Molecular Biology

The mechanisms by which microRNAs control pediatric high-grade gliomas (pHGGs) have yet to be fully elucidated. Our studies of patient-derived pHGG tissues and of the pHGG cell line KNS42 revealed down-regulation in these tumors of three microRNAs, specifically miR-107, miR-181c, and miR-29a-3p. This down-regulation increases the proliferation of KNS42 cells by de-repressing expression of the Notch2 receptor (Notch2), a validated target of miR-107 and miR-181c and a putative target of miR-29a-3p. Inhibition (either pharmacologic or genetic) of Notch2 or re-expression of the implicated microRNAs (all three combined but also individually) significantly reduced KNS42 cell proliferation. These findings suggest that Notch2 pathway activation plays a critical role in pHGGs growth and reveal a direct epigenetic mechanism that controls Notch2 expression, which could potentially be targeted by novel forms of therapy for these childhood tumors characterized by high-morbidity and high-mortality.

The amino acid sequence of the matrix protein of the human respiratory syncytial virus (RS virus) was deduced from the sequence of a cDNA insert in a recombinant plasmid harboring an almost full-length copy of this gene. It specifically hybridized to a single 1,050-base mRNA from infected cells. The recombinant containing 944 base pairs of RS viral matrix protein gene sequence lacked five nucleotides corresponding to the 5' end of the mRNA. The nucleotide sequence of the 5' end of the mRNA was determined by the dideoxy sequencing method and found to be 5' NGGGC, wherein the C residue is one nucleotide upstream of the cloned viral sequence. The initiator ATG codon for the matrix protein is embedded in an AATATGG sequence similar to the canonical PXXATGG sequence present around functional eucaryotic translation initiation codons. There is no conserved sequence upstream of the polyadenylate tail, unlike vesicular stomatitis virus and Sendai virus, in which four nucleotides ...

To study the effect of the serotonergic brain system on verbal fluency (i.e., the ability to rapidly extract necessary words from the internal vocabulary), the T102C polymorphism of the serotonin receptor type 2A (5-HTR2A) gene was tested for association with verbal fluency in 108 patients with schizophrenia or disorders of the schizophrenic spectrum and 97 mentally healthy individuals. A significant association was observed only in male schizophrenics (n = 67), with homozygotes A2A2 having lower verbal fluency. The results do not support the association between the 5-HTR2A polymorphism and verbal fluency in normalcy, and agree with the assumed contribution of genotype A2A2 to the severity of schizophrenia.

Over the last decade, liquid biopsy has gained much attention as a powerful tool in personalized medicine since it enables monitoring cancer evolution and follow-up of cancer patients in real time. Through minimally invasive procedures, liquid biopsy provides important information through the analysis of circulating tumour cells (CTCs) and circulating tumour-derived material, such as circulating tumour DNA (ctDNA), circulating miRNAs (cfmiRNAs) and extracellular vehicles (EVs). CTC analysis has already had an important impact on the prognosis, detection of minimal residual disease (MRD), treatment selection and monitoring of cancer patients. Numerous clinical trials nowadays include a liquid biopsy arm. CTC analysis is now an exponentially expanding field in almost all types of solid cancers. Functional studies, mainly based on CTC-derived cell-lines and CTC-derived explants (CDx), provide important insights into the metastatic process. The purpose of this review is to summarize the...

We directly compared two different approaches used for Circulating Tumor Cell (CTC) isolation, a size-dependent microfluidic system versus an EpCAM-dependent positive selection for downstream molecular characterization of CTC both at the gene expression and DNA methylation level in Head and Neck Squamous Cell Carcinoma (HNSCC). A size-dependent microfluidic device (Parsortix, ANGLE) and an EpCAM-dependent positive immune-magnetic isolation procedure were applied in parallel, using 10 mL PB from 50 HNSCC patients and 18 healthy donors. Total RNA was isolated from enriched CTCs and RT-qPCR was used to study the expression levels of CK-19, PD-L1, EGFR, TWIST1, CDH2 and B2M (reference gene). Real time methylation specific PCR (MSP) was used to study the methylation status of RASSF1A and MLL3 genes. In identical blood draws, the label-free size-dependent CTC-isolation system was superior in terms of sensitivity when compared to the EpCAM-dependent CTC enrichment, since a significantly hi...

Citrus Psorosis Virus (CPsV) can be detected by ELISA, but current protocols use polyclonal antisera. The availability of these antisera is limited, and new production is hampered by difficulties met with purification of the virus. Several specific monoclonal antibodies were recently produced, and we report how those have been successfully utilized for the development of an all-monoclonal antibody-based ELISA kit.

Günümüzde seri kapasitörler uzun iletim hatlarında, kayıpların azaltılması ve transfer kapasitesinin artırılmasında sıklıkla kullanılmaktadır. Kapasitörler aynı zamanda şebekenin gerilim kararlılığının iyileştirilmesinde de önemli rol oynamaktadır. Sağladığı bu olumlu katkıların yanında seri kapasitörlerin bulundukları hattaki mesafe koruma rölelerinin çalışmasını olumsuz yönde etkilediği bilinmektedir. Bu çalışmada seri kapasitör bulunan şebekelerde arıza yerinin tespiti için k-EYK sınıflandırma yöntemi kullanılarak geniş bölge izleme tabanlı bir koruma algoritması önerilmiştir. Algoritmanın testi Digsilent Power Factory programında oluşturulan radyal şebekede yapılmıştır. Test için oluşturulan kısa devre senaryolarında farklı arıza empedansları altında 3-faz dengeli ve tek-faz toprak arızaları analiz edilmiştir. Elde edilen sonuçlara göre önerilen algoritmanın hatların belirli noktalarında oluşturulan kısa devrelerde arızalı hattı doğru bir şekilde tespit edebildiği görülmüştür. Ç...

We investigated solution-grown single crystals of multidimensional 2D–3D hybrid lead bromide perovskites using spatially resolved photocurrent and photoluminescence. Scanning photocurrent microscopy (SPCM) measurements where the electrodes consisted of a dip probe contact and a back contact. The crystals revealed significant differences between 3D and multidimensional 2D–3D perovskites under biased detection, not only in terms of photocarrier decay length values but also in the spatial dynamics across the crystal. In general, the photocurrent maps indicate that the closer the border proximity, the shorter the effective decay length, thus suggesting a determinant role of the border recombination centers in monocrystalline samples. In this case, multidimensional 2D–3D perovskites exhibited a simple fitting model consisting of a single exponential, while 3D perovskites demonstrated two distinct charge carrier migration dynamics within the crystal: fast and slow. Although the first one ...

sion of 3-galactosidase incell cultures; butitisnotpossible to monitor expression inindividual cells andtoanalyze the heterogeneity ofexpression incell populations. Theuse ofa fluorogenic substrate, however, makesitpossible todetermine ,B-galactosidase activity ina verylarge numberofindividual cells bymeans offlowcytometry. Thistypeofdetermination can bemore informative withregard tothephysiology ofthe cells, since geneexpression canbecorrelated withthestagein themitotic cycle ortheviability undercertain conditions. This methodology canalso beofspecial interest fromtheviewpoint ofbiotechnology, inwhichcells transformed withunstable plasmids

p38α mitogen-activated protein (MAP) kinase is a broadly expressed signaling molecule that participates in the regulation of cellular responses to stress as well as in the control of proliferation and survival of many cell types. We have used cell lines derived from p38α knockout mice to study the role of this signaling pathway in the regulation of apoptosis. Here, we show that cardiomyocytes and fibroblasts lacking p38α are more resistant to apoptosis induced by different stimuli. The reduced apoptosis of p38α-deficient cells correlates with decreased expression of the mitochondrial proapoptotic protein Bax and the apoptosis-inducing receptor Fas/CD-95. Cells lacking p38α also have increased extracellular signal-regulated kinase (ERKs) MAP kinase activity, and the up-regulation of this survival pathway seems to be at least partially responsible for the reduced levels of apoptosis in the absence of p38α. Phosphorylation of the transcription factor STAT3 on Ser-727, mediated by the e...

Botulinum neurotoxin type E (BoNT/E), the fastest acting toxin of all BoNTs, cleaves the 25 kDa synaptosomal-associated protein (SNAP-25) in motor neurons, leading to flaccid paralysis. The specific detection and quantification of the BoNT/E-cleaved SNAP-25 neoepitope can facilitate the development of cell-based assays for the characterization of anti-BoNT/E antibody preparations. In order to isolate highly specific monoclonal antibodies suitable for the in vitro immuno-detection of the exposed neoepitope, mice and rabbits were immunized with an eight amino acid peptide composed of the C-terminus of the cleaved SNAP-25. The immunized rabbits developed a specific and robust polyclonal antibody response, whereas the immunized mice mostly demonstrated a weak antibody response that could not discriminate between the two forms of SNAP-25. An immune scFv phage-display library was constructed from the immunized rabbits and a panel of antibodies was isolated. The sequence alignment of the i...

In December 2019, the first cases of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were identified in the city of Wuhan, China. Since then, it has spread worldwide with new mutations being reported. The aim of the present study was to monitor the changes in genetic diversity and track non-synonymous substitutions (dN) that could be implicated in the fitness of SARS-CoV-2 and its spread in different regions between December 2019 and November 2020. We analyzed 2213 complete genomes from six geographical regions worldwide, which were downloaded from GenBank and GISAID databases. Although SARS-CoV-2 presented low genetic diversity, there has been an increase over time, with the presence of several hotspot mutations throughout its genome. We identified seven frequent mutations that resulted in dN substitutions. Two of them, C14408T>P323L and A23403G>D614G, located in the nsp12 and Spike protein, respectively, emerged early in the pandemic and showed a consi...

Human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G, hereinafter referred to as A3G) is an innate virus restriction factor that inhibits human immunodeficiency virus type 1 (HIV-1) replication and induces excessive deamination of cytidine residues in nascent reverse transcripts. To test the hypothesis that this enzyme can also help generate viral sequence diversification and the evolution of beneficial viral variants, we have examined the impact of A3G on the acquisition of (−)2′,3′-dideoxy-3′-thiacytidine (3TC) resistance in vitro . That characteristic resistance mutations are rapidly fixed in the presence of A3G and 3TC suggests that A3G-mediated editing can be an important source of genetic variation on which natural selection acts to shape the structure of HIV-1 populations.

Growing evidence that the CD3γ gene is specifically targeted in some T cell diseases focused our attention on the need to identify and characterize the elusive elements involved in CD3γ transcriptional control. In this study, we show that while the human CD3γ and CD3δ genes are oriented head-to-head and separated by only 1.6 kb, the CD3γ gene is transcribed from an independent but weak, lymphoid-specific TATA-less proximal promoter. Using RNA ligase-mediated rapid amplification of cDNA ends, we demonstrate that a cluster of transcription initiation sites is present in the vicinity of the primary core promoter, and the major start site is situated in a classical initiator sequence. A GT box immediately upstream of the initiator binds Sp family proteins and the general transcription machinery, with the activity of these adjacent elements enhanced by the presence of a second GC box 10 nt further upstream. The primary core promoter is limited to a sequence that extends upstream to −15 a...

The locus glc (min 64.5), associated with the glycolate utilization trait in Escherichia coli, is known to contain glcB, encoding malate synthase G, and the gene(s) needed for glycolate oxidase activity. Subcloning, sequencing, insertion mutagenesis, and expression studies showed five additional genes: glcC and in the other direction glcD, glcE, glcF, and glcG followed by glcB. The gene glcC may encode the glc regulator protein. Consistently a chloramphenicol acetyltransferase insertion mutation abolished both glycolate oxidase and malate synthase G activities. The proteins encoded from glcD and glcE displayed similarity to several flavoenzymes, the one from glcF was found to be similar to iron-sulfur proteins, and that from glcG had no significant similarity to any group of proteins. The insertional mutation by a chloramphenicol acetyltransferase cassette in either glcD, glcE, or glcF abolished glycolate oxidase activity, indicating that presumably these proteins are subunits of th...

Objectif : La chimiotherapie a fait ses preuves au fils des annees comme traitement principal permettant de detruire les cellules cancereuses presentes dans plusieurs types de cancers. La doxorubicine a longtemps ete utilisee comme un agent anti-cancerigene dans la chimiotherapie mais elle entraine des effets secondaires dans le corps tels que la suppression de plaquettes et de globules blancs, le developpement de l’alopecie, ou encore la perte de myofibrille dans les parois du coeur. De plus, la doxorubicine peut amener des effets cardiaques comme un arret du coeur. L’objectif de ce travail est de proposer des solutions pour detecter les effets cardiotoxiques de cet agent, au moyen de la spectrometrie Raman et de la rheologie, en faisant des tests moleculaires et mecaniques sur des coeurs de porc. Materiel et methodes : Des echantillons de coeur de porc ont ete recuperes d’une etude dans laquelle cinq miniporcs Yucatan femelles ont recu une dose totale de 375mg/m2 de doxorubicine a...

Fusarium head blight (FHB) is a global cereal disease caused by a complex of Fusarium species. Both Fusarium graminearum and F. asiaticum are the causal agents of FHB in China. F. asiaticum is the predominant species in the Middle–Lower Reaches of the Yangtze River (MLRYR) and southwest China. Therefore, detecting F. asiaticum in a timely manner is crucial for controlling the disease and preventing mycotoxins from entering the food chain. Here, we combined rapid genomic DNA extraction, recombinase polymerase amplification, Cas12a cleavage, and lateral flow detection techniques to develop a method for the rapid detection of F. asiaticum. The reaction conditions were optimized to provide a rapid, sensitive, and cost-effective method for F. asiaticum detection. The optimized method demonstrated exceptional specificity in detecting F. asiaticum while not detecting any of the 14 other Fusarium strains and 3 non-Fusarium species. Additionally, it could detect F. asiaticum DNA at concentra...

This study was designed with the aim to determine the impact of residual antibiotics on haematological and biochemical constituents of broiler chickens’ blood. For this, one-day-old chickens were randomly divided into four equal groups with 10 individuals in each group (n = 40). All groups of chickens fed with commercial basal diet; the first group considered as control, fed only with basal ration; the feed of the second, third and fourth groups of chicken were supplemented with amoxystin, tetracycline and chloramphenicol, respectively. Antibiotics were given daily, individually, in sub-therapeutic concentrations: amoxystin at the dosage 10 mg kg-1, tetracycline and chloramphenicol – 20 mg kg-1 of the chicken’s weight for 41 days. The blood parameters were measured at the end of experiment on the 42nd day. In comparison with the control group, decreasing of leukocytes was observed in the 2nd and 4th groups, and increasing in the 3rd group of broilers. The concentration of erythrocyt...

The dark green leaves of Sauropus androgynus (Euphorbiaceae) have various nutritive values and are commonly used for human consumption in Southeast Asia. Shoot cultures of this plant were established by adding various concentrations of kinetin (Kn) and 6-benzyl adenine (BA). The best results were recorded when Kn 0.1 ppm was used with BA 1 ppm (BA1Kn0,1 ). Spectrophotometric analysis showed two peaks of green pigment in shoot cultures, Y pigment (λmax = 663.6 - 663.8 nm, absorbance 0.1111) and Z pigment (λmax = 611.3 - 613.9 nm, absorbance 0.0390). Thin Layer Chromatography (TLC) analysis method showed two green spots (Rf Y = 0.3125 and Rf Z = 0.25) of shoot cultures on medium suplemented with BA1Kn0,1 during 10 days. Pigment profiles of shoot cultures were similar to their corresponding mother plants. Random Amplified Polymorphic DNA (RAPD), used as a preliminary technique to evaluate genetic similarity of the shoot cultures and their corresponding mother plants, showed five simila...

The dark green leaves of Sauropus androgynus (Euphorbiaceae) have various nutritive values and are commonly used for human consumption as food, medicine, and natural dye substance in South-east Asia. Shoot cultures of this plant were established by adding various concentrations of kinetin (Kn) and benzyl adenine (BA) using nodal explants. The best results were recorded when Kn 0.1 mg/L was used with BA 1 mg/L (BA1Kn0.1). Spectrophotometric analysis showed two peaks of green pigment in shoot cultures, A pigment (λmax = 663.6 - 663.8 nm, absorbance 0.1111) and B pigment (λmax= 611.3 - 613.9 nm, absorbance 0.0390). Thin Layer Chromatography (TLC) analysis showed two green spots (Rf Y = 0.31 and Rf Z = 0.25) of shoot cultures on medium supplemented with BA1Kn0.1 for 10 days. Pigment profiles of shoot culltures were similar to their corresponding mother plants. Random Amplified Polymorphic DNA (RAPD) was used as a preliminary technique to evaluate the genetic similarity of the shoot cult...

We have examined the hypothesis that tumor cells can stimulate a respiratory burst by human natural killer (NK) cells in vitro as measured by luminol-dependent chemiluminescence (CL). Percoll-purified NK cells, containing 40% HNK-1+ cells and less than 1 to 4% esterase-positive contaminating monocytes, can generate a strong CL response after stimulation with the NK-susceptible K562 tumor but not with the NK-resistant P815 tumor cells. Although the response was NK dependent, as shown by depletion with NK-directed monoclonal antibodies (HNK-1, OKT-11, and OKM-1), the cell generating the CL response was not the NK cell. On the basis of several independent experimental approaches the CL response always required the presence of monocytes in the NK preparation. a) Treatment with a monocyte-specific monoclonal antibody (MO2) and complement completely abolished CL. b) The cells producing the CL response were strongly adherent to nylon wool columns (NWC), and large granular lymphocyte prepar...

Aberrant regulation of WNT/β-catenin signaling has a crucial role in the onset and progression of cancers, where the effects are not always predictable depending on tumor context. In melanoma, for example, models of the disease predict differing effects of the WNT/β-catenin pathway on metastatic progression. Understanding the processes that underpin the highly context-dependent nature of WNT/β-catenin signaling in tumors is essential to achieve maximal therapeutic benefit from WNT inhibitory compounds. In this study, we have found that expression of the tumor suppressor, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), alters the invasive potential of melanoma cells in response to WNT/β-catenin signaling, correlating with differing metabolic profiles. This alters the bioenergetic potential and mitochondrial activity of melanoma cells, triggered through regulation of pro-survival autophagy. Thus, WNT/β-catenin signaling is a regulator of catabolic processes in cancer c...

The gene for mouse placental lactogen II (mPL-II) has been isolated and characterized. This gene contains five exons, with a transcription start site 59 nucleotides upstream of the translation initiation ATG. Introduction of a DNA construct containing 2.7 kilobases of sequence upstream of the mPL-II transcription initiation site directed the synthesis of a linked coding region for the simian virus 40 large and small tumor antigens in placental trophoblast giant cells of transgenic mice. The pattern of simian virus 40 transgene expression in the placenta was indistinguishable from that of the endogenous mPL-II gene. In contrast, the first 569 base pairs upstream of the transcription start site proved insufficient to direct placental expression. Thus, one or more elements required for placental trophoblast giant cell expression have been localized to a region between -2700 and -569 of the mPL-II gene.

Mutations at the mouse limb deformity (ld) locus result in defects of growth and patterning of the limb and kidney during embryonic development. The gene responsible for this phenotype is large and complex, with the capacity to generate a number of alternatively spliced messenger RNA transcripts encoding nuclear protein isoforms called "formins." We have made polyclonal antibodies to specific formin peptides and have confirmed the authenticity of the antibodies' reactivity, using cell lines derived from mice with molecularly defined mutations at the ld locus. In addition, we have used these antibodies to detect and characterize polypeptides encoded by both wild-type and mutant ld alleles. In so doing, we show that a formin isoform (i) is modified by posttranslational phosphorylation at serine and threonine residues and (ii) when present in a crude nuclear extract, is retained by DNA-cellulose.

To help elucidate the mechanisms by which nerve growth factor (NGF) regulates gene expression, we have identified and studied four genes (a-2, d-2, d-4, and d-5) that are positively regulated by NGF in PC12 cells, including one (d-2) which has previously been identified as a putative transcription factor (NGF I-A). Three of these genes, including d-2, were induced very rapidly at the transcriptional level, but the relative time courses of transcription and mRNA accumulation of each of these three genes were distinct. The fourth gene (d-4) displayed no apparent increase in transcription that corresponded to the increase in its mRNA, suggesting that NGF may regulate its expression at a posttranscriptional level. Thus, NGF positively regulates gene expression by more than one mechanism. These genes could also be distinguished on the basis of their response to cyclic AMP. The expression of d-2 and a-2 was increased by cholera toxin and further augmented by NGF; however, cholera toxin no...