BackgroundBenign mesenchymal tumors of the breast are rare and may mimic invasive carcinoma on imaging and morphology, thus becoming clinically challenging for clinicians, radiologists, and pathologists. To improve the understanding of these lesions and to avoid erroneous diagnosis and inappropriate treatment, we report our institution’s experience with seven cases of granular cell tumor (GCT) and myofibroblastoma (MFB) in the past 10 years.Materials and methodsSeven cases of benign mesenchymal tumors of the breast were identified at the University of Texas Medical Branch from 2008 to 2018. Breast biopsies were collected from all patients after mammography and ultrasound imaging classified their results as suspicious or highly suggestive of malignancy by the Breast Imaging Reporting and Data System (BI-RADS ≥ 4A). All cases were reviewed to study the morphologic features and their immunoprofiles. The demographic characteristics, methods of treatment, postoperative pathological results, and follow-up results of the cases were then analyzed and compared to peer-reviewed literature.ResultsThe study consisted of five females and two males with a mean age of 50 years in the GCT patients and 62 years in MFB patients. We identified four cases of GCT and three cases of MFB. The mean tumor size was 1.9 cm. Clinically, five patients presented with a palpable nontender mass, one with breast asymmetry, and one was asymptomatic. All patients underwent imaging studies prior to core needle biopsy. BI-RADS was ≥4B in patients with GCT and 4A-C in MFB. Definitive diagnosis was made by histopathology and confirmed by immunohistochemistry in accordance with the features described in the literature. Six patients underwent wide excision. The mean follow-up duration was 44.5 months. All patients remained well, without recurrence.ConclusionsMFB and GCT can mimic malignant neoplasms and the clinical significance of these entities lies primarily in their recognition as distinctive benign neoplasms. The gold standard for the diagnosis of GCT and MFB is histopathology. All the cases in our series were clinically or radiologically mistaken for carcinoma, which has been largely reported in the literature. Pathologists should bear this in mind to avoid misdiagnosis and unnecessary treatment. Benign mesenchymal breast entities may mimic invasive carcinoma on imaging and morphology, and their diagnoses can, therefore, be challenging for clinicians, radiologists, and pathologists. Among these entities, mammary myofibroblastoma (MFB) and granular cell tumor (GCT) are two rare lesions that may present as a painless palpable mass or may be clinically asymptomatic and found on routine imaging. On imaging, it can appear as a solid mass or lesion, which is suggestive of malignancy. Despite the advances in multiple imaging modalities, given the variability both within and across them, the accurate diagnosis of these entities remains a tissue diagnosis.Myofibroblastoma is a rare, benign mesenchymal tumor of the breast. It was first reported in the breast by Wargotz et al. in 1987 . Due to Both MFB and GCT typically run an indolent clinical course. Treatment by excision is recommended and curative in both etiologies. No adjuvant therapy is needed. Since both lesions have been reported to mimic carcinoma -10, it iThe data for this retrospective study were collected from our database at The University of Texas Medical Branch (UTMB). A series of four cases of GCT and three cases of MFB of the breast were identified between September 2008 and September 2018, each with a histologically confirmed diagnosis. We reviewed the pertinent medical history, imaging findings prior to breast biopsies, histologic features, treatment type, and long-term follow-up.Breast biopsies were collected from all patients after mammography and ultrasound imaging classified as suspicious or highly suggestive of malignancy by the Breast Imaging Reporting and Data System (BI-RADS ≥ 4A). All hematoxylin and eosin (H&E) and immunohistochemistry stained slides were reviewed to study the morphologic features and their immunoprofiles.Clinicopathological variables, including patient age and sex, presenting symptoms, imaging findings, histologic findings, and status of the patients at last follow-up, were analyzed. We expressed continuous variables as mean and categorical variables in frequency (percentage).Clinical featuresOur study consisted of seven subjects: five females and two males. The mean age at onset was 55 years (range from 26 to 86 years). We identified four cases (4/7) with the morphology and immunohistochemical features of GCT and three cases (3/7) were characterized as MFB. Five of the tumors were present on the right and two on the left side of the breast. The mean tumor size was 1.9 cm (ranging from 0.7 to 4 cm). At the time of presentation at UTMB, five patients complained of a palpable nontender mass, one patient complained of breast asymmetry, and one patient was asymptomatic. No symptoms suggestive of breast cancer, including skin retraction, nipple discharge, or axillary node enlargement, were seen in any of our patients , the ultrasound demonstrated a round or oval solid mass with indistinct or spiculated margins and with a hypoechoic (2/4), anechoic 1/4), or complex (1/4) echo pattern. The radiology categorization before the core needle biopsy for these four patients were: two patients as 4C, one as 4B, and one patient as BI-RADS 5 , the mammography/ultrasound showed a round or oval solid mass with circumscribed margins and a hypo 2/3) or hyperechoic (1/3) echo pattern. The radiology categorization pre-core needle biopsy in these cases was: one as 4B, one as 4A, and one patient as BI-RADS 4C , containing a tan to white in color, ill-defined, firm mass. The excised myofibroblastomas were tan-pink in color, nodular, and well-demarcated from the surrounding mammary tissue, with a variably whorling appearance. The size of the myofibroblastomas ranged from 7 mm to 40 mm. Necrosis or hemorrhage was not seen in any of the cases Figure .Histologic and immunohistochemical findingsClassic myofibroblastomas in two out of three patients consisted of uniform, slender, bipolar spindle cells haphazardly arranged in small fascicular clusters separated by bands of eosinophilic hyalinized collagen. No mitotic figures were found. The tumor in one out of three patients showed a similar morphology with increased cellularity and was called a cellular variant of myofibroblastoma. In all cases, the tumor cells were immunoreactive for CD34, desmin, smooth muscle actin (SMA), and estrogen receptor (ER) and negative for cytokeratin, p63, CD68, and beta-catenin Figures -3D.The granular cell tumors in the four patients were composed of compact nests or sheets of cells that contained abundant eosinophilic and coarse cytoplasmic granules. The cells were polygonal with indistinct cell membranes and well-defined cell borders. The nuclei were round to slightly oval with an open chromatin pattern. The tumors demonstrated an infiltrative growth pattern. No mitotic activity, necrosis, or nuclear pleomorphism was seen. A definitive diagnosis of GCT was established by using immunohistochemistry (IHC). The IHC analysis showed diffuse strong positivity for S100 protein and CD68 Figures -4D. The Treatment and follow-upSix patients  underwent a wide excision following the core needle biopsy diagnosis; five of them underwent lumpectomy and one had a simple mastectomy. One patient was recently diagnosed and has not received surgical excision yet. The prior diagnosis was confirmed for all the cases with a wide excision, and no cases were documented to have positive resection margins. The mean tumor size was 1.9 cm.The mean follow-up time was 44.5 months (ranging from one to 120 months). None of the lesions recurred or metastasized to the present date.Benign mesenchymal tumors of the breast are rare and exhibit histological features that can mimic malignant breast lesions. Among them, granular cell tumor and myofibroblastoma are two well-established entities, which are frequently confused on clinical and radiological examination with breast carcinoma -10. FailGranular cell tumor is a rare condition with a frequency of approximately 1 in 1000 breast tumors. The first report of GCT is attributed to Abrikossoff, who described a GCT as “granular cell myofibroblastoma” . GCT invThe major differential diagnostic consideration of GCT on clinical, radiological, and gross examination is invasive carcinoma. Clinically, GCT can mimic malignant disease, sometimes with skin retraction and nipple inversion in superficial lesions. Deep lesions may show fixation to the skeletal muscle of the chest wall and may be adherent to the pectoral fascia. The patients typically present with a palpable, firm, and painless mass, which was noted in three out of four patients with GCT in our study. Radiologically, they typically form a spiculated, irregular, or stellate mass on mammography or solid, poorly defined mass with indistinct margins and posterior shadowing on ultrasound ,12-16. FMacroscopically, GCT demonstrates as a gray-white to tan, firm mass, and some tumors show ill-defined borders and may infiltrate, as in our cases, into the surrounding tissues. These features may mimic malignant growth patterns. Microscopically, GCTs must be distinguished from breast cancer, especially from a histiocytic variant of invasive lobular carcinoma, apocrine carcinoma, histiocytic lesions, as well as a metastatic neoplasm in the breast that has oncocytic or clear cell features, such as renal carcinoma, malignant melanoma, and alveolar soft part sarcoma ,11,17. GImmunohistochemistry helps to distinguish GCT from invasive mammary carcinomas, including apocrine carcinoma. The tumor cells show positive immunoreactivity against S100 protein, CD68, carcinoembryonic antigen (CEA), and vimentin. Cytokeratin staining is negative and most lesions are negative for estrogen, progesterone receptors, and androgen receptor. The granular cytoplasm is positive for PAS and is diastase resistant. In our presented cases, the pathological features were supported by IHC, as reported in the literature -6,11-12.MFB is a rare benign spindle cell tumor of mammary stroma composed of myofibroblasts. This entity was first described by Wargotz et al. . The preMacroscopically, MFB tumor size can range from a few millimeters to 11 cm and is generally a well-circumscribed, ﬁrm, and rubbery, unencapsulated, round to oval mass. In some cases, the tumor may show focal to extensive mucoid- or lipomatous-appearing areas. However, cystic degeneration, necrosis, and hemorrhage are not features of MFB. The size of our MFB cases measured from 7 mm to 40 mm. The excised tumors were tan-pink in color, nodular, and well-demarcated from the surrounding mammary tissue, with a variably whorling appearance. Neither necrosis nor hemorrhage was seen in any of our cases.MFB may exhibit a wide spectrum of histological features and varied cellularity that can be misinterpreted as a malignant tumor. Histologically, the classic type of MFB is a circumscribed tumor consisting of uniform spindle cells with bland, oval nuclei and indistinct borders that are arranged in short fascicles and admixed with bands of eosinophilic hyalinized collagen ,21,24-25Immunohistochemistry may be necessary to arrive at a correct diagnosis. The MFB tumor cells show immunoreactivity for CD34, desmin, and, variably, with smooth muscle actin (SMA), estrogen receptor (ER)/progesterone/androgen receptor, CD99, B-cell lymphoma 2 (Bcl2), and CD10 ,21,28, aThe clinical significance of these entities lies primarily in its recognition as a distinctive benign neoplasm. It is important to make a deﬁnitive diagnosis preoperatively to avoid extensive resection and axillary clearance for carcinoma. For both lesions, complete local excision with free margins is the treatment of choice . SentineOur cases illustrate that although GCT and MFB of the breast are relatively rare breast neoplasms, they should always be considered in the differential diagnosis of benign and malignant lesions. Since clinically and radiologically these lesions can mimic invasive carcinoma and the definitive diagnosis is made by pathology, pathologists should bear this in mind to avoid misdiagnosing breast carcinoma, which could lead to unnecessary surgery.


The uncultured bacteria in both treatments were determined, using tagged 16S rRNA gene amplicon with Ion Torrent Sequencing Analysis. The effects of inorganic nutrients and the reservoir’s parameters on the bacterial communities were analysed. A total number of 16,828 OTUs were taxonomically classified into 89 classes and 584 genera. In the controls (sandstone cores submerged in the produced water), the dominant bacterial populations were related to Deinococcus-Thermus, and Betaproteobacteria; while in the nutrient treated samples, there was a marked increase in the relative abundance of Gammaproteobacteria in three samples. Thermus, Acinetobacter, and Pseudomonas were the most abundant genera, and these are potential microorganisms for MEOR. Analysis of correlations between physiochemical properties of the reservoir and bacterial genera, using spearman’s correlation analysis, suggested that some of the reservoir’s properties, especially of the well and the rock, could influence some bacterial genera. To our knowledge, this is the first demonstration of the effect of inorganic nutrients on alteration of bacterial communities attached to reservoir’s rock, and how the bacterial, physical, and chemical properties of a reservoir were co-analysed to serve as a basis for designing a MEOR process.Microbial Enhanced Oil Recovery (MEOR) is a promising strategy to improve recovery of residual oil in reservoirs, which can be performed by promoting specific indigenous microorganisms. In this study, we performed preliminary evaluation of the possibility of conducting MEOR at Mae Soon reservoir, an onshore reservoir in Northern Thailand. The reservoir’s physicochemical characteristics, including the characteristics of the wells, the oil-bearing sandstone cores, and the reservoir’s produced water, were determined. The microbiological characteristics of the oil wells in the reservoir were also investigated by submerging the reservoir’s sandstone core samples, obtained from 6 oil wells, in the reservoir’s produced water and in the produced water added with inorganic nutrients (KNO Petroleum resources are becoming more limited, and while other alternative energy resources are being explored, Enhanced Oil Recovery (EOR) technologies are increasingly important. These technologies have been developed in response to the fact that the conventional petroleum recovery procedures can only retrieve 10 to 45 percent of the crude oil . An EOR Bacillus, Methanobacterium, and Clostridium [Pseudomonas, were also found in subsurface strata and often in association with the presence of nitrate. These microbes can function in MEOR through different mechanisms, including the production of gases , low molecular weight acids, solvents , biosurfactants, and biopolymers. These microbial metabolites can lead to increases in the efficiency of oil recovery by viscosity reduction, permeability alteration, and emulsification of crude oil. The microbial metabolites can decrease the surface and interfacial tensions and alter wettability, resulting in increasing pore scale displacement [Many subsurface microorganisms have been reported to play crucial roles in MEOR including stridium . Aerobiclacement . One appin situ and in vitro effects of MEOR on microbial community structures, using culture-dependent methods [Several studies have reported diversity of microbial communities in onshore and offshore oil reservoirs as well as the  methods , 7. Howe methods  and, the methods , and at  methods . Most in methods , microbi methods , 11. Incin vitro nutrient treatment could affect bacterial community structures, and to analyse the correlations between some key characteristics of the reservoir and its indigenous bacterial communities. These investigations were not only intended to provide a basis for future in situ MEOR implementation at the study site , but also to demonstrate how the bacterial structure and the additional inorganic nutrients together with the physicochemical characteristics of a reservoir, are related and how these parameters can be co-analysed and manipulated, in order to serve as a basis for designing a MEOR process.With these gaps in MEOR studies, we therefore aimed to conduct an investigation on the diversity of bacteria derived from the oil-bearing rock regions of a reservoir using Ion Torrent Sequencing Analysis, to demonstrate how an All samples were collected within territories governed by the NPDC, Defence Energy Department , with the permission granted through Capt. Pinan Dawkrajai, RTN, Ph.D., .Core samples used in this study were obtained from the reference core collection of Mae Soon reservoir of Fang oil field, Chiang Mai, Thailand  . This reTemperatures and depths of oil reservoirs were retrieved from the well logs. Porosity, permeability and grain density of the core samples were determined according to the routine core analysis . The lithology of the core rock was analysed using an X-Ray fluorescence (XRF) spectrometer .Produced water  from the production wells was analysed for pH and salinity using a standard pH meter  and an electrochemical analyser . Concentrations of sulfate and nitrate in the produced water were determined using ion chromatography . Iron content was analysed using inductively coupled plasma optical emission spectrometry (ICP-OES) .3 and 0.034% (w/v) NaH2PO4, prepared from AR grade chemicals ), based on a core flooding protocol previously reported to be effective for oil recovery [To study the effect of inorganic nutrients on the changes in microbial composition within the oil sand samples, each of the six oil-bearing sandstone samples was extracted from the drilled core. Only the inner part of each core was sampled; it was placed in a sterile plastic bag, then crushed using a hammer, mixed, and divided into two 10-gram portions. The first portion was placed in a 250-ml screwed-cap DURAN bottle which was then filled completely with the produced water . The cap was then tightened to create a hypoxic condition . This treatment was performed to resemble the conditions in the formation rock layer where the core originated. The samples treated with the produced water (assigned C1P –C6P) were used as control samples. Another portion of each oil sand sample was flooded with the produced water supplemented with inorganic nutrients (0.12% (w/v) KNOrecovery . The nut4, pH 8.0, 1.5 M NaCl and 1% (v/v) CTAB) and 100 μl of proteinase K [The enriched core samples were separated from the fluids by centrifugation at 4,766 g for 5 min. The genomic DNA was extracted from 10 grams of the samples using 10 ml of lysis buffer  and E1081R (5′-CTCACGRCACGAGCTGACG-3′) attached with tagged barcode sequences [The purified metagenomic DNA was used as a template for amplification of the partial 16S rRNA gene. Universal bacterial primers E785F  version 1.9.1 . The seqrelation .A previous survey has shown that the formation pressure of the deepest oil-bearing zone of this reservoir was 1,082 psi . The physical characteristics of the sandstone cores, together with the physical properties of the oil wells from which the cores originated are summarised in 2 (80–85%). Elements including nickel, vanadium, rubidium, yttrium, niobium, chromium, strontium, barium, and zirconium were present in all samples. The chemical properties of the cores are summarised in Tables The chemical properties of the oil-bearing sandstone cores and the produced water were analysed in order to evaluate the possibility of applying MEOR to the oil wells in this reservoir. Elemental compound analysis, using an X-ray fluorescence spectrometer, revealed that the cores from this sandstone-based reservoir consisted mainly of SiOProduced water from the oil wells was analysed for chemical properties, including pH and salinity. In addition, the concentrations of sulfate, nitrate, and iron, which can be the sources of nutrient for microorganisms, were also determined. We found that the produced water from most wells  does not vary significantly in salinity, which is in the normal range of the salinity of onshore reservoirs. The pH of the produced water from the 6 wells tested showed that the wells were slightly alkaline. Sulfate was not detected in the produced water from most wells, while nitrate was present in every well, and iron in 4 out of 5 wells . An analThe purified DNAs from the sandstone core samples treated with produced water  and from those treated with produced water supplemented with inorganic nutrient (C1N-C6N) were sequenced. A total of 1,426,176 filtered reads were obtained from the ION PGM sequencing. The number of reads obtained from each sample ranged from 17,039 to 220,923 reads. The Operational Taxonomic Units (OTUs) in the samples were assigned from the filtered sequences at 97% similarity levels. The bacterial phylotypes ranged from 471 to 754 OTUs at the genetic distance of 0.05 with an average of 619 OTUs per sample. Rarefaction analysis was used to determine the microbial diversity in the filtered data set. The curves show similar numbers of OTUs obtained from both treatments, indicating that there was no significant effect of treatment on the numbers of OTUs as assessed by rarefaction . In ordeAccording to the shared OTUs chart , bacteriDeinococcus–Thermus, Proteobacteria (Beta), Acidobacteria (Solibacteres), and Firmicutes (Clostridia) were the predominant classes having mean proportions of 40.9%, 17.5%, 10.7%, and 9.2%, respectively. When the samples were treated with inorganic nutrients, the predominant classes were mainly Deinococcus–Thermus and Proteobacteria (Gamma). Deinococcus–Thermus was the major taxon found with the proportion of 41.8%, while Proteobacteria (Gamma) was the second most abundant class, contributing 36.9%. Acidobacteria, Proteobacteria (Beta), and Firmicutes (Clostridia) were found only in small proportions in the nutrient-treated samples. If considered by means of individual samples, there was a significantly increase (P < 0.05) in the relative abundance of Gammaproteobacteria in three samples  obtained from three individual wells , as a result of the inorganic nutrient treatment.Bacterial community profiles of the two treatments were analysed at the class level . From FiThermales , Solibacterales (in C6P), Burkholderiales (in C1P and C2P), Clostridiales (in C5P), and SM1H02 . For the nutrient-treated group, the top five orders were Pseudomonadales , Thermales , SM1H02 , Solibacterales (in C3N), and Pirellulales (in C2N and C5N), although the latter two had abundances smaller than 10%.At the order level, the top five orders in the control group, according to the OTU abundances, were Thermus was the most abundant genus, both in the samples submerged in produced water and in the samples treated with additional nutrients, having average distributions of 33.8% and 40.0%, respectively. Treatment with the inorganic nutrient caused a slight increase of this genus (6.2%). The populations of this genus increased in samples C2N (20.0% to 65.9%), C4N (74.5 to 78.1%), and C5N (5.2% to 76.8%); but decreased in samples C1N (27.3% to 0.6%), C3N (47.3% to 9.9%), and C6N (28.5% to 8.6%). Other bacterial genera that were obviously altered as a result of nutrient treatment were Pseudomonas and Acinetobacter. Pseudomonas increased up to 19.7%, while Acinetobacter increased up to 15.5%. Alterations of these genera were not consistent in all samples, although they increased in the majority of the samples. For example, the relative abundance of Pseudomonas increased in nutrient-treated samples C1N, C3N, C4N, and C6N. Acinetobacter increased in C1N, C2N, C3N, C4N, and C6N. The most prominent increase in Acinetobacter was observed in nutrient-treated sample C1N , and Pseudomonas in C3N .We also determined the effects of different treatment conditions on bacterial diversity at the genus level. The abundances of bacterial genera were different between the samples submerged in produced water and those treated with inorganic nutrients . In the Tepidimonas, Meiotermus, Hydrogenophilus, and Bacillus, experienced average decreases in their proportions after nutrient treatment (Tepidimonas decreased most obviously in the sample C1 (from 32.9% (C1P) to 0.6% (C1N)), Meiothermus in C2 (from 22.5% (C2P) to 0.4% (C2N)), Hydrogenophilus in C6 (from 7.9% (C6P) to 0.001% (C6N)), and Bacillus in C6 (from 3.7% (C6P) to 0.08% (C6N)).On the other hand, some genera, such as reatment . The relSpearman’s correlation analysis showed statistically significant correlations between physicochemical factors and bacterial genera in the reservoir communities   and Lactobacillus ; while permeability had a negative correlation with Rubrobacter (N). Porosity, however, did not have significant correlation with any bacterial genera. Among the chemical compositions of the reservoir rocks, there were connections between some elements and compounds such as Cr, MnO, P2O5, and Na2O and some bacterial genera, which were mostly in negative correlation patterns. Cr and MnO had effects on Acinetobacter, Corynebacterium, and Enhydobacter (C), P2O5 on Enhydobacter and Acinetobacter, and Na2O on Tepidomonas and Lactobacillus (C) and Bacillus (N). Some positive correlations were observed, such as those between P2O5 and Thermus (N), but such correlations were exceptions.As for the core characteristics, several factors, including physical and chemical characteristics, were found to affect bacterial genera in the communities. Among the physical factors, grain density positively correlated with the proportions of The produced water, in contrast to the well and rock properties, had no significant correlation with bacterial genera for both treatments.et al. (1989), who report that pressure lower than 1,400–2,800 psi does not affect on bacterial metabolism [Mae Soon reservoir is one oil-bearing reservoirs in the Fang oil field, with the oil sand at a depth between 609 and 756 metres. The depth affects the temperature and pressure of the formation. The temperature, in particular, can affect microbial growth and metabolism . The foret al. (2000), sandstone-based rock is favourable for the biotechnological oil-recovery method [et al. (2010) observed that rocks with porosity of about 20% supported recovery of additional incremental oil from the sandstone core up to 75% [The physical and chemical properties of the oil-bearing rock and the properties of the produced water from oil wells within a reservoir can determine the success of the MEOR method. Lithological analysis revealed that the rock of reservoir was sandstone-based. According to Brown y method . Core any method . Thus, fy method , the valy method . Iglauerp to 75% . The corp to 75% . Moreovep to 75% , 30.Deinococcus-Thermus (Deinococci) was the most abundant bacterial class in half (three out of six) of the samples tested. As for the other three samples, even though the Deinococcus-Thermus (Deinococci) was present, the predominant bacteria differed. Betaproteobacteria was predominant in one sample (C1P), while in samples C5P and C6P, Firmicutes (Clostridia) and Acidobacteria (Solibacteres), were more abundant than others. This is interesting because Gammaproteobacteria had been found to be a more-common predominant bacterium in many other studies of oil-reservoir microbial communities than Deinococcus-Thermus [Deinococcus-Thermus (Deinococci), was different from those related to the oil reservoirs previously reported [Besides the characteristics of the well, the rock, and the produced water, the microbiological characteristics of the reservoir were investigated. The experiment of enrichment process of 8 month period was designed as a laboratory trial to observe what might happen to the bacterial communities residing the well/reservoir rock after being treated with inorganic nutrients for an extended period of time. This enrichment process was designed according to a field experiment proposal, using enrichment/shut-in strategy, at Mae Soon site. The relative abundance of predominant taxa found in the oil-bearing sandstone core samples that had been submerged in the reservoir’s produced water (control samples) showed that -Thermus . Thus, treported , 33. TheThermus, Pseudomonas, Acinetobacter, Bacillus, and Clostridium [Thermus, which was found 27.3%, 20.0%, 47.3%, 74.5% and 28.5% in samples C1P, C2P, C3P, C4P, and C6P, respectively. This bacterium is generally found under elevated temperature conditions. It can grow at temperatures between 50 to 75°C (with the optimum temperature of 70°C), and is also found in high-temperature petroleum reservoirs (with the temperature range of 50 to 80°C) such as East Paris Basin (France) and Samotlor oil reservoir (Russia) [At the genus level, sequencing of 16S rRNA gene amplicons from the reservoir samples revealed several potential bacteria for the microbial recovery process including stridium . The mos(Russia) , 36. Man(Russia) .Bacillus genus was found in sample C1P, C3P, and C6P. This genus occurred in the environment of many oil reservoirs [Clostridium was also found, although not in a great abundance. This organism is one of the bacteria that can produce solvents and gases under anaerobic conditions, which would support the efficiency of oil recovery by reducing oil viscosity [Many facultative anaerobic bacteria found in the reservoir samples were bacteria that can potentially support the MEOR process. The servoirs . Some meservoirs . Besidesiscosity .Deinococcus-Thermus (Deinococci) did not seem to be influenced by nutrient enrichment and remained the most abundant class in the reservoir’s community; comprising 41.8%, which was similar to the control group (40.9%). However, when considering the bacterial composition in groups of individual wells, the abundances of this class were significantly different. It was more abundant in C2N, C4N, and C5N than in the controls. This group of bacteria is more commonly present in high temperature environments [Deinococcus-Thermus had not been reported to be present in sandstone-based rock; it had only been found in the rock from a coal bed methane reservoir, although in a small proportion [There were some changes in the relative abundances of predominant taxa in the core samples after being treated with inorganic nutrient. ol group .9%. HoweGammaproteobacteria, which became the second most abundant group in some nutrient-treated samples. However, because of the high level of variation in the proportions of this group among the samples, the overall changes were not significant. In contrast, Betaproteobacteria, Acidobacteria (Solibacteres), Firmicutes (Clostridia), and Firmicutes (Bacilli), were the taxa that experienced a lower abundance than in the control group.Differences of relative proportions of bacteria as a result of nutrient treatment were observed with et al. (2011), which were dominated by Delta/epsilon-Proteobacteria (sulfate-reducing bacteria) and methanogens (mostly Methanococcus species) [Delta/epsilon-Proteobacteria occurred in low abundance, while methanogens were not found. Although this is an extreme comparison between an onshore and offshore (marine) environment, between sandstone and liquid phase samples, and between the different treatments the samples received; it is clear that bacterial distribution in an environment can be greatly influenced by unique reservoir characteristics, such as NaCl concentration, types of organic compounds present, and reservoir pressure. Even the individual characteristics of the oil wells in the reservoir could also contribute to the specificity of microbial population.Bacterial community profiles of nutrient-treated sandstone samples determined in this work were clearly different from those of the liquid phase of an offshore reservoir, as reported by Kotlar species) . In our Thermus, Pseudomonas, and Acinetobacter. Again, the overall changes were not significant due to the high level of variation among the samples, but they increased significantly in some samples extracted from some wells. A previous study performed on inorganic nutrient-enriched production fluids resulted in different major microbes, including bacteria in the order Thermotogales, methanogens related to Methanobacterium and Methanococcus, and sulfur-utilising archaea of the genus Thermococcus [At the genus level, the bacterial genera that were predominant in the nutrient-treated samples were mainly mococcus . NutrienThermus, which was the most abundant genus in the treated samples, is known as thermophilic hydrocarbon-degraders [In MEOR-related terms, the major bacteria found in the cores after nutrient enrichment were considered having high potentials. egraders . This oregraders . This geegraders . It is, Pseudomonas, the second most abundant genus that became one of the predominant groups as a result of nutrient treatment, is capable of producing many kinds of biosurfactant molecules. Biosurfactants can lower interfacial tension of water, thus allowing mobilisation of bound hydrophobic molecules, which, in this case, is the oil trapped in the reservoir rock. The biosurfactants produced by this organism had a high potential for the recovery of crude oil [Pseudomonas is known as a hydrocarbon-degrading bacterium, and it is effective in stabilising oil-in-water emulsion that can improve the ability of oil recovery [Pseudomonas was applied, together with other microbes, to enhance oil recovery rate in the Bragadiru oil field, which caused the increase in oil production from 1.5 to 7.4 bbl/d per well [Pseudomonas in onshore reservoirs had also been observed in other studies [Pseudomonas was found in the produced water of Gudao oil reservoir, China [Pseudomonas, which is typically aerobic, was found in oil reservoirs was interesting. This genus could grow in an anaerobic condition, as reported by Kerschen et al. (2001), since it could use nitrate as a terminal electron acceptor for both assimilation and anaerobic respiration [rude oil . Besidesrecovery . In addiper well . The exi studies , 42. Pser, China  and of Dr, China . Fluid sr, China . The facpiration .Acinetobacter, another genus present in a large proportion as a result of nutrient treatment, is another potential bacterium for a MEOR process. Several species of Acinetobacter produce substances involved in emulsification, interfacial tension reduction, and oil viscosity reduction [eduction . These aeduction . Previoueduction , 52.Clostridium, Klebsiella, and Bacillus, known as potential producers of some metabolites that can enhance oil recovery, were also detected, although in low proportions. Clostridium and Klebsiella have been reported to increase the oil recovery process by reduction of oil viscosity [Bacillus is among the organisms with high potentials to produce biosurfactants and enzymes [Bacillus is also favourable for its survival in harsh conditions of different reservoirs [Thermus, Pseudomonas, and Acinetobacter, to prevail, obviously could not promote the growth of Bacillus. It could be possible that the organism remained in the dormant stage under the nutrient-treated conditions given in this study. Conditions that can elevate this genus to a higher relative abundance remain to be further investigated.Several other bacterial genera such as iscosity . Bacillu enzymes , 54. Memservoirs . This geSome bacterial genera in the reservoir’s communities can be influenced by reservoir parameters, such as temperature and depth of the wells, grain density and permeability of the cores, and chemical compositions of the cores. Inorganic nutrient composition could also alter bacterial population, although the patterns of change were not consistent in individual wells. Since predominant bacterial genera could greatly influence biosurfactants, gases, acids, solvents, and biopolymers production, it is important to consider the connection between physical/chemical factors of the reservoir and the microbial structure, in order to evaluate the suitability of the reservoir for MEOR application and to appropriately design a MEOR strategy.Deinococcus-Thermus (Deinococci) and Betaproteobacteria as the predominant groups. The bacterial communities in samples extracted from some wells of the reservoir were altered after the inorganic nutrient treatment, resulting in a different bacterial population, most obviously in Betaproteobacteria giving dominance to Gammaproteobacteria, while Deinococcus-Thermus remained the most abundant group. At the genus level, Thermus, Pseudomonas, and Acinetobacter were the most abundant. Members of these genera are capable of producing hydrocarbon-degrading enzymes, surfactants, and other MEOR-related substances, which are desirable for MEOR application. The microbial communities were supported by the reservoir’s physical/chemical characteristics.This study demonstrated how the bacterial structure in oil-bearing sandstone cores extracted from Mae Soon reservoir, Fang Basin, Thailand, and how the additional inorganic nutrients, together with the physical and chemical characteristics of the reservoir, were related. Bacterial community analysis of oil-bearing sandstone cores submerged in the produced water under high temperature conditions revealed in situ investigation must be performed. Nevertheless, the approach of this study in employing the information of a reservoir’s bacterial communities, fortified with inorganic nutrients as necessary, together with the reservoir’s physical and chemical properties, can be considered for a preliminary evaluation of the possibility of conducting or for designing a MEOR process.This experiment was set to address whether the inorganic nutrient treatment would have an effect on the reservoir’s bacterial community as a whole, or on the communities in the individual wells, so that MEOR could be designed according to the finding. Our results seemed to point that, for this reservoir, such nutrient treatment may be potential, but the application of MEOR processes might be considered in individual wells rather than one process for the whole reservoir’s system. However, whether or not the field application based on this laboratory finding would be effective, S1 FileThe rarefied OTUs  were obtained by subsampling method and statistical calculation was performed using the non-parametric Mann-Whitney U test.(XLSX)Click here for additional data file.S1 TableThe rarefied 16,828 OTUs  were use(XLSX)Click here for additional data file.S2 TableThe statistical results show significant correlations between physicochemical factors and bacterial genera in the reservoir communities.(XLSX)Click here for additional data file.


It is found that an optimized amount of PE fiber is beneficial for improving the tensile resistance and ductility of FLCC while a relatively large amount of microspheres can increase the entrapped air voids in the FLCC matrix and reduce its density and thermal conductivity. Microstructural analysis by scanning electron microscopy (SEM) reveals that the microspheres are distributed uniformly in the cement matrix and are subjected to triaxial compression confinement, which leads to high strength of FLCC. Segregation due to density difference of FLCC ingredients is not observed with up to 60% (by weight) of glass microspheres added. Compared to the other lightweight aggregate concretes, the proposed FLCC could be used to build floating concrete structures, insulating elements, or even load-bearing structural elements such as floor and wall panels in which self-weight is a main concern.This paper presents the development of a novel, multifunctional, floatable, lightweight cement composite (FLCC) using three different types of glass microspheres for structural engineering applications. Eight different mixtures of FLCC were produced and their matrix-related parameters were examined experimentally by adopting different types of microsphere fillers, fiber content (polyethylene fibers (PE)), and water-to-binder ratios. Along with the mechanical properties such as compressive, flexural, tensile strengths, and modulus of elasticity, the water tightness of the material was evaluated by sorptivity measurements and the energy efficiency by thermal conductivity. The optimal FLCC has an oven-dry density of 750 kg/m In the past decade, the construction industry all over the world has been exploring solutions to improve the productivity in site construction. The focus was placed on the reduction of site manpower usage at reasonable cost while maintaining design variety and high-quality work. Concrete is one of the most widely used materials in construction. Nevertheless, lately the focus has shifted to sustainable development and various industries are striving to save energy and lower the environmental impact. Concrete is more frequently required to possess more advanced characteristics to achieve low density, low cost, better thermal insulation, superior mechanical properties, and eco-friendliness. As a result, a multifunctional, lightweight, high-strength concrete is a superior alternative material used for structural elements such as building floors, exterior wall panels, and roofing tiles in which self-weight needs to be reduced significantly. The main advantage of using lightweight concrete is the reduction of self-weight of structural members and overall structures, allowing smaller sizes of structural members and less reinforcing materials. Thus, requirements for foundations and transportation cost can be reduced significantly. Additionally, it allows for easy and rapid installation at construction sites due to its lower self-weight, thereby saving time and improving productivity in construction.Lightweight onshore and offshore constructions shown in 3 while for structural lightweight concrete, ACI 213 recommends the range as 1120 to 1920 kg/m3 with compressive strength larger than 21MPa [Definitions of lightweight concrete vary among international standards and codes of practices. an 21MPa . The metan 21MPa ,15,16.  [3), high-strength (crushing strength up to 45 MPa), inert, hollow spheres made largely of silica and alumina and filled with air or inert gas, typically produced as a by-product of coal combustion at thermal power plants or artificial sintering method. Therefore, the resulting concrete using cenospheres exhibits high strength and lightweight behavior. Compared to conventional lightweight aggregates, cenospheres have lower water absorption due to their thick shell and higher crushing strength with very light unit weight. While the cenospheres provide an excellent choice for such lightweight cement composites (LCC), an important question remains on how the crushing strength of these lightweight microspheres affects the strength of the LCC. Would the strength of the LCC be higher if the crushing strength of microspheres can be increased? Artificial fine microspheres aggregates, namely, soda–lime–borosilicate-based glass microspheres, were adopted as an alternative to FAC to improve the mechanical and even functional properties of lightweight concrete [Recent development on ultralightweight cement composite (ULCC) by Wu et al. , Huang e volume) , which h volume) ,30, wall volume) ,31, and  volume) ,28,32 wi volume) ,38,39,40 volume) . Cenosphconcrete ,43,44,45concrete ,49,50,51concrete ,49,50,51This paper presents the research and development work on a floatable, lightweight cement composite (FLCC) using glass microspheres and investigates its mechanical and thermal properties, resistance to water penetration, and microstructure. Effects of different types of glass microsphere aggregate, water-binder ratio, and fiber content on compressive strength, flexural strength, tensile strength, ductility, elastic modulus, sorptivity, workability, and density of FLCC were evaluated experimentally. Potential challenges of FLCC for structural application are also discussed. This study would be useful for design and application of prefabricated and precast structural members such as partition wall panels, flooring slabs, ceilings, and insulation boards for onshore and offshore infrastructures.2 content of over 90% was used in all mixtures to strengthen the bond strength of the interface transition zone (ITZ) between the microspheres and cement paste. To gain a proper workability, a high range of polycarboxylate-based superplasticizer was used to achieve required flow around 200 mm based on flow table test [3 with relative high crushing strength from 27.6 to 68.9 MPa, while FAC had average bulk density of 450kg/m3 with relative high crushing strength of 15.0 MPa, as shown in Ordinary Portland Cement with specific gravity of 3.16 (ASTM Type I cement) was used as the binder material in the FLCC mixtures. Lightweight, high-strength glass microspheres (GM) and fly ash cenospheres (FAC) were used as microaggregates for the FLCC. Densified Elkem Microsilica 940U silica fume (SF) with SiOble test . The cheThe used FAC and GM had a relatively smooth surface and were composed of a closed external shell and internal air pores encapsulated within the shell. This could be the reason that the GM and FAC had lower water absorption (less than 2% after soaking in water for 1 h) compared to the other available lightweight aggregates (LWAs). Glass microspheres used in this paper had a diameter in the range of 2–125 microns with wall thicknesses less than 1 micron. To improve the flexural strength and ductility of FLCC, different amounts in volume of polyethylene (PE) fibers were employed. PE fiber has a tensile strength around 2900 MPa with an elastic modulus of 116 GPa. The aspect ratio of PE fiber (fiber length to diameter ratio) is 720. According to the information from the manufacturer, the PE fibers were not surface treated. A total of three mixtures were added with PE fiber ranging from 0.5% to 1.5% in an interval by volume. Floatable, lightweight cement composite was cast using above-mentioned OPC, superplasticizer, silica fume, and glass microspheres with a water/binder ratio of 0.35 to 0.65. Due to the fine particle size and corresponding larger specific surface area of the glass microspheres, more water should be used, hence a w/b of 0.6 was chosen based on trial tests. The density of lightweight fine aggregate affects the density of FLCC considerably, since the volume of aggregate is significant in the mixtures. Three types of glass microsphere aggregates with a mass ratio of 0.6 were considered in the mix proportions. Another two control mixes were ULCC using fly ash cenospheres and LWC using expanded shale aggregates. The mix proportions of FLCC, ULCC, and LWC are summarized in Unlike normal concrete, making FLCC requires longer mixing time due to the fine nature of the microspheres, while the spherical shape provides a ball-bearing effect and ensures better flowability of the FLCC. For workability measurement, the fresh FLCC mixture was placed in a truncated conical mold on a plastic board . The FLCThe flexural strength of FLCC was measured using three-point bending on 40 × 40 × 160 mm prism specimens according to BS EN 196-1 . AdditioThe thermal conductivity of the FLCC and normal concrete specimens was determined according to ASTM C518  by a heaThe sorptivity test is a useful test to examine the effectiveness of any water-repellent materials. Sorptivity of FLCC, ULCC, NWC, and LWC was determined by measuring the weight increase of a specimen due to the absorption of water as a function of time when one surface of the specimen was exposed to water according to ASTM C 1585 . Figure 3. During the mixing, it was found that a higher amount of superplasticizer was needed, which was due to the larger specific surface area of lightweight glass microspheres compared to the normal angular particles of crushed stone aggregates. Increasing the amount of the glass microspheres resulted in a corresponding decrease in the compressive strength and density. Different amounts of PE fiber addition resulted in different increases of flexural strength but in certain reductions of compressive strength. The 28-day compressive strength of FLCC ranged from 26.9 MPa to 41.0 MPa. This is a promising property that indicates FLCC could be used as a structural cement composite material in terms of compressive strength. The high strength of FLCC may be due to: (1) the high strength of the fine glass microsphere aggregate itself, and (2) the three-dimensional confinement effect from the surrounding hardened cement matrix when subjected to loading, which is attributed to uniform distribution of glass microspheres in the cement matrix. Among those mixtures, FL40 had the highest compressive strength of up to 41 Mpa, followed by FL46 and FL60 with slightly lower compressive strengths. Interestingly, it was found that while the decrease in compressive strength from FL40 to FL46 corresponded to the reduction in density, the decrease in compressive strength from FL46 to FL60 was much higher, indicating that a threshold may exist in terms of the type and density of GM that can be added to cement composites. Therefore, FL46 was chosen as the optimal type of GM for other mixes, including those containing PE fibers. For a given w/b ratio of 0.6, the FL60 sample had a higher porosity in the cement paste matrix but lower porosity in the microspheres compared with FL40 and FL46. Compared to a w/b ratio of 0.35, FL46-35 had a higher porosity of 7.51%, which may be due to the low w/b ratio. The densities of all FLCC were less than the density of water, which indicated that the FLCC concrete can float on the water. cmf). Both the test data in this paper and those from literature are plotted in the figure. Compared to other types of lightweight concrete, FLCC developed in this paper exhibited much higher specific strength . Also, among the test data with density below 1000kg/m3, FLCC showed promising performance with compressive strength (cmf) exceeding 40 MPa. For concrete with density grade between 1000 kg/m3 and 1300 kg/m3, FAC-based ULCC exhibited the highest specific strength up to 53.3 kPa/kg·m−3 [a/kg·m−3 ,35.ct) increased by about 80.3%, 223.3%, and 325.6%, respectively, compared to that without fiber addition. The average 28-day flexural strength through three-point bending test ranged from 4.0 MPa to 9.5 MPa. 3 grade ultralightweight cement composite. With the addition of 0.5%, 1.0%, and 1.5% PE fiber, the flexural strength . The test samples were soaked in isopropanol to stop the hydration, and then vacuum-dried in the oven at 40 °C before testing. 3) and compressive strength (cmf) up to 41 MPa by means of commercially available glass microspheres. A range of properties was investigated for seven different types of FLCC, and FLCC using GM46 was found to have optimal compressive and flexural strength. Addition of 1.5% PE fiber in volume improves the flexural strength by more than 3 times. Under direct tension, FLCC incorporating a low content of PE fiber (1% in volume) exhibits strain-hardening behavior with ultimate tensile strain capacity of 6%. Thus, an ultralightweight FLCC (lightweight ECC) with strain-hardening characteristics has been produced for wider engineering applications. However, the elastic modulus was lower than normal concrete and lightweight concrete due to the lower elastic modulus of the glass microsphere aggregates. The FLCC also exhibited better water absorption capacity and much superior thermal insulation properties. The use of FLCC developed in this paper enables novel metal/polymer-FLCC composite structures to be further developed with lower self-weight, which will provide alternatives for prefabricated concrete structures, marine construction, and ship hulls. This will benefit the transportation and installation of precast and mass-sensitive structures.This paper presents a new type of multifunctional, floatable, lightweight cement composite (FLCC) with densities lower than that of water (oven-dry density of 750 kg/m


Sonodynamic therapy (SDT) is a minimally invasive anticancer therapy involving a chemical sonosensitizer and high-intensity focused ultrasound (HIFU). SDT enables the reduction of drug dose and HIFU irradiation power compared to those of conventional monotherapies. In our previous study, mouse models of colon and pancreatic cancer were used to confirm the effectiveness of SDT vs. drug-only or HIFU-only therapy. To validate its usefulness, we performed a clinical trial of SDT using an anticancer micelle (NC-6300) and our HIFU system in four pet dogs with spontaneous tumors, including chondrosarcoma, osteosarcoma, hepatocellular cancer, and prostate cancer. The fact that no adverse events were observed, suggests the usefulness of SDT. NC-6300 is an antitumor micelle with a diameter of 60–70 nm that incorporates the drug epirubicin (EPI) using micellar nanoparticle technology  and Tokyo Women's Medical University . Before treatment, informed consent was obtained from the guardians of the animals.Subject recruitment at Tottori University and Tokyo Women's Medical University, were undertaken by staff veterinarians, who proposed SDT to animal guardians at outpatient clinics. Subjects were recruited to the study if the standard treatment  was impossible or undesirable to the animal guardians. Accordingly, four dogs were enrolled in the study and five SDT sessions were implemented, two sessions to subject A, and one to each of subjects B, C, and D. The weight, gender, and age of each subject at recruitment are indicated in As shown in 2 . Accordingly, the results indicated that NC-6300 at 30 mg/m2 was safer than conventional EPI at 30 mg/m2. Using 45 mg/m2 NC-6300, there were no serious adverse events, although recovery of cells, such as leukocytes, from hematotoxicity tended to be delayed relative to that in cases treated with the lower dose. In all cases, transitory changes in systemic flushing and edema were observed immediately after drug administration and disappeared within 1 h. Based on the results of this toxicity test in beagles, the dose per unit area of the drug and concentration of the infusion fluid were assumed as safe for use in this study. The NC-6300 dose in session 1 (7.5 mg/m2) was calculated from the dose (2.5 mg/kg) used in previous experiments with mouse models  in male and female beagles of 2.25 (2.07–2.49) mg/kg and 2.59 (2.42–2.91) mg/kg, respectively , a water bag, a 6-DOF robot , a dedicated robot control unit, and an ultrasound diagnostic system , as shown in The HIFU transducer was an array-type piezo-composite transducer  energized by a radiofrequency amplifier . A function generator  generated the trigger signal to the amplifier and was controlled by software we developed. As illustrated in In preparation for HIFU, the hairs around the irradiated site were shaved to prevent bubble generation, which could cause burn injury. After identifying the exact location of the tumor using a conventional diagnostic probe, the HIFU transducer covered with the water bag was placed in the appropriate position, and ultrasound gel was applied between the body surface and the water bag. The number of HIFU irradiation sequences was dependent on the size of the targeting tumor and the passing rate of the ultrasound. To confirm the safety of SDT, the irradiated region was limited to low-risk irradiation, instead of whole tumor irradiation. In particular, areas with potential appearance of serious adverse events such as large vessels or the area around those and the bones where unexpected ultrasound reflections may occur, were avoided.For hematological examination 10 parameters were assessed, red blood cell count, white blood cell count, hemoglobin, hematocrit, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, platelet count, platelet distribution width, and mean platelet volume. Biochemical parameters included albumin, alanine transaminase, aspartate transaminase, alkaline phosphatase, gamma glutamyl transferase, total bilirubin, total cholesterol, glucose level, blood urea nitrogen, creatinine, Ca, Na, K, and Cl. Blood was collected before NC-6300 administration and on the day of HIFU irradiation. Additional hematological and biochemical examinations were implemented every few days after the therapy.Tumor size was measured using 2D images that were acquired using CT, MRI, US, and X-ray. A veterinarian visually conducted the gait evaluation; common performance evaluation methods had not yet been established, thus we assessed the status of subjects with regards to gait, appetite, excretory function, and blood parameters.SDT was conducted on four pet dogs  with spontaneous tumors ; the age and weight of subjects when SDT was performed are indicated in Five SDT sessions were performed in the four dogs under the treatment conditions indicated in Upon subject A's first visit at 10 years old, an enlargement in the right hip was diagnosed as chondrosarcoma derived from the pelvis. Although therapies such as ablation and cryotherapy were performed previously, SDT was selected as a new approach due to the cancer's recurrence. Following SDT session 1, the tumor size was 48.8 × 32.2 mm 2 weeks before SDT and 47.7 × 29.2 mm 1 week post-SDT. The tumor size was reduced to 85% according to a CT image taken 2 weeks after SDT . In addiSubject B exhibited claudication of the left posterior leg and was diagnosed with osteosarcoma. Following a single SDT session (session 3), the tumor size was 1.46 × 1.42 mm before SDT and 1.29 × 1.36 mm on the 11th day post-SDT. The subject's walking gait was improved 10 days post-SDT , and theSubject C was diagnosed with hepatocellular cancer, and surgery was performed 10 months before SDT. However, recurrence was observed, with tumor growth as shown in Subject D was diagnosed with prostate cancer, and a calcified mass was observed inside the tumor in an X-ray taken 18 days before SDT. The subject also developed metastatic lung cancer. Following SDT (session 5), the tumor size was 26.0 × 21.1 mm 1 day before SDT and 35.1 × 23.3 mm on 3 months post-SDT. The urinary frequency of the subject was improved, and the calcified mass inside the tumor disappeared by 49 days post-SDT, as indicated in The information pertaining to tumor size before and after SDT in all the five sessions is arranged in Our SDT method combines an anticancer micelle (NC-6300) and a novel HIFU system. This treatment is assumed to be both effective and safe, enabling reductions in drug dose and HIFU irradiation power compared with those of conventional single therapies. The effectiveness of the proposed SDT was demonstrated previously in mouse models of colon and pancreatic cancer. In the current study, we evaluated the usefulness of our SDT approach in pet dogs to enable future clinical application. Although HIFU is considered to cause adverse events, such as burn injury, vascular obstruction, hematoma, and aching pain  and Tokyo Women's Medical University . Before treatment, informed consent was obtained from the guardians of the animals.YH contributed to coordination of SDT. MM contributed to management of treatment. YK contributed to HIFU irradiation planning. JO contributed to mechanical management. SI contributed to drug dosage selection. YO contributed to animal care and implementation of HIFU. HIsh contributed to animal care. SY contributed to setup of the HIFU transducer. SU contributed to setup of the HIFU transducer. TU contributed to setup of the HIFU robot. ST contributed to setup of the ultrasonic diagnostic equipment. AS contributed to ultrasound image diagnosis. KT contributed to drug supply and dosage selection. KM contributed advice on electrical safety and the HIFU system. HIse contributed medical advice on canine therapy. NN and KK contributed advice on the SDT mechanism. YM contributed to the generation of the SDT concept using an anticancer micelle (NC-6300) and HIFU, as well as total therapy management.MM was employed by MakeWay Merger Company. TU was employed by DENSO Corporation. ST was employed by Hitachi, Ltd. KT was employed by Kowa Company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study aimed to examine the association of late bedtime and fast eating speed, both individually and interactively, in predicting overweight and obesity in Chinese preschool-aged children. This was a cross-sectional survey among children aged 3–6 years. Overweight and obesity is defined according to the WHO, IOTF, and China criteria, respectively. Total 1123 preschool-aged children were analyzed. After multivariable adjustment, late bedtime after 11:00 pm and fast eating speed increased the risk of overweight and obesity significantly under the WHO , IOTF , and China  criteria. Relative to bedtime before 11:00 pm and eating speed ≥30 min, there was a graded increase with presence of either bedtime after 11:00 pm or eating speed 15-30 min and <15 min. Particularly, the presence of both bedtime after 11:00 and eating speed <15 min yielded the largest OR under the WHO , IOTF , and China  criteria. Taken together, our findings indicate a synergistic interaction between bedtime and eating speed in predicting overweight and obesity in Chinese preschool-aged children. Childhood overweight and obesity has escalated to epidemic proportions worldwide, and it constitutes a major public health problem . Global There is wide recognition that bedtime and dietary speed are two key modifiable components in childhood weight gain –16. TherTo test this hypothesis, we conducted a cross-sectional survey in five kindergartens from Beijing to examine the association of late bedtime after 11:00 pm and fast eating speed, both individually and interactively, in predicting the risk of overweight and obesity in Chinese preschool-aged children.The present analysis involved a total of 1123 qualified preschool-aged children (mean age: 5.12 years old), including 559 boys and 564 girls. The prevalence of overweight and obesity as a whole was 15.12% (overweight: 8.9% and obesity: 7.03%) and 15.58% (overweight: 7.12% and obesity: 8.46%) under the World Health Organization (WHO) and International Obesity Task Force (IOTF) criteria, respectively. Under the China criteria, the prevalence of overweight and obesity was as high as 22.47% (overweight: 11.13% and obesity: 11.34%).The baseline characteristics of study children are summarized in Shown in In view of individual significance for bedtime and eating speed, further explorations on their potential interaction were conducted under three different criteria . Six com2 (50 points), paternal BMI of 28 kg/m2 (37 points), pregnancy BMI of 34 kg/m2 (25 points), birth weight of 4 kg (25 points), sleep duration less than 8 hours (20 points), sleeping after 11:00 pm (20 points), and eating speed <15 min (20 points), the probability of overweight and obesity was estimated to be 63%.Finally, a nomogram model to predict the risk of overweight and obesity was constructed based on significant factors under three different criteria, as displayed in The predictive accuracy of three nomogram models was good, and the C-index was 0.718, 0.688, and 0.663 respectively under the WHO, IOTF, and China criteria .The aim of this cross-sectional study was to examine the association of late bedtime after 11:00 pm and fast eating speed, both individually and interactively, in predicting the risk of overweight and obesity among 1123 Chinese preschool-aged children. The most noteworthy finding of this study is the synergistic interaction between bedtime and eating speed in predicting overweight and obesity under three different growth criteria. To the best of our knowledge, this is thus far the first report that has interrogated the bedtime-eating speed interaction in the medical literature. Our findings highlight the importance of propagating early bedtime and slow eating speed in preschool-aged children to control or prevent the development of overweight and obesity.In the process of childhood development and growth, sleep and eating plays a key role in weight management . AlthougMoreover, our findings also showed the significant association of eating speed with overweight and obesity, in agreement with the results of some previous studies , 29, 30.As an extension of prior studies, we interestingly observed a synergistic interaction between late bedtime and fast eating speed in Chinese preschool-aged children. Specifically, children with late bedtime after 11:00 pm and eating speed less than 15 min were approximately four times more likely to become overweight or obese. There is indirect epidemiological and clinical evidence for the mechanisms behind the interaction between bedtime and eating speed. The superimposition of excessive energy intake can be a possible explanation. On one hand, late bedtime was found to be related to low diet quality . For insWhat’s more, in view of significant predictive capability of bedtime and eating speed, we constructed three predictive nomogram models by incorporating all significant factors under three different growth criteria, which can be used in routine practice to provide more accurate prediction of being overweight or obese in preschool-aged children.The clear strengths of this present study include the identification of a synergistic interaction between bedtime and eating speed, comparison of different growth criteria, and construction of an efficient prediction model. However, our study has some possible limitations. Firstly, the cross-sectional nature of our analyses limits the assumptions of any temporal relationship, which means that our data cannot be used to prove the existence of a cause-effect relationship between bedtime/eating speed and the development of childhood overweight and obesity. Secondly, anthropometric measures were obtained from questionnaires filled by child guardians, which might yield a recall bias. Thirdly, most previous studies defined late bedtime after 9:00 or 9:30 pm, yet in this present study late bedtime is defined after 11:00 pm. It is of added interest to compare two cutoff points for late bedtime. Fourthly, other information such as maternal pre-pregnancy obesity, time interval between dinner and bedtime was not recorded in this survey, and these factors are closely related to bedtime and eating habits, as well as their interaction. Nonetheless, we agree that more well-designed longitudinal studies are needed to confirm or refute the findings of this association study.Despite the above limitations in the present study, our findings indicate a synergistic interaction between bedtime and eating speed in predicting overweight and obesity in Chinese preschool-aged children. This study might help to motivate parents and kindergarten teachers to increase awareness on the detrimental impact of late bedtime and fast eating speed, and the control and prevention of childhood overweight and obesity through effective strategies should be a priority.This was a cross-sectional survey of overweight and obesity among children aged 3 to 6 years in Chaoyang District, Beijing. This study was approved by the Ethics Committee of China-Japan Friendship Hospital, and informed consent was obtained from guardians of all children prior to participation.Using a stratified cluster random sampling strategy, 1333 preschool-aged children from 5 kindergartens of Chaoyang District, Beijing were selected in this study, and a self-designed questionnaire was circulated to every child guardian from March to May in 2017. We collected 1327 questionnaires, and after excluding questionnaires with invalid gender, body height, and weight, there were 1123 validated questionnaires in the final analysis.Questionnaire data included individual data of children and parents, and children dietary and sleep habits. Children individual data contained gender, age (year and month in details), height (to the nearest 0.1 cm), weight (to the nearest 0.1 kg), birth length, and birth weight. Body height and weight of parents and maternal pregnancy were also recorded. Children dietary and sleep habits were asked, including weekly frequency of sweet food, fast food and night meal consumption, eating speed, daily sleep duration and bedtime.Before questionnaire circulation, each health physician from 5 kindergartens received the same training on investigation methods. Questionnaires were handed out and collected by kindergarten teachers, and were further checked by health physicians. Questionnaires were double checked by two authors (J.M. and S.W.), and missing information was complemented via telephone interview.Overweight and obesity is classified by BMI, which is calculated as the ratio of both weight (kg) to the square of body height (m). Some criteria have been widely adopted to define overweight and obesity in preschool-aged children, and we employed the WHO criteria (2006), the IOTF criteria (2000) and the China criteria (2009) simultaneously in this present study.Under the WHO criteria, there are two growth standards before and after 5 years old, using BMI z-scores to define overweight and obesity in our study. Before 5 years old, overweight and obesity are respectively defined as BMI z-scores >2 standard deviation (SD) and BMI z-score >3 SD . After 52 and 30 kg/m2 at age 18 [Under the IOTF criteria, overweight and obesity are respectively defined to pass through BMI of 25 kg/mt age 18 .2 and 28 kg/m2 at age 18 [Under the China criteria, overweight and obesity are respectively defined to pass through BMI of 24 kg/mt age 18 .Dietary data included weekly frequency of sweet food, fast food and night meal intake, and eating speed. Sweet food is defined as food with high sugar , and weekly intake frequency is sorted in every day, that is, often (3 to 4 times), none or once in a while. Fast food is defined as food with high energy and low nutrition (e.g. hamburger and French fries), weekly intake frequency is classified as often (3 to 4 times), and none or once in a while. Night meal is the same as the extra meal in a day, defined as eating some food within 2 hours before bedtime, and weekly intake frequency is consistent with sweet food. Eating speed is divided into fast (<15 min), moderate (15-30 min), and slow (≥30 min).Sleep data contained sleep duration and bedtime. Sleep duration is classified as short (<8 hours), moderate (8-10 hours), and long (≥10 hours). Bedtime is categorized as bedtime before and after 11:00 pm.All preschool-aged children were classified into two groups — the non-overweight group and the overweight and obesity group, according to three growth criteria mentioned above. Categorical and continuous variables were respectively described using count (percent) and median (interquartile range), and they were compared between two groups by using the Chi-squared test, t-test and rank-sum test where appropriate. Logistic regression analyses were adopted to identify significant risk factors associated with overweight or obesity before and after adjusting for age, gender, maternal BMI, paternal BMI, pregnancy BMI, birth length, birth weight, dietary and sleep habits. Effect-size estimates are expressed as OR and 95% CI.To facilitate clinical explanation, a prediction nomogram was created using significant risk factors under three different growth criteria, and predictive accuracy was determined by concordance index (C-index) and defined as the area under the receiver operating characteristics curve.Study power was estimated using the PS-Power Simple Size software (version 3.1.2). The R-language (version 3.5.2) was adopted to generate nomogram. Statistical analyses were conducted using the STATA software special edition . P value less than 0.05 was considered as statistically significant.


Many studies have shown abnormal functional connectivity in children with attention deficit hyperactivity disorder (ADHD) by using resting-state functional magnetic resonance imaging (rs-fMRI). However, few studies illustrated that to what extent these findings were consistent across different datasets. The present study aimed to assess the consistency of abnormal functional connectivity in children with ADHD across the four datasets from a public-assess rs-fMRI ADHD cohort, namely, ADHD-200. We employed the identical analysis process of previous studies and examined a few factors, including connectivity with the seed regions of the bilateral dorsal anterior cingulate cortex, bilateral inferior frontal gyrus, and bilateral middle frontal gyrus; connectivity between default mode network and executive control network; stringent and lenient statistical thresholds; and the ADHD subtypes. Our results revealed a high inconsistency of abnormal seed-based connectivity in children with ADHD across all datasets, even across three datasets from the same research site. This inconsistency could also be observed with a lenient statistical threshold. Besides, each dataset did not show abnormal connectivity between default mode network and executive control network for ADHD, albeit this abnormal connectivity between networks was intensively reported in previous studies. Importantly, the ADHD combined subtype showed greater consistency than did the inattention subtype. These findings provided methodological insights into the studies on spontaneous brain activity of ADHD, and the ADHD subtypes deserve more attention in future studies. Attention deficit hyperactivity disorder (ADHD) is one of the most common neurodevelopmental disorders in children . It is ap < 0.05, cluster size > 10 voxels) (Voxel-wise metrics mainly include amplitude of low-frequency fluctuation (ALFF) , regiona voxels) . These fFunctional connectivity was one another widely used measurement in the rs-fMRI studies on ADHD. Functional connectivity was defined as the correlation between the time course of a particular brain region (seed region) and all other voxels in the brain , and it The seed-based functional connectivity and connectivity between ECN and DMN inform our understanding about the pathological mechanism of ADHD. However, to what extent the functional connectivity results are consistent across individual datasets remains unclear. To address this issue, the present study examined the abnormal functional connectivity based on the datasets of ADHD-200. ADHD-200 is one of the most widely used multi-site MRI cohorts of ADHD, and it contains 10 independent datasets from eight different sites . These dhttp://fcon_1000.projects.nitrc.org/indi/adhd200). The ADHD-200 cohort contains both functional and anatomical MRI data contributed by eight institutions. Each dataset was approved by the research ethics review boards of each institution. Signed informed consent was obtained from all participants or their legal guardian before participation.The data we used in this study are publicly available from the ADHD-200 Consortium  were also excluded because the TR of NeuroImage was not the same as the TR of the other four datasets. The PKU2 and PKU3 datasets only had male subjects, so the female subjects in NYU and PKU1 datasets were excluded to remove potential confounding effect of gender on the results. Left-handed subjects were also excluded from each dataset. There is no significant difference in age between children with ADHD and TDC across all datasets in the present study. After case-by-case age matching between ADHD and TDC, 58 subjects from NYU, 30 from PKU1, 56 from PKU2, and 38 from PKU3 were included in the current study. Demographic information of all subjects is summarized in The datasets were first selected according to the following criteria  and Resting-State fMRI Data Analysis Toolkit (REST) (http://www.restfmri.net). The first 10 time points were removed for signal stabilization and participant adaptation. And then, the number of left time points for NYU, PKU1, PKU2, and PKU3 is 170, 230, 230, and 230, respectively. The unified number of time points (first 170 volumes) was employed for all of the four datasets. Slice timing correction and image realignment to correct head motion were followed. The head motion criteria, that is, head motion <3 mm translation or <3° rotation in any direction, were employed, referring to previous studies (3) and smoothed with an 6 × 6 × 6 full-width-at-half-maximum (FWHM) Gaussian kernel. The head motion parameter measured by Friston-24 model, global signal effect, white matter (WM), and cerebrospinal fluid (CSF) signals were further regressed out as nuisance covariates. The time course of each voxel was linearly detrended and band-pass filtered with frequencies ranging from 0.01 to 0.08 Hz.The preprocessing was carried out using the Data Processing Assistant for Resting-State fMRI (DPARSF) , which it (REST)  . Moreover, asymmetry of the human brain was mentioned intensively . The mean time course of each seed region was extracted as reference time course. Then, the functional connectivity was calculated as Fisher’s Z-transformed Pearson correlation coefficient between the reference time course and the time course of each voxel in the brain.The functional connectivity analyses were performed based on the seed regions of interest (ROIs), including R_dACC, R_IFG, and R_MFG. The three seed regions were defined as a sphere with a 6-mm radius (34 voxels) centered at the coordinates reported by Weissman et al.  converteensively , 36, so p < 0.001, Gaussian random field (GRF) corrected). Then, the mean time courses were extracted based on the ECN and DMN masks in each subject. These masks were unified for the subjects of all the datasets, and therefore, there was no network difference across the datasets. The network connectivity was further calculated through the Pearson correlation between the mean time course of DMN and the mean time course of ECN.A public DMN mask created by Yeo et al.  was emplp < 0.001, GRF corrected), and the networks were constructed for each dataset. Functional connectivity with the seed region of PCC was calculated [6-mm spherical ROI centered at MNI coordinates: x = 1, y = −55, z = 17, reported by Vincent et al. .Functional connectivity maps of the seed regions of R_dACC, R_IFG, R_MFG, L_dACC, L_IFG, and L_MFG were compared between the groups of children with ADHD and TDC. Two-sample p < 0.05, cluster size > 10 voxels) was also used for each dataset.At the same time, to reduce the possibility of false-negative results, a more lenient threshold  based on Cohen’s equation :t-test, as follows,According to equation of independent two-sample d and t-value can be obtained, as follows:The relationship of Cohen’s  t maps into SES map for each dataset. The same threshold was applied to the SES maps of each dataset. A high level SES of 0.8 was used, which corresponded to t = 3.08, 2.16, 2.96, and 2.50  for NYU, PKU1, PKU2, and PKU3, respectively.According to Equation (3), we transformedt maps and SES maps were binarized and overlapped among the four datasets. The number of overlapped voxels across four and three datasets was quantified using the Dice overlap coefficient , PKU1, PKU2, and PKU3 used the ADHD Rating Scale (ADHD-RS) IV to determine the ADHD subtypes, and NYU used Conners’ Parent Rating Scale-Revised, long version (CPRS-LV), to identify the ADHD subtypes. There were 19, 6, 12, and 7 participants with the ADHD combined subtype, and 10, 9, 16, and 12 participants with the ADHD inattention subtype in NYU, PKU1, PKU2, and PKU3, respectively. Each subtype matched the health control with age. All the above analysis procedures were repeated based on the data of each ADHD subtype.In the current study, there were two ADHD subtypes in all of the four datasets, that is, the ADHD combined subtype and the ADHD inattention subtype. The information on subtypes was provided by ADHD-200 directly. According to the diagnostics illustration provided by ADHD-200  = 2.69, p = 0.01  The abnormal seed-based functional connectivity of ADHD was not consistent across datasets. (2) For each dataset, the negative connectivity between ECN and DMN did not show any significant difference between ADHD and TDC. (3) In a subtype analysis, the combined subtype showed more consistent results across datasets as compared with the inattention subtype.p < 0.05, cluster size > 10 voxels). Notably, the inconsistency could also be observed in three datasets from the same research site, PKU. Furthermore, in each dataset, the SES of the abnormal connectivity was small, and no overlaps of the SES maps were observed from more than three datasets. These findings suggest high inconsistency of abnormal functional connectivity in children with ADHD, and such inconsistency may be related to the heterogeneity of ADHD , the National Natural Sciences Foundation of China , the National Basic Research Program of China (973 program 2014CB846104), and Zhejiang Fundamental Public Welfare Research Program (LGJ19C090001). HZ is supported by Scientific Research Staring Foundation from Hangzhou Normal University.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The expression of FOLR1, DHFR, and MTRR was evaluated in 80 cases of primary OC, 50 cases of benign ovarian tumors, and 30 normal ovarian tissues. Associations between protein expression and clinicopathological characters were assessed, and diagnostic and prognostic evaluation of FOLR1, DHFR, and MTRR was performed. Results showed that upregulated FOLR1 and MTRR and downregulated DHFR were detected in OC. Patients with abnormality of FOLR1, DHFR, and MTRR tend to have a higher percentage of platinum resistance. Moreover, the areas under receiver operating characteristic curves (AUCs-ROC) for FOLR1, DHFR, and MTRR were 0.723, 0.717, and 0.714, respectively. The combination of FOLR1, DHFR, and MTRR could produce an area of 0.864 under the receiver-operating characteristic curve in distinguishing platinum-resistant patients from platinum-sensitive patients (P < 0.0001). Correlations were present between the expression of FOLR1, DHFR, and MTRR. Furthermore, Kaplan-Meier curves indicated that the patients with overexpressed MTRR had a poorer overall survival time compared to those with low expression (P < 0.05). Thus, folate metabolic enzymes could provide a potential promising biomarker for diagnosis platinum-resistant in OC.This study was to validate changes in the levels of folate receptor- Ovarian cancer is the fourth most common cancer in women worldwide, and it has the most noteworthy lethal rate around gynecologic malignancies. Two most critical barriers to treatment of ovarian malignancy are absence of early diagnostic markers and advancement of drug resistance after therapy, especially in advanced stages. Various epigenetic changes have been recognized in ovarian cancer. Recent progresses in our understanding of molecular pathogenesis of ovarian malignancy have dramatically provided potential new targets for molecularly targeted therapies. There thus is a critical need for improved biological markers and therapies for ovarian carcinoma, which will come from a better understanding of the biology of the disease.Folate is an essential component in DNA synthesis, replication and repair, protein synthesis, and methylation reactions. This is especially true for rapidly dividing cells . Folate In the previous research in our lab, ovarian cancer-resistant cell lines were established to screening drug-resistant genes . Most ofThe aim of this study was to validate changes in the levels of FOLR, DHFR, and MTRR in the tissue of OC patients. We sought to determine whether folate metabolism enzymes could serve as a novel biomarker for early diagnosis and prognosis of platinum-resistant OC patients, as well as their clinical significance in OC.OC tissues and normal ovary tissues were collected from patients who were treated in the Department of Gynecologic Oncology of the Affiliated Tumor Hospital of Guangxi Medical University between 2004 and 2010. All the patients were pathologically diagnosed with OC. Pathological stage and histological subtype were determined according to the International Federation of Gynecology and Obstetrics (FIGO) criteria and the World Health Organization criteria. Clinical and pathological data was collected from the medical records including age, surgical stage, metastasis, ascites, tumor grade and subtype, and drug resistance. Samples were collected from 80 cases of primary OC, 50 cases of benign ovarian tumors, and 30 normal ovarian tissues. The median age was 41.1 years (range: 13-76 years) in the OC group, 40.1 years (range: 10-74 years) in the benign ovarian tumor group, and 43.1 years (range: 29-60 years) in the normal ovary group. The 80 OC patients underwent surgical intervention for OC of whom 61 patients with epithelial ovarian cancer received chemotherapy with cisplatin plus paclitaxel and 19 patients with nonepithelial ovarian cancer were treated with cisplatin, bleomycin, and vincristine. The study was approved by the Ethics Committee of Guangxi Medical University. Written informed consent was obtained from all the subjects before study.w/v) BSA in PBST  and then incubated with primary antibodies MTRR , FOLR1 , and DHFR  overnight at 4°C. These PVDF membranes were subsequently treated with PBST and incubated with peroxidase-conjugated secondary antibody (1 : 1000) (Santa Cruz Biotechnology) for 1 h. Visualization was detected by using a chemiluminescence system  according to the manufacturer's instructions. The band intensities were quantified using the ImageQuant software . Then, the membranes were stripped and reincubated with anti-GAPDH  for normalization.160 fresh specimens were sonicated with an ultrasonic tissue disrupter in lysis buffer for 30 min. The tissue debris was pelleted by centrifugation, and supernatants were collected. After measuring the protein concentration by BCA protein assay, proteins were subjected to SDS-PAGE and then transferred onto PVDF membranes. After blocking, the membranes was treated with 5% (μm) of ovarian tissues were obtained, deparaffinized, and rehydrated through a graded ethanol series. Antigen retrieval was done in 10 mM citrate buffer (pH 6.0) at 120°C for 2 min. The sections were allowed to cool to 30°C and washed with phosphate-buffered saline . After inactivating the endogenous peroxidase with 3% H2O2 for 10 min and washing with PBS, sections were incubated at 4°C overnight with primary antibodies in PBS and then washed with PBS. The primary antibodies used were polyclonal MTRR antibody , polyclonal FOLR1 antibody , and polyclonal DHFR antibody . Sections were stained with an ultrasensitive streptavidin-peroxidase kit , and visualization was performed with 3,3′-diaminobenzidine (DAB). Nuclei were stained with Harris Hematoxylin . In negative control, the primary antibody was replaced with PBS. A colon cancer sample was used as a positive control. Positive cells had brown granules in the cytoplasm. The positive cancer cells were semiquantitatively determined based on the staining intensity and percentage of positive cells. Sections were scored based on the chromatic intensity: 0, no pigmentation; 1, light yellow; 2, buff; and 3, brown. Five fields were randomly selected from each section, and the mean percentage of positive cells was determined: 0, <5%; 1, 5%-25%; 2, 26%-50%; 3, 51%-75%; and 4, d > 75%. The immunohistochemical scores were multiplied by the intensity score and percentage of positive cells: 0-2 (-); 3-4 (+); 5-8 (++); and 9-12 (+++). The sections were independently assessed by two observers.Paraffin-embedded sections  curves were established to evaluate the diagnostic value of FOLR1, MTRR, and DHFR for differentiating benign and malignant, and the cutoff values were also calculated. According to the cutoff value of the ROC curve, we defined the result of protein below the cutoff value as low expression and above the cutoff value as high expression. OS curves were plotted by the Kaplan-Meier method and compared by log-rank test. The assessment of correlation between survival time and multiple clinicopathological variables was carried out by the Cox proportional hazards regression model. Univariate and multivariate Cox proportional hazard models were used to identify variables associated with OS in the group of OC patients. When the significant variables associated with OS were obtained by univariate analysis, multivariate analysis was used for evaluating which variables were the most important in prediction of OS. A P value less than 0.05 was considered as statistically significant. All of the statistical calculations were performed using the SPSS software , and GraphPad Prism 5.0  was used to generate graphs. All of the P values <0.05 were considered to be statistically significant.Nominal variables were compared using the P < 0.05, respectively).FOLR, DHFR, and MTRR expression in tissues of 160 patients was assessed by Western blot analysis . QuantitIn order to detect the location, FOLR, DHFR, and MTRR expression in clinical samples was assessed by immunohistochemical staining of sections isolated from 10 OC, 10 patients with benign tumors OC, and 10 patients with normal ovaries. Representative examples of staining are shown in P < 0.05, respectively).The correlations of the expression of FOLR, DHFR, of MTRR with various clinical variables are listed in P < 0.001; sensitivity = 88.20%, specificity = 55.20%), 0.717 , and 0.714 , respectively  values for them were 0.723 . Correlation was also found between positive FOLR1 and positive DHFR (P < 0.05).FOLR, DHFR, and MTRR were located and coexpressed in the cytoplasm of the OC tissue . The corP > 0.05). However, FIGO Stage, grade, ascites, platinum resistance, and high expression of MTRR were significant predictive factors for the prognosis of OC patients .The results of univariate and multivariate analysis are presented in P > 0.05). However, MTRR was more sensitive for predicting prognosis in subgroups of OC patients  and high (n = 17) levels of MTRR was 40 and 19 months, respectively.To establish survival curves, continuous expression levels of FOLR1, DHFR, and MTRR were converted to a dichotomous variable, using their cutoff values from ROC curve analyses as a threshold, respectively. The Kaplan-Meier method was employed to analyze the OS times of 80 OC patients between high expression and low expression of FOLR, DHFR, and MTRR. As shown in Figures The folate metabolic pathway comprises a cycle mediated by FOLR1, DHFR, and MTRR . AberranFOLR1 overexpresses in a wide range of epithelial malignant cancers –14. SomeInterfering with the expression of DHFR is an approach in improving pharmacokinetics and reversing drug resistance in OC , 20. In MTRR (rs1801394) has been linked to many cancers –28. But Our results showed that the combined ROC analysis revealed an AUC value of 0.832 in discriminating platinum-resistant patients from platinum-sensitive patients. This analysis provided information for an effective combined prognostic approach for drug resistance. To our knowledge, this is the first report showing the relationship between platinum resistance and expressions of the three folate metabolism enzymes in OC.The limitations of this study need to be presented. First is the lack of other types of histology disease and a small sample size of OC patients, which could lead to the lack of power and the consequent imprecision. Second, although the biological functions of the three folate metabolism enzymes have been inferred by previous gene functional analysis, the mechanisms behind the predictive values of these three folate metabolism enzymes in OC are still not clear, and their functional roles should be further explored in experimental studies.In summary, the evaluation of FOLR1, DHFR, and MTRR expression may provide useful information for doctors to make optimal clinical decisions. MTRR is a better factor for determining the prognosis of OC. Combining the three enzymes diagnosis might contribute to the identification of patients who are likely to develop chemotherapy resistance and may be a novel potential target for OC therapy, which will require analysis by further validation studies.