A case of hepatic reticuloendothelial in an infant / 西安交通大学学报(医学版)

Retiform hemangioendothelioma (RH) is a rare vasogenic malignancy with a high local recurrence rate and rare distant metastasis. Hepatic RH in infants is extremely rare. Here we report a case of liver RH in a 7-month-old infant.

A Community Based Cross-Sectional Study to Assess the Socio-Economic and Health Status of Rag Pickers in Varanasi slums, North India

Background- Rag picking is one of the inferior economic activities in the urban informal sector, largely undertaken by children belonging to weaker sections of society for their survival and for supplementing their family income. Rag picking is one of the most dangerous activities in India. Rag pickers are working in a filthy environment surrounded by dust, dogs, and pigs. They suffer from many diseases, such as respiratory problems, anemia, fever, and other problems which include cuts, rashes, and injuries. Waste pickers are the general term adopted by the 1 st World Conference of Waste Pickers in 2008. Objective- [a] To assess the socio-economic condition of ragpickers in Varanasi, [b] To know the health condition and treatment behavior of ragpickers in Varanasi. Materials and Methods- A community-based descriptive cross-sectional study in which ragpickers reside in the slum area of Varanasi. All men and women present at the time of the interview who were involved in rag picking from the last 6 months were included. Seriously ill men and women were excluded. Since we had no prior knowledge about the proportion of cases (P) so a pilot study was conducted in which prevalence of health problem found in rag pickers in the last 6 months (P) = 0.90. Total estimated sample size is 100. In this study, Simple Random sampling was used. By using this sampling 100 individuals were selected randomly, fulfilling the inclusion criteria. Results-82 (82.0%) rag pickers were not educated in which 75 (91.5%) rag pickers suffered from health problems. Maximum 74 (74.0 %) rag pickers were migrants. Maximum 74 (74.0%) rag pickers were married in which 36 (48.0 %) rag pickers were married between 14- 18 ages. 80 (80.0%) rag pickers had not used any precautionary measure during rag picking in which 73 (91.2%) rag pickers faced health problems. Maximum 39(39.0%) were affected by dust during rag picking. Only 17(17%) ragpickers preferred government hospitals for treatment. 75 (75.0 %) rag pickers were addicted to intoxication in which 66 (88.0%) rag pickers faced health problems. 91 (91%) ragpickers affected by any health problems in the last 6 months in which a maximum of 63.7% of rag pickers suffered from fever, 60.4% of rag pickers complained about injuries, 56.2 % of rag pickers suffered from weakness problems, 52.7% of rag pickers affected by cough problems. There is a highly significance between educational status and any kind of intoxication. Discussion- The literacy level among the rag pickers was 18% which is much lower than the average literacy rate of Varanasi 75.60% (2011census). A study on slum dwellers in Dhaka showed the literacy rate higher than the finding of this study (18 vs 25 %). 28.0% of rag pickers adopted this work because they had no education. There is a high significance between educational status and any kind of intoxication. Chances of intoxication are high when no education among rag pickers. It is highly significant between educational status and health problems of rag pickers in the last 6 months. Uneducated rag pickers were not aware of the health effects.

Isolation and identification of primary brain microvascular endothelial cells of rat using thin-layer cell culture method / 神经解剖学杂志

Objective:To extract rat brain microvascular endothelial cells(BMECs)using thin-layer cell culture method.Methods:The brain cortex of four-week-old SD rats was obtained,which was chopped,sieved,and digested with type II collagenase to obtain microvascular segments.The amount of culture medium was strictly controlled,and it was inoculated into culture flasks for primary culture.The target cells were morphologically observed by using an invert-ed phase contrast microscope,and the factor Ⅷ related antigen(FⅧRag)was identified by using immunocytochemi-cal staining.Results:The target cells cultured in the inverted phase contrast microscope showed typical endothelial-like monolayer cobble stone-like mosaic growth;FⅧRag positive cells accounted for more than 99％of the total cells.Conclusion:Thin-layer cell culture method can successfully isolate and cultivate high-purity rat BMECs.

Establishment of hKDR+/+ Humanized and Rag1-/- Gene Knockout Double Genetically Modified Mouse Model / 实验动物与比较医学

ObjectiveThrough improving the potential of vascular endothelial growth factor receptor (VEGFR)-humanized mouse model (hKDR+/+) with C57BL/6N background to allow the growth of different mouse tumor cell lines, to establish novel tumor-bearing mouse models which can support in vivo tumorigenesis of different mouse tumor cell lines and be used to evaluate drugs targeting VEGFR.MethodsFirstly, a method to evaluate the in vivo activity of antibody targeting VEGFR based on the hKDR+/+ humanized mouse model was established. Recombinant activating gene 1 (Rag1) knockout mice (Rag1-/-) were established using CRISPR/Cas9 technology. Then these Rag1-/- mice were crossed with hKDR+/+ mice to get a double gene modified homozygous hKDR+/+/Rag1-/- mouse model by screening. Finally, tumor cell lines derived from different mouse strains were tested on the double gene-modified mouse model to compare the differences in tumor growth. ResultsAntibodies designed for VEGFR showed significant anti-tumor activity in hKDR+/+ mice, which significantly reduced tumor volume and weight compared with the PBS group (P<0.01, P<0.05). The number of B cells and T cells in the peripheral blood of Rag1-/- mice and hKDR+/+/Rag1-/- mice decreased (P<0.05, P<0.001). Tumors were observed in hKDR+/+/Rag1-/-, Rag1-/-, wild-type, and hKDR+/+ mice after 7 d of inoculation of MC38 cells derived from C57BL/6 mice. Tumors were only observed in groups of hKDR+/+/Rag1-/- and Rag1-/- mice, but not in the wild-type and hKDR+/+ mice after 10 d of inoculation with CT26 cells derived from BALB/c mice. After 3 weeks of inoculation, the tumor volume of hKDR+/+/Rag1-/- mice was significantly larger than that of Rag1-/- mice (P<0.01). ConclusionRag1 knockout mice were obtained and a novel hKDR+/+/Rag1-/- double genes modified mouse model was further screened. The tumor cell lines from different mouse strain origins were more prone to growth in mice with Rag1 gene deficiency. The results suggest that the reduced immune response of hKDR+/+ humanized mice will improve the capacity of supporting the growth of mouse tumor lines in the model. As a result, more tumor-bearing mouse models may be constructed for the evaluation of drugs targeting VEGFR in this way.

Rearrangement of T cell receptor genes Tcra and Tcrd and its regulatory mechanism / 中华微生物学和免疫学杂志

The high diversity of T cell receptors (TCRs) is the basis for recognizing antigens, playing an essential role in adaptive immunity. TCR diversity is generated from V(D)J rearrangement during the thymocyte development in the thymus. Standing out from the four TCR genes, Tcra and Tcrd genes are characterized by locating at the same locus and sharing specific V genes. Hence, their rearrangement and regulation have a certain particularity. Previous studies mainly focused on cis-regulatory elements and trans-acting factors regulating the Tcra/ Tcrd rearrangement. However, recent progress has shown that chromatin spatial organization plays an essential role in antigen receptor gene rearrangement. Chromatin organization proteins, such as CTCF-Cohesin, are involved in regulating rearrangement and enhancing the diversity of TCR repertoire by loop extrusion. Recombinase RAG also scans chromatin of antigen receptor genes for rearrangement. This review described the progress in the rearrangement of Tcra and Tcrd genes and the possible regulatory mechanism, especially the influence of the chromatin spatial organization.

Sensors for the mTORC1 pathway regulated by amino acids / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The mechanistic target of rapamycin complex 1 (mTORC1) controls cell growth and metabolism in response to various environmental inputs, especially amino acids. In fact, the activity of mTORC1 is highly sensitive to changes in amino acid levels. Over past decades, a variety of proteins have been identified as participating in the mTORC1 pathway regulated by amino acids. Classically, the Rag guanosine triphosphatases (GTPases), which reside on the lysosome, transmit amino acid availability to the mTORC1 pathway and recruit mTORC1 to the lysosome upon amino acid sufficiency. Recently, several sensors of leucine, arginine, and S-adenosylmethionine for the amino acid-stimulated mTORC1 pathway have been coming to light. Characterization of these sensors is requisite for understanding how cells adjust amino acid sensing pathways to their different needs. In this review, we summarize recent advances in amino acid sensing mechanisms that regulate mTORC1 activity and highlight these identified sensors that accurately transmit specific amino acid signals to the mTORC1 pathway.

Sensors for the mTORC1 pathway regulated by amino acids / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The mechanistic target of rapamycin complex 1 (mTORC1) controls cell growth and metabolism in response to various environmental inputs, especially amino acids. In fact, the activity of mTORC1 is highly sensitive to changes in amino acid levels. Over past decades, a variety of proteins have been identified as participating in the mTORC1 pathway regulated by amino acids. Classically, the Rag guanosine triphosphatases (GTPases), which reside on the lysosome, transmit amino acid availability to the mTORC1 pathway and recruit mTORC1 to the lysosome upon amino acid sufficiency. Recently, several sensors of leucine, arginine, and S-adenosylmethionine for the amino acid-stimulated mTORC1 pathway have been coming to light. Characterization of these sensors is requisite for understanding how cells adjust amino acid sensing pathways to their different needs. In this review, we summarize recent advances in amino acid sensing mechanisms that regulate mTORC1 activity and highlight these identified sensors that accurately transmit specific amino acid signals to the mTORC1 pathway.

CRISPR/Cas9-mediated knockout of Rag-2 causes systemic lymphopenia with hypoplastic lymphoid organs in FVB mice / 한국실험동물학회지

Recombination activating gene-2 (RAG-2) plays a crucial role in the development of lymphocytes by mediating recombination of T cell receptors and immunoglobulins, and loss of RAG-2 causes severe combined immunodeficiency (SCID) in humans. RAG-2 knockout mice created using homologous recombination in ES cells have served as a valuable immunodeficient platform, but concerns have persisted on the specificity of RAG-2-related phenotypes in these animals due to the limitations associated with the genome engineering method used. To precisely investigate the function of RAG-2, we recently established a new RAG-2 knockout FVB mouse line (RAG-2(−/−)) manifesting lymphopenia by employing a CRISPR/Cas9 system at Center for Mouse Models of Human Disease. In this study, we further characterized their phenotypes focusing on histopathological analysis of lymphoid organs. RAG-2(−/−) mice showed no abnormality in development compared to their WT littermates for 26 weeks. At necropsy, gross examination revealed significantly smaller spleens and thymuses in RAG-2(−/−) mice, while histopathological investigation revealed hypoplastic white pulps with intact red pulps in the spleen, severe atrophy of the thymic cortex and disappearance of follicles in lymph nodes. However, no perceivable change was observed in the bone marrow. Moreover, our analyses showed a specific reduction of lymphocytes with a complete loss of mature T cells and B cells in the lymphoid organs, while natural killer cells and splenic megakaryocytes were increased in RAG-2(−/−) mice. These findings indicate that our RAG-2(−/−) mice show systemic lymphopenia with the relevant histopathological changes in the lymphoid organs, suggesting them as an improved Rag-2-related immunodeficient model.

Research progress of immunodeficient animal models using gene modification techniques / 中国实验动物学报

The established immunodeficient animal models could be used as valuable resource for mechanism re-search of related disease in humans, drug discovery and development, translational research and stem cell research.How-ever, it is difficult and low-efficient to establish the genetic modified animal models using traditional technologies.The re-ports for immunodeficient animal models are few in middle-size and large animals.Recently, several effective gene-targeting tools, including ZFNs, TALENs, CRISPR/Cas9, develop quickly and provide technology basis for the establishment of im-munodeficient animal models.In this paper, the technology principles and research progresses of ZFNs, TALENs, CRISPR/Cas9 are introduced.The significant progresses of these emerging technologies achieved in immunodeficient ani-mal models are also elaborated, including KO Rag1/Rag2 rabbit, KO Rag1/Rag2 pig, KO IL2rg pig, KO Ppar-g/Rag1 monkey, and so on.In addition to being models for researching SCID-related diseases in humans, and evaluating the effica-cy and safety of stem-cell engraftment, these models may be also useful to develop surgical procedures for placement of grafts before clinical trials in humans, to produce humanized animals and bridge the gap between laboratory animal and medicical research.The immunodeficient animal models described here represent a step toward the comprehensive evalua-tion of preclinical cellular regenerative strategies.

Neuronal death signaling pathway: Involvement of Rag1 activation.

Although the transcription factor, nuclear factor-κB (NF-κB) is known to regulate cell death and survival, its precise role in cell death within the central nervous system (CNS) remains unknown. We previously reported that mice with a homozygous deficiency for NF-kBp50 spontaneously developed optic neuropathy. We examined the expression and activation of pro-opoptotic factor(s), which mediate optic neuropathy in p50-deficient (p50-/-) mice. Recombination activating gene 1 (Rag1) is known to regulate the recombination of immunoglobulin V(D)J. Experiments with genetically engineered mice revealed the involvement of Rag1 expression in the apoptosis of Brn3a-positive retinal ganglion cells (RGCs), and also showed the specific effects of a p50-deficency on the activation of Rag1 gene transcription. Furthermore, a genetic analysis of murine neuronal stem-like cells clarified the biological significance of Rag1 in NMDA (N-methyl-D-aspartat)-induced neuronal apoptosis. The apoptotic regulating factors, Bax, and cleaved caspase 3, 8, and 9 were observed in HEK293 cells expressing the external molecule of Rag1, and a human histological examination revealed the expression of Rag1 in RGCs Recent study indicated that Rag1 played a role in optic neuropathy as a pro-apoptotic candidate in p50-/- mice. This result may lead to new therapeutic targets in optic neuropathy.

Recombination-activating gene 1 and 2 (RAG1 and RAG2) in flounder (Paralichthys olivaceus).

During the development of B and T lymphocytes, Ig and TCR variable region genes are assembled from germline V, D, and J gene segments by a site-specific recombination reaction known as V(D)J recombination. The process of somatic V(D)J recombination, mediated by the recombination-activating gene (RAG) products, is the most significant characteristic of adaptive immunity in jawed vertebrates. Flounder (Paralichthys olivaceus) RAG1 and RAG2 were isolated by Genome Walker and RT-PCR, and their expression patterns were analysed by RT-PCR and in situ hybridization on sections. RAG1 spans over 7.0 kb, containing 4 exons and 3 introns, and the full-length ORF is 3207 bp, encoding a peptide of 1068 amino acids. The first exon lies in the 5′-UTR, which is an alternative exon. RAG2 full-length ORF is 1062 bp, encodes a peptide of 533 amino acids, and lacks introns in the coding region. In 6-monthold flounders, the expression of RAG1 and RAG2 was essentially restricted to the pronephros (head kidney) and mesonephros (truck kidney). Additionally, both of them were mainly expressed in the thymus. These results revealed that the thymus and kidney most likely serve as the primary lymphoid tissues in the flounder.

The study on organ weights, blood physiological and biochemical parameters and immune cells in Rag2 knockout mice / 中国比较医学杂志

Objective To measure the organ weights , blood physiological and biochemical parameters , and immune function of Specific Pathogen Free ( SPF) Rag2 knockout ( KO) mice.Methods Rag2 knockout mice were selected at five and ten weeks , and the organ weights , blood physiological and biochemical parameters were observed .The percentages of CD3+, CD4+, CD8+, CD19+, B220+, NK1.1+, and CD11b+were checked by FCM in Rag2 KO mice at six week of age in terms of its T , B lymphocyte function and NK cell activity .Results Among the same sex group of NOD/SCID mice, the weights of brain, lung, spleen, liver, heart, kidney, and the levels of TBIL, WBC in 10 weeks of age are higher than 5 weeks.At the same age, the weights of heart, kidney, liver, spleen, and the levels of AST, ALP, A/G, GLU, PLT, PCT, WBC, and LYM%in male mice are higher than females.The Rag2 KO mice lacks T cells(0.36 ±0.15)%、CD4+T cells(0.21 ±0.06)%、CD8+T cells(0.23 ±0.07)%、CD19+B cells(0.28 ±0.04)%、B220+B cells(2.03 ±0.42)%).The percentage of NK cells is(24.13 ±3.62)%, and the percentage of granulocytes is (57.20 ±3.85)%.Conclusion The study suggests that the organ weights , blood physiological and biochemical parameters are affected by age and gender in Rag2 KO mice, which main biological characteristics are similar with C 57BL/6J mice.The Rag2 KO mice show the deficiency of T , B cells function .

Tatadrama: A violência mantida em segredo que emerge na boneca de pano / Tatadrama: Violence kept secret that emerges through working with rag dolls

O método Tatadrama, com a utilização da boneca de pano do folclore brasileiro como Objeto Intermediário, foi fundamental para recuperar a identidade de mulheres que participaram de 14 oficinas, uma delas descrita nesta comunicação breve. A customização da boneca possibilitou às participantes contextualizar suas necessidades, interagir com elas mesmas desvendando segredos, inclusive de violências, o que lhes abriu novas perspectivas de vida ao resgatarem do inconsciente valores e desejos para enfrentar e transformar sua realidade...

Tatadrama utilizes rag dolls of the Brazilian folklore as Intermediary Object. This method proved to be fundamental in recovering the identity of women who participated in 14 workshops, as described in this brief paper. The use of these dolls has allowed participants to contextualize their needs and to interact with themselves unveiling secrets, such as experiences of violence; this has opened up new vistas of life by redeeming from the unconscious values and desires that can confront and transform these women's reality...

Reconstitution of Human Immune Cells with Co-transplantation of Fetal Liver/Thymus Tissues and Cultured Umbilical Cord Blood-derived Hematopoietic Stem Cells in Rag2(-/-)gamma(c)(-/-) Mice

PURPOSE: Many researchers have tried to develop animal models that mimic the human immune system, e.g. a humanized mouse model, to improve the engraftment of hematopoietic stem cells and develop human immune cells in an animal model. This study evaluated the feasibility of the cultured human umbilical cord blood (hUCB)-derived CD34(+) cells for cell expansion, in Rag2(-/-)gamma(c)(-/-) mice, and establish co-transplantation with human fetal thymus/liver tissue (Thy/Liv) under the kidney capsule. METHODS: Co-transplantation of hUCB-derived CD34(+) cells with Thy/Liv was performed. The hUCB-derived CD34(+) cells were prepared by freshly thawing (G1) and culturing for 7 days with two types of cytokine combinations (G2, G3). The CD45(+) cell populations were measured at 6, 8, 10 and 16 weeks in the peripheral blood. The splenocytes were cultured with mitogenic stimuli (PHA -L or IL-2) at 20 weeks post- transplantation, and the proliferation of human immune cells was evaluated. RESULTS: There were no significant differences in the human CD45(+) cell populations at 6, 8, 10 and 16 weeks post-transplantation between the groups. In the cultured splenocytes at 20 weeks post-transplant with PHA-L or IL-2, there was remarkable expansion of CD3(+) cells in the three groups. Although no CD19(+) cells were detected in the spleen, human Ig G was detected in the sera of these mice. CONCLUSION: The cultured and expanded hUCB-derived cells with cytokine combinations might be a feasible cell source in humanized mouse modeling. In addition, human immune cells can be reconstituted from the co-transplantation of Thy/Liv and cultured hUCB-derived CD34(+) cells.

Rapid detection of K-ras gene point mutation in pancreatic adenocarcinoma by Pyrosequencing / 中华检验医学杂志

Objective To establish a method for detecting K-rag gene point mutation in pancreatic adenocarcinoma based on the pyrosequencing and to compare its performance with that of Sanger sequencing.Methods Genomic DNA was extracted from formalin-fixed,paraffin-embedded pancreatic tissues including 49 pancreatic adenocarcinoma,10 normal pancreas,11 chronic pancreatitis,18 benign pancreatic tumor,7 insulin carcinoma,9 ampullary carcinoma,7 bile duct carcinoma and 7 duodenum papillary adenocarcinoma tissues.K-rag gene point mutations at codon 12 were analyzed by pyrosequencing and Sanger sequencing,respectively.Results No mutant K-ras gene Wag detected in normal pancreas,chronic pancreatitis,benign pancreatic tumor,insulin carcinoma,ampullary carcinoma,bile duct carcinoma and duodenum papillary adenocarcinoma tissues by either pyrosequencing or Sanger sequencing.K-rag gene point mutation was detection rate in pancreatic adenocarcinoma tissues was 71.4%(35/49)by pyrosequencing and 61.2%(30/49)by Sanger sequencing,respectively.Conclusions Pyrosequencing is more sensitive than Sanger sequencing and is also accurate,rapid and of high throushput in detecting mutant K-ras gene.It may serve as a practical method for fast batch analysis of clinical samples.

Progress in Epigenetic Regulation of V(D)J Recombination / 生物化学与生物物理进展

During lymphocyte development antigen receptor genes undergo V(D)J recombination to obtain antigen binding specificity and diversity. This process is not only controlled by genetic factors, such as tissue- and stage- specificity of RAG-1/2 protein, germline transcriptional activity and ACEs, but also regulated at epigenetic level. The chromatin accessibility of recombinase is associated with the chromatin configuration around the targeted gene segments. Thus, activation of V(D)J recombination requires the recruitment of remodeling complexes for changing the accessibility in the localized chromatin. Moreover, docking of remodeling complexes, which serve for creating active chromatin environment, relies on certain patterns of chromatin modification. Some recent findings regarding epigenetic regulation mechanisms in V(D)J recombination, such as CpG methylation, histone modification, nucleosome remodeling and nuclear topology were reviewed.

New Animal Model of Renal Cell Carcinoma / 대한비뇨기과학회지

PURPOSE: This study was undertaker in order to develop and characterize a new murine renal cell carcinoma(RCC) model which may be closely analogous to the human RCC and which may serve as a basis for the evaluation of new therapeutic modalities. MATERIALS AND METHODS: The tumor being investigated, Rag cell line, arose spont- aneously as a renal adenocarcinoma in an inbred Balb/c mouse. Tumor cells were injected into subrenal capsule and injected intravenously and intraperitoneally to induce tumors. Among the orthotopically inoculated animals, some were performed a nephrectomy to mimic the situation mostly often encountered in the human. RESULTS: 1 Tumors were transferred to all animals where inoculated into subrenal capsule. 2. The growth rates of tumor-bearing mice were retarded compared to control mice. 3. Tumors grew exponentially when inoculated into subrenal capsule. The doubling time of tumor was approximately 13 days. 4. Spontaneous lung metastasis could be better induced in the subrenal capsule model(80%). Higher rate of lung metastasis was obtained by intravenous inoculation of tumor cells(85.7%). 5. After nephrectomy performed on 14 days following subrenal capsular Inoculation of tumor cells, lung metastasis developed spontaneously in 80% of cases. CONCLUSIONS: The behavior of Rag tumor following subrenal capsular inoculation mimics that of human RCC from the viewpoint of the formation of primary tumor mass on the kidney followed by the development of spontaneous metastases. This model can be used for the evaluation of the therapeutic erects both on the primary tumor as well as on the metastatic disease.