100 research outputs found
Synthesis, Characterization, and In Vitro Photodynamic Activity of Novel Amphiphilic Zinc(II) Phthalocyanines Bearing Oxyethylene-Rich Substituents
Three novel zinc(II) phthalocyanines substituted with one or two 3,4,5-tris(3,6,9-trioxadecoxy)benzoxy group(s) have been prepared and spectroscopically characterized. These compounds are highly soluble and remain nonaggregated in N,N-dimethylformamide. Upon excitation, they exhibit a relatively weak fluorescence emission and high efficiency to generate singlet oxygen compared with the unsubstituted zinc(II) phthalocyanine. These amphiphilic photosensitizers formulated with Cremophor EL are highly photocytotoxic against HT29 human colon adenocarcinoma and HepG2 human hepatocarcinoma cells. The mono-α-substituted analogue 4 is particularly potent with IC50 values as low as 0.02 μM.
The higher photodynamic activity of this compound can be attributed to its lower aggregation tendency in the culture media as shown by absorption spectroscopy and higher cellular uptake as suggested by the stronger intracellular fluorescence, resulting in a higher efficiency to generate reactive oxygen species inside the cells
Photodynamic therapy inhibits p-glycoprotein mediated multidrug resistance via JNK activation in human hepatocellular carcinoma using the photosensitizer pheophorbide a
Characterization of aldehyde dehydrogenase isozymes in ovarian cancer tissues and sphere cultures
BACKGROUND: Aldehyde dehydrogenases belong to a superfamily of detoxifying enzymes that protect cells from carcinogenic aldehydes. Of the superfamily, ALDH1A1 has gained most attention because current studies have shown that its expression is associated with human cancer stem cells. However, ALDH1A1 is only one of the 19 human ALDH subfamilies currently known. The purpose of the present study was to determine if the expression and activities of other major ALDH isozymes are associated with human ovarian cancer and ovarian cancer sphere cultures. METHODS: Immunohistochemistry was used to delineate ALDH isozyme localization in clinical ovarian tissues. Western Blot analyses were performed on lysates prepared from cancer cell lines and ovarian cancer spheres to confirm the immunohistochemistry findings. Quantitative reverse transcription-polymerase chain reactions were used to measure the mRNA expression levels. The Aldefluor® assay was used to measure ALDH activity in cancer cells from the four tumor subtypes. RESULTS: Immunohistochemical staining showed significant overexpression of ALDH1A3, ALDH3A2, and ALDH7A1 isozymes in ovarian tumors relative to normal ovarian tissues. The expression and activity of ALDH1A1 is tumor type-dependent, as seen from immunohistochemisty, Western blot analysis, and the Aldefluor® assay. The expression was elevated in the mucinous and endometrioid ovarian epithelial tumors than in serous and clear cell tumors. In some serous and most clear cell tumors, ALDH1A1 expression was found in the stromal fibroblasts. RNA expression of all studied ALDH isozymes also showed higher expression in endometrioid and mucinous tumors than in the serous and clear cell subtypes. The expression of ALDH enzymes showed tumor type-dependent induction in ovarian cancer cells growing as sphere suspensions in serum-free medium. CONCLUSIONS: The results of our study indicate that ALDH enzyme expression and activity may be associated with specific cell types in ovarian tumor tissues and vary according to cell states. Elucidating the function of the ALDH isozymes in lineage differentiation and pathogenesis may have significant implications for ovarian cancer pathophysiology
Casein kinase I epsilon interacts with mitochondrial proteins for the growth and survival of human ovarian cancer cells
Robust estimation of bacterial cell count from optical density
Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data
Targeted gene sanger sequencing should remain the first-tier genetic test for children suspected to have the five common X-linked inborn errors of immunity
DATA AVAILABILITY STATEMENT : The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.To address inborn errors of immunity (IEI) which were underdiagnosed in resource-limited
regions, our centre developed and offered free genetic testing for the most common IEI by
Sanger sequencing (SS) since 2001. With the establishment of The Asian Primary
Immunodeficiency (APID) Network in 2009, the awareness and definitive diagnosis of
IEI were further improved with collaboration among centres caring for IEI patients from
East and Southeast Asia. We also started to use whole exome sequencing (WES) for
undiagnosed cases and further extended our collaboration with centres from South Asia
and Africa. With the increased use of Next Generation Sequencing (NGS), we have shifted
our diagnostic practice from SS to WES. However, SS was still one of the key diagnostic
tools for IEI for the past two decades. Our centre has performed 2,024 IEI SS genetic
tests, with in-house protocol designed specifically for 84 genes, in 1,376 patients with 744
identified to have disease-causing mutations (54.1%). The high diagnostic rate after just
one round of targeted gene SS for each of the 5 common IEI (X-linked
agammaglobulinemia (XLA) 77.4%, Wiskott–Aldrich syndrome (WAS) 69.2%, X-linked chronic granulomatous disease (XCGD) 59.5%, X-linked severe combined
immunodeficiency (XSCID) 51.1%, and X-linked hyper-IgM syndrome (HIGM1) 58.1%)
demonstrated targeted gene SS should remain the first-tier genetic test for the 5 common
X-linked IEI.The Hong Kong Society for Relief of Disabled Children and Jeffrey Modell Foundation.http://www.frontiersin.org/Immunologyam2023Paediatrics and Child Healt
Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples
Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts
Die Temperaturentwicklung während laserunterstützter Endodontie mit einem Diodenlaser 980nm unter Verwendung einer innovativen Pulsform
Zusammenfassung Einleitung: Ziel der In-Vitro-Studie war es, bei Anwendung eines Diodenlasers mit einer Wellenlänge von 980nm zur Wurzelkanalbehandlung zu untersuchen, ob der Temperatur-threshold von 7C im Wurzelkanal bzw.
in der Wurzelkanalumgebung während der Anwendung des Mikropuls-Modus überschritten wird.
Material und Methoden: Für die Durchführung der Studie wurden zehn extrahierte, menschliche, einwurzelige, nicht endodontisch vorbehandelte obere Frontzähne auf radiologisch verifizierte Arbeitslänge bis F4 mit ProTaper-Feilen aufbereitet und mit NaOCl, EDTA und Wasserstoffperoxid gespült. Die Zähne wurden in einer Haltevorrichtung montiert und mittels Diodenlaser (GENTLEray 980 Classic Plus, KaVo, Biberach, Deutschland) mit einer 300 m-Faser in vier unterschiedlichen Einstellungen/Gruppen bestrahlt. In Gruppe 1 (Kontrollgruppe) erfolgte die Bestrahlung im Puls-Modus mit 2,5 W. In Gruppe 2 bis 4 fand die Laserbestrahlung im Mikropuls-Modus statt (Gruppe 2 - 1,6 W, Gruppe 3 - 2,0 W und Gruppe 4 - 2,5 W). In allen Gruppen erfolgte die Bestrahlung in 6 Zyklen à 5 Sekunden mit je 20 Sekunden Pause. Die Temperaturentwicklung wurde mit einer Wärmebildkamera (EC 060; TROTEC, Heinsberg, Deutschland) über die gesamte Bestrahlungszeit gemessen und ausgewertet.
Resultate: Der höchste Temperaturanstieg wurde in Gruppe 2 mit 1,941,07C gemessen, gefolgt von Gruppe 4 (1,741,22C) und Gruppe 3 (1,581,18C). Die niedrigste Erhöhung war mit 1,061,20C in Gruppe 1 zu finden. Die Statistische Auswertung mittels ANOVA für repeated measurements ergab einen signifikanten Unterschied zwischen den Gruppen von p=0,041. Alle Gruppen wurden danach mittels t-Test paarweise miteinander verglichen und es zeigte sich ein signifikanter Unterschied zwischen den Gruppen 1&2 (p=0,007) und 1&3 (p=0,035), während der Vergleich der Gruppen 1&4 mit p=0,052 knapp zu einem nicht signifikanten Ergebnis führte. Zwischen den Gruppen 2 bis 4, das heißt zwischen Gruppen im Mikropuls-Modus, bestand kein signifikanter Unterschied. Schlussfolgerung: Die Studie zeigt auf, dass es zwar einen signifikanten Unterschied in der Temperaturerhöhung zwischen dem in der Endodontologie etablierten Pulsmodus und den Mikropuls-Modi zugunsten des Pulsmodus gibt, aber sie gibt uns auch einen eindeutigen Hinweis, dass die Bestrahlung mit den gewählten Parametern im Mikropuls-Modus für den Wurzelkanal und seine Umgebung bezüglich der Temperaturentwicklung unbedenklich ist, da der Temperatur-treshold kein einziges Mal überschritten wurde. Es bedarf jedoch weiterer Studien um den bakteriziden Effekt im Mikropuls-Modus zu untersuchen.Abstract Objective: The aim of the in vitro study was to investigate whether the temperature threshold of 7C temperature increase in the root canal and its surrounding area is exceeded by using a diode laser with a wavelength of 980nm in the micropulse mode during endodontic treatment.
Material and Methods: Ten freshly extracted human, single-rooted, not endodontically treated maxillary incisors were used. On a radiologically verified length the canals were enlarged up to an apical size of F4 with ProTaper files and irrigated with sodium hypochlorite, EDTA and hydrogen peroxide. Teeth were mounted to a holder and irradiated with a diode laser (GENTLEray 980 Classic Plus, KaVo, Biberach, Germany) with a 300 m fiber in four different settings. Group 1 (control group) was irradiated in 6 cycles of 5s irradiation / 20s pause with 2.5W in pulse mode. Groups 2 to 4 were irradiated accordingly in micropulse mode (group 2-1.6W; group 3-2.0W and group 4-2.5W). The temperature changes were continuously measured from start to end using a thermal imaging camera (EC 060; TROTEC, Heinberg, Germany).
Results: The maximum temperature rise was measured in Group 2 with 1.941.07C, followed by group 4 (1.741.22C) and group 3 (1.581.18C). The lowest increase occurred in Group 1 with 1.061.20C.
The statistical analysis via ANOVA for repeated measurements showed a significant difference (p=0.041) between the groups. All groups were then compared in pairs using t-test. Significant differences were found between groups 1&2 (p=0.007) and 1&3 (p=0.035). On the other hand a close non-significant difference between groups 1&4 (p=0.052) was noticed. Finally there was no significant difference when comparing groups 2, 3 and 4, which were all irradiated in micropulse mode.
Conclusion: The study demonstrated a significant difference in temperature rise between the endodontically established pulse mode and the micropulse modes. But it also indicates a harmless temperature development for the root canal and its surrounding area with chosen settings, because the temperature-threshold was never exceeded. However, further studies are required to investigate the bactericidal effect of the micropulse mode.eingereicht von Josip KopicAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZusammenfassung in engl. SpracheWien, Med. Univ., Dipl.-Arb., 2013(VLID)171393
Functional analysis of apoptosis-inducing factor in the human fungal pathogen Candida albicans
Introduction, aims & objectives Apoptosis is a highly organized cellular process crucial for growth and development. Apoptosis has been demonstrated in unicellular yeast Saccharomyces cerevisiae, but little is known in opportunistic human fungal pathogen Candida albicans. Previous studies showed that typical apoptotic features could be induced in C. albicans. Mitochondria are bifunctional organelles: they are energy powerhouse; but also set a central stage that leads to cell demise. Mitochondrial dysfunction has been regarded as the onset of apoptosis, manifested by depolarization of mitochondrial membrane potential (MMP), massive elevation of ROS, and release of apoptosis-inducing factor (AIF) and cytochrome c. Translocation of AIF from mitochondrial intermembrane space to nucleus causes chromatin condensation, DNA cleavage and cell death. No C. albicans AIF has been identified so far. We previously revealed that purpurin causes MMP depolarization without significant increase in intracellular ROS levels in Candida fungi, suggesting an existence of a mitochondrial-mediated cell death pathway. Searching the published C. albicans genome using S. cerevisiae AIF, we have identified three putative AIF sequences (orf19.1438, orf19.2175 and orf19.2671). The objectives of this project were: 1. To clone and express the three putative C. albicans AIF sequences; and 2. To determine the cellular localization and function role of AIF in energy production and cell death. Materials & methods The three putative C. albicans AIF sequences were cloned by PCR, expressed and purified from Escherichia coli BL21 as His-tag fusion proteins (CaAifp). Chromosomal green fluorescent protein-tagged AIF was constructed to detect cellular localization using confocal microscopy. The NADH oxidase activity of the purified CaAifp was determined by spectrophotometric method; and the cell death-related functions of the CaAifp were evaluated by examining sub-cellular translocation after apoptotic insults, ability to degrade DNA, and complementation to S. cerevisiae ∆aif1 mutant. Results The three putative C. albicans AIF sequences were cloned, expressed and purified to homogeneity from E. coli BL21 (i.e. CaAifp-orf19.1438, CaAifp-orf19.2175 and CaAifp-orf19.2671). CaAifp-orf19.2175 and CaAifp-orf19.2671 possessed NADH oxidase activity. CaAifp-orf19.2175 degraded DNA and functionally complemented S. cerevisiae ∆aif1 mutant. Conclusions The collective data of the present study suggest that orf19.2175 encodes a bona fide C. albicans AIF and the AIF-mediated cell death mechanism in C. albicans shares phylogenetic conservation to that in S. cerevisiae. Deciphering C. albicans AIF paves way for the elucidation of additional effector molecules in the mitochondrial-mediated cell death pathway and also casts light on the design of anti-Candidal strategy by promoting cell suicide
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