associated changes in functions, neuronal circuitry, and plasticity during various developmental stages. On the contrary, there is a significant knowledge gap on age-associated proteomic alterations in the hippocampus subfields. Using tandem mass tag-based high-resolution mass spectrometry and quantitative proteomics, we report here age-associated changes in the human hippocampus at the subregional level. We used formalin-fixed paraffin-embedded hippocampal tissue sections from a total of 12 healthy individuals, with 3 individuals from each of the 4 different age groups, specifically, 1-10, 21-30, 31-40, and 81-90 years. We found that lysosome and oxidative phosphorylation were the pathways enriched in the 81- to 90-year age group. On the contrray, nervous system development, synaptic plasticity and transmission, messenger RNA (mRNA) splicing, and electron transport chain (ETC) complex-I activity were the enriched biological processes observed in the younger age groups. In a hippocampus subfield context, our topline findings on age-associated proteome changes include altered expression of proteins associated with adult neurogenesis with age in the dentate gyrus and increased expression of immune response-associated proteins with age in certain cornu ammonis sectors of the hippocampus. Signal peptide analysis predicted hippocampal proteins with secretory potential. While these new findings warrant replication in larger study samples, the current data contribute to (1) our understanding of the molecular basis of proteomic changes across various age groups in hippocampus subfields in healthy individuals, and (2) the design and interpretation of future research on the age-associated neurodegenerative disorders.

Regional heterogeneity in mitochondrial function underlies region specific vulnerability in human brain ageing: Implications for neurodegeneration.

Selective neuronal vulnerability (SNV) of specific neuroanatomical regions such as frontal cortex (FC) and hippocampus (HC) is characteristic of age-associated neurodegenerative diseases (NDDs), although its pathogenetic basis remains unresolved. We hypothesized that physiological differences in mitochondrial function in neuroanatomical regions could contribute to SNV. To investigate this, we evaluated mitochondrial function in human brains (age range:1-90 y) in FC, striatum (ST), HC, cerebellum (CB) and medulla oblongata (MD), using enzyme assays and quantitative proteomics. Striking differences were noted in resistant regions- MD and CB compared to the vulnerable regions- FC, HC and ST. At younger age (25 ± 5 y), higher activity of electron transport chain enzymes and upregulation of metabolic and antioxidant proteins were noted in MD compared to FC and HC, that was sustained with increasing age (≥65 y). In contrast, the expression of synaptic proteins was higher in FC, HC and ST (vs. MD). In line with this, quantitative phospho-proteomics revealed activation of upstream regulators (ERS, PPARα) of mitochondrial metabolism and inhibition of synaptic pathways in MD. Microtubule Associated Protein Tau (MAPT) showed overexpression in FC, HC and ST both in young and older age (vs. MD). MAPT hyperphosphorylation and the activation of its kinases were noted in FC and HC with age. Our study demonstrates that regional heterogeneity in mitochondrial and other cellular functions contribute to SNV and protect regions such as MD, while rendering FC and HC vulnerable to NDDs. The findings also support the "last in, first out" hypothesis of ageing, wherein regions such as FC, that are the most recent to develop phylogenetically and ontogenetically, are the first to be affected in ageing and NDDs.

The Normal Human Adult Hypothalamus Proteomic Landscape: Rise of Neuroproteomics in Biological Psychiatry and Systems Biology.

The human hypothalamus is central to the regulation of neuroendocrine and neurovegetative systems, as well as modulation of chronobiology and behavioral aspects in human health and disease. Surprisingly, a deep proteomic analysis of the normal human hypothalamic proteome has been missing for such an important organ so far. In this study, we delineated the human hypothalamus proteome using a high-resolution mass spectrometry approach which resulted in the identification of 5349 proteins, while a multiple post-translational modification (PTM) search identified 191 additional proteins, which were missed in the first search. A proteogenomic analysis resulted in the discovery of multiple novel protein-coding regions as we identified proteins from noncoding regions (pseudogenes) and proteins translated from short open reading frames that can be missed using the traditional pipeline of prediction of protein-coding genes as a part of genome annotation. We also identified several PTMs of hypothalamic proteins that may be required for normal hypothalamic functions. Moreover, we observed an enrichment of proteins pertaining to autophagy and adult neurogenesis in the proteome data. We believe that the hypothalamic proteome reported herein would help to decipher the molecular basis for the diverse range of physiological functions attributed to it, as well as its role in neurological and psychiatric diseases. Extensive proteomic profiling of the hypothalamic nuclei would further elaborate on the role and functional characterization of several hypothalamus-specific proteins and pathways to inform future research and clinical discoveries in biological psychiatry, neurology, and system biology.

An assembly of galanin-galanin receptor signaling network.

The galanin receptor family of proteins is present throughout the central nervous system and endocrine system. It comprises of three subtypes-GalR1, GalR2, and GalR3; all of which are G-protein-coupled receptors. Galanin predominantly acts as an inhibitory, hyper-polarizing neuromodulator, which has several physiological as well as pathological functions. Galanin has a role in mediating food intake, memory, sexual behavior, nociception and is also associated with diseases such as Alzheimer's disease, epilepsy, diabetes mellitus, and chronic pain. However, the understanding of signaling mechanisms of the galanin family of neuropeptides is limited and an organized pathway map is not yet available. Therefore, a detailed literature mining of the publicly available articles pertaining to the galanin receptor was followed by manual curation of the reactions and their integration into a map. This resulted in the cataloging of molecular reactions involving 64 molecules into five categories such as molecular association, activation/inhibition, catalysis, transport, and gene regulation. For enabling easy access of biomedical researchers, the galanin-galanin receptor signaling pathway data was uploaded to WikiPathways ( https://www.wikipathways.org/index.php/Pathway:WP4970 ), a freely available database of biological pathways.

Whole Genome Sequencing of Mycobacterium tuberculosis Clinical Isolates From India Reveals Genetic Heterogeneity and Region-Specific Variations That Might Affect Drug Susceptibility.

Whole genome sequencing (WGS) of Mycobacterium tuberculosis has been constructive in understanding its evolution, genetic diversity and the mechanisms involved in drug resistance. A large number of sequencing efforts from across the globe have revealed genetic diversity among clinical isolates and the genetic determinants for their resistance to anti-tubercular drugs. Considering the high TB burden in India, the availability of WGS studies is limited. Here we present, WGS results of 200 clinical isolates of M. tuberculosis from North India which are categorized as sensitive to first-line drugs, mono-resistant, multi-drug resistant and pre-extensively drug resistant isolates. WGS revealed that 20% of the isolates were co-infected with M. tuberculosis and non-tuberculous mycobacteria species. We identified 12,802 novel genetic variations in M. tuberculosis isolates including 343 novel SNVs in 38 genes which are known to be associated with drug resistance and are not currently used in the diagnostic kits for detection of drug resistant TB. We also identified M. tuberculosis lineage 3 to be predominant in the northern region of India. Additionally, several novel SNVs, which may potentially confer drug resistance were found to be enriched in the drug resistant isolates sampled. This study highlights the significance of employing WGS in diagnosis and for monitoring further development of MDR-TB strains.

Global Proteome Profiling Reveals Drug-Resistant Traits in Elizabethkingia meningoseptica: An Opportunistic Nosocomial Pathogen.

Elizabethkingia meningoseptica is Gram-negative, rod-shaped opportunistic bacterial pathogen increasingly reported in hospital-acquired outbreaks. This bacterium is well known to thrive in the hospital environment. One of the leading causes of meningitis in pediatric and immune-compromised patients, E. meningoseptica has been noted as a "pathogen of interest" in the context of nosocomial diseases associated with device-related infections in particular. This pathogen's multidrug-resistant phenotype and attendant lack of adequate molecular mechanistic data limit the current approaches for its effective management in hospitals and public health settings. This study provides the global proteome of E. meningoseptica. The reference strain E. meningoseptica ATCC 13253 was used for proteomic analysis using high-resolution Fourier transform mass spectrometry. The study provided translational evidence for 2506 proteins of E. meningoseptica. We identified multiple metallo-ß-lactamases, transcriptional regulators, and efflux transporter proteins associated with multidrug resistance. A protein Car D, which is an enzyme of the carbapenem synthesis pathway, was also discovered in E. meningoseptica. Further, the proteomics data were harnessed for refining the genome annotation. We discovered 39 novel protein-coding genes and corrected four existing translations using proteogenomic workflow. Novel translations reported in this study enhance the molecular data on this organism, thus improving current databases. We believe that the in-depth proteomic data presented in this study offer a platform for accelerated research on this pathogen. The identification of multiple proteins, particularly those involved in drug resistance, offers new future opportunities to design novel and specific antibiotics against infections caused by E. meningoseptica.

Rise of Clinical Microbial Proteogenomics: A Multiomics Approach to Nontuberculous Mycobacterium-The Case of Mycobacterium abscessus UC22.

Nontuberculous mycobacterial (NTM) species present a major challenge for global health with serious clinical manifestations ranging from pulmonary to skin infections. Multiomics research and its applications toward clinical microbial proteogenomics offer veritable potentials in this context. For example, the Mycobacterium abscessus, a highly pathogenic NTM, causes bronchopulmonary infection and chronic pulmonary disease. The rough variant of the M. abscessus UC22 strain is extremely virulent and causes lung upper lobe fibrocavitary disease. Although several whole-genome next-generation sequencing studies have characterized the genes in the smooth variant of M. abscessus, a reference genome sequence for the rough variant was generated only recently and calls for further clinical applications. We carried out whole-genome sequencing and proteomic analysis for a clinical isolate of M. abscessus UC22 strain obtained from a pulmonary tuberculosis patient. We identified 5506 single-nucleotide variations (SNVs), 63 insertions, and 76 deletions compared with the reference genome. Using a high-resolution LC-MS/MS-based approach (liquid chromatography tandem mass spectrometry), we obtained protein coding evidence for 3601 proteins, representing 71% of the total predicted genes in this genome. Application of proteogenomic approach further revealed seven novel protein-coding genes and enabled refinement of six computationally derived gene models. We also identified 30 variant peptides corresponding to 16 SNVs known to be associated with drug resistance. These new observations offer promise for clinical applications of microbial proteogenomics and next-generation sequencing, and provide a resource for future global health applications for NTM species.

The 5-Hydroxytryptamine signaling map: an overview of serotonin-serotonin receptor mediated signaling network.

The monoamine neurotransmitter, 5-Hydroxytryptamine or serotonin, is derived from tryptophan and synthesized both centrally and systemically. Fourteen structurally and functionally distinct receptor subtypes have been identified for serotonin, each of which mediates the neurotransmitter's effects through a range of downstream signaling molecules and effectors. Although it is most frequently described for its role in the etiology of neuropsychiatric and mood disorders, serotonin has been implicated in a slew of fundamental physiological processes, including apoptosis, mitochondrial biogenesis, cell proliferation and migration. Its roles as the neurotransmitter have also emerged in pathogenic conditions ranging from anorexia nervosa to cancer. This has necessitated the understanding of the signaling mechanisms underlying the serotonergic system, which led us to construct a consolidative pathway map, which will provide as a resource for future biomedical investigation on this pathway. Using a set of stringent NetPath annotation criteria, we manually curated molecular reactions associated with serotonin and its receptors from publicly available literature; the reaction categories included molecular associations, activation/inhibition, post-translation modification, transport, and gene regulation at transcription and translational level. We identified 90 molecules in serotonin-serotonin receptor pathway. We submitted the curated data to NetPath, a publicly available database of human signaling pathways, in order to enable the wider scientific community to readily access data and contribute further to this pathway.

A network map of IL-33 signaling pathway.

Interleukin-33 (IL-33) is a member of the IL-1 family of cytokines that play a central role in the regulation of immune responses. Its release from epithelial and endothelial cells is mediated by pro-inflammatory cytokines, cell damage and by recognition of pathogen-associated molecular patterns (PAMPs). The activity of IL-33 is mediated by binding to the IL-33 receptor complex (IL-33R) and activation of NF-κB signaling via the classical MyD88/IRAK/TRAF6 module. IL-33 also induces the phosphorylation and activation of ERK1/2, JNK, p38 and PI3K/AKT signaling modules resulting in the production and release of pro-inflammatory cytokines. Aberrant signaling by IL-33 has been implicated in the pathogenesis of several acute and chronic inflammatory diseases, including asthma, atopic dermatitis, rheumatoid arthritis and ulcerative colitis among others. Considering the biomedical importance of IL-33, we developed a pathway resource of signaling events mediated by IL-33/IL-33R in this study. Using data mined from the published literature, we describe an integrated pathway reaction map of IL-33/IL-33R consisting of 681 proteins and 765 reactions. These include information pertaining to 19 physical interaction events, 740 enzyme catalysis events, 6 protein translocation events, 4 activation/inhibition events, 9 transcriptional regulators and 2492 gene regulation events. The pathway map is publicly available through NetPath ( http://www.netpath.org /), a resource of human signaling pathways developed previously by our group. This resource will provide a platform to the scientific community in facilitating identification of novel therapeutic targets for diseases associated with dysregulated IL-33 signaling. Database URL: http://www.netpath.org/pathways?path_id=NetPath_120 .

Integrated Multi-Omic Analysis of Mycobacterium tuberculosis H37Ra Redefines Virulence Attributes.

H37Ra is a virulence attenuated strain of Mycobacterium tuberculosis widely employed as a model to investigate virulence mechanisms. Comparative high-throughput studies have earlier correlated its avirulence to the presence of specific mutations or absence of certain proteins. However, a recent sequencing study of H37Ra, has disproved several genomic differences earlier reported to be associated with virulence. This warrants further investigations on the H37Ra proteome as well. In this study, we carried out an integrated analysis of the genome, transcriptome, and proteome of H37Ra. In addition to confirming single nucleotide variations (SNVs) and insertion-deletions that were reported earlier, our study provides novel insights into the mutation spectrum in the promoter regions of 7 genes. We also provide transcriptional and proteomic evidence for 3,900 genes representing ~80% of the total predicted gene count including 408 proteins that have not been identified previously. We identified 9 genes whose coding potential was hitherto reported to be absent in H37Ra. These include 2 putative virulence factors belonging to ESAT-6 like family of proteins. Furthermore, proteogenomic analysis enabled us to identify 63 novel proteins coding genes and correct 25 existing gene models in H37Ra genome. A majority of these were found to be conserved in the virulent strain H37Rv as well as in other mycobacterial species suggesting that the differences in the virulent and avirulent strains of M. tuberculosis are not entirely dependent on the expression of certain proteins or their absence but may possibly be ascertained to functional changes.

Data on whole genome sequencing of extrapulmonary tuberculosis clinical isolates from India.

This article describes the whole genome sequencing data from 5 extrapulmonary tuberculosis clinical isolates. The whole genome sequencing was carried out on Illumina MiSeq platform to identify single nucleotide variations (SNVs) associated with drug resistance. A total of 214 SNVs in the coding and promoter regions were identified in the whole genome sequencing analysis. Among the identified SNVs, 18 SNVs were identified in genes known to be associated with first and second line drug resistance. The data is related to the research article "Whole genome sequencing of Mycobacterium tuberculosis isolates from extrapulmonary sites" (Sharma et al., 2017) [1].

Whole Genome Sequencing of Mycobacterium tuberculosis Isolates From Extrapulmonary Sites.

Tuberculosis (TB) remains one of the leading causes of morbidity and mortality worldwide. Extrapulmonary tuberculosis (EPTB) constitutes around 15-20% of TB cases in immunocompetent individuals. Extrapulmonary sites that are affected by TB include bones, lymph nodes, meningitis, pleura, and genitourinary tract. Whole genome sequencing has emerged as a powerful tool to map genetic diversity among Mycobacterium tuberculosis (MTB) isolates and identify the genomic signatures associated with drug resistance, pathogenesis, and disease transmission. Several pulmonary isolates of MTB have been sequenced over the years. However, availability of whole genome sequences of MTB isolates from extrapulmonary sites is limited. Some studies suggest that genetic variations in MTB might contribute to disease presentation in extrapulmonary sites. This can be addressed if whole genome sequence data from large number of extrapulmonary isolates becomes available. In this study, we have performed whole genome sequencing of five MTB clinical isolates derived from EPTB sites using next-generation sequencing platform. We identified 1434 nonsynonymous single nucleotide variations (SNVs), 143 insertions and 105 deletions. This includes 279 SNVs that were not reported before in publicly available datasets. We found several mutations that are known to confer resistance to drugs. All the five isolates belonged to East-African-Indian lineage (lineage 3). We identified 9 putative prophage DNA integrations and 14 predicted clustered regularly interspaced short palindromic repeats (CRISPR) in MTB genome. Our analysis indicates that more work is needed to map the genetic diversity of MTB. Whole genome sequencing in conjunction with comprehensive drug susceptibility testing can reveal clinically relevant mutations associated with drug resistance.

An overview of the oxytocin-oxytocin receptor signaling network.

Oxytocin, a nine amino acid long neuropeptide hormone, is synthesized in the hypothalamus and stored and released from the neural lobe of the pituitary gland. Although commonly known for its central role in the regulation of parturition and lactation, oxytocin signaling also plays a key role in modulating social behavior, evoking contentment, initiating maternal behavior, inducing trust, generosity and bonding in humans and animals. Oxytocin signaling can prove to be of great importance in therapeutics and drug targeting because of its diverse range of actions. However, a well annotated map of oxytocin signaling pathway is currently lacking in the publicly available pathway resources. Therefore, we systematically curated the available signaling information of oxytocin from published literature and collated the data to develop a more complete map. We cataloged 66 molecules belonging to oxytocin signaling pathway, which included 9 protein-protein interactions, 39 post-translational modifications, 14 protein translocation events and 22 activation/inhibition events. Further, Oxytocin signaling network data is made freely available to academic fraternity by integrating this into NetPath ( http://www.netpath.org /), a freely available human signaling pathway resource developed previously by our group.