Macrophage-mediated tissue response evoked by subchronic inhalation of lead oxide nanoparticles is associated with the alteration of phospholipases C and cholesterol transporters.

BACKGROUND: Inhalation of lead oxide nanoparticles (PbO NPs), which are emitted to the environment by high-temperature technological processes, heavily impairs target organs. These nanoparticles pass through the lung barrier and are distributed via the blood into secondary target organs, where they cause numerous pathological alterations. Here, we studied in detail, macrophages as specialized cells involved in the innate and adaptive immune response in selected target organs to unravel their potential involvement in reaction to subchronic PbO NP inhalation. In this context, we also tackled possible alterations in lipid uptake in the lungs and liver, which is usually associated with foam macrophage formation. RESULTS: The histopathological analysis of PbO NP exposed lung revealed serious chronic inflammation of lung tissues. The number of total and foam macrophages was significantly increased in lung, and they contained numerous cholesterol crystals. PbO NP inhalation induced changes in expression of phospholipases C (PLC) as enzymes linked to macrophage-mediated inflammation in lungs. In the liver, the subchronic inhalation of PbO NPs caused predominantly hyperemia, microsteatosis or remodeling of the liver parenchyma, and the number of liver macrophages also significantly was increased. The gene and protein expression of a cholesterol transporter CD36, which is associated with lipid metabolism, was altered in the liver. The amount of selected cholesteryl esters (CE 16:0, CE 18:1, CE 20:4, CE 22:6) in liver tissue was decreased after subchronic PbO NP inhalation, while total and free cholesterol in liver tissue was slightly increased. Gene and protein expression of phospholipase PLCß1 and receptor CD36 in human hepatocytes were affected also in in vitro experiments after acute PbO NP exposure. No microscopic or serious functional kidney alterations were detected after subchronic PbO NP exposure and CD68 positive cells were present in the physiological mode in its interstitial tissues. CONCLUSION: Our study revealed the association of increased cholesterol and lipid storage in targeted tissues with the alteration of scavenger receptors and phospholipases C after subchronic inhalation of PbO NPs and yet uncovered processes, which can contribute to steatosis in liver after metal nanoparticles exposure.

Fibroblast growth factor receptor influences primary cilium length through an interaction with intestinal cell kinase.

Vertebrate primary cilium is a Hedgehog signaling center but the extent of its involvement in other signaling systems is less well understood. This report delineates a mechanism by which fibroblast growth factor (FGF) controls primary cilia. Employing proteomic approaches to characterize proteins associated with the FGF-receptor, FGFR3, we identified the serine/threonine kinase intestinal cell kinase (ICK) as an FGFR interactor. ICK is involved in ciliogenesis and participates in control of ciliary length. FGF signaling partially abolished ICK's kinase activity, through FGFR-mediated ICK phosphorylation at conserved residue Tyr15, which interfered with optimal ATP binding. Activation of the FGF signaling pathway affected both primary cilia length and function in a manner consistent with cilia effects caused by inhibition of ICK activity. Moreover, knockdown and knockout of ICK rescued the FGF-mediated effect on cilia. We provide conclusive evidence that FGF signaling controls cilia via interaction with ICK.

The Manufacture of Xeno- and Feeder-Free Clinical-Grade Human Embryonic Stem Cell Lines: First Step for Cell Therapy.

Human embryonic stem cells (hESCs) are increasingly used in clinical trials as they can change the outcome of treatment for many human diseases. They are used as a starting material for further differentiation into specific cell types and to achieve the desirable result of the cell therapy; thus, the quality of hESCs has to be taken into account. Therefore, current good manufacturing practice (cGMP) has to be implemented in the transport of embryos, derivation of inner cell mass to xeno-free, feeder-free and defined hESC culture, and cell freezing. The in-depth characterization of hESC lines focused on safety, pluripotency, differentiation potential and genetic background has to complement this process. In this paper, we show the derivation of three clinical-grade hESC lines, MUCG01, MUCG02, and MUCG03, following these criteria. We developed and validated the system for the manufacture of xeno-free and feeder-free clinical-grade hESC lines that present high-quality starting material suitable for cell therapy according to cGMP.

Direct and Indirect Biomimetic Peptide Modification of Alginate: Efficiency, Side Reactions, and Cell Response.

In the fast-developing field of tissue engineering there is a constant demand for new materials as scaffolds for cell seeding, which can better mimic a natural extracellular matrix as well as control cell behavior. Among other materials, polysaccharides are widely used for this purpose. One of the main candidates for scaffold fabrication is alginate. However, it lacks sites for cell adhesion. That is why one of the steps toward the development of suitable scaffolds for cells is the introduction of the biofunctionality to the alginate structure. In this work we focused on bone-sialoprotein derived peptide (TYRAY) conjugation to the molecule of alginate. Here the comparison study on four different approaches of peptide conjugation was performed including traditional and novel modification methods, based on 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxy succinimide (EDC/NHS), 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMTMM), thiol-Michael addition and Cu-catalyzed azide-alkyne cycloaddition reactions. It was shown that the combination of the alginate amidation with the use of and subsequent Cu-catalyzed azide-alkyne cycloaddition led to efficient peptide conjugation, which was proven with both NMR and XPS methods. Moreover, the cell culture experiment proved the positive effect of peptide presence on the adhesion of human embryonic stem cells.

Aspects of embryo selection and their preparation for the formation of human embryonic stem cells intended for human therapy.

OBJECTIVE: The work deals with a clinical part of human embryonic stem cell (hESC) research. The aim of the project is the differentiation of somatic cell types, useful in drug development, regenerative medicine and cell therapy. The aim of this work is to enable targeted therapy of yet incurable diseases. The pluripotent hESCs have unlimited self-renewal capacity. This ability is used in therapy to create missing or damaged cells in the human body. It is of interest to develop clinical-grade hESC lines useful in preclinical and clinical studies. METHODS: The derivation of the hESC must respect the legislation of the Czech Republic and the EU. The aim was to develop an informed consent of both donors for donated discarded embryos that are not suitable for treatment by in vitro fertilization according to Directive 2004/23/EC. The FNBs Center for Assisted Reproduction (CAR) participates in oocyte collection, cultivation and cryopreservation of embryos, communication with clients and ensuring the informed consent of embryo donors. A transport protocol and a methodology for handing over the thawed embryos with the original numerical code were developed. Before the embryos are handed over to the ICRC co-authors workplace (CTEF), they are thawed and, if necessary, recultivated to the blastocyst stage; afterwards, assisted hatching is performed. RESULTS: In the period from January 2018 to July 2020, 138 selected suitable clients were asked for donations, with 52 not responding, 19 terminating and 29 extending the embryo storage. Only 38 clients, i.e. 27.5%, agreed with the usage of their embryos for the preparation of hESCs. In the same period, personal communication with suitable CAR clients took place and another 17 embryo donors were obtained. A total of 160 embryos were obtained from 55 donors aged 26 to 42 years. The embryos were most often frozen in the blastocyst (53 embryos - 33.1%) and morula (74 embryos - 46.3%) stages. Of the 29 genetically examined embryos, only 5 are euploid (17.2%), 2 are mosaic and 22 are aneuploid or with translocations or carriers with a monogenic defect. CONCLUSION: We have an informed consent prepared and approved by the Ethics Committee of the Masaryk University and the University Hospital Brno; 160 donated embryos have been selected and secured. A transport protocol and handover methodology are developed. The plan for the transfer of thawed anonymized embryos in the first phase, October - December 2020, includes approximately 5 thawed blastocysts per week with assisted hatching. After their transfer to the CTEF, the embryoblast will be isolated with subsequent cultivation. The established hESCs must meet the specified criteria of safety, stability and pluripotency. We believe that, in accordance with the project plan, we will obtain at least 3 clinical-grade hESC lines, the first created in the Czech Republic, respecting the requirements for Advanced Medicinal Therapy Products   (AMTP).

HMGB2 is a negative regulator of telomerase activity in human embryonic stem and progenitor cells.

High-mobility group box (HMGB)1 and HMGB2 proteins are the subject of intensive research because of their involvement in DNA replication, repair, transcription, differentiation, proliferation, cell signaling, inflammation, and tumor migration. Using inducible, stably transfected human embryonic stem cells (hESCs) capable of the short hairpin RNA-mediated knockdown (KD) of HMGB1 and HMGB2, we provide evidence that deregulation of HMGB1 or HMGB2 expression in hESCs and their differentiated derivatives (neuroectodermal cells) results in distinct modulation of telomere homeostasis. Whereas HMGB1 enhances telomerase activity, HMGB2 acts as a negative regulator of telomerase activity in the cell. Stimulation of telomerase activity in the HMGB2-deficient cells may be related to activation of the PI3K/protein kinase B/ glycogen synthase kinase-3ß/ß-catenin signaling pathways by HMGB1, augmented TERT/telomerase RNA subunit transcription, and possibly also because of changes in telomeric repeat-containing RNA (TERRA) and TERRA-polyA+ transcription. The impact of HMGB1/2 KD on telomerase transcriptional regulation observed in neuroectodermal cells is partially masked in hESCs by their pluripotent state. Our findings on differential roles of HMGB1 and HMGB2 proteins in regulation of telomerase activity may suggest another possible outcome of HMGB1 targeting in cells, which is currently a promising approach aiming at increasing the anticancer activity of cytotoxic agents.-Kucírek, M., Bagherpoor, A. J., Jaros, J., Hampl, A., Stros, M. HMGB2 is a negative regulator of telomerase activity in human embryonic stem and progenitor cells.

Ligase 3-mediated end-joining maintains genome stability of human embryonic stem cells.

Maintenance of human embryonic stem cells (hESCs) with stable genome is important for their future use in cell replacement therapy and disease modeling. Our understanding of the mechanisms maintaining genomic stability of hESC and our ability to modulate them is essential in preventing unwanted mutation accumulation during their in vitro cultivation. In this study, we show the DNA damage response mechanism in hESCs is composed of known, yet unlikely components. Clustered oxidative base damage is converted into DNA double-strand breaks (DSBs) by base excision repair (BER) and then quickly repaired by ligase (Lig)3-mediated end-joining (EJ). If there is further induction of clustered oxidative base damage by irradiation, then BER-mediated DSBs become essential in triggering the checkpoint response in hESCs. hESCs limit the mutagenic potential of Lig3-mediated EJ by DNA break end protection involving p53 binding protein 1 (53BP1), which results in fast and error-free microhomology-mediated repair and a low mutant frequency in hESCs. DSBs in hESCs are also repaired via homologous recombination (HR); however, DSB overload, together with massive end protection by 53BP1, triggers competition between error-free HR and mutagenic nonhomologous EJ.-Kohutova, A., Raska, J., Kruta, M., Seneklova, M., Barta, T., Fojtik, P., Jurakova, T., Walter, C. A., Hampl, A., Dvorak, P., Rotrekl, V. Ligase 3-mediated end-joining maintains genome stability of human embryonic stem cells.

A Clearance Period after Soluble Lead Nanoparticle Inhalation Did Not Ameliorate the Negative Effects on Target Tissues Due to Decreased Immune Response.

The inhalation of metal (including lead) nanoparticles poses a real health issue to people and animals living in polluted and/or industrial areas. In this study, we exposed mice to lead(II) nitrate nanoparticles [Pb(NO3)2 NPs], which represent a highly soluble form of lead, by inhalation. We aimed to uncover the effects of their exposure on individual target organs and to reveal potential variability in the lead clearance. We examined (i) lead biodistribution in target organs using laser ablation and inductively coupled plasma mass spectrometry (LA-ICP-MS) and atomic absorption spectrometry (AAS), (ii) lead effect on histopathological changes and immune cells response in secondary target organs and (iii) the clearance ability of target organs. In the lungs and liver, Pb(NO3)2 NP inhalation induced serious structural changes and their damage was present even after a 5-week clearance period despite the lead having been almost completely eliminated from the tissues. The numbers of macrophages significantly decreased after 11-week Pb(NO3)2 NP inhalation; conversely, abundance of alpha-smooth muscle actin (α-SMA)-positive cells, which are responsible for augmented collagen production, increased in both tissues. Moreover, the expression of nuclear factor κB (NF-κB) and selected cytokines, such as tumor necrosis factor alpha (TNFα), transforming growth factor beta 1 (TGFß1), interleukin 6(IL-6), IL-1α and IL-1ß , displayed a tissue-specific response to lead exposure. In summary, diminished inflammatory response in tissues after Pb(NO3)2 NPs inhalation was associated with prolonged negative effect of lead on tissues, as demonstrated by sustained pathological changes in target organs, even after long clearance period.

Dishevelled is a NEK2 kinase substrate controlling dynamics of centrosomal linker proteins.

Dishevelled (DVL) is a key scaffolding protein and a branching point in Wnt signaling pathways. Here, we present conclusive evidence that DVL regulates the centrosomal cycle. We demonstrate that DVL dishevelled and axin (DIX) domain, but not DIX domain-mediated multimerization, is essential for DVL's centrosomal localization. DVL accumulates during the cell cycle and associates with NIMA-related kinase 2 (NEK2), which is able to phosphorylate DVL at a multitude of residues, as detected by a set of novel phospho-specific antibodies. This creates interfaces for efficient binding to CDK5 regulatory subunit-associated protein 2 (CDK5RAP2) and centrosomal Nek2-associated protein 1 (C-NAP1), two proteins of the centrosomal linker. Displacement of DVL from the centrosome and its release into the cytoplasm on NEK2 phosphorylation is coupled to the removal of linker proteins, an event necessary for centrosomal separation and proper formation of the mitotic spindle. Lack of DVL prevents NEK2-controlled dissolution of loose centrosomal linker and subsequent centrosomal separation. Increased DVL levels, in contrast, sequester centrosomal NEK2 and mimic monopolar spindle defects induced by a dominant negative version of this kinase. Our study thus uncovers molecular crosstalk between centrosome and Wnt signaling.

Egg maturity assessment prior to ICSI prevents premature fertilization of late-maturing oocytes.

PROPOSE: The presence of metaphase II (MII) spindle together with the polar body (PB) indicates completion of oocyte maturation. This study was designed to explore if spindle imaging can be used to optimize timing of intracytoplasmic sperm injection (ICSI). METHODS: The study involved 916 oocytes from 234 conventionally stimulated ICSI cycles with an unexpectedly poor ovarian response. All PB-displaying oocytes were subjected to polarized light microscopy (PLM) prior to ICSI. When MII spindle was absent in the majority of oocytes, ICSI was postponed and performed after additional spindle imaging. Fertilization, embryo development, and clinical outcome were evaluated with respect to the observed spindle pattern. RESULTS: The visible spindle was absent in 32.64% of PB-displaying oocytes. The late-maturing oocytes extruding PB in vitro were less likely to exhibit a spindle signal than in vivo matured MII oocytes (38.86% vs. 89.84%). When fertilization was postponed, 59.39% of initially spindle-negative oocytes developed detectable MII spindle. Spindled eggs had significantly higher developmental potential, and the presence of the spindle has been identified as an independent measure for predicting the formation of the blastocyst. Embryos derived from spindle-positive oocytes also showed a higher chance to implant and develop to term. Notably, 11 children were conceived by finely timed fertilization of late-maturing oocytes which are normally discarded. CONCLUSIONS: The study confirms the prognostic value of spindle imaging and demonstrates that immature oocytes can be clinically utilized and give rise to live births when the timing of ICSI is adjusted to their developmental stage.

The frequency and consequences of multipolar mitoses in undifferentiated embryonic stem cells.

Embryonic stem (ES) cells are pluripotent cells widely used in cell therapy and tissue engineering. However, the broader clinical applications of ES cells are limited by their genomic instability and karyotypic abnormalities. Thus, understanding the mechanisms underlying ES cell karyotypic abnormalities is critical to optimizing their clinical use. In this study, we focused on proliferating human and mouse ES cells undergoing multipolar divisions. Specifically, we analyzed the frequency and outcomes of such divisions using a combination of time-lapse microscopy and cell tracking. This revealed that cells resulting from multipolar divisions were not only viable, but they also frequently underwent subsequent cell divisions. Our novel data also showed that in human and mouse ES cells, multipolar spindles allowed more robust escape from chromosome segregation control mechanisms than bipolar spindles. Considering the frequency of multipolar divisions in proliferating ES cells, it is conceivable that cell division errors underlie ES cell karyotypic instability.

An inactivating mutation in intestinal cell kinase, ICK, impairs hedgehog signalling and causes short rib-polydactyly syndrome.

The short rib polydactyly syndromes (SRPS) are a group of recessively inherited, perinatal-lethal skeletal disorders primarily characterized by short ribs, shortened long bones, varying types of polydactyly and concomitant visceral abnormalities. Mutations in several genes affecting cilia function cause SRPS, revealing a role for cilia function in skeletal development. To identify additional SRPS genes and discover novel ciliary molecules required for normal skeletogenesis, we performed exome sequencing in a cohort of patients and identified homozygosity for a missense mutation, p.E80K, in Intestinal Cell Kinase, ICK, in one SRPS family. The p.E80K mutation abolished serine/threonine kinase activity, resulting in altered ICK subcellular and ciliary localization, increased cilia length, aberrant cartilage growth plate structure, defective Hedgehog and altered ERK signalling. These data identify ICK as an SRPS-associated gene and reveal that abnormalities in signalling pathways contribute to defective skeletogenesis.

Presence of growth/differentiation factor-15 cytokine in human follicular fluid, granulosa cells, and oocytes.

PURPOSE: The purpose of the study was to determine whether the GDF-15 is present in follicular fluid; to evaluate if there is a relation between follicular and serum levels of GDF-15 and fertility status of study subjects; and to test whether granulosa cells, oocytes, or both produce GDF-15. METHODS: This study used follicular fluid (FF, serum, and oocytes obtained under informed consent from women undergoing oocyte retrieval for in vitro fertilization. It also used ovaries from deceased preterm newborns. Collection of FF and blood at the time of oocyte retrieval, ELISA and western blot were performed to determine levels and forms of GDF-15. Concentrations of GDF-15 in FF and serum, its expression in ovarian tissue, and secretion from granulosa cells were analyzed. RESULTS: GDF-15 concentration in FF ranged from 35 to 572 ng/ml, as determined by ELISA. Western blot analysis revealed the GDF-15 pro-dimer only in FF. Both normal healthy and cancerous granulosa cells secreted GDF-15 into culture media. Primary oocytes displayed cytoplasmic GDF-15 positivity in immunostained newborn ovaries, and its expression was also observed in fully grown human oocytes. CONCLUSIONS: To the best of our knowledge, this is the first documentation of cytokine GDF-15 presence in follicular fluid. Its concentration was not associated with donor/patient fertility status. Our data also show that GDF-15 is expressed and inducible in both normal healthy and cancerous granulosa cells, as well as in oocytes.

Cancer stem cell markers in pediatric sarcomas: Sox2 is associated with tumorigenicity in immunodeficient mice.

The three most frequent pediatric sarcomas, i.e., Ewing's sarcoma, osteosarcoma, and rhabdomyosarcoma, were examined in this study: three cell lines derived from three primary tumor samples were analyzed from each of these tumor types. Detailed comparative analysis of the expression of three putative cancer stem cell markers related to sarcomas-ABCG2, CD133, and nestin-was performed on both primary tumor tissues and corresponding cell lines. The obtained results showed that the frequency of ABCG2-positive and CD133-positive cells was predominantly increased in the respective cell lines but that the high levels of nestin expression were reduced in both osteosarcomas and rhabdomyosarcomas under in vitro conditions. These findings suggest the selection advantage of cells expressing ABCG2 or CD133, but the functional tests in NOD/SCID gamma mice did not confirm the tumorigenic potential of cells harboring this phenotype. Subsequent analysis of the expression of common stem cell markers revealed an evident relationship between the expression of the transcription factor Sox2 and the tumorigenicity of the cell lines in immunodeficient mice: the Sox2 levels were highest in the two cell lines that were demonstrated as tumorigenic. Furthermore, Sox2-positive cells were found in the respective primary tumors and all xenograft tumors showed apparent accumulation of these cells. All of these findings support our conclusion that regardless of the expression of ABCG2, CD133 and nestin, only cells displaying increased Sox2 expression are directly involved in tumor initiation and growth; therefore, these cells fit the definition of the cancer stem cell phenotype.

Inhaled Cadmium Oxide Nanoparticles: Their in Vivo Fate and Effect on Target Organs.

The increasing amount of heavy metals used in manufacturing equivalently increases hazards of environmental pollution by industrial products such as cadmium oxide (CdO) nanoparticles. Here, we aimed to unravel the CdO nanoparticle destiny upon their entry into lungs by inhalations, with the main focus on the ultrastructural changes that the nanoparticles may cause to tissues of the primary and secondary target organs. We indeed found the CdO nanoparticles to be transported from the lungs into secondary target organs by blood. In lungs, inhaled CdO nanoparticles caused significant alterations in parenchyma tissue including hyperemia, enlarged pulmonary septa, congested capillaries, alveolar emphysema and small areas of atelectasis. Nanoparticles were observed in the cytoplasm of cells lining bronchioles, in the alveolar spaces as well as inside the membranous pneumocytes and in phagosomes of lung macrophages. Nanoparticles even penetrated through the membrane into some organelles including mitochondria and they also accumulated in the cytoplasmic vesicles. In livers, inhalation caused periportal inflammation and local hepatic necrosis. Only minor changes such as diffusely thickened filtration membrane with intramembranous electron dense deposits were observed in kidney. Taken together, inhaled CdO nanoparticles not only accumulated in lungs but they were also transported to other organs causing serious damage at tissue as well as cellular level.

Tumor suppressor candidate 3 (TUSC3) prevents the epithelial-to-mesenchymal transition and inhibits tumor growth by modulating the endoplasmic reticulum stress response in ovarian cancer cells.

Ovarian cancer is one of the most common malignancies in women and contributes greatly to cancer-related deaths. Tumor suppressor candidate 3 (TUSC3) is a putative tumor suppressor gene located at chromosomal region 8p22, which is often lost in epithelial cancers. Epigenetic silencing of TUSC3 has been associated with poor prognosis, and hypermethylation of its promoter provides an independent biomarker of overall and disease-free survival in ovarian cancer patients. TUSC3 is localized to the endoplasmic reticulum in an oligosaccharyl tranferase complex responsible for the N-glycosylation of proteins. However, the precise molecular role of TUSC3 in ovarian cancer remains unclear. In this study, we establish TUSC3 as a novel ovarian cancer tumor suppressor using a xenograft mouse model and demonstrate that loss of TUSC3 alters the molecular response to endoplasmic reticulum stress and induces hallmarks of the epithelial-to-mesenchymal transition in ovarian cancer cells. In summary, we have confirmed the tumor-suppressive function of TUSC3 and identified the possible mechanism driving TUSC3-deficient ovarian cancer cells toward a malignant phenotype.

Toward structured macroporous hydrogel composites: electron beam-initiated polymerization of layered cryogels.

The ability to tailor mechanical properties and architecture is crucial in creating macroporous hydrogel scaffolds for tissue engineering. In the present work, a technique for the modification of the pore size and stiffness of acrylamide-based cryogels is demonstrated via the regulation of an electron beam irradiation dose. The samples were characterized by equilibrium swelling measurements, light and scanning electron microscopy, mercury porosimetry, Brunauer-Emmett-Teller surface area analysis, and stiffness measurements. Their properties were compared to cryogels prepared by a standard redox-initiated radical polymerization. A (125)I radiolabeled azidopentanoyl-GGGRGDSGGGY-NH2 peptide was bound to the surface to determine the concentration of the adhesive sites available for biomimetic modification. The functionality of the prepared substrates was evaluated by in vitro cultivation of adipose-derived stem cells. Moreover, the feasibility of preparing layered cryogels was demonstrated. This may be the key to the future preparation of complex hydrogel-based scaffolds to mimic the extracellular microenvironment in a wide range of applications.

Use of flower-like gold nanoparticles in time-of-flight mass spectrometry.

RATIONALE: Many kinds of nanoparticles (NPs) have been used for mass spectrometry (MS) so far. Here we report the first use of flower-like gold nanoparticles (AuNPs) as a mediator to enhance ionization in MS of peptides and proteins. METHODS: Flower-like AuNPs were characterized using transmission and scanning electron microscopy, UV-VIS spectrophotometry, and laser desorption/ionization (LDI)-MS and compared with polyhedral AuNPs. Mass spectra were obtained in positive ion mode using a time-of-flight (TOF) analyzer coupled with either matrix-assisted laser desorption/ionization (MALDI) or surface-assisted laser desorption/ionization (SALDI) methods. RESULTS: The intensities of peptide peaks (m/z 500-3500) were up to 7.5× and up to 7× higher using flower-like AuNPs and flower-like AuNPs-enriched α-cyano-4-hydroxycinnamic acid (CHCA) matrix respectively, than the classical CHCA matrix. The signals of higher mass peptide/protein peaks (m/z 3600-17000) were up to 2× higher with using flower-like AuNPs-enriched CHCA matrix than conventional CHCA matrix. The signal of profile peaks generated by intact cell MALDI-TOFMS of fibroblast suspension (m/z 4000-20000) was 2× higher with using flower-like AuNPs combined with sinapinic acid (SA) compared to SA matrix alone. The use of flower-like AuNPs as internal calibration standard for the calibration of MS spectra of peptides was performed. CONCLUSIONS: Flower-like AuNPs and flower-like AuNPs combined with CHCA or SA as combined matrices for MS measurement of peptides and proteins were used. Comparison of the conventional MALDI method and our method with flower-like AuNPs was carried out. In addition, gold clusters generated from flower-like AuNPs by SALDI provide a suitable internal calibration standard for MS analysis of peptides. Copyright © 2015 John Wiley & Sons, Ltd.

MicroRNA-650 expression is influenced by immunoglobulin gene rearrangement and affects the biology of chronic lymphocytic leukemia.

MicroRNAs (miRNAs) play a key role in chronic lymphocytic leukemia as well as in normal B cells. Notably, miRNA gene encoding miR-650 and its homologs overlap with several variable (V) subgenes coding for lambda immunoglobulin (IgLλ). Recent studies describe the role of miR-650 in solid tumors, but its role in chronic lymphocytic leukemia (CLL) has not yet been studied. Our experiments demonstrate that miR-650 expression is regulated by coupled expression with its host gene for IgLλ. This coupling provides a unique yet unobserved mechanism for microRNA gene regulation. We determine that higher expression of miR-650 is associated with a favorable CLL prognosis and influences the proliferation capacity of B cells. We also establish that in B cells, miR-650 targets proteins important in cell proliferation and survival: cyclin dependent kinase 1 (CDK1), inhibitor of growth 4 (ING4), and early B-cell factor 3 (EBF3). This study underscores the importance of miR-650 in CLL biology and normal B-cell physiology.

Decrease in abundance of apurinic/apyrimidinic endonuclease causes failure of base excision repair in culture-adapted human embryonic stem cells.

The inevitable accumulation of chromosomal abnormalities in human embryonic stem cells (hESCs) during in vitro expansion represents a considerable obstacle for cell replacement therapies. To determine the source of chromosomal abnormalities, we examined hESCs maintained in culture for over 55 months for defects in telomere maintenance and DNA repair. Although prolonged culture affected neither telomerase activity nor nonhomologous end joining, the efficiency of base excision repair (BER) was significantly decreased and correlated with reduced expression of apurinic/apyrimidinic endonuclease 1 (APE1), the major nuclease required for BER. Interestingly, the expression of other BER enzymes was unchanged. Addition of human recombinant APE1 protein to nuclear extracts from late passage hESCs increased BER efficiency to the level typical of early passage hESCs. The link between BER and double-strand breaks (DSB) was demonstrated by decreased DSB release after downregulation of APE1 in early passage hESCs via siRNA. Correspondingly lower APE1 level in late passage hESC resulted in slower and less intensive but long lasting DSB release upon ionizing radiation (IR). Downregulation of APE1 in early passage hESCs also led to approximately 30% decrease in γ-H2AX signaling following IR, similar to that in late passage hESCs. We suggest that downregulation of APE1 significantly contributes to the failure of BER during long-term culture of hESCs, and further that BER failure is one of the factors affecting the genomic instability of hESCs by altering BER-dependent DSB release and cell cycle/checkpoint signaling.