A Challenging Correlation between Tumor Cellularity and Somatic Variant Allele Fraction in Lung and Colorectal Cancers-Specimens of Low Tumor Percentage Should Be Analyzed with Caution.

Background and aims: The percentage of tumor cells (tumor cellularity) in a cancerous tissue has been assumed to correlate with the variant allele fraction (VAF) of an identified pathogenic variant. Many laboratories use the tumor cellularity as part of a quality criteria for specimen processing and clinical reporting. However, a systematic study of such correlation has yet to be shown. We performed a relatively large-scale study to determine whether pathologist-estimated tumor cellularity is correlated with next-generation sequencing (NGS)-derived VAF. Materials and Methods: A total of 1511 non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) specimens, including formalin-fixed paraffin-embedded (FFPE) and fine needle aspirated (FNA) tissues, were analyzed by cancer hotspot NGS. For a given specimen, pathogenic variants of BRAF, EGFR, KRAS, and NRAS were identified and the determined VAFs were correlated with the corresponding tissue tumor cellularity. Results: The coefficient of determination R-squared (R2) values were calculated for each correlation. All R2 values were lower than 0.25, indicating poor correlations. Pathogenic variants were found, not uncommonly, in tumor specimens that carried 10% or lower tumor cellularity. There were no apparent differences of R2 values between the FFPE and FNA specimens. Conclusion: In both NSCLC and CRC, the lack of linear relationship between tumor cellularity and VAF was found across a wide range of tumor cell percentages. Caution should be used when using tumor cellularity to triage specimens for NGS testing. The tumor cellularity should be considered in relation to the limit of detection of the specific assay for the proper interpretation of a negative test result.

Biomechanical study of the male lower urinary tract: Simulation of internal and external sphincters dyssynergia.

Urethral sphincter dysfunction is an important cause of stress urinary incontinence (SUI). The most effective treatment is the insertion of an artificial urethral sphincter (AUS), which relies to a large extent on the surgeon's experience. However, there is no quantitative standard for cuff tightness, resulting in frequent postoperative complications. This study aimed to investigate the effect of internal and external sphincter dyssynergia on urodynamic parameters in the lower urinary tract. A geometric model of male lower urinary tract tissue was constructed from collodion slices, accounting for the active behavior of the internal and external sphincters. Normal and dyssynergic internal and external sphincters (active sphincter behavior was individually injured by 25%, 50%, 75%, or 100%) were simulated with fluid-structure interactions and changes in urethral stress, displacement, and urine flow rate were detected. We found that when the internal sphincter was injured by 25%, 50%, 75%, and 100%, urethral stress near the internal sphincter decreased by 8.3%, 15.6%, 24.3%, and 35.7%, respectively. Additionally, when the external sphincter was injured by 25%, 50%, 75%, and 100%, urethral stress near the external sphincter was reduced by 13.3%, 24.3%, 38.6%, and 46.6%, respectively. Internal sphincter injury primarily affects positions near the internal sphincter and prostate, while external sphincter injury affects the area between the prostate and urethral outlet. These data could facilitate the standardized evaluation of internal and external sphincter dysfunction and lead to novel methods of preoperative assessment for AUS surgery.

A static magnetic field enhances the repair of osteoarthritic cartilage by promoting the migration of stem cells and chondrogenesis.

Objective: To investigate the therapeutic effects of static magnetic field (SMF) and its regulatory mechanism in the repair of osteoarthritic cartilage. Methods: Fourteen-week-old female C57BL/6 mice were randomly divided into the sham operation group and the osteoarthritis (OA) groups with and without SMF application. SMF was applied at 200 â€‹mT for two consecutive weeks. Changes in knee cartilage were examined by histomorphometry, and the chondrogenesis and migration of endogenous stem cells were assessed. The expression of SRY-related protein 9 (SOX9), Collagen type II (COL2), matrix metallopeptidase 13 (MMP13), stromal cell-derived factor 1/C-X-C chemokine receptor type 4 (SDF-1/CXCR4), Piezo1 and other genes was evaluated, and the mechanism of SMF's action was tested using the CXCR4 inhibitor, AMD3100, and Piezo1 siRNA. Results: SMF significantly decreased the OARSI scores after induction of OA. SMF was beneficial to chondrogenesis by elevating SOX9. In the OA mouse model, an increase in MMP13 with a decrease in COL2 led to the destruction of the cartilage extracellular matrix, which was suppressed by SMF. SMF promoted the migration of cartilage-derived stem/progenitor cells and bone marrow-derived mesenchymal stem cells (MSCs). It increased SDF-1 and CXCR4, while the CXCR4 inhibitor significantly suppressed the beneficial effects of SMF. The application of Piezo1 siRNA inhibited the SMF-induced increase of CXCR4. Conclusion: SMF enhanced chondrogenesis and improved cartilage extracellular matrices. It activated the Piezo1-mediated SDF-1/CXCR4 regulatory axis and promoted the migration of endogenous stem cells. Collectively, it attenuated the pathological progression of cartilage destruction in OA mice. The Translational potential of this article: The findings in this study provided convincing evidence that SMF could enhance cartilage repair and improve OA symptoms, suggesting that SMF could have clinical value in the treatment of OA.

Three-dimensional measurement and analysis of morphological parameters of the uterus in infertile women.

Background: To determine differences in endometrial cavity anteroposterior diameter, thickness, volume, and diameter lines of uterine body and thickness, and volume of upper, middle, and lower regions of the endometrium in infertile women using a new method for three-dimensional (3D) reconstruction based on two-dimensional (2D) ultrasound images. Methods: This retrospective cross-sectional study included a total of 81 infertile women, who underwent 2D ultrasound standard examination. We created 3D models of the uterine body, endometrial cavity, and endometrium based on 2D ultrasound images. The parameters that were measured and analyzed in a 3D plane included volume and diameter lines of endometrial cavity, surface area, thickness, volume, and diameter lines of uterine body, and surface area, thickness, and volume of upper, middle, and lower region of the endometrium. These parameters were used for comparisons between normal and arcuate uterus, between non-pregnant and pregnant infertile women, and between nulliparous and multiparous infertile women. The differences between the different regions of the endometrium and the correlations between age and the parameters were also determined in this study. Results: Endometrial cavity length, and middle and lower regions of the endometrial volume in the normal uterus were 39.63±7.61 mm, 1,307.92±1,034.40 mm3, and 653.98±460.41 mm3, respectively. For arcuate uterus, these parameters were 32.96±4.69 mm, 539.89±298.94 mm3, and 347.90±129.61 mm3, respectively. The parameters were significantly higher in normal uterus compared with arcuate uterus (P=0.000, 0.001, and 0.006, respectively). Upper, middle, and lower regions of endometrial thickness in normal uterus were 7.79±3.26, 8.18±3.33, and 6.41±2.60 mm, respectively. Both upper and middle regions of endometrial thickness were significantly greater than the lower regions of endometrial thickness with P=0.009 and P=0.001, respectively. Correlation analysis revealed that age positively correlated with volume of upper endometrial regions (r=0.274, P=0.028). Conclusions: This study provides references for the volume and thickness of the endometrium in the different anatomical regions of normal and arcuate uterus. Age mainly affects the upper region of the endometrium. The 3D measurement provides a precise way to quantify the morphological parameters of gynecological diseases.

Mechanical loading mitigates osteoarthritis symptoms by regulating the inflammatory microenvironment in a mouse model.

Osteoarthritis (OA) is one of the most common chronic diseases, in which inflammatory responses in the articular cavity induce chondrocyte apoptosis and cartilage degeneration. While mechanical loading is reported to mitigate synovial inflammation, the mechanism and pathways for the loading-driven improvement of OA symptoms remain unclear. In this study, we evaluated the loading effects on M1/M2 polarization of synovial macrophages via performing histology, cytology, and molecular analyses. In the OA group, the cell layer of the synovial lining was enlarged with an increase in cell density. Also, M1 macrophages were polarized and proinflammatory cytokines were increased. In contrast, in the OA group with mechanical loading, cartilage degradation was reduced and synovial inflammation was alleviated. Notably, the M1 macrophages were diminished by mechanical loading, while M2 macrophages were increased. Furthermore, mechanical loading decreased the levels of proinflammatory cytokines, such as interleukin-1 beta and tumor necrosis factor-α, and suppressed PI3K/AKT/NF-κB signaling. Taken together, this study demonstrates that mechanical loading changes the ratio of M1 and M2 macrophages via regulation of PI3K/AKT/NF-κB signaling and provides cartilage protection in the mouse OA model.

Loading-driven PI3K/Akt signaling and erythropoiesis enhanced angiogenesis and osteogenesis in a postmenopausal osteoporosis mouse model.

Bone vasculature influences osteogenesis and haematopoiesis in the bone microenviroment. Mechanical loading has been shown to stimulate the formation of osteogenesis-related type H vessels in an ovariectomy (OVX)-induced osteoporosis mouse model. To determine the loading-driven mechanism of angiogenesis and the formation of type H vessels in bone, we evaluated the roles of PI3K/Akt signaling and erythropoiesis in the bone marrow. The daily application of mechanical loading (1 N at 5 Hz for 6 min/day) for 2 weeks on OVX mice inhibited osteoclast activity, associated with an increase in the number of osteoblasts and trabecular volume ratio. Mechanical loading enhanced bone vasculature and vessel formation, as well as PI3K/Akt phosphorylation and erythropoiesis in the bone marrow. Notably, LY294002, an inhibitor of PI3K signaling, blocked the tube formation by endothelial progenitor cells, as well as their migration and wound healing. The conditioned medium, derived from erythroblasts, also promoted the function of HUVECs with elevated levels of VEGF, CD31, and Emcn. Collectively, this study demonstrates that mechanical loading prevents osteoporotic bone loss by promoting angiogenesis and type H vessel formation. This load-driven preventing effect is in part mediated by PI3K/Akt signaling and erythropoiesis in the bone marrow.

Mechanical loading stimulates bone angiogenesis through enhancing type H vessel formation and downregulating exosomal miR-214-3p from bone marrow-derived mesenchymal stem cells.

Exosomes are important transporters of miRNAs, which play varying roles in the healing of the bone fracture. Angiogenesis is one of such critical events in bone healing, and we previously reported the stimulatory effect of mechanical loading in vessel remodeling. Focusing on type H vessels and exosomal miR-214-3p, this study examined the mechanism of loading-driven angiogenesis. MiRNA sequencing and qRT-PCR revealed that miR-214-3p was increased in the exosomes of the bone-losing ovariectomized (OVX) mice, while it was significantly decreased by knee loading. Furthermore, compared to the OVX group, exosomes, derived from the loading group, promoted the angiogenesis of endothelial cells. In contrast, exosomes, which were transfected with miR-214-3p, decreased the angiogenic potential. Notably, knee loading significantly improved the microvascular volume, type H vessel formation, and bone mineral density and contents, as well as BV/TV, Tb.Th, Tb.N, and Tb.Sp. In cell cultures, the overexpression of miR-214-3p in endothelial cells reduced the tube formation and cell migration. Collectively, this study demonstrates that knee loading promotes angiogenesis by enhancing the formation of type H vessels and downregulating exosomal miR-214-3p.

Age and tissue specific differences in the development of acute insulin resistance following injury.

Injuries, hemorrhage, sepsis, burn, and critical illnesses all induce insulin resistance, and insulin resistance is strongly associated with advancing age. However, the effect of age on injury induced insulin resistance is not well studied. We performed surgical trauma in male rats of three different ages (3-, 6-, and 10-weeks old). Rats were either hemorrhaged to a mean arterial pressure of 35-40 mmHg and subsequently maintained at that pressure for up to 90 min, or maintained without hemorrhage as controls. Results indicate that insulin-induced intracellular signaling was diminished in liver and skeletal muscle of 6- and 10-week old rats following trauma and hemorrhage. In even younger rats, immediately post-weaning ( approximately 3 weeks of age), insulin signaling was lost in liver, but not in skeletal muscle. Glucocorticoids can play a role in the chronic development of insulin resistance. Our results demonstrate that corticosterone levels were increased in 6- and 10-week old animals following hemorrhage, but little change was measured in 3-week old animals. Blockade of glucocorticoid synthesis prevented the development of insulin resistance in skeletal muscle, but not in liver of 6- and 10-week old rats. Moreover, skeletal muscle glucocorticoid receptor levels increased dramatically between 3 and 6 weeks of age. These results indicate that trauma and hemorrhage-induced hepatic insulin resistance occurs at all ages tested. However, there is no development of insulin resistance following trauma and hemorrhage in skeletal muscle of post-weaning rats. In skeletal muscle of 6- and 10-week old rats, inhibition of glucocorticoid levels prevents the development of insulin resistance.

Denonvilliers' fascia in women and its relationship with the fascia propria of the rectum examined by successive slices of celloidin-embedded pelvic viscera.

PURPOSE: The purpose of this study is to reexamine the detailed anatomy of Denonvilliers' fascia in women and to study its relationship with the fascia propria of the rectum. METHODS: Macroscopic dissection was performed and successive slices of celloidin-embedded pelvic viscera were examined. Of 25 formalin-fixed pelvic splanchnic organs removed from adult female pelves, 14 were cut midsagittally, dissected, and observed under anatomic microscopy, and 11 were embedded in celloidin and then made into successive slices. RESULTS: The rectovaginal septum was composed of anterior and posterior layers. The anterior layer was identified as Denonvilliers' fascia; the posterior layer as the fascia propria of the rectum. The bilateral insertions of Denonvilliers' fascia differ at different levels: at the cervix, Denonvilliers' fascia merged into the parametrium; at the upper vagina, it ended laterally at the paracolpium or fused with the fascia anterior to the vagina; at the middle vagina, the fasciae anchored to the arcus tendineus fasciae pelvis; at the lower vagina, it ended at the lateral side of the outlet of the levator ani muscles. CONCLUSIONS: The rectovaginal septum is formed by Denonvilliers' fascia and the fascia propria of rectum and can potentially limit the spread of malignancy.

Trauma and hemorrhage-induced acute hepatic insulin resistance: dominant role of tumor necrosis factor-alpha.

It has long been known that injury, infections, and other critical illnesses are often associated with hyperglycemia and hyperinsulinemia. Mortality of critically ill patients is greatly reduced by intensive insulin therapy, suggesting the significance of reversing or compensating for the development of acute insulin resistance. However, the development of acute injury/infection-induced insulin resistance is poorly studied, much less than the chronic diseases associated with insulin resistance, such as type 2 diabetes and obesity. We previously found that insulin resistance develops acutely in the liver after trauma and hemorrhage. The present study was designed to begin to understand the first steps in the development of trauma and hemorrhage-induced acute hepatic insulin resistance in an animal model of injury and blood loss similar to traumatic or surgical injury and hemorrhage. We present novel data that indicate that hepatic insulin resistance increased dramatically with an increasing extent of hemorrhage. With increasing extent of blood loss, there were increases in serum TNF-alpha levels, phosphorylation of liver insulin receptor substrate-1 on serine 307, and liver c-Jun N-terminal kinase activation/phosphorylation. Exogenous TNF-alpha infusion increased c-Jun N-terminal kinase phosphorylation and insulin receptor substrate-1 serine 307 phosphorylation, and inhibited insulin-induced signaling in liver. Conversely, neutralizing TNF-alpha antibody treatment reversed many of the hemorrhage-induced changes in hepatic insulin signaling. Our data indicate that the acute development of insulin resistance after trauma and hemorrhage may have some similarities to the insulin resistance that occurs in chronic diseases. However, because so little is known about this acute insulin-resistant state, much more needs to be done before we can attain a level of understanding similar to that of chronic states of insulin resistance.

[Synergetic cytotoxicity of cisplatin and etoposide to leukemia cell line K562 and its mechanism].

BACKGROUND & OBJECTIVE: Etoposide (VP-16) is one of the most common chemotherapy drugs, but its usage is limited by drug resistance. Some researches on solid tumors show that cisplatin (DDP) have synergetic effect with VP-16. This study was to evaluate synergetic cytotoxicity of VP-16 and DDP to leukemia cell line K562, and explore the mechanism. METHODS: K562 cells were treated with VP-16 (0 or 5 microg/ml) and DDP (0, 0.3, 3, 15, or 30 microg/ml) in different combination patterns. Inhibitory effect of VP-16 and DDP on survival of K562 cells was measured by MTT assay. Cell apoptosis was evaluated by AO/EB double fluorescent labeling. The expression of topoisomerase (TOPO) II alpha and II beta, and transcription factor Sp1 and Sp3 were measured by semi-quantitive reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. RESULTS: MTT assay showed significant synergetic cytotoxicity of VP-16 and DDP. VP-16 in combination with DDP obviously enhanced cell apoptosis. RT-PCR showed that DDP significantly up-regulated the expression of TOPO II and Sp1 in concentration-dependent manners (TOPO II alpha, II beta, and Sp1 were up-regulated by 36%, 25%, and 75% of control, respectively, when treated with 30 microg/ml of DDP), and down-regulated Sp3 by 49% of control; VP-16 (5 microg/ml) down-regulated TOPO II alpha by 71.9%, and up-regulated Sp3 by 14%; VP-16 (5 microg/ml) in combination with DDP (30 microg/ml) up-regulated TOPO II alpha by 83%, II beta by 11%, and Sp1 by 58% when compared with using VP-16 alone (but the levels were lower than using DDP alone), and down-regulated Sp3 by 61.3% when compared with using DDP alone. Western blot showed similar results to RT-PCR. CONCLUSION: Through up-regulating TOPO II, DDP could enhance the chemotherapeutic effect of VP-16 on K562 cells by provide more target enzyme to act on.