Efficacy of Traditional Chinese Herbs combined with bioelectrical stimulation on patients with kidney deficiency and blood stasis type thin endometrium: a retrospective observational study.

OBJECTIVE: This study aimed to investigate the efficacy of Traditional Chinese Herbs (TCH) combined with bioelectrical stimulation (BES) on patients with kidney deficiency and blood stasis type thin endometrium. PATIENTS AND METHODS: A retrospective observational study was conducted on 83 patients diagnosed with thin endometrium, treated in our hospital from August 2019 to August 2021. The clinical data of the patients were reviewed, and 60 eligible patients were categorized into two groups based on the treatment they received: the TCH-BES group (n=30, patients received Femoston, TCH and BES treatment) and the control group (n=30, patients received Femoston only). The endometrial thickness (EMT), uterine artery resistance index (RI) and pulsatility index (PI), serum reproductive hormone levels, traditional Chinese medicine (TCM) syndrome scores, and clinical pregnancy outcomes between the two groups were compared. Continuous data were described as mean ± standard deviation (X- ± S). Student's t-test was used for comparison between the two groups and paired-sample t-test was used for comparison within the same group before and after the treatment. RESULTS: A total of 60 patients with thin endometrium, aged 20-35 years (average, 31.67±3.19 years), were included in this study. After the treatment, the EMT, E2 and progesterone (P) levels of the TCH-BES group were higher than that of the control group (p<0.001, p<0.05 and p<0.001, respectively), the PI, RI level and TCM syndrome scores of the TCH-BES group were lower than those of the control group (p<0.001). The clinical efficacy and pregnancy rate in the TCH-BES group were significantly higher than those in the control group (p<0.05). CONCLUSIONS: TCH combined with EBS has a satisfactory efficacy on patients with kidney deficiency and blood stasis type thin endometrium, and improves EMT, E2 and P levels, reduces PI, RI and TCM syndrome, and eventually leads to a favorable clinical pregnancy outcome.

The diagnostic value of determination of serum GOLPH3 associated with CA125, CA19.9 in patients with ovarian cancer.

OBJECTIVE: To evaluate the value of three tumor markers serum Golgi phosphoprotein-3 (GOLPH3), cancer antigen 125 (CA125) and cancer antigen 19-9 (CA19.9) in the diagnosis and postoperative evaluation of ovarian cancer by detecting these three markers. PATIENTS AND METHODS: A total of 187 patients were studied and included in the ovarian cancer group, benign pelvic mass group, and the normal control group. The levels of serum Golgi phosphoprotein-3 (GOLPH3), cancer antigen 125 (CA125) and cancer antigen 199 (CA19.9) were detected, respectively, and their effects on the diagnosis, evaluation, pathology typing and staging of ovarian cancer were measured. RESULTS: The sensitivity of the detection of ovarian cancer by GOLPH3 combined with CA125 and CA19.9 was higher than that by a single marker (p<0.05). The level of serum GOLPH3 in patients with serous and endometrioid carcinoma was significantly higher than that in patients with mucinous carcinoma, clear-cell carcinoma and germ cell tumor (p<0.05). There was no significant statistical difference in serum GOLPH3 level between patients with ovarian malignancies at stage III-IV and those at stage I-II (p>0.05). The levels of serum GOLPH3, CA125 and CA19.9 in patients with ovarian malignancies after surgery were significantly lower than those before surgery (p<0.05). CONCLUSIONS: The combined detection by GOLPH3, CA125, and CA19.9 may improve the diagnosis rate of ovarian epithelial cancer. GOLPH3, as a new ovarian cancer tumor marker used in clinical diagnosis, is expected to become an important indicator for the early diagnosis of ovarian cancer and the determination of clinical surgery efficacy.

Single nucleotide polymorphisms in the mitochondrial displacement loop and age-at-onset of non-small cell lung cancer.

The associations between single nucleotide polymorphisms (SNPs) in the displacement loop (D-loop) of mitochondrial DNA (mtDNA) and cancer risk and disease outcome have been extensively analyzed. We investigated the association between age-at-onset and SNPs in the mitochondrial D-loop using a population-based series of non-small cell lung cancer (NSCLC) patients. The D-loop region of mtDNA from NSCLC patients was amplified and sequenced. The age-at-onset curve of NSCLC patients was calculated using the Kaplan-Meier method at each SNP site and values were compared using the log-rank test. The SNP sites of nucleotides 200G/A and 16362T/C were identified to determine their association with age-at-onset of NSCLC using the log-rank test. The nucleotide 207G/A was identified for its association with age-at-onset at a borderline significance level (P = 0.060). We found that genetic polymorphisms in the D-loop were predictive markers for age-at-onset in NSCLC patients. Accordingly, the analysis of genetic polymorphisms in the mitochondrial D-loop can be used to identify NSCLC patient subgroups at high risk of early onset.

Coupled equilibria of a self-associating drug loaded into polymeric nanoparticles.

Doxorubicin (DOX) and other anti-cancer drugs are often formulated using nanoparticles for passive or active targeting and reducing detrimental side effects. Anionic polymers have been shown to effectively facilitate loading of cationic DOX hydrochloride into nanoparticles with high efficiency. One powerful method to study DOX loading into anionic polymeric nanoparticles has been isothermal titration calorimetry (ITC), but the curves are complex and were previously interpreted in a largely qualitative manner only. Here we present detailed quantitative modelling of such ITC data, corroborated by zeta potential measurements and dynamic light scattering. The model takes into account 3 coupled equilibria. First, DOX self-associates in solution to dimers and larger aggregates. This effect is modelled in terms of the stepwise aggregation model. Second, DOX binds with a 1:1 stoichiometry to the carboxylic acids in the polymer at low salt. At about 33% saturation, the nanoparticles collapse in size and the enthalpy of further binding becomes less exothermic. Third, free DOX also stacks onto polymer-bound DOX. This stacking effect is very weak and hardly detected by ITC. It is, however, revealed by a positive zeta potential. The present work demonstrates the power of combining ITC with light scattering and zeta potential measurements for studying the thermodynamics of drug loading into polymeric nanoparticles.

Association of chemotactic factor receptor 5 gene with breast cancer.

We designed a 2-stage study to investigate chemotactic factor receptor 5 (CCR5) gene expression in breast cancer tissues and axillary lymph nodes and analyze the association between the CCR5-Î"32 gene polymorphism and the clinical features and prognosis of breast cancer patients. The first stage examined 72 cases of invasive ductal carcinoma and axillary lymph node tissue, 50 cases of breast fibroadenoma tissue, and 40 cases of normal breast tissue. The tissues specimens were embedded in paraffin, and CCR5 expression was detected using immunohistochemical methods. C-erbB-2, p53, Ki-67, estrogen receptor, and progesterone receptor expression were also detected in the breast cancer tissues. The second stage examined 35 cases of surgically removed tissue. Relative expression levels of CCR5 messenger RNA (mRNA) in primary foci, axillary lymph node, and cancer-adjacent tissues of the breast cancer and breast fibroadenoma samples were detected using real-time quantitative reverse transcription-polymerase chain reaction assay. We found that 1) CCR5 mRNA relative expression levels in breast cancer tissue were significantly higher than those in adjacent normal tissue (P < 0.01) and benign tumors (P < 0.05). The relative CCR5 mRNA relative expression level between phase II and phase III breast cancer tissues was statistically significant (P < 0.05). The CCR5 mRNA relative expression level between adjacent normal tissues and fibroadenoma tissues was not significantly different (P > 0.05). 2) Relative CCR5 mRNA expression level was significantly higher in metastatic lymph node tissues than that in non-metastatic lymph nodes (P < 0.05), and 3) CCR5 expression in breast cancer tissue was positively correlated with axillary lymph node metastasis (chi-square = 4.982, P = 0.026, r = 0.305). CCR5 expression was mildly and positively correlated with the oncogene C-erbB-2 (P < 0.05, r = 0.291). 4) CCR5 expression in breast cancer tissue was not correlated with age, menopause, maximum tumor size, tumor phase, p53, Ki-67, estrogen receptor, progesterone receptor, or other clinical features (P > 0.05). We concluded that CCR5 expression significantly increases in breast cancer tissues and metastatic lymph nodes. CCR5 plays a role in breast cancer development and axillary lymph node metastasis. It can be used indirectly as an indicator of axillary lymph node metastasis and prognosis.

Cell wall degradation and the dynamic changes of Ca2+ and related enzymes in the developing aerenchyma of wheat (Triticum aestivum L.) under waterlogging.

This research was aimed to study the cell wall degradation and the dynamic changes of Ca2+ and related enzymes in developing aerenchyma of wheat root under waterlogging. An examination of morphological development by light and electron microscope revealed that the structure of cell wall in middle cortical cells remained intact after 12 h of waterlogging and turned thinner after waterlogging for 24 h. At 48 h, the aerenchyma has been formed. The cellulase activity gradually increased in middle cortical cells within 24 h of waterlogging, and decreased with the formation of aerenchyma. Fluorescence detection and subcellular localization of Ca2+ showed the dynamic changing of Ca2+ at the cellular and subcellular levels during the development of aerenchyma. The activity of Ca2+-ATPase enhanced markedly in intercellular space, plasma membrane and tonoplast of some middle cortical cells after 8 h of waterlogging and remained high after 24 h, but it decreased after 48 h of waterlogging. All these suggests that cellulase, Ca2+ and Ca2+-ATPase show a dynamic distribution during the aerenchyma development which associated with the cell wall degradation of middle cortical cells. Moreover, there is a feedback regulation between Ca2+ and Ca2+-ATPase.

Either Kras activation or Pten loss similarly enhance the dominant-stable CTNNB1-induced genetic program to promote granulosa cell tumor development in the ovary and testis.

WNT, RAS or phosphoinositide 3-kinase signaling pathways control specific stages of ovarian follicular development. To analyze the functional interactions of these pathways in granulosa cells during follicular development in vivo, we generated specific mutant mouse models. Stable activation of the WNT signaling effector ß-catenin (CTNNB1) in granulosa cells results in the formation of premalignant lesions that develop into granulosa cell tumors (GCTs) spontaneously later in life or following targeted deletion of the tumor suppressor gene Pten. Conversely, expression of oncogenic KRAS(G12D) dramatically arrests proliferation, differentiation and apoptosis in granulosa cells, and consequently, small abnormal follicle-like structures devoid of oocytes accumulate in the ovary. Because of the potent anti-proliferative effects of KRAS(G12D) in granulosa cells, we sought to determine whether KRAS(G12D) would block precancerous lesion and tumor formation in follicles of the CTNNB1-mutant mice. Unexpectedly, transgenic Ctnnb1;Kras-mutant mice exhibited increased GC proliferation, decreased apoptosis and impaired differentiation and developed early-onset GCTs leading to premature death in a manner similar to the Ctnnb1;Pten-mutant mice. Microarray and reverse transcription-PCR analyses revealed that gene regulatory processes induced by CTNNB1 were mostly enhanced by either KRAS activation or Pten loss in remarkably similar patterns and degree. The concomitant activation of CTNNB1 and KRAS in Sertoli cells also caused testicular granulosa cell tumors that showed gene expression patterns that partially overlapped those observed in GCTs of the ovary. Although the mutations analyzed herein have not yet been linked to adult GCTs in humans, they may be related to juvenile GCTs or to tumors in other tissues where CTNNB1 is mutated. Importantly, the results provide strong evidence that CTNNB1 is the driver in these contexts and that KRAS(G12D) and Pten loss promote the program set in motion by the CTNNB1.

Molecular and functional characteristics of ovarian surface epithelial cells transformed by KrasG12D and loss of Pten in a mouse model in vivo.

Ovarian cancer is a complex and deadly disease that remains difficult to detect at an early curable stage. Furthermore, although some oncogenic (Kras, Pten/PI3K and Trp53) pathways that are frequently mutated, deleted or amplified in ovarian cancer are known, how these pathways initiate and drive specific morphological phenotypes and tumor outcomes remain unclear. We recently generated Pten(fl/fl); Kras(G12D); Amhr2-Cre mice to disrupt the Pten gene and express a stable mutant form of Kras(G12D) in ovarian surface epithelial (OSE) cells. On the basis of histopathologic criteria, the mutant mice developed low-grade ovarian serous papillary adenocarcinomas at an early age and with 100% penetrance. This highly reproducible phenotype provides the first mouse model in which to study this ovarian cancer subtype. OSE cells isolated from ovaries of mutant mice at 5 and 10 weeks of age exhibit temporal changes in the expression of specific Mullerian epithelial marker genes, grow in soft agar and develop ectopic invasive tumors in recipient mice, indicating that the cells are transformed. Gene profiling identified specific mRNAs and microRNAs differentially expressed in purified OSE cells derived from tumors of the mutant mice compared with wild-type OSE cells. Mapping of transcripts or genes between the mouse OSE mutant data sets, the Kras signature from human cancer cell lines and the human ovarian tumor array data sets, documented significant overlap, indicating that KRAS is a key driver of OSE transformation in this context. Two key hallmarks of the mutant OSE cells in these mice are the elevated expression of the tumor-suppressor Trp53 (p53) and its microRNA target, miR-34a-c. We propose that elevated TRP53 and miR-34a-c may exert negatively regulatory effects that reduce the proliferative potential of OSE cells leading to the low-grade serous adenocarcinoma phenotype.

Gene expression profiling in human insulinoma tissue: genes involved in the insulin secretion pathway and cloning of novel full-length cDNAs.

Insulinoma is a clinically common cause of organic hypoglycemia. The prominent characteristic of insulinoma is endogenous hyperinsulinism. Until now, the molecular biology of human insulinoma has been little understood. In this study, gene expression profiling of human insulinoma was established by expressed sequence tag (EST) sequencing and cDNA array. A total of 2063 clones were obtained, of these, 1589 clones were derived from EST sequencing, 975 clones were derived from cDNA array and 501 clones were shared by the two methods. G protein alpha-stimulating activity polypeptide (Gsalpha) and carboxypeptidase E (CPE) were the most highly expressed genes in human insulinoma, as derived by EST sequencing and cDNA array respectively. The genes involved in the protein/insulin secretion pathway were strongly expressed in human insulinoma tissue. Meanwhile, eight full-length cDNAs of novel genes were cloned and sequenced. The results demonstrated the molecular biology of human insulinoma tissue at the level of transcript abundance and validated the efficacy of EST sequencing combined with cDNA array in the construction of gene expression profiling. In conclusion, the predominance of the genes participating in the secretory pathway suggested that regulation of secretion might be a major mechanism by which insulin release is abnormally increased in patients with insulinomas. It was also concluded that overexpression of the Gsalpha gene played an important role in the pathogenesis of insulinoma.

Polo-like kinase-1 in porcine oocyte meiotic maturation, fertilization and early embryonic mitosis.

Polo-like kinases (Plks) are a family of serine/threonine protein kinases that regulate multiple stages of mitosis. Expression and distribution of polo-like kinase 1 (Plk1) were characterized during porcine oocyte maturation, fertilization and early embryo development in vitro, as well as after microtubule polymerization modulation. The quantity of Plk1 protein remained stable during meiotic maturation. Plk1 accumulated in the germinal vesicles (GV) in GV stage oocytes. After germinal vesicle breakdown (GVBD), Plk1 was localized to the spindle poles at metaphase I (MI) stage, and then translocated to the middle region of the spindle at anaphase-telophase I. Plk1 was also localized in MII spindle poles and on the spindle fibers and on the middle region of anaphase-telophase II spindles. Plk1 was not found in the spindle region when colchicine was used to inhibit microtubule organization, while it accumulated as several dots in the cytoplasm after taxol treatment. After fertilization, Plk1 concentrated around the female and male pronuclei. During early embryo development, Plk1 was found to be in association with the mitotic spindle at metaphase, but distributed diffusely in the cytoplasm at interphase. Our results suggest that Plk1 is a pivotal regulator of microtubule organization and cytokinesis during porcine oocyte meiotic maturation, fertilization, and early embryo cleavage in pig oocytes.

High-copy-number expression of Sub2p, a member of the RNA helicase superfamily, suppresses hpr1-mediated genomic instability.

We report on a novel role for a pre-mRNA splicing component in genome stability. The Hpr1 protein, a component of an RNA polymerase II complex and required for transcription elongation, is also required for genome stability. Deletion of HPR1 results in a 1,000-fold increase in genome instability, detected as direct-repeat instability. This instability can be suppressed by the high-copy-number SUB2 gene, which is the Saccharomyces cerevisiae homologue of the human splicing factor hUAP56. Although SUB2 is essential, conditional alleles grown at the permissive temperature complement the essential function of SUB2 yet reveal nonessential phenotypes. These studies have uncovered a role for SUB2 in preventing genome instability. The genomic instability observed in sub2 mutants can be suppressed by high-copy-number HPR1. A deletion mutant of CDC73, a component of a PolII complex, is also unstable for direct repeats. This too is suppressed by high-copy-number SUB2. Thus, defects in both the transcriptional machinery and the pre-mRNA splicing machinery can be sources of genome instability. The ability of a pre-mRNA splicing factor to suppress the hyperrecombination phenotype of a defective PolII complex raises the possibility of integrating transcription, RNA processing, and genome stability or a second role for SUB2.

Cloning and functional analysis of cDNAs with open reading frames for 300 previously undefined genes expressed in CD34+ hematopoietic stem/progenitor cells.

Three hundred cDNAs containing putatively entire open reading frames (ORFs) for previously undefined genes were obtained from CD34+ hematopoietic stem/progenitor cells (HSPCs), based on EST cataloging, clone sequencing, in silico cloning, and rapid amplification of cDNA ends (RACE). The cDNA sizes ranged from 360 to 3496 bp and their ORFs coded for peptides of 58-752 amino acids. Public database search indicated that 225 cDNAs exhibited sequence similarities to genes identified across a variety of species. Homology analysis led to the recognition of 50 basic structural motifs/domains among these cDNAs. Genomic exon-intron organization could be established in 243 genes by integration of cDNA data with genome sequence information. Interestingly, a new gene named as HSPC070 on 3p was found to share a sequence of 105bp in 3' UTR with RAF gene in reversed transcription orientation. Chromosomal localizations were obtained using electronic mapping for 192 genes and with radiation hybrid (RH) for 38 genes. Macroarray technique was applied to screen the gene expression patterns in five hematopoietic cell lines (NB4, HL60, U937, K562, and Jurkat) and a number of genes with differential expression were found. The resource work has provided a wide range of information useful not only for expression genomics and annotation of genomic DNA sequence, but also for further research on the function of genes involved in hematopoietic development and differentiation.

Activation of p53 or loss of the Cockayne syndrome group B repair protein causes metaphase fragility of human U1, U2, and 5S genes.

Infection by adenovirus 12, transfection with the Ad12 E1B 55 kDa gene, or activation of p53 cause metaphase fragility of four loci (RNU1, PSU1, RNU2, and RN5S) each containing tandemly repeated genes for an abundant small RNA (U1, U2, and 5S RNA). We now show that loss of the Cockayne syndrome group B protein (CSB) or overexpression of the p53 carboxy-terminal domain induces fragility of the same loci; moreover, p53 interacts with CSB in vivo and in vitro. We propose that CSB functions as an elongation factor for transcription of structured RNAs, including some mRNAs. Activation of p53 would inhibit CSB, stalling transcription complexes and locally blocking chromatin condensation. Impaired transcription elongation may also explain the diverse clinical features of Cockayne syndrome.

Expression and characterization of an epididymis-specific gene.

A truncated cDNA coding a rabbit epididymal protein (BE-20) was identified in a previous study. In the present study the full-length cDNA was isolated by the method of rapid amplification of cDNA ends. The BE-20 cDNA consisted of 585 bp with a poly(A) tail of 26 residues and an open reading frame composed of 369 bp encoding a deduced polypeptide containing 123 amino acid residues with a calculated molecular mass of 13 kDa. The N-terminus-contained a leucine-rich segment. BE-20 cDNA has about 76.8% homology with the HE4 gene of human epididymis. Northern blot analysis of mRNAs prepared from 17 different human tissues was performed using as probe a 0.5-kb DNA fragment corresponding to a segment of BE-20 cDNA. Positive reaction was elicited only with epididymal mRNA. A DNA fragment corresponding to a section of the open reading frame of BE-20 cDNA was cloned in Escherichia coli under the control of the T7 promoter. The cellular content of the expressed recombinant protein comprised about 55% of the total protein. The chromatographically purified bacterial product migrated as a single band with an estimated M(r) of 15 kDa on analysis by SDS-PAGE. In conclusion, BE-20 cDNA is expressed only in the epididymis. It is structurally related to the four-disulfide core family of extracellular proteinase inhibitors and may be involved in sperm maturation.

Identification of a rabbit epididymal protein gene.

A protein designated as BE-20 was purified from cauda epididymal fluid of the rabbit by preparative polyacrylamide gel electrophoresis and HPLC on a mono Q HR5/5 anion exchange column. The purified protein migrated with an estimated Mt of 20,000 when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The amino acid sequence of the N-terminus of the BE-20 protein was determined. The initial eight amino acid residues were His-Gly-Ala-Asp-Lys-Pro-Gly-Val. The corresponding 23 mer oligonucleotide (5'-CATGGCGCTGACAAGCCTGGGGT-3') was synthesized and used as sense primer with rabbit epididymal mRNA as template in the RT-PCR system. The purified BE-20 cDNA consisted of 499 bp with an open reading frame of 285 bp encoding a deduced polypeptide composed of 95 amino acids. The BE-20 cDNA had 78.5% identity in 479 bp overlap with human epididymis-specific HE4 cDNA. The amino acid sequences of the initial 30 amino acid residues of the N-terminus of the purified protein and the deduced polypeptides were as follows: N-His-Gly-Ala-Asp-Lys-Pro-Gly-Val-Cys-Pro-Gln-Leu-Ser-Ala-Asp-Leu-Asn-Cy s- Thr-Gln-Asp-Cys-Arg-Ala-Asp-Gln-Asp-Cys-Ala-Glu. The deduced polypeptide contained 16 cysteine residues and had partial sequence homology with proteins belonging to the four-disulfide core family of extracellular proteinase inhibitors. The BE-20 protein may play a role in sperm maturation and/or capacitation.

Mutations in the RNA polymerase II transcription machinery suppress the hyperrecombination mutant hpr1 delta of Saccharomyces cerevisiae.

The soh1, soh2 and soh4 mutants were isolated as suppressors of the temperature-dependent growth of the hyperrecombination mutant hpr1 of Saccharomyces cerevisiae. Cloning and sequence analysis of these suppressor genes has unexpectedly shown them to code for components of the RNA polymerase II transcription complex. SOH2 is identical to RPB2, which encodes the second largest subunit of RNA polymerase II, and SOH4 is the same as SUA7, encoding the yeast transcription initiation factor TFIIB. SOH1 encodes a novel 14-kD protein with limited sequence similarity to RNA polymerases. Interestingly, SOH1 not only interacts with factors involved in DNA repair, but transcription as well. Thus, the Soh1 protein may serve to couple these two processes. The Soh1 protein interacts with a DNA repair protein, Rad5p, in a two-hybrid system assay. Soh1p may functionally interact with components of the RNA polymerase II complex as suggested from the synthetic lethality observed in soh1 rpb delta 104, soh1 soh2-1 (rpb2), and soh1 soh4 (sua7) double mutants. Because mutations in SOH1, RPB2 and SUA7 suppress the hyperrecombination phenotype of hpr1 mutants, this suggests a link between recombination in direct repeats and transcription.