Influence of Solvent Selection on the Crystallizability and Polymorphic Selectivity Associated with the Formation of the "Disappeared" Form I Polymorph of Ritonavir.

The comparative crystallizability and polymorphic selectivity of ritonavir, a novel protease inhibitor for the treatment of acquired immune-deficiency syndrome, as a function of solvent selection are examined through an integrated and self-consistent experimental and computational molecular modeling study. Recrystallization at high supersaturation by rapid cooling at 283.15 K is found to produce the metastable "disappeared" polymorphic form I from acetone, ethyl acetate, acetonitrile, and toluene solutions in contrast to ethanol which produces the stable form II. Concomitant crystallization of the other known solid forms is not found under these conditions. Isothermal crystallization studies using turbidometric detection based upon classical nucleation theory reveal that, for an equal induction time, the required driving force needed to initiate solution nucleation decreases with solubility in the order of ethanol, acetone, acetonitrile, ethyl acetate, and toluene consistent with the expected desolvation behavior predicted from the calculated solute solvation free energies. Molecular dynamics simulations of the molecular and intermolecular chemistry reveal the presence of conformational interplay between intramolecular and intermolecular interactions within the solution phase. These encompass the solvent-dependent formation of intramolecular O-H...O hydrogen bonding between the hydroxyl and carbamate groups coupled with differing conformations of the hydroxyl's shielding phenyl groups. These conformational preferences and their relative interaction propensities, as a function of solvent selection, may play a rate-limiting role in the crystallization behavior by not only inhibiting to different degrees the nucleation process but also restricting the assembly of the optimal intermolecular hydrogen bonding network needed for the formation of the stable form II polymorph.

Circ-sh3rf3/GATA-4/miR-29a regulatory axis in fibroblast-myofibroblast differentiation and myocardial fibrosis.

The transdifferentiation from cardiac fibroblasts to myofibroblasts is an important event in the initiation of cardiac fibrosis. However, the underlying mechanism is not fully understood. Circ-sh3rf3 (circular RNA SH3 domain containing Ring Finger 3) is a novel circular RNA which was induced in hypertrophied ventricles by isoproterenol hydrochloride, and our work has established that it is a potential regulator in cardiac hypertrophy, but whether circ-sh3rf3 plays a role in cardiac fibrosis remains unclear, especially in the conversion of cardiac fibroblasts into myofibroblasts. Here, we found that circ-sh3rf3 was down-regulated in isoproterenol-treated rat cardiac fibroblasts and cardiomyocytes as well as during fibroblast differentiation into myofibroblasts. We further confirmed that circ-sh3rf3 could interact with GATA-4 proteins and reduce the expression of GATA-4, which in turn abolishes GATA-4 repression of miR-29a expression and thus up-regulates miR-29a expression, thereby inhibiting fibroblast-myofibroblast differentiation and myocardial fibrosis. Our work has established a novel Circ-sh3rf3/GATA-4/miR-29a regulatory cascade in fibroblast-myofibroblast differentiation and myocardial fibrosis, which provides a new therapeutic target for myocardial fibrosis.

TMEM151A variants associated with paroxysmal kinesigenic dyskinesia.

TMEM151A, located at 11q13.2 and encoding transmembrane protein 151A, was recently reported as causative for autosomal dominant paroxysmal kinesigenic dyskinesia (PKD). Here, through comprehensive analysis of sporadic and familial cases, we expand the clinical and mutation spectrum of PKD. In doing so, we clarify the clinical and genetic features of Chinese PKD patients harboring TMEM151A variants and further explore the relationship between TMEM151A mutations and PKD. Whole exome sequencing was performed on 26 sporadic PKD patients and nine familial PKD pedigrees without PRRT2 variants. Quantitative real-time PCR was used to assess the gene expression of frameshift mutant TMEM151A in a PKD patient. TMEM151A variants reported to date were reviewed. Four TMEM151A variants were detected in four unrelated families with 12 individuals, including a frameshift mutation [c.606_607insA (p.Val203fs)], two missense mutations [c.166G > A (p.Gly56Arg) and c.791T > C (p.Val264Ala)], and a non-pathogenic variant [c.994G > A (p.Gly332Arg)]. The monoallelic frameshift mutation [c.606_607insA (p.Val203fs)] may cause TMEM151A mRNA decay, suggesting a potential pathogenic mechanism of haploinsufficiency. Patients with TMEM151A variants had short-duration attacks and presented with dystonia. Our study provides a detailed clinical description of PKD patients with TMEM151A mutations and reports a new disease-causing mutation, expanding the known phenotypes caused by TMEM151A mutations and providing further detail about the pathoetiology of PKD.

Early autoimmunity and outcome in virus encephalitis: a retrospective study based on tissue-based assay.

To explore the autoimmune response and outcome in the central nervous system (CNS) at the onset of viral infection and correlation between autoantibodies and viruses. METHODS: A retrospective observational study was conducted in 121 patients (2016-2021) with a CNS viral infection confirmed via cerebrospinal fluid (CSF) next-generation sequencing (cohort A). Their clinical information was analysed and CSF samples were screened for autoantibodies against monkey cerebellum by tissue-based assay. In situ hybridisation was used to detect Epstein-Barr virus (EBV) in brain tissue of 8 patients with glial fibrillar acidic protein (GFAP)-IgG and nasopharyngeal carcinoma tissue of 2 patients with GFAP-IgG as control (cohort B). RESULTS: Among cohort A (male:female=79:42; median age: 42 (14-78) years old), 61 (50.4%) participants had detectable autoantibodies in CSF. Compared with other viruses, EBV increased the odds of having GFAP-IgG (OR 18.22, 95% CI 6.54 to 50.77, p<0.001). In cohort B, EBV was found in the brain tissue from two of eight (25.0%) patients with GFAP-IgG. Autoantibody-positive patients had a higher CSF protein level (median: 1126.00 (281.00-5352.00) vs 700.00 (76.70-2899.00), p<0.001), lower CSF chloride level (mean: 119.80±6.24 vs 122.84±5.26, p=0.005), lower ratios of CSF-glucose/serum-glucose (median: 0.50[0.13-0.94] vs 0.60[0.26-1.23], p=0.003), more meningitis (26/61 (42.6%) vs 12/60 (20.0%), p=0.007) and higher follow-up modified Rankin Scale scores (1 (0-6) vs 0 (0-3), p=0.037) compared with antibody-negative patients. A Kaplan-Meier analysis revealed that autoantibody-positive patients experienced significantly worse outcomes (p=0.031). CONCLUSIONS: Autoimmune responses are found at the onset of viral encephalitis. EBV in the CNS increases the risk for autoimmunity to GFAP.

Predicting the Strength of Cohesive and Adhesive Interparticle Interactions for Dry Powder Inhalation Blends of Terbutaline Sulfate with α-Lactose Monohydrate.

Grid-based systematic search methods are used to investigate molecule-molecule, molecule-surface, and surface-surface contributions to interparticle interactions in order to identify the crystal faces that most strongly affect particle behavior during powder blend formulation and delivery processes. The model system comprises terbutaline sulfate (TBS) as an active pharmaceutical ingredient (API) and α-form lactose monohydrate (LMH). A combination of systematic molecular modeling and X-ray computed tomography (XCT) is used to determine not only the adhesive and cohesive interparticle energies but, also the agglomeration behavior during manufacturing and de-agglomeration behavior during delivery after inhalation. This is achieved through a detailed examination of the balance between the adhesive and cohesive energies with the XCT results confirming the blend segregation tendencies, through the particle-particle de-agglomeration process. The results reveal that the cohesive interaction energies of TBS-TBS are higher than the adhesive energies between TBS and LMH, but that the cohesive energies of LMH-LMH are the smallest between molecule and molecule, molecule and surface, and surface and surface. This shows how systematic grid-search molecular modeling along with XCT can guide the digital formulation design of inhalation powders in order to achieve optimum aerosolization and efficacy for inhaled medicines. This will lead to faster pharmaceutical design with less variability, higher quality, and enhanced performance.

iPADD: A Computational Tool for Predicting Potential Antidiabetic Drugs Using Machine Learning Algorithms.

Diabetes mellitus is a chronic metabolic disease, which causes an imbalance in blood glucose homeostasis and further leads to severe complications. With the increasing population of diabetes, there is an urgent need to develop drugs to treat diabetes. The development of artificial intelligence provides a powerful tool for accelerating the discovery of antidiabetic drugs. This work aims to establish a predictor called iPADD for discovering potential antidiabetic drugs. In the predictor, we used four kinds of molecular fingerprints and their combinations to encode the drugs and then adopted minimum-redundancy-maximum-relevance (mRMR) combined with an incremental feature selection strategy to screen optimal features. Based on the optimal feature subset, eight machine learning algorithms were applied to train models by using 5-fold cross-validation. The best model could produce an accuracy (Acc) of 0.983 with the area under the receiver operating characteristic curve (auROC) value of 0.989 on an independent test set. To further validate the performance of iPADD, we selected 65 natural products for case analysis, including 13 natural products in clinical trials as positive samples and 52 natural products as negative samples. Except for abscisic acid, our model can give correct prediction results. Molecular docking illustrated that quercetin and resveratrol stably bound with the diabetes target NR1I2. These results are consistent with the model prediction results of iPADD, indicating that the machine learning model has a strong generalization ability. The source code of iPADD is available at https://github.com/llllxw/iPADD.

Concentrations, seasonal trends, sources, health risk and subchronic toxicity to the respiratory and immune system of PAHs in PM2.5 in Xi'an.

PM2.5-bound polycyclic aromatic hydrocarbons (PAHs) have been proved to be hazardous to health. Previous studies have focused on the distribution and sources of PAHs, whereas there is little knowledge of the damage to organs. Here we sought to investigate the pollution level and seasonal variation characteristics of PAHs in PM2.5 in Xi'an and assess the health risk, to establish a PAHs exposure model, and investigate the toxicological effects of PAHs on the respiratory and immune functions. A sub-chronic exposure model of PAHs was established by inhalation. The pathological changes of lung tissues were observed with a light microscope. Inflammatory reactions in alveolar lavage fluid were determined using the corresponding kit. The levels of interleukin-6 (IL-6) and interleukin-8 (IL-8) were detected with enzyme linked immunosorbent assay (ELISA) kit; the proliferation of lymphocytes in spleen was detected with methyl tetrazolium (MTT); DNA immune damage was determined with DNA gel electrophoresis. The results showed that (1) the total concentration of 16 PAHs ranged from 41.1 to 387 ng/m3, with a mean value of 170 ng/m3, and the concentration of PAHs in PM2.5 was higher in winter than in other seasons. (2) The sources of PAHs in the atmosphere of Xi'an urban area were mainly coal combustion, and the equivalent carcinogenic concentration of PAHs in PM2.5 was 3.9 ng/m3. (3) Foreign body granuloma formation and inflammatory cell damage were observed in the lungs of rats infected with toxin; the levels of reactive oxygen species (ROS) and mobile device assistant (MDA) increased while nitric oxide synthase (NOS) decreased with the increase of dose; the expression levels of IL-6 and IL-8 elevated with the increase of toxin dose, showing an obvious dose-effect relationship; the level of PAHs damage to cells showed a dose-effect relationship. Sub-chronic exposure to PAHs could cause sustained inflammatory injury to the organism. Measures should be taken to counter the problems of PAHs in PM2.5 in Xi'an and relevant health promotion strategies should be developed.

The structural pathway from its solvated molecular state to the solution crystallisation of the α- and ß-polymorphic forms of para amino benzoic acid.

Para amino benzoic acid (PABA) has two well-characterised α- and ß-polymorphic forms and, whilst both crystallise in the monoclinic space group P21/n, they have quite different crystal chemistry and crystallisability behaviour. Previous work has shown that the molecular conformation deformation energy in the crystalline state is higher for the ß-form than for the α-form and that the lattice energy for the former converges more slowly than for the latter overall. This suggests that not only is there a higher barrier to crystallisation for the ß-form but also that low solution supersaturations might be needed for it to preferentially nucleate. Additionally, solute cluster propensity and solute solvation energetic analysis highlight the importance of an aqueous solvation environment in inhibiting the α-form's strong OH⋯O carboxylic acid hydrogen bond (H-bond) dimer. Despite this, the detailed molecular-scale pathway from solvated molecules to 3D crystallographic structure still remains unclear, most notably regarding how the nucleation process is activated and how, as a result, this mediates the preferential formation of either of the two polymorphic forms. Molecular dynamics (MD) simulations coupled with FTIR studies and intermolecular synthon analysis address this issue through characterisation of the propensity of the incipient bulk synthons that are important in the crystallisation of the two polymorphic forms within the solution state. MD molecular trajectory analysis within crystallisation solutions reveals a greater propensity for OH⋯O synthons (both single H-bonds and homodimers) typical of the α-form and NH⋯O synthons found in both the α- and ß-forms when compared to aqueous solution but much lower propensities for the ß-form's "fingerprinting" OH⋯N and π-π stacking synthons. In contrast, data from the aqueous solution environment reveals a much greater propensity for the ß-form's π-π interaction synthons. IR dilution studies in acetonitrile in the carbonyl region reveal the presence of two CO vibrational stretching bands, whose relative intensities vary as a function of solution dilution. These were assigned to the solvated PABA monomer and a COOH dimer of PABA. Similar data in ethanol shows a main CO stretching band with a shoulder peak suggesting a similar monomer vs. dimer speciation may exist in this solvent. The IR data is consistent with the organic solvent MD data, albeit the corresponding analysis for the aqueous solution was precluded due to the latter's strong OH vibrational mode which restricted validation in aqueous solutions.

Identification of a promoter polymorphism affecting GPAT3 gene expression that is likely related to intramuscular fat content in pigs.

The intramuscular fat content (IMF) is an economically important trait in pigs and the Laiwu pig is famous for its excessively extremely high level of IMF. Our previous transcriptome study revealed that the dynamic expression of glycerol-phosphate acyltransferase 3 (GPAT3) is consistent with changes in the IMF of Laiwu pigs. In this study, we further analyzed the expression and polymorphism of GPAT3 in its promoter region. The results indicated that the expression of GPAT3 increased dramatically from 120 to 240 days and is consistent with changes in IMF deposition, and at both mRNA and protein levels, GPAT3 expression was markedly higher in the LD muscle of Laiwu pigs than that of Duroc × Landrace × Yorkshire pigs. Deletion from -1695 to -1187 of porcine GPAT3 greatly increased its transcription. Of the two SNPs identified, the transition from C to T at -1526 site increased the transcription of porcine GPAT3 and allele T mainly distributed in Laiwu pig population. These results collectively suggest that the SNP at -1526 site of GPAT3 may contribute to IMF deposition by affecting its expression in pigs.

[VCR, a Small Molecule Compound, Induces Reprogramming of Rat Fibroblasts into Neural Progenitor Cells under Hypoxic Condition].

Objective: To explore for a protocol for reprogramming rat embryonic fibroblasts (REFs) under hypoxic conditions (5% O 2) to form chemically induced rat neural progenitor cells (ciRNPCs). Methods: The reprogramming of REFs into ciNPCs was done in two stages. The first stage involved chemical induction to generate intermediate cells. The REFs were cultured in KSR medium containing valproic acid, CHIR99021, and RepSox (VCR) and 10000 U/mL leukemia inhibitory factor (LIF) for 15 days, under a physiological hypoxic condition. The formation of dense cell colonies, i.e., intermediate cells, were observed. The second stage involved the specific induction of ciRNPCs. The induced intermediate cells were digested with trypsin, seeded on a low adhesion plate, and cultured under normoxic condition to form ciRNPCs neurospheres. Then, after CM-DiI cell-labeling, the ciRNPCs were stereotactically transplanted into the substantia nigra (SN) of rats. The survival, migration, and differentiation of ciRNPCs in the host brain were examined with immunofluorescence assays. Results: After induction under hypoxic condition for 5 to 10 days, a clear trend of cell aggregation was observed. Compact cell colonies were observed in REFs treated with VCR for 15 days under a hypoxic condition. Approximately 30 colonies emerged from 1×10 5 cells, and most colonies were positive for AP staining. Moreover, when these cells were cultured further in suspension, free-floating neurospheres formed and stained positive for neural progenitor cell (NPC) markers, including Nestin, Sox2 and Pax6. These ciRNPCs could differentiate into glial cells and neurons, and express neurite marker Tuj1 and astrocyte marker GFAP. Eight weeks after transplantation, the cells could differentiate into GFAP+ and Tuj1+ cells in the rat brain. Conclusion: Our study demonstrates that VCR, a small molecule compound, can directly induce, under a hypoxic condition, the reprogramming of REFs to form ciRNPCs with the potential to be induced for differentiation into glial cells and neurons in vivo and in vitro, laying the foundation for transplanting ciRNPCs to treat neurodegenerative diseases.

Raman spectroscopic study of cervical precancerous lesions and cervical cancer.

Early detection of cervical lesions, accurate diagnosis of cervical lesions, and timely and effective therapy can effectively avoid the occurrence of cervical cancer or improve the survival rate of patients. In this paper, the spectra of tissue sections of cervical inflammation (n = 60), CIN (cervical intraepithelial neoplasia) I (n = 30), CIN II (n = 30), CIN III (n = 30), cervical squamous cell carcinoma (n = 30), and cervical adenocarcinoma (n = 30) were collected by a confocal Raman micro-spectrometer (LabRAM HR Evolution, Horiba France SAS, Villeneuve d'Ascq, France). The Raman spectra of six kinds of cervical tissues were analyzed, the dominant Raman peaks of different kinds of tissues were summarized, and the differences in chemical composition between the six tissue samples were compared. An independent sample t test (p ≤ 0.05) was used to analyze the difference of average relative intensity of Raman spectra of six types of cervical tissues. The difference of relative intensity of Raman spectra of six kinds of tissues can reflect the difference of biochemical components in six kinds of tissues and the characteristic of biochemical components in different kinds of tissues. The classification models of cervical inflammation, CIN I, CIN II, CIN III, cervical squamous cell carcinoma, and cervical adenocarcinoma were established by using a support vector machine (SVM) algorithm. Six types of cervical tissues were classified and identified with an overall diagnostic accuracy of 85.7%. This study laid a foundation for the application of Raman spectroscopy in the clinical diagnosis of cervical precancerous lesions and cervical cancer.

Proteomics and single-cell RNA analysis of Akap4-knockout mice model confirm indispensable role of Akap4 in spermatogenesis.

Sperm fibrous sheath, a unique cytoskeletal structure, is implicated in various sperm physiological functions, such as sperm maturation, motility and capacitation. AKAP4 has been described to be required for structural and functional integrity of the fibrous sheath. We generated Akap4-knockout mice line using CRISPR-Cas9 system. Cytomorphology and motility of sperm and testes were studied, confirming loss of Akap4 led to abnormal sperm morphology, motility and infertility. The proteomic components of testes were studied and Akap4 was found to be significantly decreased in the Akap4-knockout mice. Testis single-cell RNA sequencing and analysis revealed three genes with significant change in the general cell population, i.e., Akap4, Haspin, and Ccdc38. The single-cell RNA expression profiles also showed that the major difference between Akap4-knockout and wild-type testes existed in the elongating cell cluster, where in the Akap4-knockout testes, a subgroup of elongating cells with marker genes involved in cell adhesion and migration were increased, while a subgroup of elongating cells marked by mitochondrial sheath genes were decreased. Our results revealed the complex and well-coordinated procedures of spermatogenesis, and substantiated Akap4's indispensable roles in the integrity of sperm flagellum and the step-wise maturation of spermatozoa.

Enhancing the scope of in vitro maturation for fertility preservation: transvaginal retrieval of immature oocytes during endoscopic gynaecological procedures.

STUDY QUESTION: Could in vitro maturation (IVM) following transvaginal oocyte retrieval during gynaecological surgery (IVM-surgery) be an effective and safe strategy for fertility preservation? SUMMARY ANSWER: IVM-surgery on unstimulated ovaries is a novel option that can be considered for fertility preservation for women requiring gynaecological surgery, but more research is needed to identify appropriate patients who may benefit and to determine the cost-effectiveness of such an approach. WHAT IS KNOWN ALREADY: IVM followed by oocyte/embryo cryopreservation has been useful as a safe reproductive strategy for some infertile women. STUDY DESIGN, SIZE, DURATION: This prospective cohort study comprised 158 consecutive women with polycystic ovary syndrome (PCOS) who underwent laparoscopy or hysteroscopy for other reasons and had concomitant transvaginal oocyte retrieval followed by IVM between 2014 and 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 158 women with anovulatory PCOS who underwent IVM-surgery in our infertility centre were recruited for this study. Matured IVM oocytes obtained from these women were either freshly fertilized and subsequently frozen at the blastocyst stage (fresh oocyte group, n = 46) or the oocytes were frozen (frozen oocyte group, n = 112) for fertility preservation followed by later thawing for insemination and cleavage embryo transfer (ET) (n = 33). The following outcomes were then evaluated: embryological data, clinical pregnancy rate, live birth rate (LBR), neonatal outcomes, post-operative complications and post-operative ovarian function. MAIN RESULTS AND THE ROLE OF CHANCE: Among all the women who underwent IVM-surgery, the clinical pregnancy rate and LBR per initiated IVM cycle were 9.5% (15/158) and 6.9% (11/158), respectively. Women (40.6%, 20/33) who underwent the procedure with frozen-thawed oocytes (oocyte survival rate, 83.0%) obtained a high quality of cleaved embryos. In the fresh oocyte group, the clinical pregnancy rate and LBR per ET cycle were 69.2 and 53.8%, respectively. In the frozen oocyte group, the clinical pregnancy rate and LBR per ET cycle were 28.6 and 19.1%, respectively. No adverse neonatal outcomes were recorded. IVM-surgery was not associated with post-operative complications, a longer hospital stay, or impaired ovarian function. LIMITATIONS, REASONS FOR CAUTION: Because of the small sample size and the low utilization rate and cost-effectiveness per retrieval, the present findings should be interpreted with caution, and further studies are needed for the long-term follow-up of live births. WIDER IMPLICATIONS OF THE FINDINGS: This strategy can also help patients with normal ovulation to obtain available oocytes and embryos for cryopreservation and subsequent use. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the Joint Research Fund for Overseas Natural Science of China (No. 31429004), the National Key Research and Development Program of China (No. 2017YFC1002000, 2017YFC1001504, 2016YFC1000302), the Ministry of Science and Technology of China Grants (No. 2014CB943203), the Chinese Society of Reproductive Medicine Fund (No. 16020400656) and the National Natural Science Foundation of China (No. 81300456). All the authors have nothing to disclose in terms of conflicts of interest. TRIAL REGISTRATION NUMBER: chictr-ONC-17011861.

Dynamic transcriptome and DNA methylome analyses on longissimus dorsi to identify genes underlying intramuscular fat content in pigs.

BACKGROUND: The intramuscular fat content (IMF) refers to the amount of fat within muscles, including the sum of phospholipids mainly found in cell membranes, triglycerides and cholesterol, and is determined both by hyperplasia and hypertrophy of adipocyte during the development of pigs. The IMF content is an important economic trait that is genetically controlled by multiple genes. The Laiwu pig is an indigenous fatty pig breed distributed in North China, characterized by excessively higher level of IMF content (9%~12%), therefore, is suitable for the identification of genes controlling IMF variations. To identify genes underlying IMF deposition, we performed genome-wide transcriptome and methylome analyses on longissimus dorsi (LD) muscle in Laiwu pigs across four developmental stages. RESULTS: A total of 22,524 expressed genes were detected and 1158 differentially expressed genes (DEGs) were hierarchically clustered in the LD muscle over four developmental stages from 60 d to 400 d. These genes were significantly clustered into four temporal expression profiles, and genes participating in fat cell differentiation and lipid biosynthesis processes were identified. From 120 d to 240 d, the period with the maximum IMF deposition rate, the lipid biosynthesis related genes (FOSL1, FAM213B and G0S2), transcription factors (TFs) (EGR1, KLF5, SREBF2, TP53 and TWIST1) and enriched pathways (steroid biosynthesis and fatty acid biosynthesis) were revealed; and fat biosynthesis relevant genes showing differences in DNA methylation in gene body or intergenic region were detected, such as FASN, PVALB, ID2, SH3PXD2B and EGR1. CONCLUSIONS: This study provides a comprehensive landscape of transcriptome of the LD muscle in Laiwu pigs ranging from 60 to 400 days old, and methylome of the LD muscle in 120 d and 240 d Laiwu pigs. A set of candidate genes and TFs involved in fat biosynthesis process were identified, which were probably responsible for IMF deposition. The results from this study would provide a reference for the identification of genes controlling IMF variation, and for exploring molecular mechanisms underlying IMF deposition in pigs.

Detection of Atherosclerotic Plaques in the Rabbit Aorta Using Ultrasound Microbubbles Conjugated to Interleukin-18 Antibodies.

BACKGROUND The purpose of the study was to investigate the ability of microbubbles (MBs) targeting interleukin-18 (IL-18) to detect plaques in a rabbit atherosclerotic plaque model. MATERIAL AND METHODS A rabbit atherosclerotic plaque model was established. The locations of the atherosclerotic plaques were verified by two-dimensional scanning and color Doppler flow imaging. An IL-18 antibody was conjugated to naked MBs (MBc) using the biotin-streptavidin conjugation method, resulting in the formation of MBIL-18. MBc and MBIL-18 were then used for contrast-enhanced ultrasound (CEUS) studies. The locations of CD34 and IL-18 within the plaques were determined by immunohistochemistry, and IL-18 expression levels in the plaques were determined by Western blot analysis. The relationships between IL-18 expression and the contrast intensity of the 2 MBs were analyzed. RESULTS MBc and MBIL-18 were both uniformly dispersed. Fluorescence microscopy and flow cytometry revealed that IL-18 was successfully conjugated to MBs. CEUS images showed that the intensity of the MBIL-18 signal was substantially enhanced and prolonged compared with that of the MBc signal. Immunohistochemistry showed that CD34 expression was significantly increased in the plaques and that IL-18 was mainly located in the inner parts and base of the atherosclerotic plaques. Western blot analysis revealed that IL-18 expression was higher in the plaque regions. Correlation analysis showed that IL-18 expression was correlated with the contrast intensity of MBIL-18 (r=0.903, P<0.05) but not with MBc (r=0.540, P>0.05). CONCLUSIONS MBs targeting IL-18 may be a novel, noninvasive method of diagnosing atherosclerotic plaques.

Literature Review of (Q)SAR Modelling of Nanomaterial Toxicity.

Despite the clear benefits that nanotechnology can bring to various sectors of industry, there are serious concerns about the potential health risks associated with engineered nanomaterials (ENMs), intensified by the limited understanding of what makes ENMs toxic and how to make them safe. As the use of ENMs for commercial purposes and the number of workers/end-users being exposed to these materials on a daily basis increases, the need for assessing the potential adverse effects of multifarious ENMs in a time- and cost-effective manner becomes more apparent. One strategy to alleviate the problem of testing a large number and variety of ENMs in terms of their toxicological properties is through the development of computational models that decode the relationships between the physicochemical features of ENMs and their toxicity. Such data-driven models can be used for hazard screening, early identification of potentially harmful ENMs and the toxicity-governing physicochemical properties, and accelerating the decision-making process by maximising the use of existing data. Moreover, these models can also support industrial, regulatory and public needs for designing inherently safer ENMs. This chapter is mainly concerned with the investigation of the applicability of (quantitative) structure-activity relationship ((Q)SAR) methods to modelling of ENMs' toxicity. It summarizes the key components required for successful application of data-driven toxicity prediction techniques to ENMs, the published studies in this field and the current limitations of this approach.

EGF is required for cardiac differentiation of P19CL6 cells through interaction with GATA-4 in a time- and dose-dependent manner.

The regulation of cardiac differentiation is critical for maintaining normal cardiac development and function. The precise mechanisms whereby cardiac differentiation is regulated remain uncertain. Here, we have identified a GATA-4 target, EGF, which is essential for cardiogenesis and regulates cardiac differentiation in a dose- and time-dependent manner. Moreover, EGF demonstrates functional interaction with GATA-4 in inducing the cardiac differentiation of P19CL6 cells in a time- and dose-dependent manner. Biochemically, GATA-4 forms a complex with STAT3 to bind to the EGF promoter in response to EGF stimulation and cooperatively activate the EGF promoter. Functionally, the cooperation during EGF activation results in the subsequent activation of cyclin D1 expression, which partly accounts for the lack of additional induction of cardiac differentiation by the GATA-4/STAT3 complex. Thus, we propose a model in which the regulatory cascade of cardiac differentiation involves GATA-4, EGF, and cyclin D1.

Plasminogen kringle 5 induces endothelial cell apoptosis by triggering a voltage-dependent anion channel 1 (VDAC1) positive feedback loop.

Human plasminogen kringle 5 (K5) is known to display its potent anti-angiogenesis effect through inducing endothelial cell (EC) apoptosis, and the voltage-dependent anion channel 1 (VDAC1) has been identified as a receptor of K5. However, the exact role and underlying mechanisms of VDAC1 in K5-induced EC apoptosis remain elusive. In the current study, we showed that K5 increased the protein level of VDAC1, which initiated the mitochondrial apoptosis pathway of ECs. Our findings also showed that K5 inhibited the ubiquitin-dependent degradation of VDAC1 by promoting the phosphorylation of VDAC1, possibly at Ser-12 and Thr-107. The phosphorylated VDAC1 was attenuated by the AKT agonist, glycogen synthase kinase (GSK) 3ß inhibitor, and siRNA, suggesting that K5 increased VDAC1 phosphorylation via the AKT-GSK3ß pathway. Furthermore, K5 promoted cell surface translocation of VDAC1, and binding between K5 and VDAC1 was observed on the plasma membrane. HKI protein blocked the impact of K5 on the AKT-GSK3ß pathway by competitively inhibiting the interaction of K5 and cell surface VDAC1. Moreover, K5-induced EC apoptosis was suppressed by VDAC1 antibody. These data show for the first time that K5-induced EC apoptosis is mediated by the positive feedback loop of "VDAC1-AKT-GSK3ß-VDAC1," which may provide new perspectives on the mechanisms of K5-induced apoptosis.

Pigment epithelial-derived factor (PEDF)-triggered lung cancer cell apoptosis relies on p53 protein-driven Fas ligand (Fas-L) up-regulation and Fas protein cell surface translocation.

Pigment epithelium-derived factor (PEDF), a potent antiangiogenesis agent, has recently attracted attention for targeting tumor cells in several types of tumors. However, less is known about the apoptosis-inducing effect of PEDF on human lung cancer cells and the underlying molecular events. Here we report that PEDF has a growth-suppressive and proapoptotic effect on lung cancer xenografts. Accordingly, in vitro, PEDF apparently induced apoptosis in A549 and Calu-3 cells, predominantly via the Fas-L/Fas death signaling pathway. Interestingly, A549 and Calu-3 cells are insensitive to the Fas-L/Fas apoptosis pathway because of the low level of cell surface Fas. Our results revealed that, in addition to the enhancement of Fas-L expression, PEDF increased the sensitivity of A549 and Calu-3 cells to Fas-L-mediated apoptosis by triggering the translocation of Fas protein to the plasma membrane in a p53- and FAP-1-dependent manner. Similarly, the up-regulation of Fas-L by PEDF was also mediated by p53. Furthermore, peroxisome proliferator-activated receptor γ was determined to be the upstream regulator of p53. Together, these findings uncover a novel mechanism of tumor cell apoptosis induced by PEDF and provide a potential therapeutic strategy for tumors that are insensitive to Fas-L/Fas-dependent apoptosis because of a low level of cell surface Fas.

EGF Protects Cells Against Dox-Induced Growth Arrest Through Activating Cyclin D1 Expression.

It has been reported that the antitumor drug doxorubicin (Dox) exerts its toxic effects via GATA-4 depletion and that over-expression of GATA-4 reverses Dox-induced toxicity and apoptosis; however, the precise mechanisms remain unclear. In this study, we observed, for the first time, that EGF protects cells against Dox-mediated growth arrest, G2/M-phase arrest, and apoptosis. Additionally, EGF expression was down-regulated in Dox-treated cells and up-regulated in GATA-4 over-expressing cells. Utilizing real-time PCR and western blotting analysis, we found that the expression of the cell cycle-associated protein cyclin D1 was inhibited in GATA-4-silenced cells and Dox-treated cells and was enhanced in GATA-4 over-expressing cells and EGF-treated cells. Furthermore, EGF treatment reversed the inhibited expression of cyclin D1 that was mediated by GATA-4 RNAi or Dox. Our results indicate that EGF, as a downstream target of Dox, may be involved in Dox-induced toxicity as well as in the protective role of GATA-4 against toxicity induced by Dox via regulating cyclin D1 expression, which elucidates a new molecular mechanism of Dox toxicity with important clinical implications.