Non-induced Cells Research Articles

Preliminary Study of the Characterization of the Viable but Noncultivable State of Yersinia enterocolitica Induced by Chloride and UV Irradiation.

The viable but non-culturable (VBNC) state is a survival strategy for many foodborne pathogens under adverse conditions. Yersinia enterocolitica (Y. enterocolitica) as a kind of primary foodborne pathogen, and it is crucial to investigate its survival strategies and potential risks in the food chain. In this study, the effectiveness of ultraviolet (UV) irradiation and chlorine treatment in disinfecting the foodborne pathogen Y. enterocolitica was investigated. The results indicated that both UV irradiation and chlorine treatment can induce the VBNC state in Y. enterocolitica. The bacteria completely lost culturability after being treated with 25 mg/L of NaClO for 30 min and a UV dose of 100 mJ/cm². The number of culturable and viable cells were detected using plate counting and a combination of fluorescein and propidium iodide (live/dead cells). Further research found that these VBNC cells exhibited reduced intracellular Adenosine Triphosphate (ATP) levels, and increased levels of reactive oxygen species (ROS) compared to non-induced cells. Morphologically, the cells changed from a rod shape to a shorter, coccobacillary shape with small vacuoles forming at the edges, indicating structural changes. Both condition-induced VBNC-state cells were able to resuscitate in tryptic soy broth (TSB) medium supplemented with Tween 80, sodium pyruvate, and glucose. These findings contribute to a better understanding of the survival mechanisms of Y. enterocolitica in the environment and are of significant importance for the development of effective disinfection strategies.

Cytotoxicity Effect of Aqueous Propolis Extract of Geniotrigona thoracica Sumatrans on Colo-201 Colon Cancer Cell Line and Senescence Colo-201 Colon Cancer Cell Line Induced by Low-dose Doxorubicin

Propolis, a resinous compound honeybees produce, demonstrates an extensive spectrum of powerful biological properties. However, the anti-cancer activity of propolis derived from Geniotrigona thoracica Sumatrans has yet to be reported. Thus, we sought to investigate the cytotoxicity of aqueous propolis extracts from Geniotrigona thoracica Sumatrans against Colo-201 colon cancer cell line and senescence Colo-201 colon cancer cell line induced by low-dose doxorubicin. This study was conducted at the Parasitology Laboratory of Human, Safety, and Environment, Universitas Padjadjaran Bandung from January to May 2024. This study assessed cell viability using the WST-1 test. Non-induced Colo-201 cells were treated with an aqueous extract of propolis (AEP) 100 ppm, or 5-fluorouracil (5-FU) 5 mg/ml as the positive control or water as a vehicle on untreated control. Colo-201 senescence was induced by doxorubicin 0.1 µM for three days. Doxorubicin-induced Colo-201 senescence was then treated with AEP 100 ppm, with 5-FU 5 mg/ml as the positive control, or with the combination of AEP 100 ppm and 5-FU 5 mg/ml, or water as a vehicle on untreated control. The data were analyzed using SPSS version 25.0, a one-way ANOVA, and Tukey’s post hoc test. The results showed that AEP has cancer-killing effects on Colo-201 cells and Colo-201 senescent cells induced by low-dose doxorubicin. AEP-treated Colo-201 cells and Colo-201 senescent cells induced by low-dose doxorubicin viability were significantly reduced to 37.15% and 13.72%, respectively, although slightly higher than those of the 5-FU-treated one at this concentration. There was also a decrease in the cancer-killing effect of 5-FU from 88.55% in non-induced Colo-201 cells to 41.5% in the doxorubicin-induced Colo-201 senescence model. In conclusion, aqueous extract of propolis from Geniotrigona thoracica Sumatrans showed cancer-killing-effects both on the Colo-201 colon cancer cell line and senescence Colo-201 colon cancer cell line induced by low-dose doxorubicin.

Decolorization of anthraquinone dye by Brevibacillus laterosporus R81 via polysaccharides excreted

AbstractThe aim of this research was to assess the efficacy of different microbial strains in the decolorization of anthraquinone dyes. Strain R81 was obtained from a textile company's wastewater discharge for its remarkable ability to decolorize reactive blue 19 (RB19). By employing physiological and biochemical analyses, along with 16S rRNA gene sequencing, strain R81 was determined to be Brevibacillus laterosporus. After optimization, the decolorization rate achieved a peak of 86.24% over a 48‐h timeframe, utilizing an initial dye concentration of 100 mg L–1. The decolorization capacity of strain R81 was observed to be impeded by heightened levels of salt and temperature in culture solutions, yet remained unaltered when R81 cells were directly introduced into dye solutions. Furthermore, cells that were induced through prior cultivation in a medium containing RB19 demonstrated enhanced efficacy in decolorization compared to noninduced cells. Subsequent analysis indicated that the development of biofilms and the synthesis of polysaccharides by strain R81 were augmented in a concentration‐dependent fashion by RB19. Nevertheless, the decolorization efficacy of R81 was impeded by the existence of sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB), both of which possess the capacity to eliminate polysaccharides. The decolorization capabilities were reinstated by the SDS or CTAB eluent containing polysaccharides, suggesting a reliance on the presence of polysaccharides. The employment of stepwise diethylaminoethyl (DEAE)‐cellulose chromatography and decolorization experiments elucidated the importance of a specific polysaccharide in the decolorization process. This study proposes a bacteria‐derived polysaccharide as a promising remedy for treating dyeing wastewater contaminated with anthraquinones.

In vitro 3D microfluidic peritoneal metastatic colorectal cancer model for testing different oxaliplatin-based HIPEC regimens.

Treatment of colorectal peritoneal metastases with cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (HIPEC) is still evolving. Conducting a randomized trial is challenging due to the high heterogeneity in the presentation of peritoneal disease and various surgical approaches. Biological research may facilitate more rapid translation of information into clinical practice. There is an emerging need for a preclinical model to improve HIPEC treatment protocols in terms of drug doses and treatment durations. The aim of the study is to design a tool that serves as an invitro three-dimensional (3D) microfluidic peritoneal metastatic colorectal cancer model to test the efficacy of different HIPEC treatments. We determined the effects of current therapy options using a 3D static disease model on human colon carcinoma cell lines (HCT 116) and transforming growth factor-β1 induced epithelial-to-mesenchymal transition (EMT) HCT 116 lines at 37 °C and 42 °C for 30, 60, and 120 min. We determined oxaliplatin's half maximal inhibitory concentrations in a 3D static culture by using viability assay. Clinical practices of HIPEC were applied in the developed model. EMT-induced HCT 116 cells were less sensitive to oxaliplatin treatment compared to non-induced cells. We observed increased cytotoxicity when increasing the temperature from 37 °C to 42 °C and extending the treatment duration from 30 to 120 min. We found that 200 mg/m2 oxaliplatin administered for 120 min is the most effective HIPEC treatment option within the framework of clinic applications. The tool map provide insights into creating more realistic pre-clinical tools that could be used for a patient-based drug screening.

Achaete-scute family bHLH transcription factor 2 activation promotes hepatoblastoma progression.

Achaete-scute family bHLH transcription factor 2 (ASCL2) is highly expressed in hepatoblastoma (HB) tissues, but its role remains unclear. Thus, biological changes in the HB cell line HepG2 in response to induced ASCL2 expression were assessed. ASCL2 expression was induced in HepG2 cells using the Tet-On 3G system, which includes doxycycline. Cell viability, proliferation activity, mobility, and stemness were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony-formation, migration, invasion, and sphere-formation assays. Quantitative reverse-transcription polymerase chain reaction was used to assess the expression of markers for proliferation (CCND1 and MYC), epithelial-mesenchymal transition (EMT; SNAI1, TWIST1, and ZEB1), mesenchymal-epithelial transition (CDH1), and stemness (KLF4, POU5F1, and SOX9). Compared with the non-induced HepG2 cells, cells with induced ASCL2 expression showed significant increases in viability, colony number, migration area (%), and sphere number on days 7, 14, 8, and 7, respectively, and invasion area (%) after 90 h. Furthermore, induction of ASCL2 expression significantly upregulated CCND1, MYC, POU5F1, SOX9, and KLF4 expression on days 2, 2, 3, 3, and 5, respectively, and increased the ratios of SNAI1, TWIST1, and ZEB1 to CDH1 on day 5. ASCL2 promoted the formation of malignant phenotypes in HepG2 cells, which may be correlated with the upregulation of the Wnt signaling pathway-, EMT-, and stemness-related genes. ASCL2 activation may therefore be involved in the progression of HB.

Impact of vitamin B12 on rhamnose metabolism, stress defense and in-vitro virulence of Listeria monocytogenes

Listeria monocytogenes is a facultative anaerobe which can cause a severe food-borne infection known as listeriosis. L. monocytogenes is capable of utilizing various nutrient sources including rhamnose, a naturally occurring deoxy sugar abundant in foods. L. monocytogenes can degrade rhamnose into lactate, acetate and 1,2-propanediol. Our previous study showed that addition of vitamin B12 stimulated anaerobic growth of L. monocytogenes on rhamnose due to the activation of bacterial microcompartments for 1,2-propanediol utilization (pdu BMC) with concomitant production of propionate and propanol. Notably, anaerobic 1,2-propanediol metabolism has been linked to virulence of enteric pathogens including Salmonella spp. and L. monocytogenes. In this study we investigated the impact of B12 and BMC activation on i) aerobic and anerobic growth of L. monocytogenes on rhamnose and ii) the level of virulence. We observed B12-induced pdu BMC activation and growth stimulation only in anaerobically grown cells. Comparative Caco-2 virulence assays showed that these pdu BMC-induced cells have significantly higher translocation efficiency compared to non-induced cells (anaerobic growth without B12; aerobic growth with or without B12), while adhesion and invasion capacity is similar for all cells. Comparative proteome analysis showed specific and overlapping responses linked to metabolic shifts, activation of stress defense proteins and virulence factors, with RNA polymerase sigma factor SigL, teichoic acid export ATP-binding protein TagH, DNA repair and protection proteins, RadA and DPS, and glutathione synthase GshAB, previously linked to activation of virulence response in L. monocytogenes, uniquely upregulated in anaerobically rhamnose grown pdu-induced cells. Our results shed light on possible effects of B12 on L. monocytogenes competitive fitness and virulence activation when utilizing rhamnose in anaerobic conditions encountered during transmission and the human intestine.

Rifabutin but not rifampicin can partly out-balance P-glycoprotein induction by concurrent P-glycoprotein inhibition through high affinity binding to the inhibitory site

Physiology-based pharmacokinetic modeling suggests that rifabutin can out-balance P-glycoprotein (P-gp) induction by concurrent P-gp inhibition. However, clinical or experimental evidence for this Janus-faced rifabutin effect is missing. Consequently, LS180 cells were exposed to a moderately (2 µM) and strongly (10 µM) P-gp-inducing concentration of rifampicin or rifabutin for 6 days. Cellular accumulation of the fluorescent P-gp substrate rhodamine 123 was evaluated using flow cytometry, either without (induction only) or with adding rifamycin drug to the cells during the rhodamine 123 efflux phase (induction + potential inhibition). Rhodamine 123 accumulation was decreased similarly by both drugs after 6-day exposure (2 µM: 55% residual fluorescence compared to non-induced cells, P < 0.01; 10 µM: 30% residual fluorescence compared to non-induced cells, P < 0.001), indicating P-gp induction. Rhodamine 123 influx transporters mRNA expressions were not affected, excluding off-target effects. Acute re-exposure to rifabutin, however, considerably re-increased rhodamine 123 accumulation (2 µM induction: re-increase by 55%, P < 0.01; 10 µM induction: 49% re-increase, P < 0.001), suggesting P-gp inhibition. In contrast, rifampicin only had weak effects (2 µM induction: no re-increase; 10 µM induction: 16% re-increase; P < 0.05). Molecular docking analysis eventually revealed that rifabutin has a higher binding affinity to the inhibitor binding site of P-gp than rifampicin (ΔG (kcal/mol) = −11.5 vs −5.3). Together, this study demonstrates that rifabutin can at least partly mask P-gp induction by P-gp inhibition, mediated by high affinity binding to the inhibitory site of P-gp.

Retinoic Acid-Induced HL-60 Cell Differentiation Is Augmented by Copper Supplementation1–4

The objective of this study was to determine how copper influences the ability of HL-60 cells to differentiate into cells of the granulocytic lineage. We hypothesized that granulopoiesis requires copper because copper-deficient humans become neutropenic. Differentiation of HL-60 cells along the granulocytic lineage with retinoic acid was enhanced by copper. The results showed a greater number of cells were more differentiated when copper was added to the medium for 96 h. The respiratory burst activity of retinoic acid-induced cells was increased by copper supplementation, but intracellular superoxide anion generation was not affected. Supplementation with copper resulted in more cell-associated copper in both noninduced and induced cells; however, the induced cells accumulated three times more copper than the noninduced cells. Even though the amount of copper associated with retinoic acid-treated cells was greater than in untreated cells, the activity of a copper-requiring enzyme, copper/zinc superoxide dismutase, was significantly lower. Copper supplementation increased the activity of this enzyme in both retinoic acid-treated and untreated cells. Cytochrome c oxidase activity was not affected by retinoic acid treatment or by copper supplementation. Copper seems to play a specific role during the early stages of granulocyte differentiation.

Methylated HNRNPK acts on RPS19 to regulate ALOX15 synthesis in erythropoiesis.

Post-transcriptional control is essential to safeguard structural and metabolic changes in enucleated reticulocytes during their terminal maturation to functional erythrocytes. The timely synthesis of arachidonate 15-lipoxygenase (ALOX15), which initiates mitochondria degradation at the final stage of reticulocyte maturation is regulated by the multifunctional protein HNRNPK. It constitutes a silencing complex at the ALOX15 mRNA 3′ untranslated region that inhibits translation initiation at the AUG by impeding the joining of ribosomal 60S subunits to 40S subunits. To elucidate how HNRNPK interferes with 80S ribosome assembly, three independent screens were applied. They consistently demonstrated a differential interaction of HNRNPK with RPS19, which is localized at the head of the 40S subunit and extends into its functional center. During induced erythroid maturation of K562 cells, decreasing arginine dimethylation of HNRNPK is linked to a reduced interaction with RPS19 in vitro and in vivo. Dimethylation of residues R256, R258 and R268 in HNRNPK affects its interaction with RPS19. In noninduced K562 cells, RPS19 depletion results in the induction of ALOX15 synthesis and mitochondria degradation. Interestingly, residue W52 in RPS19, which is frequently mutated in Diamond-Blackfan Anemia (DBA), participates in specific HNRNPK binding and is an integral part of a putative aromatic cage.

Reliable, scalable functional genetics in bloodstream-form Trypanosoma congolense in vitro and in vivo.

Animal African trypanosomiasis (AAT) is a severe, wasting disease of domestic livestock and diverse wildlife species. The disease in cattle kills millions of animals each year and inflicts a major economic cost on agriculture in sub-Saharan Africa. Cattle AAT is caused predominantly by the protozoan parasites Trypanosoma congolense and T. vivax, but laboratory research on the pathogenic stages of these organisms is severely inhibited by difficulties in making even minor genetic modifications. As a result, many of the important basic questions about the biology of these parasites cannot be addressed. Here we demonstrate that an in vitro culture of the T. congolense genomic reference strain can be modified directly in the bloodstream form reliably and at high efficiency. We describe a parental single marker line that expresses T. congolense-optimized T7 RNA polymerase and Tet repressor and show that minichromosome loci can be used as sites for stable, regulatable transgene expression with low background in non-induced cells. Using these tools, we describe organism-specific constructs for inducible RNA-interference (RNAi) and demonstrate knockdown of multiple essential and non-essential genes. We also show that a minichromosomal site can be exploited to create a stable bloodstream-form line that robustly provides >40,000 independent stable clones per transfection-enabling the production of high-complexity libraries of genome-scale. Finally, we show that modified forms of T. congolense are still infectious, create stable high-bioluminescence lines that can be used in models of AAT, and follow the course of infections in mice by in vivo imaging. These experiments establish a base set of tools to change T. congolense from a technically challenging organism to a routine model for functional genetics and allow us to begin to address some of the fundamental questions about the biology of this important parasite.

Chromosomal localization of Ewing sarcoma EWSR1/FLI1 protein promotes the induction of aneuploidy

Ewing sarcoma is a pediatric bone cancer that expresses the chimeric protein EWSR1/FLI1. We previously demonstrated that EWSR1/FLI1 impairs the localization of Aurora B kinase to the midzone (the midline structure located between segregating chromosomes) during anaphase. While localization of Aurora B is essential for faithful cell division, it is unknown whether interference with midzone organization by EWSR1/FLI1 induces aneuploidy. To address this, we generated stable Tet-on inducible cell lines with EWSR1/FLI1, using CRISPR/Cas9 technology to integrate the transgene at the safe-harbor AAVS1 locus in DLD-1 cells. Induced cells expressing EWSR1/FLI1 displayed an increased incidence of aberrant localization of Aurora B, and greater levels of aneuploidy, compared with noninduced cells. Furthermore, the expression of EWSR1/FLI1-T79A, containing a threonine (Thr) to alanine (Ala) substitution at amino acid 79, failed to induce these phenotypes, indicating that Thr 79 is critical for EWSR1/FLI1 interference with mitosis. In contrast, the phosphomimetic mutant EWSR1/FLI1-T79D (Thr to aspartic acid (Asp)) retained the high activity as wild-type EWSR1/FLI1. Together, these findings suggest that phosphorylation of EWSR1/FLI1 at Thr 79 promotes the colocalization of EWSR1/FLI1 and Aurora B on the chromosomes during prophase and metaphase and, in addition, impairs the localization of Aurora B during anaphase, leading to induction of aneuploidy. This is the first demonstration of the mechanism for EWSR1/FLI1-dependent induction of aneuploidy associated with mitotic dysfunction and the identification of the phosphorylation of the Thr 79 of EWSR1/FLI1 as a critical residue required for this induction.

Targeted histone demethylation improves somatic cell reprogramming into cloned blastocysts but not postimplantation bovine concepti†.

Correct reprogramming of epigenetic marks in the donor nucleus is a prerequisite for successful cloning by somatic cell transfer (SCT). In several mammalian species, repressive histone (H) lysine (K) trimethylation (me3) marks, in particular H3K9me3, form a major barrier to somatic cell reprogramming into pluripotency and totipotency. We engineered bovine embryonic fibroblasts (BEFs) for the doxycycline-inducible expression of a biologically active, truncated form of murine Kdm4b, a demethylase that removes H3K9me3 and H3K36me3 marks. Upon inducing Kdm4b, H3K9me3 and H3K36me3 levels were reduced about 3-fold and 5-fold, respectively, compared with noninduced controls. Donor cell quiescence has been previously associated with reduced somatic trimethylation levels and increased cloning efficiency in cattle. Simultaneously inducing Kdm4b expression (via doxycycline) and quiescence (via serum starvation) further reduced global H3K9me3 and H3K36me3 levels by a total of 18-fold and 35-fold, respectively, compared with noninduced, nonstarved control fibroblasts. Following SCT, Kdm4b-BEFs reprogrammed significantly better into cloned blastocysts than noninduced donor cells. However, detrimethylated donors and sustained Kdm4b-induction during embryo culture did not increase the rates of postblastocyst development from implantation to survival into adulthood. In summary, overexpressing Kdm4b in donor cells only improved their reprogramming into early preimplantation stages, highlighting the need for alternative experimental approaches to reliably improve somatic cloning efficiency in cattle.

The differential effects of commercial specialized media on cell growth and transforming growth factor beta 1-induced epithelial-mesenchymal transition in bronchial epithelial cells.

Epithelial-mesenchymal transition (EMT) is one of the mechanisms that contribute to bronchial remodelling which underlie chronic inflammatory airway diseases such as chronic obstructive pulmonary disorder (COPD) and asthma. Bronchial EMT can be triggered by many factors including transforming growth factor β1 (TGFβ1). The majority of studies on TGFβ1-mediated bronchial EMT used BEGM as the culture medium. LHC-9 medium is another alternative available which is more economical but a less common option. Using normal human bronchial epithelial cells (BEAS-2B) cultured in BEGM as a reference, this study aims to validate the induction of EMT by TGFβ1 in cells cultured in LHC-9. Briefly, the cells were maintained in either LHC-9 or BEGM, and induced with TGFβ1 (5, 10 and 20ng/ml) for 48h. EMT induction was confirmed by morphological analysis and EMT markers expression by immunoblotting. In both media, cells induced with TGFβ1 displayed spindle-like morphology with a significantly higher radius ratio compared to non-induced cells which displayed a cobblestone morphology. Correspondingly, the expression of the epithelial marker E-cadherin was significantly lower, whereas the mesenchymal marker vimentin expression was significantly higher in induced cells, compared to non-induced cells. By contrast, a slower cell growth rate was observed in LHC-9 compared to that of BEGM. This study demonstrates that neither LHC-9 nor BEGM significantly influence TGFβ1-induced bronchial EMT. However, LHC-9 is less optimal for bronchial epithelial cell growth compared to BEGM. Thus, LHC-9 may be a more cost-effective substitute for BEGM, provided that time is not a factor.

Transcriptional and Translational Leakiness in Expression of a Fungal Cyclopropane Fatty Acid Synthase in the Escherichia coli pET-vector Expression System

We expressed the putative cyclopropane synthase Cfs1 from the mushroom Coprinopsis cinerea and the native bacterial Cfa enzyme in Escherichia coli as fusion proteins with an Nterminal 10xHis-tag and a proteolytic Factor Xa site, using the commercial pET-16b vector with the T7lac promoter for expression. Proteomics detected both proteins with high Mascot scores via trypsin-digested peptides in total cytoplasmic protein samples from transformed BL21(DE3) bacteria, induced by IPTG (isopropyl β-D-1- thiogalactopyranoside) for T7 RNA polymerase production from theT7 phage gene 1 present in the host genome under control by the lacUV5 promoter. In lower amounts, Cfs1 and Cfa were also found in protein samples from non-induced cells based on the known transcriptional leakiness of the pET-system in expression of T7 RNA polymerase. As a novelty, the results on Cfs1 from proteomics suggest that the sequence at the pET-16b-cfs1 fusion point CTCGAGGAT CCTATG (BamHI cloning site in italic, native cfs1 ATG start codon in bold) serves in transcripts as a minor RBS (ribosome binding site) to inadvertently initiate protein synthesis on the native cfs1 start codon. The first peptide MPAHHHPSSSAPCVSFPSSSK of the native Cfs1 protein was found in E. coli which is only possible if the normal cfs1 ATG was recognized as the start codon in protein biosynthesis. The GAGG-stretch in the above DNA sequence with a 7-bases-aligned spacing to the native cfs1 start codon features a partial Shine-Dalgarno (SD) sequence.

Prostate Cancer Epithelial Mesenchymal Transition by TGF-β1 exposure monitored with a Photonic Crystal Biosensor.

During prostate cancer progression, cancerous epithelial cells can undergo epithelial-mesenchymal transition (EMT). EMT is a crucial mechanism for the invasion and metastasis of epithelial tumors characterized by the loss of cell-cell adhesion and increased cell mobility. It is associated with biochemical changes such as epithelial cell markers E-cadherin and occludins being down-regulated, and mesenchymal markers vimentin and N-cadherin being upregulated. These changes in protein expression, specifically in the cell membrane, may be monitored via biophysical principles, such as changes in the refractive index (RI) of the cell membrane. In our previous research, we demonstrated the feasibility of using cellular RI as a unique contrast parameter to accomplish label-free detection of prostate cancer cells. In this paper, we report the use of our Photonic-Crystal biosensor in a Total-Internal-Reflection (PC-TIR) configuration to construct a label-free biosensing system, which allows for ultra-sensitive quantification of the changes in cellular RI due to EMT. We induced prostate cancer cells to undergo EMT by exposing these cells to soluble Transforming Growth Factor Beta 1 (TGF-β1). The biophysical characteristics of the cellular RI were quantified extensively in comparison to non-induced cancer cells. Our study shows promising clinical potential in utilizing the PC-TIR biosensing system not only to detect prostate cancer cells, but also to evaluate changes in prostate cancer cells due to EMT.

Kaposi's Sarcoma-Associated Herpesvirus-Encoded circRNAs Are Expressed in Infected Tumor Tissues and Are Incorporated into Virions.

Kaposi's sarcoma-associated herpesvirus (KSHV) has recently been found to generate circular RNAs (circRNAs) from several KSHV genes, most abundantly from K10 (viral interferon regulatory factor 4 [vIRF4]), K7.3, and polyadenylated nuclear (PAN) RNA. To define expression of these circRNAs, KSHV-infected cell lines, patient tissues, and purified virions were examined. KSHV circRNA expression was universally detected in tests of six primary effusion lymphoma (PEL) cell lines but ranged from low-level expression in BC-1 cells dually infected with tightly latent KSHV and Epstein-Barr virus to abundant expression in KSHV-only BCBL-1 cells with spontaneous virus production. Generally, the PAN/K7.3 locus broadly and bidirectionally generated circRNA levels that paralleled the corresponding linear RNA levels. However, RNA corresponding to a particular KSHV circularization site (circ-vIRF4) was minimally induced, despite linear vIRF4 RNA being activated by virus induction. In situ hybridization showed abundant circ-vIRF4 in noninduced PEL cells. All three KSHV circRNAs were isolated as nuclease-protected forms from gradient-purified virions collected from BrK.219 cells infected with a KSHV molecular clone. For circ-vIRF4, the fully processed form that is exported to the cytoplasm was incorporated into virus particles but the nuclear, intron-retaining form was not. The half-life of circ-vIRF4 was twice as long as that of its linear counterpart. The KSHV circRNAs could be detected at a higher rate than their corresponding linear counterparts by in situ hybridization in archival tissues and by reverse transcription-PCR (RT-PCR) in sera stored for over 25 years. In summary, KSHV circRNAs are expressed in infection-associated diseases, can be regulated depending on virus life cycle, and are incorporated into viral particles for preformed delivery, suggesting a potential function in early infection.IMPORTANCE KSHV has recently been found to encode circRNAs. circRNAs result from back-splicing of an upstream pre-mRNA splice donor exon-intron junction to an acceptor site, generating a covalently closed circle. This study revealed that for one KSHV region, the PAN/K7.3 locus, broadly and bidirectionally generated circRNA levels parallel corresponding linear RNA levels. Another KSHV circularization site (circ-vIRF4), however, showed expression that differed from that of the corresponding linear RNA. All KSHV circRNAs are incorporated into KSHV virions and are potentially expressed as immediate early products in newly infected cells.

DIBI, a novel polymeric iron chelator modulates IL-6 and IL-8 secretion from Cystic Fibrosis airway epithelial cells in response to endotoxin induction

Iron chelators have been utilized clinically to treat patients with iron overload conditions. There is a growing body of evidence linking iron dysregulation and reactive oxygen species (ROS) overproduction as underlying factors in Cystic Fibrosis (CF) disease. The chronic inflammation can lead to progressive airway destruction. Alleviation of this chronic inflammation is a potential target for CF treatment and thus, this research investigated the dose-response effects of DIBI, a novel iron chelator, on inflammation in CF nasal epithelial cells. Polarized CF cells were stimulated with, lipopolysaccharide (LPS), co-treated with DIBI (LPS+DIBI), or DIBI alone (DIBI). We demonstrated that DIBI modulated the release of IL-6 and IL-8 in CF cells in a dose-dependent manner. Reduction of extracellular iron with the lower doses of DIBI (25 and 50μM), increased IL-6 secretion in non-induced cells. LPS challenge increased IL-6 and IL-8 secretion which was suppressed by high dose (200μM) DIBI administration. This study demonstrates the therapeutic potential of iron chelation therapy to treat the dysregulation of the immune response in CF patients.

Disease Phenotype and Clonal Amplification in Calreticulin del52 and ins5 Knock-in Mice Are Dependent on the Type of Mutations and Gene Dosage

Introduction BCR-ABL-negative myeloproliferative neoplasms (MPNs) result from the transformation of a hematopoietic stem cell (HSC). Somatic mutations in the calreticulin (CALR) gene are associated with approximately 30% of essential thrombocythemia (ET) and primary myelofibrosis (PMF). All CALR mutations induce a frameshift to the same alternative reading frame generating a new C-terminal tail. The two most frequent CALR mutations are a 52 bp deletion (del52) or type 1 and a 5 bp insertion (ins5) or type 2. In patients, del52 and ins5 are equally found in ET but del52 is more frequent in PMF. In mouse retroviral model, del52 mice progress from ET to myelofibrosis (MF) while ins5 mice remain mostly with an ET. Methods In order to study the effect of endogenous levels of del52 and ins5 in hematopoiesis, we generated conditional knock-in (KI) mice expressing the murine CALR del52 or ins5 with the human mutated C-terminal tail under the control of a Scl-driven tamoxifen-inducible Cre recombinase (Scl-CreERT). We have also used Ubi-GFP transgenic mice to perform competitive engraftments. Results After tamoxifen-induction, both del52 and ins5 KI mice developed a rapid thrombocytosis, more severe in the homozygous than the heterozygous setting. In contrast, leukocytosis was observed only in homozygous setting. At similar zygosity, del52 induced a higher thrombocytosis compared to ins5. After 10 months of induction, both the bone marrow (BM) and the spleen of homozygous del52 KI mice and, to a much lower extent of homozygous ins5 KI mice, presented a significant increase in megakaryocytes (MKs) and in MK progenitors by flow cytometry. Von Willebrand factor staining showed that both del52 and ins5 homozygous mice displayed giant polylobulated MKs, associated with a similar increase in ploidy (mean ploidy 32N-33N). Heterozygous del52 presented also an increase ploidy of MK (mean of 25N) compared to controls (mean of 17N), whereas the MK ploidy of heterozygous ins5 mice was similar to control mice. The increase in number and size of MKs in homozygous del52 mice partially explained the significant decrease in BM cellularity and the splenomegaly. Moreover, we observed a decrease in BM erythroblasts and, in spleen, an increase in both erythroblast and granulocytic precursors together with a decrease in lymphocytes associated with a major disorganization of white pulp territories. Thus, the del52 homozygous KI mice developed features of a MF-like disease further illustrated by the presence of reticulin fibers stained with silver, mainly in spleen. Presence of fibrosis was not as pronounced in heterozygous del52 mice and more rarely observed in spleen of homozygous ins5 KI mice. In homozygous del52 KI mice, there was a significant amplification of the HSC compartment in both BM and spleen that was stronger than in homozygous ins5 mice. To study whether del52 and ins5 could provide a competitive advantage to HSCs, we performed BM transplantation with increasing percentages of non-induced homozygous del52 or ins5 cells with wild-type GFP+ cells into lethally-irradiated recipient mice. The homozygous del52 BM cells strongly competed wild-type hematopoiesis from an initial engraftment as low as 10% of mutated clones, reaching 100% in both blood myeloid cells and BM HSC compartments at 4 months. In contrast, out-competition of wild-type hematopoiesis by homozygous ins5 cells was slower, especially when less than 50% of mutated cells were initially engrafted suggesting that del52 provides a stronger advantage to the HSCs than ins5. Conclusion In conclusion, these results demonstrate that modeling CALRdel52 and ins5 mutations in mice can successfully recapitulate the differences in phenotype observed in patients, i.e del52 KI mice recapitulate an ET progressing to MF while ins5 KI mice only mimic an ET. This might be explained by a more profound effect of del52 than ins5 at the level of HSC. These KI mice offer solid in vivo models to investigate the mechanism of action of both types of mutations on HSCs and MKs and will be used to test new therapeutic approaches. Disclosures: No relevant conflicts of interest to declare. Disclosures Constantinescu: AlsaTech: Other: Co-Founde; Wiley &amp; Sons: Other: Editor in Chief, Journal of Cellular and Molecular Medicine; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AgenDix GmbH: Other: Co-Founder, MyeloPro Research and Diagnostics.

Microvesicles promote megakaryopoiesis by regulating DNA methyltransferase and methylation of Notch1 promoter.

Megakaryopoiesis is the process of formation of mature megakaryocytes that takes place in the bone marrow niche resulting in the release of platelets into the peripheral blood. It has been suggested that cell to cell communication in this dense bone marrow niche may influence the fate of the cells. Numerous studies point to the role of exosomes and microvesicles not only as a messenger of the cellular crosstalk but also in growth and developmental process of various cell types. In the current study, we explored the effects of megakaryocyte-derived microvesicles in hematopoietic cell lines in the context of differentiation. Our study demonstrated that microvesicles isolated from the induced megakaryocytic cell lines have the ability to stimulate noninduced cells specifically into that particular lineage. We showed that this lineage commencement comes from the change in the methylation status of Notch1 promoter, which is regulated by DNA methyltransferases.

Short communication: Pegbovigrastim treatment in vivo does not affect granulocyte ability to migrate to endometrial cells and kill bacteria in vitro in healthy cows

In periparturient dairy cows, immune suppression, resulting in decreased neutrophil numbers and function, leads to increased susceptibility to postpartum conditions such as mastitis, retained placenta, and metritis. Administration of polyethylene glycol-conjugated bovine granulocyte colony stimulating factor (pegbovigrastim, Imrestor; Elanco Animal Health, Greenfield, IN) 7 d before and within 24 h of calving, effectively improves granulocyte production and function in vivo as well as in milk. A recently developed coculture assay was adapted for use with endometrial epithelial cells to assess the effects of pegbovigrastim application on directed granulocyte migration and bactericidal activity in vitro on a per-cell basis in endometrial cell cultures. Granulocytes from treated and untreated periparturient cows (6 and 5 per group, respectively) were evaluated for their ability to migrate to and kill bacteria after treatment, in context of the infected endometrium. We hypothesized that in addition to increasing the absolute concentration of circulating neutrophil granulocytes, pegbovigrastim treatment in vivo alters the ability of granulocytes to migrate to endometrial cells in vitro. The results clearly show a marked increase in the total concentration of granulocytes and monocytes between the 2 treatment groups as early as 2 d after the first injection, and this increased between the samples taken 2 d after calving. No migratory or killing differences were identified between granulocytes of both groups, suggesting that pegbovigrastim-induced granulocytes were as effective as non-induced cells. This may also be due to the absence of negative energy balance in the study animals and leads us to conclude that the positive effects seen in vivo are most likely based on the larger number of granulocytes present rather than a direct effect of pegbovigrastim treatment on the functionality of cells for the parameters tested in this study.