Small Number Of Cells Research Articles

2cChIP-seq and 2cMeDIP-seq: The Carrier-Assisted Methods for Epigenomic Profiling of Small Cell Numbers or Single Cells.

Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) can profile genome-wide epigenetic marks associated with regulatory genomic elements. However, conventional ChIP-seq is challenging when examining limited numbers of cells. Here, we developed a new technique by supplementing carrier materials of both chemically modified mimics with epigenetic marks and dUTP-containing DNA fragments during conventional ChIP procedures (hereafter referred to as 2cChIP-seq), thus dramatically improving immunoprecipitation efficiency and reducing DNA loss of low-input ChIP-seq samples. Using this strategy, we generated high-quality epigenomic profiles of histone modifications or DNA methylation in 10-1000 cells. By introducing Tn5 transposase-assisted fragmentation, 2cChIP-seq reliably captured genomic regions with histone modification at the single-cell level in about 100 cells. Moreover, we characterized the methylome of 100 differentiated female germline stem cells (FGSCs) and observed a particular DNA methylation signature potentially involved in the differentiation of mouse germline stem cells. Hence, we provided a reliable and robust epigenomic profiling approach for small cell numbers and single cells.

Condensing of Low-Density Fat by Mechanical Process Improves Fat Retention and Reduces Oil Cyst Formation in Breast Reconstruction.

Although autologous fat grafting is a useful adjunct for breast reconstruction, its indications remain limited as large-volume fat grafting results in high absorption and complication rates. Low-density fat includes small numbers of viable cells and considerable oil, resulting in nodules and oil cysts. This study evaluated the volumization effect and complications with combined fat grafting of condense low-density fat and high-density fat. This retrospective, single-center study included 25 patients who underwent combined grafting of condensed low-density fat and high-density fat (CLDF + HDF) and 20 patients who underwent conventional Coleman fat grafting for breast reconstruction from December 2017 to January 2022. Retention rates and complications were evaluated by magnetic resonance imaging and ultrasound rates. Patient satisfaction was evaluated using a typical Likert scale. Photographs were taken and imageological examinations were performed before and after treatment. Graft retention rate was higher in patients who underwent CLDF + HDF than Coleman fat grafting for breast reconstruction (38.40 ± 4.41% vs. 31.43 ± 5.43%, p <0.05). One patient in the CLDF + HDF grafting group, compared with twelve in the Coleman fat grafting group, developed oil cysts exceeding 1 cm. Patient satisfaction rate was higher in the CLDF + HDF grafting group. Mechanical processes can concentrate the cellular content of LDF and remove oil, condensing LDF to the level of HDF. Combined grafting of CLDF optimized by mechanical processing and HDF is effective for breast reconstruction, with a higher retention rate and a lower incidence of complications than conventional Coleman fat grafting. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Whole-embryonic identification of maternal microchimeric cell types in mouse using single-cell RNA sequencing

Even though the mother and the fetus of placental mammals are immunologically non-self with respect to one other, mutual exchange of small numbers of cells between them is known to occur. Maternal cells entering the fetus, called maternal microchimeric cells (MMc cells), are thought to be involved in different physiological phenomena, such as establishing immune tolerance, tissue repair, and the pathogenesis or deterioration of some inflammatory diseases and congenital malformations. While specific MMc cell types have been reported as associated with these phenomena, the contribution of MMc cells to these different outcomes remains unknown. As one possibility, we hypothesized that different embryos have differing repertoires of MMc cell types, leading to or biasing embryos toward different fates. To date, no studies have succeeded in identifying the MMc cell type repertoire of a single embryo. Accordingly, here, we isolated MMc cells from whole mouse embryos, determined their types, and analyzed their MMc cell type variability. By combining our previously established, whole-embryonic MMc isolation method with single-cell RNA sequencing, we successfully estimated the cell type repertoires of MMc cells isolated from 26 mouse embryos. The majority of MMc cells were immune-related cells, such as myeloid cells and granulocytes. We also detected stem cell-like MMc cells expressing proliferation marker genes and terminally differentiated cells. As hypothesized, we noted statistically significant inter-individual variation in the proportion of immune-related cells in the different embryos. We here successfully estimated MMc cell types in individual whole mouse embryos. The proportion of immune-related cells significantly differed among the individual embryos, suggesting that the variations are one of the potential mechanisms underlying the differing MMc-related physiological phenomena in offspring. These findings provide insight into cell-level epigenetics by maternal cells.

Ageing-resembling phenotype of long-term allogeneic hematopoietic cells recipients compared to their donors

BackgroundAgeing is a complex phenomenon that leads to decreased proliferative activity, loss of function of the cells, and cellular senescence. Senescence of the immune system exacerbates individual’s immune response, both humoral and cellular but increases the frequency of infections. We hypothesized that physiological ageing of adaptive immune system occurs in recipients of allogeneic hematopoietic cells transplant (allo-HCT) at faster rate when compared to their respective donors since the small number of donor cells undergo immense proliferative stress restoring recipients hematopoiesis. We compared molecular characterizations of ageing between recipients and donors of allo-HCT: telomeric length and immunophenotypic changes in main lymphocyte subsets – CD4+, CD8+, CD19+, CD56+.ResultsMedian telomeric length (TL) of CD8+ lymphocytes was significantly longer in donors compared to recipients (on average 2,1 kb and 1,7 kb respectively, p = 0,02). Similar trends were observed for CD4+ and CD19+ although the results did not reach statistical significance. We have also found trends in the immunophenotype between recipients and donors in the subpopulations of CD4+ (naïve and effector memory), CD8+ Eomes+ and B-lymphocytes (B1 and B2). Lower infection risk recipients had also a significantly greater percentage of NK cells (22,3%) than high-risk patients (9,3%) p = 0,04.ConclusionOur data do not support the initial hypothesis of accelerated aging in the long term all-HCT recipients with the exception of the recipients lymphocytes (mainly CD8+) which present some molecular features, characteristic for physiological ageing (telomeric shortening, immunophenotype) when compared to their respective donors. However, a history of lower infection numbers in HCT recipients seems to be associated with increased percentage of NK cells. The history of GVHD seems not to affect the rate of ageing. Therefore, it is safe to conclude that the observed subtle differences between recipients’ and donors’ cells result mainly from the proliferative stress in the early period after allo-HCT and the difference between hosts’ and recipients’ microenvironments.

Highly Efficient CRISPR/Cas9-Mediated Genome Editing in Human Pluripotent Stem Cells.

Human pluripotent stem cells hold tremendous potential for both basic biology and cell-based therapies for a wide variety of diseases. The ability to manipulate the genome of these cells using the CRISPR/Cas9 technology has expanded this potential by providing a valuable tool to engineer or correct disease-associated mutations. Because of the high efficiency with which CRISPR/Cas9 creates targeted double-strand breaks, a major challenge has been the introduction of precise genetic modifications on one allele without indel formation on the non-targeted allele. To overcome this obstacle, we describe use of two oligonucleotide repair templates: one expressing the sequence change and the other maintaining the normal sequence. In addition, we have streamlined both the transfection and screening methodologies to make the protocols efficient, with small numbers of cells and a limited amount of labor-intensive clone passaging. This article provides a technically simple approach for generating valuable tools to model human disease in stem cells. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Application and optimization of CRISPR-based genome editing in human pluripotent stem cells Basic Protocol 2: Genetic modification of human pluripotent stem cells using a double-oligonucleotide CRISPR/Cas9 recombination system.

Comparison of ligamentization potential between anterior cruciate ligament-derived cells and adipose-derived mesenchymal stem cells reseeded to acellularized tendon allograft.

To test the hypothesis that reseeded anterior cruciate ligament (ACL)-derived cells have a better ability to survive and integrate into tendon extracellular matrix (ECM) and accelerate the ligamentization process, compared to adipose-derived mesenchymal stem cells (ADMSCs). Acellularized tibialis allograft tendons were used. Tendons were randomly reseeded with ACL-derived cells or ADMSCs. ACL-derived cells were harvested and isolated from remnants of ruptured ACLs during reconstruction surgery and cultured at passage three. Cell suspensions (200 µl) containing 2 × 106 ACL-derived cells or ADMSCs were prepared for the purpose of reseeding. At days 1, 3, and 7 post-reseeding, graft composites were assessed for repopulation with histological and immunohistochemical analysis. Matrix protein contents and gene expression levels were analyzed. In the graft reseeded with ACL-derived cells, a large number of elongated cells that integrated into the matrix were evident at day 3 and day 7. However, in the graft reseeded with ADMSCs, only a small number of elongated cells were found integrated into the matrix. Immunofluorescence for Ki-67 and type I collagen confirmed the pronounced production of type I collagen by Ki-67-positive ACL-derived cells integrated into the ECM. A messenger RNA (mRNA) expression assay demonstrated significantly higher gene expression levels of types I (p = 0.013) and III (p = 0.050) collagen in the composites reseeded with ACL-derived cells than ADMSCs. ACL-derived cells, when reseeded to acellularized tendon graft, demonstrated earlier better survival and integration in the tendon ECM and resulted in higher gene expression levels of collagen, which may be essential to the normal ligamentization process compared to ADMSCs.Cite this article: Bone Joint Res2022;11(11):777-786.

2-Cell-like Cells: An Avenue for Improving SCNT Efficiency.

After fertilization, the zygote genome undergoes dramatic structural reorganization to ensure the establishment of totipotency, and then the totipotent potential of the zygote or 2-cell-stage embryo progressively declines. However, cellular potency is not always a one-way street. Specifically, a small number of embryonic stem cells (ESCs) occasionally overcome epigenetic barriers and transiently convert to a totipotent status. Despite the significant potential of the somatic cell nuclear transfer (SCNT) technique, the establishment of totipotency is often deficient in cloned embryos. Because of this phenomenon, the question arises as to whether strategies attempting to induce 2-cell-like cells (2CLCs) can provide practical applications, such as reprogramming of somatic cell nuclei. Inspired by strategies that convert ESCs into 2CLCs, we hypothesized that there will be a similar pathway by which cloned embryos can establish totipotent status after SCNT. In this review, we provide a snapshot of the practical strategies utilized to induce 2CLCs during investigations of the development of cloned embryos. The 2CLCs have similar transcriptome and chromatin features to that of 2-cell-stage embryos, and we propose that 2CLCs, already a valuable in vitro model for dissecting totipotency, will provide new opportunities to improve SCNT efficiency.

Fracture performance of epoxy foam: Low density to bulk polymer

Epoxy foams with densities ranging from 180 to 500 kg/m3 were prepared and mechanically tested in compression, tension, and single-edge notched bending (SENB) configurations. Fracture results revealed a marked transition in behaviour at a critical density, between 227 kg/m3 and 249 kg/m3. Lower density foams failed at low SENB displacement, producing low toughness and fracture energy results, whereas higher density foams failed at higher SENB displacements, with correspondingly higher values of toughness and fracture energy. The stress-intensity factor increased monotonically with density, from 0.1 to 0.79 MPa m1/2. The fracture energy, GIc, of the foams reached values of up to 3.5 times that of the bulk polymer, 268 J/m2. Lower density foams below the transition in fracture behaviour exhibited a small number of large cells, caused by cell coalescence, and a wider cell size distribution than the denser foams. This distribution appears linked to the transition in fracture behaviour. The behaviour revealed in this paper raises the point whether in future design criteria, where foams are now often used in composite sandwich structures, allowance should be made for denser foams to be used as appreciable increases in fracture energy of the foam core are achievable.

Variable allelic expression of imprinted genes at the Peg13, Trappc9, Ago2 cluster in single neural cells.

Genomic imprinting is an epigenetic process through which genes are expressed in a parent-of-origin specific manner resulting in mono-allelic or strongly biased expression of one allele. For some genes, imprinted expression may be tissue-specific and reliant on CTCF-influenced enhancer-promoter interactions. The Peg13 imprinting cluster is associated with neurodevelopmental disorders and comprises canonical imprinted genes, which are conserved between mouse and human, as well as brain-specific imprinted genes in mouse. The latter consist of Trappc9, Chrac1 and Ago2, which have a maternal allelic expression bias of ∼75% in brain. Findings of such allelic expression biases on the tissue level raise the question of how they are reflected in individual cells and whether there is variability and mosaicism in allelic expression between individual cells of the tissue. Here we show that Trappc9 and Ago2 are not imprinted in hippocampus-derived neural stem cells (neurospheres), while Peg13 retains its strong bias of paternal allele expression. Upon analysis of single neural stem cells and in vitro differentiated neurons, we find not uniform, but variable states of allelic expression, especially for Trappc9 and Ago2. These ranged from mono-allelic paternal to equal bi-allelic to mono-allelic maternal, including biased bi-allelic transcriptional states. Even Peg13 expression deviated from its expected paternal allele bias in a small number of cells. Although the cell populations consisted of a mosaic of cells with different allelic expression states, as a whole they reflected bulk tissue data. Furthermore, in an attempt to identify potential brain-specific regulatory elements across the Trappc9 locus, we demonstrate tissue-specific and general silencer activities, which might contribute to the regulation of its imprinted expression bias.

From multitude to singularity: An up-to-date overview of scRNA-seq data generation and analysis.

Single cell RNA sequencing (scRNA-seq) is today a common and powerful technology in biomedical research settings, allowing to profile the whole transcriptome of a very large number of individual cells and reveal the heterogeneity of complex clinical samples. Traditionally, cells have been classified by their morphology or by expression of certain proteins in functionally distinct settings. The advent of next generation sequencing (NGS) technologies paved the way for the detection and quantitative analysis of cellular content. In this context, transcriptome quantification techniques made their advent, starting from the bulk RNA sequencing, unable to dissect the heterogeneity of a sample, and moving to the first single cell techniques capable of analyzing a small number of cells (1–100), arriving at the current single cell techniques able to generate hundreds of thousands of cells. As experimental protocols have improved rapidly, computational workflows for processing the data have also been refined, opening up to novel methods capable of scaling computational times more favorably with the dataset size and making scRNA-seq much better suited for biomedical research. In this perspective, we will highlight the key technological and computational developments which have enabled the analysis of this growing data, making the scRNA-seq a handy tool in clinical applications.

Biological functions and clinical significance of calprotectin. Review

Recently, in addition to clinical, endoscopic and morphological studies, the algorithm for the diagnosis of inflammatory bowel disease (IBD) also includes some serum and fecal biomarkers that help select patients for endoscopy, as well as distinguish between organic and functional (non‑inflammatory) diseases (for example, irritable bowel syndrome). Among the latter, the definition of fecal calprotectin (CP) is used mostly often, it is a well‑studied biomarker of inflammation due to its stability, reproducibility of analysis, low cost, and high diagnostic value. The presented review highlights the latest data on the main biological CP functions and the clinical application of its definition in inflammatory diseases of intestine, skin, joints, etc.&#x0D; It is shown that CP belongs to the family of calcium‑binding leukocyte S100 proteins and consists of two monomers: S100A8 and S100A9. CP is constitutively expressed by monocytes, dendritic cells, activated macrophages, keratinocytes of the oral cavity and squamous epithelium of the mucosa. Thus, the CP expression in a healthy person is limited by a small number of specialized cells and is usually activated during inflammation. Both subunits of CP, S100A8 and S100A9, have a wide range of intracellular and extracellular immunomodulatory properties, control the intracellular pathways of innate immune cells and are responsible for organizing the response to the inflammatory process.&#x0D; Determination of fecal CP enables distinguishing between non‑inflammatory and inflammatory bowel diseases, it is a non‑invasive inexpensive method, and the CP itself remains stable at room temperature in the feces for at least 3 days. In the diagnosis of IBD, fecal CP is a more sensitive marker than C‑reactive protein, which has made it an excellent (i.e., highly sensitive) biomarker for detecting intestinal inflammation in IBD and has contributed to its widespread use worldwide. The value of the level of fecal Cp &lt; 40 mg/g can reliably exclude IBD, and a value &gt; 250 mg/g dictates the need for endoscopic examination of the patient for IBD or raises the suspicion of its recurrence.&#x0D;

Study on the mechanism of low shear stress restoring the viability of damaged breast tumor cells

Blood vessel is one of the ‘pathways’ for cancer to metastasize, in which cells are exposed to the fluid shear stress. Although most cells are damaged by fluid shear stress, a small number of tumor cells survive and metastasize when they are exposed to low shear stress (LSS) of tiny capillary. It is important to study the survival state of damaged cells during LSS. In this study, high shear stress (HSS) was applied to simulate the blood circulation and damage cells. The viability and mitochondrial function of cells were detected after HSS and LSS loading, respectively. Further, the expression of mitochondrial related proteins and genes were detected by western blot and real-time quantitative polymerase chain reaction, respectively. The role of cytochrome c (Cyt C) was also verified in this process. The experimental results showed that the viability of HSS damaged cells was increased significantly when they were exposed to LSS subsequently. The function of mitochondria was improved via reducing the release of Cyt C by LSS during this process. This study is expected to provide potential target for suppressing hematogenous metastasis.

Quantitative proteomics of small numbers of closely-related cells: Selection of the optimal method for a clinical setting.

Mass spectrometry (MS)-based proteomics profiling has undoubtedly increased the knowledge about cellular processes and functions. However, its applicability for paucicellular sample analyses is currently limited. Although new approaches have been developed for single-cell studies, most of them have not (yet) been standardized and/or require highly specific (often home-built) devices, thereby limiting their broad implementation, particularly in non-specialized settings. To select an optimal MS-oriented proteomics approach applicable in translational research and clinical settings, we assessed 10 different sample preparation procedures in paucicellular samples of closely-related cell types. Particularly, five cell lysis protocols using different chemistries and mechanical forces were combined with two sample clean-up techniques (C18 filter- and SP3-based), followed by tandem mass tag (TMT)-based protein quantification. The evaluation was structured in three phases: first, cell lines from hematopoietic (THP-1) and non-hematopoietic (HT-29) origins were used to test the approaches showing the combination of a urea-based lysis buffer with the SP3 bead-based clean-up system as the best performer. Parameters such as reproducibility, accessibility, spatial distribution, ease of use, processing time and cost were considered. In the second phase, the performance of the method was tested on maturation-related cell populations: three different monocyte subsets from peripheral blood and, for the first time, macrophages/microglia (MAC) from glioblastoma samples, together with T cells from both tissues. The analysis of 50,000 cells down to only 2,500 cells revealed different protein expression profiles associated with the distinct cell populations. Accordingly, a closer relationship was observed between non-classical monocytes and MAC, with the latter showing the co-expression of M1 and M2 macrophage markers, although pro-tumoral and anti-inflammatory proteins were more represented. In the third phase, the results were validated by high-end spectral flow cytometry on paired monocyte/MAC samples to further determine the sensitivity of the MS approach selected. Finally, the feasibility of the method was proven in 194 additional samples corresponding to 38 different cell types, including cells from different tissue origins, cellular lineages, maturation stages and stimuli. In summary, we selected a reproducible, easy-to-implement sample preparation method for MS-based proteomic characterization of paucicellular samples, also applicable in the setting of functionally closely-related cell populations.

Optimization Methods for Feasible Deployment Planning of Future Mobile Networks

The exponential growth of mobile traffic requires mobile operators to update their network infrastructure to provide greater capacity and better connections for end-users. A promising alternative is to deploy heterogeneous networks (HetNets) that combine macrocells and small cells; however, this alternative increases the complexity and cost of transport (connections between the small cells and the operator’s control center). Most planning strategies outlined in the literature are aimed at reducing the number of small cells without considering important aspects involving transport (access backbone). With the advent of centralized architectures, this point becomes essential, since it is necessary to consider the potential impact of the transport segment on the deployment cost of the network (with the advent of the fronthaul). In this sense, this work proposes an optimal multiobjective model of radio and transport allocation based on linear programming to minimize the total cost of the network and two efficient heuristics to obtain a near-optimal solution. Considering a real case study of the literature, we show the cost (financial and computational) of the optimal placement of radio and transport infrastructure and the limitations of the solution. We also compare the proposed function placement heuristic with the optimal solution in terms of cost efficiency and execution time and demonstrate that it can provide a good estimation of the deployment cost in a much shorter time, with an approximation of up to 10% in relation to the optimal model.

High quality mapping of chromatin at or near the nuclear lamina from small numbers of cells reveals cell cycle and developmental changes of chromatin at the nuclear periphery.

The chromatin associated with the nuclear lamina (NL) is referred to as lamina-associated domains (LADs). Here, we present an adaptation of the tyramide-signal amplification sequencing (TSA-seq) protocol, which we call chromatin pull down-based TSA-seq (cTSA-seq), that can be used to map chromatin regions at or near the NL from as little as 50 000 cells. The cTSA-seq mapped regions are composed of previously defined LADs and smaller chromatin regions that fall within the Hi-C defined B-compartment containing nuclear peripheral heterochromatin. We used cTSA-seq to map chromatin at or near the assembling NL in cultured cells progressing through early G1. cTSA-seq revealed that the distal ends of chromosomes are near or at the reassembling NL during early G1, a feature similar to those found in senescent cells. We expand the use of cTSA-seq to the mapping of chromatin at or near the NL from fixed-frozen mouse cerebellar tissue sections. This mapping reveals a general conservation of NL-associated chromatin and identifies global and local changes during cerebellar development. The cTSA-seq method reported here is useful for analyzing chromatin at or near the NL from small numbers of cells derived from both in vitro and in vivo sources.

Astrocyte Progenitors Derived From Patients With Alzheimer Disease Do Not Impair Stroke Recovery in Mice.

Species-specific differences in astrocytes and their Alzheimer disease-associated pathology may influence cellular responses to other insults. Herein, human glial chimeric mice were generated to evaluate how Alzheimer disease predisposing genetic background in human astrocytes contributes to behavioral outcome and brain pathology after cortical photothrombotic ischemia. Neonatal (P0) immunodeficient mice of both sexes were transplanted with induced pluripotent stem cell-derived astrocyte progenitors from Alzheimer disease patients carrying PSEN1 exon 9 deletion (PSEN1 ΔE9), with isogenic controls, with cells from a healthy donor, or with mouse astrocytes or vehicle. After 14 months, a photothrombotic lesion was produced with Rose Bengal in the motor cortex. Behavior was assessed before ischemia and 1 and 4 weeks after the induction of stroke, followed by tissue perfusion for histology. Open field, cylinder, and grid-walking tests showed a persistent locomotor and sensorimotor impairment after ischemia and female mice had larger infarct sizes; yet, these were not affected by astrocytes with PSEN1 ΔE9 background. Staining for human nuclear antigen confirmed that human cells successfully engrafted throughout the mouse brain. However, only a small number of human cells were positive for astrocytic marker GFAP (glial fibrillary acidic protein), mostly located in the corpus callosum and retaining complex human-specific morphology with longer processes compared with host counterparts. While host astrocytes formed the glial scar, human astrocytes were scattered in small numbers close to the lesion boundary. Aβ (beta-amyloid) deposits were not present in PSEN1 ΔE9 astrocyte-transplanted mice. Transplanted human cells survived and distributed widely in the host brain but had no impact on severity of ischemic damage after cortical photothrombosis in chimeric mice. Only a small number of transplanted human astrocytes acquired GFAP-positive glial phenotype or migrated toward the ischemic lesion forming glial scar. PSEN1 ΔE9 astrocytes did not impair behavioral recovery after experimental stroke.

Comprehensive Immunopeptidome Analysis of I-Ab-bound peptides from Thymus, Splenic B cells and Dendritic cells in C57BL/6 mice

Antigen presentation in the thymus is central to selection of CD4+ and CD8+ T cells. Despite postulated roles for differential central and peripheral antigen presentation pathways for MHC-I and MHC-II, comprehensive MHC Class II immunopeptidome analysis of the thymus has not been reported. Advances in mass spectrometry (MS) technology now allow analysis of the MHC-presented peptidomes of tissue samples containing small numbers of antigen-presenting cells. To map the immunopeptidome in the thymus of C57/BL6 mice presented by I-Ab, we used immunoaffinity coupled to MS. We identified >1000 peptides using a conventional data-dependent acquisition strategy (DDA). We compared the immunopeptidome of thymus to that of the splenic B cells and dendritic cells. We also identified >1000 peptides in splenic B cells and dendritic cells. Some of the core epitopes were unique to thymus or splenic B cells or dendritic cells, although most were shared. We observed differences in the general characteristics of the peptidomes presented by thymus as compared to splenic B cells and dendritic cells, including length distribution and hydrophobicity. To help understand the differences, we selected 57 peptides representative of thymic-derived, splenic B-derived, or shared peptidomes, and studied their binding affinity to I-Ab. Results suggest that the pool of peptides presented by I-Ab in thymus has lower MHC binding affinities than does the pool presented by splenic B cells. We are further mapping the immunopeptidome of mTEC’s to understand the peptide length differences in thymus peptidome and if they are regulated by differential DM mediated editing. We are also exploring a data-independent acquisition strategy (DIA) for quantitative comparison of peptides identified in thymus, splenic B cells and dendritic cells to further characterize antigen presentation differences in thymus and periphery.

Clinicopathological features of angioimmunoblastic T-cell lymphoma pattern Ⅰ

Objective: To investigate the clinicopathological features of angioimmunoblastic T-cell lymphoma pattern Ⅰ (AITL Pattern Ⅰ). Methods: The clinicopathological data of 11 AITL Pattern Ⅰ cases that were diagnosed at the Beijing Friendship Hospital Affiliated to Capital Medical University (10 cases) and Beijing Lu Daopei Hospital (1 cases) from January 2019 to October 2021 were retrospectively collected. Immunophenotype, Epstein-Barr virus infection status and T cell receptor (TCR) clonality of the tumor cells were tested, and clinicopathological features of cases were analyzed. Results: Among the 11 AITL Pattern Ⅰ cases, the male to female ratio was 1.2∶1.0. The median age was 59 years (range 47-78 years). Seven cases had B symptoms, while eleven cases presented with systemic lymphadenopathy. According to Ann Arbor system staging, two cases were classified as stage Ⅰ-Ⅱ, and 9 cases as stage Ⅲ-Ⅳ. Hepatosplenomegaly was present in two cases (2/11), three cases (3/11) had skin rash and pruritus, and two cases (2/11) had pleural effusion. Previously, 6 cases (6/11) were diagnosed as reactive hyperplasia, 1 case (1/11) as EBV-associated lymphoproliferative disorder, and 4 cases (4/11) as hyperplasia of lymphoid tissue, which was unable to exclude lymphoma. Histologically, all the 11 cases showed hyperplastic follicles in the paracortical regions with well-formed germinal centers. The hyperplastic follicles showed ill-defined borders and attenuated mantle zones in 7 cases. Mantle zones completely disappeared in 4 cases. The follicles were surrounded by a thin layer of atypical lymphocytes with bright or faintly stained cytoplasm. In 2 cases, the clear cells were located between the germinal centers and the thin residual mantle cell layers, showing a circular growth pattern. The cells were medium in size, with irregular karyotype, coarse chromatin and indistinct nucleoli. Immunohistochemically, CD21 staining showed that the meshworks of follicular dendritic cells(FDC)were mainly confined to the follicles. There was a subtle expansion of the meshworks of FDC in 4 cases with ill-defined borders. The atypical cells surrounding the follicles expressed CD3 (11/11), CD4 (11/11), PD-1 (11/11), CXCL13 (6/11), ICOS (10/11) and CD10 (7/11). PD-1 staining showed a strong perifollicular pattern, and a small number of positive cells were scattered around the high endothelial veins in the interfollicular region. CXCL13, ICOS and CD10 showed similar distribution patterns. EBV-encoded small RNA probe (EBER) in situ hybridization showed that EBER positive B lymphocytes were scattered in the interfollicular region (5-20/HPF) in all cases. T cell receptor gene rearrangement was monoclonal in all cases. Conclusions: Diagnosing AITL Pattern Ⅰ may be challenging and requires comprehensive analysis of clinical manifestations, histological morphology, immunophenotype and gene rearrangement results.

Histone Modification Analysis of Low-Mappability Regions.

Posttranslational modifications of histone are intimately related to chromatin/chromosome-mediated cellular events. Among all, the roles of histone modifications including acetylation, methylation, ubiquitination, and SUMOylation of lysine or arginine residue of nucleosome core histones in gene expression have been intensively studied. Genome-wide profiles of histone modification marks revealed their combinatorial organization in the functional features of chromatin. Analysis of histone modification by chromatin immunoprecipitation (ChIP) is one of the standard assays to examine chromatin states. Although high-throughput sequencing analysis (ChIP-seq) is now widely conducted, classical ChIP-qPCR analysis has advantages in investigation of multiple histone modification marks at a target site simply through theuse ofrelatively small numbers of cells. Since ChIP-qPCR is devoid of biases caused by overamplification and inaccurate mapping of sequencing reads, it is a more reliable quantification method than genome-wide ChIP-seq especially for analyses of the low-mappability regions, which harbor many repetitive sequences and/or highly homologous segmental multiplications as found in gene clusters. We have recently analyzed histone H3 and H4 modifications of the Zscan4 family gene loci in an 880kb gene cluster and found that the atypical enhancer-like structure is formed upon derepression of Zscan4. In this chapter, we describe the detailed protocols for histone modification ChIP-assay of repeat-enriched gene cluster regions. The protocol here we applied to mouse ES cells, but the protocol is perfectly applicableto humanculturedcellsand specimens.

Research on mechanism of curcumin carried with PLGA-DSPE-PEG hybridized nanoparticles on regulating the nuclear receptor coactivator5 presentation, oxidative stress and presentation of alfa-fetoprotein in rats with liver cancer

This study intends to discuss the mechanism of curcumin carried with poly (lactic-co-glycolic acid)-1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000] (PLGA-DSPE-PEG) hybridized nanoparticles on regulating the Nuclear receptor coactivator5 (NCOA5) expression, oxidative stress and level of Alfa-fetoprotein (AFP) in rats with liver cancer. Curcumin carried with PLGA-DSPE-PEG hybridized nanoparticles was prepared. These rats were divided into normal set, model set, curcumin set and set of curcumin and nanoparticles randomly. The presentation of alanine transaminase (ALT) and aspartate aminotransferase (AST) in every set was detected. The pathological change in liver tissue with hematoxylin and eosin (H&amp;E) staining method, level of malondialdehyde (MDA) and superoxide dismutase (SOD) was detected. Presentation of AFP and NCOA5 was detected with Western Blotting assay and real-time reverse transcription–polymerase chain reaction (RT-PCR). Level of ALT, AST, MDA and AFP in normal set was the highest, but lowest in the set of curcumin and nanoparticles. The SOD presentation in the set of curcumin and nanoparticles was the highest. There was distinct surface texture, good glossiness and orderly arrangement along edge in normal set. There were grey nodular nodules, vacuole and dark-red lumps in part of live tissue in model set. There was slightly distinct liver plate texture, punctate particles and a small amount of congestion in curcumin set. There was distinct liver plate texture with orderly arrangement and a small number of inflammatory cells in set of curcumin and nanoparticles. The level of ALT and AST in rats with liver cancer was reduced by curcumin carried with PLGA-DSPE-PEG hybridized nanoparticles. The SOD activity was increased and MDA was reduced. The AFP presentation was reduced and NCOA5 expression was increased.