Low-intensity pulsed ultrasound activated the anti-tumor immunity by irradiating the spleen of mice in 4 T-1 breast cancer.

Tumor immunotherapy is booming around the world. However, strategies to activate the immune system and alleviate the immunosuppression still need to be refined. Here, we demonstrate for the first time that low-intensity pulsed ultrasound (LIPUS, spatial average time average intensity (Isata) is 200 mW/cm2, frequency is 0.3 MHz, repetition frequency is 1 kHz, and duty cycle is 20%) triggers the immune system and further reverses the immunosuppressive state in the mouse models of breast cancer by irradiating the spleen of mice. LIPUS inhibited tumor growth and extended survival in mice with 4 T-1 tumors. Further studies had previously shown that LIPUS enhanced the activation of CD4+ and CD8+ T cells in the spleen and led to significant changes in cytokines, as well as induced upregulation of mRNA levels involved in multiple immune regulatory pathways in the spleen. In addition, LIPUS promoted tumor-infiltrating lymphocyte accumulation and CD8+ T cell activation and improved the dynamics of cytokines/chemokines in the tumor microenvironment, resulting in a reversal of the immunosuppressive state of the tumor microenvironment. These results suggest a novel approach to activate the immune response by irradiating the spleen with LIPUS.

Anti-inflammatory Effect of Low-Intensity Ultrasound in Septic Rats.

OBJECTIVE: Sepsis is a severe systemic inflammatory response caused by infection. Here, the spleen region of Sprague-Dawley (SD) rats with sepsis was irradiated with low-intensity ultrasound (LIUS) to explore the regulation of inflammation and its mechanism by LIUS. METHODS: In this study, 30 rats used for survival analysis were randomly divided into the sham-operated group (Sham, n = 10), the group in which sepsis was induced by cecal ligation and puncture (CLP, n = 10) and the group treated with LIUS immediately after CLP (LIUS, n = 10). The other 120 rats were randomly divided into the aforementioned three groups for detection at each time point. The parameters used in the LIUS group were 200 mW/cm2, 0.37 MHz, 20% duty cycle and 20 min, and no ultrasonic energy was produced in the Sham and CLP groups. Seven-day survival rate, histopathology and expression of inflammatory factors and proteins were evaluated in the three groups. RESULTS: LIUS was able to improve the survival rate of septic SD rats (p < 0.05), significantly inhibit the expression of tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß), interleukin 6 (IL-6) and nuclear factor-κB p65 (NF-κB p65) (p < 0.05) and restore the ultrastructure of the spleen. CONCLUSION: Our study determined that LIUS can relieve spleen damage and alleviate severe cytokine storm to improve survival outcomes in septic SD rats, and its mechanism may be related to the inhibition of the NF-κB signaling pathway by downregulation of IL-1ß.

Altered lung physiology in two cohorts after COVID-19 infection as assessed by computed cardiopulmonography.

The longer-term effects of COVID-19 on lung physiology remain poorly understood. Here, a new technique, computed cardiopulmonography (CCP), was used to study two COVID-19 cohorts (MCOVID and C-MORE-LP) at both ∼6 and ∼12 mo after infection. CCP is comprised of two components. The first is collection of highly precise, highly time-resolved measurements of gas exchange with a purpose-built molecular flow sensor based around laser absorption spectroscopy. The second component is estimation of physiological parameters by fitting a cardiopulmonary model to the data set. The measurement protocol involved 7 min of breathing air followed by 5 min of breathing pure O2. One hundred seventy-eight participants were studied, with 97 returning for a repeat assessment. One hundred twenty-six arterial blood gas samples were drawn from MCOVID participants. For participants who had required intensive care and/or invasive mechanical ventilation, there was a significant increase in anatomical dead space of ∼30 mL and a significant increase in alveolar-to-arterial Po2 gradient of ∼0.9 kPa relative to control participants. Those who had been hospitalized had reductions in functional residual capacity of ∼15%. Irrespectively of COVID-19 severity, participants who had had COVID-19 demonstrated a modest increase in ventilation inhomogeneity, broadly equivalent to that associated with 15 yr of aging. This study illustrates the capability of CCP to study aspects of lung function not so easily addressed through standard clinical lung function tests. However, without measurements before infection, it is not possible to conclude whether the findings relate to the effects of COVID-19 or whether they constitute risk factors for more serious disease.NEW & NOTEWORTHY This study used a novel technique, computed cardiopulmonography, to study the lungs of patients who have had COVID-19. Depending on severity of infection, there were increases in anatomical dead space, reductions in absolute lung volumes, and increases in ventilation inhomogeneity broadly equivalent to those associated with 15 yr of aging. However, without measurements taken before infection, it is unclear whether the changes result from COVID-19 infection or are risk factors for more severe disease.

Identifying putative ventilation-perfusion distributions in COVID-19 pneumonia.

Busana et al. (doi.org/10.1152/japplphysiol.00871.2020) published 5 patients with COVID-19 in whom the fraction of non-aerated lung tissue had been quantified by computed tomography. They assumed that shunt flow fraction was proportional to the non-aerated lung fraction, and, by randomly generating 106 different bimodal distributions for the ventilation-perfusion ([Formula: see text]) ratios in the lung, specified as sets of paired values {[Formula: see text]}, sought to identify as solutions those that generated the observed arterial partial pressures of CO2 and O2 (PaCO2 and PaO2). Our study sought to develop a direct method of calculation to replace the approach of randomly generating different distributions, and so provide more accurate solutions that were within the measurement error of the blood-gas data. For the one patient in whom Busana et al. did not find solutions, we demonstrated that the assumed shunt flow fraction led to a non-shunt blood flow that was too low to support the required gas exchange. For the other four patients, we found precise solutions (prediction error < 1x10-3 mmHg for both PaCO2 and PaO2), with distributions qualitatively similar to those of Busana et al. These distributions were extremely wide and unlikely to be physically realisable, because they predict the maintenance of very large concentration gradients in regions of the lung where convection is slow. We consider that these wide distributions arise because the assumed value for shunt flow is too low in these patients, and we discuss possible reasons why the assumption relating to shunt flow fraction may break down in COVID-19 pneumonia.

Potential Application of Low-Intensity Pulsed Ultrasound in Delaying Aging for Mice.

INTRODUCTION: The low-intensity pulsed ultrasound (LIPUS) is one of the popular treatment modalities allowing to boost the proliferation, differentiation, and migratory activity of cells, which might be a powerful strategy for anti-aging. Seeking a novel setup for LIPUS would benefit the development of ultrasound therapeutics. METHODS: Here, we proposed a novel underwater exposure setup of LIPUS. C57BL/6 mice were reared in the designated age-groups, which consisted of a middle-aged group (12-14 months) and an old-age group (20-23 months). The age-related changes of body composition, imbalance of energy supply and demand, imbalance of signal network maintaining internal stability, and representative phenotypes of neurodegeneration and neuroplasticity with the presence and absence of underwater LIPUS in middle-aged and aged groups were evaluated. RESULTS: The results showed that there were obvious aging changes, imbalance of energy supply and demand, imbalance of signal network maintaining homeostasis, neurodegeneration, and damage of neural plasticity in the middle-aged and aged group with or without the LIPUS. Although middle-aged group and aged group responded differently to LIPUS, they mostly generated positive results in relieving bone loss, improving ovarian structure, regulated immune system, and enhanced endurance ability, which should have declined over age. DISCUSSION: These findings indicate that underwater extracorporeal LIPUS exposure could be employed as single or combined anti-aging strategies that generated positive outcomes against the process of aging.

Low-Intensity Pulsed Ultrasound Promotes Repair of 4-Vinylcyclohexene Diepoxide-Induced Premature Ovarian Insufficiency in SD Rats.

Women with premature ovarian insufficiency (POI) may be more vulnerable to a variety of health risks. To seek a new method to treat the disease, the effects of low-intensity pulsed ultrasound (LIPUS) on promoting repair of ovarian injury in female SD rats induced by 4-vinylcyclohexene diepoxide (VCD) were explored in this research. A total of 24 female SD rats were subjected to intraperitoneal injection of VCD to induce POI. Successful modeling was achieved in 22 rats, which were then randomized into VCD + LIPUS group (n = 13) and VCD group (n = 9). The control group (n = 5) was injected with equal normal saline. Hematoxylin and eosin staining, enzyme-linked immunosorbent assay, Western blot analysis, scanning electron microscope, immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated nick end labeling assay were applied to detect the results. The results indicated that rats in the VCD group showed disorder in the estrous cycle, the number of atresia follicles and apoptosis granulosa cells increased (p < .05). After the LIPUS treatment, the estrous cycle recovered, the number of follicles increased (p < .05), the level of E2 and anti-Müllerian hormone enhanced (p < .05), and the follicle-stimulating hormone decreased (p < .05). The expression of NF-κB p65, TNFα, Bax, ATF4, and caspase-3 in ovarian tissue was significantly decreased (p < .05). These findings showed that LIPUS could promote the repair of the VCD-induced ovarian damage in SD rats, which has the potential to be further applied in the clinic.

Effects of low intensity pulsed ultrasound on expression of B-cell lymphoma-2 and BCL2-Associated X in premature ovarian failure mice induced by 4-vinylcyclohexene diepoxide.

BACKGROUND: Premature ovarian failure (POF) is a common disease in the field of Gynecology. Low intensity pulsed ultrasound (LIPUS) can promote tissue repair and improve function. This study was performed to determine the effects of LIPUS on granulosa cells (GCs) apoptosis and protein expression of B-cell lymphoma-2 (Bcl-2) and BCL2-Associated X (Bax) in 4-vinylcyclohexene diepoxide (VCD)-induced POF mice and investigate the mechanisms of LIPUS on ovarian function and reserve capacity. METHODS: The current POF mice model was administrated with VCD (160 mg/kg) by intraperitoneal injection for 15 consecutive days. The mice were divided into the POF group, LIPUS group and control group. In the LIPUS group, the right ovary of mice was treated by LIPUS (acoustic intensity was 200 mW/cm2, frequency was 0.3 MHz, and duty cycle was 20%) for 20 min, 15 consecutive days from day 16. The mice of the POF group and control group were treated without ultrasonic output. The basic observation and body weight were recorded. Hematoxylin and eosin staining (H&E staining) and enzyme-linked immunosorbent assay (ELISA) were applied to detect ovarian follicle development, ovarian morphology and sex hormone secretion. Ovarian GCs apoptosis was detected by TUNEL assay and immunohistochemistry. RESULTS: The results showed that VCD can induce estrus cycle disorder, follicular atresia, sex hormone secretion decreased and GCs apoptosis in mice to establish POF model successfully. LIPUS significantly promoted follicular development, increased sex hormone secretion, inhibited excessive follicular atresia and GCs apoptosis. The mechanism might be achieved by increasing the protein expression of Bcl-2 and decreasing the expression of Bax in ovaries. CONCLUSIONS: LIPUS can improve the POF induced by VCD. These findings have the potential to provide novel methodological foundation for the future research, which help treat POF patients in the clinic.

Gut microbiota dysbiosis in stable coronary artery disease combined with type 2 diabetes mellitus influences cardiovascular prognosis.

BACKGROUND AND AIMS: Host-microbiota interactions involving metabolic pathways have been linked to the pathogenesis of atherosclerotic disease and type 2 diabetes. As stable coronary artery disease (SCAD) patients combined with type 2 diabetes have significantly increased risk for cardiac event, we focused on elucidating the role of microbiota affecting cardiometabolic disease development. METHODS AND RESULTS: We used multi-omics analyses (metagenomics and metabolomics) of fecal and serum samples from a prospective cohort including stable coronary artery disease combined with diabetes mellitus (SCAD + T2DM, n = 38), SCAD (n = 71), and healthy control (HC, n = 55). We linked microbiome features to disease severity in a three-pronged association analysis and identified prognostic bacterial biomarkers. We identified that bacterial and metabolic signatures varied significantly between SCAD and SCAD + T2DM groups. SCAD + T2DM individuals were characterized by increased levels of aromatic amino acids and carbohydrates, which correlate with a gut microbiome with enriched biosynthetic potential. Our study also addressed how metformin may confound gut dysbiosis and increase the potential for nitrogen metabolism. In addition, we found that specific bacterial taxa Ruminococcus torques [HR: 2.363 (08-4.56), P = 0.03] was predictive of cardiac survival outcomes. CONCLUSION: Overall, our study identified relationships between features of the gut microbiota (GM) and circulating metabolites, providing a new direction for future studies aiming to understand the host-GM interplay in atherosclerotic cardiovascular pathogenesis.

Low-intensity pulsed ultrasound alleviating myelosuppression of Sprague-Dawley rats after combined treating by paclitaxel and carboplatin.

BACKGROUND: Our team has previously reported that low intensity pulsed ultrasound (LIPUS) can alleviate myelosuppression in rats induced by single chemotherapy drugs. But in clinics, chemotherapy is often performed with multiple drugs simultaneously. To be closer to the clinical status quo, this experiment was designed to show whether it was the same effect of LIPUS on myelosuppression caused by combination therapy of chemotherapy drugs. METHODS: The rat model of myelosuppression was established by continuous injection of paclitaxel and carboplatin for 4 days. These myelosuppressive rats were randomly divided into LIPUS group (n=40) and control group (n=40). The LIPUS group was given continuous LIPUS irradiation for 7 days, while the control group was given sham irradiation (no energy output). The evaluation of blood cells counts, Hematoxylin-Eosin staining (H&E staining), scanning electron microscopy, enzyme-linked immunosorbent assay (ELISA) and real-time quantitative PCR (qPCR) was then performed. RESULTS: The results showed in the LIPUS group the blood cells count, hematopoietic tissue of bone marrow, the colonies formed from adhering of bone marrow stromal cells, levels of hematopoietic regulators and adhesion molecules all increased (LIPUS group vs. control group, P<0.05). CONCLUSIONS: The results indicated that LIPUS can relieve myelosuppression induced by combined treatment of paclitaxel and carboplatin. The mechanisms may be LIPUS can increase the levels of hematopoietic regulators and adhesion molecules.

Effects of Low-Intensity Pulsed Ultrasound on Myelosuppression of Rats Induced by Chemotherapy Drugs With Cell Cycle Specificity.

OBJECTIVES: To explore the ameliorating effects of low-intensity pulsed ultrasound (LIPUS) on Sprague Dawley rat myelosuppression induced by cell cycle specificity drugs (docetaxel, mitotic phase sensitive; and etoposide, gap 2 phase sensitive). METHODS: Rats were respectively administered docetaxel (100 mg/kg) or etoposide (110 mg/kg) by intraperitoneal injection for 4 consecutive days. Then the rats were divided randomly into a LIPUS group and a non-LIPUS group. In the LIPUS group, the right femoral metaphysis of rats was treated by LIPUS (acoustic intensity, 200 mW/cm2 ; frequency, 0.3 MHz; and duty cycle, 20%) for 20 minutes on 7 consecutive days from day 5. The rats of the non-LIPUS group were treated without ultrasound output. A blood cell count, an enzyme-linked immunosorbent assay, a real-time quantitative polymerase chain reaction, and hematoxylin-eosin staining were applied to detect the results. RESULTS: Low-intensity pulsed ultrasound significantly promoted the counts of bone marrow nucleated cells, white blood cells, immunoglobulin A (IgA), IgG, granulocyte colony-stimulating factor, stem cell factor, and intercellular cell adhesion molecule 1 and reduced the counts of vascular cell adhesion molecule 1 whether in the docetaxel or etoposide group (P < .05). Low-intensity pulsed ultrasound only increased the expression level of IgM in the docetaxel group but decreased the level of interleukin 6 in the etoposide group (P < .05). CONCLUSIONS: Low-intensity pulsed ultrasound has potential to be a noninvasive treatment for myelosuppression caused by different cell cycle-sensitive chemotherapy drugs.

Clinical characteristics and survival analysis in critical and non-critical patients with COVID-19 in Wuhan, China: a single-center retrospective case control study.

Since the outbreak of COVID-19 in China at the end of 2019, the world has experienced a large-scale epidemic caused by the SARS-CoV-2. The epidemiological and clinical course of COVID-19 patients has been reported, but there have been few analyses about the characteristics, predictive risk factors, and outcomes of critical patients. In this single-center retrospective case-control study, 90 adult inpatients hospitalized at Tongji Hospital (Wuhan, China) were included. Demographic, clinical, laboratory tests, and treatment data were obtained and compared between critical and non-critical patients. We found that compared with non-critical patients, the critical patients had higher SOFA score and qSOFA scores. Critical patients had lower lymphocyte and platelet count, elevated D-dimer, decreased fibrinogen, and elevated high-sensitivity C-reactive protein (hsCRP), and interleukin-6(IL-6). More critical patients received treatment including antibiotics, anticoagulation, corticosteroid, and oxygen therapy than non-critical ones. Multivariable regression showed higher qSOFA score and elevation of IL-6 were related to critical patients. Antibiotic usage and anticoagulation were associated with decreased in-hospital mortality. And critical grouping contributed greatly to in-hospital death. Critical COVID-19 patients have a more severe clinical course. qSOFA score and elevation of IL-6 are risk factors for critical condition. Non-critical grouping, positive antibiotic application, and anticoagulation may be beneficial for patient survival.

Time-Restricted Feeding Reduces the Detrimental Effects of a High-Fat Diet, Possibly by Modulating the Circadian Rhythm of Hepatic Lipid Metabolism and Gut Microbiota.

Background: Time-restricted feeding, also known as intermittent fasting, can confer various beneficial effects, especially protecting against obesity, and related metabolic disorders, but little is known about the underlying mechanisms. Therefore, the present study aims to investigate the effects of time-restricted feeding on the circadian rhythm of gut microbiota and hepatic metabolism. Methods: Eight-week-old male Kunming mice received either a normal diet ad libitum, a high-fat diet ad libitum, or a high-fat diet restricted to an 8-h temporal window per day for an experimental period of 8 weeks. Weight gain and calorie intake were measured weekly. Serum metabolites, hepatic sections and lipid metabolites, gut microbiota, and the hepatic expression of Per1, Cry1, Bmal1, SIRT1, SREBP, and PPARα were measured at the end of the experimental period. The composition of gut microbiota and the expression of hepatic genes were compared between four timepoints. Results: Mice that received a time-restricted high-fat diet had less weight gain, milder liver steatosis, and lower hepatic levels of triglycerides than mice that received a high-fat diet ad libitum (p < 0.05). The numbers of Bacteroidetes and Firmicutes differed between mice that received a time-restricted high-fat diet and mice that received a high-fat diet ad libitum (p < 0.05). Mice fed a time-restricted high-fat diet showed distinct circadian rhythms of hepatic expression of SIRT1, SREBP, and PPARα compared with mice fed a normal diet ad libitum, as well as the circadian rhythm of the abundance of Bacteroidetes and Firmicutes. Conclusions: Time-restricted feeding is associated with better metabolic conditions, perhaps owing to alterations in gut microbiota and the circadian pattern of molecules related to hepatic lipid metabolism, which were first to report.

Ethical dilemma of identity disclosure faced by medical students in clinical clerkships: A nationwide multicenter study in China.

OBJECTIVE: Medical students in China are currently facing a dilemma of whether to clarify their identity as students to patients. Further investigation is needed to support policy-making. The aim was to identify factors influencing medical students' decision on whether or not to clarify their identity to patients and to examine the effects of their decision. METHODS: The study was a cross-sectional nationwide multicenter survey consisting of 947 medical students. A self-designed questionnaire was composed of 19 structured questions investigating the present situation and participants' perception of the ethical dilemma surrounding medical student identity. The questionnaires were distributed randomly in teaching hospitals affiliated with 13 medical schools across China from June 2015 to January 2016. RESULTS: A total of 947 valid questionnaires were retrieved with a valid response rate of 83.7%. Most medical students (71.4%) tended to be ambiguous about their student identity in front of patients. The frequency of encountering distrust and patients' or patient relatives' refusal to allow students to perform procedures was significantly lower for students who explicitly stated their identity than for those who were ambiguous about their identity (p<0.001). Less experience in clinical rotations (<0.5 y/0.5-1 y, OR 2.7, 95% CI 1.7-4.3; <0.5 y/>1 y, OR 3.6, 95% CI 2.0-6.5), preceptors' straightforward introduction of the students (OR 8.7, 95% CI 5.4-13.8) and students' acknowledgment of patients' right to know (OR 2.3, 95% CI 1.2-4.5) were related to students' clear self-introduction to patients. CONCLUSION: It is beneficial for medical students to clearly explain their identity to patients in order to decrease patient distrust and prevent the refusal to have certain appropriate procedures performed. Several methods, including emphasizing the role of mentors, developing curriculum for medical students, and creating clear regulations and guidelines for revealing the identity of medical students on the healthcare team can help address and ideally resolve this ethical dilemma of identity disclosure.

Methylation of WTH3, a possible drug resistant gene, inhibits p53 regulated expression.

BACKGROUND: Previous results showed that over-expression of the WTH3 gene in MDR cells reduced MDR1 gene expression and converted their resistance to sensitivity to various anticancer drugs. In addition, the WTH3 gene promoter was hypermethylated in the MCF7/AdrR cell line and primary drug resistant breast cancer epithelial cells. WTH3 was also found to be directly targeted and up regulated by the p53 gene. Furthermore, over expression of the WTH3 gene promoted the apoptotic phenotype in various host cells. METHODS: To further confirm WTH3's drug resistant related characteristics, we recently employed the small hairpin RNA (shRNA) strategy to knockdown its expression in HEK293 cells. In addition, since the WTH3 promoter's p53-binding site was located in a CpG island that was targeted by methylation, we were interested in testing the possible effect this epigenetic modification had on the p53 transcription factor relative to WTH3 expression. To do so, the in vitro methylation method was utilized to examine the p53 transgene's influence on either the methylated or non-methylated WTH3 promoter. RESULTS: The results generated from the gene knockdown strategy showed that reduction of WTH3 expression increased MDR1 expression and elevated resistance to Doxorubicin as compared to the original control cells. Data produced from the methylation studies demonstrated that DNA methylation adversely affected the positive impact of p53 on WTH3 promoter activity. CONCLUSION: Taken together, our studies provided further evidence that WTH3 played an important role in MDR development and revealed one of its transcription regulatory mechanisms, DNA methylation, which antagonized p53's positive impact on WTH3 expression.

Differential methylation of TSP50 and mTSP50 genes in different types of human tissues and mouse spermatic cells.

Earlier studies identified human TSP50 as a testis-specific gene that encoded a threonine protease. Most importantly, TSP50 could be a cancer/testis antigen since there was a high frequency of reactivation in breast cancer biopsies. It was also found to be negatively regulated by the p53 gene. To further characterize this gene, we recently examined the DNA methylation patterns of the TSP50 gene promoter in normal human testis, as well as breast tissue and a testicular embryonic carcinoma cell line (HTECCL). Bisulfite genomic sequencing results demonstrated that the promoter exhibited mixed DNA methylation patterns in normal human testis, mainly non-methylation versus slight methylation, which could be attributed to the different stages spermatic cells go through during spermatogenesis. In contrast, it was methylated to a much greater extent in both breast tissue and HTECCL. To find out whether DNA methylation status was related to spermatogenesis stages, we analyzed DNA methylation patterns of the mTSP50 (the mouse ortholog of TSP50) promoter in spermatocytes and spermatozoa isolated from sexually mature mice. The results clearly demonstrated that each group of cells exhibited its preferential DNA methylation pattern that apparently was consistent with the gene expression status observed before. Taken together, our findings suggested that DNA methylation might regulate the TSP50 and mTSP50 gene expressions in different types of tissues and spermatic cells.

TSP50 encodes a testis-specific protease and is negatively regulated by p53.

Earlier studies suggested that TSP50 is a testis-specific gene that encodes a protein, which is homologous to serine proteases but differs in that threonine replaces serine in its catalytic triad. Most importantly, it was abnormally reactivated in many breast cancer biopsies tested. While further investigating its biochemical and cell biological natures, we found that TSP50 exhibited enzyme activity and was located in the endoplasmic reticulum and cytosol membrane. During our studies to elucidate the regulatory mechanisms related to its differential expression, we discovered a putative p53-binding site and several Sp1-binding sites in the TSP50 promoter, which led us to test if it was regulated by the p53 gene. We found that the p53 transgene negatively regulated the TSP50 promoter in diverse types of cell lines. This result was consistent with other observations: (a) p53 overexpression reduced endogenous TSP50 expression; and (b) breast cancer cell lines containing mutated p53, such as MCF7/Adr, or normal p53, such as MCF7, produced high or low levels of TSP50 transcripts, which was consistent with the fact that TSP50 promoter activity was much higher in MCF7/Adr than that in MCF7 cells. We also found that the quantity of Sp1 transcription factor was lower in MCF7/Adr than in MCF7 cells, which suggested that another mechanism (i.e., transcription factor modulation) was also involved in TSP50 differential expression.

Screening of single-chain variable fragments against TSP50 from a phage display antibody library and their expression as soluble proteins.

A novel gene, testes-specific protease 50 (TSP50), is abnormally activated and differentially expressed in most patients with breast cancer, suggesting it as a novel biomarker for this disease. The possibility that TSP50 may be an oncogene is presently under investigation. In this study, the single-chain variable fragments (scFvs) against TSP50 were panned from a phage display antibody library using TSP50-specific peptide, pep-50, as a target antigen. After 4 rounds of panning, 3 clones (A1, A11, and C8) from the library were verified to show strong binding affinities for TSP50 by enzyme-linked immunosorbent assay (ELISA) and to contain the variable region genes of the light and heavy chains of scFv antibodies but different complementary determining regions by sequencing. The genes of scFv-A1 and scFv-A11 were cloned into expression vector pPELB and successfully expressed as a soluble protein inEscherichia coli Rosetta. The yields of expressions were about 4.0 to 5.0 mg of protein from 1 L of culture. The expressed proteins were purified by a 2-step procedure consisting of ion-exchange chromatography, followed by immobilized metal affinity chromatography. The purified proteins were shown a single band at the position of 31 KDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sandwich ELISA demonstrated that the expressed scFv proteins were able to specifically react with pep-50, laying a foundation for the investigation of the function of TSP50 in the development and treatment of breast cancer.

Epigenetic regulation of WTH3 in primary and cultured drug-resistant breast cancer cells.

Previous studies showed that the WTH3 gene functioned as a negative regulator during multidrug resistance (MDR) development in vitro. To understand whether this gene is also involved in clinical drug resistance, hypermethylation at its promoter region observed in cultured MDR MCF7/AdrR cells was examined in primary drug-resistant breast cancer epithelial cells isolated from effusions of breast cancer patients. The results showed that this event also occurred in drug-resistant breast cancer epithelial cells and a newly induced drug-resistant cell line, MCF7/inR. Interestingly, we found that a CpG (CpG 23) that was close to the TATA-like box was constantly methylated in the WTH3 promoter of drug-resistant breast cancer epithelial and cultured MDR cells. Mutagenic study suggested that this CpG site had a functional effect on promoter activity. We also discovered that MCF7/AdrR cells treated with trichostatin A, a histone deacetylase inhibitor, exhibited higher WTH3, but lower MDR1, expression. A reverse correlation between WTH3 and MDR1 gene expression was also observed in MCF7/AdrR, and its non-MDR parental cell line, MCF7/WT. This result indicated that both DNA methylation and histone deacetylase could act in concert to inhibit WTH3 and consequently stimulate MDR1 expression. This hypothesis was supported by data obtained from introducing the WTH3 transgene into MDR cell lines, which reduced endogenous MDR1 expression. Therefore, our studies suggested that the behavior of WTH3 in primary drug-resistant breast cancer epithelial cells was similar to that in a model system where epigenetic regulation of the WTH3 gene was linked to the MDR phenotype.

WTH3, which encodes a small G protein, is differentially regulated in multidrug-resistant and sensitive MCF7 cells.

The WTH3 gene's biological characteristics and relationship to multidrug resistance (MDR) were investigated further. Results showed that WTH3 was mainly located in the cytosol and capable of binding to GTP. In addition, WTH3's promoter function was significantly attenuated in MDR (MFC7/AdrR) relative to non-MDR (MCF7/WT) cells. Advanced analyses indicated that two mechanisms could be involved in WTH3's down-regulation: DNA methylation and trans-element modulations. It was found that the 5' end portion of a CpG island in WTH3's promoter was hypermethylated in MCF7/AdrR but not MCF7/WT cells, which could have a negative effect on the WTH3 promoter. This idea was supported by the observation that a 45-bp sequence (DMR45) in this differentially methylated region positively influenced promoter activity. We also discovered that different nuclear proteins in MCF7/AdrR and MCF7/WT cells bound to methylated or nonmethylated DMR45. Moreover, a sequence containing a unique repeat that was also a positive cis-element for the promoter was attached by different transcription factors depending on whether they were prepared from MCF7/AdrR or MCF7/WT cells. These molecular changes, apparently induced by drug treatment, resulted in WTH3's down regulation in MDR cells. Therefore, present studies support previous observations that WTH3, as a negative regulator, participates in MDR development in MCF7/AdrR cells.