Цитологические эффекты в лимфатических узлах зоны абдоминальной лимфодиссекции после интраоперационной фотодинамической терапии при злокачественных новообразованиях желудочно-кишечного тракта

Cytological studies on lymph nodes of abdominal lymphodissection zone after local intraoperative photodynamic therapy (IOPDT) of gastrointestinal cancers were carried out. As a result of the PDT, the metastatic cells are destroyed, their cytoplasmic membranes and the cytoplasm disappears, leaving behind interphase nuclei ("naked nuclei") (p<0,0001). Cytological confrmation of apoptosis (the presence of apoptotic bodies) in metastatic lymph nodes after IOPDT sessions on the lymph nodes of the abdominal lymphodissection zone is also presented.Проведены цитологические исследования материала лимфатических узлов зоны абдомининальной лимфодиссекции после интраоперационной фотодинамической терапии (ИОФДТ) при злокачественных новообразованиях желудочно‑кишечного тракта. Установлено, что в результате проведения ФДТ клетки метастатических опухолей разрушаются с исчезновением цитоплазматической мембраны и цитоплазмы, при этом остаются только интерфазные ядра («голые ядра») (р<0,0001). Также представлено цитологическое подтверждение апоптоза (наличие апоптотических телец) в лимфатических узлах с метастазами после курсов ИОФДТ в лимфатических узлах зоны абдоминальной лимфодиссекции

ИЗУЧЕНИЕ АПОПТОЗА В ЛИМФАТИЧЕСКИХ УЗЛАХ ЗОНЫ АБДОМИНАЛЬНОЙ ЛИМФОДИССЕКЦИИ ПОСЛЕ ИНТРАОПЕРАЦИОННОЙ ФОТОДИНАМИЧЕСКОЙ ТЕРАПИИ ПРИ ЗЛОКАЧЕСТВЕННЫХ НОВООБРАЗОВАНИЯХ ЖЕЛУДОЧНО-КИШЕЧНОГО ТРАКТА

The method of DNA-electrophoresis was used to study apoptosis in the lymph nodes of the abdominal lymphodissection zone after local intraoperative photodynamic therapy (PDT) for the gastrointestinal cancers. Apoptosis (apoptotic ladder) was determined in the four cell populations of lymph nodes:– in the lymph nodes with metastases that have not been irradiated;– in the irradiated during PDT lymph nodes with metastases;– in the lymph nodes without metastases that have not been irradiated;– in the irradiated during PDT lymph nodes without metastases.The implementation of this study was possible due to intraoperative division of lymph nodes into two parts, one of which was removed while another one intentionally wasn’t removed and was irradiated (in human). It was also removed 60-90 minutes after PDT treatment and examined for apoptosis. As a result, it was found that apoptosis is detected in the irradiated during PDT lymph nodes with metastases. The obtained results show the fact that in the lymph nodes of the abdominal lymphodissection zone tumor cells selectively die by apoptosis as a result of the local intraoperative photodynamic therapy (PDT) for the gastrointestinal cancers while in non-malignant cells apoptosis doesn’t occur after PDT (p <0.01). Thus, the obtained clinical and experimental data made it possible to evaluate the target PDT effect and confirm the safety of the photodynamic effect on healthy tissues of the lymphoid system located in the irradiation zone.Методом ДНК-электрофореза исследован апоптоз в лимфоузлах после интраоперационной фотодинамической терапии (ФДТ) зоны абдоминальной лимфодиссекции при злокачественных новообразованиях желудочно-кишечного тракта. Апоптоз («апоптотическую лестницу») определяли в четырех клеточных популяциях лимфоузлов:– в лимфоузлах с метастазами, не подвергавшихся ФДТ;– в тех же лимфоузлах с метастазами после ФДТ;– в лимфоузлах без метастазов, не подвергавшихся ФДТ;– в тех же лимфоузлах без метастазов после ФДТ. Дизайн исследования включал интраоперационное разделение лимфоузлов на две части, одна из которых была сразу удалена и не подвергалась воздействию ФДТ, а вторая оставалась неудаленной и подвергалась ФДТ, затем через 60–90 мин после сеанса ФДТ также была удалена и исследована на апоптоз. В результате оказалось, что апоптоз обнаруживается только в лимфоузлах с метастазами, облучённых во время ФДТ. Полученные результаты указывают на тот факт, что в лимфоузлах зоны абдоминальной лимфодиссекции в результате местной интраоперационной ФДТ при злокачественных новообразованиях желудочно-кишечного тракта злокачественные клетки избирательно гибнут путём апоптоза, а незлокачественные клетки при этом в апоптоз не вступают (р<0,01). Таким образом, полученные клинические данные позволили оценить таргетный эффект ФДТ и подтвердить безопасность фотодинамического воздействия на здоровые ткани лимфоидной системы, находящихся в зоне облучения

A Pre-mRNA–Associating Factor Links Endogenous siRNAs to Chromatin Regulation

In plants and fungi, small RNAs silence gene expression in the nucleus by establishing repressive chromatin states. The role of endogenous small RNAs in metazoan nuclei is largely unknown. Here we show that endogenous small interfering RNAs (endo-siRNAs) direct Histone H3 Lysine 9 methylation (H3K9me) in Caenorhabditis elegans. In addition, we report the identification and characterization of nuclear RNAi defective (nrde)-1 and nrde-4. Endo-siRNA–driven H3K9me requires the nuclear RNAi pathway including the Argonaute (Ago) NRDE-3, the conserved nuclear RNAi factor NRDE-2, as well as NRDE-1 and NRDE-4. Small RNAs direct NRDE-1 to associate with the pre-mRNA and chromatin of genes, which have been targeted by RNAi. NRDE-3 and NRDE-2 are required for the association of NRDE-1 with pre-mRNA and chromatin. NRDE-4 is required for NRDE-1/chromatin association, but not NRDE-1/pre-mRNA association. These data establish that NRDE-1 is a novel pre-mRNA and chromatin-associating factor that links small RNAs to H3K9 methylation. In addition, these results demonstrate that endo-siRNAs direct chromatin modifications via the Nrde pathway in C. elegans

The ERI-6/7 Helicase Acts at the First Stage of an siRNA Amplification Pathway That Targets Recent Gene Duplications

Endogenous small interfering RNAs (siRNAs) are a class of naturally occuring regulatory RNAs found in fungi, plants, and animals. Some endogenous siRNAs are required to silence transposons or function in chromosome segregation; however, the specific roles of most endogenous siRNAs are unclear. The helicase gene eri-6/7 was identified in the nematode Caenorhabditis elegans by the enhanced response to exogenous double-stranded RNAs (dsRNAs) of the null mutant. eri-6/7 encodes a helicase homologous to small RNA factors Armitage in Drosophila, SDE3 in Arabidopsis, and Mov10 in humans. Here we show that eri-6/7 mutations cause the loss of 26-nucleotide (nt) endogenous siRNAs derived from genes and pseudogenes in oocytes and embryos, as well as deficiencies in somatic 22-nucleotide secondary siRNAs corresponding to the same loci. About 80 genes are eri-6/7 targets that generate the embryonic endogenous siRNAs that silence the corresponding mRNAs. These 80 genes share extensive nucleotide sequence homology and are poorly conserved, suggesting a role for these endogenous siRNAs in silencing of and thereby directing the fate of recently acquired, duplicated genes. Unlike most endogenous siRNAs in C. elegans, eri-6/7–dependent siRNAs require Dicer. We identify that the eri-6/7–dependent siRNAs have a passenger strand that is ∼19 nt and is inset by ∼3–4 nts from both ends of the 26 nt guide siRNA, suggesting non-canonical Dicer processing. Mutations in the Argonaute ERGO-1, which associates with eri-6/7–dependent 26 nt siRNAs, cause passenger strand stabilization, indicating that ERGO-1 is required to separate the siRNA duplex, presumably through endonucleolytic cleavage of the passenger strand. Thus, like several other siRNA–associated Argonautes with a conserved RNaseH motif, ERGO-1 appears to be required for siRNA maturation

A Conserved PHD Finger Protein and Endogenous RNAi Modulate Insulin Signaling in Caenorhabditis elegans

Insulin signaling has a profound effect on longevity and the oxidative stress resistance of animals. Inhibition of insulin signaling results in the activation of DAF-16/FOXO and SKN-1/Nrf transcription factors and increased animal fitness. By studying the biological functions of the endogenous RNA interference factor RDE-4 and conserved PHD zinc finger protein ZFP-1 (AF10), which regulate overlapping sets of genes in Caenorhabditis elegans, we identified an important role for these factors in the negative modulation of transcription of the insulin/PI3 signaling-dependent kinase PDK-1. Consistently, increased expression of pdk-1 in zfp-1 and rde-4 mutants contributed to their reduced lifespan and sensitivity to oxidative stress and pathogens due to the reduction in the expression of DAF-16 and SKN-1 targets. We found that the function of ZFP-1 in modulating pdk-1 transcription was important for the extended lifespan of the age-1(hx546) reduction-of-function PI3 kinase mutant, since the lifespan of the age-1; zfp-1 double mutant strain was significantly shorter compared to age-1(hx546). We further demonstrate that overexpression of ZFP-1 caused an increased resistance to oxidative stress in a DAF-16–dependent manner. Our findings suggest that epigenetic regulation of key upstream signaling components in signal transduction pathways through chromatin and RNAi may have a large impact on the outcome of signaling and expression of numerous downstream genes.Leukemia & Lymphoma Society of America (3260-07 Special Fellow Award)Arnold and Mabel Beckman Foundation (Young Investigator Award)United States. National Institutes of Health (Director's New Innovator Award (1 DP2 OD006412-01))United States. National Institutes of Health (grant GM66269)modENCODE (grant U01 HG004270)United States. National Institutes of Health (training grant 5T32 GM07088-34

RNAi Effector Diversity in Nematodes

While RNA interference (RNAi) has been deployed to facilitate gene function studies in diverse helminths, parasitic nematodes appear variably susceptible. To test if this is due to inter-species differences in RNAi effector complements, we performed a primary sequence similarity survey for orthologs of 77 Caenorhabditis elegans RNAi pathway proteins in 13 nematode species for which genomic or transcriptomic datasets were available, with all outputs subjected to domain-structure verification. Our dataset spanned transcriptomes of Ancylostoma caninum and Oesophagostomum dentatum, and genomes of Trichinella spiralis, Ascaris suum, Brugia malayi, Haemonchus contortus, Meloidogyne hapla, Meloidogyne incognita and Pristionchus pacificus, as well as the Caenorhabditis species C. brenneri, C. briggsae, C. japonica and C. remanei, and revealed that: (i) Most of the C. elegans proteins responsible for uptake and spread of exogenously applied double stranded (ds)RNA are absent from parasitic species, including RNAi-competent plant-nematodes; (ii) The Argonautes (AGOs) responsible for gene expression regulation in C. elegans are broadly conserved, unlike those recruited during the induction of RNAi by exogenous dsRNA; (iii) Secondary Argonautes (SAGOs) are poorly conserved, and the nuclear AGO NRDE-3 was not identified in any parasite; (iv) All five Caenorhabditis spp. possess an expanded RNAi effector repertoire relative to the parasitic nematodes, consistent with the propensity for gene loss in nematode parasites; (v) In spite of the quantitative differences in RNAi effector complements across nematode species, all displayed qualitatively similar coverage of functional protein groups. In summary, we could not identify RNAi effector deficiencies that associate with reduced susceptibility in parasitic nematodes. Indeed, similarities in the RNAi effector complements of RNAi refractory and competent nematode parasites support the broad applicability of this research genetic tool in nematodes

Cytological effects in lymph nodes of abdominal lymphodissection zone after intraoperative photodynamic therapy of gastrointestinal cancers

Cytological studies on lymph nodes of abdominal lymphodissection zone after local intraoperative photodynamic therapy (IOPDT) of gastrointestinal cancers were carried out. As a result of the PDT, the metastatic cells are destroyed, their cytoplasmic membranes and the cytoplasm disappears, leaving behind interphase nuclei ("naked nuclei") (p<0,0001). Cytological confrmation of apoptosis (the presence of apoptotic bodies) in metastatic lymph nodes after IOPDT sessions on the lymph nodes of the abdominal lymphodissection zone is also presented

APOPTOSIS IN THE LYMPH NODES OF THE ABDOMINAL LYMPHODISSECTION ZONE AFTER LOCAL INTRAOPERATIVE PHOTODYNAMIC THERAPY OF GASTROINTESTINAL CANCERS

The method of DNA-electrophoresis was used to study apoptosis in the lymph nodes of the abdominal lymphodissection zone after local intraoperative photodynamic therapy (PDT) for the gastrointestinal cancers. Apoptosis (apoptotic ladder) was determined in the four cell populations of lymph nodes:– in the lymph nodes with metastases that have not been irradiated;– in the irradiated during PDT lymph nodes with metastases;– in the lymph nodes without metastases that have not been irradiated;– in the irradiated during PDT lymph nodes without metastases.The implementation of this study was possible due to intraoperative division of lymph nodes into two parts, one of which was removed while another one intentionally wasn’t removed and was irradiated (in human). It was also removed 60-90 minutes after PDT treatment and examined for apoptosis. As a result, it was found that apoptosis is detected in the irradiated during PDT lymph nodes with metastases. The obtained results show the fact that in the lymph nodes of the abdominal lymphodissection zone tumor cells selectively die by apoptosis as a result of the local intraoperative photodynamic therapy (PDT) for the gastrointestinal cancers while in non-malignant cells apoptosis doesn’t occur after PDT (p <0.01). Thus, the obtained clinical and experimental data made it possible to evaluate the target PDT effect and confirm the safety of the photodynamic effect on healthy tissues of the lymphoid system located in the irradiation zone

Differential Impact of the HEN1 Homolog HENN-1 on 21U and 26G RNAs in the Germline of Caenorhabditis elegans

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