Novel Application of Photo-Crosslinked Urocanic-Acid-Modified Chitosan in Corneal Wounds.

In the last few years, the use of tissue adhesives in corneal perforation has gained immense popularity in clinical practices. The present study aimed to devise a new application of urocanic-acid-modified chitosan (CS) with methylene blue (MB) as a photosensitizer for the development of a photo-crosslinked tissue adhesive. In particular, the curing time was controlled with the aid of a 650 nm red diode. Under the same irradiation condition, the mechanical properties were tuned using the photosensitizer at different concentrations. In vitro tests revealed that the gel was ductile and biocompatible. The application of the gel to a perforated cornea model stopped the leakage of aqueous humor, immediately after the gel was photo-crosslinked. The blue appearance of the gel provided high precision when applied to corneal wounds. Importantly, the crosslinked gel became transparent within 24 h, owing to the dissipation of MB from tears, and the gel spontaneously sloughed off without artificial removal. Altogether, the study reported the development of a novel photo-crosslinkable urocanic-acid-modified CS gel that exhibited significant potential to be utilized in the healing of corneal perforation.

Antifibrotic Effect of Bletilla striata Polysaccharide-Resveratrol-Impregnated Dual-Layer Carboxymethyl Cellulose-Based Sponge for The Prevention of Epidural Fibrosis after Laminectomy.

The application of antifibrotic materials can alleviate epidural fibrosis by restricting excessive fibroblast proliferation and mitigating scar tissue formation. Here, a biodegradable carboxymethyl cellulose (CMC)-Bletilla striata polysaccharide (BSP)-resveratrol (RES) sponge was fabricated to inhibit scar tissue formation post laminectomy surgery. Fibroblasts NIH/3T3, myoblasts C2C12, neural cells PC-12, and Schwann cells RSC96 were used to evaluate the in vitro cytocompatibility. Laminectomies on 10 Sprague-Dawley rats with/without the application of the CMC-BSP-RES sponge were performed. The severity of adhesion between the dura mater and formed scar tissue was qualitatively scored. All cell lines exhibited good viability with no significant difference in cytotoxicity when cultured with variable extractions of the CMC-BSP-RES sponge. S100a4 and P4hb expressions were downregulated in NIH/3T3 cultured in the CMC-BSP-RES sponge, implying that this sponge potentially inhibits fibroblast activity. No post-operative shrinkage or dura mater expansion along the surgical site was detected. The peel-off tests revealed that the tenacity of adhesion de-creased. Histopathological examinations verified that the average number of fibroblasts in the CMC-BSP-RES group considerably decreased. The CMC-BSP-RES sponge is a biocompatible and effective material for alleviating post-operative epidural fibrosis and mitigating fibroblast expression following laminectomy.

Head and Neck Mesenchymal Neoplasms With GLI1 Gene Alterations: A Pathologic Entity With Distinct Histologic Features and Potential for Distant Metastasis.

Soft tissue tumors with GLI1 gene fusions or amplifications have been recently described as a unique pathologic entity with an established risk of malignancy. We herein expand these findings by investigating a cohort of 11 head and neck lesions with GLI1 alterations, including 8 from the tongue, for their clinicopathologic and molecular features. The tumors commonly affected males in their 30s (male:female ratio 2.7:1; range: 1 to 65). Tumors showed a multinodular growth pattern, nested architecture separated by a delicate, arborizing vascular network, monotonous round to ovoid nuclei, and clear cytoplasm. Tumor protrusion into vascular spaces was common. Genetic alterations were investigated by fluorescence in situ hybridization and/or targeted RNA sequencing. Seven tumors harbored GLI1 fusions with the following partners: ACTB (n=4), PTCH1 (n=2), or MALAT1 (n=1). The remaining 4 cases showed coamplifications of GLI1 with CDK4 and MDM2 genes. Tumors were commonly positive for S100 protein and CD56. CDK4, MDM2, and STAT6 were positive in GLI1-amplified tumors. Two of 6 patients with available follow-up (1 each with GLI1 amplification and PTCH1-GLI1 fusion) developed distant metastases. Both tumors showed a high mitotic index and tumor necrosis. The head and neck region, particularly tongue, is a common location for GLI1-related mesenchymal tumors. Although a morphologic overlap was noted with the previously reported "pericytoma with t(7,12) translocation," often occurring in the tongue, our findings expand the original findings, to include a more variable immunophenotype, propensity for late distant metastases, and alternative mechanisms of GLI1 oncogenic activation, such as various GLI1 fusion partners or GLI1 coamplifications with MDM2 and CDK4 genes.

Tumor-to-Tumor Metastasis: Lung Carcinoma Metastasizing to Thyroid Neoplasms.

Tumor-to-tumor metastasis is extremely rare in the thyroid glands, and only seven cases of lung carcinoma metastasizing to thyroid tumors have been reported in the literature. We report another two cases of lung carcinoma metastasizing to thyroid neoplasms and review of the literature. The first case was a 64-year-old man presenting with neck mass, hoarseness, and easy choking for 2 months. Image studies showed several nodular lesions within bilateral thyroid glands. A histological examination after radical thyroidectomy revealed lung small cell carcinoma metastasizing to a thyroid follicular adenoma. The second case was a 71-year-old woman with a history of lung adenosquamous carcinoma. The PET/CT scan showed left lower lung cancer and a hypermetabolic area in the right thyroid lobe, highly suspicious for malignancy. Radical thyroidectomy and left lung lobectomy were performed, and the thyroid gland revealed lung adenosquamous carcinoma metastasizing to a papillary thyroid carcinoma.

Monoclonal antibody against cell surface GRP78 as a novel agent in suppressing PI3K/AKT signaling, tumor growth, and metastasis.

PURPOSE: The ER chaperone GRP78 translocates to the surface of tumor cells and promotes survival, metastasis, and resistance to therapy. An oncogenic function of cell surface GRP78 has been attributed to the activation of the phosphoinositide 3-kinase (PI3K) pathway. We intend to use a novel anti-GRP78 monoclonal antibody (MAb159) to attenuate PI3K signaling and inhibit tumor growth and metastasis. EXPERIMENTAL DESIGN: MAb159 was characterized biochemically. Antitumor activity was tested in cancer cell culture, tumor xenograft models, tumor metastasis models, and spontaneous tumor models. Cancer cells and tumor tissues were analyzed for PI3K activity. MAb159 was humanized and validated for diagnostic and therapeutic application. RESULTS: MAb159 specifically recognized surface GRP78, triggered GRP78 endocytosis, and localized to tumors but not to normal organs in vivo. MAb159 inhibited tumor cell proliferation and enhanced tumor cell death both in vitro and in vivo. In MAb159-treated tumors, PI3K signaling was inhibited without compensatory MAPK pathway activation. Furthermore, MAb159 halted or reversed tumor progression in the spontaneous PTEN-loss-driven prostate and leukemia tumor models, and inhibited tumor growth and metastasis in xenograft models. Humanized MAb159, which retains high affinity, tumor specific localization, and the antitumor activity, was nontoxic in mice, and had desirable pharmacokinetics. CONCLUSIONS: GRP78-specific antibody MAb159 modulates the PI3K pathway and inhibits tumor growth and metastasis. Humanized MAb159 will enter human trials shortly.

Desmoplastic medulloblastoma arising from an ovarian teratoma: a case report and review of the literature.

The primary neuroectodermal tumor of the ovary is extremely rare, and it manifests in 3 forms: differentiated, primitive, and anaplastic. The medulloblastoma belongs to the subgroup of primitive neuroectodermal tumor of the ovary. Only 3 cases of ovarian medulloblastoma have been reported in the literature, and all of them are presented without information about the specific pathological subtype. We present the fourth case of a 26-year-old woman who presented with abdominal fullness for months. Ultrasound exam showed a right adnexal mass with cystic feature and foci solid components. A partial oophorectomy was performed, and the mass was a desmoplastic medulloblastoma arising from an ovarian teratoma.

Acute inducible ablation of GRP78 reveals its role in hematopoietic stem cell survival, lymphogenesis and regulation of stress signaling.

GRP78, a master regulator of the unfolded protein response (UPR) and cell signaling, is required for inner cell mass survival during early embryonic development. However, little is known about its role in adult hematopoietic stem cells (HSCs) and hematopoiesis. Here we generated a conditional knockout mouse model that acutely deletes Grp78 in the adult hematopoietic system. Acute GRP78 ablation resulted in a significant reduction of HSCs, common lymphoid and myeloid progenitors, and lymphoid cell populations in the mutant mice. The GRP78-null induced reduction of the HSC pool could be attributed to increased apoptosis. Chimeric mice with Grp78 deletion only in the hematopoietic cells also showed a loss of HSCs and lymphopenia, suggesting a cell intrinsic effect. Analysis of GRP78 deficient bone marrow (BM) cells showed constitutive activation of all the major UPR signaling pathways, including activation of eIF2α, ATF6, xbp-1 splicing, as well as caspase activation. A multiplex cytokine assay further revealed alteration in select cytokine and chemokine serum levels in the mutant mice. Collectively, these studies demonstrate that GRP78 plays a pleiotropic role in BM cells and contributes to HSC survival and the maintenance of the lymphoid lineage.

Inducible knockout of GRP78/BiP in the hematopoietic system suppresses Pten-null leukemogenesis and AKT oncogenic signaling.

Traditionally, GRP78 is regarded as protective against hypoxia and nutrient starvation prevalent in the microenvironment of solid tumors; thus, its role in the development of hematologic malignancies remains to be determined. To directly elucidate the requirement of GRP78 in leukemogenesis, we created a biallelic conditional knockout mouse model of GRP78 and PTEN in the hematopoietic system. Strikingly, heterozygous knockdown of GRP78 in PTEN null mice is sufficient to restore the hematopoietic stem cell population back to the normal percentage and suppress leukemic blast cell expansion. AKT/mTOR activation in PTEN null BM cells is potently inhibited by Grp78 heterozygosity, corresponding with suppression of the PI3K/AKT pathway by GRP78 knockdown in leukemia cell lines. This is the first demonstration that GRP78 is a critical effector of leukemia progression, at least in part through regulation of oncogenic PI3K/AKT signaling. In agreement with PI3K/AKT as an effector for cytosine arabinoside resistance in acute myeloid leukemia, overexpression of GRP78 renders human leukemic cells more resistant to cytosine arabinoside-induced apoptosis, whereas knockdown of GRP78 sensitizes them. These, coupled with the emerging association of elevated GRP78 expression in leukemic blasts of adult patients and early relapse in childhood leukemia, suggest that GRP78 is a novel therapeutic target for leukemia.

Poorly differentiated thyroid carcinoma in a 9-year-old boy: case report.

Poorly differentiated thyroid carcinoma (PDTC) is a rare disease with a poor prognosis in children. We describe a 9-year-old boy with a thyroid nodule composed of cystic and solid components, which became completely solid and hypoechoic and was subsequently proved to be PDTC. The tumor consisted of small- to intermediate-size round cells in a trabecular or insular pattern with hyperchromatic nuclei and mitotic figures. The tumor cells were positive for thyroid transcription factor 1 and thyroglobulin. PDTC is morphologically and prognostically between the well-differentiated and anaplastic carcinomas. It must be distinguished from the solid variant of papillary carcinoma and well-differentiated follicular carcinoma with a predominantly solid/trabecular growth pattern. The tumor stage was T2N0M0. The patient was treated with total thyroidectomy, left-sided neck level VI lymph node dissection, recombinant human thyrotropin-stimulated 131I ablation therapy, and thyroid-stimulating hormone suppression. Malignancy should be suspected in a cystic thyroid nodule that becomes solid and hypoechoic.

The endoplasmic reticulum chaperone protein GRP94 is required for maintaining hematopoietic stem cell interactions with the adult bone marrow niche.

Hematopoietic stem cell (HSC) homeostasis in the adult bone marrow (BM) is regulated by both intrinsic gene expression products and interactions with extrinsic factors in the HSC niche. GRP94, an endoplasmic reticulum chaperone, has been reported to be essential for the expression of specific integrins and to selectively regulate early T and B lymphopoiesis. In GRP94 deficient BM chimeras, multipotent hematopoietic progenitors persisted and even increased, however, the mechanism is not well understood. Here we employed a conditional knockout (KO) strategy to acutely eliminate GRP94 in the hematopoietic system. We observed an increase in HSCs and granulocyte-monocyte progenitors in the Grp94 KO BM, correlating with an increased number of colony forming units. Cell cycle analysis revealed that a loss of quiescence and an increase in proliferation led to an increase in Grp94 KO HSCs. This expansion of the HSC pool can be attributed to the impaired interaction of HSCs with the niche, evidenced by enhanced HSC mobilization and severely compromised homing and lodging ability of primitive hematopoietic cells. Transplanting wild-type (WT) hematopoietic cells into a GRP94 null microenvironment yielded a normal hematology profile and comparable numbers of HSCs as compared to WT control, suggesting that GRP94 in HSCs, but not niche cells, is required for maintaining HSC homeostasis. Investigating this, we further determined that there was a near complete loss of integrin α4 expression on the cell surface of Grp94 KO HSCs, which showed impaired binding with fibronectin, an extracellular matrix molecule known to play a role in mediating HSC-niche interactions. Furthermore, the Grp94 KO mice displayed altered myeloid and lymphoid differentiation. Collectively, our studies establish GRP94 as a novel cell intrinsic factor required to maintain the interaction of HSCs with their niche, and thus regulate their physiology.

Targeted mutation of the mouse Grp94 gene disrupts development and perturbs endoplasmic reticulum stress signaling.

Glucose-regulated protein 94 (GRP94) is one of the most abundant endoplasmic reticulum (ER) resident proteins and is the ER counterpart of the cytoplasmic heat shock protein 90 (HSP90). GRP94, a component of the GRP78 chaperone system in protein processing, has pro-survival properties with implicated function in cancer progression and autoimmune disease. Previous studies on the loss of GRP94 function showed that it is required for embryonic development, regulation of toll-like receptors and innate immunity of macrophages. Here we report the creation of mouse models targeting exon 2 of the Grp94 allele that allows both traditional and conditional knockout (KO) of Grp94. In this study, we utilized the viable Grp94+/+ and +/- mice, as well as primary mouse embryonic fibroblasts generated from them as experimental tools to study its role in ER chaperone balance and ER stress signaling. Our studies reveal that while Grp94 heterozygosity reduces GRP94 level it does not alter ER chaperone levels or the ER stress response. To study the effect of complete loss of GRP94 function, since homozygous GRP94 KO leads to embryonic lethality, we generated Grp94-/- embryonic stem cells. In contrast to Grp94 heterozygosity, complete knockout of GRP94 leads to compensatory upregulation of the ER chaperones GRP78, calnexin and calreticulin but not protein disulphide isomerase. Unexpectedly, loss of GRP94 leads to significant decrease in the level of ER-stress induced spliced form of XBP-1 protein, a downstream target of the IRE1 signaling pathway. Furthermore, from analysis of microarray database and immunohistochemical staining, we present predictions where GRP94 may play an important role in specific adult organ homeostasis and function.

Regulation of PERK signaling and leukemic cell survival by a novel cytosolic isoform of the UPR regulator GRP78/BiP.

The unfolded protein response (UPR) is an evolutionarily conserved mechanism to allow cells to adapt to stress targeting the endoplasmic reticulum (ER). Induction of ER chaperone GRP78/BiP increases protein folding capacity; as such it represents a major survival arm of UPR. Considering the central importance of the UPR in regulating cell survival and death, evidence is emerging that cells evolve feedback regulatory pathways to modulate the key UPR executors, however, the precise mechanisms remain to be elucidated. Here, we report the fortuitous discovery of GRP78va, a novel isoform of GRP78 generated by alternative splicing (retention of intron 1) and alternative translation initiation. Bioinformatic and biochemical analyses revealed that expression of GRP78va is enhanced by ER stress and is notably elevated in human leukemic cells and leukemia patients. In contrast to the canonical GRP78 which is primarily an ER lumenal protein, GRP78va is devoid of the ER signaling peptide and is cytosolic. Through specific knockdown of endogenous GRP78va by siRNA without affecting canonical GRP78, we showed that GRP78va promotes cell survival under ER stress. We further demonstrated that GRP78va has the ability to regulate PERK signaling and that GRP78va is able to interact with and antagonize PERK inhibitor P58(IPK). Our study describes the discovery of GRP78va, a novel cytosolic isoform of GRP78/BiP, and the first characterization of the modulation of UPR signaling via alternative splicing of nuclear pre-mRNA. Our study further reveals a novel survival mechanism in leukemic cells and other cell types where GRP78va is expressed.

Role of the unfolded protein response regulator GRP78/BiP in development, cancer, and neurological disorders.

GRP78/BiP is a major endoplasmic reticulum (ER) chaperone protein critical for protein quality control of the ER, as well as controlling the activation of the ER-transmembrane signaling molecules. Through creation of mouse models targeting the Grp78 allele, the function of GRP78 in development and disease has been investigated. These led to the discovery that GRP78 function is obligatory for early embryonic development. However, in adult animals, GRP78 is preferably required for cancer cell survival under pathologic conditions such as tumor progression and drug resistance. The discovery of surface localization of GRP78 in cancer cells reveals potential novel function, interaction with cell-surface receptors, and possible therapeutic implications. Mouse models also reveal that GRP78 controls maturation and secretion of neuronal factors for proper neural migration and offers neuroprotection.

Pten null prostate tumorigenesis and AKT activation are blocked by targeted knockout of ER chaperone GRP78/BiP in prostate epithelium.

GRP78/BiP has recently emerged as a novel biomarker for aggressive prostate cancer. Here, we report that homozygous deletion of Grp78 specifically in mouse prostate epithelium suppresses prostate tumorigenesis without affecting postnatal prostate development and growth. Mouse prostates with double conditional knockout of Grp78 and Pten exhibit normal histology and cytology, in contrast to the invasive adenocarcinoma in mouse prostates with Pten inactivation. AKT activation in Pten null prostate epithelium is inhibited by Grp78 homozygous deletion, corresponding with suppression of AKT phosphorylation by GRP78 knockdown in prostate cancer cell line. Thus, inactivation of GRP78 may represent a previously undescribed approach to stop prostate cancer and potentially other cancers resulting from the loss of PTEN tumor suppression and/or activation of the oncogenic AKT.

Three dimensional rendering of the mitochondrial sheath morphogenesis during mouse spermiogenesis.

In the middle piece of mouse sperm tail, the idea of the mitochondria wrapping in a sinistral (left-handed) double helical structure was generally accepted. In the existing model, mitochondria aligned in four longitudinal rows (stage 1) and twisted dextrally (right-handed) (stage 2) and began to stagger, where opposing rows of mitochondria contacted each other to form a sinistral double helix (stage 3), finally, the end-on touching mitochondria further elongated to their mature length (stage 4). However, in this model, mitochondria need to shift a long distance and reposition themselves. Since no gaps have been found in mitochondrial sheath, repositioning of most mitochondria along the middle piece is unlikely to happen. Therefore, we reapproached the questions through three-dimensional rendering to provide a new model for mitochondrial sheath formation. In our proposed model, four dextrally arranged spherical mitochondrial arrays were considered stage 1 (resembles stage 2 of the old model). In stage 2 (resembles stage 3 of the old model), a critical difference was found that pairs of mitochondria from the opposing arrays formed ring-like structures instead of a sinistral double helix. In stage 3, which was not observed in the old model, mitochondria staggered in a specific pattern to form the sinistral double helix. In stage 4, mitochondria elongated from crescent-shaped to rod-shaped structures. The new model proposed here would allow each mitochondrion to stay at where they attached first and elongate laterally from two directions to reach their final double helical structure without unreasonable great distance shift along the outer dense fibers.

Incremental hierarchical discriminant regression.

This paper presents incremental hierarchical discriminant regression (IHDR) which incrementally builds a decision tree or regression tree for very high-dimensional regression or decision spaces by an online, real-time learning system. Biologically motivated, it is an approximate computational model for automatic development of associative cortex, with both bottom-up sensory inputs and top-down motor projections. At each internal node of the IHDR tree, information in the output space is used to automatically derive the local subspace spanned by the most discriminating features. Embedded in the tree is a hierarchical probability distribution model used to prune very unlikely cases during the search. The number of parameters in the coarse-to-fine approximation is dynamic and data-driven, enabling the IHDR tree to automatically fit data with unknown distribution shapes (thus, it is difficult to select the number of parameters up front). The IHDR tree dynamically assigns long-term memory to avoid the loss-of-memory problem typical with a global-fitting learning algorithm for neural networks. A major challenge for an incrementally built tree is that the number of samples varies arbitrarily during the construction process. An incrementally updated probability model, called sample-size-dependent negative-log-likelihood (SDNLL) metric is used to deal with large sample-size cases, small sample-size cases, and unbalanced sample-size cases, measured among different internal nodes of the IHDR tree. We report experimental results for four types of data: synthetic data to visualize the behavior of the algorithms, large face image data, continuous video stream from robot navigation, and publicly available data sets that use human defined features.

Auditory learning: a developmental method.

Motivated by the human autonomous development process from infancy to adulthood, we have built a robot that develops its cognitive and behavioral skills through real-time interactions with the environment. We call such a robot a developmental robot. In this paper, we present the theory and the architecture to implement a developmental robot and discuss the related techniques that address an array of challenging technical issues. As an application, experimental results on a real robot, self-organizing, autonomous, incremental learner (SAIL), are presented with emphasis on its audition perception and audition-related action generation. In particular, the SAIL robot conducts the auditory learning from unsegmented and unlabeled speech streams without any prior knowledge about the auditory signals, such as the designated language or the phoneme models. Neither available before learning starts are the actions that the robot is expected to perform. SAIL learns the auditory commands and the desired actions from physical contacts with the environment including the trainers.