scholarly journals Characterisation of Neurospheres-Derived Cells from Human Olfactory Epithelium

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1690
Author(s):  
Elena A. Zelenova ◽  
Nikolay V. Kondratyev ◽  
Tatyana V. Lezheiko ◽  
Grigoriy Y. Tsarapkin ◽  
Andrey I. Kryukov ◽  
...  
Keyword(s):  
Expression Profiling ◽  
Immunofluorescence Microscopy ◽  
Differential Expression Analysis ◽  
Olfactory Receptors ◽  
Psychiatric Research ◽  
Sample Collection ◽  
Additional Protocol ◽  
Neuronal Progenitors ◽  
Olfactory Neuroepithelium

A major problem in psychiatric research is a deficit of relevant cell material of neuronal origin, especially in large quantities from living individuals. One of the promising options is cells from the olfactory neuroepithelium, which contains neuronal progenitors that ensure the regeneration of olfactory receptors. These cells are easy to obtain with nasal biopsies and it is possible to grow and cultivate them in vitro. In this work, we used RNAseq expression profiling and immunofluorescence microscopy to characterise neurospheres-derived cells (NDC), that simply and reliably grow from neurospheres (NS) obtained from nasal biopsies. We utilized differential expression analysis to explore the molecular changes that occur during transition from NS to NDC. We found that processes associated with neuronal and vascular cells are downregulated in NDC. A comparison with public transcriptomes revealed a depletion of neuronal and glial components in NDC. We also discovered that NDC have several metabolic features specific to neuronal progenitors treated with the fungicide maneb. Thus, while NDC retain some neuronal/glial identity, additional protocol alterations are needed to use NDC for mass sample collection in psychiatric research.

Progress and Challenges in Laboratory-Based Diagnostic and Screening Approaches for Aneuploidy Detection during Pregnancy

2021 ◽  
pp. 247263032110217
Author(s):  
Lindsay Schneider ◽  
Anubhav Tripathi
Keyword(s):  
Sample Collection ◽  
Aneuploidy Screening ◽  
Preimplantation Genetic Testing ◽  
Cellular Division ◽  
Vitro Fertilization ◽  
Genetic Abnormalities ◽  
The Social ◽  
Diagnostic Technologies ◽  
Screening Approaches

Aneuploidy is caused by problems during cellular division and segregation errors during meiosis that lead to an abnormal number of chromosomes and initiate significant genetic abnormalities during pregnancy or the loss of a fetus due to miscarriage. Screening and diagnostic technologies have been developed to detect this genetic condition and provide parents with critical information about their unborn child. In this review, we highlight the complexities of aneuploidy as a disease as well as multiple technological advancements in testing that help to identify aneuploidy at various time points throughout pregnancy. We focus on aneuploidy diagnosis during preimplantation genetic testing that is performed during in vitro fertilization as well as prenatal screening and diagnosis during pregnancy. This review focuses on DNA-based analysis and laboratory techniques for aneuploidy detection through reviewing molecular- and engineering-based technical advancements. We also present key challenges in aneuploidy detection during pregnancy, including sample collection, mosaic embryos, economic factors, and the social implications of this testing. The goal of this review is to synthesize broad information about aneuploidy screening and diagnostic sample collection and analysis during pregnancy and discuss major challenges the field is still facing despite decades of advancements.

Basic fibroblast growth factor induces retinal pigment epithelium to generate neural retina in vitro

Development ◽  
1991 ◽  
Vol 113 (2) ◽  
pp. 577-588 ◽  
Author(s):  
C. Pittack ◽  
M. Jones ◽  
T.A. Reh
Keyword(s):  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Basic Fibroblast Growth Factor ◽  
Retinal Pigment ◽  
Neural Retina ◽  
Phenotypic Switching ◽  
Surgical Removal ◽  
Fibroblast Growth ◽  
Neuronal Progenitors

During embryogenesis, the cells of the eye primordium are initially capable of giving rise to either neural retina or pigmented epithelium (PE), but become restricted to one of these potential cell fates. However, following surgical removal of the retina in embryonic chicks and larval amphibians, new neural retina is generated by the transdifferentiation, or phenotypic switching, of PE cells into neuronal progenitors. A recent study has shown that basic fibroblast growth factor (bFGF) stimulates this process in chicks in vivo. To characterize further the mechanisms by which this factor regulates the phenotype of retinal tissues, we added bFGF to enzymatically dissociated chick embryo PE. We found that bFGF stimulated proliferation and caused several morphological changes in the PE, including the loss of pigmentation; however, no transdifferentiation to neuronal phenotypes was observed. By contrast, when small sheets of PE were cultured as aggregates on a shaker device, preventing flattening and spreading on the substratum, we found that a large number of retinal progenitor cells were generated from the PE treated with bFGF. These results indicate that bFGF promotes retinal regeneration in vitro, as well as in ovo, and suggest that the ability of chick PE to undergo transdifferentiation to neuronal progenitors appears to be dependent on the physical configuration of the cells.

PSIII-11 The effect of feeding supplemental zeolite (clinoptilolite) of two different particle sizes on measures of nitrogen utilization and nutrient digestibility in finishing beef heifers

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 287-287
Author(s):  
Cheyanne A Myers ◽  
Mario de Haro Marti ◽  
Mireille Chahine ◽  
Gwinyai E Chibisa
Keyword(s):  
Particle Size ◽  
Nutrient Intake ◽  
Nutrient Digestibility ◽  
Particle Sizes ◽  
Sample Collection ◽  
Beef Heifers ◽  
Plasma Urea ◽  
Ruminal Ph ◽  
N Utilization

Abstract Clinoptilolite (CLN), could potentially improve nitrogen (N) utilization when fed to beef cattle as it can bind ruminal-ammonia-N (NH3-N), limiting its loss and subsequent detoxification into urea-N, which is released into blood and is excreted in urine. However, the effectiveness of CLN is influenced by physical properties such as particle size. Although decreasing the particle size has been shown to increase the binding of ammonium in-vitro, this remains to be evaluated in vivo. Therefore, the objective of this study was to determine the effects of feeding CLN of two different particle sizes (30 and 400 µm) on ruminal NH3-N and plasma-urea-N (PUN) concentrations, ruminal pH, and nutrient intake and apparent total-tract digestibility. Six ruminally-cannulated beef heifers (mean initial BW± SD, 620.8 ± 30.15) were used in a replicated 3 × 3 Latin square design with 21 d periods (sample collection from d 15 to 21). Dietary treatments were: 1) finishing ration with no supplement (CON), 2) CON +30-µm CLN (CL-30), and 3) CON + 400-µm CLN (CL-400). Clinoptilolite was top-dressed (2.5% of diet DM) during morning feeding. Intake was measured daily. Ruminal fluid was collected on d 19 for NH3-N analysis and blood was collected 3 h post-feeding on d 21 for PUN analysis. Indwelling pH loggers were used to measure ruminal pH (d 15 to 21) and grab fecal samples were collected from d 19 to 21 to determine total-tract nutrient digestibility. Statistical analysis was conducted using PROC MIXED in SAS. There was no treatment effect (P ≥ 0.13) on ruminal NH3-N and PUN concentrations, ruminal pH, and nutrient (DM, OM, NDF, ADF and CP) intake and apparent total tract digestibility. In conclusion, feeding CLN to finishing heifers had no effect on measures of N utilization, ruminal pH and nutrient intake and apparent total-tract digestibility.

scholarly journals Occupational safety of pressurized intraperitoneal aerosol chemotherapy (PIPAC)

2017 ◽  
Vol 2 (3) ◽  
pp. 121-128 ◽  
Author(s):  
Wouter Willaert ◽  
Paul Sessink ◽  
Wim Ceelen
Keyword(s):  
Detection Limit ◽  
Occupational Safety ◽  
Health Hazard ◽  
University Hospital ◽  
Sample Collection ◽  
Position Detection ◽  
Outer Pair ◽  
Environmental Air ◽  
Water Seal

AbstractBackgroundPressurized intraperitoneal aerosol chemotherapy (PIPAC) has emerged as a novel method to treat extensive, small volume peritoneal metastases. The clinical use of chemotherapy containing aerosols represents a potential occupational health hazard. We report the results of toxicological analysis during the first two clinical PIPAC procedures performed at Ghent University Hospital.MethodsAfter extensive preparation and in vitro testing, two patients were treated with PIPAC: the first using doxorubicin (2.86 mg in 51.43 mL) and cisplatin (14.28 mg in 164.3 mL), the second using oxaliplatin (182.10 mg in 186.42 mL). A standardized safety checklist was developed and used. Aerosol delivery was combined with electrostatic precipitation (ePIPAC). The following samples were obtained at several time points and locations: environmental air, floor surface wipes, surgeon’s gloves, surgeon’s hand wipes, circuit filters, and fluid from the water seal collection chamber container placed along the closed aerosol waste evacuating line. Platinum concentration was measured in these samples using voltammetry. Sample collection and analysis were performed by an independent external laboratory.ResultsPlatinum was not detected on the four floor locations after both procedures (detection limit 0.02 ng/cm2). Similarly, no platinum was detected in environmental air during both PIPACs at the surgeon’s or anesthesiologist’s position (detection limit 4.0–27 ng/m3). No platinum contamination was detected on the hands, outer pair of gloves, or inner pair of gloves of the surgeon (detection limit 70 and 50 ng respectively). Platinum was not detected on the filters and in the air-seal container liquid.ConclusionsWith adequate preparation and precautions, a clinical PIPAC program can be established without measurable chemotherapy exposure to the operating room environment or healthcare workers.

scholarly journals Relative messenger RNA expression profiling of collagenases and aggrecanases in human articular chondrocytes in vivo and in vitro

2002 ◽  
Vol 46 (10) ◽  
pp. 2648-2657 ◽  
Author(s):  
Brigitte Bau ◽  
Pia M. Gebhard ◽  
Jochen Haag ◽  
Thomas Knorr ◽  
Eckart Bartnik ◽  
...  
Keyword(s):  
Expression Profiling ◽  
Messenger Rna ◽  
Articular Chondrocytes ◽  
Human Articular Chondrocytes ◽  
Rna Expression ◽  
Rna Expression Profiling

scholarly journals 256CONSTRUCTION OF A BOVINE CDNA ARRAY TO MONITOR GENE EXPRESSION PROFILES IN BOVINE PREIMPLANTATION EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 248
Author(s):  
C. Wrenzycki ◽  
T. Brambrink ◽  
D. Herrmann ◽  
J.W. Carnwath ◽  
H. Niemann
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Cdna Array ◽  
Preimplantation Embryos ◽  
Average Insert Size ◽  
Rt Pcr ◽  
Public Data

Array technology is a widely used tool for gene expression profiling, providing the possibility to monitor expression levels of an unlimited number of genes in various biological systems including preimplantation embryos. The objective of the present study was to develop and validate a bovine cDNA array and to compare expression profiles of embryos derived from different origins. A bovine blastocyst cDNA library was generated. Poly(A+)RNA was extracted from in vitro-produced embryos using a Dynabead mRNA purification kit. First-strand synthesis was performed with SacIT21 primer followed by randomly primed second-strand synthesis with a DOP primer mix (Roche) and a global PCR with 35 cycles using SacIT21 and DOP primers. Complementary DNA fragments from 300 to 1500bp were extracted from the gel and normalized via reassoziation and hydroxyapatite chromatography. Resulting cDNAs were digested with SacI and XhoI, ligated into a pBKs vector, and transfected into competent bacteria (Stratagene). After blue/white colony selection, plasmids were extracted and the inserts were subjected to PCR using vector specific primers. Average insert size was determined by size idenfication on agarose gels stained with ethidium bromide. After purification via precipitation and denaturation, 192 cDNA probes were double-spotted onto a nylon membrane and bound to the membrane by UV cross linking. Amplified RNA (aRNA) probes from pools of three or single blastocysts were generated as described recently (Brambrink et al., 2002 BioTechniques, 33, 3–9) and hybridized to the membranes. Expression profiles of in vitro-produced blastocysts cultured in either SOF plus BSA or TCM plus serum were compared with those of diploid parthenogenetic ones generated by chemical activation. Thirty-three probes have been sequenced and, after comparison with public data bases, 26 were identified as cDNAs or genes. Twelve out of 192 (6%) seem to be differentially expressed within the three groups;; 7/12 (58%) were down-regulated, 3/12 (25%) were up-regulated in SOF-derived embryos, and 2/12 (20%) were up-regulated in parthenogenetic blastocysts compared to their in vitro-generated counterparts. Three of these genes involved in calcium signaling (calmodulin, calreticulin) and regulation of actin cytoskeleton (destrin) were validated by semi-quantitative RT-PCR (Wrenzycki et al., 2001 Biol. Reprod. 65, 309–317) employing poly(A+) RNA from a single blastocyst as starting material. No differences were detected in the relative abundance of the analysed gene transcripts within the different groups. These findings were confirmed employing the aRNA used for hybridization in RT-PCR and showed a good representativity of the selected transcripts. Results indicate that it is possible to construct a homologous cDNA array which could be used for gene expression profiling of bovine preimplantation embryos. Supported by the Deutsche Forschungsgemeinschaft (DFG Ni 256/18-1).

scholarly journals Genome-Wide microRNA Expression Profiling in Molecular Subgroups of Peripheral T-Cell Lymphoma Identified Role of Mir-126 in T-Cell Lymphomagenesis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2767-2767
Author(s):  
Waseem Lone ◽  
Alyssa Bouska ◽  
Tyler Herek ◽  
Catalina Amador ◽  
Mallick Saumyaranajn ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Expression Profiling ◽  
Cell Lymphoma ◽  
Ectopic Expression ◽  
T Cell Lymphoma ◽  
Molecular Subgroups ◽  
Peripheral T Cell Lymphoma ◽  
Tfh Cells

Peripheral T-cell lymphoma (PTCL) is a heterogeneous group of non-Hodgkin lymphomas and approximately 30% of PTCLs are designated as not-otherwise specified (PTCL-NOS). Gene expression profiling (GEP) identified molecular classifiers for PTCL entities and identified 2 novel biological subgroups within PTCL-NOS (PTCL-GATA3 and PTCL-TBX21), associated with T-cell differentiation subsets. To further investigate molecular oncogenesis, we performed microRNA expression profiling (miR-EP) in several molecular subtypes of PTCL including angioimmunoblastic T-cell lymphoma (AITL), PTCL-GATA3 and PTCL-TBX21 using formalin fixed paraffin embedded tissues. We also performed miR-EP of normal T-cell subsets polarized to represent different differentiation stages (TFH, TH1 and TH2). We performed miR-EP on 102 PTCL cases using either quantitative real time PCR (ABI, Biosystem) or ultra-sensitive direct miRNA counting (nCounter, NanoString). Corresponding GEP (mRNA) were available for 67 PTCL cases. Normal T-cells were polarized in-vitro with different cytokine milieu and examined by flow cytometry. We observed distinct miRNA profiles, with miRNA being uniquely expressed in TFH polarized cells (miR-26a-5p, miR-17-5p, miR-30d-5p, miR-22-3p, miR-222-3p, miR-142-3p, let-7i-5p and miR-29b-3p). In contrast, the TH1 lineage was enriched for expression of miR-155-5p, miR-146a-5p, miR-1246, miR-93-5p, miR-16-5p, miR-21-5p, miR-363-3p, miR-1260a, miR-186-5p, miR-148a-3p and miR-579-3p, whereas TH2 polarized cells expressed miR-181a-5p, let-7a-5p, miR-191-5p, miR-15b-5p, let-7d-5p, let-7b-5p, miR-140-5p, miR-98-5p, miR-423-5p and miR-630. Several of these miRNA expressed in the T-cells subsets showed corresponding expression in their respective PTCL entity such as miR-142-3p, let7i-5p, miR-21-5p and miR-29b-3p with AITL, miR-146-5p, miR-155-5p and miR-16-5p in PTCL-TBX21 and miR-181a-5p, miR-630 and let7a-5p in PTCL-GATA3. We also performed the MiRNA Enrichment Analysis and Annotation (miEAA) for miRNA signatures and observed an enrichment of miRNA regulating epigenetic modifications in TFH cells (p=0.028), whereas TH1 showed an enrichment of miRNA regulating IFN-g signaling (p=0.0024), and miRNA signatures in TH2 showed negative regulation of TGF-b signaling (p=0.023). Supervised analysis (p=0.05) of the miRNA profiles identified significant association of miR-126, miR-145, and let-7c-5p with AITL, when compared to other PTCLs. Similarly, miR-92a, miR-25, miR-636, miR-210, miR-222 and miR-491-5p significantly associated with PTCL-GATA3 and miRNA 126-3p, 145-5p, miR-26a-5p and miR-34a-5p associated with PTCL-TBX21. The miEAA for tumor miRNA signatures revealed enrichment of miRNAs regulating histone methylation (h3 k4 methylation) and chemokine receptor signaling in AITL, whereas miRNA regulating T-cell receptor were enriched in PTCL-TBX21 and TP53 signaling pathway in PTCL-GATA3. We validated the expression of miR-126 in AITL by qRT-PCR and also observed its increased expression in IL21 stimulated CD4+ T-cells. Ectopic expression of miR-126 resulted in a ~3 fold increased expression in T-cell lines and led to reduced proliferation and increased apoptosis with expression of T-cell exhaustion makers PD1 and TIM3. Computational algorithmic programs identified relevant biological targets of miR-126, including p85/PIK3R2, S1PR2 and DNMT3A that were further validated in-vitro. We observed an inverse correlation of miR-126 expression with S1PR2 expression (r=-0.64). S1PR2 is a crucial G protein-coupled receptor regulating B and T-cell migration in the germinal center (GC) reaction. Migration assays demonstrated significant decreases in T to B-cell migration, when B-cells (Raji) were co-cultured with Jurkat cells with ectopic expression of miR-126. With the GC reaction holding an important role in AITL, we investigated the biological significance of miRNA-126 in the context of the AITL microenvironment. High expression of miRNA-126 significantly associated with inferior survival in AITL (p=0.008) and significant differences in tumor microenvironment signatures. We identified distinct miRNA signatures for AITL and molecular subgroups of PTCL-NOS. Furthermore, elevated expression of miR-126 may contribute to the dysregulation and the homing of TFH cells in GC reaction through S1PR2 and warrants further mechanistic investigation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Detection of UV-Induced Thymine Dimers in Individual Cryptosporidium parvum and Cryptosporidium hominis Oocysts by Immunofluorescence Microscopy

2006 ◽  
Vol 73 (3) ◽  
pp. 947-955 ◽  
Author(s):  
B. H. Al-Adhami ◽  
R. A. B. Nichols ◽  
J. R. Kusel ◽  
J. O'Grady ◽  
H. V. Smith
Keyword(s):  
Cryptosporidium Parvum ◽  
Immunofluorescence Microscopy ◽  
Uv Light ◽  
Fluorescein Isothiocyanate ◽  
Immunofluorescence Assay ◽  
Cryptosporidium Hominis ◽  
Specific Fluorescence ◽  
Thymine Dimers ◽  
Texas Red

ABSTRACT To investigate the effect of UV light on Cryptosporidium parvum and Cryptosporidium hominis oocysts in vitro, we exposed intact oocysts to 4-, 10-, 20-, and 40-mJ�cm−2 doses of UV irradiation. Thymine dimers were detected by immunofluorescence microscopy using a monoclonal antibody against cyclobutyl thymine dimers (anti-TDmAb). Dimer-specific fluorescence within sporozoite nuclei was confirmed by colocalization with the nuclear fluorogen 4′,6′-diamidino-2-phenylindole (DAPI). Oocyst walls were visualized using either commercial fluorescein isothiocyanate-labeled anti-Cryptosporidium oocyst antibodies (FITC-CmAb) or Texas Red-labeled anti-Cryptosporidium oocyst antibodies (TR-CmAb). The use of FITC-CmAb interfered with TD detection at doses below 40 mJ�cm−2. With the combination of anti-TDmAb, TR-CmAb, and DAPI, dimer-specific fluorescence was detected in sporozoite nuclei within oocysts exposed to 10 to 40 mJ�cm−2 of UV light. Similar results were obtained with C. hominis. C. parvum oocysts exposed to 10 to 40 mJ�cm−2 of UV light failed to infect neonatal mice, confirming that results of our anti-TD immunofluorescence assay paralleled the outcomes of our neonatal mouse infectivity assay. These results suggest that our immunofluorescence assay is suitable for detecting DNA damage in C. parvum and C. hominis oocysts induced following exposure to UV light.

scholarly journals Complete biosynthetic pathways of ascofuranone and ascochlorin inAcremonium egyptiacum

2019 ◽  
Vol 116 (17) ◽  
pp. 8269-8274 ◽  
Author(s):  
Yasuko Araki ◽  
Takayoshi Awakawa ◽  
Motomichi Matsuzaki ◽  
Rihe Cho ◽  
Yudai Matsuda ◽  
...  
Keyword(s):  
Differential Expression Analysis ◽  
Cost Effective ◽  
Drug Candidate ◽  
P450 Monooxygenase ◽  
Genome Wide ◽  
Common Precursor ◽  
A Genome ◽  
Membrane Bound ◽  
The Cost

Ascofuranone (AF) and ascochlorin (AC) are meroterpenoids produced by various filamentous fungi, includingAcremonium egyptiacum(synonym:Acremonium sclerotigenum), and exhibit diverse physiological activities. In particular, AF is a promising drug candidate against African trypanosomiasis and a potential anticancer lead compound. These compounds are supposedly biosynthesized through farnesylation of orsellinic acid, but the details have not been established. In this study, we present all of the reactions and responsible genes for AF and AC biosyntheses inA. egyptiacum, identified by heterologous expression, in vitro reconstruction, and gene deletion experiments with the aid of a genome-wide differential expression analysis. Both pathways share the common precursor, ilicicolin A epoxide, which is processed by the membrane-bound terpene cyclase (TPC) AscF in AC biosynthesis. AF biosynthesis branches from the precursor by hydroxylation at C-16 by the P450 monooxygenase AscH, followed by cyclization by a membrane-bound TPC AscI. All genes required for AC biosynthesis (ascABCDEFG) and a transcriptional factor (ascR) form a functional gene cluster, whereas those involved in the late steps of AF biosynthesis (ascHIJ) are present in another distantly located cluster. AF is therefore a rare example of fungal secondary metabolites requiring multilocus biosynthetic clusters, which are likely to be controlled by the single regulator, AscR. Finally, we achieved the selective production of AF inA. egyptiacumby genetically blocking the AC biosynthetic pathway; further manipulation of the strain will lead to the cost-effective mass production required for the clinical use of AF.

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