Dendritic Cell-Derived Artificial Microvesicles Inhibit RLS₄₀ Lymphosarcoma Growth in Mice via Stimulation of Th1/Th17 Immune Response

Cell-free antitumor vaccines represent a promising approach to immunotherapy of cancer. Here, we compare the antitumor potential of cell-free vaccines based on microvesicles derived from dendritic cells (DCs) with DC- and cationic-liposome-based vaccines using a murine model of drug-resistant lymphosarcoma RLS40 in vivo. The vaccines were the following: microvesicle vaccines—cytochalasin B-induced membrane vesicles (CIMVs) obtained from DCs loaded with total tumor RNA using cholesterol/spermine-containing cationic liposomes L or mannosylated liposomes ML; DC vaccines—murine DCs loaded with total tumor-derived RNA using the same liposomes; and liposomal vaccines—lipoplexes of total tumor-derived RNA with liposomes L or ML. Being non-hepatotoxic, CIMV- and DC-based vaccines administered subcutaneously exhibited comparable potential to stimulate highly efficient antitumor CTLs in vivo, whereas liposomal vaccines were 25% weaker CTL inducers. Nevertheless, the antitumor efficiencies of the different types of the vaccines were similar: sizes of tumor nodes and the number of liver metastases were significantly decreased, regardless of the vaccine type. Notably, the booster vaccination did not improve the overall antitumor efficacy of the vaccines under the study. CIMV- and DC- based vaccines more efficiently than liposome-based ones decreased mitotic activity of tumor cells and induced their apoptosis, stimulated accumulation of neutrophil inflammatory infiltration in tumor tissue, and had a more pronounced immunomodulatory activity toward the spleen and thymus. Administration of CIMV-, DC-, and liposome-based vaccines resulted in activation of Th1/Th17 cells as well as the induction of positive immune checkpoint 4-1BBL and downregulation of suppressive immune checkpoints in a raw PD-1 >>> TIGIT > CTLA4 > TIM3. We demonstrated that cell-free CIMV-based vaccines exhibited superior antitumor and antimetastatic activity in a tumor model in vivo. The obtained results can be considered as the basis for developing novel strategies for oncoimmunotherapy

A toolset to study functions of Cytosolic non-specific dipeptidase 2 (CNDP2) using Drosophila as a model organism

Abstract Background Expression of the CNDP2 gene is frequently up- or down-regulated in different types of human cancers. However, how the product of this gene is involved in cell growth and proliferation is poorly understood. Moreover, our knowledge of the functions of the CNDP2 orthologs in well-established model organisms is scarce. In particular, the function of the D. melanogaster ortholog of CNDP2, encoded by the CG17337 gene (hereafter referred to as dCNDP2), is still unknown. Results This study was aimed at developing a set of genetic and molecular tools to study the roles of dCNDP2. We generated a dCNDP2 null mutation (hereafter ∆dCNDP2) using CRISPR/Cas9-mediated homologous recombination (HR) and found that the ∆dCNDP2 mutants are homozygous viable, morphologically normal and fertile. We also generated transgenic fly lines expressing eGFP-tagged and non-tagged dCNDP2 protein, all under the control of the UAS promoter, as well as polyclonal antibodies specific to dCNDP2. Using these tools, we demonstrate that only one of the two predicted dCNDP2 isoforms is expressed throughout the different tissues tested. dCNDP2 was detected in both the cytoplasm and the nucleus, and was found to be associated with multiple sites in the salivary gland polytene chromosomes. Conclusions The dCNDP2 gene is not essential for fly viability under standard laboratory conditions. The subcellular localization pattern of dCNDP2 suggests that this protein might have roles in both the cytoplasm and the nucleus. The genetic and molecular tools developed in this study will allow further functional characterization of the conserved CNDP2 protein using D. melanogaster as a model system

RNAi-mediated depletion of the NSL complex subunits leads to abnormal chromosome segregation and defective centrosome duplication in Drosophila mitosis.

The Drosophila Nonspecific Lethal (NSL) complex is a major transcriptional regulator of housekeeping genes. It contains at least seven subunits that are conserved in the human KANSL complex: Nsl1/Wah (KANSL1), Dgt1/Nsl2 (KANSL2), Rcd1/Nsl3 (KANSL3), Rcd5 (MCRS1), MBD-R2 (PHF20), Wds (WDR5) and Mof (MOF/KAT8). Previous studies have shown that Dgt1, Rcd1 and Rcd5 are implicated in centrosome maintenance. Here, we analyzed the mitotic phenotypes caused by RNAi-mediated depletion of Rcd1, Rcd5, MBD-R2 or Wds in greater detail. Depletion of any of these proteins in Drosophila S2 cells led to defects in chromosome segregation. Consistent with these findings, Rcd1, Rcd5 and MBD-R2 RNAi cells showed reduced levels of both Cid/CENP-A and the kinetochore component Ndc80. In addition, RNAi against any of the four genes negatively affected centriole duplication. In Wds-depleted cells, the mitotic phenotypes were similar but milder than those observed in Rcd1-, Rcd5- or MBD-R2-deficient cells. RT-qPCR experiments and interrogation of published datasets revealed that transcription of many genes encoding centromere/kinetochore proteins (e.g., cid, Mis12 and Nnf1b), or involved in centriole duplication (e.g., Sas-6, Sas-4 and asl) is substantially reduced in Rcd1, Rcd5 and MBD-R2 RNAi cells, and to a lesser extent in wds RNAi cells. During mitosis, both Rcd1-GFP and Rcd5-GFP accumulate at the centrosomes and the telophase midbody, MBD-R2-GFP is enriched only at the chromosomes, while Wds-GFP accumulates at the centrosomes, the kinetochores, the midbody, and on a specific chromosome region. Collectively, our results suggest that the mitotic phenotypes caused by Rcd1, Rcd5, MBD-R2 or Wds depletion are primarily due to reduced transcription of genes involved in kinetochore assembly and centriole duplication. The differences in the subcellular localizations of the NSL components may reflect direct mitotic functions that are difficult to detect at the phenotypic level, because they are masked by the transcription-dependent deficiency of kinetochore and centriolar proteins

Determination of the Mineral Composition of the Lake Bottom Sediments by X-Ray Diffraction Method and Physico-Chemical Modelling

Сопоставлены три способа определения содержания минералов и минеральных групп в карбонатно-силикатных озерных донных отложениях. Два способа основаны на методе рентгеновской порошковой дифрактометрии. Первый использует обработку дифрактограмм методом Ритвельда в программном обеспечении DIFFRAC Plus дифрактометра D8 Advance (база данных PDF-2). Второй применяет метод ссылочных интенсивностей (корундовых отношений) и оптимизацию модельной дифрактограммы из рентгенофазовых эталонов базы PDF-2 и уравнений элементного баланса с использованием регуляризированного метода наименьших квадратов. Третий способ, основанный на физико-химическом моделировании, выполняет подбор вероятных мономинеральных и многокомпонентных фаз с помощью модели твердых растворов и использует данные об элементном составе, полученные с помощью рентгенофлуоресцентного метода анализа и данные рентгеновской дифрактометрии о качественном минеральном составе. 30 образцов керна донных отложений оз. Зун-Торей (Восточная Сибирь) были проанализированы тремя упомянутыми способами. Содержания минеральных групп (полевые шпаты, кварц, глинистые минералы, карбонаты) варьировали в диапазоне приблизительно 10-40 мас. %. Расхождения между результатами определений тремя способами характеризуются стандартным отклонением в диапазоне 2-9 мас. %. Относительное стандартное отклонение, как правило, составляло величину менее 30 %, поэтому такие определения можно считать количественными. На основании полученных данных трудно отдать предпочтение какому- либо из рассмотренных способов. Приведенные данные позволили оценить погрешность рентгенофазового порошкового анализа при определении содержания минеральных групп в карбонатно-силикатных осадочных породах в отсутствие стандартных образцов сравнения с аттестованным минеральным составомThe paper reports comparison of three approaches to define the contents of minerals and mineral groups in the carbonate-silicate lake bottom sediments. The two approaches are based on the method of X-ray powder diffraction. The first one treats with the Rietveld Method in the software DIFFRAC Plus diffractometer D8 Advance (PDF-2 database). The second one uses the method of reference intensities (corundum ratios) and optimization of the model powder patterns from the X-ray phase standards of PDF-2 database and equations of the element balance with regularization of the least square functional. The third approach of physic-chemical modeling selects probable single mineral and multi-component phases through modelling the sold solutions, and it uses the data on the element composition obtained by XRF technique, as well as the data of X-ray diffraction on the qualitative mineral composition. Thirty samples of bottom sediment cores taken in the Zun-Torey Lake in East Siberia were analyzed by the three approaches described herein. The contents of mineral groups (feldspars, quartz, clay minerals and carbonates) varied within the range 10-40 mass %. The discrepancies between obtained results show the standard deviation ranging from 2 to 9 mass %. A relative standard deviation commonly provides the value below 30 %, so such determinations could be considered quantitative ones. With regard to the acquired data, it is hard to prefer this or that approach. Available data was employed to assess the error of X-ray phase powder analysis in measuring the abundance of mineral groups in the carbonate-silicate sedimentary rocks in the absence of reference materials to compare with certified mineral compositio