scholarly journals Γενετική χαρτογράφηση στο κυπαρίσσι (Cupressus sempervirens L.)

2014 ◽  
Author(s):  
Ευαγγελία Αβραμίδου
Keyword(s):  
De Novo ◽  
Cupressus Sempervirens ◽  
Seiridium Cardinale

Το κυπαρίσσι (Cupressus sempervirens L.) αποτελεί σημαντικό είδος της Μεσογειακής χλωρίδας και αποτελεί ένα από τα κύρια είδη των ευαίσθητων μεσογειακών οικοσυστημάτων που παρουσιάζει ευρεία αντοχή σε αντίξοες περιβαλλοντικές συνθήκες. Απαντάται σε δύο κύριες μορφές λόγω της ιδιαιτερότητας της μορφής της κόμης που παρουσιάζει: την οριζοντιόκλαδη (Cupressus sempervirens var. horizontalis) και την ορθόκλαδη μορφή (Cupressus sempervirens var. pyramidalis). Η παρούσα διδακτορική διατριβή εστιάστηκε στη λεπτομερή χαρτογράφηση του γονιδιώματος του κυπαρισσιού. Με τη χρήση 24 f-AFLP και οκτώ f-SSR εκκινητών προέκυψαν 1332 γονιδιακές θέσεις εκ των οποίων οι 1260 ήταν πολυμορφικές. Δημιουργήθηκαν τέσσερεις γενετικοί χάρτες με χρήση χαρτογραφικής οικογένειας που προήλθε από ελεγχόμενη διασταύρωση ενός οριζοντιόκλαδου θηλυκού και ενός ορθόκλαδου αρσενικού γονέα και αποτελούνταν από 382 απογόνους. Ο χάρτης πλαισίου της οριζοντιόκλαδης ποικιλίας (μητρικός) περιελάμβανε έξι ομάδες σύνδεσης συνολικού μήκους 818.58 cM και ο αντίστοιχος της ορθόκλαδης ποικιλίας (πατρικός) πέντε ομάδες σύνδεσης συνολικού μήκους γονιδιώματος 560.33 cM. Ο ενιαίος χάρτης πλαισίου που δημιουργήθηκε χρησιμοποιώντας τις γονιδιακές θέσεις f-AFLP με αναλογία διαχωρισμού 1:1 και τις f-SSR γονιδιακές θέσεις, αποτελείται από 11 ομάδες σύνδεσης συνολικού μήκους 1278.74 cM. Προσθέτοντας τις f-AFLP γονιδιακές θέσεις με αναλογία διαχωρισμού 3:1 το συνολικό μήκος του ολοκληρωμένου χάρτη ήταν 2.374,41 cM. Επιπρόσθετα με τη μέθοδο χαρτογράφησης διαστημάτων βρέθηκε ένα QTL που είναι συνδεδεμένο με μια f-AFLP γονιδιακή θέση στο 11ο χρωμόσωμα και σχετίζεται στατιστικά σημαντικά (LOD=3.30) με τη μορφή της κόμης. Ο δείκτης αυτός ενδέχεται να αποτελέσει μοριακό δείκτη πρώιμης επιλογής για τη μορφή της κόμης, μπορεί δηλαδή να εφαρμοστεί η υποβοηθούμενη από γονίδια σημάνσεως επιλογή (MAS) και η διερεύνηση ύπαρξης αντίστοιχων QTL σε άλλα είδη. Επιπλέον ανιχνεύθηκαν δύο QTLs με ασθενέστερη σύνδεση σε δείκτες f-AFLP στο 3ο και 11ο χρωμόσωμα για τα ποσοτικό γνώρισμα του ύψους (LOD= 1.65 και LOD=1.63, 0.001<p<0.004). Το έλκος του κυπαρισσιού προκαλείται από το μύκητα Seiridium cardinale και είναι μια από τις πιο καταστρεπτικές ασθένειες για την οικογένεια των Cupressaceae. Η αντοχή της χαρτογραφικής οικογένειας στο μύκητα εξετάστηκε χρησιμοποιώντας την τεχνική του εμβολιασμού με τον μύκητα. Μετά την πάροδο έξι μηνών καταγράφηκαν τα ποσοστά ανθεκτικών, μερικώς ανθεκτικών και ευαίσθητων στο μύκητα ατόμων που ήταν 22.64%, 70.94% και 6.42% αντίστοιχα. Στην περαιτέρω διερεύνηση, μέσω γενετικής χαρτογράφησης QTL βρέθηκαν δύο QTL ασθενώς συνδεδεμένα με δύο δείκτες f-AFLP, με τιμές LOD=1.78 (0.01<p<0.04) και LOD=1.48 (0.01<p<0.04) στo 10ο και στο 3ο χρωμόσωμα αντίστοιχα. Σημαντικό ήταν επίσης το γεγονός ότι βρέθηκε ένας γενότυπος που παρουσίασε 100% ανθεκτικότητα στο μύκητα Seiridium cardinale. Πραγματοποιήθηκε επίσης βελτιστοποίηση in-vitro πρωτοκόλλου (συνδυασμό MS και SH πρωτοκόλλων), η οποία προσφέρει τη δυνατότητα αγενούς αναπαραγωγής του ανθεκτικού κλώνου με δυνατότητα μαζικής φύτευσης του σε περιοχές της Ελλάδας με σοβαρά προβλήματα προσβολής. Τέλος μελετήθηκε η επιγενετική κληρονομησιμότητα στη χαρτογραφική οικογένεια του κυπαρισσιού χρησιμοποιώντας f-MSAP μοριακούς δείκτες. Χρησιμοποιώντας τέσσερις συνδυασμούς f-MSAP προέκυψαν 266 πολυμορφικές γονιδιακές θέσεις. Τα αποτελέσματα έδειξαν ότι η μέση τιμή της συνολικής μεθυλίωσης του DNA στους απογόνους (28.2%) ήταν υψηλότερη από τη μέση τιμή των γονικών τύπων. Επίσης βρέθηκε υψηλότερη μητρική κληρονομησιμότητα της μεθυλίωσης του DNA (5.65%) έναντι της πατρικής (3.01%). Η πιστή Μενδελική κληρονομησιμότητα μεθυλιωμένων θέσεων παρουσιάστηκε σε ένα μικρό ποσοστό (4.29%). Βρέθηκε ένα μεγάλο ποσοστό de novo μεθυλίωσης (19.65%) στους απογόνους σε σχέση με τους γονικούς τύπους.

In vitro expression of resistance responses to Seiridium species in micropropagated shoots of Cupressus sempervirens and Chamaecyparis lawsoniana

1997 ◽  
Vol 75 (7) ◽  
pp. 1103-1109 ◽  
Author(s):  
K. A. Spanos ◽  
A. Pirrie ◽  
S. Woodward
Keyword(s):  
Cell Walls ◽  
Guard Cells ◽  
Cupressus Sempervirens ◽  
Fungal Hyphae ◽  
In Vitro Inoculation ◽  
Host Tree Species ◽  
Stomatal Guard Cells ◽  
Seiridium Cardinale ◽  
Chamaecyparis Lawsoniana

Wounded and nonwounded micropropagated shoots of Cupressus sempervirens and Chamaecyparis lawsoniana were inoculated in vitro with the canker-causing pathogens Seiridium cardinale (Wag.) Sutton & Gibson, Seiridium cupressi (Guba) Boeswinkel and Seiridium unicorne (Cke & Ell.) Sutton. Seiridium cardinale was significantly more pathogenic on Cupressus sempervirens than on Chamaecyparis lawsoniana (Murr.) Parlatore, irrespective of the presence of wounds on the shoots. On wounded shoots, both S. cupressi and S. unicorne caused significantly larger lesions on Chamaecyparis lawsoniana than on Cupressus sempervirens by 20 days after inoculation. Superficial wounding of shoots prior to inoculation caused a significant increase in the lengths of lesions and numbers of shoots girdled by the pathogens on both hosts. These results broadly correlate with known virulence of the three pathogens on these two host tree species in field and glasshouse tests. Using histological methods, penetration of fungal hyphae through stomatal pores of both shoots and leaves into the substomatal cavity and the mesophyll space was observed. Penetration directly through the cuticle was also seen. Defence-related responses, including accumulation of oxidized polyphenols compounds and deposition of lignin and suberin in cell walls, were detected in inoculated tissues. These responses occurred predominantly in the epidermis, including stomatal guard cells, and the hypodermis and were particularly marked in Chamaecyparis lawsoniana inoculated with S. cardinale. The possible utility of these methods in the study and detection of host genotypes resistant to Seiridium spp. is discussed. Key words: Seiridium, Cupressus, Chamaecyparis, micropropagation, in vitro inoculation, defence.

Einfluß der Glucose auf die [14C] Thymidin-Einbaurate von Ehrlich-Ascitescarcinomzellen in vitro

1969 ◽  
Vol 08 (02) ◽  
pp. 196-206 ◽  
Author(s):  
Dieter. Kummer
Keyword(s):  
De Novo

ZusammenfassungIn nahezu glucosefreier Suspension von Ehrlich-Ascitescarcinomzellen bewirkt die Zufuhr von Glucose 2,5 × 10–4 bis 10–2 M:1. Hemmung der [14C] Thymidin-Einbaurate in die Zellen.2. Aktivierung des Ribonucleotid-Reductase-Systems und damit Stimulierung der Desoxyribonucleotidsynthese (auch der Thymidintriphosphat-de-novo-Synthese).3. Blockierung der Thymidinkinase über Endprodukthemmung, wodurch die Minderung des [14C] Thymidin-Einbaus in die Zellen erklärbar ist.

Особенности морфогенеза редкого вида Allium neriniflorum (Herb.) Backer в условиях in vitro

2019 ◽  
pp. 37-41 ◽  
Author(s):  
Альбина Шамсуновна Ахметова ◽  
Альфия Ануровна Зарипова
Keyword(s):  
De Novo

Показана возможность эффективного применения метода культуры тканей для размножения Allium neriniflorum (Herb.) Backer. Исследуемый вид является декоративным растением, размножение которого затруднено из-за низкой всхожести семян и ослабленной способности к формированию дочерних луковиц. Разработана технология клонального микроразмножения из стерильных луковиц. В качестве исходного материала использовали семена A. neriniflorum. Подобраны условия стерилизации, позволяющие достичь максимального числа (75 %) жизнеспособных эксплантов. Поверхностную стерилизацию проводили в ламинар-боксе с использованием в качестве стерилизующего агента 0,1 % раствор диацида. Семена сначала обрабатывали 70 % этанолом, затем стерилизующим раствором. Экспозиция стерилизующих растворов составляла от 5 до 9 мин. Показано, что способность к индуцированному морфогенезу существенно зависит от состава питательной среды. Максимальное число луковиц образовывалось на среде QL — 9 шт./эксплант. Исследуемые виды обладали высокой способностью к мультипликации и формированию полноценных растений при подобранных условиях культивирования in vitro. Выявленная морфогенетическая активность зачаточного побега, сегментов чешуй и донца стерильной луковицы A. neriniflorum, проявляющаяся в способности регенерировать побеги de novo, что возможно только в культуре in vitro, обеспечивает формирование полноценных луковиц. Луковицы, полученные in vitro, включали в последующие циклы микроразмножения. Культура тканей и органов in vitro позволяет размножать A. neriniflorum с более высоким коэффициентом размножения. От одной стерильной луковицы можно получить до 7000 луковиц в год. При традиционном вегетативном способе размножения материнская луковица формирует 1, редко 2 дочерние луковицы.

Study of Phosphocalcic Glasses from SiO2-CaO-P2O5 System with and Without Silver II. The bioactivity analysis by FTIR, SEM methods and microbiological study of silver-doped glasses

2017 ◽  
Vol 68 (6) ◽  
pp. 1188-1192
Author(s):  
Daniela Avram ◽  
Nicolae Angelescu ◽  
Dan Nicolae Ungureanu ◽  
Ionica Ionita ◽  
Iulian Bancuta ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Body Fluid ◽  
Pathogenic Bacteria ◽  
De Novo ◽  
Microbiological Examination ◽  
Doped Glasses ◽  
P2o5 System ◽  
Scanning Electron

The study in vitro of the glass powders bioactivity was performed by soaking them in simulated body fluid for 3 to 21 days at a temperature of 37�C and pH = 7.20. The synthesis de novo of hydroxyapatite, post soaking was confirmed by Fourier Transform Infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The study of the antimicrobial activity was performed by microbiological examination on two strains of pathogenic bacteria involved in postoperative nosocomial infections.

scholarly journals Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in COVID-19 patients and healthy individuals

2021 ◽  
pp. eabd6990
Author(s):  
Sang Il Kim ◽  
Jinsung Noh ◽  
Sujeong Kim ◽  
Younggeun Choi ◽  
Duck Kyun Yoo ◽  
...  
Keyword(s):  
B Cells ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
De Novo ◽  
Binding Domain ◽  
Host Cells ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Healthy Individuals

Stereotypic antibody clonotypes exist in healthy individuals and may provide protective immunity against viral infections by neutralization. We observed that 13 out of 17 patients with COVID-19 had stereotypic variable heavy chain (VH) antibody clonotypes directed against the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. These antibody clonotypes were comprised of immunoglobulin heavy variable (IGHV)3-53 or IGHV3-66 and immunoglobulin heavy joining (IGHJ)6 genes. These clonotypes included IgM, IgG3, IgG1, IgA1, IgG2, and IgA2 subtypes and had minimal somatic mutations, which suggested swift class switching after SARS-CoV-2 infection. The different immunoglobulin heavy variable chains were paired with diverse light chains resulting in binding to the RBD of SARS-CoV-2 spike protein. Human antibodies specific for the RBD can neutralize SARS-CoV-2 by inhibiting entry into host cells. We observed that one of these stereotypic neutralizing antibodies could inhibit viral replication in vitro using a clinical isolate of SARS-CoV-2. We also found that these VH clonotypes existed in six out of 10 healthy individuals, with IgM isotypes predominating. These findings suggest that stereotypic clonotypes can develop de novo from naïve B cells and not from memory B cells established from prior exposure to similar viruses. The expeditious and stereotypic expansion of these clonotypes may have occurred in patients infected with SARS-CoV-2 because they were already present.

Computational study for suppression of CD25/IL-2 interaction

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Moein Dehbashi ◽  
Zohreh Hojati ◽  
Majid Motovali-bashi ◽  
Mazdak Ganjalikhani-Hakemi ◽  
Akihiro Shimosaka ◽  
...  
Keyword(s):  
Small Molecules ◽  
Cancer Progression ◽  
Immune Escape ◽  
De Novo ◽  
Computational Study ◽  
Treg Cells ◽  
Homology Search ◽  
Small Interfering Rnas

AbstractCancer recurrence presents a huge challenge in cancer patient management. Immune escape is a key mechanism of cancer progression and metastatic dissemination. CD25 is expressed in regulatory T (Treg) cells including tumor-infiltrating Treg cells (TI-Tregs). These cells specially activate and reinforce immune escape mechanism of cancers. The suppression of CD25/IL-2 interaction would be useful against Treg cells activation and ultimately immune escape of cancer. Here, software, web servers and databases were used, at which in silico designed small interfering RNAs (siRNAs), de novo designed peptides and virtual screened small molecules against CD25 were introduced for the prospect of eliminating cancer immune escape and obtaining successful treatment. We obtained siRNAs with low off-target effects. Further, small molecules based on the binding homology search in ligand and receptor similarity were introduced. Finally, the critical amino acids on CD25 were targeted by a de novo designed peptide with disulfide bond. Hence we introduced computational-based antagonists to lay a foundation for further in vitro and in vivo studies.

scholarly journals EXTH-51. ANTI-INVASIVE EFFICACY AND SURVIVAL BENEFIT OF THE YAP-TEAD INHIBITOR VERTEPORFIN IN PRECLINICAL GLIOBLASTOMA MODELS

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii98-ii98
Author(s):  
Anne Marie Barrette ◽  
Alexandros Bouras ◽  
German Nudelman ◽  
Zarmeen Mussa ◽  
Elena Zaslavsky ◽  
...  
Keyword(s):  
Survival Benefit ◽  
De Novo ◽  
Epithelial Mesenchymal Transition ◽  
Intraperitoneal Administration ◽  
Standard Of Care ◽  
Tumor Burden ◽  
Mesenchymal Transition ◽  
Protein Levels

Abstract Glioblastoma (GBM) remains an incurable disease, in large part due to its malignant infiltrative spread, and current clinical therapy fails to target the invasive nature of tumor cells in disease progression and recurrence. Here, we use the YAP-TEAD inhibitor Verteporfin to target a convergence point for regulating tumor invasion/metastasis and establish the robust anti-invasive therapeutic efficacy of this FDA-approved drug and its survival benefit across several preclinical glioma models. Using patient-derived GBM cells and orthotopic xenograft models (PDX), we show that Verteporfin treatment disrupts YAP/TAZ-TEAD activity and processes related to cell adhesion, migration and epithelial-mesenchymal transition. In-vitro, Verteporfin impairs tumor migration, invasion and motility dynamics. In-vivo, intraperitoneal administration of Verteporfin in mice with orthotopic PDX tumors shows consistent drug accumulation within the brain and decreased infiltrative tumor burden, across three independent experiments. Interestingly, PDX tumors with impaired invasion after Verteporfin treatment downregulate CDH2 and ITGB1 adhesion protein levels within the tumor microenvironment. Finally, Verteporfin treatment confers survival benefit in two independent PDX models: as monotherapy in de-novo GBM and in combination with standard-of-care chemoradiation in recurrent GBM. These findings indicate potential therapeutic value of this FDA-approved drug if repurposed for GBM patients.

scholarly journals Natural Variation in Plant Pluripotency and Regeneration

Plants ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 1261
Author(s):  
Robin Lardon ◽  
Danny Geelen
Keyword(s):  
De Novo ◽  
Shoot Organogenesis ◽  
In Vitro Regeneration ◽  
Relative Role ◽  
Root Explants ◽  
Experimental Systems ◽  
Complex Process ◽  
Insight Into ◽  
Molecular Framework

Plant regeneration is essential for survival upon wounding and is, hence, considered to be a strong natural selective trait. The capacity of plant tissues to regenerate in vitro, however, varies substantially between and within species and depends on the applied incubation conditions. Insight into the genetic factors underlying this variation may help to improve numerous biotechnological applications that exploit in vitro regeneration. Here, we review the state of the art on the molecular framework of de novo shoot organogenesis from root explants in Arabidopsis, which is a complex process controlled by multiple quantitative trait loci of various effect sizes. Two types of factors are distinguished that contribute to natural regenerative variation: master regulators that are conserved in all experimental systems (e.g., WUSCHEL and related homeobox genes) and conditional regulators whose relative role depends on the explant and the incubation settings. We further elaborate on epigenetic variation and protocol variables that likely contribute to differential explant responsivity within species and conclude that in vitro shoot organogenesis occurs at the intersection between (epi) genetics, endogenous hormone levels, and environmental influences.

scholarly journals PAICS contributes to gastric carcinogenesis and participates in DNA damage response by interacting with histone deacetylase 1/2

2020 ◽  
Vol 11 (7) ◽  
Author(s):  
Nan Huang ◽  
Chang Xu ◽  
Liang Deng ◽  
Xue Li ◽  
Zhixuan Bian ◽  
...  
Keyword(s):  
Dna Damage ◽  
Histone Deacetylase ◽  
Dna Damage Response ◽  
De Novo ◽  
Dna Damage Repair ◽  
Histone Deacetylase 1 ◽  
Damage Repair ◽  
Damage Response

AbstractPhosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS), an essential enzyme involved in de novo purine biosynthesis, is connected with formation of various tumors. However, the specific biological roles and related mechanisms of PAICS in gastric cancer (GC) remain unclear. In the present study, we identified for the first time that PAICS was significantly upregulated in GC and high expression of PAICS was correlated with poor prognosis of patients with GC. In addition, knockdown of PAICS significantly induced cell apoptosis, and inhibited GC cell growth both in vitro and in vivo. Mechanistic studies first found that PAICS was engaged in DNA damage response, and knockdown of PAICS in GC cell lines induced DNA damage and impaired DNA damage repair efficiency. Further explorations revealed that PAICS interacted with histone deacetylase HDAC1 and HDAC2, and PAICS deficiency decreased the expression of DAD51 and inhibited its recruitment to DNA damage sites by impairing HDAC1/2 deacetylase activity, eventually preventing DNA damage repair. Consistently, PAICS deficiency enhanced the sensitivity of GC cells to DNA damage agent, cisplatin (CDDP), both in vitro and in vivo. Altogether, our findings demonstrate that PAICS plays an oncogenic role in GC, which act as a novel diagnosis and prognostic biomarker for patients with GC.

scholarly journals Localization of ferrochelatase in Plasmodium falciparum

2004 ◽  
Vol 384 (2) ◽  
pp. 429-436 ◽  
Author(s):  
Sundaramurthy VARADHARAJAN ◽  
B. K. Chandrashekar SAGAR ◽  
Pundi N. RANGARAJAN ◽  
Govindarajan PADMANABAN
Keyword(s):  
Plasmodium Falciparum ◽  
De Novo ◽  
Enzymic Activity ◽  
Malarial Parasite ◽  
Parasite Genome ◽  
Marker Proteins ◽  
Parasite Plasmodium ◽  
Theoretical Predictions ◽  
Parasite Plasmodium Falciparum

Our previous studies have demonstrated de novo haem biosynthesis in the malarial parasite (Plasmodium falciparum and P. berghei). It has also been shown that the first enzyme of the pathway is the parasite genome-coded ALA (δ-aminolaevulinate) synthase localized in the parasite mitochondrion, whereas the second enzyme, ALAD (ALA dehydratase), is accounted for by two species: one species imported from the host red blood cell into the parasite cytosol and another parasite genome-coded species in the apicoplast. In the present study, specific antibodies have been raised to PfFC (parasite genome-coded ferrochelatase), the terminal enzyme of the haem-biosynthetic pathway, using recombinant truncated protein. With the use of these antibodies as well as those against the hFC (host red cell ferrochelatase) and other marker proteins, immunofluorescence studies were performed. The results reveal that P. falciparum in culture manifests a broad distribution of hFC and a localized distribution of PfFC in the parasite. However, PfFC is not localized to the parasite mitochondrion. Immunoelectron-microscopy studies reveal that PfFC is indeed localized to the apicoplast, whereas hFC is distributed in the parasite cytoplasm. These results on the localization of PfFC are unexpected and are at variance with theoretical predictions based on leader sequence analysis. Biochemical studies using the parasite cytosolic and organellar fractions reveal that the cytosol containing hFC accounts for 80% of FC enzymic activity, whereas the organellar fraction containing PfFC accounts for the remaining 20%. Interestingly, both the isolated cytosolic and organellar fractions are capable of independent haem synthesis in vitro from [4-14C]ALA, with the cytosol being three times more efficient compared with the organellar fraction. With [2-14C]glycine, most of the haem is synthesized in the organellar fraction. Thus haem is synthesized in two independent compartments: in the cytosol, using the imported host enzymes, and in the organellar fractions, using the parasite genome-coded enzymes.

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