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Paired Box (Pax) gene family, a group of transcription regulators have been implicated in diverse physiological processes. However, their role during hematopoiesis which generate a plethora of blood cells remains largely unknown. Using a previously reported single cell transcriptomics data, we analyzed the expression of individual Pax family members in hematopoietic cells in zebrafish. We have identified that Pax9, which is an essential regulator for odontogenesis and palatogenesis, is selectively localized within a single cluster of the hematopoietic lineage. To further analyze the function of Pax9 in hematopoiesis, we generated two independent pax9 knock-out mutants using the CRISPR-Cas9 technique. We found that Pax9 appears to be an essential regulator for granulopoiesis but dispensable for erythropoiesis during development, as lack of pax9 selectively decreased the number of neutrophils with a concomitant decrease in the expression level of neutrophil markers. In addition, embryos, where pax9 was functionally disrupted by injecting morpholinos, failed to increase the number of neutrophils in response to pathogenic bacteria, suggesting that Pax9 is not only essential for developmental granulopoiesis but also emergency granulopoiesis. Due to the inability to initiate emergency granulopoiesis, innate immune responses were severely compromised in pax9 morpholino-mediated embryos, increasing their susceptibility and mortality. Taken together, our data indicate that Pax9 is essential for granulopoiesis and promotes innate immunity in zebrafish larvae.

The purpose of this study was to examine the relationships among coaches’ authentic leadership and athletes’ psychological capital (PsyCap), performance satisfaction, and psychological well-being. The authors recruited 224 athletes participating in intercollegiate athletics in the United States. The results indicated that the authentic leadership of the head coaches positively influenced the athletes’ PsyCap levels. The enhanced PsyCap, in turn, positively influenced both performance satisfaction and psychological well-being. The interaction effect of student development was found in the relationship between authentic leadership and PsyCap. The findings contribute to an improved understanding of a head coach’s authentic behaviors in sport teams, and also illuminate how athletes’ performance satisfaction and psychological well-being can be augmented by PsyCap enhancement.

The quality of a student-athlete’s experience can be a product of the services provided by their sponsoring sport organization. In an attempt to improve the student-athlete experience, this study was positioned to examine how collegiate sport services could use academic psychological capital (PsyCap) and student-athlete engagement to promote school satisfaction and psychological well-being. A total of 248 National Collegiate Athletic Association Division I student-athletes participated in this study. Results indicated that academic classification moderated academic PsyCap’s influence on engagement. In addition, the academic PsyCap of the student-athletes positively influenced school satisfaction and psychological well-being, but student-athlete engagement fully mediated the relationship between academic PsyCap and psychological well-being. This empirical evidence provides new knowledge on the relationships among student-athletes’ motivational cognitive constructs, educational engagement, school satisfaction, and psychological well-being in the context of highly competitive collegiate sports. Theoretical and practical implications are discussed, including incorporating the results with services provided to student-athletes.

Neuropathic pain is a chronic pain state characterized by nerve damage, inflammation, and nociceptive neuron hyperactivity. As the underlying pathophysiology is complex, a more effective therapy for neuropathic pain would be one that targets multiple elements. Here, we generated recombinant adeno-associated viruses (AAVs) encoding three therapeutic genes, namely, glutamate decarboxylase 65, glial cell-derived neurotrophic factor, and interleukin-10, with various combinations. The efficacy for pain relief was evaluated in a rat spared nerve injury model of neuropathic pain. The maximal analgesic effect was achieved when the AAVs expressing all three genes were administered to rats with neuropathic pain. The combination of two virus constructs expressing the three genes was named KLS-2031 and evaluated as a potential novel therapeutic for neuropathic pain. Single transforaminal epidural injections of KLS-2031 into the intervertebral foramen to target the appropriate dorsal root ganglion produced notable long-term analgesic effects in female and male rats. Furthermore, KLS-2031 mitigated the neuroinflammation, neuronal cell death, and dorsal root ganglion hyperexcitability induced by the spared nerve injury. These results suggest that KLS-2031 represents a promising therapeutic option for refractory neuropathic pain., Graphical Abstract, Kim and colleagues demonstrated that single transforaminal injection of KLS-2031 (AAV vectors containing GAD65, GDNF, and IL-10) could produce long-term analgesic effects in rats with neuropathic pain by diminishing the neuroinflammation, neuronal cell death, and hyperexcitability. These observations suggest that KLS-2031 could be a promising treatment option for neuropathic pain.

Social isolation during the juvenile critical window is detrimental to proper functioning of the prefrontal cortex (PFC) and establishment of appropriate adult social behaviors. However, the specific circuits that undergo social experience-dependent maturation to regulate social behavior are poorly understood. We identify a specific activation pattern of parvalbumin-positive interneurons (PVIs) in dorsal-medial PFC (dmPFC) prior to an active bout, or a bout initiated by the focal mouse, but not during a passive bout when mice are explored by a stimulus mouse. Optogenetic and chemogenetic manipulation reveals that brief dmPFC-PVI activation triggers an active social approach to promote sociability. Juvenile social isolation decouples dmPFC-PVI activation from subsequent active social approach by freezing the functional maturation process of dmPFC-PVIs during the juvenile-to-adult transition. Chemogenetic activation of dmPFC-PVI activity in the adult animal mitigates juvenile isolation-induced social deficits. Therefore, social experience-dependent maturation of dmPFC-PVI is linked to long-term impacts on social behavior., Isolation during critical periods of development prevents development of normal social behaviours in mice, and this is thought to involve the prefrontal cortex. Here, the authors identify an activation pattern in parvalbumin-positive interneurons in the dorsal medial prefrontal cortex that when activated promotes sociability behaviours in mice.

We investigated the clinical relevance of urinary cytokines/chemokines reflecting intrarenal immunologic micromilieu as prognostic markers and the optimal measurement timing after living donor kidney transplantation (LDKT). This prospective cohort study included 77 LDKT patients who were followed for ≥ 5 years. Patients were divided into control (n = 42) or acute rejection (AR, n = 35) group. Early AR was defined as AR occurring within 3 months. Serum and urine cytokines/chemokines were measured serially as follows: intraoperative, 8/24/72 h, 1 week, 3 months, and 1 year after LDKT. Intrarenal total leukocytes, T cells, and B cells were analyzed with immunohistochemistry followed by tissueFAXS. Urinary MCP-1 and fractalkine were also analyzed in a validation cohort. Urinary MCP-1 after one week was higher in the AR group. Urinary MCP-1, fractalkine, TNF-α, RANTES, and IL-6 after one week were significantly higher in the early AR group. Intrarenal total leukocytes and T cells were elevated in the AR group compared with the control group. Urinary fractalkine, MCP-1, and IL-10 showed positive correlation with intrarenal leukocyte infiltration. Post-KT 1 week urinary MCP-1 showed predictive value in the validation cohort. One-week post-KT urinary MCP-1 may be used as a noninvasive diagnostic marker for predicting AR after LDKT.

BRAF plays an indispensable role in activating the MEK/ERK pathway to drive tumorigenesis. Receptor tyrosine kinase and RAS-mediated BRAF activation have been extensively characterized, however, it remains undefined how BRAF function is fine-tuned by stimuli other than growth factors. Here, we report that in response to proinflammatory cytokines, BRAF is subjected to lysine 27-linked poly-ubiquitination in melanoma cells by the ITCH ubiquitin E3 ligase. Lysine 27-linked ubiquitination of BRAF recruits PP2A to antagonize the S365 phosphorylation and disrupts the inhibitory interaction with 14–3–3, leading to sustained BRAF activation and subsequent elevation of the MEK/ERK signaling. Physiologically, proinflammatory cytokines activate ITCH to maintain BRAF activity and to promote proliferation and invasion of melanoma cells, whereas the ubiquitination-deficient BRAF mutant displays compromised kinase activity and reduced tumorigenicity. Collectively, our study reveals a pivotal role for ITCH-mediated BRAF ubiquitination in coordinating the signals between cytokines and the MAPK pathway activation in melanoma cells., BRAF drives MEK/ERK activation to facilitate tumorigenesis. Here, the authors show that in response to pro-inflammatory cytokines, ITCH mediates a non-proteolytic ubiquitination and activation of BRAF, which in turn sustains MEK/ERK signaling to facilitate melanoma cell growth.

Melanoma is one of the most highly mutated cancer types. To identify functional drivers of melanoma, we searched for cross-species conserved mutations utilizing a mouse melanoma model driven by loss of PTEN and CDKN2A, and identified mutations in Kras, Erbb3, and Ptpn11. PTPN11 encodes the SHP2 protein tyrosine phosphatase that activates the RAS/RAF/MAPK pathway. Although PTPN11 is an oncogene in leukemia, lung, and breast cancers, its roles in melanoma are not clear. In this study, we found that PTPN11 is frequently activated in human melanoma specimens and cell lines and is required for full RAS/RAF/MAPK signaling activation in BRAF wild-type (either NRAS mutant or wild-type) melanoma cells. PTPN11 played oncogenic roles in melanoma by driving anchorage-independent colony formation and tumor growth. In Pten- and Cdkn2a-null mice, tet-inducible and melanocyte-specific PTPN11E76K expression significantly enhanced melanoma tumorigenesis. Melanoma cells derived from this mouse model showed doxycycline-dependent tumor growth in nude mice. Silencing PTPN11E76K expression by doxycycline withdrawal caused regression of established tumors by induction of apoptosis and senescence, and suppression of proliferation. Moreover, the PTPN11 inhibitor (SHP099) also caused regression of NRASQ61K-mutant melanoma. Using a quantitative tyrosine phosphoproteomics approach, we identified GSK3α/β as one of the key substrates that were differentially tyrosine-phosphorylated in these experiments modulating PTPN11. This study demonstrates that PTPN11 plays oncogenic roles in melanoma and regulates RAS and GSK3β signaling pathways. Implications: This study identifies PTPN11 as an oncogenic driver and a novel and actionable therapeutic target for BRAF wild-type melanoma.

Traditionally, the diagnostic mainstay of recurrent urinary tract infection has been urinary culture. However, the causative uropathogen of recurrent cystitis has not been well established. Urine DNA next-generation sequencing (NGS) can provide additional information on these infections. Herein, we compared urine NGS results and urine cultures in patients with acute uncomplicated cystitis (AUC) and recurrent cystitis (RC), and evaluated the difference in microbiome patterns in the NGS results. Patients who underwent urine culture and NGS due to AUC or RC were retrospectively reviewed. All urine samples were collected via a transurethral catheter and studied utilizing a type of NGS called 16S ribosomal RNA gene amplification and sequencing. The sensitivity of urine NGS was significantly higher than that of conventional urine culture (69.0% vs. 16.7%, p &lt, 0.05). The detection rate of urine NGS was slightly lower in the RC group than in the AUC group (67.7% vs. 72.7%). Microbiome diversity was significantly higher in the RC group compared to the AUC group (p = 0.007), and the microbiome composition was significantly different between the AUC and RC groups. In the urine NGS results, Pseudomonas, Acinetobacter, and Enterobacteriaceae were found in the AUC group, and Sphingomonas, Staphylococcus, Streptococcus, and Rothia spp. were detected in the RC group. Urine NGS can significantly increase the diagnostic sensitivity compared to traditional urine culture methods, especially in RC patients. AUC and RC patients had significant differences in bacterial diversity and patterns. Therefore, recurrent cystitis might be approached from a different perspective.

Bone Morphogenetic Protein (BMP) signaling regulates diverse biological processes. Upon ligand binding, BMP receptors (BMPRs) phosphorylate SMAD1/5 and other noncanonical downstream effectors to induce transcription of downstream targets. However, the precise role of individual BMP receptors in this process remains largely unknown due to the complexity of downstream signaling and the innate promiscuity of ligand-receptor interaction. To delineate unique downstream effectors of individual BMPR1s, we analyzed the transcriptome of human umbilical endothelial cells (HUVECs) expressing three distinct constitutively active BMPR1s of which expression was detected in endothelial cells (ECs). From our analyses, we identified a number of novel downstream targets of BMPR1s in ECs. More importantly, we found that each BMPR1 possesses a distinctive set of downstream effectors, suggesting that each BMPR1 is likely to retain unique function in ECs. Taken together, our analyses suggest that each BMPR1 regulates downstream targets non-redundantly in ECs to create context-dependent outcomes of the BMP signaling.

Uncontrolled activation of transforming growth factor (TGF)-β results in a wide range of pathologic conditions. Therapeutic interventions to regulate TGF-β signaling during fibrosis have been developed but the effectiveness is still limited. Here, we show that developmental endothelial locus-1 (Del-1) ameliorates fibrosis in mice by inhibiting αv integrin-mediated activation of TGF-β. Del-1 bound to αvβ6 integrin, an important activator of TGF-β, and inhibited the binding of αvβ6 integrin to the latency-associated peptide (LAP), thereby suppressing αv integrin-mediated activation of TGF-β. Lack of Del-1 increased colocalization of αv integrin and LAP in the lungs, which was reversed by Del-1 supplementation. The crucial role of Del-1 in regulating TGF-β activity was recapitulated in a mouse model of fibrosis using an adenovirus expressing inactive TGF-β1. Del-1 supplementation improved the pathological characteristics of the mice and reduced mortality. Thus, we propose that Del-1 is a negative regulator of TGF-β activation and a potential anti-fibrotic factor.

The GATA and PAX-SIX-EYA-DACH (PSEDN) transcriptional networks are essential for proper development across taxa. Here we demonstrate novel PSEDN roles in vivo in Drosophila hematopoiesis and in human erythropoiesis in vitro. Using Drosophila genetics, we show that PSEDN members function with GATA to block lamellocyte differentiation and maintain the prohemocyte pool. Overexpression of human SIX1 stimulated erythroid differentiation of human erythroleukemia TF1 cells and primary hematopoietic stem-progenitor cells. Conversely, SIX1 knockout impaired erythropoiesis in both cell types. SIX1 stimulation of erythropoiesis required GATA1, as SIX1 overexpression failed to drive erythroid phenotypes and gene expression patterns in GATA1 knockout cells. SIX1 can associate with GATA1 and stimulate GATA1-mediated gene transcription, suggesting SIX1-GATA1 physical interactions contribute to the observed functional interactions. In addition, both fly and human SIX proteins regulated GATA protein levels. Collectively, our findings demonstrate that SIX proteins enhance GATA function at multiple levels and reveal evolutionarily conserved cooperation between the GATA and PSEDN networks that may regulate developmental processes beyond hematopoiesis.

As one of the emerging themes in research on sport management leadership, servant leadership focuses on facilitating individual growth and moral development. The present study tested a hypothesized research model that demonstrates support for the effects of a head coach's servant leadership on athletes' ethical development and team outcome confidence through the quality that characterizes the coach–athlete relationship. We recruited 347 student-athletes of football teams and men's basketball teams who play under the Division I system of the US National Collegiate Athletic Association. Whereas the quality of the coach–athlete relationship partially mediated the association between servant leadership and ethical development, it fully mediated the paths from servant leadership to team outcome confidence. This study provides empirical support for the positive influence of servant leadership behaviors and advances an improved understanding of the role played by the aforementioned relationship quality in coaching leadership research.

Cancer-specific promoter driven replication of oncolytic adenovirus (Ad) is cancer-specific, but shows low transcriptional activity. Thus, we generated several chimeric α-fetoprotein (AFP) promoter variants, containing reconstituted enhancer and silencer regions, to preferentially drive Ad replication in hepatocellular carcinoma (HCC). Modified AFP promoter, containing 2 enhancer A regions and a single enhancer B region (a2bm), showed strong and HCC-specific transcription. In AFP-positive HCCs, gene expression was 43- to 456-fold higher than those of control AFP promoter lacking enhancers. a2bm promoter was further modified by inserting multiple hypoxia-responsive elements (HRE) to generate Ha2bm promoter, which showed stronger transcriptional activity than a2bm promoter under hypoxic conditions. Ha2bm promoter-regulated oncolytic Ad (Ha2bm-d19) showed a stronger antitumor and proapoptotic effect than did a2bm promoter-regulated oncolytic Ad (a2bm-d19) in HCC xenograft tumors. Systemically administered Ha2bm-d19 caused no observable hepatotoxicity, whereas control replication-competent Ad, lacking cancer specificity (d19), induced significant hepatic damage. Ha2bm-d19 caused significantly lower expression of interleukin-6 than d19, showing that HCC-targeted delivery of Ad attenuates induction of the innate immune response against Ad. This chimeric AFP promoter enabled Ad to overcome the hypoxic tumor microenvironment and target HCC with high specificity, rendering it a promising candidate for the treatment of aggressive HCCs.

T-cell receptor (TcR) recombinations can be recovered from tumor specimen, whole exome sequences (WXS) files. However, it is not yet clear how these recombinations represent lymphocytes or an anti-tumor immune response. Here we report the identification of productive TcR-β recombinations in WXS files representing primary and metastatic melanoma. The recombinations are identifiable in about 20% of the cancer genome atlas melanoma samples. This frequency of detection is lower than the frequency of TcR-α VJ recombinations, consistent with the occurrence of biallelic TcR-α recombinations and possibly consistent with the fact that only one junctional recombination is required for TcR-α whereas two recombinations are required to form a TcR-β gene. Nevertheless, the ratio of productive TcR-β to unproductive TcR-β samples, in comparison to the ratio of productive to unproductive TcR-α or TcR-γ positive-samples, is very high. This result indicates that detection of a productive TcR-β VDJ recombination represents a comparatively high standard for potential antigen binding capacity, when employing a tumor specimen exome file for the assessment. Additionally, PD-1 expression and antigen presentation functions correlated with the co-detection of TcR-α and -β recombinations (e.g., p

Hematopoiesis is a highly-regulated development process orchestrated by lineage-specific transcription factors that direct the generation of all mature blood cells types, including red blood cells, megakaryocytes, granulocytes, monocytes, and lymphocytes. Under homeostatic conditions, the hematopoietic system of the typical adult generates over 1011 blood cells daily throughout life. In addition, hematopoiesis must be responsive to acute challenges due to blood loss or infection. MicroRNAs (miRs) cooperate with transcription factors to regulate all aspects of hematopoiesis, including stem cell maintenance, lineage selection, cell expansion, and terminal differentiation. Distinct miR expression patterns are associated with specific hematopoietic lineages and stages of differentiation and functional analyses have elucidated essential roles for miRs in regulating cell transitions, lineage selection, maturation, and function. MiRs function as downstream effectors of hematopoietic transcription factors and as upstream regulators to control transcription factor levels. Multiple miRs have been shown to play essential roles. Regulatory networks comprised of differentially expressed lineage-specific miRs and hematopoietic transcription factors are involved in controlling the quiescence and self-renewal of hematopoietic stem cells as well as proliferation and differentiation of lineage-specific progenitor cells during erythropoiesis, myelopoiesis, and lymphopoiesis. This review focuses on hematopoietic miRs that function as upstream regulators of central hematopoietic transcription factors required for normal hematopoiesis. This article is categorized under: RNA in Disease and Development > RNA in Development Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs.

In this study we present polymeric microneedles composed of multiple layers to control drug release kinetics. Layered microneedles were fabricated by spraying poly(lactic-co-glycolic acid) (PLGA) and polyvinylpyrrolidone (PVP) in sequence, and were characterized by mechanical testing and ex vivo skin insertion tests. The compression test demonstrated that no noticeable layer separation occurred, indicating good adhesion between PLGA and PVP layers. Histological examination confirmed that the microneedles were successfully inserted into the skin and indicated biphasic release of dyes incorporated within microneedle matrices. Structural changes of a model protein drug, bovine serum albumin (BSA), in PLGA and PVP matrices were examined by circular dichroism (CD) and fluorescence spectroscopy. The results showed that the tertiary structure of BSA was well maintained in both PLGA and PVP layers while the secondary structures were slightly changed during microneedle fabrication. In vitro release studies showed that over 60% of BSA in the PLGA layer was released within 1 h, followed by continuous slow release over the course of the experiments (7 days), while BSA in the PVP layer was completely released within 0.5 h. The initial burst of BSA from PLGA was further controlled by depositing a blank PLGA layer prior to forming the PLGA layer containing BSA. The blank PLGA layer acted as a diffusion barrier, resulting in a reduced initial burst. The formation of the PLGA diffusion barrier was visualized using confocal microscopy. Our results suggest that the spray-formed multilayer microneedles could be an attractive transdermal drug delivery system that is capable of modulating a drug release profile.

The PAX-SIX-EYA-DACH network (PSEDN) is a central developmental transcriptional regulatory network from Drosophila to humans. The PSEDN is comprised of four conserved protein families; including paired box (PAX), sine oculis (SIX), eyes absent (EYA), and dachshund (DACH). Aberrant expression of PSEDN members, particularly SIX1, has been observed in multiple human cancers, where SIX1 expression correlates with increased aggressiveness and poor prognosis. In conjunction with its transcriptional activator EYA, the SIX1 transcription factor increases cancer stem cell (CSC) numbers and induces epithelial-mesenchymal transition (EMT). SIX1 promotes multiple hallmarks and enabling characteristics of cancer via regulation of cell proliferation, senescence, apoptosis, genome stability, and energy metabolism. SIX1 also influences the tumor microenvironment, enhancing recruitment of tumor-associated macrophages and stimulating angiogenesis, to promote tumor development and progression. EYA proteins are multifunctional, possessing a transcriptional activation domain and tyrosine phosphatase activity, that each contributes to cancer stem cell properties. DACH proteins function as tumor suppressors in solid cancers, opposing the actions of SIX-EYA and reducing CSC prevalence. Multiple mechanisms can lead to increased SIX1 expression, including loss of SIX1-targeting tumor suppressor microRNAs (miRs), whose expression correlates inversely with SIX1 expression in cancer patient samples. In this review, we discuss the major mechanisms by which SIX1 confers CSC and EMT features and other important cancer cell characteristics. The roles of EYA and DACH in CSCs and cancer progression are briefly highlighted. Finally, we summarize the clinical significance of SIX1 in cancer to emphasize the potential therapeutic benefits of effective strategies to disrupt PSEDN protein interactions and functions.

Hepatitis B virus X protein (HBx) contributes to the development of hepatocellular carcinoma (HCC), probably by regulating activities of many host or viral proteins through protein-protein interactions. In this study, we identified poly(ADP-ribose) polymerase (PARP1), a crucial factor in DNA repair, as an HBx-interacting protein using a proteomics approach. Coimmunoprecipitation and proximity ligation assays confirmed the binding and colocalization of HBx and PARP1 in the nucleus. The carboxyl-terminus of HBx protein bound to the catalytic domain of PARP1, and this binding reduced the enzymatic activity of PARP1 in both in vitro and in vivo assays. HBx interrupted the binding of PARP1 to Sirt6, which catalyzes the mono-ADP-ribosylation required for DNA repair. Consistently, overexpression of HBx inhibited the clearance of γH2AX DNA repair foci generated under oxidative stress in Chang liver cells. Recruitment of the DNA repair complex to the site-specific double-strand breaks was inhibited in the presence of HBx, when measured by laser microirradiation assay and damage-specific chromatin immunoprecipitation assays. Consequently, HBx increased signs of DNA damage such as accumulation of 8-hydroxy-2'-deoxyguanosine and comet formation, which were reversed by overexpression of PARP1 and/or Sirt6. Finally, the interaction between PARP1 and Sirt6 was markedly lower in the livers of HBx-transgenic mice and specimens obtained from HCC patients to compare with the corresponding control. Our data suggest that the physical interaction of HBx and PARP1 accelerates DNA damage by inhibiting recruitment of the DNA repair complex to the damaged DNA sites, which may lead to the onset of hepatocarcinogenesis.

Antibiotic-resistant bacterial infection is a serious threat to public health. Peptidoglycan biosynthesis is a well-established target for antibiotic development. MraY (phospho-MurNAc-pentapeptide translocase) catalyses the first and an essential membrane step of peptidoglycan biosynthesis. It is considered a very promising target for the development of new antibiotics, as many naturally occurring nucleoside inhibitors with antibacterial activity target this enzyme. However, antibiotics targeting MraY have not been developed for clinical use, mainly owing to a lack of structural insight into inhibition of this enzyme. Here we present the crystal structure of MraY from Aquifex aeolicus (MraYAA) in complex with its naturally occurring inhibitor, muraymycin D2 (MD2). We show that after binding MD2, MraYAA undergoes remarkably large conformational rearrangements near the active site, which lead to the formation of a nucleoside-binding pocket and a peptide-binding site. MD2 binds the nucleoside-binding pocket like a two-pronged plug inserting into a socket. Further interactions it makes in the adjacent peptide-binding site anchor MD2 to and enhance its affinity for MraYAA. Surprisingly, MD2 does not interact with three acidic residues or the Mg(2+) cofactor required for catalysis, suggesting that MD2 binds to MraYAA in a manner that overlaps with, but is distinct from, its natural substrate, UDP-MurNAc-pentapeptide. We have determined the principles of MD2 binding to MraYAA, including how it avoids the need for pyrophosphate and sugar moieties, which are essential features for substrate binding. The conformational plasticity of MraY could be the reason that it is the target of many structurally distinct inhibitors. These findings can inform the design of new inhibitors targeting MraY as well as its paralogues, WecA and TarO.

// Hyeran Sung 1, 2 , Krishna L. Kanchi 3 , Xue Wang 1, 2 , Kristen S. Hill 1, 2 , Jane L. Messina 2, 4, 5 , Ji-Hyun Lee 6 , Youngchul Kim 7 , Nathan D. Dees 3 , Li Ding 3, 8, 9 , Jamie K. Teer 7 , Shengyu Yang 2, 10 , Amod A. Sarnaik 2, 4 , Vernon K. Sondak 2, 4 , James J. Mule 2, 4 , Richard K. Wilson 3, 9 , Jeffrey S. Weber 11 , Minjung Kim 1, 2, 4 1 Department of Molecular Oncology, Moffitt Cancer Center, Tampa, FL, USA 2 Comprehensive Melanoma Research Center, Moffitt Cancer Center, Tampa, FL, USA 3 The Genome Institute, Washington University, St. Louis, MO, USA 4 Department of Cutaneous Oncology, Moffitt Cancer Center, Tampa, FL, USA 5 Department of Pathology, Moffitt Cancer Center, Tampa, FL, USA 6 Department of Internal Medicine, University of New Mexico Comprehensive Cancer Center, Albuquerque, NM, USA 7 Department of Biostatistics and Bioinformatics, Moffitt Cancer Center, Tampa, FL, USA 8 Department of Medicine, Washington University, St. Louis, MO, USA 9 Department of Genetics, Washington University, St. Louis, MO, USA 10 Department of Tumor Biology, Moffitt Cancer Center, Tampa, FL, USA 11 Department of Medicine, NYU Langone Medical Center, New York, NY, USA Correspondence to: Minjung Kim, e-mail: Minjung.kim@moffitt.org Keywords: melanoma, RASA1, RasGAP, R-Ras, whole genome sequencing Received: January 15, 2016 Accepted: February 28, 2016 Published: March 16, 2016 ABSTRACT Inactivation of Ras GTPase activating proteins (RasGAPs) can activate Ras, increasing the risk for tumor development. Utilizing a melanoma whole genome sequencing (WGS) data from 13 patients, we identified two novel, clustered somatic missense mutations (Y472H and L481F) in RASA1 (RAS p21 protein activator 1, also called p120RasGAP). We have shown that wild type RASA1, but not identified mutants, suppresses soft agar colony formation and tumor growth of BRAF mutated melanoma cell lines via its RasGAP activity toward R-Ras (related RAS viral (r-ras) oncogene homolog) isoform. Moreover, R-Ras increased and RASA1 suppressed Ral-A activation among Ras downstream effectors. In addition to mutations, loss of RASA1 expression was frequently observed in metastatic melanoma samples on melanoma tissue microarray (TMA) and a low level of RASA1 mRNA expression was associated with decreased overall survival in melanoma patients with BRAF mutations. Thus, these data support that RASA1 is inactivated by mutation or by suppressed expression in melanoma and that RASA1 plays a tumor suppressive role by inhibiting R-Ras, a previously less appreciated member of the Ras small GTPases.

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Long noncoding RNAs (lncRNAs) are emerging as an important controller affecting metabolic tissue development, signaling, and function. However, little is known about the function and profile of lncRNAs in osteoblastic differentiation in mice. Here, we analyzed the RNA-sequencing (RNA-Seq) datasets obtained for 18 days in two-day intervals from neonatal mouse calvarial pre-osteoblast-like cells. Over the course of osteoblast differentiation, 4058 mRNAs and 3948 lncRNAs were differentially expressed, and they were grouped into 12 clusters according to the expression pattern by fuzzy c-means clustering. Using weighted gene coexpression network analysis, we identified 9 modules related to the early differentiation stage (days 2–8) and 7 modules related to the late differentiation stage (days 10–18). Gene ontology and KEGG pathway enrichment analysis revealed that the mRNA and lncRNA upregulated in the late differentiation stage are highly associated with osteogenesis. We also identified 72 mRNA and 89 lncRNAs as potential markers including several novel markers for osteoblast differentiation and activation. Our findings provide a valuable resource for mouse lncRNA study and improves our understanding of the biology of osteoblastic differentiation in mice.

Background & Aim Since numbers of harvested hematopoietic stem cells (HSCs) continues to limit not only laboratory research but also clinical HSC transplantation and gene therapies, development of ex vivo culture systems to expand harvested human hematopoietic stem-progenitor cells (HSPCs) remains a critical translational research quest. To address the barrier that HSCs generally die or differentiate in current ex vivo culture media, we have developed an xeno-free, serum-free medium - StemPro™ HSC Expansion Medium (Prototype) by extensive evaluation of iterative modification of medium constituents. Methods, Results & Conclusion Cultures of primary human CD34+ HSPCs immunopurified from cord blood, mobilized peripheral blood and bone marrow harvests generated greatly increased numbers of immunphenotyped-defined HSPC in StemPro™ HSC Expansion Medium (Prototype) supplemented with FLT3L, KITL (also known as SCF), TPO, IL-3, and IL-6 (FKT36), as compared to either uncultured day 0 cells or cells cultured in industry-standard culture media containing FKT36. For example, culture of primary human CD34+ cells from mobilized peripheral blood for 7 days in FKT36-containing StemPro™ HSC Expansion Medium (Prototype) resulted in ∼100-fold increased numbers of CD34+CD45+Lin− cells and ∼2000-fold increased numbers of CD34+Lin−CD90+CD45RA− cells (an early HSPC immunophenotype), as compared to uncultured day 0 cells. The ex vivo-cultured cells contained high frequencies of aldehyde dehydrogenase-containing cells and formed erythroid and non-erythroid hematopoietic colonies in vitro. In an ongoing in vivo hematopoietic chimera experiment, ex vivo-cultured mPB CD34+ HSPCs harbored robust in vivo-engrafting capacity at the 8-week post-transplant short-term HSC time point evaluated to date. Thus, it appears that StemPro™ HSC Expansion Medium (Prototype) supports superior HSPC expansion that includes self-renewal of short-term-HSCs. Evaluation of long-term-HSC capacity is in progress.

A main characteristic of aging is the debilitating, progressive and generalized impairment of biological functions, resulting in an increased vulnerability to disease and death. Skeletal muscle comprises approximately 40% of the human body; thus, it is the most abundant tissue. At the age of 30 onwards, 0.5‑1% of human muscle mass is lost each year, with a marked acceleration in the rate of decline after the age of 65. Thus, novel strategies that effectively attenuate skeletal muscle loss and enhance muscle function are required to improve the quality of life of older subjects. The aim of the present study was to determine whether loquat (Eriobotrya japonica) leaf extract (LE) can prevent the loss of skeletal muscle function in aged rats. Young (5-month-old) and aged (18‑19-month-old) rats were fed LE (50 mg/kg/day) for 35 days and the changes in muscle mass and strength were evaluated. The age‑associated loss of grip strength was attenuated, and muscle mass and muscle creatine kinase (CK) activity were enhanced following the administration of LE. Histochemical analysis also revealed that LE abrogated the age‑associated decrease in cross‑sectional area (CSA) and decreased the amount of connective tissue in the muscle of aged rats. To investigate the mode of action of LE, C2C12 murine myoblasts were used to evaluate the myogenic potential of LE. The expression levels of myogenic proteins (MyoD and myogenin) and functional myosin heavy chain (MyHC) were measured by western blot analysis. LE enhanced MyoD, myogenin and MyHC expression. The changes in the expression of myogenic genes corresponded with an increase in the activity of CK, a myogenic differentiation marker. Finally, LE activated the Akt/mammalian target of rapamycin (mTOR) signaling pathway, which is involved in muscle protein synthesis during myogenesis. These findings suggest that LE attenuates sarcopenia by promoting myogenic differentiation and subsequently promoting muscle protein synthesis.

A simple, sensitive, and rapid HPLC method was developed to analyze bakuchiol and two furocoumarins (psoralen and angelicin) simultaneously in bakuchiol extracts from Psoralea corylifolia seeds. The analysis was performed within 30 min on a phenyl-hexyl column using gradient elution with a mobile phase composed of water and methanol with UV detection at 260 nm for bakuchiol and 246 nm for psoralen and angelicin. The method was validated with respect to linearity (r2 > 0.99 for all components), accuracy (>95% for all components), and precision (

Prevotella intermedia has long been known to be as the principal etiologic agent of periodontal diseases and associated with various systemic diseases. Previous studies showed that the intra-species difference exists in capacity of biofilm formation, antibiotic resistance, and serological reaction among P. intermedia strains. Here we report the genome sequence of P. intermedia SUNY aB G8-9K-3 (designated ATCC49046) that displays a relatively high antimicrobial resistant and biofilm-forming capacity. Genome sequencing information provides important clues in understanding the genetic bases of phenotypic differences among P. intermedia strains.

Calcium signaling mediated by STIM1 and Orai1 activates Src to promote invadopodium assembly while simultaneously promoting MT1-MMP recycling to the plasma membrane to promote ECM degradation., Ca2+ signaling has been increasingly implicated in cancer invasion and metastasis, and yet, the underlying mechanisms remained largely unknown. In this paper, we report that STIM1- and Orai1-mediated Ca2+ oscillations promote melanoma invasion by orchestrating invadopodium assembly and extracellular matrix (ECM) degradation. Ca2+ oscillation signals facilitate invadopodial precursor assembly by activating Src. Disruption of Ca2+ oscillations inhibited invadopodium assembly. Furthermore, STIM1 and Orai1 regulate the proteolysis activity of individual invadopodia. Mechanistically, Orai1 blockade inhibited the recycling of MT1–matrix metalloproteinase (MMP) to the plasma membrane and entrapped MT1-MMP in the endocytic compartment to inhibit ECM degradation. STIM1 knockdown significantly inhibited melanoma lung metastasis in a xenograft mouse model, implicating the importance of this pathway in metastatic dissemination. Our findings provide a novel mechanism for Ca2+-mediated cancer cell invasion and shed new light on the spatiotemporal organization of store-operated Ca2+ signals during melanoma invasion and metastasis.

Dunaliella successfully survives and photosynthesizes in hypersaline environments. To better understand the physiological and photosynthetic characteristics of Dunaliella exposed to long-term hypersaline conditions, we compared two morphologically distinct species, Dunaliella tertiolecta and Dunaliella salina. Despite similar glycerol accumulation patterns and maintenance of the maximum photosynthetic quantum yield, photosynthetic oxygen evolution was enhanced by increased salinity in D. tertiolecta but remained constant in D. salina. Total chlorophyll content was dramatically reduced in D. tertiolecta but did not change significantly in D. salina. In D. salina, β-carotene content increased with increasing salinity and reached 9.2 ± 0.15 fmol cell−1 at 4.5 M NaCl; in contrast, that of D. tertiolecta was reduced. Expression of carbonic anhydrase and Rubisco activase, enzymes related to photosynthetic carbon assimilation, increased with increasing salinity in D. tertiolecta but not in D. salina. The expression of carotenoid biosynthesis-related protein, which is possibly involved in photoprotection, was increased by high salt. De-epoxidation of xanthophyll pigments in D. tertiolecta and the increased β-carotene content in D. salina may play an important role as protective mechanisms to prevent photodamage in response to hypersaline conditions. We conclude that the two Dunaliella species likely employ different strategies to adapt to long-term hypersaline conditions.

Apigenin (4',5,7-trihydroxyflavone) is a natural flavonoid, shown to have chemopreventive and/or anticancer properties in a variety of human cancer cells. The involvement of autophagy in apigenin-induced apoptotic cell death of HCT116 human colon cancer cells was investigated. Apigenin induced suppression of cell growth in a concentration-dependent manner in HCT116 cells. Flow cytometric analyses indicated that apigenin resulted in G2/M phase arrest. This flavone also suppressed the expression of both cyclin B1 and its activating partners, Cdc2 and Cdc25c, whereas the expression of cell cycle inhibitors, such as p53 and p53-dependent p21(CIP1/WAF1), was increased after apigenin treatment. Apigenin induced poly (ADP-ribose) polymerase (PARP) cleavage and decreased the levels of procaspase-8, -9 and -3. In addition, the apigenin-treated cells exhibited autophagy, as characterized by the appearance of autophagosomes under fluorescence microscopy and the accumulation of acidic vesicular organelles by flow cytometry. Furthermore, the results of the western blot analysis revealed that the levels of LC3-II, the processed form of LC3-I, was increased by apigenin. Treatment with the autophagy inhibitor 3-methyladenine (3-MA) significantly enhanced the apoptosis induced by apigenin, which was accompanied by an increase in the levels of PARP cleavage. These results indicate that apigenin has apoptosis- and autophagy-inducing effects in HCT116 colon cancer cells. Autophagy plays a cytoprotective role in apigenin-induced apoptosis, and the combination of apigenin and an autophagy inhibitor may be a promising strategy for colon cancer control.

Increasing the numbers of donor hematopoietic stem cells (HSCs) would accelerate hematopoietic recovery in many HSC transplant recipients, thereby reducing mortality, morbidity and costs. HSC gene therapies would further benefit from high transplant donor HSC numbers, due to inefficiencies in genetic modification of HSCs and losses during transfection protocols. Unfortunately, worldwide attempts to optimize culture parameters, such as hematopoietic growth factor combinations, feeder cells and bioengineered chambers, have failed to result in the substantial HSC self-renewal needed clinically, although it was reported recently that substitution of polyvinyl alcohol for albumin resulted in massive expansion of mouse HSCs (Wilkinson et al. Nature 571:117;2019).Since the same few base hematopoietic culture media have been used for decades, we set out to develop a culture medium specifically for ex vivo expansion of human HSCs. In a Design of Experiments approach, media constituents were systematically varied and each iteration evaluated with the goal to maximize ex vivo expansion of hematopoietic stem-progenitor cell (HSPC) immunophenotypes. After 1 week (wk) culture of human mPB-, CB- or BM-derived CD34+ cells in optimized xeno-free, serum-free StemPro™ HSC Expansion Medium (Prototype) (SPHSC), supplemented with FLT3L, KITL, TPO, IL3, and IL6 (FKT36), there were on average 96-, 178- or 80-fold, respectively, greater numbers (yields) of viable nucleated cells, as compared to day (d)0 (Fig 1). Average yields of the CD34+CD45+Lin- HSPC immunophenotype were increased similarly, by 80-, 104- or 42-fold, respectively. Average yields of the CD34+CD45+CD45RA+CD90+ early HSPC immunophenotype were much more highly increased, by 2300-, 6047- or 1248-fold, respectively. CB-derived CD34+ cells typically generated greater fold increases in these parameters than did CD34+ cells from mPB or BM. In addition, we consistently observed (a) the presence of very early CD34+++CD90+ cells and (b) few Lin+ cells in the ex vivo cultured populations. Furthermore, compared to 2 different FKT36-containing commercial standard media, SPHSC supported statistically greater yields of nucleated cells, CD34+CD45+Lin- HSPCs and CD34+CD45+CD45RA+CD90+ early HSPCs. We focused on the challenge of expanding HSCs from mPB, the most common clinical source. Yields of nucleated cells, CD34+CD45+Lin- HSPCs, and CD34+CD45+CD45RA+CD90+ early HSPCs expanded progressively from d4-d10. Transplant of 1 wk-cultured mPB CD34+ HSPCs generated donor cell dose-dependent increases in %hCD45+mCD45- cells in sublethally-irradiated NRG mouse bone marrows and spleens at 26 wks post-transplant. Human HSPCs and CD33+ myeloid and CD19+ lymphoid progeny were present in hematopoietic organs of the transplanted mice (Fig 2). The long-term (LT)-HSC engraftment capacity of the entire 1 wk-cultured cell population was 10-fold greater than at d0. These results for 1 wk ex vivo expansion of mPB CD34+ cells in SPHSC are similar or superior to reported 12d or 15d expansions of CB CD34+ cells in cytokine-supplemented standard media containing UM171 or SR1 (Fares et al. Science 345:1509;2014; Cohen et al. Biol Blood Marrow Transplant 24:S190;2018; Wagner et al.Cell Stem Cell 18:144;2016). Thus, SPHSC medium may by itself be impactful in clinical transplantation and gene therapy and would also be an optimized platform medium for additional approaches to further enhance ex vivo expansion of human HSCs for transplant and gene therapies. Disclosures Sei: Thermo Fisher Scientific Inc: Employment. Harris-Becker:Thermo Fisher Scientific Inc: Employment. Kaur:Thermo Fisher Scientific Inc: Employment. Vemuri:Thermo Fisher Scientific Inc: Employment.

Erythropoiesis is orchestrated by the coordinated action of multiple transcription factors. The master erythropoietic regulator GATA1 is itself modulated via interactions with multiple co-regulatory factors, such as FOG1, KLF1, and LMO2. Though the PAX-SIX-EYA-DACH network (PSEDN) of conserved transcription factors has been well characterized in the formation of eyes, kidney, branchial structures, and skeletal muscles, a role for PSEDN members in hematopoietic systems has only recently been recognized (Liu et al., Nature 2019 PMID:30894749). Here, we studied the PSEDN member SIX1 and discovered its ability to drive erythroid differentiation of human hematopoietic cells. Enforced overexpression (OE) of SIX1 in human TF1 erythroleukemia cells or primary CD34+ hematopoietic stem-progenitor cells (HSPCs) stimulated the generation of erythroid cells, as determined by increased numbers of cells expressing erythroid-selective surface markers (CD235ahiCD71hiCD34-) and hemoglobin (HBB). Conversely, SIX1 knockout in TF1 cells or primary HSPCs reduced erythroid cell generation in response to erythropoietin (EPO). SIX1 OE could also stimulate TF1 cell erythroid differentiation in the absence of EPO. Further analysis of SIX1 OE in TF1 cells revealed that SIX1 stimulated the expression of multiple functionally important erythroid molecules including ALAS2, SLC4a1, EPOR, SPTA1, KLF1 and ANK1. By gene set enrichment analysis (GSEA) of global RNA-seq data, SIX1 OE stimulated heme metabolism genes as well as many genes known to be regulated by GATA1, including FOG1-dependent and -independent genes. SIX1 OE reduced GATA2 and increased GATA1 protein and RNA expression, resembling GATA switching downstream of EPO signaling. To determine whether GATA1 was necessary for SIX1 to stimulate erythropoiesis, we generated GATA1 knockout cells using CRISPR/Cas9 technology. In contrast to control cells, SIX1 OE in GATA1 knockout cells failed to stimulate erythropoiesis, indicating that SIX1 stimulation of erythropoiesis requires GATA1. To gain further insight into the mechanism by which SIX1 stimulates erythropoiesis, the promiscuous biotin ligase, BirA (Choi-Rhee et al., Protein Sci. 2004 PMID:15459338), was fused in-frame to SIX1 to determine the SIX1 proximal interactome. Streptavidin-enrichment of biotinylated proteins in SIX1-BirA OE lysates revealed GATA1 and FOG1 as proximal interactors of SIX1-BirA, but not of BirA alone. When co-expressed in HEK293T cells GATA1 and SIX1 were found to co-immunoprecipitate, suggesting the two proteins can physically interact in a complex. We demonstrated the functional consequence of the SIX1 interaction with GATA1 using a GATA1-dependent luciferase reporter gene harboring three copies of GATA binding sites. Cells in which SIX1 and GATA1 were co-expressed exhibited significantly higher levels of luciferase expression compared to cells expressing only GATA1, suggesting SIX1 could stimulate GATA1-dependent transcription. Introduction of mutations in SIX known to cause Branchio-Oto-Renal (BOR) (Ruf et al., PNAS 2004 PMID:15141091) syndrome did not inhibit the ability of SIX to bind GATA1 nor its ability to drive erythropoiesis. Taken together our results suggest that SIX1 can stimulate erythropoiesis via multiple mechanisms, including increased GATA1 expression and function. Our findings provide the first demonstration of a role for the PSEDN in erythropoiesis and reveal unknown physical and functional interactions between two central developmental transcriptional networks (GATA:FOG network and PSEDN). Disclosures No relevant conflicts of interest to declare.

Background Periodontitis is the most common chronic inflammatory disease caused by complex interaction between the microbial biofilm and host immune responses. In the present study, high-throughput RNA sequencing was utilized to systemically and precisely identify gene expression profiles and alternative splicing. Methods The pooled RNAs of 10 gingival tissues from both healthy and periodontitis patients were analyzed by deep sequencing followed by computational annotation and quantification of mRNA structures. Results The differential expression analysis designated 400 up-regulated genes in periodontitis tissues especially in the pathways of defense/immunity protein, receptor, protease, and signaling molecules. The top 10 most up-regulated genes were CSF3, MAFA, CR2, GLDC, SAA1, LBP, MME, MMP3, MME-AS1, and SAA4. The 62 down-regulated genes in periodontitis were mainly cytoskeletal and structural proteins. The top 10 most down-regulated genes were SERPINA12, MT4, H19, KRT2, DSC1, PSORS1C2, KRT27, LCE3C, AQ5, and LCE6A. The differential alternative splicing analysis revealed unique transcription variants in periodontitis tissues. The EDB exon was predominantly included in FN1, while exon 2 was mostly skipped in BCL2A1. Conclusions These findings using RNA sequencing provide novel insights into the pathogenesis mechanism of periodontitis in terms of gene expression and alternative splicing. Electronic supplementary material The online version of this article (doi:10.1186/s40246-016-0084-0) contains supplementary material, which is available to authorized users.

It has been shown that inhibition of GTPase-activating protein of ADP-ribosylation factor (Arf), ArfGAP, with a small molecule (QS11) results in synergistic activation of Wnt/β-catenin signaling. However, the role of Arf in Wnt/β-catenin signaling has not yet been elucidated. Here, we show that activation of Arf is essential for Wnt/β-catenin signaling. The level of the active form of Arf (Arf-GTP) transiently increased in the presence of Wnt, and this induction event was abrogated by blocking the interaction between Wnt and Frizzled (Fzd). In addition, knockdown of Fzds, Dvls or LRP6 blocked the Wnt-mediated activation of Arf. Consistently, depletion of Arf led to inhibition of Wnt-mediated membrane PtdIns (4,5)P2 (phosphatidylinositol 4, 5-bisphosphate) synthesis and LRP6 phosphorylation. Overall, our data suggest that transient activation of Arf modulates LRP6 phosphorylation for the transduction of Wnt/β-catenin signaling.

The p53 gene plays a determinant role in radiation-induced cell death and its protein product is negatively regulated by MDM2. We investigated whether adenovirus-mediated modified p53 gene transfer, which blocks p53-MDM2 binding, is effective for radiation-induced cell death in hepatocellular carcinoma (HCC) at different MDM2 cellular levels. Human hepatocellular carcinoma cell lines expressing MDM2 at low levels (Huh7) and high levels (SK-Hep1) were used. Ad-p53 and Ad-p53vp are replication-deficient adenoviral vectors containing human wild-type or modified p53, respectively. The anti-tumor effect was highest for Ad-p53 + radiotherapy (RT) in the low-level MDM2 cells, whereas this effect was highest for Ad-p53vp + RT in the MDM2-overexpressing cells. In Huh-7 cells, Ad-p53 + RT decreased cell viability (32%) in vitro and inhibited tumor growth (enhancement factor, 1.86) in vivo. Additionally, p21 expression and apoptosis were increased. In contrast, in SK-Hep1 cells, Ad-p53vp + RT showed decreased cell viability (51%) in vitro and inhibition of tumor growth (enhancement factor, 3.07) in vivo. Caspase-3 expression and apoptosis were also increased. Adenovirus-expressing modified p53, which blocks p53-MDM2 binding, was effective in killing tumor cells overexpressing MDM2. Furthermore, the combination strategy for disruption of the p53-MDM2 interaction with RT demonstrated enhanced anti-tumor effects both in vitro and in vivo.

Cancer cells constantly reprogram their metabolism to meet anabolic and catabolic demands in real time. Increased aerobic glycolysis (the Warburg effect) is associated with highly proliferative tumors, since it confers several advantages to proliferating tumor cells by providing rapid ATP production from glycolysis and macromolecules (building blocks) for cell division. On the other hand, mitochondria-dependent oxidative phosphorylation (OxPhos) generates the maximal amount of ATP from glucose. Although underappreciated, OxPhos is also utilized to support growth of cancer cells, especially for “oxidative tumors” such as lymphomas, glioblastomas, and melanomas. This article is protected by copyright. All rights reserved.

Fascin, an actin-bundling protein overexpressed in all carcinomas, has been associated with poor prognosis, shorter survival, and more metastatic diseases. It is believed that fascin facilitates tumor metastasis by promoting the formation of invasive membrane protrusions. However, the mechanisms by which fascin is overexpressed in tumors are not clear. TGFβ is a cytokine secreted by tumor and mesenchymal cells and promotes metastasis in many late stage tumors. The pro-metastasis mechanisms of TGFβ remain to be fully elucidated. Here we demonstrated that TGFβ induced fascin expression in spindle-shaped tumor cells through the canonical Smad-dependent pathway. Fascin was critical for TGFβ-promoted filopodia formation, migration, and invasion in spindle tumor cells. More importantly, fascin expression significantly correlates with TGFβ1 and TGFβ receptor I levels in a cohort of primary breast tumor samples. Our results indicate that elevated TGFβ level in the tumor microenvironment may be responsible for fascin overexpression in some of the metastatic tumors. Our data also suggest that fascin could play a central role in TGFβ-promoted tumor metastasis.

Summary MAPK and AKT pathways are frequently co-activated in melanoma through overexpression of receptor tyrosine kinases, mutations in their signaling surrogates, such as RAS and BRAF, or loss of negative regulators such as PTEN. Since RAS can be a positive upstream regulator of PI3-K, it has been proposed that the loss of PTEN and the activation of RAS are redundant events in melanoma pathogenesis (Tsao et al., 2000). Here, in genetically engineered mouse models of cutaneous melanomas, we sought to better understand the genetic interactions between HRAS activation and PTEN inactivation in melanoma genesis and progression in vivo. We showed that HRAS activation cooperates with Pten+/- and Ink4a/Arf-/- to increase melanoma penetrance and promote metastasis. Correspondingly, gain- and loss-of-function studies established that Pten loss increases invasion and migration of melanoma cells and non-transformed melanocytes, and that such biological activity correlates with a shift to phosphorylation of AKT2 isoform and E-cadherin down-regulation. Thus, Pten inactivation can drive the genesis and promote the metastatic progression of RAS activated Ink4a/Arf deficient melanomas.

The RAS/RAF/MEK/ERK signaling pathway, which is intricately regulated by multiple proteins including PTPN11 (Tyrosine-Protein Phosphatase Non-Receptor Type 11, encoding SHP2), is frequently activated in melanoma. Although implicated as an oncogene in multiple cancer types, the oncogenic role of PTPN11 has not been established in melanoma. Recently, we preformed whole exome sequencing on tumors generated by a mouse model driven by loss of PTEN and CDKN2A (INK4A/ARF) and identified several conserved cross-species orthologous mutations in Kras, Erbb3, and Ptpn11. In this study, we addressed the functional roles of PTPN11 in melanoma tumorigenesis and tumor maintenance; as well as, PTPN11's effect on the RAS/RAF/MEK/ERK signaling pathway and its activation status in human melanoma. PTPN11 can be activated by receptor tyrosine kinases (RTKs) or by point mutations. We observed activating phosphorylation on Tyr 542 of PTPN11 in 40% (n=15/38) of melanoma specimens and the majority of human melanoma cell lines (n=14), supporting frequent activation of PTPN11 in human melanoma even though the mutation rate is low (1~3%). PTPN11 knock-down suppressed ERK activation in NRAS mutant and BRAF/NRAS wild-type melanoma cells, but not in BRAF mutant cells. Moreover, we have shown that expression of active PTPN11E76K mutant drives soft-agar colony growth in vitro, tumor growth in nude mice, RAS/RAF/MEK/ERK activation, and resistance to MEK inhibition. Alternatively, knock-down of Ptpn11 reduces colony growth and ERK activation. We generated a tet-inducible, melanocyte-specific, PTPN11E76K transgenic mouse model in a PTEN and CDKN2A null background and observed melanoma formation. Implantation of melanoma cells derived from this model showed doxycycline dependent tumor growth in nude mice. Doxycycline withdrawal and subsequent extinction of PTPN11E76K caused regression of established tumors, supporting the tumor maintenance role of PTPN11. Subsequently, tumor tissue from this model underwent phosphor-tyrosine proteomic analysis to identify downstream effectors of PTPN11's protein tyrosine phosphatase activity. The proteins identified in this analysis are currently being confirmed. These data support the oncogenic roles of PTPN11 in melanoma by regulating RAS/RAF/MAPK pathway activation and the value of PTPN11 as a novel and actionable therapeutic target. Citation Format: Kristen Suzanne Hill, Evan Roberts, Ellen Marin, Xue Wang, Jamie Teer, Jane Messina, Jerry Wu, Minjung Kim. The oncogenic role and therapeutic potential of PTPN11 in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2387.

Melanoma is a deadly disease carrying many genetic mutations. A major challenge to the development of effective targeted therapies in melanoma is the identification of true “driver” mutations among numerous “passenger” alterations. Several previous studies support using cross-species comparative oncogenomic approaches for cancer gene discovery. Specifically, it has been shown that mice and humans share several genetic events in the development of cancer and that these events that are conserved across different species may point to functionally important and evolutionary conserved alterations targeting “driver” genes. Recently, we analyzed melanoma genomes from a mouse model driven by the loss of PTEN and CDKN2A (INK4A/ARF), commonly observed alterations in human melanoma patients, by whole-exome sequencing. This study identified several conserved cross-species orthologous mutations in Kras, Erbb3, and Ptpn11. In this study, we addressed the functional roles of PTPN11 in melanoma tumorigenesis and tumor maintenance, its effect on RAS/RAF/MEK/ERK signaling pathway, and its activation status in human melanoma. Melanoma displays frequent activation of the RAS/RAF/MEK/ERK signaling pathway, which is intricately regulated by multiple proteins including PTPN11 (Tyrosine-Protein Phosphatase Non-Receptor Type 11, encoding SHP2). Although implicated as an oncogene in multiple cancer types, the oncogenic role of PTPN11 has not been fully established in melanoma. PTPN11 can be activated by receptor tyrosine kinases (RTKs) and/or by point mutations. Although the mutation rate is low (1~3%), we observed activating phosphorylation on Tyr 542 of PTPN11 in 40% (n=15/38) of melanoma specimens and the majority of human melanoma cell lines (n=14), indicating the potential frequent activation of PTPN11 in human melanoma. PTPN11 knock-down suppressed ERK activation in NRAS mutant (WM1361A, 1366, 1346) and BRAF/NRAS wt (WM3211, MeWo, CHL1) melanoma cells, but not in BRAF mutant (1205Lu, IGR1, 983C) cells. Moreover, we have shown that the expression of active PTPN11 E76K mutant drives soft-agar colony growth in vitro, tumor growth in nude mice, RAS/RAF/MEK/ERK activation, and resistance to MEK inhibition, whereas knock-down of Ptpn11 reduces colony growth and ERK activation. We generated a tet-inducible, melanocyte-specific, PTPN11 E76K transgenic mouse model in a Pten and Cdkn2a null background and observed melanoma formation. Implantation of melanoma cells derived from this model showed doxycycline-dependent tumor growth in nude mice; additionally, withdrawal of doxycycline and subsequent extinction of PTPN11 E76K caused regression of established tumors, supporting a tumor-maintenance role of PTPN11. These data support the oncogenic roles of PTPN11 in melanoma by regulating RAS/RAF/MAPK pathway activation and the value of PTPN11 as a novel and actionable therapeutic target. Citation Format: Kristen S. Hill, Xue Wang, Evan R. Roberts, Ellen M. Marin, Jamie K. Teer, Youngchul Kim, Jane Messina, Jie Wu, Minjung Kim. Cross-species oncogenomics approach identifies PTPN11 as an oncogene and potential therapeutic target in melanoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A04.

SUMMARY To investigate which genes may be important for growth under extreme conditions such as very low or high salinities, a survey of the Dunaliella sp. transcriptome was performed with a cDNA microarray which had been generated previously representing 778 expressed sequence tags. The comparative microarray analysis indicated that 142 genes differed in expression levels by more than twofold in cells grown at extreme salinities (0.08 M and 4.5 M NaCl) when compared with cells grown at intermediate salinity (1.5 M NaCl). Of these genes, 28 had increased expression and 57 were suppressed in cells grown at low salinity. In cells grown at high salinity, 43 genes showed increased expression and 69 genes showed suppressed expression. However, we did observe a large overlap in the expression of extreme salinity-responsive genes based on Venn diagram analysis, which found 55 genes that responded to both of the two extreme salinity conditions. Further, we found that several genes had similar expression levels under low and high salinities, including some general stress response genes that were upregulated in both extreme salinity conditions. For confirmation of the validity of the cDNA microarray analysis, expression of several genes was independently confirmed by the use of gene-specific primers and real-time polymerase chain reaction. The present study is the first large-scale comparative survey of the transcriptome from the microalga Dunaliella sp. acclimated to extreme salinities, thus providing a platform for further functional investigation of differentially expressed genes in Dunaliella.

Teucrium chamaedrys (Gemander) has been reported as an adulterant of Scutellaria lateriflora (American skullcap) herbal preparations and is also known to be hepatotoxic. A quick and simple high-performance thin-layer chromatographic (HPTLC) method was developed for the detection of T. chamaedrys (Germander) in S. baicalensis (Chinese skullcap) extract, an ingredient of the proprietary blend product, Univestin. The HPTLC profile of T. chamaedrys was distinguished from that of S. baicalensis by its bright green fluorescence bands. This simple method can be completed in an hour for the quality control of Univestin and its raw material, S. baicalensis. The method is sensitive and can detect T. chamaedrys at levels as low as 0.5 (w/w).

LIM-domain binding protein 1 (Ldb1) is a multiadaptor protein that mediates the action of transcription factors, including LIM-homeodomain proteins. To elucidate the functional role of Ldb1 in the neuronal differentiation of embryonic stem (ES) cells, we have generated Ldb1-null mutant (Ldb1−/−) ES cells and examined neuronal differentiation potentials in vitro using two different neuronal differentiation protocols. When subjected to a five-stage protocol that recapitulates in vivo conditions of neuronal differentiation, wild-type ES cells differentiated into a wide spectrum of neuronal cell types. However, Ldb1−/− ES cells did not differentiate into neuronal cells; instead, they differentiated into sarcomeric α-actinin-positive muscle cells. In contrast, when an adherent monolayer culture procedure (which is based on the default mechanism of neural induction and eliminates environmental influences) was applied, both wild-type and Ldb1−/− ES cells differentiated into MAP2-positive mature neurons. Comparison of the results obtained when two different neuronal differentiation protocols were used suggests that Ldb1−/− ES cells have an innate potential to differentiate into neuronal cells, but this potential can be inhibited by environmental influences. Disclosure of potential conflicts of interest is found at the end of this article.

Notch activation inhibits neuronal differentiation during development of the nervous system; however, the dynamic role of Notch signaling in individual cell lineages remains poorly understood. We have characterized 3.4 kb 5′-regulatory sequence of a Notch target gene, her4, and used it to drive fluorescent gene expression in transgenic lines where the spatiotemporal pattern of Notch activation can be examined in vivo. The 3.4 kb her4 promoter contains five predicted Su(H) binding sites of which two proximal sites were confirmed to be required for Notch-mediated transcriptional activation. Without Notch, Su(H) effectively represses transcription regulated by the promoter. Analyses of transgenic zebrafish showed that while the expression of proneural genes and Notch activation were both critical for endogenous her4 expression, reporter gene expression was primarily regulated by Notch activity. This study also showed that her4 may be differently regulated in sensory cranial ganglia, where Notch activity is not essential for her4 expression and where Su(H) may repress her4 expression. The establishment of a reporter line with Notch-Su(H)-dependent fluorescent gene expression provides a tool to explore the complex role of Notch signaling in the development of vertebrate nervous system.

Polyphosphate (polyP) has gained a wide interest in the food industry due to its potential as a decontaminating agent. In this study, we examined the effect of sodium tripolyphosphate (polyP3; Na 5 P 3 O 10 ) against planktonic and biofilm cells of Prevotella intermedia , a major oral pathogen. The MIC of polyP3 against P. intermedia ATCC 49046 determined by agar dilution method was 0.075%, while 0.05% polyP3 was bactericidal against P. intermedia in time-kill analysis performed using liquid medium. A crystal violet binding assay for the assessment of biofilm formation by P. intermedia showed that sub-MICs of polyP3 significantly decreased biofilm formation. Under the scanning electron microscope, decreased numbers of P. intermedia cells forming the biofilms were observed when the bacterial cells were incubated with 0.025% or higher concentrations of polyP3. Assessment of biofilm viability with LIVE/DEAD staining and viable cell count methods showed that 0.05% or higher concentrations of polyP3 significantly decreased the viability of the preformed biofilms in a concentration-dependent manner. The zone sizes of alpha-hemolysis formed on horse blood agar produced by P. intermedia were decreased in the presence of polyP3. The expression of the genes encoding hemolysins and the genes of the hemin uptake ( hmu ) locus was downregulated by polyP3. Collectively, our results show that polyP is an effective antimicrobial agent against P. intermedia in biofilms as well as planktonic phase, interfering with the process of hemin acquisition by the bacterium.

Folic acid is a water-soluble vitamin in the B-complex group, and an exogenous intake is required for health, growth and development. As a precursor to co-factors, folic acid is required for one-carbon donors in the synthesis of DNA bases and other essential biomolecules. A lack of dietary folic acid can lead to folic acid deficiency and can therefore result in several health problems, including macrocytic anemia, elevated plasma homocysteine levels, cardiovascular disease, birth defects, carcinogenesis, muscle weakness and difficulty in walking. Previous studies have indicated that folic acid exerts a positive effect on skeletal muscle functions. However, the precise role of folic acid in skeletal muscle cell differentiation remains poorly understood. Thus, in the present study, we examined the effects of folic acid on neo-myotube maturation and differentiation using C2C12 murine myoblasts. We found that folic acid promoted the formation of multinucleated myotubes, and increased the fusion index and creatine kinase (CK) activity in a concentration-dependent manner. In addition, western blot analysis revealed that the expression levels of the muscle-specific marker, myosin heavy chain (MyHC), as well as those of the myogenic regulatory factors (MRFs), MyoD and myogenin, were increased in the folic acid-treated myotubes during myogenic differentiation. Folic acid also promoted the activation of the Akt pathway , and this effect was inhibited by treatment of the C2C12 cells with LY294002 (Akt inhibitor). Blocking of the Akt pathway with a specific inhibitor revealed that it was necessary for mediating the stimulatory effects of folic acid on muscle cell differentia-tion and fusion. Taken together, our data suggest that folic acid promotes the differentiation of C2C12 cells through the activa -tion of the Akt pathway.

Summary Most surfaces reflect light from external sources, but others emit light: they glow . Glowing surfaces are often a sign of an important feature of the environment, such as a heat source or a bioluminescent life form, but we know little about how the human visual system identifies them. Previous work has shown that luminance and luminance gradients are important in glow perception [1,2]. While a link between glow and shape has been suggested in the literature [3], there has been no systematic investigation of this relationship. Here we show that perceived three-dimensional shape plays a decisive role in glow perception; vivid percepts of glow can be toggled on and off, simply by changing cues to three-dimensional shape while holding other image features constant.

Several mouse models of human prostate cancer were studied to identify and characterize potential precursor lesions containing foci of atypical epithelial cells. These lesions exhibit a sequence of changes suggesting progressive evolution toward malignancy. Based on these observations, a grading system is proposed to classify prostatic intraepithelial neoplasia (PIN) in genetically engineered mice (GEM). Four grades of GEM PIN are proposed based on their architecture, differentiation pattern, and degree of cytological atypia. PIN I lesions have one or two layers of atypical cells. PIN II has two or more layers of atypical cells. PIN III has large, pleomorphic nuclei with prominent nucleoli and the cells tend to involve the entire lumen with expansion of the duct outlines. PIN IV lesions contain atypical cells that fill the lumen and bulge focally into, and frequently compromise, the fibromuscular sheath. Within the same cohorts, the lower grade PINs first appear earlier than the higher grades. Morphometric and immunohistochemical analyses confirm progressive change. Although the malignant potential of PIN IV in mice has not been proven, GEM PIN is similar to human PIN. This PIN classification system is a first step toward a systematic evaluation of the biological potential of these lesions in GEM.

Thioltransferase, also known as glutaredoxin, is an enzyme that catalyzes the reduction of a variety of disulfide compounds. In Schizosaccharomyces pombe, two thioltransferases were reported and the cDNA of one of the thioltransferases (thioltransferase-1) was cloned. Using a Northern blot assay, we investigated the thioltransferase transcription in response to various stress conditions. When the culture was shifted to a high temperature, the thioltransferase transcription was not significantly changed compared to the unshifted 30 degrees culture. Treatment of zinc chloride to exponentially-growing cells remarkably increased the thioltransferase transcription, whereas the treatment of mercury chloride greatly reduced the transcription. Treatment of hydrogen peroxide and cadmium chloride caused no significant effects on the transcription of the thioltransferase. These results suggest that the transcription of thioltransferase-1 in S. pombe is induced in response to metal stress that is caused by zinc chloride, but not in response to heat stress or oxidative stress that is caused by hydrogen peroxide.