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Results for “Genomic characterization”

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PubMed1993

Strain characterization, genome size and plasmid content in the Lactobacillus acidophilus group (Hansen and Mocquot).

Chromosomal DNA of nine strains of the Lactobacillus acidophilus (Hansen and Mocquot) group in which heterogeneity had previously been revealed by biochemical characteristics and DNA-DNA hybridization studies were further investigated by restriction analysis, Southern hybridization, conventional and pulsed-field gel electrophoresis (PFGE) analyses. Industrial strains were characterized and identified as Lact. acidophilus. For this species, the number of rRNA gene clusters was estimated to be at least four from analyses of rRNA gene restriction patterns. The chromosome size of type-strains of Lact. acidophilus and Lact. gasseri was estimated from PFGE analysis to be 1.85 and 2.02 Mb respectively and Lact. gasseri strains were found to contain large-sized linear plasmids.

Bacterial Typing Techniques↗

PubMed2026

Genomic characterization of a hypervirulent Aeromonas veronii NN0115 from Nile tilapia and head kidney transcriptome of infected fish reveals B-cell-dominated immune response with specific immunoglobulin downregulation.

Aeromonas veronii is a pathogen of multiple fish species, yet systematic understanding of its infection in Nile tilapia (Oreochromis niloticus) remains limited. A dominant strain, NN0115, was isolated from a natural outbreak and identified as A. veronii by 16S rRNA and whole-genome average nucleotide identity (ANI, 96.33%). Experimental infection revealed high virulence (LD50 = 3.41 × 106 CFU/mL, equivalent to 8.53 × 104 CFU/fish). The genome is 4.58 Mb (58.57% GC) and encodes 4216 proteins. Virulence factor analysis identified 1253 genes, dominated by motility-related (264) and immune modulation (208) factors. Genomic island GI2 harbors 7 virulence genes and two dual-function resistance-virulence genes. The strain is resistant to 9 of 25 agents tested but carries three RND efflux pump genes whose predicted resistance was not phenotypically observed. The head kidney transcriptome of tilapia at 24 h post-bacterial infection identified 773 differentially expressed genes; among them, 57 were immunoglobulin (Ig) genes, and 56 were down-regulated. Integration of published single-cell transcriptomic data showed that non-Ig B-cell marker genes were down-regulated by 32%, whereas Ig genes were reduced by 63%, indicating selective transcriptional suppression of Ig genes rather than a general decrease in B-cell transcriptional activity. Together, this study provides a comprehensive characterization of a highly virulent A. veronii from Nile tilapia and reveals that selective downregulation of B-cell Ig genes is the dominant transcriptional feature of the host head kidney response.

Animals↗

PubMed1995

Cloning and genomic characterization of LST1: a new gene in the human TNF region.

The leucocyte specific transcript - 1 (LST1) represents the human homolog of the mouse B144 transcript, encoded within the tumor necrosis factor (TNF) region of the human major histocompatibility complex class III interval. The gene is localized about 4 kilobases upstream of the lymphotoxin beta gene. It spans a polymorphic genomic region encompassing the microsatellites TNFd and TNFe in intron 3 and a polymorphic Pvu II restriction site 260 base pairs downstream of the polyadenylation signal. Isolation of a full-length cDNA clone revealed that LST1 codes for IFN-gamma-inducible 800 nt transcripts, which are present in lymphoid tissues, T cells, macrophages, and histiocyte cell lines. The cDNA contains three long open reading frames (ORF) with the most likely ORF encoding a transmembrane protein. Its close linkage to the TNF genes and pattern of expression point toward a possible role for LST1 in the immune response.

Amino Acid Sequence↗

PubMed1998

Molecular cloning, genomic characterization and expression of novel human alpha1A-adrenoceptor isoforms.

We have isolated and characterized from human prostate novel splice variants of the human alpha1A-adrenoceptor, several of which generate truncated products and one isoform, alpha(1A-4), which has the identical splice site as the three previously described isoforms. Long-PCR on human genomic DNA showed that the alpha(1A-4) exon is located between those encoding the alpha(1A-1) and alpha(1A-3) variants. CHO-K1 cells stably expressing alpha(1A-4) showed ligand binding properties similar to those of the other functional isoforms as well as agonist-stimulated inositol phosphate accumulation. Quantitative PCR analyses revealed that alpha(1A-4) is the most abundant isoform expressed in the prostate with high levels also detected in liver and heart.

Adrenergic alpha-Antagonists↗

PubMed2000

The KLK7 (PRSS6) gene, encoding for the stratum corneum chymotryptic enzyme is a new member of the human kallikrein gene family - genomic characterization, mapping, tissue expression and hormonal regulation.

The human stratum corneum chymotryptic enzyme (HSCCE; PRSS6, KLK7 gene) has been purified from human stratum corneum and is known to participate in the cell shedding process. The cDNA of the gene has previously been reported. Here, we describe the identification of 5' and 3' extensions of the published mRNA, and the complete genomic organization of the gene. KLK7 is composed of five coding exons which have similar lengths to exons of other kallikrein-like genes. The intron phases are completely conserved between this gene and other members of the kallikrein-like gene family. Precise mapping of KLK7 has indicated that it is located at chromosomal locus 19q13. 3-q13.4 between the already known genes zyme (KLK6) (centromere) and neuropsin (KLK8) (telomere). Until recently, it was thought that this gene is expressed only in the skin. We here provide evidence that KLK7 is also expressed at relatively high levels in the central nervous system, kidney, mammary and salivary glands. Its expression is up-regulated by estrogens and glucocorticoids in the breast carcinoma cell line BT-474. The cDNA and protein of this gene are homologous to sequences of other kallikrein-like genes. The gene encodes for a secreted protein. Phylogenetic analysis, the close structural similarities, and its co-localization in the same chromosomal region, suggest that the gene encoding for the stratum corneum chymotryptic enzyme is a new member of the expanded human kallikrein gene family.

Amino Acid Sequence↗

PubMed2026

Development and evaluation of an ARTIC-based amplicon sequencing assay for whole-genome characterization of respiratory syncytial virus.

Respiratory syncytial virus (RSV), a ~15.2 kb negative-sense RNA virus, causes acute respiratory infections in infants and older adults. Its two subtypes, RSV-A and RSV-B, evolve rapidly, making ongoing monitoring of circulating strains essential. The Georgia Public Health Laboratory (GPHL) developed and evaluated an amplicon-based whole-genome sequencing (WGS) assay for RSV surveillance. A total of 214 de-identified remnant clinical specimens (102 RSV-A and 112 RSV-B) with RT-PCR Cq values <31 were included. RSV genomes were amplified using ARTIC-style and custom primer sets, with the ARTIC set showing superior performance. Libraries were prepared using a modified Illumina COVIDSeq protocol, sequenced on NextSeq 1000/2000 instruments, and analyzed using the GPHL-RSV-PIPE bioinformatics pipeline. Among genomes meeting validation criteria, sequencing depth was slightly higher for RSV-A (median 53,433×; mean 51,076×) than RSV-B (median 49,699×; mean 46,945×), whereas genomic coverage was slightly lower for RSV-A (median 97.5%; mean 96.6%) than RSV-B (median 98.3%; mean 97.6%). Predominant lineages were A.D.3.1 and A.D.5.2 for RSV-A and B.D.E.1 for RSV-B. For RSV-A, the assay showed 92.8% accuracy, 96.2% sensitivity, 87.2% specificity, 92.6% positive predictive value, and 93.2% negative predictive value. Intra- and inter-run precision assessed using 16 and 53-57 genomes, respectively, showed nearly 100% consensus genome identity with 0-5 nucleotide differences. Specificity testing of 31 non-RSV specimens produced no false-positive detections. Limits of detection were 4.4 TCID50/mL for RSV-A and 18.6 TCID50/mL for RSV-B. These results demonstrate that the ARTIC-based RSV WGS assay enables near real-time surveillance and strengthens data-driven public health responses to future outbreaks.IMPORTANCERSV, with two major subtypes, RSV-A and RSV-B, causes acute respiratory infections that can be severe in infants under 6 months and older adults. Current RSV surveillance at the GPHL relies on the Thermo Fisher TaqMan Gene Expression Capillary assay, which detects and subtypes RSV but lacks resolution for lineage classification and identification of emerging variants. To address this critical gap, GPHL developed and evaluated an amplicon-based WGS assay using 214 de-identified RSV clinical specimens. Genomes were amplified using ARTIC-style and custom-primer sets, with ARTIC primers showing superior performance. The assay demonstrated strong sequencing depth, genomic coverage, specificity, repeatability, reproducibility, and low limits of detection. RSV lineages were accurately determined based on genetic variation. These results establish that the ARTIC-based WGS assay enables near real-time genomic surveillance, supporting monitoring of circulating RSV strains and informing data-driven public health responses.

bioinformatics pipeline↗

PubMed1999

Genomic characterization of the sheep vasopressin V1a receptor gene and promoter, with assignment to bands q23-24 of sheep chromosome 3 and cattle chromosome 5.

Arginine vasopressin interacts with the vasopressin type 1a receptor (V1aR) to initiate physiological effects such as vasoconstriction of blood vessels and glycogenolysis. AVP is also involved in central nervous effects such as body homeostasis and blood pressure control. The complete genomic organization of the sheep V1aR gene has been determined, including the presence of one major and two minor transcriptional start sites at -321, -206 and -91bp respectively, relative to the ATG codon. Another more distal minor transcriptional start site was also localized between nucleotides -997 and -892 relative to the ATG codon. One intron exists in the sheep V1aR gene and potential cis- and trans- acting sites were identified in the sheep V1aR promoter. The promoter was also compared to the rat V1aR promoter. The sheep V1aR promoter displays features typical of housekeeping genes, although tissue-specific expression does not support this. V1aR mRNA is absent in the adult sheep liver but not the kidney. One copy of the V1aR gene exists in the sheep genome, which was localized to chromosome 3q23-24, and to the homoeologous position, 5q23-24 in cattle.

Amino Acid Sequence↗

PubMed2003

Full-length genome characterization of HIV type 1 subtype C isolates from two slow-progressing perinatally infected siblings in South Africa.

Isolation and characterization of HIV-1 from asymptomatic, slow-progressing individuals are important in studying viral pathogenesis and facilitate the development of vaccines and antivirals. In this study we identified two slow-progressing HIV-1-infected siblings, isolated viruses, and sequenced the full-length genome, to identify virus attenuations that may contribute to their altered rate of disease progression. Proviral DNA from strains 99ZATM10 and 01ZATM45 was isolated from peripheral blood mononuclear cells (PBMC) coculture.Virtually full-length genomes and long terminal repeat (LTR) regions were polymerase chain reaction (PCR) amplified, sequenced, and assembled to generate the complete genomes. Phylogenetic analysis confirmed that both isolates were subtype C throughout their genome. Predicted amino acid sequence analysis for all the HIV-1 proteins showed that both viruses had open reading frames for all genes, and encoded proteins of the expected length, except for the rev gene. The 3' end of rev exon 2 did not have the 16-amino acid (aa) truncation characteristic of subtype C viruses, and in addition, had a three-aa extension (GlyCysCys). Rev is a necessary regulatory factor for HIV expression, and changes in the protein may affect viral replication. These results suggest that slower HIV disease progression in these children may be attributed, at least in part, to an altered Rev protein.

Amino Acid Sequence↗

PubMed2026

Genomic Characterization of Classic Adamantinoma, Osteofibrous Dysplasia, and Osteofibrous Dysplasia-like Adamantinoma.

Classic adamantinoma, osteofibrous dysplasia (OFD), and OFD-like adamantinoma are rare bone tumors arising primarily in the tibiae. Their distinction can be challenging; data on their molecular pathogenesis remain limited. We searched our pathology files in 2004-2024 for available cases and performed targeted next-generation sequencing along with whole-genome single-nucleotide polymorphism arrays and 3-dimensional genomics/Hi-C sequencing in selected cases. Our cohort included 3 classic adamantinomas (2 females and 1 male; age, 14-56 years), 5 OFDs (3 females and 2 males; age, 9-25 years), and 2 OFD-like adamantinomas (1 female and 1 male; age, 30-41 years). Of the 10 tumors, 9 arose from the tibiae; 1 classic adamantinoma originated from the radius. The 3 classic adamantinomas harbored multiple copy number gains involving chromosome 7, 8, 10, 12, and/or 19. Focal deletion of chromosome 17, intergenic rearrangement involving FGFR1, and NRAS p.G12D were each present in 1 classic adamantinoma. Of the 5 OFDs, KMT2A p.C2441F, KMT2D p.S1040P, PHOX2B p.G213D, and RIF1 deletion were each present in 1 case; no additional copy number/single-nucleotide variants were identified. Of the 2 OFD-like adamantinomas, one case with tumor clusters visible only on cytokeratin immunostain harbored no variants, whereas another case with tumor clusters visible on light microscopy and cytokeratin/p40 immunostains showed gains of chromosome 7, 8, 19, and 20. By Hi-C, 1 classic adamantinoma harbored an approximately 9 Mb tandem duplication on chromosome 12q, 1 OFD harbored a rearrangement with breakpoints near MECOM and HOOK3, and the OFD-like adamantinoma with tumor clusters visible only on cytokeratin immunostain harbored no structural variant. In conclusion, classic adamantinomas and OFD might be genetically distinct. Classic adamantinomas harbored multiple alterations, including chromosome/arm-level copy number gains, the detection of which could aid their distinction from OFDs. Using genomics as the benchmark, OFD-like adamantinomas might be better delineated by light microscopy or p40 than by cytokeratin immunohistochemistry. These data expanded our molecular understanding of these rare bone tumors.

Humans↗

PubMed2026

Clinical and genomic characterization of Influenza A co-infection with SARS-CoV-2 and Influenza B: a respiratory surveillance study in Assam, India.

Influenza and SARS-CoV-2 are the primary contributors to seasonal respiratory infections and frequently co-circulate, creating significant health challenges. The present respiratory surveillance study was conducted in Dibrugarh, Assam, India from January 2025 to August 2025 to investigate the genomic characteristics of circulating viruses and identify potential co-infections. Overall, 4,948 respiratory samples were screened using multiplex real-time PCR, followed by subtyping of Influenza A and Influenza B. Next-generation sequencing (NGS) was performed in selected positives of SARS-CoV-2 and Influenza A. Genomic analysis included mutational profiling, phylogenetic analysis and N-glycosylation site prediction using bioinformatics tools. Two co-infection cases were detected: one involving Influenza A (H3N2) with SARS-CoV-2 (Omicron XFG lineage) and another involving Influenza A (H3N2) with Influenza B (Victoria lineage). Both patients experienced mild illness without hospitalisation. NGS revealed that the Influenza A (H3N2) viruses belonged to clade 3C.2a1b.2a.2a.3a.1 while SARS-CoV-2 sequence was classified under the Omicron XFG lineage. Mutational analysis of the HA gene showed several amino acid differences compared to the reference vaccine strain A/Darwin/6/2021. N-glycosylation analysis predicted conserved sites at positions 79, 181, 262, and 301 in all strains along with an additional predicted site at position 110 in both co-infection cases. Although the co-infection cases presented with mild clinical manifestations, the observed genomic variations indicate a potential role of co-infecting viruses in shaping viral evolution. Given the limited genomic data available from Northeast India, the study underscores the need for sustained large scale follow up and genomic surveillance to monitor emerging mutations and target future vaccine strategies.

Humans↗

PubMed2005

Genomic characterization of non-O1, non-O139 Vibrio cholerae reveals genes for a type III secretion system.

Non-O1, non-O139 Vibrio cholerae can cause gastroenteritis and extraintestinal infections, but, unlike O1 and O139 strains of V. cholerae, little is known about the virulence gene content of non-O1, non-O139 strains and their phylogenetic relationship to other pathogenic V. cholerae. Comparative genomic microarray analysis of four pathogenic non-O1, non-O139 strains indicates that these strains are quite divergent from O1 and O139 strains. Genomic sequence analysis of a non-O1, non-O139 strain (AM-19226) that appeared particularly pathogenic in experimental animals suggests that this strain carries a type III secretion system (TTSS) that is related to the TTSS2 gene cluster found in a pandemic clone of Vibrio parahaemolyticus. The genes for this V. cholerae TTSS system appear to be present in many clinical and environmental non-O1, non-O139 strains, including at least one clone that is globally distributed. We hypothesize that the TTSS present in some pathogenic strains of non-O1, non-O139 V. cholerae may be involved in the virulence and environmental fitness of these strains.

Animals↗

PubMed2026

Description and genomic characterization of Aquipuribacter aurantiacus sp. nov., isolated from saline lake sediment.

Strains MA13-6T and MA13-13, two Gram-stain-positive, aerobic, short rod-shaped actinobacteria, were isolated from a saline lake in Ngari Prefecture, Xizang Autonomous Region, China. Phylogenetic analysis based on 16S rRNA gene sequences indicated that these two strains belonged to the genus Aquipuribacter, with the closest relationship to Aquipuribacter hungaricus IV-75T (98.47% sequence similarity) and Aquipuribacter nitratireducens AMV4T (97.36% sequence similarity). Phylogenetic analysis based on genomes further confirmed their classification as a distinct cluster within the genus Aquipuribacter. The average nucleotide identity and digtal DNA-DNA hybridization values between these two strains and their closest relative Aquipuribacter hungaricus IV-75T, were 82.44-82.49% and 23.00%, respectively, clearly indicating that strains MA13-6T and MA13-13 represent a novel species. The 16S rRNA gene sequence similarity, average nucleotide identity and digital DNA-DNA hybridization values between these two strains were 99.79%, 99.97% and 99.40%, respectively, unequivocally confirming their classification within the same species. However, DNA fingerprinting analysis distinguished them as non-clonal variants. The polar lipids comprised phosphatidylglycerol, two unidentified phospholipids, two unidentified glycolipids, and two unidentified lipids. The predominant respiratory quinone was MK-10 (H4). The major fatty acids were anteiso-C15:0, C18:1ω9c, isoC16:0 and anteiso-C17:0. The cell wall diagnostic diamino acid was meso-diaminopimelic acid. Based on phylogenetic analyses combined with phenotypic and chemotaxonomic characterization, strains MA13-6T and MA13-13 represent a novel species of the genus Aquipuribacter, for which the name Aquipuribacter aurantiacus sp. nov. is proposed. The type strain is MA13-6T (=MCCC 1K10045T = KCTC 59572T).

Phylogeny↗

PubMed2000

Whole-genome characterization of embryonic stage inbreeding depression in a selfed loblolly pine family.

Inbreeding depression is important in the evolution of plant populations and mating systems. Previous studies have suggested that early-acting inbreeding depression in plants is primarily due to lethal alleles and possibly epistatic interactions. Recent advances in molecular markers now make genetic mapping a powerful tool to study the genetic architecture of inbreeding depression. We describe a genome-wide evaluation of embryonic viability loci in a selfed family of loblolly pine (Pinus taeda L.), using data from AFLP markers from an essentially complete genome map. Locus positions and effects were estimated from segregation ratios using a maximum-likelihood interval mapping procedure. We identified 19 loci showing moderately deleterious to lethal embryonic effects. These loci account for >13 lethal equivalents, greater than the average of 8.5 lethal equivalents reported for loblolly pine. Viability alleles show predominantly recessive action, although potential overdominance occurs at 3 loci. We found no evidence for epistasis in the distribution of pairwise marker correlations or in the regression of fitness on the number of markers linked to deleterious alleles. The predominant role of semilethal alleles in embryonic inbreeding depression has implications for the evolution of isolated populations and for genetic conservation and breeding programs in conifers.

Crosses, Genetic↗

PubMed2003

Refinement of the physical location and the genomic characterization of the CRSP2 (EXLM1) gene on Xp11.4.

In the course of our search for the gene responsible for X-linked cone-rod dystrophy (COD1), we constructed a physical map and contig (encompassing the region between DXS556 and DXS228), and identified sequences showing homologies to the expressed sequence tags (ESTs) that matched CRSP2 (EXLM1) transcript. We confirmed the expression of the CRSP2 gene in the retina and refined its exact genomic location between DXS1368 and DXS993. We demonstrated that the entire transcript is encoded within 31 exons. Primers were designed for mutation analysis of the exons by direct sequencing of PCR products from genomic DNA, and revealed no mutations in COD1 families. We subsequently excluded CRSP2 as a candidate for COD1 by demonstrating the causative mutations in the RPGR. However, due to its expression in different tissues and its contribution to transcriptional regulation, CRSP2 may be a candidate for other diseases that map to this region of the X chromosome.

Chromosome Mapping↗

PubMed1998

Genomic characterization of the coding region of the human type II 5'-deiodinase gene.

Type II 5'-Deiodinase (5'DII) is a key element in the maintenance of peripheral thyroid hormone homeostasis through the regulation of local T4 to T3 conversion in pituitary, brain, brown adipose tissue and placenta. The cDNA containing the coding region of the human 5'DII (HDII) has been recently cloned from infant brain. In the present paper we report the genomic structure, chromosomal localization and restriction map of the coding region of HDII. The presence of a single intron located at codon 75 was demonstrated using a PCR-based strategy; the exon-intron junctions were then cloned and partially sequenced. Chromosomal localization was performed by radiation hybrid mapping. This study demonstrated that the entire coding region of the HDII gene is contained in two exons spliced at codon 75 by a 7.4 Kb intron and that the HDII chromosomal location is 14q24.3. These data will allow further studies of the role of HDII in the pathophysiology of thyroid homeostasis.

Base Sequence↗

PubMed2005

Complementary DNA cloning, genomic characterization and expression analysis of a mammalian gene encoding histidine-rich calcium binding protein.

A protein complex present at the junctional sarcoplasmic reticulum (SR) membrane is implicated in the Ca(2+) release process during muscle contraction. The histidine-rich Ca(2+)-binding protein (HRC) is an emerging component associated into the SR protein complex. We cloned cDNAs for rat and monkey HRCs, showing a conserved sequence organization in common with other mammalian HRCs. Genomic analysis revealed that each mammalian HRC gene is present as a single copy in the genome, consisting of 6 exons and 5 introns. Developmental expression analysis using mouse embryos and postnatal hearts demonstrated that Hrc transcription begins at 12.5 days postcoitum and its level increases gradually, reaching an adult level in the range 5-20 days after birth. Comparing the Hrc gene and other SR genes, we found that the timing and pattern of gene expression vary among the SR genes and the full-level expression of these genes is achieved in the heart after postnatal day 20. Collectively, our study provides comprehensive information about the structure and expression of the mammalian HRC gene, together with the comparative expression data of the related SR genes.

Amino Acid Sequence↗

PubMed2005

Identification and genomic characterization of a new virus (Tymoviridae family) associated with citrus sudden death disease.

Citrus sudden death (CSD) is a new disease that has killed approximately 1 million orange trees in Brazil. Here we report the identification of a new virus associated with the disease. RNAs isolated from CSD-affected and nonaffected trees were used to construct cDNA libraries. A set of viral sequences present exclusively in libraries of CSD-affected trees was used to obtain the complete genome sequence of the new virus. Phylogenetic analysis revealed that this virus is a new member of the genus Marafivirus. Antibodies raised against the putative viral coat proteins allowed detection of viral antigens of expected sizes in affected plants. Electron microscopy of purified virus confirmed the presence of typical isometric Marafivirus particles. The screening of 773 affected and nonaffected citrus trees for the presence of the virus showed a 99.7% correlation between disease symptoms and the presence of the virus. We also detected the virus in aphids feeding on affected trees. These results suggest that this virus is likely to be the causative agent of CSD. The virus was named Citrus sudden death-associated virus.

Amino Acid Sequence↗

PubMed2003

Genomic characterization of the S-adenosylmethionine decarboxylase genes from soybean.

A full-length gene GmSAMDC1, encoding the S-adenosylmethionine decarboxylase (SAMDC), a key enzyme involved in polyamine biosynthesis, was identified from soybean expressed sequence tags and was characterized. GmSAMDC1 encoded a peptide of 355 amino acids. When compared with other plant SAMDCs, the GmSAMDC1 protein had several highly conserved regions including a putative pro-enzyme cleavage site and a PEST sequence. The 5' leader sequence of the the GmSAMDC1 mRNA contained two additional open reading frames (ORFs), which may regulate the translational process. The genomic sequence of the GmSAMDC1 gene contained three introns in the 5' leader sequence, but no intron in the 3'-UTR or the main pro-enzyme ORF. A simple sequence repeat (SSR) was found in intron 2, and the GmSAMDC1 gene was mapped to linkage group D1 using this SSR. The genomic organization of the GmSAMDC1 gene in the subgenus Glycine and the subgenus Soja was found to be different by Southern-blot and PCR analysis. A pseudogene, GmSAMDC2, was also identified. This gene contained no intron and lost its two uORFs. Northern-blot analysis showed that the GmSAMDC1 gene expression was induced by salt, drought and cold, but not induced by wounding; suggesting that the gene was implicated in response to multiple-stress conditions.

3' Untranslated Regions↗