Characterization of ovarian progenitor cells for their potential to generate steroidogenic theca cells in vitro.

In brief: Progenitor cells with ovulation-related tissue repair activity were identified with defined markers (LGR5, EPCR, LY6A, and PDGFRA), but their potentials to form steroidogenic cells were not known. This study shows that the cells can generate progenies with different steroidogenic activities. Abstract: Adult mammalian ovaries contain stem/progenitor cells necessary for folliculogenesis and ovulation-related tissue rupture repair. Theca cells are recruited and developed from progenitors during the folliculogenesis. Theca cell progenitors were not well defined. The aim of current study is to compare the potentials of four ovarian progenitors with defined markers (LY6A, EPCR, LGR5, and PDGFRA) to form steroidogenic theca cells in vitro. The location of the progenitors with defined makers was determined by immunohistochemistry and immunofluorescence staining of ovarian sections of adult mice. Different progenitor populations were purified by magnetic-activated cell sorting (MACS) and/or fluorescence-activated cell sorting (FACS) techniques from ovarian cell preparation and were tested for their abilities to generate steroidogenic theca cells in vitro. The cells were differentiated with a medium containing LH, ITS, and DHH agonist for 12 days. The results showed that EPCR+ and LGR5+ cells primarily distributed along the ovarian surface epithelium (OSE), while LY6A+ cells distributed in both the OSE and parenchyma. However, PDGFRA+ cells were exclusively located in interstitial compartment. When the progenitors were purified by these markers and differentiated in vitro, LY6A+ and PDGFRA+ cells formed steroidogenic cells expressing both CYP11A1 and CYP17A1 and primarily producing androgens, showing characteristics of theca-like cells, while LGR5+ cells generated steroidogenic cells devoid of CYP17A1 expression and androgen production, showing a characteristic of progesterone-producing cells (granulosa- or lutea-like cells). In conclusion, progenitors from both OSE and parenchyma of adult mice are capable of generating steroidogenic cells with different steroidogenic capacities, showing a possible lineage preference.

Pubertal lead exposure affects ovary development, folliculogenesis and steroidogenesis by activation of IRE1α-JNK signaling pathway in rat.

Epidemic studies showed that lead exposures are associated with various female reproductive dysfunctions, including infertility, miscarriage, preterm delivery, and early menopause. However, the mechanism involved is still unclear. In the current study, SD rats were exposed to lead at doses of 0, 5, 25, 50 or 250 mg/L through drinking water from postnatal day 21-56. Lead exposures did not affect the body weight or ovary weight. However, the puberty initiation (ages by which vagina opens and estrous cycle occurs) was significantly delayed by as many as 5.8 and 6.8 days respectively (P < 0.05). Also, lead exposures disrupted the estrous cycles, reduced the numbers of primordial and primary follicles and increased the number of atretic follicles by adult. Furthermore, for the highest does group, serum levels of progesterone and testosterone decreased by 80.2% (P < 0.01) and 49.9% (P < 0.05) respectively, while estradiol level increased by 69.8% (P < 0.01). Western blot analyses indicated that lead exposures specifically down-regulated the expressions of steroidogenic protein STAR, CYP17A1, and HSD3B1, while up-regulated FSHR and CYP19A1. Also, the exposure stimulated the endoplasmic reticulum stress (ERS)-related IRE1α-JNK signaling pathway members. Such activation may also result in apoptosis since the death-signaling molecules CHOP and cleaved-CASP3 were up-regulated while BCL2 was down-regulated. In conclusion, lead exposure during juvenile and puberty significantly affected ovary development and functions. The effects may relate to ERS response since the 6 members related to the pathway were all consistently activated.

Three-dimensional analysis of the relationship between mandibular retromolar space and positional traits of third molars in non-hyperdivergent adults.

BACKGROUND: The anatomical position of the mandibular third molars (M3s) is located in the distal-most portions of the molar area. In some previous literature, researchers evaluated the relationship between retromolar space (RS) and different classifications of M3 in three­dimensional (3D) cone-beam computed tomography (CBCT). METHODS: Two hundred six M3s from 103 patients were included. M3s were grouped according to four classification criteria: PG-A/B/C, PG-I/II/III, mesiodistal angle and buccolingual angle. 3D hard tissue models were reconstructed by CBCT digital imaging. RS was measured respectively by utilizing the fitting WALA ridge plane (WP) which was fitted by the least square method and the occlusal plane (OP) as reference planes. SPSS (version 26) was used to analyze the data. RESULTS: In all criteria evaluated, RS decreased steadily from the crown to the root (P < 0.05), the minimum was at the root tip. From PG-A classification, PG-B classification to PG-C classification and from PG-I classification, PG-II classification to PG-III classification, RS both appeared a diminishing tendency (P < 0.05). As the degree of mesial tilt decreased, RS appeared an increasing trend (P < 0.05). RS in classification criteria of buccolingual angle had no statistical difference (P > 0.05). CONCLUSIONS: RS was associated with positional classifications of the M3. In the clinic, RS can be evaluated by watching the Pell&Gregory classification and mesial angle of M3.

Pyogenic liver abscess related to intrahepatic bile duct stones: Difficulties in infectious control and diagnosis of concomitant cholangiocarcinoma.

BACKGROUND AND AIM: Cholangitis, bacteremia, and pyogenic liver abscess (PLA) can be often caused by intrahepatic bile ducts stone (IBDS), which is endemic to South-East Asia. The association between IBDS and cholangiocarcinoma has been well recognized. Concomitant cholangiocarcinoma in the PLA related to IBDS is often missed. METHODS: A case-control study consisting of 64 patients with PLA related to IBDS and 256 control patients with PLA not related to IBDS was used to investigate clinical features of PLA and incidence of concomitant cholangiocarcinoma in patients with PLA related to IBDS. RESULTS: The main imaging manifestations of PLA related to IBDS was cystic-solid lesions and solid lesions. Of seven patients (10.9%) with pathology-proven cholangiocarcinoma in the same area of PLA related to IBDS among 64 patients, only two patients were initially diagnosed as having concomitant cholangiocarcinoma by biopsy, and other five patients diagnosed as acute inflammatory lesion. Within 60 days after onset, the infection-related death rate and recurrence rate in patients with PLA related to IBDS were 12.9% and 20.3%, respectively, whereas in patients with PLA not related to IBDS were 3.9% and 3.1%, respectively. Only 25% of patients with PLA related to IBDS underwent surgery after admission. The main pathogens in PLA patients related to IBDS were Escherichia coli and extended-spectrum beta-lactamase-producing Enterobacteriaceae. CONCLUSIONS: The imaging manifestations of PLA related to IBDS often present cystic-solid or solid lesions. PLA related to IBDS is characterized by high rate of recurrence and infection-related death, difficulty in diagnosis of concomitant cholangicarcinoma.

Detection of exogenous gene doping of IGF-I by a real-time quantitative PCR assay.

Gene doping can be easily concealed since its product is similar to endogenous protein, making its effective detection very challenging. In this study, we selected insulin-like growth factor I (IGF-I) exogenous gene for gene doping detection. First, the synthetic IGF-I gene was subcloned to recombinant adeno-associated virus (rAAV) plasmid to produce recombinant rAAV2/IGF-I-GFP vectors. Second, in an animal model, rAAV2/IGF-I-GFP vectors were injected into the thigh muscle tissue of mice, and then muscle and blood specimens were sampled at different time points for total DNA isolation. Finally, real-time quantitative PCR was employed to detect the exogenous gene doping of IGF-I. In view of the characteristics of endogenous IGF-I gene sequences, a TaqMan probe was designed at the junction of exons 2 and 3 of IGF-I gene to distinguish it from the exogenous IGF-I gene. In addition, an internal reference control plasmid and its probe were used in PCR to rule out false-positive results through comparison of their threshold cycle (Ct) values. Thus, an accurate exogenous IGF-I gene detection approach was developed in this study.

[Treatment of Infective Ulcer Patients with Yang Syndrome by External Use of Shengji Ointment: a Multicenter Randomized Controlled Trial].

OBJECTIVE: To evaluate the safety and effectiveness of Shengji Ointment (SO) (by external use) in treating infective ulcer patients with yang syndrome (YS). METHODS: A multicenter, stratified, randomized controlled clinical study was used in this study. A random digit table was formed in the ratio of 3:1,440 infective ulcer patients of YS from 5 centers were recruited, 330 in the treatment group (external use of SO, exchange once every 24 h) and 110 in the control group (external use of Votalin, exchange once every 24 h). The therapeutic course consisted of 21 days. The ulcer area, wound depth, pain degree were observed before treatment at day 3, 7, 14, and 21, and safety indices monitored at the same time. RESULTS: The ulcer area was significantly reduced in the treatment group after 21-day medication, obviously superior to that of the control group (P = 0.002). Ulcer areas at different time points were analyzed. From the 7th day of medication the reduced area was obviously less in the treatment than in the control group (P = 0.002). Besides, the longer the more obvious difference. Compared with the control group, the healing rate of ulcer area, the pain disappearance rate, and the healing rate of ulcer depth were all obviously improved in the treatment group (all P < 0.01). No adverse event occurred in the two groups. CONCLUSIONS: External use of SO was safe and effective in treating infective ulcer patients with YS. It could accelerate wound healing with better analgesic effect.

Ultralow-cost piezoelectric sensor constructed by thermal compression bonding for long-term biomechanical signal monitoring in chronic mental disorders.

Wearable bioelectronic devices, which circumvent issues related to the large size and high cost of clinical equipment, have emerged as powerful tools for the auxiliary diagnosis and long-term monitoring of chronic psychiatric diseases. Current devices often integrate multiple intricate and expensive devices to ensure accurate diagnosis. However, their high cost and complexity hinder widespread clinical application and long-term user compliance. Herein, we developed an ultralow-cost poly(vinylidene fluoride)/zinc oxide nanofiber film-based piezoelectric sensor in a thermal compression bonding process. Our piezoelectric sensor exhibits remarkable sensitivity (13.4 mV N-1), rapid response (8 ms), and exceptional stability over 2000 compression/release cycles, all at a negligibly low fabrication cost. We demonstrate that pulse wave, blink, and speech signals can be acquired by the sensor, proposing a single biomechanical modality to monitor multiple physiological traits associated with bipolar disorder. This ultralow-cost and mass-producible piezoelectric sensor paves the way for extensive long-term monitoring and immediate feedback for bipolar disorder management.

A novel molecular subtyping based on multi-omics analysis for prognosis predicting in colorectal melanoma: A 16-year prospective multicentric study.

Colorectal melanoma (CRM) is a rare malignant tumor with severe complications, and there is currently a lack of systematic research. We conducted a study that combined proteomics and mutation data of CRM from a cohort of three centers over a 16-years period (2005-2021). The patients were divided into a training set consisting of two centers and a testing set comprising the other center. Unsupervised clustering was conducted on the training set to form two molecular subtypes for clinical characterization and functional analysis. The testing set was used to validate the survival differences between the two subtypes. The comprehensive analysis identified two subtypes of CRM: immune exhausted C1 cluster and DNA repair C2 cluster. The former subtype exhibited characteristics of metabolic disturbance, immune suppression, and poor prognosis, along with APC mutations. A machine learning algorithm named Support Vector Machine (SVM) was applied to predict the classification of CRM patients based on protein expression in the external testing cohort. Two subtypes of primary CRM with clinical and proteomic characteristics provides a reference for subsequent diagnosis and treatments.

In-Memory Tactile Sensor with Tunable Steep-Slope Region for Low-Artifact and Real-Time Perception of Mechanical Signals.

A tactile sensor needs to perceive static pressures and dynamic forces in real-time with high accuracy for early diagnosis of diseases and development of intelligent medical prosthetics. However, biomechanical and external mechanical signals are always aliased (including variable physiological and pathological events and motion artifacts), bringing great challenges to precise identification of the signals of interest (SOI). Although the existing signal segmentation methods can extract SOI and remove artifacts by blind source separation and/or additional filters, they may restrict the recognizable patterns of the device, and even cause signal distortion. Herein, an in-memory tactile sensor (IMT) with a dynamically adjustable steep-slope region (SSR) and nanocavity-induced nonvolatility (retention time >1000 s) is proposed on the basis of a machano-gated transistor, which directly transduces the tactile stimuli to various dope states of the channel. The programmable SSR endows the sensor with a critical window of responsiveness, realizing the perception of signals on demand. Owing to the nonvolatility of the sensor, the mapping of mechanical cues with high spatiotemporal accuracy and associative learning between two physical inputs are realized, contributing to the accurate assessment of the tissue health status and ultralow-power (about 25.1 µW) identification of an occasionally occurring tremor.

Computational investigation of a 3D-printed skin substitute with orthotropy in mechanical property.

As a promising treatment for third-degree burns, grafting with bioengineering skin substitutes shows a capability to overcome the deficiency of donor skin. Similar mechanical properties with human skin are required for employed skin substitutes to avoid secondary damage to patients. Given the representativeness of orthotropy in mechanical properties, there is a need for developing orthotropic skin substitutes. This paper presents computational investigation as well as structural design for the fabrication of orthotropic skin substitutes. A finite element method (FEM) based mechanics simulation model for analyzing the stress field in the skin substitute was developed, by which the stress distribution in mimetic structures of the epidermis and dermis can be acquired. Moreover, the equation of Young's modulus was deduced based on the simulation result, which expressed the mechanical property of designed skin substitutes. Furthermore, several structures of skin substitutes were proposed and their calculated Young's modulus ranged from 21.87 kPa to 213.32 kPa, which was similar to the human skin. Ultimately, uniaxial tensile tests were performed for three types of 3D-printed orthotropic skin substitutes, which validates the feasibility to regulate Young's modulus by regulating the structure of fabricated skin substitutes.

Involvement of guanylate cyclase and K+ channels in relaxation evoked by ferulate nitrate in rat aorta artery.

Vasorelaxant properties of N-2-(ferulamidoethyl)-nitrate (ferulate nitrate, FLNT), a newly synthesized nitrate, were compared with those of isosorbide dinitrate, nicorandil, nitroglycerin, and 8-bromoguanosine 3,5-cyclic monophosphate (8-Br-cGMP) in rat aorta pre-contracted by phenylephrine. FLNT produced vasorelaxation in a concentration-dependent manner (0.1 - 100 µM). The degree of relaxation induced by FLNT was similar to that induced by isosorbide dinitrate. In addition, removal of endothelium did not affect the relaxant effect of FLNT. FLNT caused a rightward shift of the cumulative concentration-response curves of phenylephrine and reduced the maximal efficacy of contraction. 1H-[1,2,4]Oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 10 µM) and K(+)-channel blockers charybdotoxin (CHT, 0.1 µM) and BaCl(2) (1 µM) reduced the relaxant effect of FLNT in the endothelium-denuded arteries, whereas glibenclamide (1 µM) and 4-aminopyridine (1 mM) failed to influence FLNT-induced vasorelaxation. Furthermore, in the presence of ODQ, both CHT (0.1 µM) and BaCl(2) (1 µM) still significantly reduced the relaxation evoked by FLNT. Pretreatment of vessels with hydroxocobalamin, a nitric oxide scavenger, abolished the FLNT effect. These findings demonstrate that FLNT induces relaxation of the rat aorta rings endothelium-independently. Furthermore, we demonstrated that FLNT-induced vasorelaxation is related to its stimulation of soluble guanylate cyclase and activation of K(+) channels.

A Distinct Tobamovirus Associated With Trichosanthes kirilowii Mottle Mosaic Disease.

Trichosanthes kirilowii is one of the most important perennial herbaceous vines that have been used in traditional Chinese medicine. In this study, a novel RNA virus was discovered in T. kirilowii plants showing leaf mottling and mosaic symptoms. The complete genome of this virus is 6,524 nucleotides long and encodes four open reading frames which are arranged in a manner typical of tobamoviruses. Phylogenetic analysis based on the complete genome sequence revealed that the virus was clustered into a branch with the tobamoviruses whose natural host are plants belonging to the family Cucurbitaceae. A full-length infectious cDNA clone was then constructed and demonstrated to establish a systemic infection with typical symptoms in Nicotiana benthamiana, T. kirilowii, and five other cucurbitaceous crops including Cucumis melo, C. lanatus, C. sativus, Luffa aegyptiaca, and Cucurbita pepo via agrobacterium-mediated infectivity assays. Further experiments provided evidence that the rod-shaped viral particles derived from the infectious clone could be mechanically transmitted and reproduce indistinguishable symptoms in the tested plants. Taken together, the mottle mosaic disease of T. kirilowii is caused by a distinct tobamovirus, for which the name Trichosanthes mottle mosaic virus (TrMMV) is proposed. As the infectious cDNA clone of TrMMV could also infect five other cucurbit crops, this distinct tobamovirus could be a potential threat to other cucurbitaceous crops.

Three-Dimensional Analysis of Alveolar Bone Morphological Characteristics in Skeletal Class II Open Bite Malocclusion: A Cone-Beam Computed Tomography Study.

This cross-sectional research evaluated the morphological characteristics of alveolar bone in skeletal class II open-bite individuals compared to skeletal class II and class I non-open-bite individuals. A sample of 82 patients (all were in cervical vertebral stage 6) was divided into three groups (class II OB group, n = 29; class II NOB group, n = 29; class I NOB group, n = 24) according to bilateral molar relationship, ANB angle, and anterior overbite measured by cephalometric analysis. The evaluation was performed with specialized software (Mimics 21.0) and initial cone-beam computed tomography (CBCT) data. The alveolar bone height and thickness of the whole dentition area and alveolar crest level and root apex position of the incisors were measured with a series of appropriate CBCT images. One-way analysis of variance followed by the Tukey post hoc test and the Kruskall−Wallis test were performed for statistical comparisons. The class II open-bite group had increased alveolar bone height for the maxillary first molar and decreased alveolar bone height for mandibular molars compared to the class II non-open-bite group (p < 0.05 for both). Furthermore, there were significant negative correlations between the alveolar bone heights of the upper first and second molars (A6-height, A7-height) and overbite (both p < 0.01). The alveolar thicknesses of all measured teeth were generally reduced in the class II OB group.

Impact of 25-Hydroxy Vitamin D on White Matter Hyperintensity in Elderly Patients: A Systematic Review and Meta-Analysis.

Some studies show that low serum vitamin D levels are associated with white matter hyperintensity (WMH), while other studies report no association. This meta-analysis aimed to investigate the presence of an association between serum 25-hydroxy vitamin D [25(OH)D] levels and WMH. PubMed, Embase, the Cochrane Library, CNKI, WANFANG, and VIP were searched for available papers published up to December 2020. The outcomes were the odds ratios (ORs) with 95% confidence intervals (CIs) for the association between different vitamin D statuses and WMH. All meta-analyses were performed using a random-effects model. Five studies (4393 patients) were included. Compared with sufficient 25(OH)D levels, 25(OH)D deficiency was not associated with WMH (OR = 1.67, 95%CI: 0.92-3.04; I2 = 70.2%, P heterogeneity = 0.009), nor was 25(OH)D insufficiency (OR = 1.21, 95%CI: 0.89-1.65; I2 = 48.1%, P heterogeneity = 0.103). A decrease of 25 nmol/L in 25(OH)D levels was associated with WMH (OR = 1.83, 95%CI: 1.34-2.49; I2 = 0%, P heterogeneity= 0.512). The sensitivity analyses showed that the results were robust. 25(OH)D deficiency and insufficiency are not associated with WMH. A decrease of 25 nmol/L in 25(OH)D levels was associated with WMH, but this result will have to be confirmed. Prospective trials, both cross-sectional and longitudinal, are necessary to examine the association between 25(OH)D levels and WMH.

[MRI scan of striatum volume in healthy adults].

OBJECTIVE: To examine the age, sex, and hemispheric differences in volume of the striatum by MRI in healthy adults. METHODS: The volumes of the bilateral caudate nucleus and putamen were measured on MR images in 100 healthy right-handed adults (18-70 y). RESULTS: The volume of bilateral caudate nucleus and putamen in healthy adults was (8.42 +/-0.88) cm(3) and (8.90 +/-0.89) cm(3), which were decreased with aging (for caudate nucleus r=-0.727, P<0.001; for putamen r=-0.709, P<0.001). The average annual shrinkage rate was 0.52 % in the caudate nucleus and 0.50 % in the putamen. There were no gender differences in the volume of the striatum, however, the age-related shrinkage of the striatum was more evident in men than that in women. The volume of the left caudate nucleus (t=4.43, P<0.001) and the putamen (t=4.88, P<0.001) was greater than that of its right counterpart. CONCLUSION: Bilateral age-related shrinkage of the striatum is found in healthy adults, which is more evident in men than that in women. In both sexes, significant leftward asymmetry in volume of the caudate nucleus and the putamen is found.

Imaging features, clinicopathological analysis and diagnostic strategy of IgG4-related hypertrophic pachymeningitis.

BACKGROUND: This study was conducted to summarize the clinical, magnetic resonance imaging (MRI) and pathological features of IgG4-realated hypertrophic pachymeningitis (IgG4-RHP) and its differential diagnosis from similar diseases. METHODS: Data of IgG4-RHP patients admitted to Department of Neurology, Neurosurgery and Infection, the First Affiliated Hospital of Medical School of Zhejiang University from January 1, 2015 to July 31, 2019 were collected and their clinical symptoms, laboratory examinations, imaging and pathological features were investigated. At the same time, the clinicopathological and imaging findings of other dura thickening diseases diagnosed in our hospital were compared and analyzed. RESULTS: The clinical symptoms of 4 IgG4-RHP patients include chronic headache and cranial nerves injury, etc. Levels of serum IgG4 and cerebrospinal fluid (CSF) IgG4 increased in all patients. Focal enhancement of dura mater could be seen on plain and enhanced cranial MRI. Pathological results were consistent with IgG4-RHP symptoms. Among other diseases that cause dural thickening, the content of serum C-reactive protein in patients with Rosai-Dorfman disease declined. Patients with intracranial hypotension syndrome often have postural headache. Patients with tuberculous meningitis can have previous pulmonary tuberculosis. The diagnosis of patients with atypical meningioma depends on the results of operation and pathology. Patients with central nervous system leukemia can be diagnosed with reference to the results of laboratory results. CONCLUSIONS: The clinicopathological and imaging manifestations of IgG4-RHP are summarized in this study. Meanwhile, the clinical data of several other diseases with similar imaging characteristics are analyzed in order to clarify the diagnostic strategy of IgG4-RHP and provide help for the next treatment.

Sanggenon C Ameliorates Cerebral Ischemia-Reperfusion Injury by Inhibiting Inflammation and Oxidative Stress through Regulating RhoA-ROCK Signaling.

Sanggenon C (SC), a natural flavonoid extracted from Cortex Mori (Sang Bai Pi), is reported to possess anti-inflammatory and antioxidant properties in hypoxia. The present study aimed to investigate the therapeutic potential and the underlying mechanisms of SC in cerebral ischemia-reperfusion (I/R) injury. A rat model of reversible middle cerebral artery occlusion (MCAO) was used to induce cerebral I/R injury in vivo, and SC was administrated intragastrically. Brain injuries were evaluated using Bederson scores, brain water content, and 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. The levels of inflammatory factors and oxidative stress were examined using corresponding kits. Cell apoptosis was evaluated by TUNEL. Moreover, the expressions of apoptosis-related and RhoA/ROCK signaling-related proteins were detected through western blotting. In vitro, RhoA was overexpressed in oxygen-glucose deprivation and reperfusion (OGD/R)-induced PC12 cells to confirm the contribution of RhoA-ROCK signaling inhibition by SC to the neuroprotective effects post OGD/R. Pretreatment with SC significantly ameliorated the neurologic impairment, brain edema, and cerebral infarction post MCAO-reperfusion, associated with reductions of inflammation, oxidative stress, and cell apoptosis in the brain. Furthermore, SC remarkably downregulated the expression of RhoA/ROCK signaling-related proteins post MCAO-reperfusion in rats, while overexpression of RhoA reversed the beneficial effects of SC on protecting against inflammation and oxidative stress in OGD/R-induced PC12 cells. Taken together, these findings demonstrated that SC exerts neuroprotective effects after cerebral I/R injury via inhibiting inflammation and oxidative stress through regulating RhoA-ROCK signaling, suggesting a therapeutic potential of SC in cerebral I/R injury.

Quantitative volumetric assessment of the solid portion percentage on CT images to predict ROS1/ALK rearrangements in lung adenocarcinomas.

In the present study, the predictive role of the percentage of the solid portion volume (PSV) in patients with lung adenocarcinoma was investigated. The PSV was obtained through quantitative volumetric assessments based on reconstructed CT images of lung adenocarcinoma by comparing the index among tumors with c-ros oncogene 1 (ROS1) rearrangement, epidermal growth factor receptor (EGFR) mutations, echinoderm anaplastic lymphoma kinase (ALK) rearrangements or wild-type (WT) status for the three genes. Among 1,120 patients with lung adenocarcinoma, 28 patients with ROS1 rearrangement lung adenocarcinoma, 71 with ALK rearrangement and 578 with EGFR mutations were diagnosed. PSV was quantitatively measured by semi-automated nodule assessment software and compared in patients with different mutation statuses. The PSV (presented as the median with interquartile range) in the ROS1 rearrangement group [87.9 (82.7-92.3)%] was higher than that in the EGFR mutation group [70.4 (51.4-83.4%)] and the WT group [63.0 (50.9-83.2)%; P<0.001], but was similar to that in the ALK rearrangement group [84.0 (70.3-90.0)%; P=0.251]. The area under the receiver operating characteristic curve (AUC) for the PSV to predict ROS1 or ALK rearrangement combined was 0.702 (95% CI: 0.631-0.773; P<0.001); at a cut-off value of 0.805 (when the Youden index was maximal), the predictive sensitivity was 0.697 and the specificity was 0.702. Younger age and higher PSV values were independent predictors of ROS1/ALK rearrangements. The AUC for the predictive model combined with age and PSV was 0.785. In conclusion, the PSV in the lung adenocarcinomas with ROS1 rearrangement was significantly higher compared with that in the EGFR-mutated and WT lung adenocarcinoma, but was similar to that in lung adenocarcinoma with ALK rearrangement. Younger age and higher PSV values on CT in patients with lung adenocarcinomas were predictive factors for ROS1/ALK rearrangement.

Phospholipids-based microbubbles sonoporation pore size and reseal of cell membrane cultured in vitro.

OBJECTIVE: To investigate phospholipids-based microbubbles induced sonoporation and cell membrane reseal in vitro under various conditions. METHODS: A breast cancer cell line SK-BR-3 was used to investigate ultrasonic sonoporation under various conditions. Atomic force microscopy (AFM) scanning techniques were employed to observe the change of membrane pores. RESULTS: Normal SK-BR-3 cells membrane pores were evenly distributed and less than 1 microm. After ultrasound exposure, membrane pores were enlarged at different degree depending on ultrasound exposure durations, filling gas species and microbubble suspension concentration. With microbubble suspension concentration being increased to 5% or ultrasound exposure reached 30 s, membrane pores in fluorocarbon (C(3)F(8) or SF(6))-filled microbubble groups exceeded 1 microm, which were significantly larger than that of air-filled microbubble group. Membrane pores were about 2-3 microm under ultrasound 60 s with 5% fluorocarbon-filled microbubble suspension. After 24 h of incubation, most of the enlarged membrane pores could reseal to normal size, which corresponded to cell viability. CONCLUSIONS: Membrane pores can be obviously enlarged by ultrasonic sonoporation of fluorocarbon-filled microbubbles, whose reseal time depended on ultrasound exposure duration and microbubble suspension concentration.

[Optimizing the operating variables that affect the transfection experiment of antisense oligodeoxyribonucleotide by gas-filled microbubbles].

To optimize the operating variables that affect the transfection of antisense oligodeoxyribonucleotide (AS-ODNs) by insonated gas-filled lipid microbubbles, SF6-filled microbubbles were prepared by sonication-lyophilization method. An AS-ODNs sequence and a breast cancer cell line SK-BR-3 were used to define the various operating variables determining the transfection efficiency of SF6-filled microbubbles. Three levels of mixing speed, different durations of mixing and various delay time before ultrasound were examined, separately. Transfection efficiency was detected by fluorescence microscopy. Transfection results with and without incubation of AS-ODNs and microbubbles before mixing cells were compared. From the results, there is no significant difference between the transinfection efficiency with or without incubation of AS-ODNs and microbubbles before mixing cells. AS-ODNs transfection efficiency showed an increasing trend with mixing speed and mixing duration, but there is a negative relationship with delay time before ultrasound. The optimum parameters for AS-ODNs transfection by SF6-filled microbubbles were found at a mixing speed of 40-50 r x min(-1) for 30-60 s with less than 60 s delay before ultrasound. For a successful transfection, long time of incubation with gene is essential for normal nonviral vectors such as liposomes or cationic lipid-polymer hybrids, because these vectors depend on endocytosis and membrane fusion to realize transfection. Unlike liposomes and cationic lipid-polymer hybrids, gas-filled lipid microbubbles depend on sonorporation effect to realize transfection. Therefore, the incubation of gene and microbubbles before mixing cells may not be necessary. Ultrasound-mediated AS-ODNs transfection enhanced by gas-filled lipid microbubbles represents an effective avenue for gene transfer.