RESUMO
BACKGROUND: The South China landmass has been characterized by a complex geological history, including mountain lifting, climate changes, and river capture/reversal events. To determine how this complexity has influenced the landmass's phylogeography, our study examined the phylogeography of Garra orientalis, a cyprinid widely distributed in South China, using sequences from the mitochondrial DNA control region and cytochrome b gene (1887 bp) and polymorphisms of thirteen microsatellite loci. RESULTS: In total, 157 specimens were collected from eight populations. All 88 mtDNA haplotypes were identified as belonging to three major lineages, and these lineages were almost allopatric in their distributions. The results of a statistical dispersal-vicariance analysis suggested that the ancestral populations of G. orientalis were distributed south of the Yunkai Mountains, including on Hainan Island. The mtDNA data revealed a strong relationship between phylogeny and geography. In the microsatellite analysis, a total of 339 alleles with an average of 26 alleles per locus were observed across thirteen microsatellite loci. A clustering algorithm for microsatellite data revealed an admixture-like genetic structure. Although the mtDNA and microsatellite data sets displayed a discordant population structure, the results of an approximate Bayesian computation approach showed that these two markers revealed congruent historical signals. The population history of G. orientalis reflects vicariance events and dispersal related to the complex geological history of South China. CONCLUSION: Our results (i) found that the discordances between mtDNA and microsatellite markers were accounted for by admixtures; (ii) showed that the Wuzhishan and Yinggeling mountain ranges and Qiongzhou Strait were important barriers limiting gene exchange between populations on both sides; (iii) indicated that during glaciation and inter-glacial periods, the strait and continental shelves were exposed and sank, which contributed with the dispersion and differentiation of populations; and (iv) displayed that the admixtures between lineages took place in coastal populations and then colonized the tributaries of the Pearl River.
Assuntos
Cyprinidae/genética , Animais , Teorema de Bayes , China , Citocromos b/genética , DNA Mitocondrial/genética , Genética Populacional , Repetições de Microssatélites , Dados de Sequência Molecular , Filogenia , Filogeografia , Polimorfismo GenéticoRESUMO
Background: Multiple sclerosis (MS) is a condition that can impact the central nervous system (CNS) and cause damage to the myelin, which is responsible for facilitating the normal transmission of electrical impulses along the nerves. We performed a bibliometric analysis of the scientific publications on myelin imaging in MS to reveal the development trends in this field and to evaluate research trends in myelin imaging in MS. Methods: The Web of Science Core Collection was searched for articles related to myelin imaging in MS published between January 2000 and December 2022. CiteSpace, VOSviewer, and R language were used to evaluate and visualize contributions by and co-occurrence relationships among countries and institutions, authors, journals, citations, keywords, and so on. Results: A total of 1,639 articles addressed the topic of myelin imaging in MS. The United States had the largest number of annual publications. The University of London was the institution with the highest number of publications (n=118) and citations (n=9,885). The top 3 productive authors were all from the University of British Columbia in Canada. An article published by Mackay et al. in 1994 had the most citations (n=272). Neuroimage [impact factor (IF) =7.40, Journal Citation Reports quartile 1 (Q1)] was the most productive journal in terms of the number of articles relating to myelin imaging in MS (n=149). In recent years, myelin water imaging, synthetic magnetic resonance imaging (SyMRI), inhomogeneous magnetization, positron emission tomography (PET) imaging, and aquaporin-4 (AQP4) have been researched hotspots of myelin imaging in MS. Conclusions: With advancements in the pathophysiological research on myelin changes in MS, myelin imaging is playing an important role in the diagnosis and treatment of MS. In addition, the use of new sequences of myelin imaging to distinguish MS from other inflammatory demyelinating diseases is a future development trend in this field.
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Studies have shown that clock gene Cry1 may have important roles in the endocrine process of seasonal reproduction in mammals. In this study, Duolang sheep (non-seasonal reproduction sheep breed) and Chinese Merino (seasonal reproduction sheep breed) were used to determine the expression change of Cry1 in hypothalamus-pituitary-ovary axis in different stage of estrous cycle by quantitative real-time PCR. The results showed that the Cry1 mRNA was expressed in all tested tissues, in which the expression levels in pineal gland and thyroid gland were higher than in other tissues. As far as different sheep breeds were concerned, the tissue expression profiles of Cry1 at different stage of estrous cycle were broadly similar. Besides hypothalamus, the expression levels of Cry1 in ovary, uterus, pineal gland, pituitary gland, and thyroid gland were all reached to peak in proestrus. The differences of expression change extent for Cry1 in vary, uterus, pineal gland, and pituitary gland in proestrus and oestrus were significant between different sheep breeds. The results suggested that Cry1 may play roles in switching on the estrus and seasonal reproduction.
Assuntos
Criptocromos/genética , Ciclo Estral , Hipotálamo/metabolismo , Ovário/metabolismo , Hipófise/metabolismo , RNA Mensageiro/genética , Ovinos/genética , Animais , Criptocromos/metabolismo , Feminino , Masculino , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Ovinos/fisiologia , Transcrição GênicaRESUMO
Formosaniaimmaculata, a new species, is described from the Ou-Jiang basin in Zhejiang Province, Southeast China. It is distinguished from other species of the genus by having a combination of the following characteristics: body without obvious mottling; snout length longer than postorbital length; abdominal scaleless area extending to middle of pectoral-fin base; shorter rostral barbels, the outermost pair length 112.9%-140.0% of eye diameter; and shorter lower lip papillae, length 19.9%-24.4% of eye diameter. Its validity is also affirmed by its distinct Cytb gene sequence divergence from all congeners and its monophyly recovered in a Cytb gene-based phylogenetic analysis.
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Coilia nasus is widely distributed in the Yangtze River, the coastal waters of China, Korea and the Ariake Sound of Japan. Several ecotypes exist and this provides a useful model for the study of comparative diversity between molecular markers. Here we analyze and compare the nucleotide sequences between single-copy ribosomal protein S7 gene intron 1 (rpS7) and multiple-copy ribosomal internal transcribed spacer 1 (ITS1) in this species to compare the phylogenetic signal of the two nuclear genes. Nucleotide substitutions among the two gene sequences and partial sequence of mitochondrial cytochrome c oxidase subunit I (COI) gene were also analyzed. A total of 115 clones for rpS7 and 122 clones for ITS1 were obtained from 37 specimens. The nucleotide sequence length is 741 to 743 bp for rpS7 and 334 to 348 bp for ITS1. Intra- and inter-specimen variation in rpS7 results from nucleotide substitution, while such variation in ITS1 is mainly due to different numbers of short base repeats. The content of G + C is lower in rpS7 (43.5%) than in ITS1 (68.2%). Our results indicate that the proportion of the sequence variable sites is higher in rpS7 (61) than in ITS1 (23); the informative parsimony of rpS7 is evidently higher than that of ITS1 (26 vs. 2); the overall ratio between transitions and transversions in ITS1 is slightly lower than in rpS7, but remarkably lower than in COI. These results suggest that rpS7 is more suitable than ITS1 as a marker for genetic divergence of this group. Furthermore, gene flow is observed between the different geographic populations of C. nasus from the phylogeny of this species based on rpS7, showing that rpS7 has more evolutionary characteristics for understanding the processes of genomic evolution at the intraspecific level.
Assuntos
Proteínas de Peixes/genética , Peixes/genética , Proteínas Ribossômicas/genética , Animais , Sequência de Bases , DNA Espaçador Ribossômico/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Evolução Molecular , Ásia Oriental , Variação Genética , Haplótipos , Íntrons , Dados de Sequência Molecular , Filogenia , Rios , Homologia de Sequência do Ácido NucleicoRESUMO
Phylogeographical analyses on Squalidus argentatus samples from thirteen localities within mainland China and Taiwan were conducted for biogeographic studies, as their dispersal strictly depends on geological evolution of the landmasses. A total of 95 haplotypes were genotyped for mtDNA cyt b gene in 160 specimens from nine river systems. Relatively high levels of haplotype diversity (h = 0.984) and low levels of nucleotide diversity (π = 0.020) were detected in S. argentatus. Two major phylogenetic haplotype groups, A and B, were revealed via phylogenetic analysis. The degree of intergroup divergence (3.96%) indicates that these groups diverged about 4.55 myr (million years) ago. Haplotype network and population analyses indicated significant genetic structure (F(ST) = 0.775), largely concordant with the geographical location of the populations. According to SAMOVA analysis, we divided these populations into four units: Yangtze-Pearl, Qiantang-Minjiang, Jiulong-Beijiang and Taiwan groups. Mismatch distribution analysis, neutrality tests and Bayesian skyline plots indicated a significant population expansion for lineage A and B, approximately dated 0.35 and 0.04 myr ago, respectively. We found strong geographical organization of the haplotype clades across different geographic scales that can be explained by episodes of dispersal and population expansion followed by population fragmentation and restricted gene flow.
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Cyprinidae/genética , Citocromos b/genética , DNA Mitocondrial/genética , Proteínas de Peixes/genética , Filogenia , Animais , China , Fluxo Gênico , Haplótipos , Filogeografia , TaiwanRESUMO
Squalidus argentatus (Sauvage and Dabry de Thiersant 1874) is a small-sized freshwater fish which is distributed in Mainland China, Hainan Island and Taiwan. The populations of S. argentatus have dropped sharply probably due to overharvesting and water pollution recently. Eleven polymorphic microsatellite markers were developed for the cyprinid fish S. argentatus. These new markers were tested on 43 individuals collected from Yangtze River and Qiantang River. The number of alleles, observed and expected heterozygosity per locus, in two populations ranged from 3 to 14, from 0.333 to 0.954 and from 0.480 to 0.928, respectively. Only two loci are significantly deviated from Hardy-Weinberg expectations due to the heterozygote deficiency. No significant linkage disequilibrium was detected between the pairwise comparisons of these loci. These polymorphic microsatellite loci will enable us to study the genetic variation, population structure, and conservation genetics of this species in the future.
Assuntos
Cyprinidae/genética , Loci Gênicos/genética , Repetições de Microssatélites/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase/métodos , Polimorfismo Genético , Alelos , Animais , China , Frequência do Gene , Genótipo , Desequilíbrio de Ligação , RiosRESUMO
The cyprinid genus Onychostoma Günther, 1896 consists of 24 valid species distributed in Southeast Asia, including Taiwan, Hainan, mainland China and the Indochina region. In the present study, we determined the complete mitochondrial genome of O. lepturum, which is 16,598 bp in length, containing 13 protein-coding genes, two rRNA genes, 22 tRNA genes and a typical control region (D-loop). To verify the molecular phylogeny of the subfamily Acrossocheilinae, we provide new insights to better understand the taxonomic status of Acrossocheilus, Onychostoma and Folifer brevifilis. The phylogenetic trees presented three major clades based on the 13 protein-coding genes from 28 Acrossocheilinae species. Clades I and II represent the Onychostoma and Acrossocheilus groups, respectively. Species of Acrossocheilus, Onychostoma and F. brevifilis are included in Clade III, which is considered as an ancestral group. This work provides genomic variation information and improves our understanding of the Acrossocheilinae mitogenome, which will be most valuable in providing new insights for phylogenetic analysis and population genetics research.
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Mitochondrial DNA cytochrome b and d-loop sequences (2,137 bp) in 65 specimens of Sarcocheilichthys sinensis from five populations were identified as two lineages (I and II). The pairwise genetic distance between lineages I and II was 1.94%. SAMOVA analyses suggested that the best grouping occurred at three groups, Yangtze, Qiantang and Minjiang Rivers. High haplotype diversity (0.949) and low nucleotide diversity (θπ = 1.067%) were detected. The results of the neutrality tests, mismatch distribution and approximate Bayesian computation (ABC) did not support demographic expansions. The results of phylogenetic analysis, statistical dispersal-vicariance analysis (S-DIVA), ABC, MIGRATE-N and the time to the most recent common ancestor (TMRCA) indicated two colonization routes. First, before the Wuyi Mountains lifted, S. sinensis dispersed from the Yangtze River to the Minjiang River. Second, during glaciation, the continental shelf was exposed, which contributed to the dispersion of populations from the Yangtze River.
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Cyprinidae/classificação , Citocromos b/genética , Animais , Cyprinidae/genética , Evolução Molecular , Variação Genética , Lagos , Filogenia , Dinâmica PopulacionalRESUMO
The mechanism(s) underlying the beneficial effects of adult mesenchymal stem cells (MSCs) after myocardial infarction (MI) is poorly understood. One possible explanation is the ability of MSCs to secrete cytokines, which modulate cardiomyocyte survival and function. MSCs express at least two cytoprotective cytokines, hepatocyte growth factor (HGF) and stromal cell-derived factor-1 alpha (CXCL12). The aim of our study was to compare the effects of these two cytokines administered acutely post-MI. We subjected adult male Lewis rats to myocardial ischemia/reperfusion injury. Immediately upon reperfusion, polymers saturated with HGF or CXCL12 were placed onto the infarcted anterior wall and the rats were allowed to recover. Echocardiographic analysis at 4 wk post-MI to assess left ventricular (LV) function revealed that LV ejection fraction was increased in the HGF treated group compared with the phosphate-buffered saline (PBS) control group. Likewise, LV end diastolic dimension was reduced in the HGF treated group compared with the PBS control group. Similarly, invasive hemodynamics at 12 wk showed improved contractility and relaxation in the HGF treated group compared with the PBS control group. In contrast, no significant effect on LV function was seen in the CXCL12 treated group. To determine the potential mechanism for this effect, infarct size (IFS) at 72 h was determined. IFS was decreased 4.2-fold in the HGF treated group compared with the PBS control group. Thus, HGF acutely post-MI using polymer delivery reduces IFS, leading to beneficial effects on post-MI LV remodeling.
Assuntos
Quimiocina CXCL12/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Isquemia Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Células Cultivadas , Quimiocina CXCL12/farmacologia , Fator de Crescimento de Hepatócito/farmacologia , Fator de Crescimento de Hepatócito/uso terapêutico , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Isquemia Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Ratos , Ratos Endogâmicos Lew , Volume Sistólico/efeitos dos fármacos , Volume Sistólico/fisiologia , Remodelação Ventricular/efeitos dos fármacos , Remodelação Ventricular/fisiologiaRESUMO
The Coilia nasus is an important fish species, which is of commercial values in China. In order to manage the fisheries resources and the development of sustainable fishing strategies to protect this species, 11 microsatellite loci polymorphisms and mtDNA COI sequences were used to examine the genetic diversity of C. nasus in Japan and China. In total, the 40 COI haplotypes and 93 microsatellite alleles were detected. The mtDNA phylogeny did not support population grouping, but the distribution patterns of mtDNA haplotypes and the results of STRUCTURE analysis based on microsatellite indicated a degree of genetic isolation in this species. Our study suggested that the lack of a population genetic structure might result in its amphidromous life cycle, and the geographical distance and habitat fragments might cause isolated populations. Thus, the sampling populations of C. nasus in Japan and China could be divided as four geographical/ecological populations.
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Peixes/genética , Variação Genética , Genoma Mitocondrial , Geografia , Filogenia , Animais , China , Complexo IV da Cadeia de Transporte de Elétrons/genética , Genes Mitocondriais , Japão , Repetições de Microssatélites , Análise de Sequência de DNARESUMO
Mitochondrial DNA cytochrome b sequences (1141 bp) in 229 specimens of Acrossocheilus paradoxus from 26 populations were identified as four lineages. The pairwise genetic distances among these four lineages ranged from 1.57 to 2.37% (mean= 2.00%). Statistical dispersal-vicariance analysis suggests that the ancestral populations were distributed over mainland China and Northern and Western Taiwan. Approximate Bayesian computation approaches show that the three lineages in Taiwan originated from the lineage in mainland China through three colonization routes during two glaciations. The results indicated that during the glaciation and inter-glacial periods, the Taiwan Strait was exposed and sank, which contributed to the dispersion and differentiation of populations. Furthermore, the populations of A. paradoxus colonized Taiwan through a land bridge to the north of the Formosa Bank, and the Miaoli Plateau in Taiwan was an important barrier that limited gene exchange between populations on both the sides.
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Cyprinidae/genética , DNA Mitocondrial/genética , Evolução Molecular , Especiação Genética , Filogenia , Polimorfismo Genético , Animais , Cyprinidae/classificação , Fenômenos Geológicos , FilogeografiaRESUMO
Alpha-fetoprotein (AFP) is a self protein expressed by fetal liver at high levels, but is transcriptionally repressed at birth. AFP is up-regulated in hepatocellular carcinomas, and patients with active disease could have plasma levels as high as 1 mg/mL. We previously identified four immunodominant HLA-A*0201-restricted peptides [hAFP(137-145) (PLFQVPEPV), hAFP(158-166) (FMNKFIYEI), hAFP(325-334) (GLSPNLNRFL), and hAFP(542-550) (GVALQTMKQ)] derived from human AFP that could stimulate specific T cell responses in healthy donor peripheral blood lymphocytes in vitro. We conducted a phase I/II clinical trial in which HLA-A*0201 patients with AFP-positive hepatocellular carcinoma were immunized with three biweekly intradermal vaccinations of the four AFP peptides pulsed onto autologous dendritic cells (DC). DCs were prepared from adherent peripheral blood mononuclear cells cultured with granulocyte-macrophage colony-stimulating factor and interleukin-4 for 7 days. Sixteen subjects were enrolled and 10 were treated. Peripheral blood lymphocytes were isolated from these patients before, during, and after AFP peptide/DC immunization and were tested ex vivo with MHC tetramer and IFNgamma ELISPOT analysis. Six of 10 subjects expanded statistically significant levels of AFP-specific T cells postvaccine to at least one peptide by MHC tetramer. Also, 6 of 10 subjects increased IFNgamma producing AFP-specific T cell responses to at least one of the peptides postvaccination, by ELISPOT. We conclude that the human T cell repertoire is capable of responding to the AFP self antigen after the administration of AFP peptide-pulsed DC even in an environment of high circulating levels of this oncofetal antigen.
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Carcinoma Hepatocelular/terapia , Células Dendríticas/transplante , Neoplasias Hepáticas/terapia , Fragmentos de Peptídeos/uso terapêutico , alfa-Fetoproteínas/uso terapêutico , Adulto , Idoso , Sequência de Aminoácidos , Carcinoma Hepatocelular/imunologia , Progressão da Doença , Feminino , Humanos , Neoplasias Hepáticas/imunologia , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/toxicidade , alfa-Fetoproteínas/toxicidadeRESUMO
Population genetic structure of Onychostoma lepturum on Hainan Island was investigated based on mitochondrial CR + cyt b region in 63 specimens collected from four populations. Population analyses indicated significant genetic structure (FST = 0.749) and displayed a significant relationship between phylogeny and geography (NST = 0.750 and GST = 0.140). Thirty-one mtDNA haplotypes were classified into four lineages, and these lineages had an almost allopatric distribution. The results of a statistical dispersal-vicariance analysis suggest that the ancestral populations were distributed widely on Hainan Island, and the rising of the central mountainous area of Hainan Island, the Wuzhi and Yinggeling Mountain Range, separated these four drainages into independent lineages. According to a spatial analysis of molecular variance analysis, we divided these populations into three units: ND, CH and WQ + LS, running into Qiongzhou Strait, the Gulf of Tokin and the South China Sea, respectively. According to our study, the exposure of straits and shelf under water retreat gave chances for population dispersion during the glaciations.
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Cyprinidae/genética , Variação Genética , Genoma Mitocondrial , Filogenia , Animais , China , Genética Populacional , Haplótipos , Ilhas , Filogeografia , Análise de Sequência de DNARESUMO
Six main mitochondrial DNA (mtDNA) lineages have been described in minnow (Zacco platypus) samples obtained from northern, western and southern China. Perdices et al. (2004) predicted that further sampling of other tributaries might discover more lineages of this species. In this study, we collected 26 Zacco platypus individuals in the Huangshan area of eastern China and determined the cytochrome b (cytb) sequence variations. Combined with reported data in GenBank, we identified ten matrilines (Zacco A-J) in a total of 169 samples, with relatively high molecular divergence found among them. The Huangshan population had the greatest genetic variation among all sampled regions and hosted six of the ten matrilines. Our results highlight the significance of the Huangshan area for the conservation of Zacco platypus.
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Cyprinidae/genética , Variação Genética , Animais , China , Cyprinidae/classificação , DNA Mitocondrial/genética , Haplótipos , FilogeniaRESUMO
The oriental sucking barb, Garra orientalis, is a small to moderate-sized freshwater fish. In this study, the complete mitochondrial genome of G. oriental was successfully sequenced for the first time with total length of 17,288 bp. The genome structure consists of 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA (tRNA) genes and a control region. Moreover, tandem repeat unit ranged from 259 to 260 bp (repeated 3-4 times) was identified in the control region among G. orientalis individuals.
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Cyprinidae/genética , Genoma Mitocondrial , Animais , Composição de Bases , Genes Mitocondriais , Fases de Leitura Aberta , Sequências Reguladoras de Ácido Nucleico , Sequências Repetitivas de Ácido NucleicoRESUMO
The spotted scat, Scatophagus argus (Perciformes, Scatophagidae), is a recreational and commercial fish in China. In this paper, the complete mitochondrial genome of S. argus was firstly determined. It is 16,783 bp in length and consists of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and 1 control region. Except for the eight tRNA and ND6 genes, all other mitochondrial genes are encoded on the heavy strand. The mitochondrial DNA information would be useful in species identification and natural resources conservation.
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Genes Mitocondriais/genética , Genoma Mitocondrial/genética , Perciformes/genética , Animais , Composição de Bases , Sequência de Bases , China , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
In this study, we sequenced the complete mitochondrial genome of Scaphiodonichthys acanthopterus (Cypriniformes, Cyprinidae). This mitochondrial genome, consisting of 16,612 base pairs (bp), encoded genes for 13 protein-coding genes, two ribosomal RNAs, 22 transfer RNAs, and one non-coding control region as those found in other vertebrates, with the gene order identical to that of typical vertebrates. The control region, of 941 bp in length, is located between tRNA (Pro) and tRNA (Phe) . The overall base composition of the heavy strand shows T 25.23%, C 26.85%, A 31.96%, and G 15.95%, with a slight AT bias of 57.19%.
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Cyprinidae/genética , DNA Mitocondrial/genética , Animais , RNA Ribossômico/genética , RNA de Transferência/genéticaRESUMO
Osteochilus salsburyi (Cypriniformes, Cyprinidae) is a small-sized fish of significant economic value. In this paper, the complete mitogenome sequence of O. salsburyi was first determined. It is 16,599 bp in length and consists of a typical vertebrate genome structure including 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA (tRNA) genes, and a control region. Except for eight tRNA and ND6 genes, all other mitochondrial genes are encoded on the heavy strand. The overall base composition of the heavy strand in descending order is A (33.0%), T (26.5%), C (25.4%), and G (15.0%), with a slight AT bias of 59.5%. This information of O. salsburyi mitogenome could contribute not only to the development of efficient conservation strategy on vulnerable genetic diversity but also to the identification of useful genetic markers for distinction across species boundary.
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Cipriniformes/genética , Genoma Mitocondrial , Animais , Proteínas/genética , RNA Ribossômico/genética , RNA de Transferência/genéticaRESUMO
We determined the complete mitochondrial genome (mitogenome) sequence of Sarcocheilichthys nigripinnis by using long polymerase chain reaction method. The total length of S. nigripinnis mitogenome is 16,680 bp, including 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs, and a noncoding control region. The overall base composition of S. nigripinnis is 26.36% for T, 26.67% for C, 30.07% for A, and 16.90% for G, with a slight AT bias of 56.43%.