Cell membrane-covered drug-delivery nanoplatforms have been garnering attention because of their enhanced biointerfacing capabilities that originate from source cells. In this top-down technique, nanoparticles (NPs) are covered by various membrane coatings, including membranes from specialized cells or hybrid membranes that combine the capacities of different types of cell membranes. Here, hybrid membrane-coated doxorubicin (Dox)-loaded poly(lacticco-glycolic acid) (PLGA) NPs (DPLGA@[RAW-4T1] NPs) were fabricated by fusing membrane components derived from RAW264.7(RAW) and 4T1 cells (4T1). These NPs were used to treat lung metastases originating from breast cancer. This study indicates that the coupling of NPs with a hybrid membrane derived from macrophage and cancer cells has several advantages, such as the tendency to accumulate at sites of inflammation, ability to target specific metastasis, homogenous tumor targeting abilities in vitro, and markedly enhanced multi-target capability in a lung metastasis model in vivo. The DPLGA@[RAW-4T1] NPs exhibited excellent chemotherapeutic potential with approximately 88.9% anti-metastasis efficacy following treatment of breast cancer-derived lung metastases. These NPs were robust and displayed the multi-targeting abilities of hybrid membranes. This study provides a promising biomimetic nanoplatform for effective treatment of breast cancer metastasis.

Cancer vaccines based on resected tumors from patients have gained great interest as an individualized cancer treatment strategy. However, eliciting a robust therapeutic effect with personalized vaccines remains a challenge because of the weak immunogenicity of autologous tumor antigens. Utilizing exogenous prokaryotic constituents that act as adjuvants to enhance immunogenicity is a promising strategy to overcome this limitation. However, nonspecific stimulation of the immune system may elicit an undesirable immunopathological state. To specifically trigger sufficient antitumor reactivity without notable adverse effects, we developed an antigen and adjuvant codelivery nanoparticle vaccine based on Escherichia coli cytoplasmic membranes (EMs) and tumor cell membranes (TMs) from resected autologous tumor tissue. Introduction of the EM into the hybrid membrane nanoparticle vaccines (HM-NPs) induced dendritic cell maturation, thus activating splenic T cells. HM-NPs showed efficacy in immunogenic CT26 colon and 4T1 breast tumor mouse models and also efficiently induced tumor regression in B16-F10 melanoma and EMT6 breast tumor mouse models. Furthermore, HM-NPs provoked a strong tumor-specific immune response, which not only extended postoperative animal survival but also conferred long-term protection (up to 3 months) against tumor rechallenge in a CT26 colon tumor mouse model. Specific depletion of different immune cell populations revealed that CD8+ T and NK cells were crucial to the vaccine-elicited tumor regression. Individualized autologous tumor antigen vaccines based on effective activation of the innate immune system by bacterial cytoplasmic membranes hold great potential for personalized treatment of postoperative patients with cancer.

Toll-like receptor 9 (TLR9) senses bacterial DNA characteristic of unmethylated CpG motifs to induce innate immune response. TLR9 is de novo expressed in podocytes of some patients with glomerular diseases, but its role in podocyte injury remains undetermined. Since TLR9 activates p38 MAPK and NFkB that are known to mediate podocyte apoptosis, we hypothesized that TLR9 induces podocyte apoptosis in glomerular diseases. We treated immortalized podocytes with puromycin aminonucleosides (PAN) and observed podocyte apoptosis, accompanied by TLR9 upregulation. Prevention of TLR9 upregulation by siRNA significantly attenuated NFκB p65 or p38 activity and apoptosis, demonstrating that TLR9 mediates podocyte apoptosis. We next showed that endogenous mitochondrial DNA (mtDNA), whose CpG motifs are also unmethylated, is the ligand for TLR9, because PAN induced mtDNA accumulation in endolysosomes where TLR9 is localized, overexpression of endolysosomal DNase 2 attenuated PAN-induced p38 or p65 activity and podocyte apoptosis, and DNase 2 silencing was sufficient to activate p38 or p65 and induce apoptosis. In PAN-treated rats, TLR9 was upregulated in the podocytes, accompanied by increase of apoptosis markers. Thus, de novo expressed TLR9 may utilize endogenous mtDNA as the ligand to facilitate podocyte apoptosis, a novel mechanism underlying podocyte injury in glomerular diseases.

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This study reports practices implemented in over 2,000 minutes by 16 middle school special education and general education co-teaching pairs in English language arts classes. We report the extent to which teachers integrated literacy activities that support reading comprehension, the co-teaching models used, and the frequency with which each teacher led instruction. We also report the types of grouping structures teachers used and the extent to which teachers interacted with students with disabilities. Finally, we report the types of text used. Observations revealed that more than half of time spent on literacy activities involved reading aloud or silently with no co-occurring literacy instruction that supports reading comprehension. Students with disabilities spent a majority of their time in whole-class instruction or working independently with little teacher interaction. Special education teachers spent most of their time supporting whole-class instruction led by the content-area teacher. Implications and directions for future research are provided.

Carcinoma cell lines are frequently refractory to transforming growth factor-␤ (TGF␤)-mediated cell cycle arrest. Whether and how TGF␤ signaling is disrupted in the majority of human tumors, however, remains unclear. To investigate whether TGF␤ signaling might be disrupted by inactivation of the key signaling molecule, the TGF␤ type I (T␤R-I) receptor, and whether or not T␤R-I inactivation is associated with late stage disease, we conducted a comprehensive structural analysis of the T␤R-I gene in fine-needle aspirates of 23 head-&-neck cancer metastases. We encountered 4 different mutations of T␤R-I, 3 of which have not been previously identified. In 1 case, we found a somatic intragenic 4-bp deletion predicting for a truncation of the receptor protein. This is the first example of a true loss-of-function mutation of T␤R-I in a human epithelial neoplasm. In 2 other cases, we identified missense mutations located between the juxtamembrane-and serine-threonine kinase domains. One of these resulted in an alanine-to-threonine substitution (A230T), which disrupts receptor signaling activity by causing rapid protein degradation within the endoplasmatic reticulum. This represents a novel mechanism of inactivation of a TGF␤ signaling intermediate. Finally, we identified a serineto-tyrosine substitution at codon 387 (S387Y) in a metastasis but not in the corresponding primary tumor. We had previously shown this S387Y mutant to be predominantly associated with breast cancer metastases and to have a diminished ability to mediate TGF␤-dependent signaling. In aggregate, these findings provide further support for the hypothesis that inactivation of the TGF␤ signaling pathway occurs in a significant subset of human cancers. © 2001 Wiley-Liss, Inc. Key words: transforming growth factor-␤ receptor; mutation; headand-neck cancerThe 25 kDa dimeric polypeptide, transforming growth factor-␤ (TGF␤) is the most potent known inhibitor of normal epithelial cell replication in vitro. 1,2 The TGF␤ signal is transduced by a pair of transmembrane serine-threonine kinase receptors. 3,4 TGF␤ binds primarily to TGF␤ type II receptor (T␤R-II) homodimers, which then form heterotetrameric complexes with 2 TGF␤ type I receptor (T␤R-I) molecules. As a consequence, the T␤R-II kinase phosphorylates the T␤R-I receptor, thereby activating its serinethreonine kinase. In response to T␤R-I activation, 2 cytosolic proteins, Smad2 and Smad3, become transiently associated with and phosphorylated by the T␤R-I kinase. After their activation and release from T␤R-I, Smad 2 and -3 form heteromeric complexes with a third homologue, Smad4. These multimeric Smad complexes are then translocated to the nucleus, bind to DNA in a sequence-specific manner and regulate gene transcription. [3][4][5] The resulting induction of cyclin-dependent kinase (cdk) inhibitors and decreased expression of cyclins, cdks, and the CDK tyrosine phosphatase, Cdc25A, lead to G1 phase cell cycle arrest. 3

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Retinoblastoma is a common intraocular malignancy that occurs during childhood. MicroRNAs play critical roles in the regulation of retinoblastoma initiation and progression, and aberrant expression of miR-613 had been reported in various types of cancer. However, the role and mechanism of its function in retinoblastoma are still unclear. In this study, we found that miR-613 was downregulated in retinoblastoma tissues and cell lines. Overexpression of miR-613 suppressed retinoblastoma cell proliferation, migration, and invasion and induced cell cycle arrest in vitro. Additionally, overexpressed miR-613 also inhibited tumor formation of retinoblastoma cells in vivo. We further identified E2F5 as a direct target of miR-613. Reintroduction of E2F5 without 3'-untranslated region reversed the inhibitory effects of miR-613 on cell proliferation and invasion. Our data collectively indicate that miR-613 functions as a tumor suppressor in retinoblastoma through downregulating E2F5, supporting the targeting of the novel miR-613/E2F5 axis as a potentially effective therapeutic approach for retinoblastoma.