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PDF file - 26K, Figure S2. HER2 overexpression in MCF7 cells does not increase Fn14 mRNA expression.

PDF file - 72K, Figure S3. Fn14 expression is elevated in letrozole-resistant MCF7 cells and NIH3T3 cells stably overexpressing HER2.

PDF file - 514K, Figure S5. U0126 or Wortmannin treatment of MCF7/HER2 and AU565 cells decreases Fn14 expression.

PDF file - 30K, Figure S6. HRG1-β1 stimulation of MCF7 cells induces Fn14 mRNA expression.

PDF file - 111K, Figure S4. Lapatinib treatment or HER2/HER3 siRNA co-transfection of MCF7/HER2-18 cells decreases Fn14 expression levels.

PDF file - 103K

HER2 overexpression occurs in 15% to 20% of all breast cancers and is associated with increased metastatic potential and poor patient survival. Abnormal HER2 activation, either through HER2 overexpression or heregulin (HRG):HER3 binding, elicits the formation of potent HER2–HER3 heterodimers and drives breast cancer cell growth and metastasis. In a previous study, we found that fibroblast growth factor-inducible 14 (Fn14), a member of the TNF receptor superfamily, was frequently overexpressed in human HER2+ breast tumors. We report here that HER2 and Fn14 are also coexpressed in mammary tumors that develop in two different transgenic mouse models of breast cancer. In consideration of these findings, we investigated whether HER2 activation in breast cancer cells could directly induce Fn14 gene expression. We found that transient or stable transfection of MCF7 cells with a HER2 expression plasmid increased Fn14 protein levels. Also, HRG1-β1 treatment of MCF7 cells transiently induced Fn14 mRNA and protein expression. Both the HER2- and HRG1-β1–induced increase in Fn14 expression in MCF7 cells as well as basal Fn14 expression in HER2 gene-amplified AU565 cells could be reduced by HER2 kinase inhibition with lapatinib or combined HER2 and HER3 depletion using siRNA. We also report that Fn14-depleted, HER2-overexpressing MCF7 cells have reduced basal cell migration capacity and reduced HRG1-β1–stimulated cell migration, invasion, and matrix metalloproteinase (MMP)-9 expression. Together, these results indicate that Fn14 may be an important downstream regulator of HER2/HER3–driven breast cancer cell migration and invasion. Mol Cancer Res; 11(4); 393–404. ©2013 AACR.

PDF file - 308K, Figure S1. Analysis of Fn14 and EGFR family member expression in breast cancer cell lines.

PDF file - 177K, Figure S7. HRG1-β1 stimulation increases MCF7/HER2-18 cell migration and invasion.

Supplementary Figure 1. Induction profiles of various known DNA sensors and adaptors in the glioma microenvoironment. Supplementary Figure 2. STING agonist promotes type-I IFN signaling in BILs.

Although type I IFNs play critical roles in antiviral and antitumor activity, it remains to be elucidated how type I IFNs are produced in sterile conditions of the tumor microenvironment and directly affect tumor-infiltrating immune cells. Mouse de novo gliomas show increased expression of type I IFN messages, and in mice, CD11b+ brain-infiltrating leukocytes (BIL) are the main source of type I IFNs that are induced partially in a STING (stimulator of IFN genes)-dependent manner. Consequently, glioma-bearing StingGt/Gt mice showed shorter survival and lower expression levels of Ifns compared with wild-type mice. Furthermore, BILs of StingGt/Gt mice showed increased CD11b+ Gr-1+ immature myeloid suppressor and CD25+ Foxp3+ regulatory T cells (Treg) and decreased IFNγ-producing CD8+ T cells. CD4+ and CD8+ T cells that received direct type I IFN signals showed lesser degrees of regulatory activity and increased levels of antitumor activity, respectively. Finally, intratumoral administration of a STING agonist (cyclic diguanylate monophosphate; c-di-GMP) improved the survival of glioma-bearing mice associated with enhanced type I IFN signaling, Cxcl10 and Ccl5, and T-cell migration into the brain. In combination with subcutaneous OVA peptide vaccination, c-di-GMP increased OVA-specific cytotoxicity of BILs and prolonged their survival. These data demonstrate significant contributions of STING to antitumor immunity via enhancement of type I IFN signaling in the tumor microenvironment and suggest a potential use of STING agonists for the development of effective immunotherapy, such as the combination with antigen-specific vaccinations. Cancer Immunol Res; 2(12); 1199–208. ©2014 AACR.

The surgical clerkship is the primary surgical learning experience for medical students. This study aims to understand student perspectives on the surgery clerkship both before and after the core surgical rotation.Medical students at 4 academic hospitals completed pre and postclerkship surveys that included open-ended questions regarding (1) student learning goals and concerns and (2) how surgical clerkship learning could be enhanced. Thematic analysis was performed, and interrater reliability was calculated.Ninety-one percent of students completed both a pre and postclerkship survey (n =162 of 179), generating 320 preclerkship and 270 postclerkship responses. Mean kappa coefficients were 0.83 and 0.82 for pre and postclerkship primary themes, respectively. Thematic analysis identified 5 broad themes: (1) core learning expectations, (2) understanding surgical careers, culture, and work, (3) inhabiting the role of a surgeon, (4) inclusion in the surgical team, and (5) the unique role of the medical student on clinical clerkships. Based on these themes, we propose a learner-centered model of a successful surgical clerkship that satisfies discrete student learning and goals and career objectives while ameliorating the challenges of high-stakes clinical surgical environments such as the operating room.Understanding student perspectives on the surgery clerkship, including preclerkship motivations and concerns and postclerkship reflections on surgical learning, revealed potential targets of intervention to improve the surgery clerkship. Future investigation may elucidate whether the proposed model of the elements of a successful surgery clerkship learning facilitates improvement of the surgical learning environment and enhanced surgical learning.

This study describes the relationship between medical student perception of surgery, frequency of positive surgery clerkship activities, and overall surgical clerkship experience.Medical students at four academic hospitals completed pre- and post-clerkship surveys assessing 1) surgery clerkship activities/experiences and 2) perceptions of surgery during the 2017-2018 academic year.Ninety-one percent of students completed both a pre- and post-clerkship survey (n = 162 of 179). Student perception of surgery significantly improved across the clerkship overall (P 0.0001) and for 7 of 21 specific items. Eighty-six percent of students agreed that the clerkship was a meaningful experience. Sixty-six percent agreed that the operating room was a positive learning environment. Multivariable logistic regression identified one-on-one mentoring from a resident (OR [95% CI] = 2.12 [1.11-4.04], P = 0.02) and establishing a meaningful relationship with a surgical patient (OR = 2.21 [1.12-4.37], P = 0.02) as activities predictive of student agreement that the surgical clerkship was meaningful. Making an incision (OR = 2.92 [1.54-5.56], P = 0.001) and assisting in dissection (OR = 1.67 [1.03-2.69], P = 0.035) were predictive of student agreement that the operating room was a positive learning environment. Positive student perception of surgery before the clerkship was associated with increased frequency of positive clerkship activities including operative involvement (r = 0.26, P = 0.001) and relationships with surgical attendings (r = 0.20, P = 0.01), residents (r = 0.41, P 0.0001), and patients (r = 0.24, P = 0.003).Interventions to improve surgery clerkship quality should target enhancing student relationships with residents and surgical patients as well as providing opportunity for student operative involvement beyond just suturing. In addition, fostering positive perceptions of surgery in the preclinical period may increase meaningfulness and experience with the later surgery clerkship.

RNA interference (RNAi) is a universal phenomenon of RNA silencing or gene silencing with broader implications in important physiological and developmental processes of most eukaryotes, including plants. Small RNA (sRNA) are the critical drivers of the RNAi machinery that ensures down-regulation of the target genes in a homology-dependent manner and includes small-interfering RNAs (siRNAs) and micro RNAs (miRNAs). Plant researchers across the globe have exploited the powerful technique of RNAi to execute targeted suppression of desired genes in important crop plants, with an intent to improve crop protection against pathogens and pests for sustainable crop production. Biotic stresses cause severe losses to the agricultural productivity leading to food insecurity for future generations. RNAi has majorly contributed towards the development of designer crops that are resilient towards the various biotic stresses such as viruses, bacteria, fungi, insect pests, and nematodes. This review summarizes the recent progress made in the RNAi-mediated strategies against these biotic stresses, along with new insights on the future directions in research involving RNAi for crop protection.

Oligoadenylate synthetases (OAS) are a family of interferon (IFN)-stimulated genes known to inhibit viral replication through the enzymatic synthesis of 2'-5' oligoadenylates and activation of Ribonuclease L. However, this canonical mechanism does not explain the antiviral properties of enzymatically inactive OAS proteins. Here we describe an enzyme activity-independent function of OAS1 that restricts West Nile virus (WNV) growth. The human OAS1 P46 isoform, generated by alternative splicing of the G allele of rs10774671 SNP, upregulates translation of specific host mRNA including the DNA-sensor cGAS. Increased cGAS expression by OAS1 P46 augments the type I IFN response and inhibits WNV infection. A mouse Oas1 gene, Oas1b, also inhibits WNV growth through a similar mechanism. Specific residues in the RNA binding domains of P46 and Oas1b that regulate association of cGAS mRNA with OAS1, also control the antiviral activity. Our findings explain how some enzymatically inactive OAS1 proteins function as antiviral molecules.

Highlights • Goblet cell carcinoid (GCC) is a rare appendiceal tumor of the appendix. • Our patient is the youngest reported case of GCC, at 20 years old. • Patient presented with appendicitis and was diagnosed post-operatively. • Neoplasm must be kept in mind when offering non-operative care for appendicitis., Introduction Goblet cell carcinoid (GCC) is an extraordinarily rare appendiceal tumor that is usually an incidental diagnosis on post-operative histology. It typically presents in the fifth or sixth decade of life. Our patient is the only reported case study of GCC in a pediatric-young adult. Due to its potentially poor prognosis, GCC is surgically treated as an adenocarcinoma, with right hemicolectomy as the mainstay of treatment. Presentation of case The patient was a 20-year-old male who presented with a history, physical exam, and work up consistent with acute appendicitis. He underwent an uneventful laparoscopic appendectomy and was diagnosed with a high grade GCC post-operatively. Discussion GCC is a rare tumor of the appendix with unique histological features including small rosettes with crescentic nuclei distended with mucin. It is often retroactively diagnosed with histology after a majority of patients present with acute appendicitis symptoms. The behavior of this tumor in pediatric-young adults is very poorly understood. Conclusion We review the literature for GCC of the appendix and illustrate a case report of a young, otherwise healthy 20-year-old who presented as appendicitis. Although rare, neoplasm must be kept in mind while offering non-operative management for acute appendicitis.

Alternative Lengthening of Telomeres (ALT) is a homology-directed repair (HDR) mechanism of telomere elongation that controls proliferation in aggressive cancers. We show that the disruption of RAD51-associated protein 1 (RAD51AP1) in ALT+ cancer cells leads to generational telomere shortening. This is due to RAD51AP1’s involvement in RAD51 dependent homologous recombination (HR) and RAD52-POLD3 dependent break induced DNA synthesis. RAD51AP1 KO ALT+ cells exhibit telomere dysfunction and cytosolic telomeric DNA fragments that are sensed by cGAS. Intriguingly, they activate ULK1-ATG7 dependent autophagy as a survival mechanism to mitigate DNA damage and apoptosis. Importantly, RAD51AP1 protein levels are elevated in ALT+ cells due to MMS21 associated SUMOylation. Mutation of a single SUMO-targeted lysine residue perturbs telomere dynamics. These findings indicate that RAD51AP1 is an essential mediator of the ALT mechanism and is co-opted by post-translational mechanisms to maintain telomere length and ensure proliferation of ALT+ cancer cells.

Epilepsy is prevalent with about 50 million patients affected worldwide. There are many treatment gaps in the management of epilepsy in India. Within Anti-epileptic drugs (AED), Brivaracetam, which is a high-affinity, selective, and reversible ligand for synaptic vesicle 2A is approved by the Food and Drug Administration for monotherapy as well as adjunctive treatment of focal seizures. A series of meeting occurring during April 2020 inviting neurologist across India as panel members, reviewed the efficacy and safety of brivaracetam and discussed the use of brivaracetam in clinical settings and the drivers and barriers for the use of brivaracetam in the management of epilepsy. Brivaracetam has good efficacy and tolerability as adjunctive therapy in the treatment of focal (partial onset) seizures in patients 16 years of age and older. Brivaracetam is safe for prolonged use in patients with epilepsy and in children with epilepsy. The most common adverse events with brivaracetam are related to central nervous system and include fatigue, dizziness, and somnolence; these may improve or resolve during treatment. A consensus was sought for the use of brivaracetam in epilepsy management in routine neurology practices Brivaracetam is a safer AED with lesser behavioral AEs, lack of cognitive impairment, and no clinically relevant drug-drug interactions or dose adjustment for renal patients.

Moloney leukemia virus 10, homolog (MOV10) is an IFN-inducible RNA helicase, associated with small RNA-induced silencing. In this article, we report that MOV10 exhibits antiviral activity, independent of its helicase function, against a number of positive- and negative-strand RNA viruses by enhancing type I IFN induction. Using a number of genome-edited knockout human cells, we show that IFN regulatory factor 3–mediated IFN induction and downstream IFN signaling through IFN receptor was necessary to inhibit virus replication by MOV10. MOV10 enhanced IFN regulatory factor 3–mediated transcription of IFN. However, this IFN induction by MOV10 was unique and independent of the known retinoic acid–inducible gene I/mitochondrial antiviral-signaling protein–mediated RNA-sensing pathway. Upon virus infection, MOV10 specifically required inhibitor of κB kinase ε, not TANK-binding kinase 1, for its antiviral activity. The important role of MOV10 in mediating antiviral signaling was further supported by the finding that viral proteases from picornavirus family specifically targeted MOV10 as a possible innate immune evasion mechanism. These results establish MOV10, an evolutionary conserved protein involved in RNA silencing, as an antiviral gene against RNA viruses that uses an retinoic acid–inducible gene I–like receptor–independent pathway to enhance IFN response.

SummaryBackground The Harvard Medical School Cambridge Integrated Clerkship longitudinal cancer curriculum directly facilitates students’ engagement with cancer patients to develop a comprehensive understanding of the disease and the patient's experience of illness. Third-year medical students follow newly diagnosed cancer patients over the course of a year, across all disciplines, and make formal presentations to a multidisciplinary forum at the end of the year. The aim of the study was to discover which aspects of longitudinal care were most meaningful to the students themselves. Method Researchers performed a qualitative thematic analysis of students’ presentations. Basing the analysis on principles of grounded theory, researchers took an inductive approach using the constant comparative method to discover core themes and to cluster themes into encompassing domains. Result Researchers identified 33 individual themes among 60 presentations, reflecting five major domains: clinical issues; patient characteristics; systems problems; psychosocial response to cancer; and existential decision making. Conclusions In this qualitative study of students’ year-end final presentations after a year of cancer care experiences, two areas stood out: students perceived the complexities of medical decision making and students considered the impact of psychosocial factors on patients facing this disease over time. Which aspects of longitudinal care were most meaningful to the students(?)

Interferons inhibit virus replication through the expression of interferon stimulated genes (ISGs). We have found that a specific isoform of one such ISG, Oligoadenylate Synthetase 1 (OAS1) limits host susceptibility to West Nile Virus (WNV) infection through a non-canonical mechanism. This OAS1 isoform (OAS1 P46) in humans is generated due to an alternative splice acceptor site at the C-terminus of OAS1 gene. The SNP rs10774671 at this site has been associated with disease severity to WNV. We show that human OAS1-KO cells have lower basal levels of cGAS protein and can be rescued by OAS1 P46 independent of its enzyme activity. Additionally, through RNA-seq, SILAC, polysome profiling and radiolabeling experiments, we show that OAS1 does not regulate mRNA transcription but instead enhances protein translation of a select set of mRNAs, thereby increasing the steady state and induced levels of specific proteins with antiviral properties. Inducible expression of OAS1 P46 in cGAS-KO cells does not suppress WNV replication, suggesting that the antiviral activity of OAS1 is mediated through cGAS. We also have established functional equivalence between OAS1 P46 and a mouse ortholog, Oas1b (no enzyme activity), which similarly affects WNV susceptibility. Oas1b inhibits WNV infection and pathogenesis in vivo and inhibits WNV infection in vitro in cGAS-dependent manner. Through RNA-protein crosslinking experiments we have identified target mRNAs that bind to OAS1 and have demonstrated increased sensitivity of WNV in Oas1b RNA binding mutants. Our findings suggest a novel mechanism of OAS1 in which it binds to target mRNAs, enhances the translation of these RNAs and limits virus infection.

A 26-year-old, otherwise healthy female presented to the Emergency Room for the evaluation of abdominal pain. It was immediately apparent that she had a massively distended abdomen. History revealed progressive abdominal distension over several years. Evaluation for pregnancy was negative and a computed tomography (CT) scan demonstrated a 38 × 32 × 23 cm septated cystic mass. Careful controlled partial needle decompression of the cyst, removing 18.5 l of fluid, was followed by a mini-laparotomy with complete removal of a multi-loculated cystic ovarian mass approximately 45 lb in weight. Pathology was consistent with mucinous cystadenoma of the ovary in association with a mature cystic teratoma. This surgical technique of percutaneous drainage of the cyst, followed by mini-laparotomy is a valuable example of a safe and effective minimally invasive treatment modality for giant ovarian mucinous cystadenomas.

Interferon-inducible human oligoadenylate synthetase-like (OASL) and its mouse ortholog, Oasl2, enhance RNA-sensor RIG-I-mediated type I interferon (IFN) induction and inhibit RNA virus replication. Here, we show that OASL and Oasl2 have the opposite effect in the context of DNA virus infection. In Oasl2-/- mice and OASL-deficient human cells, DNA viruses such as vaccinia, herpes simplex, and adenovirus induced increased IFN production, which resulted in reduced virus replication and pathology. Correspondingly, ectopic expression of OASL in human cells inhibited IFN induction through the cGAS-STING DNA-sensing pathway. cGAS was necessary for the reduced DNA virus replication observed in OASL-deficient cells. OASL directly and specifically bound to cGAS independently of double-stranded DNA, resulting in a non-competitive inhibition of the second messenger cyclic GMP-AMP production. Our findings define distinct mechanisms by which OASL differentially regulates host IFN responses during RNA and DNA virus infection and identify OASL as a negative-feedback regulator of cGAS.

The cellular innate immune system plays a critical role in mounting the initial resistance to virus infection. It is comprised of various pattern-recognition receptors that induce type I interferon production, which further shapes the adaptive immunity. However, to overcome this resistance and promote replication, viruses have evolved mechanisms to evade this host innate immune response. Here we discuss a recently described mechanism of boosting the innate immunity by oligoadenylate synthetase-like (OASL) protein, which can potentially be used to overcome viral evasion and enhance innate immunity.

Health systems worldwide are increasingly unable to meet individual and population health needs. The shortage of healthcare workers in rural and other underserved communities is compounded by inadequate primary care infrastructure and maldistribution of services. At the same time, the medical education system has not changed to address the growing mismatch between population health needs and care delivery capacity. Internationally, leaders are calling for change to address these challenges. Substantive changes are needed in medical education’s stance, structure, and curricula. Educational continuity and symbiosis are two guiding principles at the center of current clinical educational redesign discourse. These principles rely on empirically-derived science to guide educational structure and improve outcomes. Educational continuity and symbiosis may improve student learning and support population health through workforce transformation. Longitudinal integrated clerkships (LICs), growing out of workforce imperatives in the 1970s, have demonstrated sustainable educational and workforce outcomes. Alongside the success of LICs, more innovation and more reaching innovation are needed. We propose restructuring clinical medical education specifically to address workforce needs and develop science-minded (rigorous, inquisitive, and innovative) and service-minded (humanistic, community-engaged, and socially accountable) graduates.

During the present investigation pollen morphological studies of 66 species belonging to 19 families of monocots in Paschim Medinipur district have been worked out by light microscopy. The studied families are Agavaceae, Amaryllidaceae, Arecaceae, Asphodelaceae, Cannaceae, Colchicaceae, Commelinaceae, Costaceae, Cyperaceae, Hemerocallidaceae, Hydrocharitaceae, Iridaceae, Liliaceae, Limnocharitaceae, Musaceae, Poaceae, Pontederiaceae, Typhaceae and Zingiberaceae. The apertural patterns are mostly belong to two different categories viz. monosulcate form (Agavaceae, Amaryllidaceae, Arecaceae, Asphodelaceae, Colchicaceae, Commelinaceae, Costaceae, Hemerocallidaceae, Hydrocharitaceae, Iridaceae, Liliaceae, Limnocharitaceae, Musaceae, Poaceae, Pontederiaceae) and anaporate type (Cyperaceae, Poaceae and Typhaceae). The shape of the pollen grains with monosulcate apertures are mostly oblate to peroblate type whereas taxa showing anaporate apertures are more or less spheroidal. Regarding the mode of pollen dispersal plant taxa with monosulcate apertures and apiculate surface ornamentations (e.g., reticulate, rugulate, spinulate, verrucate) are entomophilous (mainly melittophilous) whilest anaporate with smooth or minutely apiculate surface features are anemophilous. Here, entomophilous taxa provide rewards as pollen grains and nectar to the honeybee speecies, therefore, contribute as resource mobilizer for sustainance of honeybee colonies.

The popularity of Building Information Modeling (BIM) as an integral concept in the Architecture, Engineering, and Construction (AEC) industries has motivated its necessary inclusion in Construction Management (CM) education. Implementing BIM as a mainstream, elective or integrated subject within an established undergraduate curriculum is challenging due to limitations such as available teaching time, knowledge retention in students and the flexibility of the curriculum to adapt with a fast developing technology. Pedagogy must combine fundamental learning of theory, practical experience and the use of technology in a collaborative environment to effectively implement BIM. Vertical integration is one such method that was implemented and evaluated to promote BIM education in the undergraduate Construction Management program at Arizona State University. This article discusses the evolution of the BIM curriculum and focuses on the vertical integration of upper-division and lower-division students for a Site L...

Virus infection is sensed in the cytoplasm by retinoic acid-inducible gene I (RIG-I, also known as DDX58), which requires RNA and polyubiquitin binding to induce type I interferon (IFN) and activate cellular innate immunity. We show that the human IFN-inducible oligoadenylate synthetases-like (OASL) protein has antiviral activity and mediates RIG-I activation by mimicking polyubiquitin. Loss of OASL expression reduced RIG-I signaling and enhanced virus replication in human cells. Conversely, OASL expression suppressed replication of a number of viruses in a RIG-I-dependent manner and enhanced RIG-I-mediated IFN induction. OASL interacted and colocalized with RIG-I, and through its C-terminal ubiquitin-like domain specifically enhanced RIG-I signaling. Bone-marrow-derived macrophages from mice deficient for Oasl2 showed that among the two mouse orthologs of human OASL, Oasl2 is functionally similar to human OASL. Our findings show a mechanism by which human OASL contributes to host antiviral responses by enhancing RIG-I activation.

Arginase I is a marker of murine M2 macrophages and is highly expressed in many inflammatory diseases. The basis for high arginase I expression in macrophages in vivo is incompletely understood but likely reflects integrated responses to combinations of stimuli. Our objective was to elucidate mechanisms involved in modulating arginase I induction by IL-4, the prototypical activator of M2 macrophages. IL-4 and 8-bromo-cAMP individually induce arginase I, but together they rapidly and synergistically induce arginase I mRNA, protein, and promoter activity in murine macrophage cells. Arginase I induction by IL-4 requires binding of the transcription factors STAT6 and C/EBPβ to the IL-4 response element of the arginase I gene. Chromatin immunoprecipitation showed that the synergistic response involves binding of both transcription factors to the IL-4 response element at levels significantly greater than in response to IL-4 alone. The results suggest that C/EBPβ is a limiting factor for the level of STAT6 bound to the IL-4 response element. The enhanced binding in the synergistic response was not due to increased expression of either STAT6 or C/EBPβ but was correlated primarily with increased nuclear abundance of C/EBPβ. Our findings also suggest that induction of arginase I expression is stochastic; that is, differences in induction reflect differences in probability of transcriptional activation and not simply differences in rate of transcription. Results of the present study also may be useful for understanding mechanisms underlying regulated expression of other genes in macrophages and other myeloid-derived cells in health and disease.

Naturally occurring isothiocyanates (ITCs) from cruciferous vegetables are widely studied for their cancer chemopreventive effects. In this study, we investigated the effects of ITCs on TLR signaling, and found that the two most promising ITCs, phenethyl ITCs (PEITC) and D,L-sulforaphane (SFN), have differential effects on dsRNA-mediated innate immune signaling through TLR3. PEITC preferentially inhibited TLR3-mediated IFN regulatory factor 3 (IRF3) signaling and downstream gene expression in vivo and in vitro, whereas SFN caused inhibition of TLR3-mediated NF-κB signaling and downstream gene expression. Mechanistically, PEITC inhibited ligand (dsRNA)-dependent dimerization of TLR3, resulting in inhibition of signaling through IFN regulatory factor 3. In contrast, SFN did not disrupt TLR3 dimerization, indicating that it affects further downstream pathway resulting in NF-κB inhibition. To examine the biological significance of these findings in the context of antitumor activities of these compounds, we used two approaches: first, we showed that dsRNA-mediated apoptosis of tumor cells via TLR3 was inhibited in the presence of PEITC, whereas this response was augmented by SFN treatment; second, in a separate assay measuring anchorage-independent growth and colony formation by immortalized fibroblasts, we made similar observations. Again in this study, PEITC antagonized dsRNA-mediated inhibition of colony formation, whereas SFN enhanced the inhibition. These results indicate biologically relevant functional differences between two structurally similar ITCs and may provide important insights in therapeutic development of these compounds targeted to specific cancer.

HER2 overexpression occurs in 15% to 20% of all breast cancers and is associated with increased metastatic potential and poor patient survival. Abnormal HER2 activation, either through HER2 overexpression or heregulin (HRG):HER3 binding, elicits the formation of potent HER2–HER3 heterodimers and drives breast cancer cell growth and metastasis. In a previous study, we found that fibroblast growth factor-inducible 14 (Fn14), a member of the TNF receptor superfamily, was frequently overexpressed in human HER2+ breast tumors. We report here that HER2 and Fn14 are also coexpressed in mammary tumors that develop in two different transgenic mouse models of breast cancer. In consideration of these findings, we investigated whether HER2 activation in breast cancer cells could directly induce Fn14 gene expression. We found that transient or stable transfection of MCF7 cells with a HER2 expression plasmid increased Fn14 protein levels. Also, HRG1-β1 treatment of MCF7 cells transiently induced Fn14 mRNA and protein expression. Both the HER2- and HRG1-β1–induced increase in Fn14 expression in MCF7 cells as well as basal Fn14 expression in HER2 gene-amplified AU565 cells could be reduced by HER2 kinase inhibition with lapatinib or combined HER2 and HER3 depletion using siRNA. We also report that Fn14-depleted, HER2-overexpressing MCF7 cells have reduced basal cell migration capacity and reduced HRG1-β1–stimulated cell migration, invasion, and matrix metalloproteinase (MMP)-9 expression. Together, these results indicate that Fn14 may be an important downstream regulator of HER2/HER3–driven breast cancer cell migration and invasion. Mol Cancer Res; 11(4); 393–404. ©2013 AACR.

Background: EBP1, an ErbB3-binding protein, sensitises breast cancer cells to tamoxifen in part by decreasing ErbB2 protein levels. The p21-regulated serine/threonine kinase PAK1, implicated in tamoxifen resistance, phosphorylates EBP1 in vitro and in vivo at T261. Phosphorylation of EBP1 at this site induces tamoxifen resistance. We thus postulated that inhibition of PAK1 activity, by restoring EBP1 function, could ameliorate the hormone refractory phenotype of ErbB2-overexpressing breast cancer cells. Methods: Effects of EBP1 on ErbB2 levels were measured by western blotting. Effects of EBP1 and IPA-3 on tamoxifen sensitivity were measured using a tetrazolium based cell viability assay. Results: Transient transfection studies indicated that an EBP1 T261E mutant, which mimics EPB1 phosphorylated by PAK1, increased ErbB2 protein levels. An EBP1 T261A mutant, unable to be phosphorylated by PAK1, ameliorated PAK1-induced tamoxifen resistance, suggesting that phosphorylation of EBP1 by PAK1 contributes to tamoxifen resistance. We then tested if pharmacological inhibition of PAK1 activity might render hormone resistant cells, which endogenously overexpress PAK1, tamoxifen sensitive. IPA-3, a specific small MW PAK1 inhibitor, sensitised cells to tamoxifen only when EBP1 was ectopically expressed. IPA had no effect on tamoxifen resistance in T47D cells in which EBP1 protein had been ablated by shRNA. The IPA-induced increase in tamoxifen sensitivity was accompanied by a decrease in ErbB2 levels only in EBP1-overexpressing cells. Conclusion: These studies suggest that phosphorylation of EBP1 may be one mechanism of PAK1-induced hormone resistance and that PAK1 inhibitors may be useful in cells in which EBP1 is overexpressed.

Ectopic expression of EBP1, an ErbB3-interacting protein, reduces the expression of the ErbB2 protein and mRNA. However, the mechanism of EBP1-induced decrease in ErbB2 mRNA levels has not yet been determined. Since EBP1 affects both transcriptional and post-transcriptional processes, we evaluated the ability of EBP1 to regulate ErbB2 transcription and RNA stability. We discovered that while wild-type EBP1 decreased the activity of a proximal ErbB2 promoter, EBP1 mutants unable to interact with the Sin3A transcriptional repressor inhibited activity to a lesser extent. EBP1 also decreased the activity of distal ErbB2 promoters. Chromatin immunoprecipitation analysis indicated that EBP1 bound both distal and proximal endogenous ErbB2 promoters in serum-starved conditions. The ErbB3 ligand heregulin (HRG) at growth-promoting concentrations reduced EBP1 binding to the ErbB2 promoter. Although endogenous EBP1 bound ErbB2 mRNA, EBP1 overexpression or ablation of EBP1 protein by shRNA failed to alter ErbB2 mRNA stability. These results suggest that the major effect of EBP1 on ErbB2 mRNA levels is at the transcriptional level.

We designed and execute a longitudinal curriculum that provides a comprehensive understanding of cancer illness and its impact upon the patient.The Harvard Medical School-Cambridge Integrated Clerkship is a redesign of the 3rd year where the traditional rotations are replaced by a single integrated year-long experience. Students are required to follow a patient with newly diagnosed gastrointestinal cancer and breast cancer, across all venues and disciplines. Twenty-nine of 34 students responded to a survey. On average patients were followed for 7 months, through 12 encounters across 4 different specialties. Students responded that this experience facilitated their understanding of cancer in a way not feasible in a traditional clerkship model.Medical students perceive that this longitudinal model of cancer education improves integration of the surgical, medical, scientific, emotional, and social issues. Traditional "block rotation" students and even residents are rarely afforded such an educational opportunity.

Active learning is based on self-directed and autonomous teaching methods, whereas passive learning is grounded in instructor taught lectures. An animal physiology course was studied over a two-year period (Year 1, n = 42 students; Year 2, n = 30 students) to determine the effects of student-led seminar (andragogical) and lecture (pedagogical) teaching methods on students' retention of information and performance. For each year of the study, the course was divided into two time periods. The first half was dedicated to instructor-led lectures, followed by a control survey in which the students rated the efficiency of pedagogical learning on a five-point Likert scale from one (strongly disagree) to five (strongly agree). During the second period, students engaged in andragogical learning via peer-led seminars. An experimental survey was then administered to students using the same scale as above to determine students' preferred teaching method. Raw examination scores and survey results from both halves of the course were statistically analyzed by ANOVA with Newman-Keuls multiple comparison test. By the end of the study, student preference for peer-led seminars increased [mean ± SD: (2.47 ± 0.94)/(4.03 ± 1.36), P < 0.04], and examination scores significantly increased [mean ± SD: (73.91% ± 13.18)/(85.77 ± 5.22), P < 0.001]. A majority of students (68.8%) preferred a method that contained peer-led seminars and instructor-led lectures. These results may indicate that integration of active and passive learning into undergraduate courses may have greater benefit in terms of student preference and performance than either method alone.

Summary Inflammasome-activated caspase-1 cleaves gasdermin D to unmask its pore-forming activity, the predominant consequence of which is pyroptosis. Here, we report an additional biological role for gasdermin D in limiting cytosolic DNA surveillance. Cytosolic DNA is sensed by Aim2 and cyclic GMP-AMP synthase (cGAS) leading to inflammasome and type I interferon responses, respectively. We found that gasdermin D activated by the Aim2 inflammasome suppressed cGAS-driven type I interferon response to cytosolic DNA and Francisella novicida in macrophages. Similarly, interferon-β (IFN-β) response to F. novicida infection was elevated in gasdermin D-deficient mice. Gasdermin D-mediated negative regulation of IFN-β occurred in a pyroptosis-, interleukin-1 (IL-1)-, and IL-18-independent manner. Mechanistically, gasdermin D depleted intracellular potassium (K+) via membrane pores, and this K+ efflux was necessary and sufficient to inhibit cGAS-dependent IFN-β response. Thus, our findings have uncovered an additional interferon regulatory module involving gasdermin D and K+ efflux.

Platelets express TLR4 receptors, but its ligand LPS does not directly activate thrombotic functions nor, obviously, transcription by these anucleate cells. Platelets, however, store information that changes their phenotype over a few hours in the form of unprocessed RNA transcripts. We show even low concentrations of LPS in the presence of soluble CD14 initiated splicing of unprocessed IL-1β RNA, with translation and accumulation of IL-1β protein. LPS was a more robust agonist for this response than thrombin. Platelets also contained cyclooxygenase-2 pre-mRNA, which also was spliced and translated after LPS stimulation. Flow cytometry and immunocytochemistry of platelets extensively purified by negative immunodepletion showed platelets contained IL-1β, and quantitative assessment of white blood cell contamination by CD14 real time PCR confirms that leukocytes were not the IL-1β source, nor were they required for platelet stimulation. LPS did not initiate rapid platelet responses, but over time did prime platelet aggregation to soluble agonists, induced actin rearrangement, and initiated granule secretion with P-selectin expression that resulted the coating of quiescent leukocytes with activated platelets. LPS is a direct agonist for platelets that allows these cells to directly participate in the innate immune response to bacteria.

Head and neck squamous cell carcinoma (HNSCC) is a devastating disease for which new treatments, such as immunotherapy are needed. Synthetic double-stranded RNAs, which activate toll-like receptor 3 (TLR3), have been used as potent adjuvants in cancer immunotherapy by triggering a proapoptotic response in cancer cells. A better understanding of the mechanism of TLR3-mediated apoptosis and its potential involvement in controlling tumor metastasis could lead to improvements in current treatment. Using paired, autologous primary and metastatic HNSCC cells we previously showed that metastatic, but not primary tumor-derived cells, were unable to activate prosurvival NF-κB in response to p(I):p(C) resulting in an enhanced apoptotic response. Here, we show that transcriptional downregulation of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) in metastatic HNSCC cells causes a loss of TLR3-mediated NF-κB signaling, resulting in enhanced apoptosis. Loss of RIPK1 strongly correlates with metastatic disease in a cohort of HNSCC patients. This downregulation of RIPK1 is possibly mediated by enhanced methylation of the RIPK1 promoter in tumor cells and enhances protumorigenic properties such as cell migration. The results described here establish a novel mechanism of TLR3-mediated apoptosis in metastatic cells and may create new opportunities for using double stranded RNA to target metastatic tumor cells.

© 2015 Taylor & Francis Group, LLC. We recently reported that STING contributes to antiglioma immunity by triggering type I IFN induction in glioma microenvironment. Moreover, intratumoral administration of STING agonist improved the efficacy of peptide vaccination in a mouse glioma model, suggesting the rational use of STING agonists in the immunotherapy of brain tumor.

Unlike other RNA polymerases, 2'-5' oligoadenylate synthetases, a family of interferon-induced enzymes, catalyze the formation of 2'-5', not 3'-5', phosphodiester bonds. Moreover, to be active, these proteins require double-stranded RNA as a cofactor. We have been identifying the specific residues of these proteins that impart their novel properties. Here, we report the identity of three such residues that underwent natural mutations in a transgenic mouse line. When deliberately introduced into recombinant proteins, each of these mutations rendered the protein enzymatically inactive. In an effort to understand the roles of these residues in enzyme activity, new mutants carrying other residues in one of these three sites were generated. Detailed characterization of the properties of the mutant proteins revealed that Lys 404 is needed for proper binding of the acceptor substrate, Pro 500 provides structural flexibility to the protein, and Ser 471 is probably required for its proper folding. This study illustrates the power of using natural mutations in transgenes as guides for studying structure-function relationships of proteins.

Prostate-specific membrane antigen (PSMA), a type II transmembrane glycoprotein, is overexpressed in prostate cancer. PSMA is a unique cell surface marker, negatively regulated by androgen and extensively used for imaging of hormone refractory carcinomas and metastatic foci. PSMA is a carboxypeptidase with two important enzymatic functions, namely, folate hydrolase and NAALADase. PSMA also exhibits an endocytic function, in which it spontaneously recycles through endocytic vesicles. PSMA is overexpressed at various stages of prostate cancer, including androgen-sensitive and -independent disease, increased in expression with early relapse after therapy. We have used in vitro invasion assays to explore the possible role of PSMA in the metastasis of prostate cancer cells. Androgen-dependent prostate cancer lines, which express PSMA endogenously (e.g., LNCaP, MDA PCa2b, and CWR22Rv1) are less invasive compared with androgen-independent PC3 or DU145 cells, neither of which expresses PSMA. Ectopic expression of PSMA in PC3 cells reduced the invasiveness of these cells, suggesting that this reduction in the invasion capability of PSMA-expressing cells is due to PSMA expression and not to intrinsic properties of different prostate cancer cell lines. Furthermore, knockdown of PSMA expression increased invasiveness of LNCaP cells by 5-fold. Finally, expression of PSMA mutants lacking carboxypeptidase activity reduced the impact of PSMA expression on invasiveness. Thus, it seems that the enzymatic activity is associated with the effect of PSMA on invasiveness.

Prostate specific membrane antigen (PSMA), is a unique membrane bound glycoprotein, which is overexpressed manifold on prostate cancer as well as neovasculature of most of the solid tumors, but not in the vasculature of the normal tissues. This unique expression of PSMA makes it an important marker as well as a large extracellular target of imaging agents. PSMA can serve as target for delivery of therapeutic agents such as cytotoxins or radionuclides. PSMA has two unique enzymatic functions, folate hydrolase and NAALADase and found to be recycled like other membrane bound receptors through clathrin coated pits. The internalization property of PSMA leads one to consider the potential existence of a natural ligand for PSMA. In this review we have discussed the regulation of PSMA expression within the cells, and significance of its expression in prostate cancer and metastasis.

BACKGROUND Prostate specific membrane antigen or PSMA has been recognized as one of the important cellular markers for prostate cancer, the expression of which is enhanced many fold in prostate cancer and other tumor neovasculature. PSMA is a type II membrane glycoprotein with a short cytoplasmic N-terminal region, a transmembrane domain, and a 701 amino acid extracellular portion with 10 potential N-linked glycosylation sites. PSMA is a folate hydrolase, which cleaves terminal glutamates from poly- and gamma-glutamated folates; and NAALADase, which hydrolyses alpha-glutamate-linked dipeptide, N-acetyl-aspartyl-glutamate (NAAG) and is a glutamate carboxypeptidase. METHODS In our study we have used various enzymes or site directed mutagenesis to remove sugar molecules from PSMA protein and studied its folate hydrolase function. We have performed a biochemical characterization of N-linked glycosylation of the various mutant proteins. RESULTS PSMA protein expressed in different prostate cancer cell lines is differentially glycosylated. Removal of sugar residues either enzymatically or by mutagenesis abolishes the enzyme activity of PSMA protein completely. CONCLUSION N-linked carbohydrate structures are important for the folate hydrolase function of the protein. Removal of sugars partially or completely causes PSMA to be enzymatically inactive, improperly folded, resulting in increased rate of degradation. Prostate 57: 140–151, 2003. © 2003 Wiley-Liss, Inc.

Mycena galopus is among the most important leaf litter decomposers in UK coniferous and angiosperm woodlands, having the potential to utilise all the major constituents of plant litter. Even so, the enzyme or combination of enzymes produced by M. galopus responsible for lignin depolymerisation was previously unknown. A range of media from liquid and semi-solid cultures to more natural substrata was tested to determine whether laccase was produced by an isolate of M. galopus, M9053. Malt extract liquid medium (MEL) with 2,5-xylidine favoured laccase production as compared with the same medium containing the inducers veratryl alcohol, veratryl aldehyde, veratric acid, homoveratric acid, vanillic acid or p-anisic acid. A semi-solid medium of cereal bran in phosphate buffer and a solid medium of Picea sitchensis F1 horizon needle litter were also not as effective as MEL with 2,5-xylidine as an inducer. Compared with six other isolates of the same species grown in MEL without inducers, M9053 exhibited rates of laccase activity fairly typical for M. galopus. An isolate from a dark coloured basidiome of M. galopus, but not var. nigra, exhibited the greatest activity while var. candida showed relatively low laccase activity. Marasmius androsaceus exhibited peak laccase production several days later than M. galopus. In addition, a manganese-dependent peroxidase that was responsible for 15% (in MEL culture fluid) and 39% (in needle litter extract III) of ligninolytic activity was produced by M9053. A further peroxidase was found to be the major ligninolytic constituent in MEL extracts (53%), and had a similar contribution to total activity (29%) as laccase (32%) in needle litter fraction III. Mycena galopus produced water- and buffer-extractable mannases and xylanases when grown on needle litter.