Data obtained from high-density oligonucleotide tiling arrays present new computational challenges for users. This chapter presents ACME (Algorithm for Capturing Microarray Enrichment), a computer ...program developed for the analysis of data obtained using NimbleGen-tiled microarrays. ACME identifies signals or "peaks" in tiled array data using a simple sliding window and threshold strategy and assigns a probability value (p value) to each and every probe on the array. We present data indicating that this approach can be applied successfully to at least two different genomic applications involving tiled arrays: ChIP-chip and DNase-chip. In addition to highlighting previously described methods for analyzing tiled array data, we provide recommendations for assessing the quality of ChIP-chip and DNase-chip data, suggestions for optimizing the use of ACME, and descriptions of several of ACME features designed to facilitate interpretation of processed tiled array data. ACME is written in R language and is freely available upon request or through Bioconductor.
•Biomechanical stability plays a critical role in fracture healing.•Although stability is often described to be essential for both prevention and treatment of FRI, the underlying mechanisms and the ...optimal degree of stability remain unknown.•Clinical studies on the topic are challenging and therefore the scientific evidence on this topic is limited to experimental studies in laboratory animals.•Future research is required, as improvements in the surgical management of FRI may be achieved by a better understanding of the role of stability.
Bone healing is a complicated process of tissue regeneration that is influenced by multiple biological and biomechanical processes. In a minority of cases, these physiological processes are complicated by issues such as nonunion and/or fracture-related infection (FRI). Based on a select few in vivo experimental animal studies, construct stability is considered an important factor influencing both prevention and treatment of FRI. Stephan Perren played a pivotal role in the evolution of our current understanding of the critical relationship between biomechanics, fracture healing and infection. Furthermore, his concept of strain theory and the process of fracture healing is familiar to several generations of surgeons and has influenced implant development and design for the past 50 years. In this review we describe the role of biomechanical stability on fracture healing, and provide a detailed analysis of the preclinical studies addressing this in the context of FRI. Furthermore, we demonstrate how Perren's concepts of stability are still applied to current surgical techniques to aid in the prevention and treatment of FRI. Finally, we highlight the key knowledge gaps in the underlying basic research literature that need to be addressed as we continue to optimize patient care.
2,3,7,8-Tetrachlordibenzo-p-dioxin (TCDD) is an environmental pollutant that can cause various toxic effects, including chloracne, metabolic syndrome, and immune suppression. Most of the toxicity ...associated with TCDD is mediated through activation of the aryl hydrocarbon receptor (AHR). Recent research has suggested the presence of a wide-range of interindividual variability in TCDD-mediated suppression of the Immunoglobulin-M (IgM) response across the human population. In an attempt to identify putative modifiers of AHR-mediated immunosuppression beyond the AHR, B cells were isolated from a panel of genetically diverse mouse strain to scan for modulators that drive interstrain differences in TCDD-mediated suppression of the IgM response. Results implicated a region of mouse Chromosome 1 near a gene encoding serine peptidase inhibitor, clade B, member 2 (Serpinb2) whose human ortholog is plasminogen activator inhibitor 2 (PAI2). Further downstream analyses indicated that Serpinb2 is dysregulated by TCDD and, furthermore, that B cells from Serpinb2 –/– mice are significantly more sensitive to TCDD-mediated suppression as compared to littermate controls. This study suggests a protective role of Serpinb2 within TCDD-mediated immunosuppression and, furthermore, a novel function of Serpinb2-related activity in the IgM response.
Patients with the dedicator of cytokinesis 8 (DOCK8) immunodeficiency syndrome suffer from recurrent viral and bacterial infections, hyper–immunoglobulin E levels, eczema, and greater susceptibility ...to cancer. Because natural killer T (NKT) cells have been implicated in these diseases, we asked if these cells were affected by DOCK8 deficiency. Using a mouse model, we found that DOCK8 deficiency resulted in impaired NKT cell development, principally affecting the formation and survival of long-lived, differentiated NKT cells. In the thymus, DOCK8-deficient mice lack a terminally differentiated subset of NK1.1+ NKT cells expressing the integrin CD103, whereas in the liver, DOCK8-deficient NKT cells express reduced levels of the prosurvival factor B-cell lymphoma 2 and the integrin lymphocyte function-associated antigen 1. Although the initial NKT cell response to antigen is intact in the absence of DOCK8, their ongoing proliferative and cytokine responses are impaired. Importantly, a similar defect in NKT cell numbers was detected in DOCK8-deficient humans, highlighting the relevance of the mouse model. In conclusion, our data demonstrate that DOCK8 is required for the development and survival of mature NKT cells, consistent with the idea that DOCK8 mediates survival signals within a specialized niche. Accordingly, impaired NKT cell numbers and function are likely to contribute to the susceptibility of DOCK8-deficient patients to recurrent infections and malignant disease.
•The development and survival of mature NKT cells are impaired in DOCK8-deficient mice.•DOCK8 is required for antigen-induced NKT cell proliferation and cytokine production.
•Naturally occurring CD7 negative T cells are functional effector T cells that can be used for chimeric antigen receptor therapy.•CD7 negative T cells expressing a CD7-CAR have robust antitumor ...activity and bypass fratricide.
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Chimeric antigen receptor (CAR) T-cell therapy targeting T-cell acute lymphoblastic leukemia (T-ALL) faces limitations such as antigen selection and limited T-cell persistence. CD7 is an attractive antigen for targeting T-ALL, but overlapping expression on healthy T cells leads to fratricide of CD7-CAR T cells, requiring additional genetic modification. We took advantage of naturally occurring CD7− T cells to generate CD7-CAR (CD7-CARCD7−) T cells. CD7-CARCD7− T cells exhibited a predominantly CD4+ memory phenotype and had significant antitumor activity upon chronic antigen exposure in vitro and in xenograft mouse models. Based on these encouraging results, we next explored the utility of CD7− T cells for the immunotherapy of CD19+ hematological malignancies. Direct comparison of nonselected (bulk) CD19-CAR and CD19-CARCD7− T cells revealed that CD19-CARCD7− T cells had enhanced antitumor activity compared with their bulk counterparts in vitro and in vivo. Lastly, to gain insight into the behavior of CD19-CAR T cells with low levels of CD7 gene expression (CD7lo) in humans, we mined single-cell gene and T-cell receptor (TCR) expression data sets from our institutional CD19-CAR T-cell clinical study. CD19-CARCD7lo T cells were present in the initial CD19-CAR T-cell product and could be detected postinfusion. Intriguingly, the only functional CD4+ CD19-CAR T-cell cluster observed postinfusion exhibited CD7lo expression. Additionally, samples from patients responsive to therapy had a higher proportion of CD7lo T cells than nonresponders (NCT03573700). Thus, CARCD7− T cells have favorable biological characteristics and may present a promising T-cell subset for adoptive cell therapy of T-ALL and other hematological malignancies.
CD7 is expressed on many T-cell malignancies and is an attractive target for CAR T-cell therapy for T-cell leukemia/lymphoma, but concurrent CD7 expression on CAR T cells predisposes to cell loss through fratricide. Freiwan and colleagues report on selection to enrich CD7-negative T cells as targets for CAR T-cell engineering and demonstrate that they have enhanced antitumor activity, suggesting a potentially attractive strategy for optimizing CAR T-cell therapy for T-cell malignancies.
Immune evasion is indispensable for cancer initiation and progression, although its underlying mechanisms in pancreatic ductal adenocarcinoma (PDAC) are not fully known. Here, we characterize the ...function of tumor-derived PGRN in promoting immune evasion in primary PDAC. Tumor- but not macrophage-derived PGRN is associated with poor overall survival in PDAC. Multiplex immunohistochemistry shows low MHC class I (MHCI) expression and lack of CD8
T cell infiltration in PGRN-high tumors. Inhibition of PGRN abrogates autophagy-dependent MHCI degradation and restores MHCI expression on PDAC cells. Antibody-based blockade of PGRN in a PDAC mouse model remarkably decelerates tumor initiation and progression. Notably, tumors expressing LCMV-gp33 as a model antigen are sensitized to gp33-TCR transgenic T cell-mediated cytotoxicity upon PGRN blockade. Overall, our study shows a crucial function of tumor-derived PGRN in regulating immunogenicity of primary PDAC.
Despite its relatively low incidence, pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer deaths because of the aggressive growth/metastasis of the tumor, the lack of early symptoms, ...and the poor treatment options. Basic research to identify potential therapeutic targets for PDAC is greatly needed.
We used a negative-selection genome-wide CRISPR screen to identify essential genes in the PANC-1 human pancreatic carcinoma cell line. We validated the top hits with follow-up siRNA screens, using the HPNE, HPAF-II, AsPC-1, and Mia PaCa-2 cell lines.
The PSMA6 gene was an identified candidate hit after the CRISPR screen, siRNA validation screen, and siRNA deconvolution screen. Spheroid formation assays and flow cytometry analysis showed that PSMA6 is critical for survival in many pancreatic ductal carcinoma cell models. Lastly, as PSMA6 protein is a proteosomal subunit of the 20S core complex, we showed that bortezomib, a proteasome inhibitor, was especially toxic in PANC-1 cells.
Further study of PSMA6 and the proteasome subunit that it encodes, along with other hits identified in our CRISPR screens, may provide valuable insights into potential therapeutic targets for PDAC.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The Cape Gannet Morus capensis is one of several seabird species endemic to the Benguela upwelling ecosystem (BUS) but whose population has recently decreased, leading to an unfavourable IUCN Red ...List assessment. Application of 'JARA' ('Just Another Red-List Assessment,' a Bayesian state-space tool used for IUCN Red List assessments) to updated information on the areas occupied by Cape Gannets and the nest densities of breeding birds at their six colonies, suggested that the species should be classified as Vulnerable. However, the rate of decrease of Cape Gannets in their most-recent generation exceeded that of the previous generation, primarily as a result of large decreases at Bird Island, Lambert's Bay, and Malgas Island, off South Africa's west coast (the western part of their range). Since the 1960s, there has been an ongoing redistribution of the species from northwest to southeast around southern Africa, and ∼70% of the population now occurs on the south coast of South Africa, at Bird Island in Algoa Bay, on the eastern border of the BUS. Recruitment rather than adult survival may be limiting the present population; however, information on the seabird's demographic parameters and mortality in fisheries is lacking for colonies in the northern part of the BUS. Presently, major threats to Cape Gannet include: substantially decreased availability of their preferred prey in the west; heavy mortalities of eggs, chicks and fledglings at and around colonies, inflicted by Cape Fur Seals Arctocephalus pusillus and other seabirds; substantial disturbance at colonies caused by Cape Fur Seals attacking adult gannets ashore; oiling; and disease.
Bromodomains are acetyllysine recognition domains present in a variety of human proteins. Bromodomains also bind small molecules that compete with acetyllysine, and therefore bromodomains have been ...targets for drug discovery efforts. Highly potent and selective ligands with good cellular permeability have been proposed as chemical probes for use in exploring the functions of many of the bromodomain proteins. We report here the discovery of a class of such inhibitors targeting the family VIII bromodomains of SMARCA2 (BRM) and SMARCA4 (BRG1), and PBRM1 (polybromo-1) bromodomain 5. We propose one example from this series, GNE-064, as a chemical probe for the bromodomains SMARCA2, SMARCA4, and PBRM1(5) with the potential for in vivo use.
Alzheimer’s disease (AD) is a leading cause of mortality among the elderly. We performed a whole-genome sequencing study of AD in the Chinese population. In addition to the variants identified in or ...around the APOE locus (sentinel variant rs73052335, P = 1.44 × 10−14), two common variants, GCH1 (rs72713460, P = 4.36 × 10−5) and KCNJ15 (rs928771, P = 3.60 × 10−6), were identified and further verified for their possible risk effects for AD in three small non-Asian AD cohorts. Genotype–phenotype analysis showed that KCNJ15 variant rs928771 affects the onset age of AD, with earlier disease onset in minor allele carriers. In addition, altered expression level of the KCNJ15 transcript can be observed in the blood of AD subjects. Moreover, the risk variants of GCH1 and KCNJ15 are associated with changes in their transcript levels in specific tissues, as well as changes of plasma biomarkers levels in AD subjects. Importantly, network analysis of hippocampus and blood transcriptome datasets suggests that the risk variants in the APOE, GCH1, and KCNJ15 loci might exert their functions through their regulatory effects on immune-related pathways. Taking these data together, we identified common variants of GCH1 and KCNJ15 in the Chinese population that contribute to AD risk. These variants may exert their functional effects through the immune system.
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