[Expression of ProEXC and PRMT5 in cervical adenocarcinoma and their clinical significance].

This study observed the expression of ProEXC protein and PRMT5 protein in cervical adenocarcinoma and adjacent tissues, exploring the relationship between the expression of ProEXC and PRMT5 and the auxiliary diagnosis of cervical adenocarcinoma, as well as the clinical pathological parameters. A total of 88 specimens diagnosed with cervical adenocarcinoma from the Second Affiliated Hospital of Soochow University between 2015 and 2020 were collected. Immunohistochemistry was employed to detect the expression of ProEXC and PRMT5 in cervical adenocarcinoma and adjacent tissues, and statistical analysis was conducted. The Cancer Genome Atlas (TCGA) database was utilized to analyze the correlation between the prognosis of cervical adenocarcinoma patients and the expression of ProEXC and PRMT5, as well as their related gene pathways. The GSE39293 dataset from the Gene Expression Omnibus (GEO) was selected to compare the expression levels of ProEXC and PRMT5 in cervical adenocarcinoma cell lines (HELA) before and after antiviral drug treatment.In cervical adenocarcinoma tissues, the expression of ProEXC protein (95.5% vs 4.6%, P<0.001) and PRMT5 protein (81.8% vs 26.1%, P<0.001) was significantly higher than in surrounding adjacent tissues. Their expression was correlated with the tumor's T stage, lymph node metastasis, and human papillomavirus (HPV) infection (P<0.05). TCGA database analysis showed that patients with high expression of MCM2 in PRMT5 and ProEXC had a lower overall survival rate (P<0.05), while the expression of TOP2A was not significantly correlated with survival. In the GSE39293 dataset, the expression of MCM2 (9.34 vs 9.68, P<0.001) and PRMT5 (8.16 vs 8.26, P=0.087) in cells decreased after treatment with cidofovir, while TOP2A (8.54 vs 8.42, P=0.056) expression did not change significantly. In the drug-resistant group, the expression of PRMT5 (8.42 vs 8.16, P=0.002) and MCM2 (9.51 vs 9.34, P=0.029) increased, while TOP2A (8.06 vs 8.54, P<0.001) expression decreased. Gene set enrichment analysis (GSEA) suggested that high expression of ProEXC mainly affected the cell cycle pathway, while high expression of PRMT5 mainly affected the RNA splicing pathway.This study found that ProEXC protein and PRMT5 protein were highly expressed in cervical adenocarcinoma tissues, and the high-expression group had a poorer prognosis, showing a certain correlation with the clinical and pathological characteristics of cervical adenocarcinoma. This may be related to their influence on the cell cycle and RNA synthesis pathways, suggesting their potential significant roles in the progression of cervical adenocarcinoma.

[Clinicopathological and gene mutation characteristics of uterine carcinosarcoma].

To explore the clinicopathological characteristics, immunophenotype, diagnosis and differential diagnosis of uterine carcinosarcoma (UCS), and to explore the gene mutation characteristics and tumor mutation burden (TMB) of UCS. The clinical imaging, pathomorphological data and immunohistochemical expression of 4 cases of UCS, which were archived in the Department of Pathology of the Second Affiliated Hospital of Soochow University from January 2021 to May 2022 were retrospectively analyzed. All exon groups of 4 cases of UCS were sequenced. All the 4 patients were female, aged 47-81 years. The maximum diameter of the tumor was 4.0-13.0 cm, and the boundary was unclear. Microscopically, the tumor was composed of malignant epithelium and sarcoma. Immunohistochemistry showed that the epithelial components of 4 patients expressed broad-spectrum cytokeratin (AE1/E3), the sarcoma components expressed Vimentin, PAX8, ER, PR were expressed to varying degrees, and Ki-67 positive index was high (60%-90%). There were 3 p53 missense mutations, 1 nonsense mutation, 4 MLH1, PMS2, MSH2, MSH6 were positive and PD-L1 was negative. The sequencing results of the whole exon group of 4 UCS patients showed that TP53, BCL9L, BRD4, CLTCLI, PSMD1I, PLEC genes showed a high mutation ratio, which was 3/4, 2/4, 2/4, 2/4, 2/4, 2/4, respectively. TMB analysis showed that the TMB of 4 cases of UCS was<5 mut/Mb. UCS is a rare and highly malignant endometrial tumor. The sequencing results of the whole exon group suggested that TP53, BCL9L, BRD4 and other genes had high mutation rates, suggesting that the occurrence and development of UCS may be closely related to Wnt signaling pathway. Molecular typing indicated that 3 cases of UCS were of high copy number type/p53 mutation type, and 1 case had POLD1 mutation. Microsatellite stability, low PD-L1 expression and TMB results suggested that UCS patients have no obvious advantage in immunotherapy.

[Thyroglobulin measurement in fine-needle aspiration washout fluid for detection of differentiated thyroid cancer lymph nodes metastases].

Objective: To investigate the feasibiity of detecting lymph node metastasis in differentiated thyroid cancer patients by thyroglobin（Tg）measurement in fine-needle aspiration washout fluid. Method: Forty-seven differentiated thyroid cancer patients with cervical lymph node metastasis were included in this study. Fine-needle aspirations were performed on lymph nodes which were surgically resected for measurement of Tg in washout fluid. Diagnostic sensitivity, specificity and accuracy were evaluated according to three predetermined Tg threshold values: FNA-Tg/serum Tg＞1, 10 ng/ml and 100 ng/ml. Result: The meadian value of FNA-Tg in metastatic lymph node (n=46) was 5 177 ng/ml (1.79-408 300 ng/ml)，while lymph node without metastasis (n=11) showed a meadian level of 0.63 ng/ml (0.04-125.4 ng/ml). With the threshold of FNA-Tg level at FNA-Tg/serum Tg＞1, 10 ng/ml and 100 ng/ml respectively, the diagnostic sensitivity was 91.3%, 97.83% and 93.48% respectively,the specificity was 81.82%,63.64% and 90.90% respectively,and the accuracy was 89.47%,91.22% and 92.98% respectively. Conclusion: Thyroglobin measurement in fine-needle aspiration washout fluid is effective to diagnose lymph node metastasis in differentiated thyroid cancer.

Investigating conversion of endplate chondrocytes induced by intermittent cyclic mechanical unconfined compression in three-dimensional cultures.

Mechanical stimulation is known to regulate the calcification of endplate chondrocytes. The ANK protein has a strong influence on anti-calcification by transports intracellular inorganic pyrophosphate (PPi) to the extracellular. It is known that TGF-ß1 is able to induced Ank gene expression and protect chondrocyte calcification. Intermittent cyclic mechanical tension (ICMT) could induce calcification of endplate chondrocytes by decrease the expression of Ank gene. In this study, we investigated the relation of intermittent cyclic mechanical unconfined compression (ICMC) and Ank gene expression. We found that ICMC decreased the Ank gene expression in the endplate chondrocytes, and there was an decreased in the  TGF-ß1 expression after ICMC stimulation. The Ank gene expression significantly increased when treated by transforming growth factor alpha 1 (TGF-ß1) in a dose-dependent manner and decreased when treated by SB431542 (ALK inhibitor) in a dose-dependent manner. Our results implicate that ICMC-induced downregulation of Ank gene expression may be regulated by TGF-ß1 in endplate chondrocytes.

Effects of Bacillus subtilis natto on milk production, rumen fermentation and ruminal microbiome of dairy cows.

Two experiments were conducted to evaluate the effects of Bacillus subtilis natto, which was initially isolated from fermented soybeans on milk production, rumen fermentation and ruminal microbiome in dairy cows. In Experiment 1, 36 early lactation Chinese Holstein dairy cows (56 ± 23 days in milk) were randomly assigned to three groups: Control, cows were fed total mixed ration (TMR); BSNLOW, TMR plus 0.5 × 1011 colony-forming units (cfu) of B. subtilis natto/cow per day; and BSNHIGH, TMR plus 1.0 × 1011 cfu of B. subtilis natto/cow per day. During the 70-day treatment period, daily milk production and daily milk composition were determined in individual cows. The results showed that supplementing dairy cows with 0.5 × 1011 and 1.0 × 1011 cfu of B. subtilis natto linearly increased (P < 0.01) milk production (25.2 and 26.4 kg/day v. 23.0 kg/day), 4% fat-corrected milk (27.3 and 28.1 kg/day v. 24.2 kg/day), energy-corrected milk (27.3 and 28.2 kg/day v. 24.2 kg/day), as well as milk fat (1.01 and 1.03 kg/day v. 0.88 kg/day), protein (0.77 and 0.82 kg/day v. 0.69 kg/day) and lactose yield (1.16 and 1.22 kg/day v. 1.06 kg/day) but decreased milk somatic cell counts (SCC) by 3.4% to 5.5% (P < 0.01) in BSNLOW and BSNHIGH treatments compared with Control. In Experiment 2, four rumen-cannulated dairy cows were fed the basal diet from 1 to 7 days (pre-trial period) and rumen samples were collected on days 6 and 7; the same cows then were fed 1.0 × 1011 cfu/day B. subtilis natto from days 8 to 21 (trial period) and rumen samples were collected on days 20 and 21. B. subtilis natto was discontinued from days 22 to 28 (post-trial period) and rumen samples were collected on days 27 and 28. Compared with the pre- and post-periods, ruminal pH decreased by 2.7% to 3.0% during the trial period (P < 0.01), whereas ammonia nitrogen (NH3-N), total volatile fatty acids and molar proportion of propionate (P < 0.01) and valerate (P < 0.05) increased. Molar proportion of acetate decreased and the acetate to propionate ratio was lower (P < 0.01) during the trial period. However, no differences for 24-h in sacco dry matter digestibility were detected among different periods (treatments) though NDF digestibility was reduced in the trial and post-trial periods (P < 0.01). Compared with pre-trial period, total ruminal bacteria, proteolytic and amylolytic bacteria in rumen enumerated by culture methods increased by 15.0%, 16.2% and 11.7%, respectively (P < 0.01) but protozoa decreased to 5.35 log10 cfu/ml (P < 0.01) during the trial period. These results demonstrate that B. subtilis natto improves milk production and milk components yield, decreases SCC and promotes the growth of total ruminal bacteria, proteolytic and amylolytic bacteria, which indicate that B. subtilis natto has potential to be applied as a probiotic for dairy cows.

Mechanical properties of femoral cortical bone following cemented hip replacement.

Femoral bone remodeling following total hip replacement is a big concern and has never been examined mechanically. In this study, six goats underwent unilateral cemented hip hemiarthroplasty with polymethyl methacrylate (PMMA) bone cement. Nine months later animals were sacrificed, and the femoral cortical bone slices at different levels were analysed using microhardness testing and microcomputed tomography (micro-CT) scanning. Implanted femurs were compared to contralateral nonimplanted femurs. Extensive bone remodeling was demonstrated at both the proximal and middle levels, but not at the distal level. Compared with the nonimplanted side, significant decreases were found in the implanted femur in cortical bone area, bone mineral density, and cortical bone hardness at the proximal level, as well as in bone mineral density and bone hardness at the middle level. However, no significant difference was observed in either variable for the distal level. In addition, similar proximal-to-distal gradient changes were revealed both in cortical bone microhardness and bone mineral density. From the mechanical point of view, the results of the present study suggested that stress shielding is an important mechanical factor associated with bone adaptation following total hip replacement.

In vivo cancellous bone remodeling on a strontium-containing hydroxyapatite (sr-HA) bioactive cement.

The purpose of this study was to investigate the in vivo bone response to the strontium-containing hydroxyapatite (Sr-HA) bioactive bone cement injected into the cancellous bone. Sr-HA cement was injected into the iliac crest of rabbits for 1, 3, and 6 months. Active bone formation and remodeling were observed after 1 month. Newly formed bone was observed to grow onto the bone cement after 3 months. Thick osteoid layer with osteoblasts formed along the bone and guided over the bone cement surface reflected the stimulating effect of Sr-HA. From scanning electron microscopy (SEM) and energy-dispersive X-ray (EDX) analysis, high calcium and phosphorus levels were detected at the interface with a thick layer of 70 microm in width, and fusion of Sr-HA with the bone was observed. Blood vessels were found developing in remodeling sites. The affinity of bone on Sr-HA cement was increased from 73.55 +/- 3.50% after 3 months up to 85.15 +/- 2.74% after 6 months (p < 0.01). In contrast to Sr-HA cement, poly(methyl methacrylate) (PMMA) bone cement was neither osteoconductive nor bioresorbable. Results show that the Sr-HA cement is biocompatible and osteoconductive, which is suitable for use in treating osteoporotic vertebral fractures.

[Extension of life-span of normal human fibroblasts by reconstitution of telomerase activity].

Most of normal human somatic cells can divide only a finite number of times and inevitably become senescent. Telomerase is an enzyme that imparts replicative immortality by maintaining the length of the telomeres when expressed in reproductive and cancer cells. Cells that are mortal do not express the telomerase. Recently it was reported that the life-span of the normal human cells could be successfully extended by introduction of telomerase into these cells. We have found, in the previous work, that fibroblasts exhibited an osteogentic potential, and therefore, can be considered as a type of "seed cells" in tissue engineering for bone repairing and reconstruction. But this potential was impaired by the limitation in life-span and proliferative capacity of the normal fibroblasts. In the present work, plasmid pGRN145 bearing a cDNA insert of human telomerase reverse transcriptase (hTERT) was introduced into the fibroblasts with osteogenic potential by electroporation. The stable hTERT+ fibroblast clones was established and cultured for long-term in a medium containing hygromycin-B. The exogenous hTERT mRNA expression and telomerase activity were detected. The hTERT+ fibroblasts showed shorter population doubling time and no beta-galactosidase stain, which indicated a stronger proliferative capacity and fewer signs of cell senescence, compared to their hTERT- counterpart. These evidenced that the life-span of hTERT+ fibroblasts was extended. The assays for DNA euploidy by flow cytometry and chromosome karyotype by cytogenetic technique showed no signs of heteroploidy, providing the data for cell carcinogenesis and utilization safety. The results of the present study suggested that the introduction of hTERT could make the life-span of normal fibroblast extended without causing their malignant transformation, and such type of "longevous" fibroblasts might be clinically useful in tissue engineering for bone repairing and reconstruction.