Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) sample preparation methods, including the direct, on-plate formic acid, and ethanol/formic acid tube extraction methods, were evaluated for their ability to render highly pathogenic organisms nonviable and safe for handling in a biosafety level 2 laboratory. Of these, the tube extraction procedure was the most successful, with none of the tested strains surviving this sample preparation method. Tube extracts from several agents of bioterrorism and their near neighbors were analyzed in an eight-laboratory study to examine the utility of the Bruker Biotyper and Vitek MS MALDI-TOF MS systems and their in vitro diagnostic (IVD), research-use-only, and Security-Relevant databases, as applicable, to accurately identify these agents. Forty-six distinct strains of Bacillus anthracis, Yersinia pestis, Francisella tularensis, Burkholderia mallei, Burkholderia pseudomallei, Clostridium botulinum, Brucella melitensis, Brucella abortus, Brucella suis, and Brucella canis were extracted and distributed to participating laboratories for analysis. A total of 35 near-neighbor isolates were also analyzed.

Background: Diabetes and periodontitis have a bi-directional relationship. And yet, collaborations between primary healthcare practitioners in diabetes and oral health care are minimal. This study explored the views of general practice and oral health professionals on the link between diabetes and periodontitis, and interprofessional diabetes and oral health management. Methods: A sequential mixed-methods exploratory research design was used. General practice and oral health professionals were recruited from four community health centres in Melbourne. Quantitative surveys explored participants' experiences, attitudes and knowledge of diabetes and oral health management and interprofessional collaboration; qualitative follow-up interviews explored survey responses with selected participants. Results: 58 participants completed the online surveys; 22 then participated in semi-structured interviews. Participants generally had strong intentions to collaborate interprofessionally in diabetes and oral health management. Most general practice and oral health professional participants were willing to perform simple screening for periodontitis or diabetes respectively. Themes from the interviews were grouped under three domains: 'a ttitude towards diabetes and oral health management', 'subjective norms' and 'perceived behavioural control'; and an overarching domain to describe participants' 'current practice'. Existing siloed primary healthcare practices and lack of formal referral pathways contribute to poor interprofessional collaboration. Most participants were unsure of each other's responsibilities and roles. Their lack of training in the relationship between general and oral health, compounded by systemic barriers including time constraint, high dental costs, long public dental waiting list and unintegrated health information systems, also impeded interprofessional care. Conclusions: The diabetes and oral health link is not properly recognised or managed collaboratively by relevant primary healthcare professionals in Australia. There is, nonetheless, strong intentions to engage in interprofessional diabetes and oral health care to contribute to improved patient outcomes. Primary healthcare professionals need dedicated and accredited interprofessional training and competencies, formal referral systems and sustainable health policies to facilitate collaboration.

Medulloblastomas are the most common malignant pediatric brain tumor and have been divided into four major molecular subgroups. Animal models that mimic the principal molecular aberrations of these subgroups will be important tools for preclinical studies and allow greater understanding of medulloblastoma biology. We report a new transgenic model of medulloblastoma that possesses a unique combination of desirable characteristics including, among others, the ability to incorporate multiple and variable genes of choice and to produce bioluminescent tumors from a limited number of somatic cells within a normal cellular environment. This model, termed BarTeL, utilizes a Barhl1 homeobox gene promoter to target expression of a bicistronic transgene encoding both the avian retroviral receptor TVA and an eGFP-Luciferase fusion protein to neonatal cerebellar granule neuron precursor (cGNP) cells, which are cells of origin for the sonic hedgehog (SHH) subgroup of human medulloblastomas. The Barhl1 promoter-driven transgene is expressed strongly in mammalian cGNPs and weakly or not at all in mature granule neurons. We efficiently induced bioluminescent medulloblastomas expressing eGFP-luciferase in BarTeL mice by infection of a limited number of somatic cGNPs with avian retroviral vectors encoding the active N-terminal fragment of SHH and a stabilized MYCN mutant. Detection and quantification of the increasing bioluminescence of growing tumors in young BarTeL mice was facilitated by the declining bioluminescence of their uninfected maturing cGNPs. Inclusion of eGFP in the transgene allowed enriched sorting of cGNPs from neonatal cerebella. Use of a single bicistronic avian vector simultaneously expressing both Shh and Mycn oncogenes increased the medulloblastoma incidence and aggressiveness compared to mixed virus infections. Bioluminescent tumors could also be produced by ex vivo transduction of neonatal BarTeL cerebellar cells by avian retroviruses and subsequent implantation into nontransgenic cerebella. Thus, BarTeL mice provide a versatile model with opportunities for use in medulloblastoma biology and therapeutics.

Atypical teratoid rhabdoid tumor (AT/RT) is among the most fatal of all pediatric brain tumors. Aside from loss of function mutations in the SMARCB1 (BAF47/INI1/SNF5) chromatin remodeling gene, little is known of other molecular drivers of AT/RT. LIN28A and LIN28B are stem cell factors that regulate thousands of RNAs and are expressed in aggressive cancers. We identified high-levels of LIN28A and LIN28B in AT/RT primary tumors and cell lines, with corresponding low levels of the LIN28-regulated microRNAs of the let-7 family. Knockdown of LIN28A by lentiviral shRNA in the AT/RT cell lines CHLA-06-ATRT and BT37 inhibited growth, cell proliferation and colony formation and induced apoptosis. Suppression of LIN28A in orthotopic xenograft models led to a more than doubling of median survival compared to empty vector controls (48 vs 115 days). LIN28A knockdown led to increased expression of let-7b and let-7g microRNAs and a down-regulation of KRAS mRNA. AT/RT primary tumors expressed increased mitogen activated protein (MAP) kinase pathway activity, and the MEK inhibitor selumetinib (AZD6244) decreased AT/RT growth and increased apoptosis. These data implicate LIN28/RAS/MAP kinase as key drivers of AT/RT tumorigenesis and indicate that targeting this pathway may be a therapeutic option in this aggressive pediatric malignancy.

Phosphotyrosine Interaction Domain containing 1 (PID1; NYGGF4) inhibits growth of medulloblastoma, glioblastoma and atypical teratoid rhabdoid tumor cell lines. PID1 tumor mRNA levels are highly correlated with longer survival in medulloblastoma and glioma patients, suggesting their tumors may have been more sensitive to therapy. We hypothesized that PID1 sensitizes brain tumors to therapy. We found that PID1 increased the apoptosis induced by cisplatin and etoposide in medulloblastoma and glioblastoma cell lines. PID1 siRNA diminished cisplatin-induced apoptosis, suggesting that PID1 is required for cisplatin-induced apoptosis. Etoposide and cisplatin increased NFκB promoter reporter activity and etoposide induced nuclear translocation of NFκB. Etoposide also increased PID1 promoter reporter activity, PID1 mRNA, and PID1 protein, which were diminished by NFκB inhibitors JSH-23 and Bay117082. However, while cisplatin increased PID1 mRNA, it decreased PID1 protein. This decrease in PID1 protein was mitigated by the proteasome inhibitor, bortezomib, suggesting that cisplatin induced proteasome dependent degradation of PID1. These data demonstrate for the first time that etoposide- and cisplatin-induced apoptosis in medulloblastoma and glioblastoma cell lines is mediated in part by PID1, involves NFκB, and may be regulated by proteasomal degradation. This suggests that PID1 may contribute to responsiveness to chemotherapy.

Quantitative ultrasound (QUS) techniques such as pixel intensity, ultrasound strain, and shear wave elastography have made it possible to identify the echogenicity (brightness) and mechanical properties (stiffness) of normal and pathological tissues. These techniques can be utilized as an alternative diagnosis tool to assess post stroke spasticity. Current clinical assessment methods include the Modified Ashworth Scale (MAS) and the Modified Tardieu Scale (MTS), which can result in inconsistencies due to their subjective nature. QUS provides robust approaches to assessing muscle stiffness associated with post stroke spasticity. Computer-aided pixel count quantifies tissue echogenicity in grayscale image. A strain ratio in ultrasound strain imaging compares the stiffness and movement (lengthening or shortening) of a spastic muscle with nonspecific muscle. In addition, shear wave elastography provides the shear wave velocity of an affected muscle that directly associated with the muscle stiffness before and after treatment for spasticity. This article reviews the theory behind these aforementioned concepts and discuss the relations between QUS and skeletal muscles in post stroke spasticity.

We recently reported that atypical teratoid rhabdoid tumors (ATRTs) comprise at least two transcriptional subtypes with different clinical outcomes; however, the mechanisms underlying therapeutic heterogeneity remained unclear. In this study, we analyzed 191 primary ATRTs and 10 ATRT cell lines to define the genomic and epigenomic landscape of ATRTs and identify subgroup-specific therapeutic targets. We found ATRTs segregated into three epigenetic subgroups with distinct genomic profiles, SMARCB1 genotypes, and chromatin landscape that correlated with differential cellular responses to a panel of signaling and epigenetic inhibitors. Significantly, we discovered that differential methylation of a PDGFRB-associated enhancer confers specific sensitivity of group 2 ATRT cells to dasatinib and nilotinib, and suggest that these are promising therapies for this highly lethal ATRT subtype.

Recombinant protein production in the methylotrophic yeast Pichia pastoris largely relies on integrative vectors. Although the stability of integrated expression cassettes is well appreciated for most applications, the availability of reliable episomal vectors for this host would represent a useful tool to expedite cloning and high-throughput screening, ameliorating also the relatively high clonal variability reported in transformants from integrative vectors caused by off-target integration in the P. pastoris genome. Recently, heterologous and endogenous autonomously replicating sequences (ARS) were identified in P. pastoris by genome mining, opening the possibility of expanding the available toolbox to include efficient episomal plasmids. The aim of this technical report is to validate a 452-bp sequence ("panARS") in context of P. pastoris expression vectors, and to compare their performance to classical integrative plasmids. Moreover, we aimed to test if such episomal vectors would be suitable to sustain in vivo recombination, using fragments for transformation, directly in P. pastoris cells.

Inhibition of integrins αvβ3/αvβ5 by the cyclic function-blocking peptide, RGDfV (Arg-Gly-Asp-Phe-Val) can induce apoptosis in both normal cells and tumor cells. We show that RGDfV induced apoptosis in ECV-304 carcinoma cells, increased activity and mRNA expression of acid sphingomyelinase (ASM), and increased ceramides C(16), C(18:0), C(24:0) and C(24:1) while decreasing the corresponding sphingomyelins. siRNA to ASM decreased RGDfV-induced apoptosis as measured by TUNEL, PARP cleavage, mitochondrial depolarization, and caspase-3 and caspase-8 activities, as well as by annexinV in a 3D collagen model. These findings indicate a causal role for ASM in RGDfV-induced apoptosis in ECV-304. We have shown that c-Abl, a non-receptor tyrosine kinase, also mediates RGDfV-induced apoptosis. However, c-Abl, has not been previously linked to ASM in any system. Here we show that STI-571 (imatinib, inhibitor of c-Abl) inhibited RGDfV-induced ASM activity. Furthermore, STI-571 and c-Abl-siRNA both inhibited RGDfV-induced increase in ASM mRNA, but ASM-siRNA did not affect c-Abl phosphorylation or expression, supporting that c-Abl regulates the RGDfV-induced increase in ASM expression. These studies implicate ASM as a mediator of apoptosis induced by inhibition of integrins αvβ3/αvβ5, and for the first time place c-Abl as an upstream regulator of ASM expression and activity.

The releasable factor adenosine blocks the formation of long-term potentiation (LTP). These experiments used this observation to uncover the synaptic processes that stabilize the potentiation effect. Brief adenosine infusion blocked stimulation-induced actin polymerization within dendritic spines along with LTP itself in control rat hippocampal slices but not in those pretreated with the actin filament stabilizer jasplakinolide. Adenosine also blocked activity-driven phosphorylation of synaptic cofilin but not of synaptic p21-activated kinase (PAK). A search for the upstream origins of these effects showed that adenosine suppressed RhoA activity but only modestly affected Rac and Cdc42. A RhoA kinase (ROCK) inhibitor reproduced adenosine's effects on cofilin phosphorylation, spine actin polymerization, and LTP, whereas a Rac inhibitor did not. However, inhibitors of Rac or PAK did prolong LTP's vulnerability to reversal by latrunculin, a toxin which blocks actin filament assembly. Thus, LTP induction initiates two synaptic signaling cascades: one (RhoA-ROCK-cofilin) leads to actin polymerization, whereas the other (Rac-PAK) stabilizes the newly formed filaments.

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Our previous studies showed that the angiogenic integrin αvβ3 was increased in high-risk metastatic (stage 4) NB compared with localized neuroblastomas. Herein, we show that integrin αvβ3 was expressed on 68% of microvessels in MYCN-amplified stage 3 neuroblastomas, but only on 34% (means) in MYCN-non-amplified tumors (p < 0.001; n = 54). PTEN, a tumor suppressor involved in αvβ3 signaling, was expressed in neuroblastomas either diffusely, focally or not at all (immunohistochemistry). Integrin αvβ3 was expressed on 60% of tumor microvessels when PTEN was negative or focal, as compared to 32% of microvessels in tumors with diffuse PTEN expression (p < 0.001). In a MYCN transgenic mouse model, loss of one allele of PTEN promoted tumor growth, illustrating the potential role of PTEN in neuroblastoma pathogenesis. Interestingly, we report the novel dual PI-3K/BRD4 activity of SF1126 (originally developed as an RGD-conjugated pan PI3K inhibitor). SF1126 inhibits BRD4 bromodomain binding to acetylated lysine residues with histone H3 as well as PI3K activity in the MYCN amplified neuroblastoma cell line IMR-32. Moreover, SF1126 suppressed MYCN expression and MYCN associated transcriptional activity in IMR-32 and CHLA136, resulting in overall decrease in neuroblastoma cell viability. Finally, treatment of neuroblastoma tumors with SF1126 inhibited neuroblastoma growth in vivo. These data suggest integrin αvβ3, MYCN/BRD4 and PTEN/PI3K/AKT signaling as biomarkers and hence therapeutic targets in neuroblastoma and support testing of the RGD integrin αvβ3-targeted PI-3K/BRD4 inhibitor, SF1126 as a therapeutic strategy in this specific subgroup of high risk neuroblastoma.

G-protein coupled receptor kinase 2 (GRK2; ADRBK1, BARK1) is most known as a regulator of G-protein coupled receptors. However, GRK2 also has other functions. Medulloblastomas are the most common malignant brain cancers in children. GRK2 has not been implicated in medulloblastoma biology. Here we report that GRK2 knockdown slowed cell growth, diminished proliferation, and enhanced cisplatin- and etoposide-induced apoptosis in medulloblastoma cell lines UW228-2 and Daoy. Reciprocally, GRK2 overexpression attenuated apoptosis induced by these chemotherapy drugs. Cisplatin and etoposide increased phosphorylation of AKT (S473) and GRK2 knockdown mitigated this increase. Cisplatin and etoposide attenuated ERK phosphorylation, but GRK2 knockdown did not alter this effect. Wildtype GRK2 reversed the increase in cisplatin- and etoposide-induced apoptosis caused by GRK2 knockdown. GRK2-K220R (kinase dead) and GRK2-S670A (unphosphorylated, constitutively active) conferred protection from cisplatin that was similar to wildtype GRK2, suggesting that this protection may be mediated though a kinase-independent activity of GRK2. These data demonstrate that GRK2 contributes to proliferation and survival of these medulloblastoma cell lines and to their protection from cisplatin- and etoposide-induced apoptosis.

We reviewed relevant syphilis diagnostic literature and conducted a meta-analysis to address the question, "What is the sensitivity and specificity of the Syphilis Health Check, a rapid qualitative test for the detection of human antibodies to Treponema pallidum." The Syphilis Health Check is the only rapid syphilis test currently cleared by the Food and Drug Administration (FDA). We conducted a systematic review and a meta-analysis using Bayesian bivariate random-effects and fixed-effect models to create pooled estimates of sensitivity and specificity of the Syphilis Health Check. We identified 5 test evaluations published in the literature and 10 studies submitted to the FDA and for a Clinical Laboratory Improvement Amendments waiver application. The pooled sensitivity (95% CI) from the laboratory evaluations (n = 5) was 98.5% (92.1-100%), while pooled specificity was 95.9% (81.5-100.0%). The pooled sensitivity for prospective studies (n = 10) was 87.7% ( 71.8-97.2%), while pooled specificity was 96.7% (91.9-99.2%). Using nontreponemal supplemental testing, the sensitivity improved to a pooled sensitivity of 97.0% (94.8-98.6%). The Syphilis Health Check may provide accurate detection of treponemal antibody.

We conducted a systematic review of relevant syphilis diagnostic literature to address the question, "What is the sensitivity and specificity of the treponemal tests currently approved by the Food and Drug Administration (FDA) for the diagnosis of syphilis (by stage)?" There were 16 treponemal assays evaluated: 13 immunoassays and 3 manual assays (fluorescent treponemal antibody absorbed test [FTA-ABS], microhemagglutination assay for Treponema pallidum antibodies [MHA-TP], Treponema pallidum particle agglutination assay [TP-PA]). MHA-TP and FTA-ABS were less sensitive in primary and secondary syphilis than TP-PA; TP-PA is the most specific manual treponemal assay. There is insufficient evidence to recommend one particular treponemal immunoassay (eg, enzyme immunoassays, chemiluminescence immunoassays, microbead immunoassays) over another based on published performance data. For diagnosis of neurosyphilis, cerebrospinal fluid (CSF) TP-PA has similar performance to CSF FTA-ABS in studies with patients with definitive or presumptive neurosyphilis. However, CSF treponemal testing has limitations in its sensitivity and specificity and should be interpreted within the context of the clinical scenario, additional CSF test results and syphilis prevalence.

MYC family oncoproteins MYC, MYCN, and MYCL are deregulated in diverse cancers and via diverse mechanisms. Recent studies established a novel form of MYCN regulation in MYCN-overexpressing retinoblastoma and neuroblastoma cells in which the MDM2 oncoprotein promotes MYCN translation and MYCN-dependent proliferation via a p53-independent mechanism. However, it is unclear if MDM2 also promotes expression of other MYC family members and has similar effects in other cancers. Conversely, MYCN has been shown to induce MDM2 expression in neuroblastoma cells, yet it is unclear if MYC shares this ability, if MYC family proteins upregulate MDM2 in other malignancies, and if this regulation occurs during tumorigenesis as well as in cancer cell lines. Here, we report that intrinsically high MDM2 expression is required for high-level expression of MYCN, but not for expression of MYC, in retinoblastoma, neuroblastoma, small cell lung cancer, and medulloblastoma cells. Conversely, ectopic overexpression of MYC as well as MYCN induced high-level MDM2 expression and gave rise to rapidly proliferating and MDM2-dependent cone-precursor-derived masses in a cultured retinoblastoma genesis model. These findings reveal a highly specific collaboration between the MDM2 and MYCN oncoproteins and demonstrate the origin of their oncogenic positive feedback circuit within a normal neuronal tissue.

Glioblastoma (GBM) is a highly invasive brain tumor. Perivascular invasion, autovascularization and vascular co-option occur throughout the disease and lead to tumor invasion and progression. The molecular basis for perivascular invasion, i.e., the interaction of glioma tumor cells with endothelial cells is not well characterized. Recent studies indicate that glioma cells have increased expression of CXCR4. We investigated the in-vivo role of CXCR4 in perivascular invasion of glioma cells using shRNA-mediated knock down of CXCR4. We show that primary cultures of human glioma stem cells HF2303 and mouse glioma GL26-Cit cells exhibit significant migration towards human (HBMVE) and mouse (MBVE) brain microvascular endothelial cells. Blocking CXCR4 on tumor cells with AMD3100 in-vitro, inhibits migration of GL26-Cit and HF2303 toward MBVE and HBMVE cells. Additionally, genetic down regulation of CXCR4 in mouse glioma GL26-Cit cells inhibits their in-vitro migration towards MBVE cells; in an in-vivo intracranial mouse model, these cells display reduced tumor growth and perivascular invasion, leading to increased survival. Quantitative analysis of brain sections showed that CXCR4 knockdown tumors are less invasive. Lastly, we tested the effects of radiation on CXCR4 knock down GL26-Cit cells in an orthotopic brain tumor model. Radiation treatment increased apoptosis of CXCR4 downregulated tumor cells and prolonged median survival. In summary, our data suggest that CXCR4 signaling is critical for perivascular invasion of GBM cells and targeting this receptor makes tumors less invasive and more sensitive to radiation therapy. Combination of CXCR4 knock down and radiation treatment might improve the efficacy of GBM therapy.

While major advances have been made in improving the quality of life and survival of children with most forms of medulloblastoma (MB), those with MYC-driven tumors (Grp3-MB) still suffer significant morbidity and mortality. There is an urgent need to explore multimodal therapeutic regimens which are effective and safe for children. Large-scale studies have revealed abnormal cancer epigenomes caused by mutations and structural alterations of chromatin modifiers, aberrant DNA methylation, and histone modification signatures. Therefore, targeting epigenetic modifiers for cancer treatment has gained increasing interest, and inhibitors for various epigenetic modulators have been intensively studied in clinical trials. Here, we report a cross-entity, epigenetic drug screen to evaluate therapeutic vulnerabilities in MYC amplified MB, which sensitizes them to macrophage-mediated phagocytosis by targeting the CD47-signal regulatory protein α (SIRPα) innate checkpoint pathway.

Interleukin 17 producing CD4 T cells contribute to control of Mycobacterium tuberculosis (Mtb) infection in humans; whether infection with Human Immunodeficiency Virus (HIV) disproportionately affects distinct Th17 cell subsets that respond to Mtb are incompletely defined.

During the summer of 2015, New York, New York, USA, had one of the largest and deadliest outbreaks of Legionnaires' disease in the history of the United States. A total of 138 cases and 16 deaths were linked to a single cooling tower in the South Bronx. Analysis of environmental samples and clinical isolates showed that sporadic cases of legionellosis before, during, and after the outbreak could be traced to a slowly evolving, single-ancestor strain. Detection of an ostensibly virulent Legionella strain endemic to the Bronx community suggests potential risk for future cases of legionellosis in the area. The genetic homogeneity of the Legionella population in this area might complicate investigations and interpretations of future outbreaks of Legionnaires' disease.

Medulloblastoma, the most common malignant brain tumor in children, occurs with increased frequency in individuals with Fanconi anemia who have biallelic germline mutations in BRCA2. We describe an 8-year-old child who had disseminated anaplastic medulloblastoma and a deleterious heterozygous BRCA2 6174delT germline mutation. Molecular profiling was consistent with Group 4 medulloblastoma. The posterior fossa mass was resected and the patient received intensive chemotherapy and craniospinal irradiation. Despite this, the patient succumbed to a second recurrence of his medulloblastoma, which presented 8 months after diagnosis as malignant pleural and peritoneal effusions. Continuous medulloblastoma cell lines were isolated from the original tumor (CHLA-01-MED) and the malignant pleural effusion (CHLA-01R-MED). Here, we provide their analyses, including in vitro and in vivo growth, drug sensitivity, comparative genomic hybridization, and next generation sequencing analysis. In addition to the BRCA2 6174delT, the medulloblastoma cells had amplification of MYC, deletion at Xp11.2, and isochromosome 17, but no structural variations or overexpression of GFI1 or GFI1B. To our knowledge, this is the first pair of diagnosis/recurrence medulloblastoma cell lines, the only medulloblastoma cell lines with BRCA2 6174delT described to date, and the first reported case of a child with medulloblastoma associated with a germline BRCA2 6174delT who did not also have Fanconi anemia.