No abstract

Gap junctions are highly conductive channels that allow the direct transfer of intracellular messengers such as Ca 2ϩ and inositol triphosphate (IP 3 ) between interconnected cells. In brain, astrocytes are coupled extensively by gap junctions. We found here that gap junctions among astrocytes in acutely prepared brain slices as well as in culture remained open during ischemic conditions. Uncoupling first occurred after the terminal loss of plasma membrane integrity. Gap junctions therefore may link ischemic astrocytes in an evolving infarct with the surroundings. The free exchange of intracellular messengers between dying and potentially viable astrocytes might contribute to secondary expansion of ischemic lesions.

The study on circulating tumor cells (CTCs) has great significance for cancer prognosis, treatment monitoring, and metastasis diagnosis, in which isolation and enrichment of CTCs are key steps due to their extremely low concentration in peripheral blood. Herein, magnetic nanospheres (MNs) were fabricated by a convenient and highly controllable layer-by-layer assembly method. The MNs were nanosized with fast magnetic response, and nearly all of the MNs could be captured by 1 min attraction with a commercial magnetic scaffold. In addition, the MNs were very stable without aggregation or precipitation in whole blood and could be re-collected nearly at 100% in a monodisperse state. Modified with anti-epithelial-cell-adhesion-molecule (EpCAM) antibody, the obtained immunomagnetic nanospheres (IMNs) successfully captured extremely rare tumor cells in whole blood with an efficiency of more than 94% via only a 5 min incubation. Moreover, the isolated cells remained viable at 90.5 ± 1.2%, and they could be directly used for culture, reverse transcription-polymerase chain reaction (RT-PCR), and immunocytochemistry (ICC) identification. ICC identification and enumeration of the tumor cells in the same blood samples showed high sensitivity and good reproducibility. Furthermore, the IMNs were successfully applied to the isolation and detection of CTCs in cancer patient peripheral blood samples, and even one CTC in the whole blood sample was able to be detected, which suggested they would be a promising tool for CTC enrichment and detection.

Transplantation of hematopoietic stem cells (HSCs) with a naturally occurring CCR5 mutation confers a loss of detectable HIV-1 in the patient, making ablation of the CCR5 gene in HSCs an ideal therapy for an HIV-1 cure. Although CCR5 disruption has been attempted in CD4 T cells and hematopoietic stem/progenitor cells (HSPCs), efficient gene editing with high specificity and long-term therapeutic potential remains a major challenge for clinical translation. Here, we established a CRISPR/Cas9 gene editing system in human CD34 HSPCs and achieved efficient CCR5 ablation evaluated in long-term reconstituted NOD/Prkdc/IL-2Rγ mice. The CCR5 disruption efficiency in our system remained robust in secondary transplanted repopulating hematopoietic cells. More importantly, an HIV-1 resistance effect was observed as indicated by significant reduction of virus titration and enrichment of human CD4 T cells. Hence, we successfully established a CRISPR/Cas9 mediated CCR5 ablating system in long-term HSCs, which confers HIV-1 resistance in vivo. Our study provides evidence for translating CCR5 gene-edited HSC transplantation for an HIV cure to the clinic.

No abstract

In cerebellar granule neurons, a BH3-only Bcl-2 family member, death protein 5/harakiri, is up-regulated in a JNK-dependent manner during apoptosis induced by potassium deprivation. However, it is not clear whether c-Jun is directly involved in the induction of dp5. Here, we showed that the up-regulation of dp5, but not fas ligand and bim, after potassium deprivation was suppressed by the expression of a dominant negative form of c-Jun. Deletion analysis of the 5-flanking sequence of the dp5 gene revealed that a major responsive element responsible for the induction by potassium deprivation is an ATF binding site located at ؊116 to ؊109 relative to the transcriptional start site. Mutation of this site completely abolished promoter activation. Furthermore, a gel shift assay showed that a specific complex containing c-Jun and ATF2 recognized this site and increased in potassium-deprived cerebellar granule neurons. Chromatin immunoprecipitation demonstrated that c-Jun was able to bind to this site in vivo. Finally, we demonstrated that knockdown of Dp5 by small interfering RNA rescued neurons from potassium deprivation-induced apoptosis. Taken together, these results suggest that dp5 is a target gene of c-Jun and plays a critical role in potassium deprivation-induced apoptosis in cerebellar granule neurons.The Bcl-2 family proteins can be divided into three major subgroups (1). Antiapoptotic proteins, such as Bcl-2, Bcl-X L , and Mcl-1, typically share four conserved motifs termed Bcl-2 homology (BH) 3 domains and inhibit mitochondrial cytochrome c release and apoptosis. Multidomain proapoptotic proteins, the second subgroup, such as Bax, Bak, and Bok, typically have three BH domains but promote cytochrome c release and apoptosis. The third, and the most structurally diverse subgroup, is the BH3-only proteins, including Dp5/HRK (death protein 5/harakiri), Bim (Bcl2-interacting mediator of cell death), Bid, Bad, Puma, and Noxa, which share the BH3 domain. The BH3-only proteins are critical initiators of apoptosis. Upon challenge, BH3-only proteins translocate to mitochondria and promote the chromec release by neutralizing the antiapoptotic action of Bcl-2 family members. BH3-only proteins are stringently regulated at the transcriptional and post-translational levels during apoptosis, such as Dp5, Bim, and Puma, depending on the cell type and apoptotic stimulus (2-6). Among the BH3-only proteins, Dp5 is of particular interest to studies of apoptosis in the nervous system. In rodents, the expression of Dp5 is largely restricted to and is developmentally regulated in the nervous system (2, 7). Dp5 is the first found BH3-only protein to be induced by NGF deprivation in sympathetic neurons (2). dp5 is highly homologous to the human gene harakiri (HRK) cloned by a two-hybrid screen with Bcl-2 and Bcl-X L (3). As well as being induced in NGF-deprived sympathetic neurons, the induction of dp5 is also observed in cerebellar granule neurons (CGNs) deprived of potassium, cortical neurons exposed to toxic concentrations of amyl...

This review presents a pattern recognition approach for the diagnosis of malignant effusions. The cytomorphologic features of reactive mesothelial proliferation, mesothelioma and metastatic carcinoma are presented. In addition, the role of ancillary studies in challenging cases and the importance of integrating clinical findings are stressed. An algorithmic approach to the workup of serous effusions as well as pitfalls for false-positive diagnosis are discussed.

Histomorphologic features and routine endocrine immunohistochemical (IHC) markers do not differentiate neuroendocrine tumors (NETs) in relation to their location, making it difficult to establish the site of origin of a metastatic neoplasm. Site-specific markers would be useful, particularly when examining small biopsies. CDX-2 and thyroid transcription factor-1 (TTF-1) are transcription factors that have been recently proposed as IHC markers of intestinal and pulmonary adenocarcinomas, respectively. However, their expression in NETs has not been widely studied. The objective of this study is to evaluate the expression of TTF-1 and CDX-2 in NETs and their potential usefulness in distinguishing gastrointestinal and pulmonary NETs from other sites. We performed an IHC study on formalin-fixed, paraffin-embedded sections from 155 primary NETs, including 60 pulmonary, 60 gastrointestinal, 30 pancreatic, and 5 NETs from other sites. In addition, we evaluated 13 metastatic NETs, including 11 cases of gastrointestinal and 2 of pulmonary origin. In this study, CDX-2 was expressed in 28/60 (47%) of gastrointestinal NETs with the following results: 11/11 (100%) appendiceal, 12/14 (86%) small intestinal, 3/4 (75%) colonic, 2/11 (18%) rectal, and 0/20 (0%) gastric. TTF-1 was expressed in pulmonary carcinoid tumors in 13/30 (43%) and in 27/30 (90%) pulmonary small cell carcinomas. NETs of other origins (pancreas, skin, ovary, and thymus) were negative for both TTF-1 and CDX-2. Metastatic neuroendocrine neoplasms of intestinal origin were positive for CDX-2 and negative for TTF-1. In conclusion, CDX-2 expression is highly specific in identifying NETs of intestinal origin and TTF-1 expression is helpful in identifying NETs of pulmonary origin, which can be quite useful in the diagnosis of metastatic NETs of unknown origin.