Container End Closure Sealing Liner and Methods of Preparing the Same

A container having a body and an improved, resealable end closure. The end closure includes a polymer film on a closure inner surface of the end closure. The end closure is selectively attachable to the body such that the end closure covers a sealable opening of the body. The polymer film of the end closure may contact the body of the container, and the contact may form a seal that seals a receiving area defined by the body

Principles to Guide the Office of Legal Counsel

Former members of Office of Legal Counsel ( OLC ) in the Department of Justice offer guidance for their successors. Among the document\u27s recommendations are suggestions that the OLC provide an accurate and honest appraisal of applicable law, even if that advice will constrain the administration’s pursuit of desired policies; and publicly disclose its written legal opinions in a timely manner, absent strong reasons for delay or nondisclosure

Lipidomimetic Compounds Act as HIV-1 Entry Inhibitors by Altering Viral Membrane Structure

The envelope of Human Immunodeficiency Virus type 1 (HIV-1) consists of a liquid-ordered membrane enriched in raft lipids and containing the viral glycoproteins. Previous studies demonstrated that changes in viral membrane lipid composition affecting membrane structure or curvature can impair infectivity. Here, we describe novel antiviral compounds that were identified by screening compound libraries based on raft lipid-like scaffolds. Three distinct molecular structures were chosen for mode-of-action studies, a sterol derivative (J391B), a sphingosine derivative (J582C) and a long aliphatic chain derivative (IBS70). All three target the viral membrane and inhibit virus infectivity at the stage of fusion without perturbing virus stability or affecting virion-associated envelope glycoproteins. Their effect did not depend on the expressed envelope glycoproteins or a specific entry route, being equally strong in HIV pseudotypes carrying VSV-G or MLV-Env glycoproteins. Labeling with laurdan, a reporter of membrane order, revealed different membrane structure alterations upon compound treatment of HIV-1, which correlated with loss of infectivity. J582C and IBS70 decreased membrane order in distinctive ways, whereas J391B increased membrane order. The compounds' effects on membrane order were reproduced in liposomes generated from extracted HIV lipids and thus independent both of virion proteins and of membrane leaflet asymmetry. Remarkably, increase of membrane order by J391B required phosphatidylserine, a lipid enriched in the HIV envelope. Counterintuitively, mixtures of two compounds with opposite effects on membrane order, J582C and J391B, did not neutralize each other but synergistically inhibited HIV infection. Thus, altering membrane order, which can occur by different mechanisms, constitutes a novel antiviral mode of action that may be of general relevance for enveloped viruses and difficult to overcome by resistance development

An alternative CYB5A transcript is expressed in aneuploid ALL and enriched in relapse

Background: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a genetically heterogenous malignancy with poor prognosis in relapsed adult patients. The genetic basis for relapse in aneuploid subtypes such as near haploid (NH) and high hyperdiploid (HeH) BCP-ALL is only poorly understood. Pathogenic genetic alterations remain to be identified. To this end, we investigated the dynamics of genetic alterations in a matched initial diagnosis-relapse (ID-REL) BCP-ALL cohort. Here, we firstly report the identification of the novel genetic alteration CYB5Aalt, an alternative transcript of CYB5A, in two independent cohorts. Methods: We identified CYB5alt in the RNAseq-analysis of a matched ID-REL BCP-ALL cohort with 50 patients and quantified its expression in various molecular BCP-ALL subtypes. Findings were validated in an independent cohort of 140 first diagnosis samples from adult BCP-ALL patients. Derived from patient material, the alternative open reading frame of CYB5Aalt was cloned (pCYB5Aalt) and pCYB5Aalt or the empty vector were stably overexpressed in NALM-6 cells. RNA sequencing was performed of pCYB5Aalt clones and empty vector controls followed by differential expression analysis, gene set enrichment analysis and complementing cell death and viability assays to determine functional implications of CYB5Aalt. Results: RNAseq data analysis revealed non-canonical exon usage of CYB5Aalt starting from a previously undescribed transcription start site. CYB5Aalt expression was increased in relapsed BCP-ALL and its occurrence was specific towards the shared gene expression cluster of NH and HeH BCP-ALL in independent cohorts. Overexpression of pCYB5Aalt in NALM-6 cells induced a distinct transcriptional program compared to empty vector controls with downregulation of pathways related to reported functions of CYB5A wildtype. Interestingly, CYB5A wildtype expression was decreased in CYB5Aalt samples in silico and in vitro. Additionally, pCYB5Aalt NALM-6 elicited a more resistant drug response. Conclusions: Across all age groups, CYB5Aalt was the most frequent secondary genetic event in relapsed NH and HeH BCP-ALL. In addition to its high subgroup specificity, CYB5Aalt is a novel candidate to be potentially implicated in therapy resistance in NH and HeH BCP-ALL. This is underlined by overexpressing CYB5Aalt providing first evidence for a functional role in BCL2-mediated apoptosis

Circulating exosomes inhibit B cell proliferation and activity

(1) Background: Head and neck squamous cell carcinoma (HNSCC) is characterized by a distinctive suppression of the anti-tumor immunity, both locally in the tumor microenvironment (TME) and the periphery. Tumor-derived exosomes mediate this immune suppression by directly suppressing T effector function and by inducing differentiation of regulatory T cells. However, little is known about the effects of exosomes on B cells. (2) Methods: Peripheral B cells from healthy donors and HNSCC patients were isolated and checkpoint receptor expression was analyzed by flow cytometry. Circulating exosomes were isolated from the plasma of HNSCC patients (n = 21) and healthy individuals (n = 10) by mini size-exclusion chromatography. B cells from healthy individuals were co-cultured with isolated exosomes for up to 4 days. Proliferation, viability, surface expression of checkpoint receptors, and intracellular signaling were analyzed in B cells by flow cytometry. (3) Results: Expression of the checkpoint receptors PD-1 and LAG3 was increased on B cells from HNSCC patients. The protein concentration of circulating exosomes was increased in HNSCC patients as compared to healthy donors. Both exosomes from healthy individuals and HNSCC patients inhibited B cell proliferation and survival, in vitro. Surface expression of inhibitory and stimulatory checkpoint receptors on B cells was modulated in co-culture with exosomes. In addition, an inhibitory effect of exosomes on B cell receptor (BCR) signaling was demonstrated in B cells. (4) Conclusions: Plasma-derived exosomes show inhibitory effects on the function of healthy B cells. Interestingly, these inhibitory effects are similar between exosomes from healthy individuals and HNSCC patients, suggesting a physiological B cell inhibitory role of circulating exosomes

PITX1 is a regulator of TERT expression in prostate cancer with prognostic power

Simple Summary Most prostate cancer is of an indolent form and is curable. However, some prostate cancer belongs to rather aggressive subtypes leading to metastasis and death, and immediate therapy is mandatory. However, for these, the therapeutic options are highly invasive, such as radical prostatectomy, radiation or brachytherapy. Hence, a precise diagnosis of these tumor subtypes is needed, and the thus far applied diagnostic means are insufficient for this. Besides this, for their endless cell divisions, prostate cancer cells need the enzyme telomerase to elongate their telomeres (chromatin endings). In this study, we developed a gene regulatory model based on large data from transcription profiles from prostate cancer and chromatin-immuno-precipitation studies. We identified the developmental regulator PITX1 regulating telomerase. Besides observing experimental evidence of PITX1′s functional role in telomerase regulation, we also found PITX1 serving as a prognostic marker, as concluded from an analysis of more than 15,000 prostate cancer samples. Abstract The current risk stratification in prostate cancer (PCa) is frequently insufficient to adequately predict disease development and outcome. One hallmark of cancer is telomere maintenance. For telomere maintenance, PCa cells exclusively employ telomerase, making it essential for this cancer entity. However, TERT, the catalytic protein component of the reverse transcriptase telomerase, itself does not suit as a prognostic marker for prostate cancer as it is rather low expressed. We investigated if, instead of TERT , transcription factors regulating TERT may suit as prognostic markers. To identify transcription factors regulating TERT , we developed and applied a new gene regulatory modeling strategy to a comprehensive transcriptome dataset of 445 primary PCa. Six transcription factors were predicted as TERT regulators, and most prominently, the developmental morphogenic factor PITX1. PITX1 expression positively correlated with telomere staining intensity in PCa tumor samples. Functional assays and chromatin immune-precipitation showed that PITX1 activates TERT expression in PCa cells. Clinically, we observed that PITX1 is an excellent prognostic marker, as concluded from an analysis of more than 15,000 PCa samples. PITX1 expression in tumor samples associated with (i) increased Ki67 expression indicating increased tumor growth, (ii) a worse prognosis, and (iii) correlated with telomere length

Immune Suppressive Effects of Plasma-Derived Exosome Populations in Head and Neck Cancer

Plasma-derived exosomes of head and neck squamous cell carcinoma (HNSCC) patients carry inhibitory factors mediating immune suppression. Separation of tumor-derived exosomes (TEX) and non-TEX may assist in a better understanding of their respective parental cells. Here, we evaluate the impact of TEX or hematopoietic-derived exosomes on immune suppression. We evaluated apoptosis in CD8+ T cells, conversion of CD4+ T cells to regulatory T cells (Treg), and adenosine production by TEX, non-TEX, or total exosomes. Exosome protein cargo was significantly higher in total and CD45(−) exosomes from high stage compared to low stage patients. Furthermore, total and CD45(−) exosomes of high stage patients induced more apoptosis in CD8+ T cells than their low stage counterparts. CD69 suppression, a marker of reduced CD8+ T cell activation, was only mediated by CD45(−) exosomes. All fractions induced Treg differentiation, defined by CD39 expression, but only CD45(−) exosomes showed a stage-dependent conversion. CD45(−) exosomes produced higher adenosine concentrations than CD45(+) exosomes, concluding that adenosine production measured in total exosomes mainly derives from TEX. The presented results show significant induction of immune suppression by TEX in HNSCC. This immunosuppressive effect supports the potential role of exosomes as liquid biomarkers for disease stage and level of immune suppression

Patterns of antibody responses to nonviral cancer antigens in head and neck squamous cell carcinoma patients differ by human papillomavirus status

There have been hints that nonviral cancer antigens are differentially expressed in human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinoma (HNSCC). Antibody responses (AR) to cancer antigens may be used to indirectly determine cancer antigen expression in the tumor using a noninvasive and tissue-saving liquid biopsy. Here, we set out to characterize AR to a panel of nonviral cancer antigens in HPV-positive and HPV-negative HNSCC patients. A fluorescent microbead multiplex serology to 29 cancer antigens (16 cancer-testis antigens, 5 cancer-retina antigens and 8 oncogenes) and 29 HPV-antigens was performed in 382 HNSCC patients from five independent cohorts (153 HPV-positive and 209 HPV-negative). AR to any of the cancer antigens were found in 272/382 patients (72%). The ten most frequent AR were CT47, cTAGE5a, c-myc, LAGE-1, MAGE-A1, -A3, -A4, NY-ESO-1, SpanX-a1 and p53. AR to MAGE-A3, MAGE-A9 and p53 were found at significantly different prevalences by HPV status. An analysis of AR mean fluorescent intensity values uncovered remarkably different AR clusters by HPV status. To identify optimal antigen selections covering a maximum of patients with ≤10 AR, multiobjective optimization revealed distinct antigen selections by HPV status. We identified that AR to nonviral antigens differ by HPV status indicating differential antigen expression. Multiplex serology may be used to characterize antigen expression using serum or plasma as a tissue-sparing liquid biopsy. Cancer antigen panels should address the distinct antigen repertoire of HPV-positive and HPV-negative HNSCC

Adjunctive Dexamethasone Affects the Expression of Genes Related to Inflammation, Neurogenesis and Apoptosis in Infant Rat Pneumococcal Meningitis

Streptococcus pneumoniae is the most common pathogen causing non-epidemic bacterial meningitis worldwide. The immune response and inflammatory processes contribute to the pathophysiology. Hence, the anti-inflammatory dexamethasone is advocated as adjuvant treatment although its clinical efficacy remains a question at issue. In experimental models of pneumococcal meningitis, dexamethasone increased neuronal damage in the dentate gyrus. Here, we investigated expressional changes in the hippocampus and cortex at 72 h after infection when dexamethasone was given to infant rats with pneumococcal meningitis. Nursing Wistar rats were intracisternally infected with Streptococcus pneumoniae to induce experimental meningitis or were sham-infected with pyrogen-free saline. Besides antibiotics, animals were either treated with dexamethasone or saline. Expressional changes were assessed by the use of GeneChip® Rat Exon 1.0 ST Arrays and quantitative real-time PCR. Protein levels of brain-derived neurotrophic factor, cytokines and chemokines were evaluated in immunoassays using Luminex xMAP® technology. In infected animals, 213 and 264 genes were significantly regulated by dexamethasone in the hippocampus and cortex respectively. Separately for the cortex and the hippocampus, Gene Ontology analysis identified clusters of biological processes which were assigned to the predefined categories “inflammation”, “growth”, “apoptosis” and others. Dexamethasone affected the expression of genes and protein levels of chemokines reflecting diminished activation of microglia. Dexamethasone-induced changes of genes related to apoptosis suggest the downregulation of the Akt-survival pathway and the induction of caspase-independent apoptosis. Signalling of pro-neurogenic pathways such as transforming growth factor pathway was reduced by dexamethasone resulting in a lack of pro-survival triggers. The anti-inflammatory properties of dexamethasone were observed on gene and protein level in experimental pneumococcal meningitis. Further dexamethasone-induced expressional changes reflect an increase of pro-apoptotic signals and a decrease of pro-neurogenic processes. The findings may help to identify potential mechanisms leading to apoptosis by dexamethasone in experimental pneumococcal meningitis