Cellular Intrinsic Mechanism Affecting the Outcome of AML Treated with Ara-C in a Syngeneic Mouse Model

The mechanisms underlying acute myeloid leukemia (AML) treatment failure are not clear. Here, we established a mouse model of AML by syngeneic transplantation of BXH-2 derived myeloid leukemic cells and developed an efficacious Ara-C-based regimen for treatment of these mice. We proved that leukemic cell load was correlated with survival. We also demonstrated that the susceptibility of leukemia cells to Ara-C could significantly affect the survival. To examine the molecular alterations in cells with different sensitivity, genome-wide expression of the leukemic cells was profiled, revealing that overall 366 and 212 genes became upregulated or downregulated, respectively, in the resistant cells. Many of these genes are involved in the regulation of cell cycle, cellular proliferation, and apoptosis. Some of them were further validated by quantitative PCR. Interestingly, the Ara-C resistant cells retained the sensitivity to ABT-737, an inhibitor of anti-apoptosis proteins, and treatment with ABT-737 prolonged the life span of mice engrafted with resistant cells. These results suggest that leukemic load and intrinsic cellular resistance can affect the outcome of AML treated with Ara-C. Incorporation of apoptosis inhibitors, such as ABT-737, into traditional cytotoxic regimens merits consideration for the treatment of AML in a subset of patients with resistance to Ara-C. This work provided direct in vivo evidence that leukemic load and intrinsic cellular resistance can affect the outcome of AML treated with Ara-C, suggesting that incorporation of apoptosis inhibitors into traditional cytotoxic regimens merits consideration for the treatment of AML in a subset of patients with resistance to Ara-C.     

Prognostic value and immune cell infiltration of hypoxic phenotype‐related gene signatures in glioblastoma microenvironment

Glioblastoma (GBM) is a malignant intracranial tumour with the highest proportion and lethality. It is characterized by invasiveness and heterogeneity. However, the currently available therapies are not curative. As an essential environmental cue that maintains glioma stem cells, hypoxia is considered the cause of tumour resistance to chemotherapy and radiation. Growing evidence shows that immunotherapy focusing on the tumour microenvironment is an effective treatment for GBM; however, the current clinicopathological features cannot predict the response to immunotherapy and provide accurate guidance for immunotherapy. Based on the ESTIMATE algorithm, GBM cases of The Cancer Genome Atlas (TCGA) data set were classified into high‐ and low‐immune/stromal score groups, and a four‐gene tumour environment‐related model was constructed. This model exhibited good efficiency at forecasting short‐ and long‐term prognosis and could also act as an independent prognostic biomarker. Additionally, this model and four of its genes (CLECL5A, SERPING1, CHI3L1 and C1R) were found to be associated with immune cell infiltration, and further study demonstrated that these four genes might drive the hypoxic phenotype of perinecrotic GBM, which affects hypoxia‐induced glioma stemness. Therefore, these might be important candidates for immunotherapy of GBM and deserve further exploration.     

Individual Microcystis colonies harbour distinct bacterial communities that differ by Microcystis oligotype and with time

Interactions between bacteria and phytoplankton in the phycosphere have impacts at the scale of whole ecosystems, including the development of harmful algal blooms. The cyanobacterium Microcystis causes toxic blooms that threaten freshwater ecosystems and human health globally. Microcystis grows in colonies that harbour dense assemblages of other bacteria, yet the taxonomic composition of these phycosphere communities and the nature of their interactions with Microcystis are not well characterized. To identify the taxa and compositional variance within Microcystis phycosphere communities, we performed 16S rRNA V4 region amplicon sequencing on individual Microcystis colonies collected biweekly via high-throughput droplet encapsulation during a western Lake Erie cyanobacterial bloom. The Microcystis phycosphere communities were distinct from microbial communities in whole water and bulk phytoplankton seston in western Lake Erie but lacked ‘core’ taxa found across all colonies. However, dissimilarity in phycosphere community composition correlated with sampling date and the Microcystis 16S rRNA oligotype. Several taxa in the phycosphere were specific to and conserved with Microcystis of a single oligotype or sampling date. Together, this suggests that physiological differences between Microcystis strains, temporal changes in strain phenotypes, and the composition of seeding communities may impact community composition of the Microcystis phycosphere.     

Ribavirin targets eIF4E dependent Akt survival signaling

The eukaryotic translation initiation factor eIF4E is dysregulated in many cancers. eIF4E, through its mRNA export and translation functions, combinatorially modulates the expression of genes involved in Akt dependent survival signaling. For these activities, eIF4E must bind the 7-methyl guanosine (m⁷G) cap moiety on the 5′-end of mRNAs. We demonstrate that a physical mimic of the m⁷G cap, ribavirin, inhibits eIF4E dependent Akt survival signaling. Specifically, ribavirin impairs eIF4E mediated Akt activation via inhibiting the production of an upstream activator of Akt, NBS1. Consequently, ribavirin impairs eIF4E dependent apoptotic rescue. A ribavirin analog with distinct physico-chemical properties, tiazofurin, does not impair eIF4E activity indicating that only analogs that mimic the m⁷G cap will inhibit eIF4E function. Ribavirin represents a first-in-class strategy to inhibit eIF4E dependent cancers, through competition for m⁷G cap binding. Thus, ribavirin coordinately impairs eIF4E dependent pathways and thereby, potently inhibits its biological effects.     

Productive infection of a cervical epithelial cell line with human immunodeficiency virus: implications for sexual transmission.

The human cervix-derived epithelial cell line (ME180) used in this study displays a characteristics epithelial morphology, including numerous desmosomes, tonofilaments, and epidermal filaments. When T-cell lines infected with human immunodeficiency virus (HIV) are added to epithelial cultures, they rapidly adhere to the epithelial monolayer. Within a few minutes, the T cells shed numerous virions into narrow spaces formed between the epithelial cell and the adherent T cells. Virions subsequently enter the ME180 cells via large vesicles. A few days after infection, cytopathic effects and syncytium formation were observed. Infected clones of ME180 cells have remained infected for 8 months. p24 enzyme-linked immunosorbent assay and infectivity assays show that one subclone of the cell line produces virus titers equivalent to those of high-secreting HIV-infected T-cell lines. Electron microscopy reveals numerous virions budding from both the basal and apical surfaces of the epithelium. These observations suggest that cervical epithelium has the potential to serve as a site of HIV infection.     

The magnitude of dengue virus NS1 protein secretion is strain dependent and does not correlate with severe pathologies in the mouse infection model

There are conflicting data on the relationship between the level of secreted NS1 (sNS1), viremia, and disease severity upon dengue virus (DENV) infection in the clinical setting, and therefore, we examined this relationship in the widely accepted AG129 mouse model. Because of the failure of a routinely used NS1 detection kit to detect sNS1 of the mouse-adapted DENV2 strain, we screened 15 previously undescribed NS1 monoclonal antibodies and developed a robust capture enzyme-linked immunosorbent assay (ELISA) with detection sensitivity at the low nanogram level (0.2 ng/ml) using recombinant baculovirus-expressed sNS1 as well as sNS1 that was immunoaffinity purified from the various DENV2 strains employed in this study. Using this test, we demonstrated that increased viremia paralleled severe pathologies; however, sNS1 level did not correlate with viremia or severity. Furthermore, among the DENV2 strains that were tested, the level of NS1 secretion did not correspond to virus replication rate in vitro, at the cellular level. Together, our data indicate that the magnitude of NS1 secretion appears to be strain dependent and does not correlate with viral virulence in the AG129 mouse model.     

Improved protein A resin for antibody capture in a continuous countercurrent tangential chromatography system

Continuous countercurrent tangential chromatography (CCTC) enables steady-state continuous bioprocessing with low-pressure operation and high productivity. CCTC has been applied to initial capture of monoclonal antibodies (mAb) from clarified cell culture harvest and postcapture polishing of mAb; however, these studies were performed with commercial chromatography resins designed for conventional column chromatography. In this study, a small particle size prototype agarose resin (20–25 µm) with lower cross-linking was co-developed with industrial partner Purolite and tested with CCTC. Due to increased binding capacity and faster kinetics, the resulting CCTC process showed more than a 2X increase in productivity, and a 2X reduction in buffer consumption over commercial protein A resins used in previous CCTC studies, as well as more than a 10X productivity increase versus conventional column operation. Single-pass tangential flow filtration was integrated with the CCTC system, enabling simple control of eluate concentration. A scale-up exercise was conducted to provide a quantitative comparison of CCTC and batch column chromatography. These results clearly demonstrate opportunities for using otherwise unpackable soft small particle size resins with CCTC as the core of a continuous bioprocessing platform.     

USP12 promotes CD4+ T cell responses through deubiquitinating and stabilizing BCL10

Deubiquitinases (DUBs) regulate diverse biological processes and represent a novel class of drug targets. However, the biological function of only a small fraction of DUBs, especially in adaptive immune response regulation, is well-defined. In this study, we identified DUB ubiquitin-specific peptidase 12 (USP12) as a critical regulator of CD4⁺ T cell activation. USP12 plays an intrinsic role in promoting the CD4⁺ T cell phenotype, including differentiation, activation, and proliferation. Although USP12-deficient CD4⁺ T cells protected mice from autoimmune diseases, the immune response against bacterial infection was subdued. USP12 stabilized B cell lymphoma/leukemia 10 (BCL10) by deubiquitinating, and thereby activated the NF-κB signaling pathway. Interestingly, this USP12 regulatory mechanism was identified in CD4⁺ T cells, but not in CD8⁺ T cells. Our study results showed that USP12 activated CD4⁺ T cell signaling, and targeting USP12 might help develop therapeutic interventions for treating inflammatory diseases or pathogen infections.Subject terms: Ubiquitins, T cells