Traditional Chinese medicine invigorating the spleen and kidney promotes HBsAg seroclearance in the mouse model.

Traditional Chinese medicine (TCM) is often used as an adjuvant or alternative therapy for abnormal liver biochemistry or liver fibrosis associated with chronic hepatitis B (CHB). However, the role of TCM in HBsAg seroclearance remains unclear. We aimed at exploring the role and possible mechanisms of TCM in HBsAg seroclearance. Fifteen widely used TCM granules invigorating the spleen and kidneys were screened. C57BL/6J mice were administered daily with TCM granules by gavage for 1 week. The effect of TCM on the M1 polarization of macrophages was measured using a CD86 assay. According to the principles of formulating prescriptions, three single TCM with the most noticeable effect on M1 polarization, accompanied by two other TCM granules, were used to develop a TCM formula. The hepatitis B virus-expressing mouse model was constructed by hydrodynamic injection of the pAAV/HBV1.2 plasmid. Hepatitis B virus-expressing mice were gavaged daily with phosphate-buffered saline (PBS), TCM formula, or Codonopsis Radix, for 1 week. HBsAg, HBeAg, and hepatitis B virus DNA levels were measured. In addition, gut microbiota was profiled using 16S rDNA sequencing. Several TCM granules showed significant effects on M1 polarization. The TCM formula accelerated HBsAg seroclearance compared with the Codonopsis Radix and PBS groups. Intrahepatic M1 polarization, as indicated by flow cytometry and immunohistochemistry, was induced in the TCM formula and Codonopsis Radix groups. The abundance of Alloprevotella significantly increased in the TCM formula and Codonopsis Radix groups. These results demonstrate that the TCM formula for invigorating the spleen and kidney can accelerate HBsAg seroclearance. This effect can be attributed, at least in part, to M1 polarization of intrahepatic macrophages.

[Quality evaluation of Galli Gigerii Endothelium Corneum based on HPLC fingerprints and content determination of nucleosides].

Galli Gigerii Endothelium Corneum(GGEC), the dried gizzard membrane of Gallus gallus domesticus is a Chinese medicinal material commonly used for digestion. However, due to the particularity of texture and composition, its active ingre-dients have not been clarified so far, and there is also a lack of quality evaluation indicators. In this study, UPLC-Q-TOF-MS was used to analyze the chemical components from the water extract of GGEC, and ten nucleosides were identified for the first time. HPLC fingerprints of the water extracts of GGEC were established and the content of seven nucleosides was determined. The fingerprint similarities of 40 batches of GGEC samples ranged from 0.765 to 0.959, indicating that there were great differences among the GGEC products processed with different methods. In addition, SPSS 22.0 and SIMCA 14.1 were used for hierarchical cluster analysis(HCA) and principal component analysis(PCA) on the 19 common peaks of the HPLC fingerprints of GGEC, and the 40 batches of samples were divided into three categories: raw GGEC, fried GGEC and vinegar-processed GGEC. Eight differential components in GGEC were marked by orthogonal partial least squares discrimination analysis(OPLS-DA), two of which were adenine and thymine. The results of content determination showed that the total content of the seven nucleosides in raw GGEC, fried GGEC and vinegar-processed GGEC were 182.5-416.8, 205.3-368.7, and 194.2-283.0 µg·g~(-1), respectively. There were significant differences in the content of hypoxanthine, thymine and thymidine among the GGEC products processed with different methods(P<0.05), which were graded in the order of fried GGEC>vinegar-processed GGEC>raw GGEC. This suggested that the content of hypoxanthine, thymine and thymidine tended to increase during the frying process, and the variation range might be related to the degree of heat exposure. The established methods in this study were simple and reproducible, and could be used for qualitative and quantitative analysis of GGEC and its processed pro-ducts. This study also provided reference for the establishment of quality standards of GGEC with chemical components as control index.

[Qualitative and quantitative analysis of nucleosides and nucleobases in Phylloporia ribis by UPLC-Q-TOF-MS and HPLC].

The dried fruit body of Phylloporia ribis(Hymenochaetaceae), which prefers to live on the stumps of Lonicera japonica(Caprifoliaceae), has a variety of activities, whereas its pharmacodynamic material basis is not completely clear and there are few reports on its quality control and evaluation. In this study, an UPLC-Q-TOF-MS method was used to analyze the nucleosides and nucleobases in P. ribis and a HPLC method was established for simultaneous determination of 10 nucleosides and nucleobases. MS and MS/MS data were acquired in positive ion mode. Based on the data comparison of the sample and the reference substance, the literature data and the compound databases of ChemSpider and PubChem, 18 nucleosides and nucleobases were identified qualitatively from the water extract of P. ribis for the first time. After optimization, the HPLC was performed using a Welch Ultimate AQ C_(18) column(4.6 mm×250 mm, 5 µm) by gradient elution with acetonitrile and water as mobile phase, the flow rate of 1.0 mL·min~(-1), the detection wavelength of 260 nm, and the column temperature of 30 ℃. Through the investigation of the extraction method, solvent and time, it was determined that the test solution should be obtained by cold water extraction for 18 h. At the present HPLC conditions, 10 components of uracil, cytidine, hypoxanthine, uridine, thymine, inosine, guanosine, 2'-deoxyinosine, 2'-deoxyguanosine and thymidine could be well separated(R > 1.5) and showed good linearity(r > 0.999 9) in the concentration ranges of 0.247-24.7, 0.283-28.3, 0.273-27.3, 0.256-25.6, 0.257-25.7, 0.318-31.8, 0.245-24.5, 0.267-26.7, 0.250-25.0 and 0.267-26.7 mg·L~(-1), respectively. The average reco-veries of 10 components were 95.78%-104.5%, and the RSDs were 2.2%-5.2%(n=6). The contents of 10 nucleosides and nucleobases in different samples of P. ribis varied greatly, which were 0.021-0.122, 0.004-0.029, 0.014-0.226, 0.009-0.442, 0.003-0.014, 0.002-0.146, 0.007-0.098, 0-0.054, 0.005-0.069, 0.004-0.081 and 0.072-1.28 mg·g~(-1) for uracil, cytidine, hypoxanthine, uridine, thymine, inosine, guanosine, 2'-deoxyinosine, 2'-deoxyguanosine, thymidine and total 10 components, respectively. These results demonstrated that the components had significant differences in the internal quality, and good quality control was needed to ensure the medical efficacy. This study provides a scientific basis for the discovery of pharmacodynamic ingredients, quality control and evaluation of P. ribis.

[Qualitative and quantitative analysis of Paris polyphylla var. chinensis by UPLC-Q-TOF-MS/MS and HPLC].

Paridis Rhizoma(PR) is prepared from the dried rhizome of Paris polyphylla var. yunnanensis(PPY) or P. polyphylla var. chinensis(PPC) in Liliaceae family. The rapid development of PPY or PPC planting industry resulted from resource shortage has caused the waste of a large number of non-medicinal resources. To clarify the chemical compositions in rhizomes, fibrous roots, stems, leaves, seeds and pericarps of PPC, and explore the comprehensive application value and development prospect of these parts, the qualitative and quantitative analyses on the different parts of PPC were carried out by ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS/MS) and high performance liquid chromatography(HPLC). A total of 136 compounds were identified, including 112 steroidal saponins, 6 flavonoids, 11 nitrogen-containing compounds and 7 phytosterols. Rhizomes, fibrous roots, and seeds mainly contained protopennogenyl glycosides and pennogenyl glycosides; leaves and stems mainly contained protodiosgenyl glycosides and diosgenyl glycosides; pericarps mainly contained pennogenyl glycosides, followed by diosgenyl glycosides. The total level of four saponins was the highest in fibrous roots and rhizomes, followed by those in the pericarps and arillate seeds, and the lowest in the stems and exarillate seeds. This study can provide data support for the comprehensive development and rational application of non-medicinal parts of PPC.

[Rapid discovery of tyrosinase inhibitors from Sophora flavescens:significance for comprehensive utilization of its non-medicinal resources].

Sophorae Flavescentis Radix,derived from the root of Sophora flavescens in the Leguminosae family,has been widely used in the medicine,agriculture,animal husbandry,and daily chemical industry. A pharmacophore model-based method for rapid discovery of tyrosinase inhibitors( TIs) from S. flavescens was established by molecular docking under Lipinski rules,and verified by enzyme assays. Briefly,the chemical constituent database of S. flavescens( CDSF) was established based on the previous papers. Theoptimal pharmacophore model( OPM) was constructed by DS 2019 on the basis of known active TIs. Eighty-three hits predominated by flavonoids having higher fitting scores with OPM than the positive control were screened out,and subjected to molecular docking based on the three-dimensional structure of tyrosinase crystal protein. The potential TIs such as kurarinone and nor-kurarinone were rapidly discovered from the compounds with higher docking scores than the positive control under the Lipinski rules. The results were verified by the in vitro enzyme assays. The inhibition activities of tyrosinase from non-medicinal parts of S. flavescens were also tested to explore the relationship between the inhibition activity and chemical compositions. This study is expected to provide data support for the comprehensive application and development of S. flavescens and also a new method for the rapid discovery of active substances or functional constituents in the complex systems.

[Determination of ß-nicotinamide mononucleotide and nicotinamide adenine dinucleotide in Dendrobium officinale and congeneric species by UPLC-MS/MS].

As anti-aging ingredients, ß-nicotinamide mononucleotide(NMN) and nicotinamide adenine dinucleotide(NAD~+) have attracted worldwide attention in recent years. After oral administration, NMN can be converted into NAD~+ in vivo and the latter is the actual ingredient which exerts anti-aging effect. In order to explore the "rejuvenating and anti-aging" effect of Dendrobium officinale, which was firstly recorded in Shennong's Herbal Classic of Materia Medica, this study established the quantitative method of UPLC-MS/MS for simultaneous determination of NMN and NAD~+ in D. officinale and the congeneric species for the first time, and 34 batches of samples were detected. UPLC conditions are as follows: ACQUITY UPLC HSS T3 column(2.1 mm × 100 mm, 1.8 µm), gradient elution with acetonitrile-0.1% formic acid in water at the flow rate of 0.3 mL·min~(-1), and column temperature of 40 ℃. MS conditions were scanned electrospray ionization source and multiple reaction monitoring mode. The method was verified by systematic methodology. The mean recoveries of NMN and NAD~+ were 77.58% and 80.70%, respectively, with RSD of 3.6% and 4.3%, separately. All results showed that the content of NMN was higher in D. officinale than in the other congeneric species. Particularly, the content in fresh D. officinale stems was as high as 0.931 9 µg·g~(-1). NAD~+ was only found in D. officinale and the content was three times higher than that of NMN. This may be the reason that D. officinale topped the "nine famous anti-aging herbs". In addition, processing method influences the content of NMN and NAD~+ in Dendrobium. Specifically, the content of NMN and NAD~+ was in the order of fresh Dendrobium stems > dried Dendrobium stem segments > spiral or spring-like dried Dendrobium stems.

[Optimization of determination of astragaloside â £ in Astragali Radix by continuous single-factor method].

This paper aims to solve the problems of complicated-unstable test solution preparation process and insufficient extraction of the active ingredient astragaloside Ⅳ in the legal method for the determination of astragaloside Ⅳ in Astragali Radix. The continuous single-factor analysis of seven main factors affecting the content of astragaloside Ⅳ was carried out by HPLC-ELSD, and then the pre-paration method of test solution was optimized. This optimized method exhibited excellent performance in precision, repeatability and stability. The average recovery rate of astragaloside Ⅳ was 99.65% with RSD 2.2%. Astragaloside Ⅳ showed a good linearity between the logarithm of peak area and the logarithm of injection quantity in the range of 0.46-9.1 µg(r=0.999 6). The contents of astragaloside Ⅳ in 29 batches of Astragali Radix were determined by the new and the legal methods. The results showed that the average content of astragaloside Ⅳ in these Astragali Radix samples determined by the former method was 1.458 times than that of the latter one, indicating the new method was simple, reliable and more adequate to extract target compound. According to the results, it is suggested to improve the content standard of astragaloside Ⅳ in Astragali Radix in the new edition of Chinese Pharmacopeia.

[Effects of different harvesting and processing methods on quality of Andrographis Herba].

The effects of the growing periods, processing methods, nutritive organs and cut parts of the herbs on the quality of Andrographis Herba have been studied, which could provide scientific basis for producing high-quality crude materials of Andrographis Herba. A quantitative analysis of multi-components by single-marker(QAMS) method was used to simultaneously determine 4 diterpene lactones of andrographolide(AD), neoandrographolide(NAD), 14-deoxyandrographolide(14-DAD), and dehydroandrographolide(DHAD) in 78 batches of Andrographis Herba samples. Visual analysis, clustering analysis and partial least squares discrimination analysis(PLS-DA) were used for data processing and mining. The growing periods of Andrographis Herba were divide into the rapid growing stage(Ⅰ), the luxuriant foliage stage(Ⅱ), the bud stage(Ⅲ), the flower stage(Ⅳ), the fruiting stage(Ⅴ) and the later fruiting stage(Ⅵ), six ones in total. The results showed that the total contents of 4 diterpene lactones(TC) kept almost constant in growing periods of Ⅰ-Ⅲ, began to fall off in growing period Ⅳ, decreased rapidly in growing period Ⅴ and reduced to minimum in growing period Ⅵ. Therefore, the best harvesting period for Andrographis Herba was the bud stage. For the processing methods, none significant difference occurred for TC between drying by the sun and drying in the shade. However, in comparison of the samples dried by the sun, the samples dried in the shade exhibited lower content of AD, nonetheless, the higher contents of DHAD and NAD, indicating that the process of drying in the shade may lead to the decomposition and transformation of AD. Therefore, when the weather conditions permitted, the method drying by the sun should be used. The distributions of AD, DHAD and TC in the different cut parts and nutritive organs were determined as follows: upper cut parts > middle cut parts ≈ entire herbs > lower cut parts, leaves > entire herbs > stems. Furthermore, the contents of 4 diterpene lactones in leaves and herbs were much higher than those in stems. Therefore, it was suggested that the upper and middle cut parts of the herbs or the parts containing leaves should be preferred for harvesting to ensure the proportion of the leaves in Andrographis Herba.

[Study on revision of quality standard of Sophorae Flavescentis Radix and its processed slices].

Sophorae Flavescentis Radix is prepared from the root of Sophora flavescens. The comprehensive knowledge of the effective components in S. flavescens makes its quality standard improvement more possible. TLC identification method for main flavonoids and alkaloids in one test was established using GF_(254) thin layer plate and the lower solution of chloroform-methanol-water-formic acid(4∶2∶1∶0.6) as developing solvent. The quantitative method of marker alkaloids was revised including the simplified sample preparation procedure and the chromatographic conditions. The determination of four alkaloids in the samples indicated that 32 batches of tested samples were qualified ones. The total oxymatrine and matrine, total oxysophocarpine and sophocarpine, as well as total amount of four tested components in the different samples were 1.08%-2.55%, 0.369%-0.860%, 1.67%-3.40%, respectively. There was a significantly positive correlation between oxymatrine and oxysophocarpine. Total oxymatrine and matrine had same correlation with total oxysophocarpine and sophocarpine. The moisture content and extractive in 32 batches of samples fulfilled the require of not more than 11.0% and not less than 20.0%, respectively. Thirteen tested pesticides were not detected in 12 batches of samples. The present study provided the evidence for the revision of quality standard of Sophora Flavescentis Radix.

[Recommendations on quality standards of Andrographis Herba and its processed slices in Chinese Pharmacopoeia (2020 edition)].

To improve and perfect the quality standards and propose recommendations for the revision of quality standards for Andrographis Herba and its processed slices in Chinese Pharmacopoeia(ChP)(2020 edition) based on the problems and limitations in ChP(2015 edition). TLC identification method with andrographolide and control herbs as references was established using silica gel G thin layer plate, with chloroform-methylbenzene-methanol(8∶1∶1) as developing solvent, and 10% sulfuric acid ethanol solution as colour-developing agent. This method has good reproducibility, strong specificity and high sensitivity. As compared with the original method in ChP 2015, this method has better development effect and clearer spots. Based on the previous research, a quantitative analysis of multi-components by single-marker(QAMS) method with andrographolide as the internal reference substance was developed to simultaneously determine the contents of 4 diterpene lactones: andrographolide(S), neoandrographolide(A), 14-deoxyandrographolide(B), and dehydroandrographolide(C). The relative correction factors of f_(A/S), f_(B/S), and f_(C/S) were determined as 1.12, 0.79, and 0.63, respectively. The relative retention time of t_(A/S), t_(B/S), and t_(C/S) was 1.95, 2.18, and 2.25, respectively. According to the content determination results in 46 batches of crude drugs and 38 batches of processed slices, it was stipulated that the total contents of 4 diterpene lactones should not be less than 1.5% and 1.2% in crude drugs and processed slices, respectively. As compared with the original method in ChP 2015, the present QAMS method could not only reduce the detection cost and improve the efficiency, but also can be used to evaluate the quality of Andrographis Herba and its processed slices more comprehensively and objectively. Diterpene lactones are generally recognized as the effective components in Andrographis Herba, and their contents in leaves were much higher than those in stems. However, almost all of the current commercial processed slices are processed from stems, so their quality is gene-rally poor and the efficacy is hard to be guaranteed. Therefore, the weight percentage of leaves should be added into the inspection items of the processed slices and it should not be less than 25%.

[Scientificity of traditional commodity grade of Chinese medicinal materials--taking Lonicerae Japonicae Flos as example].

Taking Lonicerae Japonicae Flos as an example, the method of "expert consensus of different regions" was used to screen the representative samples and evaluate their commodity grades. The correlation analysis, hierarchical cluster analysis and partial least squares discriminant analysis(PLS-DA) of "commodity grade-appearance characteristic-component content" were carried out to reveal the scientificity of traditional commodity grade of Chinese medicinal material. By referring to the existing literature and the grade investigation from the sample collection regions, 78 "initial grade" samples were screened out from 118 collected samples. Authoritative experts from four regions(n=4) including Linyi(Shangdong province), Bozhou(Anhui province), Anguo(Hebei province) and Beijing were organized to evaluate their commodity grades, separately. Based on the grade consistency rate(R_i≥70%), 69 "local grade" samples were screened out from the "initial grade" samples. Based on the average grade consistency rate ■ "authoritative grade" samples were screened out from the "local grade" samples, including15 first-grade samples, 9 second-grade samples, 11 third-grade samples and 17 fourth-grade samples. For these "authoritative grade" samples, the main appea-rance characteristics were quantified and the contents of 13 components were determined by ultra performance liquid chromatography(UPLC). Furthermore, the total contents of 6 phenolic acids, 4 flavonoids and 3 iridoids were calculated, respectively. The results of correlation analysis showed that 4 appearance characteristics indices were correlated with the commodity grades: color, rate of yellow bars(including blooming flowers), rate of black heads(including black bars), and rate of stems and leaves(including bud debris). Five component content indices were correlated with the commodity grades: chlorogenic acid, isochlorogenic acid C, sweroside, loganin and the total contents of six phenolic acids. Furthermore, chlorogenic acid, loganin and the total contents of six phenolic acids showed significantly negative correlation with the main appearance characteristics, indicating that the appearance characteristics of Lonicerae Japonicae Flos can reflect its internal quality, and these 3 indices can be used as quality markers(Q-markers). The results of hierarchical cluster analysis showed that the samples of four grades were classified into four categories, and the samples with the same grades and the same categories accounted for 80.8% of the total samples, while the samples with the different grades were obviously classified into different categories. The results of PLS-DA analysis showed that the samples of different grades showed obvious intragroup aggregation and intergroup dispersion. The above results indicated that it was feasible to evaluate the traditional commodity grade of Lonicerae Japonicae Flos by the method of "expert consensus of different regions". For the evaluation of traditional commodity grade of Chinese medicinal material, the samples should be representative, expert conclusions should have enough consensuses, and grade determination should be authoritative. As the crystallization of clinical experience, traditional commodity grade can scientifically reflect the internal quality of Chinese medicinal material.

The genetic architecture of Down syndrome phenotypes revealed by high-resolution analysis of human segmental trisomies.

Down syndrome (DS), or trisomy 21, is a common disorder associated with several complex clinical phenotypes. Although several hypotheses have been put forward, it is unclear as to whether particular gene loci on chromosome 21 (HSA21) are sufficient to cause DS and its associated features. Here we present a high-resolution genetic map of DS phenotypes based on an analysis of 30 subjects carrying rare segmental trisomies of various regions of HSA21. By using state-of-the-art genomics technologies we mapped segmental trisomies at exon-level resolution and identified discrete regions of 1.8-16.3 Mb likely to be involved in the development of 8 DS phenotypes, 4 of which are congenital malformations, including acute megakaryocytic leukemia, transient myeloproliferative disorder, Hirschsprung disease, duodenal stenosis, imperforate anus, severe mental retardation, DS-Alzheimer Disease, and DS-specific congenital heart disease (DSCHD). Our DS-phenotypic maps located DSCHD to a <2-Mb interval. Furthermore, the map enabled us to present evidence against the necessary involvement of other loci as well as specific hypotheses that have been put forward in relation to the etiology of DS-i.e., the presence of a single DS consensus region and the sufficiency of DSCR1 and DYRK1A, or APP, in causing several severe DS phenotypes. Our study demonstrates the value of combining advanced genomics with cohorts of rare patients for studying DS, a prototype for the role of copy-number variation in complex disease.

[Characteristics and driving forces of wetland landscape pattern evolution of the city belt along the Yellow River in Ningxia, China]. / å®å¤æ²¿é»„åŸŽå¸‚å¸¦æ¹¿åœ°æ™¯è§‚æ ¼å±€æ¼”å˜ç‰¹å¾åŠé©±åŠ¨åŠ›.

Wetlands are one of the most productive ecosystems in the world, with functions of water purification, climate regulation, and carbon sinks. Due to the stresses caused by human social development and changes of natural conditions, wetlands have been seriously damaged. We examined the evolutionary law of landscape pattern of wetland along the Yellow River, and acquainted the current situation of wetland resources and dynamic change. Based on satellite images of year 2000, 2009, and 2018 from Landsat, we used landscape indices and geographic detectors to quantitatively analyze the characteristics and driving forces of wetland landscape pattern evolution of the city belt along the Yellow River in Ningxia from 2000 to 2018. The results showed that the wetland area of the city belt along the Yellow River in Ningxia enlarged first and then decreased from 2000 to 2018. The wetland area increased by 52.2 km2 in the early stage of the study with an increasing rate of 8.2%, and decreased by 26.8 km2 with a reduction rate of 3.9% in the later stage. The wetland was mainly transformed to construction land and unused land, with transfer out area being 166.7 and 158.4 km2 respectively. New wetland was mainly transformed from unused land, forest, and grassland, with an area of 543.1 km2. The fragmentation degree of wetland landscape in city belt was increasing, the balanced distribution of all kinds of wetlands was gradually strengthened, the landscape diversity was increasing, and the dominant landscape types were gradually weakening. Natural factors and socio-economic factors jointly affected the evolution of wetland landscape pattern in city belt. Among all socio-economic factors population was the most important one. Among natural factors, precipitation and temperature were important. Other driving factors were relatively weak, but could not be ignored.

William's syndrome: gene expression is related to parental origin and regional coordinate control.

William's syndrome (WS) features a spectrum of neurocognitive and behavioral abnormalities due to a rare 1.5 MB deletion that includes about 24-28 genes on chromosome band 7q11.23. Study of the expression of these genes from the single normal copy provides an opportunity to elucidate the genetic and epigenetic controls on these genes as well as their roles in both WS and normal brain development and function. We used quantitative RT-PCR to determine the transcriptional level of 14 WS gene markers in a cohort of 77 persons with WS and 48 normal controls. Results reported here: (1) show that the expression of the genes deleted in WS is decreased in some but not all cases, (2) demonstrate that the parental origin of the deletion contributes to the level of expression of GTF2I independently of age and gender and (3) indicate that the correlation of expression between GTF2I and some other genes in the WS region differs in WS subjects and normal controls, which in turn points toward a regulatory role for this gene. Interspecies comparisons suggest GTF2I may play a key role in normal brain development.

Estrogen receptor-alpha and -beta regulate the human corticotropin-releasing hormone gene through similar pathways.

Corticotropin-releasing hormone (CRH) plays a central role in controlling stress response. In this study, we aimed to identify the regulatory effect of estrogen receptor (ER) on CRH and the underlying mechanism. We investigated the regulation of CRH mRNA in the BE(2)-C cell line, a human neuroblastoma cell line which express endogenous CRH. Quantitative reverse transcriptase-polymerase chain reactions showed that in the presence of estradiol overexpressing ER alpha or ER beta in BE(2)-C cells increased the transcription of CRH. Chromatin immunoprecipitation assays in this cell line also showed that both ER alpha and ER beta can be recruited to the CRH promoter with the treatment of estradiol. However, electrophoretic mobility shift assays did not show direct binding between estrogen receptors and two estrogen response elements (ERE) half sites in the CRH promoter. To clarify the regulatory mechanism, site-directed mutagenesis and reporter gene assay in the CHO cell line were used. When the ERE half sites and the cAMP regulatory element (CRE) in the CRH promoter were disrupted, ER-mediated up-regulation of CRH promoter activity reduced. Between the two ERE half sites studied, the -316 ERE half site contributed more to the constitutive CRH expression induced by ER. In summary, our results confirm the stimulation of ER alpha and ER beta on CRH expression and demonstrate the important roles of the ERE half sites and CRE for the action of ER alpha and ER beta.

Detection of chromosome aberrations in Chinese children with autism using G-banding and BAC FISH.

OBJECTIVE: To detect the characteristic chromosomal changes in Chinese children with infantile autism. METHODS: Chromosome aberrations in 68 cases of infantile autism were analyzed by high-resolution G-banding and fluorescence in situ hybridization (FISH) with bacterial artificial chromosome (BAC) clones. RESULTS: Chromosomal changes were detected in 4 cases by high-resolution G-banding: one case with t(4;6)(q23-24;p21), one case with longer p arm of chromosome 21 (21p+), and two cases with pericentric inversion of chromosome 9 (inv(9)) which was confirmed by C-banding. BAC FISH analysis was performed to confirm these observations and changes in chromosomes 2, 7 and 15, which are often found in autistic children. There could exist the translocation of t(4;6) (q25-26;p21.1). Chromosome changes often reported previously in chromosomes 2, 7 and 15 were not detected in this study. Inv(9) and 21p+ were not confirmed with present BAC clones. CONCLUSION: Chromosomal changes were detected in four cases of infantile autism, with a detectability of 5.9% , far lower than that (10% to 48%) reported in literature. The breakpoint of translocation could be detected more accurately using BAC FISH method.

Refined physical map of the human PAX2/HOX11/NFKB2 cancer gene region at 10q24 and relocalization of the HPV6AI1 viral integration site to 14q13.3-q21.1.

BACKGROUND: Chromosome band 10q24 is a gene-rich domain and host to a number of cancer, developmental, and neurological genes. Recurring translocations, deletions and mutations involving this chromosome band have been observed in different human cancers and other disease conditions, but the precise identification of breakpoint sites, and detailed characterization of the genetic basis and mechanisms which underlie many of these rearrangements has yet to be resolved. Towards this end it is vital to establish a definitive genetic map of this region, which to date has shown considerable volatility through time in published works of scientific journals, within different builds of the same international genomic database, and across the differently constructed databases. RESULTS: Using a combination of chromosome and interphase fluorescent in situ hybridization (FISH), BAC end-sequencing and genomic database analysis we present a physical map showing that the order and chromosomal orientation of selected genes within 10q24 is CEN-CYP2C9-PAX2-HOX11-NFKB2-TEL. Our analysis has resolved the orientation of an otherwise dynamically evolving assembly of larger contigs upstream of this region, and in so doing verifies the order and orientation of a further 9 cancer-related genes and GOT1. This study further shows that the previously reported human papillomavirus type 6a DNA integration site HPV6AI1 does not map to 10q24, but that it maps at the interface of chromosome bands 14q13.3-q21.1. CONCLUSIONS: This revised map will allow more precise localization of chromosome rearrangements involving chromosome band 10q24, and will serve as a useful baseline to better understand the molecular aetiology of chromosomal instability in this region. In particular, the relocation of HPV6AI1 is important to report because this HPV6a integration site, originally isolated from a tonsillar carcinoma, was shown to be rearranged in other HPV6a-related malignancies, including 2 of 25 genital condylomas, and 2 of 7 head and neck tumors tested. Our finding shifts the focus of this genomic interest from 10q24 to the chromosome 14 site.

The 200-kb segmental duplication on human chromosome 21 originates from a pericentromeric dissemination involving human chromosomes 2, 18 and 13.

Regions close to human centromeres contain DNA fragments spanning hundreds of kilobases that exhibit a high degree of sequence identity (>95%). Here we report the genomic structure and evolution of a family of four paralogous regions related to a 220-kb genomic fragment present on the long arm of human chromosome 21 (21q22.1). Phylogenetic classification of the paralogous sequences obtained from the draft of the Human Genome Project are in agreement with results from comparative fluorescence in situ hybridization on metaphase chromosomes from human and great apes. The original copy present in 21q22.1 in human was duplicated in great apes after the divergence of the orang-utan and inserted in a pericentromeric region, most likely the ancestor of HSA2q, then disseminated by transposition of a larger fragment to other pericentromeric locations: HSA18p11, HSA13q11 and HSA21q11.1. The degree of dissemination varies among species.

Molecular cytogenetic characterization of a subtle interstitial del(3)(p25.3p26.2) in a patient with deletion 3p syndrome.

Deletion 3p syndrome is associated with characteristic facial features, growth failure, and mental retardation. Typically, individuals with deletion 3p syndrome have terminal deletions that result in loss of material from 3p25 to 3pter. We present a child with a clinical phenotype consistent with deletion 3p syndrome (ptosis, microcephaly, growth retardation, and developmental delay) and a subtle interstitial deletion in the distal portion of the short arm of chromosome 3, del(3)(p25.3p26.2). Fluorescence in situ hybridization (FISH) studies using 3p subtelomeric probes confirmed the terminal region of chromosome 3 was present. Sequence tagged sites (STS)-linked BAC clones mapping to chromosomal region 3p25-p26 were used to characterize the interstitial deletion by FISH. The results indicate the deletion is within a region of approximately 4.5 Mb between STS markers D3S3630 and D3S1304. This interstitial deletion lies within all previously reported terminal deletions in deletion 3p syndrome individuals, and represents the smallest reported deletion associated with deletion 3p syndrome. Characterization of the deletion may help identify genes important to growth and development that contribute to the deletion 3p syndrome phenotype when present in a hemizygous state.