Coral cell separation and isolation by fluorescence-activated cell sorting (FACS).

BACKGROUND: Generalized methods for understanding the cell biology of non-model species are quite rare, yet very much needed. In order to address this issue, we have modified a technique traditionally used in the biomedical field for ecological and evolutionary research. Fluorescent activated cell sorting (FACS) is often used for sorting and identifying cell populations. In this study, we developed a method to identify and isolate different cell populations in corals and other cnidarians. METHODS: Using fluorescence-activated cell sorting (FACS), coral cell suspension were sorted into different cellular populations using fluorescent cell markers that are non-species specific. Over 30 different cell markers were tested. Additionally, cell suspension from Aiptasia pallida was also tested, and a phagocytosis test was done as a downstream functional assay. RESULTS: We found that 24 of the screened markers positively labeled coral cells and 16 differentiated cell sub-populations. We identified 12 different cellular sub-populations using three markers, and found that each sub-population is primarily homogeneous. Lastly, we verified this technique in a sea anemone, Aiptasia pallida, and found that with minor modifications, a similar gating strategy can be successfully applied. Additionally, within A. pallida, we show elevated phagocytosis of sorted cells based on an immune associated marker. CONCLUSIONS: In this study, we successfully adapted FACS for isolating coral cell populations and conclude that this technique is translatable for future use in other species. This technique has the potential to be used for different types of studies on the cellular stress response and other immunological studies.

The BAG-1 isoform BAG-1M regulates keratin-associated Hsp70 chaperoning of aPKC in intestinal cells during activation of inflammatory signaling.

Atypical PKC (ι/λ and ζ; hereafter referred to as aPKC) is a key player in the acquisition of epithelial polarity and participates in other signaling cascades including the control of NF-κB signaling. This kinase is post-translationally regulated through Hsp70-mediated refolding. Previous work has shown that such a chaperoning activity is specifically localized to keratin intermediate filaments. Our work was performed with the goal of identifying the molecule(s) that block Hsp70 activity on keratin filaments during inflammation. A transcriptional screen allowed us to focus on BAG-1, a multi-functional protein that assists Hsp70 in nucleotide exchange but also blocks its activity at higher concentrations. We found the BAG-1 isoform BAG-1M upregulated threefold in human Caco-2 cells following stimulation with tumor necrosis factor receptor α (TNFα) to induce a pro-inflammatory response, and up to sixfold in mouse enterocytes following treatment with dextran sodium sulfate (DSS) to induce colitis. BAG-1M, but no other isoform, was found to co-purify with intermediate filaments and block Hsp70 activity in the keratin fraction but not in the soluble fraction within the range of concentrations found in epithelial cells cultured under control and inflammation conditions. Constitutive expression of BAG-1M decreased levels of phosphorylated aPKC. By contrast, knockdown of BAG-1, blocked the TNFα-induced decrease of phosphorylated aPKC. We conclude that BAG-1M mediates Hsp70 inhibition downstream of NF-κB.

Inflammasome Activation Induces Pyroptosis in the Retina Exposed to Ocular Hypertension Injury.

Mechanical stress and hypoxia during episodes of ocular hypertension (OHT) trigger glial activation and neuroinflammation in the retina. Glial activation and release of pro-inflammatory cytokines TNFα and IL-1ß, complement, and other danger factors was shown to facilitate injury and loss of retinal ganglion cells (RGCs) that send visual information to the brain. However, cellular events linking neuroinflammation and neurotoxicity remain poorly characterized. Several pro-inflammatory and danger signaling pathways, including P2X7 receptors and Pannexin1 (Panx1) channels, are known to activate inflammasome caspases that proteolytically activate gasdermin D channel-formation to export IL-1 cytokines and/or induce pyroptosis. In this work, we used molecular and genetic approaches to map and characterize inflammasome complexes and detect pyroptosis in the OHT-injured retina. Acute activation of distinct inflammasome complexes containing NLRP1, NLRP3 and Aim2 sensor proteins was detected in RGCs, retinal astrocytes and Muller glia of the OHT-challenged retina. Inflammasome-mediated activation of caspases-1 and release of mature IL-1ß were detected within 6 h and peaked at 12-24 h after OHT injury. These coincided with the induction of pyroptotic pore protein gasdermin D in neurons and glia in the ganglion cell layer (GCL) and inner nuclear layer (INL). The OHT-induced release of cytokines and RGC death were significantly decreased in the retinas of Casp1-/-Casp4(11)del, Panx1-/- and in Wild-type (WT) mice treated with the Panx1 inhibitor probenecid. Our results showed a complex spatio-temporal pattern of innate immune responses in the retina. Furthermore, they indicate an active contribution of neuronal NLRP1/NLRP3 inflammasomes and the pro-pyroptotic gasdermin D pathway to pathophysiology of the OHT injury. These results support the feasibility of inflammasome modulation for neuroprotection in OHT-injured retinas.

Characterization of twenty-five ovarian tumour cell lines that phenocopy primary tumours.

Currently available human tumour cell line panels consist of a small number of lines in each lineage that generally fail to retain the phenotype of the original patient tumour. Here we develop a cell culture medium that enables us to routinely establish cell lines from diverse subtypes of human ovarian cancers with >95% efficiency. Importantly, the 25 new ovarian tumour cell lines described here retain the genomic landscape, histopathology and molecular features of the original tumours. Furthermore, the molecular profile and drug response of these cell lines correlate with distinct groups of primary tumours with different outcomes. Thus, tumour cell lines derived using this methodology represent a significantly improved platform to study human tumour pathophysiology and response to therapy.

Par-complex aPKC and Par3 cross-talk with innate immunity NF-κB pathway in epithelial cells.

Components of the Par-complex, atypical PKC and Par3, have been found to be downregulated upon activation of NF-κB in intestinal epithelial cells. To determine their possible role in pro-inflammatory responses we transduced Caco-2 human colon carcinoma cells with constitutively active (ca) PKCι or anti-Par3 shRNA-expressing lentiviral particles. Contrary to previous reports in other cell types, ca-PKCι did not activate, but rather decreased, baseline NF-κB activity in a luminiscence reporter assay. An identical observation applied to a PB1 domain deletion PKCι, which fails to localize to the tight-junction. Conversely, as expected, the same ca-PKCι activated NF-κB in non-polarized HEK293 cells. Likewise, knockdown of Par3 increased NF-κB activity and, surprisingly, greatly enhanced its response to TNFα, as shown by transcription of IL-8, GRO-1, GRO-2 and GRO-3. We conclude that aPKC and Par3 are inhibitors of the canonical NF-κB activation pathway, although perhaps acting through independent pathways, and may be involved in pro-inflammatory responses.

Evidence of novel fine-scale structural variation at autism spectrum disorder candidate loci.

BACKGROUND: Autism spectrum disorders (ASD) represent a group of neurodevelopmental disorders characterized by a core set of social-communicative and behavioral impairments. Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain, acting primarily via the GABA receptors (GABR). Multiple lines of evidence, including altered GABA and GABA receptor expression in autistic patients, indicate that the GABAergic system may be involved in the etiology of autism. METHODS: As copy number variations (CNVs), particularly rare and de novo CNVs, have now been implicated in ASD risk, we examined the GABA receptors and genes in related pathways for structural variation that may be associated with autism. We further extended our candidate gene set to include 19 genes and regions that had either been directly implicated in the autism literature or were directly related (via function or ancestry) to these primary candidates. For the high resolution CNV screen we employed custom-designed 244 k comparative genomic hybridization (CGH) arrays. Collectively, our probes spanned a total of 11 Mb of GABA-related and additional candidate regions with a density of approximately one probe every 200 nucleotides, allowing a theoretical resolution for detection of CNVs of approximately 1 kb or greater on average. One hundred and sixty-eight autism cases and 149 control individuals were screened for structural variants. Prioritized CNV events were confirmed using quantitative PCR, and confirmed loci were evaluated on an additional set of 170 cases and 170 control individuals that were not included in the original discovery set. Loci that remained interesting were subsequently screened via quantitative PCR on an additional set of 755 cases and 1,809 unaffected family members. RESULTS: Results include rare deletions in autistic individuals at JAKMIP1, NRXN1, Neuroligin4Y, OXTR, and ABAT. Common insertion/deletion polymorphisms were detected at several loci, including GABBR2 and NRXN3. Overall, statistically significant enrichment in affected vs. unaffected individuals was observed for NRXN1 deletions. CONCLUSIONS: These results provide additional support for the role of rare structural variation in ASD.

Microarray-based detection of CYP1A1, CYP2C9, CYP2C19, CYP2D6, GSTT1, GSTM1, MTHFR, MTRR, NQO1, NAT2, HLA-DQA1, and AB0 allele frequencies in native Russians.

Xenobiotic-metabolizing genes (e.g., Cytochromes P450, GST, NAT2, and NQO1), folate metabolism genes (e.g., MTHFR and MTRR), and major histocompatibility complex genes (e.g., HLA-DQA1) play multiple roles in the organism functioning. In addition, AB0 is the most clinically significant high-polymorphic gene in transfusion and transplantation medicine. Epidemiological data show that allele frequencies of these genes exhibit ethnic and geographic diversity. Besides, little is known about frequency distribution of the major polymorphic variants in native Russians. We developed biological microchips that allow us to analyze a spectrum of allelic variants in 12 different genes: CYP1A1, CYP2D6, CYP2C9, CYP2C19, GSTT1, GSTM1, MTHFR, MTRR, NQO1, NAT2, HLA-DQA1, and AB0. Using this composite methodological platform we have studied 352 DNA samples from healthy native Russian volunteers. The allelic frequencies of gene polymorphisms obtained are close to allelic frequencies observed in some European populations, as published earlier. These data were used in comparative studies to determine predisposition to tuberculosis, lymphoma, and leukemia in adults and to childhood acute leukemia. The HLA-DQA1 and AB0 allele frequencies were used to estimate forensic population parameters for these loci.