BACKGROUND: Digital immunohistochemistry (IHC) is one of the most promising applications brought by new generation image analysis (IA). While conventional IHC staining quality is monitored by semi-quantitative visual evaluation of tissue controls, IA may require more sensitive measurement. We designed an automated system to digitally monitor IHC multi-tissue controls, based on SQL-level integration of laboratory information system with image and statistical analysis tools. METHODS: Consecutive sections of TMA containing 10 cores of breast cancer tissue were used as tissue controls in routine Ki67 IHC testing. Ventana slide label barcode ID was sent to the LIS to register the serial section sequence. The slides were stained and scanned (Aperio ScanScope XT), IA was performed by the Aperio/Leica Colocalization and Genie Classifier/Nuclear algorithms. SQL-based integration ensured automated statistical analysis of the IA data by the SAS Enterprise Guide project. Factor analysis and plot visualizations were performed to explore slide-to-slide variation of the Ki67 IHC staining results in the control tissue. RESULTS: Slide-to-slide intra-core IHC staining analysis revealed rather significant variation of the variables reflecting the sample size, while Brown and Blue Intensity were relatively stable. To further investigate this variation, the IA results from the 10 cores were aggregated to minimize tissue-related variance. Factor analysis revealed association between the variables reflecting the sample size detected by IA and Blue Intensity. Since the main feature to be extracted from the tissue controls was staining intensity, we further explored the variation of the intensity variables in the individual cores. MeanBrownBlue Intensity ((Brown+Blue)/2) and DiffBrownBlue Intensity (Brown-Blue) were introduced to better contrast the absolute intensity and the colour balance variation in each core; relevant factor scores were extracted. Finally, tissue-related factors of IHC staining variance were explored in the individual tissue cores. CONCLUSIONS: Our solution enabled to monitor staining of IHC multi-tissue controls by the means of IA, followed by automated statistical analysis, integrated into the laboratory workflow. We found that, even in consecutive serial tissue sections, tissue-related factors affected the IHC IA results; meanwhile, less intense blue counterstain was associated with less amount of tissue, detected by the IA tools.
Abstract The qualified presumption of safety (QPS) process was developed to provide a safety assessment approach for microorganisms intended for use in food or feed chains. In the period covered by this Statement, no new information was found that would change the status of previously recommended QPS taxonomic units (TUs). The TUs in the QPS list were updated based on a verification, against their respective authoritative databases, of the correctness of the names and completeness of synonyms. Of 54 microorganisms notified to EFSA between April and September 2024 (33 as feed additives, 17 as food enzymes or additives, 4 as novel foods), 50 were not evaluated because: 12 were filamentous fungi, 1 was Enterococcus faecium and 8 were Escherichia coli (all excluded from the QPS evaluation), and 29 were TUs that already have a QPS status. One notification (Ensifer adhaerens) was already evaluated in a previous Panel Statement. Another notification (Enterococcus lactis) was already evaluated in the previous 3‐year QPS cycle and was reassessed within this document. Two TUs were notified for the first time and were assessed for a possible QPS status: Serratia plymuthica and Lacticaseibacillus huelsenbergensis. Bacillus thuringiensis and Bacillus nakamurai have been assessed for a possible QPS status in response to internal requests. The following was concluded on the five assessed TUs. L. huelsenbergensis can be granted the QPS status based on its close relatedness to several other QPS Lacticaseibacillus species. E. lactis is not recommended for the QPS status due to insufficient information on safety. S. plymuthica and B. thuringiensis are not recommended for the QPS status due to safety concerns. B. nakamurai cannot be recommended for the QPS list due to a lack of body of knowledge for its use in the food and feed chain.
Abstract Electron microscopy of biological tissue has recently seen an unprecedented increase in imaging throughput moving the ultrastructural analysis of large tissue blocks such as whole brains into the realm of the feasible. However, homogeneous, high quality electron microscopy staining of large biological samples is still a major challenge. To date, assessing the staining quality in electron microscopy requires running a sample through the entire staining protocol end-to-end, which can take weeks or even months for large samples, rendering protocol optimization for such samples to be inefficient. Here we present an in situ time-lapsed X-ray assisted staining procedure that opens the “black box” of electron microscopy staining and allows observation of individual staining steps in real time. Using this novel method we measured the accumulation of heavy metals in large tissue samples immersed in different staining solutions. We show that the measured accumulation of osmium in fixed tissue obeys empirically a quadratic dependence between the incubation time and sample size. We found that potassium ferrocyanide, a classic reducing agent for osmium tetroxide, clears the tissue after osmium staining and that the tissue expands in osmium tetroxide solution, but shrinks in reduced osmium solution. X-ray assisted staining gave access to the in situ staining kinetics and allowed us to develop a diffusion-reaction-advection model that accurately simulates the measured accumulation of osmium in tissue. These are first steps towards in silico staining experiments and simulation-guided optimization of staining protocols for large samples. Hence, X-ray assisted staining will be a useful tool for the development of reliable staining procedures for large samples such as entire brains of mice, monkeys or humans.
Age-related macular degeneration (AMD) is the leading cause of certified vision loss worldwide. The standard treatment for neovascular AMD involves repeated intravitreal injections of therapeutic proteins directed against vascular endothelial growth factor, such as ranibizumab. Biodegradable polymers, such as poly(lactic-co-glycolic acid) (PLGA), form delivery vehicles which can be used to treat posterior segment eye diseases, but suffer from poor protein loading and release. This work describes a "system-within-system", PLGA microparticles incorporating chitosan-based nanoparticles, for improved loading and sustained intravitreal delivery of ranibizumab. Chitosan-N-acetyl-l-cysteine (CNAC) was synthesized and its synthesis confirmed using FT-IR and (1)H NMR. Chitosan-based nanoparticles composed of CNAC, CNAC/tripolyphosphate (CNAC/TPP), chitosan, chitosan/TPP (chit/TPP), or chit/TPP-hyaluronic acid (chit/TPP-HA) were incorporated in PLGA microparticles using a modified w/o/w double emulsion method. Nanoparticles and final nanoparticles-within-microparticles were characterized for their protein-nanoparticle interaction, size, zeta potential, morphology, protein loading, stability, in vitro release, in vivo antiangiogenic activity, and effects on cell viability. The prepared nanoparticles were 17-350 nm in size and had zeta potentials of -1.4 to +12 mV. Microscopic imaging revealed spherical nanoparticles on the surface of PLGA microparticles for preparations containing chit/TPP, CNAC, and CNAC/TPP. Ranibizumab entrapment efficiency in the preparations varied between 13 and 69% and was highest for the PLGA microparticles containing CNAC nanoparticles. This preparation also showed the slowest release with no initial burst release compared to all other preparations. Incorporation of TPP to this formulation increased the rate of protein release and reduced entrapment efficiency. PLGA microparticles containing chit/TPP-HA showed the fastest and near-complete release of ranibizumab. All of the prepared empty particles showed no effect on cell viability up to a concentration of 12.5 mg/mL. Ranibizumab released from all preparations maintained its structural integrity and in vitro activity. The chit/TPP-HA preparation enhanced antiangiogenic activity and may provide a potential biocompatible platform for enhanced antiangiogenic activity in combination with ranibizumab. In conclusion, the PLGA microparticles containing CNAC nanoparticles showed significantly improved ranibizumab loading and release profile. This novel drug delivery system may have potential for improved intravitreal delivery of therapeutic proteins, thereby reducing the frequency, risk, and cost of burdensome intravitreal injections.
Herein, the molecular pathogenic pathways implicated in renal injury triggered by amikacin (AK), together with the alleviating actions of β ‐caryophyllene (BCP), were investigated. Adult male Wistar rats ( n = 32) were disseminated to the four following groups ( n = 8/group): normal group, positive control animals (PC) that received AK intraperitoneal injections for 14 days (500 mg/kg/day), and rats that received AK simultaneously with small (200 mg/kg/day) and high doses (400 mg/kg/day) of BCP. The PC renal tissues revealed abnormal histology alongside increased apoptosis and significantly elevated serum creatinine and urea with marked proteinuria and oliguria relative to the normal rats. Moreover, renal tissues from the PC animals also showed substantial upregulations in NF‐ κ B/TGF‐ β /KIM‐1, whilst Nrf2/AMPK/AKT/PCNA declined, at the gene and protein levels in comparison to the normal rats. Additionally, the levels of markers of oxidative stress (MDA/H 2 O 2 /protein adducts) and inflammation (TNF‐ α /IL‐1 β /IL‐6/IL‐18/TLR/HSP25) were substantially higher in the PC renal specimens, whereas the antioxidants (GSH/GPx/SOD1/CAT) and interleukin‐10 decreased, relative to the NC group. Both BCP protocols improved the biochemical markers of renal functions, alleviated renal histopathology and apoptosis, and decreased NF‐ κ B/TGF‐ β /KIM‐1 alongside the concentrations of oxidative stress and proinflammatory markers, whilst promoting Nrf2/AMPK/AKT/IL‐10/PCNA and the targeted antioxidants. However, the improving effects in the high‐dose regimen were markedly stronger than those observed in animals treated with low dose of BCP. In conclusion, the present report is the first to connect NF‐ κ B/TGF‐ β /KIM‐1 proinflammatory and Nrf2/AMPK/AKT antioxidative stress pathways with the pathogenesis of AK‐induced nephrotoxicity. Additionally, the current report is the first to disclose alleviating activities for BCP against AK‐triggered nephrotoxicity by modulating multiple antioxidative stress with anti‐inflammatory molecular pathways.
The article presents the results of treatment of 157 patients with generalized periodontitis depending on blood type using polypeptide drugs. The concentration of total protein, C – reactive protein, IgG, TRACP, BSALP, albumin, amylase, lipase, Ca, Fe was determined in the oral fluid. The treatment allowed to reduce the content of total protein, C – reactive protein, TRACP in the oral fluid with increasing levels of calcium, Fe, BSALP, in relation to the relevant data before treatment. And also, polypeptide drugs helped to increase the level of albumin in representatives of 0 (I) and A (II) blood type in the oral fluid; contributed to a decrease: glucose, IgG with increasing activity, amylase and lipase in carriers of B (III) and AB (IV) blood type in the oral fluid, in relation to the values before treatment. The dynamics of indicators of oral fluid metabolism with the help of polypeptide drugs, convincingly proves the effectiveness of their use in the treatment of generalized periodontitis in patients with different blood type.
Leptin plays a key role in the pathogenesis of obesity and depression via the long form of leptin receptor (LepRb). An animal model of comorbid obesity and depression induced by high-fat diet (HFD) combined with chronic unpredictable mild stress (CUMS) was developed to study the relationship between depression/anxiety-like behavior, levels of plasma leptin and LepRb in the brains between four groups of rats, the combined obesity and CUMS (Co) group, the obese (Ob) group, the CUMS group and controls. Our results revealed that the Co group exhibited most severe depression-like behavior in the open field test (OFT), anxiety-like behavior in elevated plus maze test (EMT) and cognitive impairment in the Morris water maze (MWM). The Ob group had the highest weight and plasma leptin levels while the Co group had the lowest levels of protein of LepRb in the hypothalamus and hippocampus. Furthermore, depressive and anxiety-like behaviors as well as cognitive impairment were positively correlated with levels of LepRb protein and mRNA in the hippocampus and hypothalamus. The down-regulation of leptin/LepRb signaling might be associated with depressive-like behavior and cognitive impairment in obese rats facing chronic mild stress.
Acquiring information on the precise distribution of a tumor is essential to evaluate intratumoral heterogeneity. Conventional hematoxylin and eosin staining, which has been used by pathologists for more than 100 years, is the gold standard of tumor diagnosis. However, it is difficult to stain entire tumor tissues with hematoxylin and eosin and then acquire the three-dimensional distribution of cells in solid tumors due to difficulties in the staining and rinsing. In this paper, we propose a modified hematoxylin and eosin staining method, in which delipidation and ultrasound waves were applied to enhance tissue permeability and accelerate dye diffusion. This improved hematoxylin and eosin staining method is termed iHE (intact tissue hematoxylin and eosin staining). We applied the iHE method to stain intact organs of mice, which were then sectioned and imaged sequentially. The results showed that the whole tissue was stained homogeneously. Combined with micro-optical sectioning tomography (MOST), the iHE method can be used for 3D volume imaging and to evaluate the intratumoral heterogeneity of the entire tumor tissue spatially. Therefore, this method may help to accurately diagnose the invasion stage of tumors and guide clinical treatments.
Abstract Background Pulmonary fibrosis (PF) is a devastating disease characterized by remodeling of lung architecture and abnormal deposition of fibroblasts in parenchymal tissue and ultimately results in respiratory failure and death. Preclinical studies suggest that mesenchymal stem cell (MSC) administration may be a safe and promising option in treating PF. The objective of our meta-analysis is to assess the efficacy of MSC therapy in preclinical models of PF. Methods We performed a comprehensive literature search in PubMed, EMBASE, Web of Science, and Cochrane Library databases from inception to March 17, 2021. Studies that assessed the efficacy of MSC therapy to animals with PF were included. The SYRCLE bias risk tool was employed to evaluate the bias of included studies. The primary outcomes included survival rate and pulmonary fibrosis scores. Meta-analysis was conducted via Cochrane Collaboration Review Manager (version 5.4) and Stata 14.0 statistical software. Results A total of 1120 articles were reviewed, of which 24 articles met inclusion criteria. Of these, 12 studies evaluated the survival rate and 20 studies evaluated pulmonary fibrosis scores. Compared to the control group, MSC therapy was associated with an improvement in survival rate (odds ratios (OR) 3.10, 95% confidence interval (CI) 2.06 to 4.67, P < 0.001, I 2 = 0%) and a significant reduction in pulmonary fibrosis scores (weighted mean difference (WMD) 2.05, 95% CI −2.58 to −1.51, P < 0.001, I 2 = 90%). Conclusions MSC therapy is a safe and effective method that can significantly improve the survival and pulmonary fibrosis of PF animals. These results provide an important basis for future translational clinical studies.
Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms including wound healing disorder. The physiological process of wound healing is highly complex. The formation of granulation tissue is a key step in this process resulting in a preliminary wound closure and providing a network of new capillary blood vessels - either through vasculogenesis (novel formation) or angiogenesis (sprouting of existing vessels). Both vasculo- and angiogenesis require functional, directed migration of endothelial cells. Thus, investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of chemical induced wound healing disorders and to potentially identify novel strategies for therapeutic intervention. We assessed impaired wound healing after alkylating agent exposure and tested potential candidate compounds for treatment. We used a set of techniques outlined in this protocol. A modified Boyden chamber to quantitatively investigate chemokinesis of EEC is described. Moreover, the use of the wound healing assay in combination with track analysis to qualitatively assess migration is illustrated. Finally, we demonstrate the use of the fluorescent dye TMRM for the investigation of mitochondrial membrane potential to identify underlying mechanisms of disturbed cell migration. The following protocol describes basic techniques that have been adapted for the investigation of EEC.
Using morphological, histological, and TEM analyses of the cranium, we provide a detailed description of bone and suture growth in zebrafish. Based on expression patterns and localization, we identified osteoblasts at different degrees of maturation. Our data confirm that, unlike in humans, zebrafish cranial sutures maintain lifelong patency to sustain skull growth. The cranial vault develops in a coordinated manner resulting in a structure that protects the brain. The zebrafish cranial roof parallels that of higher vertebrates and contains five major bones: one pair of frontal bones, one pair of parietal bones, and the supraoccipital bone. Parietal and frontal bones are formed by intramembranous ossification within a layer of mesenchyme positioned between the dermal mesenchyme and meninges surrounding the brain. The supraoccipital bone has an endochondral origin. Cranial bones are separated by connective tissue with a distinctive architecture of osteogenic cells and collagen fibrils. Here we show RNA in situ hybridization for col1a1a, col2a1a, col10a1, bglap/osteocalcin, fgfr1a, fgfr1b, fgfr2, fgfr3, foxq1, twist2, twist3, runx2a, runx2b, sp7/osterix, and spp1/ osteopontin, indicating that the expression of genes involved in suture development in mammals is preserved in zebrafish. We also present methods for examining the cranium and its sutures, which permit the study of the mechanisms involved in suture patency as well as their pathological obliteration. The model we develop has implications for the study of human disorders, including craniosynostosis, which affects 1 in 2,500 live births.
Conventional microdiscectomy treatment for intervertebral disc herniation alleviates pain but does not repair the annulus fibrosus, resulting in a high incidence of recurrent herniation and persistent dysfunction. The lack of repair and the acute inflammation that arise after injury can further compromise the disc and result in disc-wide degeneration in the long term. To address this clinical need, we developed tension-activated repair patches (TARPs) for annulus fibrosus repair and local delivery of the anti-inflammatory factor anakinra (a recombinant interleukin-1 receptor antagonist). TARPs transmit physiologic strain to mechanically activated microcapsules embedded within the patch, which release encapsulated bioactive molecules in direct response to spinal loading. Mechanically activated microcapsules carrying anakinra were loaded into TARPs, and the effects of TARP-mediated annular repair and anakinra delivery were evaluated in a goat model of annular injury in the cervical spine. TARPs integrated with native tissue and provided structural reinforcement at the injury site that prevented aberrant disc-wide remodeling resulting from detensioning of the annular fibrosus. The delivery of anakinra by TARP implantation increased matrix deposition and retention at the injury site and improved maintenance of disc extracellular matrix. Anakinra delivery additionally attenuated the inflammatory response associated with TARP implantation, decreasing osteolysis in adjacent vertebrae and preserving disc cellularity and matrix organization throughout the annulus fibrosus. These results demonstrate the therapeutic potential of TARPs for the treatment of intervertebral disc herniation.
Fluoroquinolones (FQs) are synthetic and broad-spectrum antimicrobial drugs derived from nalidixic acid. FQs are used against SARS-CoV-2 in our country, and for the treatment of some urinary tract diseases, gastrointestinal diseases, respiratory tract diseases, sexually transmitted diseases, and dermatological diseases. The present study investigated the effect of 1-,7-,14-day treatments of three different FQ derivatives; ciprofloxacin (CIP) 80 mg/kg/day, levofloxacin (LVX) 40 mg/kg/day, and moxifloxacin (MXF) 40 mg/kg/day, on biochemical parameters, lipid peroxidation, antioxidant enzymes, and immunotoxicity. 72 Wistar albino male rats were distributed to four groups including 18 rats in each group and were sacrificed on three different time points. The 14-day treatment of MXF significantly reduced the levels of aspartate aminotransferase (AST), glucose, reduced glutathione (GSH), malondialdehyde (MDA), catalase (CAT), myeloperoxidase (MPO), adenosine deaminase (ADA), and glutathione peroxidase (GPx). Furthermore, 14-day treatment of LVX increased liver [GSH, MPO, ADA, superoxide dismutase (SOD)], and GSH (erythrocyte) levels; whereas it significantly reduced the levels of AST, TG (triglycerides) and associated parameters levels in all the tissues (MDA), erythrocytes, and liver (MPO, CAT, SOD, GPx). After 14-day treatment of CIP; the erythrocyte levels of GSH, MPO, GPx, and CAT significantly decreased; whereas the levels of glucose, creatinine, MPO (liver), and GST (kidney and erythrocyte) significantly increased. It has been concluded that FQ derivatives used in this experiment did not display any correlation in terms of the efficacies in the different time points and tissues. Thus, it is recommended to use such FQ derivatives considering the duration of use and target tissue.
// Bangwen Xie 1 , Marieke A. Stammes 1, 2, 3 , Pieter B.A.A. van Driel 1, 2 , Luis J. Cruz 1 , Vicky T. Knol-Blankevoort 1, 2 , Martijn A.M. Löwik 1 , Laura Mezzanotte 1 , Ivo Que 1 , Alan Chan 2 , Jeroen P.H.M. van den Wijngaard 4 , Maria Siebes 4 , Sven Gottschalk 5, 6 , Daniel Razansky 5, 6 , Vasilis Ntziachristos 5, 6 , Stijn Keereweer 1 , Richard W. Horobin 7 , Mathias Hoehn 1, 2, 3 , Eric L. Kaijzel 1 , Ermond R. van Beek 1, 8 , Thomas J.A. Snoeks 1 , Clemens W.G.M. Löwik 1 1 Department of Radiology, Leiden University Medical Center, Leiden, The Netherlands 2 Percuros BV, Enschede, The Netherlands 3 In-vivo -NMR Laboratory, Max Planck Institute for Neurological Research, Cologne, Germany 4 Department of Biomedical Engineering and Physics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands 5 Faculty of Medicine, Technical University of Munich, Munich, Germany 6 Institute for Biological and Medical Imaging, Helmholtz Center Munich, Munich, Germany 7 School of Life Sciences, College of Medical, Veterinary and Life Sciences, The University of Glasgow, University Avenue, Glasgow, Scotland, UK 8 Medres, Cologne, Germany Correspondence to: Clemens W.G.M. Löwik, e-mail: c.lowik@erasmusmc.nl Keywords: cell death, imaging, cyanines, necrosis avid contrast agents, cancer Received: June 29, 2015 Accepted: September 30, 2015 Published: October 12, 2015 ABSTRACT Quantification of tumor necrosis in cancer patients is of diagnostic value as the amount of necrosis is correlated with disease prognosis and it could also be used to predict early efficacy of anti-cancer treatments. In the present study, we identified two near infrared fluorescent (NIRF) carboxylated cyanines, HQ5 and IRDye 800CW (800CW), which possess strong necrosis avidity. In vitro studies showed that both dyes selectively bind to cytoplasmic proteins of dead cells that have lost membrane integrity. Affinity for cytoplasmic proteins was confirmed using quantitative structure activity relations modeling. In vivo results, using NIRF and optoacoustic imaging, confirmed the necrosis avid properties of HQ5 and 800CW in a mouse 4T1 breast cancer tumor model of spontaneous necrosis. Finally, in a mouse EL4 lymphoma tumor model, already 24 h post chemotherapy, a significant increase in 800CW fluorescence intensity was observed in treated compared to untreated tumors. In conclusion, we show, for the first time, that the NIRF carboxylated cyanines HQ5 and 800CW possess strong necrosis avid properties in vitro and in vivo . When translated to the clinic, these dyes may be used for diagnostic or prognostic purposes and for monitoring in vivo tumor response early after the start of treatment.
Fourier Transform Infrared (FTIR) micro-spectroscopy is an emerging technique for the biochemical analysis of tissues and cellular materials. It provides objective information on the holistic biochemistry of a cell or tissue sample and has been applied in many areas of medical research. However, it has become apparent that how the tissue is handled prior to FTIR micro-spectroscopic imaging requires special consideration, particularly with regards to methods for preservation of the samples. We have performed FTIR micro-spectroscopy on rodent heart and liver tissue sections (two spectroscopically very different biological tissues) that were prepared by desiccation drying, ethanol substitution and formalin fixation and have compared the resulting spectra with that of fully hydrated freshly excised tissues. We have systematically examined the spectra for any biochemical changes to the native state of the tissue caused by the three methods of preparation and have detected changes in infrared (IR) absorption band intensities and peak positions. In particular, the position and profile of the amide I, key in assigning protein secondary structure, changes depending on preparation method and the lipid absorptions lose intensity drastically when these tissues are hydrated with ethanol. Indeed, we demonstrate that preserving samples through desiccation drying, ethanol substitution or formalin fixation significantly alters the biochemical information detected using spectroscopic methods when compared to spectra of fresh hydrated tissue. It is therefore imperative to consider tissue preparative effects when preparing, measuring, and analyzing samples using FTIR spectroscopy.
The effect of myeloid leukemia, especially cute myeloid leukemia (AML), has been widely noticed on the parameters of liver and kidney functions and the levels of certain hormones. This study aimed to evaluate a number of biochemical parameters of liver and kidney functions and hormones in Iraqi subjects with newly diagnosed acute myeloid leukemia. Eighty newly diagnosed AML adult patients (40 males and 40 females) and forty healthy individuals (20 males and 20 females) with an age range of 16-75 years were involved in this study during their attendance at the Hematology Department of Baghdad Teaching Hospital/ Medical city in Baghdad province from March 2019 to February 2020. Blood samples were collected from all subjects for the determination of serum levels of the parameters of liver function parameters., kidney function , lactate dehydrogenase (LDH), and Erythropoietin (EPO). The results showed that the serum levels of liver function parameters (alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) had highly significant increases (p< 0.01) in AML patients (85.87±2.49 , 53.93±1.76, 150.87±7.04 U/L, respectively) as compared to the control (30.58 ±2.04, 22.89 ±0.97, 75.51 ±2.12 U/L, respectively ). Also, the level of kidney function parameters (blood urea, creatinine and uric acid) showed highly significant increases (p< 0.01) in AML patients (58.82 ±1.49, 1.831 ±0.05, 8.34 ±0.15 mg/dl, respectively) as compared to the control (31.10 ±1.03, 0.850 ±0.02, 4.81 ±0.14 mg/dl, respectively). In addition, the level of LDH showed a highly significant increase (p< 0.01) in the patients with AML (657.72 ±80.76 U/L) as compared to the control (166.05 ±6.15 U/L). Moreover, the level of EPO showed a highly significant increase (p<0.01) in the patients with AML (11763.80 ±329.46 pg/ml ) as compared to the control (316.94 ±34.42 pg/ml).
OBJECTIVE: The aim of the study was to investigate molecular mechanisms underlying the role of miR-126-5p in cisplatin (DDP) sensitivity of non-small-cell lung cancer (NSCLC). METHODS: The expression of miR-126-5p and ADAM9 in NSCLC cancer tissues and adjacent tissues, cisplatin-sensitive and drug-resistant NSCLC patient tissues, human normal lung epithelial cells (BESA-2B), human lung adenocarcinoma cell lines A549 and H1560, and cisplatin-resistant mutant cell lines A549/DDP and H1560/DDP was detected by qRT-PCR. After overexpression of miR-126-5p or ADAM9 in A549/DDP and H1560/DDP, MTT and clone formation were used to detect the cell proliferation ability of each treatment group. Flow cytometry was used to detect changes in cell apoptosis. The protein expression of ADAM9 and key molecules of PTEN/PI3K/Akt pathways in cells was measured by western blot. RESULTS: Compared with NSCLC adjacent tissues and NSCLC cisplatin-sensitive tissues, miR-126-5p expression was downregulated in NSCLC tissues and cisplatin-resistant NSCLC tissues and ADAM9 was upregulated. qRT-PCR further detected that miR-126-5p was downregulated in A549, H1560, and their cisplatin-resistant strains A549/DDP and H1560/DDP, while ADAM9 was upregulated. Moreover, overexpression of miR-126-5p inhibited A549/DDP and H1560/DDP cell proliferation and promoted cell apoptosis. The results of dual luciferase showed that miR-126-5p targeted and negatively regulated ADAM9. We also found that overexpression of ADAM9 could reverse the effects of miR-126-5p on NSCLC cell proliferation, apoptosis, and cisplatin sensitivity, and this effect may be achieved by inhibiting the activity of the PTEN/PI3K/Akt signaling pathway. CONCLUSION: Our data indicated that miR-126-5p may negatively regulate ADAM9 to promote the sensitivity of clinical DDP treatment of NSCLC and be a potential therapeutic target for NSCLC treatment.
Rapid and adequate vascularization is vital to the long-term success of porous orbital enucleation implants. In this study, porous hydroxyapatite (HA) scaffolds coated with vascular endothelial growth factor (VEGF)-functionalized collagen (COL)/heparin (HEP) multilayers (porosity 75%, pore size 316.8 ± 77.1 μm, VEGF dose 3.39 ng/mm(3)) were fabricated to enhance vascularization by inducing the differentiation of mesenchymal stem cells (MSCs) to endothelial cells. The in vitro immunofluorescence staining, quantitative real-time polymerase chain reaction (qRT-PCR), and western blotting results demonstrated that the expression of the endothelial differentiation markers CD31, Flk-1, and von Willebrand factor (vWF) was significantly increased in the HA/(COL/HEP)5/VEGF/MSCs group compared with the HA/VEGF/MSCs group. Moreover, the HA/(COL/HEP)5 scaffolds showed a better entrapment of the MSCs and accelerated cell proliferation. The in vivo assays showed that the number of newly formed vessels within the constructs after 28 d was significantly higher in the HA/(COL/HEP)5/VEGF/MSCs group (51.9 ± 6.3/mm(2)) than in the HA (26.7 ± 2.3/mm(2)) and HA/VEGF/MSCs (38.2 ± 2.4/mm(2)) groups. The qRT-PCR and west rn blotting results demonstrated that the HA/(COL/HEP)5/VEGF/MSCs group also had the highest expression of CD31, Flk-1, and vWF at both the mRNA and protein levels.
Since the majority of protein-coding genes in vertebrates have intra-genomic homologues, it has been difficult to eliminate the potential of functional redundancy from analyses of mutant phenotypes, whether produced by genetic lesion or transient knockdown. Further complicating these analyses, not all gene products have activities that can be assayed in vitro, where the efficiency of the various family members can be compared against constant substrates. Two vertebrate UNC-45 homologues, unc45a and unc45b, affect distinct stages of muscle differentiation when knocked down in cell culture and are functionally redundant in vitro. UNC-45 proteins are members of the UCS (UNC-45/CRO1/She4p) protein family that has been shown to regulate myosin-dependent functions from fungi to vertebrates through direct interaction with the myosin motor domain. To test whether the same functional relationship exists between these unc45 paralogs in vivo, we examined the developmental phenotypes of doubly homozygous unc45b(-/-); unc45a(-/-) mutant zebrafish embryos. We focused specifically on the combined effects on morphology and gene expression resulting from the zygotic lack of both paralogs. We found that unc45b(-/-) and unc45b(-/-); unc45a(-/-) embryos were phenotypically indistinguishable with both mutants displaying identical cardiac, skeletal muscle, and jaw defects. We also found no evidence to support a role for zygotic Unc45a function in myoblast differentiation. In contrast to previous in vitro work, this rules out a model of functional redundancy between Unc45a and Unc45b in vivo. Instead, our phylogenetic and phenotypic analyses provide evidence for the role of functional divergence in the evolution of the UCS protein family.
BACKGROUND: Temperature, humidity, vision, and particularly odor, are external cues that play essential roles to mosquito blood feeding and oviposition. Entomological and behavioral studies employ well-established methods to evaluate mosquito attraction or repellency and to identify the source of the blood meal. Despite the efficacy of such methods, the costs involved in the production or acquisition of all parts, components and the chemical reagents involved are unaffordable for most researchers from poor countries. Thus, a simple and relatively low-cost method capable of evaluating mosquito preferences and the blood volume ingested is desirable. PRINCIPAL FINDINGS: By using Evans blue (EB) vital dye and few standard laboratory supplies, we developed and validated a system capable of evaluating mosquito's choice between two different host sources of blood. EB-injected and PBS-injected mice submitted to a number of situations were placed side by side on the top of a rounded recipient covered with tulle fabric and containing Aedes aegypti mosquitoes. Homogenates from engorged mosquitoes clearly revealed the blood source (EB- or PBS-injected host), either visually or spectrometrically. This method was able to estimate the number of engorded mosquitoes, the volume of blood ingested, the efficacy of a commercial repellent and the attractant effects of black color and human sweat. SIGNIFICANCE: Despite the obvious limitations due to its simplicity and to the dependence of a live source of blood, the present method can be used to assess a number of host variables (diet, aging, immunity, etc) and optimized for several aspects of mosquito blood feeding and vector-host interactions. Thus, it is proposed as an alternative to field studies, and it could be used for initial screenings of chemical compound candidates for repellents or attractants, since it replicates natural conditions of exposure to mosquitoes in a laboratory environment.