Hr Of Exposure Research Articles

Changes of cellular stress response related hsp70 and abcb1 transcript and Hsp70 protein levels in Siberian freshwater amphipods upon exposure to cadmium chloride in the lethal concentration range.

The induction of cellular stress response systems, heat shock protein hsp70/Hsp70 and multixenobiotic transporter abcb1, by cadmium chloride (CdCl2) was explored in amphipod species with different stress adaptation strategies from the Lake Baikal area. Based on the lethal concentrations (LC) of CdCl2, the sensitivities of the different species to CdCl2 were ranked (24 hr LC50 in mg/L CdCl2 (mean/95% confidence interval)): Gammarus lacustris (1.7/1.3–2.4) < Eulimnogammarus cyaneus (2.9/2.1–4.0) < Eulimnogammarus verrucosus (5.7/3.8–8.7) < Eulimnogammarus vittatus (18.1/12.4–26.6). Conjugated dienes, indicating lipid peroxidation, were significantly increased after 24 hr exposures to 5 mg/L CdCl2 only in the more CdCl2-sensitive species G. lacustris and E. cyaneus. Upon treatment with 0.54 to 5.8 mg/L CdCl2 for 1, 6 and 24 hrs, hsp70 transcript levels were generally more increased after the longer exposure times and in the more CdCl2-sensitive species. Relating the CdCl2 exposure concentrations to LCx values revealed that across the species the increases of hsp70 transcript levels were comparatively low (up to 2.6-fold) at CdCl2 concentrations ≤LC50. Relative hsp70 transcript levels were maximally increased in E. cyaneus by 5 mg/L CdCl2 (n}{}hat {=}LC70) at 24 hrs (9.1-fold increase above the respective control). When G. lacustris was exposed to 5 mg/L CdCl2 (n}{}hat {=}LC90) for 24 hrs, the increase in hsp70 was in comparison to E. cyaneus considerably less pronounced (3.0-fold increase in hsp70 levels relative to control). Upon exposure of amphipods to 5 mg/L CdCl2, increases in Hsp70 protein levels compared to untreated controls were highest in E. cyaneus at 1 and 6 hrs (5 mg/L CdCl2 n}{}hat {=} LC70) and in E. verrucosus at 24 hrs (5 mg/L CdCl2 n}{}hat {=} LC45). Thus, when the fold increases in Hsp70 protein levels in the different amphipod species were related to the respective species-specific LCx values a similar bell-shaped trend as for hsp70 transcript levels was seen across the species. Transcript levels of abcb1 in CdCl2exposed individuals of the different amphipod species varied up to 4.7-fold in relation to the respective controls. In contrast to hsp70/Hsp70, abcb1 transcripts in CdCl2 exposed individuals of the different amphipod species did not indicate similar levels of induction of abcb1 at equal LCx levels across the species. Induction of hsp70 and abcb1 genes and Hsp70 proteins by CdCl2 in the lethal concentration range shows that these cellular responses are rather insensitive to CdCl2 stress in the examined amphipod species. Furthermore, the increase of expression of these cellular defense systems at such high stress levels suggests that induction of these genes is not related to the maintenance of normal metabolism but to mitigation of the effects of severe toxic stress.

Rainbow Darter (Etheostoma caeruleum, Storer, 1845) predation on early ontogenetic stages of Lake Sturgeon (Acipenser fulvescens, Rafinesque, 1817)

Previous molecular diet analysis identified lake sturgeon (Acipenser fulvescens, Rafinesque, 1817) DNA in the gastrointestinal tracts of stream-resident rainbow darters (Etheostoma caeruleum, Storer, 1845) during the egg incubation, free embryo, and larval drift stages. The objectives of this experimental study were to: (a) quantify levels of predation by rainbow darters on lake sturgeon at the egg and free-embryo stages; and (b) evaluate whether predation varied as a function of substrate size and rainbow darter body size. We conducted experimental trials in 23-L polycarbonate tanks 0.41 m (L) × 0.33 m (W) × 0.30 m (D) with a standardized benthic area of 0.14 m2. The tanks were randomly assigned one of two different substrate size classes: large rock (51.35 mm ± 0.91 mm) or small rock (27.68 mm ± 0.57 mm). We stocked individual rainbow darter, which were deprived of feed for 48 hr, with lake sturgeon (133 individuals/m2) in each of 12 replicates per ontogenetic stage and substrate type. The number of surviving lake sturgeon was quantified following a 24-hr predation exposure period. We used a generalized linear model with a binomial distribution to assess the influence of ontogenetic stage, substrate size, and rainbow darter body size on proportional lake sturgeon survival. Predation on lake sturgeon occurred at both egg (6.25 ± 1.16 individuals, mean ± 2SE) and free embryo (3.08 ± 1.08 individuals, mean ± 2SE) stages. Egg proportional survival was generally lower than at the free embryo stage in both substrate sizes; however, free embryo proportional survival was greater in small substrate trials. Rainbow darter total length did not affect the probability of lake sturgeon survival at either developmental stage. Results demonstrate that rainbow darters prey on early ontogenetic stages of lake sturgeon, corroborating previous results based on genetic diet analysis. Results fill a major knowledge gap concerning the vulnerability of pre-drift sturgeon to predation by an abundant river resident species that was previously discounted as a predator for early ontogenetic stages of lake sturgeon due to its small body size.

GPCR Stimulation Modulates Camkii Translocation and Targeting in Cardiomyocytes

Calcium/Calmodulin-dependent protein kinase II (CaMKIIδ) is a key regulator of cardiac function via its influence on ion channels, calcium handling, contraction, and transcription. Our group recently showed that pacing-induced activation of CaMKIIδ in cardiomyocytes enhances its mobility and rapid translocation to its extra-dyadic targets, providing a mechanism for broad myocyte target phosphorylation. It is not known whether GPCR-induced CaMKIIδ activation likewise stimulates CaMKII mobility. Here we used immunocytochemistry and protein expansion microscopy of endogenous CaMKII and fluorescence recovery after photobleach (FRAP) of GFP-tagged CaMKIIδ isoforms to measure CaMKIIδ mobility and intracellular targeting in intact adult cardiomyocytes. At low [Ca] (BAPTA-treated cells) and at rest, CaMKII was concentrated at the Z-lines but spread throughout the sarcomere with isoproterenol (ISO, 1 µM) or Angiotensin II (Ang II,100 nM) stimulation for 10 min. FRAP experiments likewise showed increased mobility for δC-GFP (τ for BAPTA ∼ 35s, ISO ∼ 16s and AngII ∼13s) and δB-GFP (τ for respectively BAPTA ∼ 21s, ISO ∼ 10s and AngII ∼10s). These effects were more pronounced upon chronic (20-24 hr) exposure to 0.5 Hz pacing with or without AngII or ISO. FRAP was significantly faster with pacing (τ ∼ 12s vs. rest ∼ 17s) and even more so with ISO or AngII exposure for δC-GFP (τ ISO ∼ 10s and AngII ∼10s) and δB-GFP (ISO ∼ 7s). The contribution of specific activating posttranslational modifications were also assessed using PTM-resistant CaMKIIδ constructs. In conclusion, acute and chronic activation of CaMKII via GPCR stimulation enhances its translocation which could explain CaMKII's regulation of pathological conditions, and widespread signaling within the myocyte.

Virulence of entomopathogenic nematodes against castor capsule borer Conogethes punctiferalis (Guenée)

Two EPN species, Heterorhabditis indica Poinar and H. bacteriophora Poinar were evaluated for their virulence and reproductive potential against late instar larvae of castor capsule borer Conogethes punctiferalis (Guenee) with wax moth Galleria mellonella (L.) used for comparison. Late instar larvae were found highly susceptible at all six infective juvenile (IJ) concentrations (30, 60, 90, 120, 150 and 200 IJ larva−1) of both the EPNs evaluated. At 48 hr post infection, complete larval mortality was observed in both capsule borer and wax moth. LC50 values of 570 and 40.1 IJ larva−1 for capsule borer and 15.1 and 5.4 IJ larva−1 forwax moth were observed with H. bacteriophora and H. indica, respectively at 24 hr exposure. These values indicated the highly virulent nature of the EPNs. In capsule borer, maximum IJ production/cadaver for H. bacteriophora and H. indica was 84 ± 4.6 x 103 IJs larva−1 and 98 ± 6.7 x 103 IJs larva−1 at an inoculation concentration of 30 IJs larva−1. In wax moth, it was 262.3 ± 27.2 x 103 IJs larva−1 and 231.9 ± 8.1 x 103 IJs larva−1 for H. bacteriophora and H. indica, at an inoculation concentration of 90 IJs larva−1. These results reveal that host size is very important in fixing optimal inoculation concentration and final IJ production, and that both the EPNs have good potential for the management of capsule borer in castor.

Graphene oxide-based nanomaterial interaction with human breast cancer cells.

Graphene and graphene-based nanomaterials have great potential for various biomedical applications due to their unique physicochemical properties. However, how graphene-based nanomaterials interact with biological systems has not been thoroughly studied. This study shows that 24, 48, and 72 hr exposure of 2.4 μg/cm2 of graphene oxide (GOX) and GOX modified with DAB-AM-16 and PAMAM dendrimers (GOXD and GOXP, respectively) did not exhibit toxicity to MCF-7 cells. However, higher graphene concentrations, such as 24 and 48 μg/cm2 , induced low cytotoxic effects. The GOX, GOXD, and GOXP particles have a strong affinity with the cellular membrane. Cells that internalized the nanomaterials presented morphological alterations and modifications in the organization of microfilaments and microtubules compared with control cells. Then, cells were treated with 24 μg/cm2 of GOX, GOXD or GOXP for 24 hr and recovered for an additional period of 24 hr in normal medium. Nanoparticles remained in the cytoplasm of some cells, apparently with no effect on cellular morphology, being consistent with the data found in the cell proliferation experiment, which showed that the cells remained alive up to 72 hr.

PSVII-36 Examining the effect of a physiological dose of the polyamine; spermine, on myogenic regulatory transcription factor expression

Abstract Examining the effect of a physiological dose of the polyamine; spermine, on myogenic regulatory transcription factor expression. Spermine is a micronutrient derived from amino acids. It affects cell growth, proliferation, differentiation and gene regulation in many cell types. Polyamines have long been overlooked with respect to their biological effects on muscle growth. Myogenic regulatory factors (myoD, myf5) initiate, promote and regulate myogenesis. Supporting myogenic transcription factors transcription and by such enhancing muscle production in livestock, through nutrition may be possible with dietary spermine supplementation. We examined the effect of spermine (0.5mM) in undifferentiated c2c12 muscle cells at two time points (8hr and 16hr). Using TaqMan-MGB qRT-PCR we quantified mRNA for key myogenic regulatory factors in a minimum of three experiments each containing 3 technical replicates. We report a significant increase in myoD (P = 0.02) and myf5 (P = 0.05) mRNA at 8hr following spermine treatment as compared to controls (no spermine). After 16 hr exposure to spermine (0.5mM) treatment myoD (P = 0.01) remained significantly different from controls. Our results indicate that spermine supports myogenesis through expression of increased myogenic regulatory factors at early stages of myogenesis. These findings support the need to further test the hypothesis that spermine promotes increased muscle growth and ultimately may represent a dietary means to maximize muscle growth in livestock species.

Anti‐glaucomatous preparations and effect on human conjunctival goblet cells

AbstractPurposeTo investigate goblet cell (GC) survival after treatment with benzalkonium chloride (BAC)‐preserved anti‐glaucomatous eye drops. Conjunctival GCs produce mucin which stabilizes the tear film and protects against dry eyes. Eyedrop‐induced GC death may cause ocular surface disorders.MethodLatanoprost Teva (LT; prostaglandin analogue), Optimol® (OP; beta‐blocker), Dorzolamid Stada (DS; carbonic anhydrase inhibitor) and Brimondine tartrat Teva (BT; alfa2‐agonist) BAC‐preserved eye drops were used for treatment of ex vivo cultivated human GCs. The cell survival was evaluated by lactate dehydrogenase assay on human GCs after 30 min and 6 hr exposure to the different treatments and compared to the untreated control group. pH‐value and osmolality were also measured.ResultsAfter 30 min of treatment, the human GC survival was 69.05% for LT, 69.78% for OP, 71.12% for DS and 93.42% for BT. The survival was significantly reduced only for the treatment with LT compared to the controls (p = 0.044). After 6 hr of exposure, GC survival was 31.08% for LT, 33.81 % for OP, 51.64 % for DS and 74.49 % for BT. The survival was significantly reduced for LT, OP, and DS compared to controls (p = 0.002, p = 0.004, p = 0.022). DS had the lowest pH (5.58), while OP the highest (6.84). LT had the lowest osmolality (267.6 mOsm/kg), while BT had the highest (293.2). There was no apparent correlation between GC survival, pH and osmolality. However, there seemed to be a connection between GC survival and BAC concentration. LT caused the lowest survival with a BAC concentration of 0.02%, while OP had a concentration of 0.01%, DS of 0.0075% and BT of 0.005%.ConclusionLatanoprost Teva was the most cytotoxic, causing significant GC death after 30 min and 6 hr of treatment. OP and DS caused significant GC death only after 6 hr of exposure. BT did not affect the GC survival. The cytotoxicity appeared to correlate with the BAC concentration, but not pH and osmolality.

Multiorgan microfluidic platform with breathable lung chamber for inhalation or intravenous drug screening and development.

Efficient and economical delivery of pharmaceuticals to patients is critical for effective therapy. Here we describe a multiorgan (lung, liver, and breast cancer) microphysiological system ("Body-on-a-Chip") designed to mimic both inhalation therapy and/or intravenous therapy using curcumin as a model drug. This system is "pumpless" and self-contained using a rocker platform for fluid (blood surrogate) bidirectional recirculation. Our lung chamber is constructed to maintain an air-liquid interface and contained a "breathable" component that was designed to mimic breathing by simulating gas exchange, contraction and expansion of the "lung" using a reciprocating pump. Three cell lines were used: A549 for the lung, HepG2 C3A for the liver, and MDA MB231 for breast cancer. All cell lines were maintained with high viability (>85%) in the device for at least 48 hr. Curcumin is used to treat breast cancer and this allowed us to compare inhalation delivery versus intravenous delivery of the drug in terms of effectiveness and potentially toxicity. Inhalation therapy could be potentially applied at home by the patient while intravenous therapy would need to be applied in a clinical setting. Inhalation therapy would be more economical and allow more frequent dosing with a potentially lower level of drug. For 24 hr exposure to 2.5 and 25 µM curcumin in the flow device the effect on lung and liver viability was small to insignificant, while there was a significant decrease in viability of the breast cancer (to 69% at 2.5 µM and 51% at 25 µM). Intravenous delivery also selectively decreased breast cancer viability (to 88% at 2.5 µM and 79% at 25 µM) but was less effective than inhalation therapy. The response in the static device controls was significantly reduced from that with recirculation demonstrating the effect of flow. These results demonstrate for the first time the feasibility of constructing a multiorgan microphysiological system with recirculating flow that incorporates a "breathable" lung module that maintains an air-liquid interface.

Superior Pre-Clinical Efficacy of Novel Protac Based BET Degrader in a Large Acute Myeloid Leukemia Cohort

Bromodomain and Extra Terminal (BET) proteins which include ubiquitously expressed BRD2, BRD3, BRD4, and testis-specific BRDT, are epigenetic "readers" and play a major role in epigenetic regulation of gene transcription. BET family proteins are crucial for acute myeloid leukemia (AML) maintenance and expression of BRD4 is a poor prognostic indicator in AML. BET inhibitor activity is associated with repression of oncogenic c-MYC and their efficacy in pre-clinical models of cancer has led to rapid development of BET inhibitors entering clinical trials. Depletion of key oncogenic proteins in tumor cells could achieve much better clinical efficacy than inhibition of the same proteins. Therefore, we have developed novel and highly potent BET degrader, QCA570, based on the proteolysis targeting chimera (PROTAC) concept. This "BET-degrader" is based upon a new class of BET inhibitor, HYD276. QCA570 effectively degrades all BET-BRD proteins at concentrations as low as 30 pM within a few hours of treatment in the RS4;11 leukemia cell line and achieves IC50 values of 50 pM in inhibition of RS4;11 cell growth. We tested QCA570 (BET-degrader) in a panel of 102 primary AML samples in ex-vivo cultures. Leukemia stem cells (LSC) were enriched by positive selection with CD34 antibody. Cells were plated in LSC conducive medium containing growth factors. Cell death and apoptosis induction was measured by flow-cytometry for DAPI-Annexin V binding after incubation with BET-inhibitor and BET-degrader for 3 days in the presence of drug. The AML cells were obtained from newly diagnosed AML (N=87) or relapse/refractory AML (N=15). The median IC50s was 120 pM (range 32 pm to 23 nM). Only 13 of 102 AML samples had an IC50 &gt;1 nM with the BET degrader. In contrast median IC50 for the inhibitor HYD276 was 30,000 fold higher at 3.5 µM (range 8 nM to 151 µM). An IC50 of greater than 10 µM HYD276 was considered as resistant to BET-inhibitor. BET-inhibitor resistant CD34 enriched LSCs were sensitive to the degrader QCA570, irrespective of mutation status and chromosomal aberrations, suggesting that there are additional mechanisms of action of the degrader as compared to the inhibitor. The AML cells were heterogeneous for the expression of mutant FLT-3 (36%), NPM1 (22%), IDH1/2 (16%), CEBPalpha (2%), DNMT3A (2%) and other mutations (10%). Based on classical karyotype 30% of them had a normal karyotype and 55% had abnormal karyotype (15% data missing). The degradation of BRD4 and repression of c-MYC was confirmed by immunoblotting after a 4 hr exposure of LSCs to degrader. Moreover, treatment of normal CD34 positive hematopoietic cells in apoptosis assay and colony formation assays suggests lack of toxicity to normal hematopoiesis. In conclusion, QCA570 represents a highly potent and efficacious panBET-BRD degrader undergoing extensive pre-clinical evaluation for the treatment of acute leukemias. Disclosures Peterson: Penrose TherapeuTx: Employment. Wang:Oncopia Therapeutics: Consultancy, Equity Ownership, Patents &amp; Royalties. Talpaz:Samus Therapeutics: Research Funding; Imago BioSciences: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; CTI BioPharma: Research Funding; Constellation: Research Funding; Incyte: Research Funding; Novartis: Research Funding.

Improved Anti-Leukemic Pre-Clinical Efficacy of a Protac Based MDM2 Degrader in a Large AML Cohort

Mouse double minute 2 homolog (MDM2) is a E3 ubiquitin ligase that promotes p53 tumor suppressor degradation. Many tumors harbor mutations in TP53 that don't bind MDM2, nevertheless, MDM2 has emerged as a therapeutic target in the treatment of TP53 wild-type cancers. Although in acute myeloid leukemia (AML) incidence of TP53 mutations is low (15-20%) inactivation of wild-type p53 protein frequently occurs through overexpression of its negative regulator MDM2. Thus MDM2 is an attractive target for novel therapeutics in AML tailored to treat TP53-wild type AML overexpressing MDM2. Several MDM2 inhibitors are currently in clinical trials. However, p53 stabilization upregulates MDM2, which limits the clinical efficacy of the inhibitors. Proteolysis Targeting Chimeric (PROTAC) molecules recruit targeted proteins for degradation and therefore can improve efficacy by removing the targeted protein. We developed a MDM2 degrader, MI-265, that binds to MDM2 and targets it for degradation thus removing the repression of p53, which results in apoptosis induction in leukemic cells. We tested MI-265 in a panel of 96 primary AML samples in ex-vivo cultures. AML blood and bone marrow samples were collected after obtaining University of Michigan IRB approved informed consent from AML patients at Michigan Medicine. Leukemia stem cells (LSC) were enriched by positive selection with CD34 antibody. Cells were plated in LSC conducive medium containing growth factors. Cell death and apoptosis induction was measured by flow-cytometry for DAPI-Annexin V binding after incubation with MDM2-inhibitor and MDM2-degrader for 3 days in the presence of drug. The AML cells were obtained from newly diagnosed AML (N=82) or relapse/refractory AML (N=14). The median IC50s was 120 pM (range 32 pm to 23 nM). In contrast median IC50 for the inhibitor MI-1061 was 20,000 fold higher at 2.3 µM (range 3 nM to 455 µM). Sensitivity and resistance to MDM2-degrader and inhibitor were similar in most of the samples suggesting that the mechanism of action of both drugs are also similar. The AML cells were heterogeneous for the expression of mutant FLT-3 (36%), NPM1 (22%), IDH1/2 (16%), CEBPalpha (2%), DNMT3A (2%) and other mutations (10%). Based on classical karyotype 30% of them had a normal karyotype and 55% had abnormal karyotype (15% data missing). The MDM2-degrader resistant LSCs (IC50 greater than 1 µM) were analyzed for TP53 mutation status and 8 of 14 were found to harbor mutations in TP53. The degradation of MDM2 and overexpression of p53 was confirmed by immunoblotting after a 4 hr exposure of LSCs to degrader. Moreover, treatment of normal CD34 positive hematopoietic cells in apoptosis assay and colony formation assays suggests lack of toxicity to normal hematopoiesis. In conclusion, MI-265 represents a highly potent and efficacious MDM2 degrader, and is currently undergoing extensive preclinical evaluation for the treatment of acute leukemias. Disclosures Peterson: Penrose TherapeuTx: Employment. Wang:Oncopia Therapeutics: Consultancy, Equity Ownership, Patents &amp; Royalties. Talpaz:Imago BioSciences: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; CTI BioPharma: Research Funding; Samus Therapeutics: Research Funding; Novartis: Research Funding; Constellation: Research Funding; Incyte: Research Funding.

MCL-1 Inhibition By the Selective MCL-1 Inhibitor AMG-176 Induces in Vitro Activity Against Burkitt Lymphoma Cell Lines and Synergistically Enhances the Cytotoxic Effect of Chemotherapy and BH3 Mimetics

Background: Although &gt;90% of Burkitt Lymphoma (BL) patients will be cured by intensive multi-agent immunochemotherapy, relapsed and refractory BL patients have poor prognoses demonstrating the need to identify novel therapies. Myeloid cell leukemia-1 (MCL-1) is a pro-survival protein within the BCL-2 family that has been investigated as a therapeutic target in several leukemia and lymphoma pre-clinical models and clinical studies. Analysis of pediatric BL tumors has implicated MCL-1 in BL pathogenesis (Giulino-Roth et al., Blood 2012) and targeting MCL-1 may be effective in MYC-driven lymphomas (Kelly et al., Genes Dev 2014). High MCL-1 expression has also been associated with therapy resistance and can confer resistance to BH3 mimetic agents (Carrington et al., Cell Death Differ 2017). AMG176, a selective MCL-1 inhibitor, is an effective promoter of apoptosis and is currently under investigation in clinical trials for relapsed and refractory multiple myeloma, lymphoma, and acute myeloid leukemia. Hypothesis: We hypothesized that MCL-1 inhibition with AMG176 will promote apoptosis and increase sensitivity of BL therapy-sensitive (TSCL) and therapy-resistant (TRCL) cell lines to other chemotherapy drugs. Methods: A panel of TSCLs (Raji, Ramos and Daudi) and one TRCL (Raji 4RH) BL cell lines were exposed to increasing concentrations of AMG176 as a single agent and in combination with Doxorubicin or Venetoclax at different time points. Cell viability was analyzed using PrestoBlue. Synergy in drug combination exposures was determined by Calcusyn software and reported as a combination index (CI) value with values increasingly &lt;1 indicating increasing synergistic activity. Apoptosis induction was analyzed by flow cytometry for Annexin V (AV) and Sytox-7AAD and by western blotting for PARP. Effects of MCL-1 inhibition on the expression of other pro- and anti-apoptotic BCL-2 family member proteins was assessed by western blotting. AMG176 mechanism(s) of action was evaluated by co-immunoprecipitation followed by western blotting for pro-apoptotic BCL-2 family members known to be bound by MCL-1. Results: Single agent AMG176 exposure of BL cell lines exhibited a dose and time-dependent decrease in viable cells. 48hr IC50 values of Ramos (0.3097uM) and Raji (1.652uM) showed an increased sensitivity to AMG176 as compared to Daudi (9.616uM) and Raji 4RH (12.45uM). Exposure of cells to AMG176 also resulted in altered expression levels of pro-survival and pro-apoptotic proteins of the BCL-2 family proteins. Co-immunoprecipitation performed after 48hrs of AMG176 exposure exhibited a decrease in MCL-1:BIM and MCL-1:BAK complexes in Raji and Ramos cells promoting a pro-apoptotic environment. After 48hr exposure to AMG176, induction of apoptosis was observed in a dose-dependent manner in all cell lines as evidenced by increased PARP cleavage on western blot and AV-Sytox positivity on flow cytometry. To investigate the ability of MCL-1 inhibition to enhance the activity of cytotoxic chemotherapy, BL cells were exposed to AMG176 in combination with Doxorubicin. Daudi and Raji exhibited strong synergy with peak synergistic activity observed at 72hrs (Daudi CI values &lt;0.1 at all AGM176 concentrations with 0.1-0.2uM Doxorubicin; Raji CI ranged from 0.233-0.295 at multiple AMG176 concentrations with &lt;0.4uM Doxorubicin) while a minimal effect was observed in Ramos and Raji 4RH. As high MCL-1 is associated with resistance to Venetoclax by sequestering free BIM following Venetoclax exposure, the combination of AMG176 and Venetoclax was investigated and also showed significant synergy in BL cells. A minimal effect was observed in Daudi and Raji 4RH in Venetoclax combination but Ramos and Raji, the more AMG176 sensitive cell lines, showed peak synergy at 24hrs at high doses of Venetoclax (Ramos CI values ranged from 0.3-0.478; Raji CI ranged from 0.511 to 0.67). Conclusion: MCL-1 inhibition with AMG176 demonstrates dose- and time-dependent anti-lymphoma activity impairing in vitro proliferation and inducing apoptosis in BL cells. AMG176 exhibits synergistic activity in combination with cytotoxic chemotherapy in both therapy-sensitive and therapy-resistant BL cell lines and synergistically enhances response to the BH3 mimetic agent Venetoclax. These findings highlight the potential of MCL-1 inhibition as a therapeutic option in BL. This work was supported by T35 NIH training grant (T35AI089693). Disclosures No relevant conflicts of interest to declare.

Preclinical Development of an Anti-CD38 Antibody-Drug Conjugate for Treatment of Hematological Malignancies

CD38 is a validated target for the treatment of multiple myeloma (MM). Daratumumab (Darzalex®), an anti-CD38 monoclonal antibody (mAb), has shown great clinical efficacy and has been approved for multiple myeloma treatment. However, both primary refractoriness and development of resistance to daratumumab therapy have been reported. Based on the therapeutic benefits of this CD38 antibody, we developed a CD38-targeting antibody-drug conjugate (ADC), employing a fully human anti-CD38 antibody STI-6129, identified from Sorrento's G-MAB® antibody library, and proprietary linker-toxin technology. The toxin payload is duostatin 5.2 (Duo.5.2), a microtubule inhibitor, conjugated to STI-6129 via a non-polyethylene glycol linker resulting in our lead ADC CD38-077. Cell binding studies showed that it specifically binds to CD38-positive tumor cells but not CD38-negative cell lines. The cell binding was proportional to the CD38 expression level on the cell surface. The ADC was internalized into CD38-positive cells at a rate comparable to that of the unconjugated antibody, indicating that conjugation did not change the binding characteristics of STI-6129 to its antigen. In cytotoxicity studies, CD38-077 exhibited a CD38-dependent cytotoxic activity against a panel of CD38-expressing tumor cell lines and was more potent in cells with high CD38 expression. The cytotoxic effect of CD38-077 was also examined against human PBMC cells, as it has been reported that certain types of the immune cells express CD38. The result indicated that normal PBMC cells were generally insensitive to the ADC up to 1 µM following 120 hr exposure. We investigated the anti-tumor activity of CD38-077 in xenograft animal models of Burkitt's lymphoma and two different multiple myeloma (MM) cell lines. The studies evaluated different dose levels and dosing regimens, including single dose and multiple doses at various intervals. The data showed that the ADC has a broad, potent and CD38-dependent in vivo efficacy in all three xenograft tumor models examined. In a pharmacokinetic study in naïve mice, CD38-077 was found to be stable, with T1/2 of about 7-11 days, comparable to that of the unconjugated STI-6129 antibody. In summary, CD38-077 exhibits strong anti-tumor activity in vitro and in vivo. The ADC showed specific activity towards CD38-expressing tumors but was less active against CD38-expressing normal PBMC cells, which express relatively low levels of CD38 level and where internalization was not detectable. These results warrant further development exploration of CD38-077. Disclosures Li: Concortis Biotherapeutics: Employment, Equity Ownership. Lau:Levena Biopharma: Employment, Equity Ownership. Fells:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Zhu:Levena Biopharma: Employment, Equity Ownership, Patents &amp; Royalties. Sun:Levena Biopharma: Employment, Equity Ownership. Kovacs:Levena Biopharma: Employment, Equity Ownership. Khasanov:Levena Biopharma: Employment, Equity Ownership. Yan:Levena Biopharma: Employment, Equity Ownership. Deng:Levena Biopharma: Employment, Equity Ownership. Takeshita:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Kaufmann:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents &amp; Royalties. Ji:Sorrento Therapeutics Inc: Employment, Equity Ownership, Patents &amp; Royalties; Celularity, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Li:Levena Biopharma: Employment, Equity Ownership, Patents &amp; Royalties; Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents &amp; Royalties. Zhang:Concortis Biotherapeutics: Employment, Equity Ownership, Patents &amp; Royalties.

Effects of microcystins-LR on genotoxic responses in human intestinal epithelial cells (NCM460)

ABSTRACTMicrocystin-LR (MC-LR), a cyclic heptapeptide toxin produced by cyanobacteria, was found to induce genotoxic actions in various types of cells. Some investigators reported that microcystin-LR acted as tumor initiator in the observed genotoxic action mediated by this cyanotoxin. However, the underlying mechanisms underlying MC-induced DNA damage in the human intestine epithelium cell line (NCM460) are not known. The purpose of this study was to examine the influence of 24 hr exposure to 5 or 10 µM MC-LR on intestinal DNA damage using NCM460 intestine cell line as a model. Data showed that MC-LR increased Olive tail moment (OTM) as evidenced by the comet assay, inhibited protein phosphatase 2A (PP2A) activity, elevated reactive oxygen species levels (ROS) and enhanced γ-H2AX and p-p53 protein expression levels. Results indicated that MC-LR produced intestinal DNA damage by inhibiting PP2A activity, activating p53 protein and subsequently initiating excess generation of ROS. These observations suggest that MC-LR–induced intestinal DNA damage involves a complex series of events that include oxidant stress, PP2A enzymic inhibition and activation of p53 protein.

Chronic Exposure to High Concentrations of Road Salt Decreases the Immune Response of Dragonfly Larvae

Salinization of freshwater ecosystems, due to the application of road salts, is recognized as a potential threat to aquatic communities. Much of the research on the impact of salinity has focused on performance metrics in vertebrates, including respiration and osmoregulation. Here we focus on immune function in of the dragonfly Anax junius, a top predator in fishless aquatic habitats. Impacts on this top predator have the potential to cascade through the community, and immune function is known to be both plastic and sensitive to stress. We injected larvae with monofilaments (simulating a parasite) and placed them in one of three environmentally relevant concentrations of deicing road salt: control (dechlorinated tap water with no added salt), low (1000 mgL-1), or high salt (3000 mgL-1), for either acute (24hr) chronic or (96hr) exposures. We hypothesized that elevated salinity would suppress the immune response and that longer exposure magnifies this effect. As predicted, chronic exposure to high salt concentrations resulted in a significantly reduced larval immune response, however, there was no detectable treatment effects in larvae exposed to low concentrations of road salt or to acute high concentrations. Our results demonstrate that prolonged exposure to high levels of road salt can compromise the immune response of dragonfly larvae. Our findings suggest that insects in aquatic environments that experience sustained environmental salt pollution will be more susceptible to parasites and pathogens, which in turn may affect the impact of this major predator on aquatic community dynamics.

Detecting Thin Stellar Streams in External Galaxies: Resolved Stars and Integrated Light

Abstract The morphology of thin stellar streams can be used to test the nature of dark matter. It is therefore crucial to extend searches for globular cluster (GC) streams to other galaxies than the Milky Way. In this paper, we investigate the current and future prospects of detecting GC streams in external galaxies in resolved stars (e.g., with Wide Field InfraRed Survey Telescope (WFIRST)) and using integrated light (e.g., with Hyper Suprime-cam (HSC), the Large Synoptic Survey Telescope (LSST), and Euclid). In particular, we inject mock streams to data from the PAndAS M31 survey and produce simulated M31 backgrounds mimicking what WFIRST will observe in M31. Additionally, we estimate the distance limit to which GC streams will be observable. Our results demonstrate that for a 1 hr (1000 s) exposure, using conservative estimates, WFIRST should detect GC streams in resolved stars in galaxies out to distances of ∼3.5 Mpc (∼2 Mpc). This volume contains 199 (122) galaxies, of which &gt;90% are dwarfs. With integrated light, thin streams can be resolved out to ∼100 Mpc with HSC and LSST and to ∼600 Mpc with WFIRST and Euclid. The low surface brightness of the streams (typically &gt;30 mag arcsec−2), however, will make them difficult to detect, unless the streams originate from very young clusters. We emphasize that if the external galaxies do not host spiral arms or galactic bars, gaps in their stellar streams provide an ideal test case for evidence of interactions with dark matter subhalos. Furthermore, obtaining a large samples of thin stellar streams can help constrain the orbital structure and hence the potentials of external halos.

Mechanisms of electrical remodeling in lipotoxic guinea pig heart

ObjectivesTo develop an adult guinea pig model of lipotoxicity and explore the underlying mechanisms associated with changes in the expression of the delayed rectifier potassium current (IK). BackgroundLipotoxicity may represent a common link among metabolic disorders and a higher vulnerability to arrhythmias. MethodsWhole-cell patch clamp, and palmitic acid (PA, a potent inducer of lipotoxicity), were used to assess mechanisms of short-term (∼50 days) high-fat diet (HFD) feeding on atrial electrophysiology in guinea pig hearts and myocytes. ResultsHFD fed guinea pigs were significantly heavier, displayed hypertriglyceridemia and hypercholesterolemia; but no signs of hyperglycemia or inflammation compared to low-fat diet fed controls. Increasing cardiac PA levels, resulted in shortened atrial action potential duration, and increased IK density. Inhibition of phosphoinositide 3-kinase (PI3K) prevented increases in IK due to PA. Acute (≥1hr) exposure of atrial myocytes to exogenous PA (1 mM) increased the density of the rapid delayed rectifier potassium current IKr, while it was decreased with the unsaturated oleic acid (OA, 1 mM). Serine-threonine protein phosphatase-2 (PP2A) inhibition with cantharidin reversed the effect of OA on IKr. ConclusionOur data provide evidence of a novel lipotoxic guinea pig model with signs of vulnerability to arrhythmias. Inhibition of PA/PI3K/IK and/or activation of the OA/PP2A/IKr pathways may be therapeutically beneficial for lipotoxic arrhythmias.

Discovery of natural naphthoquinones as sortase A inhibitors and potential anti-infective solutions against Staphylococcus aureus.

Three natural naphthoquinones were screened to find new anti-virulence agents as inhibitors against sortase A from Staphylococcus aureus (SaSrtA) by quantifying the increase in fluorescence intensity upon substrate cleavage at various concentrations. The 5-hydroxy-1,4-naphthalenedione derivatives, juglone and plumbagin, demonstrated a potent inhibitory effect, with IC50 values of 1.78 μM, respectively, 16.71 μM. The related 2-hydroxy-1,4-naphthalenedione derivative, lawsone, demonstrated the selectivity of the chemical scaffold having no significant effect on SaSrtA. The experimental assay was reinforced by molecular docking experiments, antimicrobial, and toxicological studies. Molecular docking studies and the electrophilic character analysis suggest bonding to the enzyme active cysteine residue by a Michael addition reaction. None of the compounds had a significant effect on the concentration of total thiol proteins in the Daphnia magna toxicological assay after 24 hr exposure. Juglone and plumbagin moderately inhibited biofilm formation with no significant effect on bacterial growth of S. aureus, Enterococcus faecalis, and Staphylococcus epidermidis, indicating a selective anti-virulence profile.

IL6 and STAT-3 pathway highlight the differences in molecular responses in myometrium and uterine fibroids

Human fibroids are highly prevalent and symptomatic uterine tumors. Phenotypically, they are different from normal myometrium because of massive production of extracellular matrix (ECM), which is a hallmark of these benign tumors resulting in symptoms including abnormal bleeding and pain. Dysregulated production of inflammatory cytokine, IL6 and its regulated JAK/STAT-3 pathway are known to contribute to the fibrotic process. Our objective was to understand the effect of IL6 on fibroid and myometrium cells and the role of JAK/STAT pathway in production of matrix proteins that play a major role in fibroid pathogenesis. Laboratory study. Leiomyoma and patient-matched myometrium cell lines exposed to different concentrations of IL6 and JAK/STAT-3 modulators for various exposure time periods, in a 2D culture model system. Changes in expression of ECM proteins and regulating pathways were assessed using western blot. In leiomyoma cells, IL6 activation of STAT-3 peaked (2.32+/- 0.21 fold) as early as 1.5hr of continuous exposure, and was 1.4+/- 0.04 fold increased at end of 3hr of exposure. The maximum increase (1.59+/- 0.07 fold) in the feedback loop protein, Suppressor of Cytokine Signaling (SOCS3) required a short exposure to IL6 (30min) followed by collection after 3hr. We also observed an increase (1.9+/- 0.11 fold) in the ECM structural collagen-1 after continuous exposure to IL6 (3hr) in leiomyoma cells. No significant effect of IL6 was observed in myometrium cells. Collagen-1 protein was also affected significantly in the leiomyoma cells on use of JAK/STAT-3 modulators. An increase of 2.21+/-0.052 fold was observed on direct activation of the pathway in leiomyoma cells. Increase were also observed in ECM protein fibronectin in response to the JAK/STAT-3 modulators. Response of transforming growth factor beta3 (TGFβ3) protein, involved in fibrotic specific pathway, on use of JAK/STAT-3 modulators indicated that both pathways may interact to regulate the production of the ECM proteins in leiomyomas. Direct effect of inflammatory cytokine IL6, on leiomyoma and not myometrium cells indicate that this differential response to IL6 and JAK/STAT3 pathway may be the key to increased ECM production and growth in uterine leiomyomas.

Response of short-term heat shock on photosynthetic activity of soil crust cyanobacteria.

Short-term heat exposure in tropical regions can generate severe stress in the photosynthetic activity of soil crust cyanobacteria. We investigated the responses of two filamentous cyanobacteria, Scytonema tolypothrichoides and Tolypothrix bouteillei, to 1hr exposure at 35, 45, and 55°C using variable chlorophyll fluorescence. Protocols for maximum quantum yield (FV/FM) and dark recovery of chlorophyll a fluorescence (OJIP) transient were applied. Heat exposure caused damage to the donor side of PSII, indicated by a decrease in FV/FM and a rapid increase in F0. After heat stress, photochemical energy utilization (φPo, φETo, and φRE1o) declined and energy dissipation (φDIo) increased. At 45°C, the photosynthetic apparatus was reversibly damaged, since full recovery was observed after 7days of relaxation. S. tolypothrichoides was more resistant to heat stress than T. bouteillei, confirming better adaptation to higher temperatures as observed in growth experiments.

Biomedical Applications of &lt;em&gt;Chasmanthera dependens&lt;/em&gt; stem extract mediated silver nanoparticles as Antimicrobial, Antioxidant, Anticoagulant, thrombolytic, and Larvicidal agents

The stem extract of Chasmanthera dependens was used in the biofabrication of silver nanoparticles (AgNPs) in this study. The AgNPs was characterised using UV-visible spectroscopy, Field Emission Scanning Electron Microscope (FESEM), EDX, and the Fourier Transform Infrared Spectroscopy (FTIR). Antibacterial, antioxidant, anticoagulant, thrombolytic and larvicidal activities of the biosynthesised nanoparticles were carried out. There was a peak at 418 nm with a strong silver peak observed around 2.7KeV when the EDX analysis was carried out. The FESEM showed a large number of cubically shaped nanoparticles with sizes ranging from 24.53 to 92.38 nm. CDE-AgNPs was effective against Klebsiella pneumoniae at 80 µg/ml and 100 µg/ml concentrations while little or no inhibition observed with other bacterial isolates used. CDE-AgNPs displayed potent activity with LC50 and LC90 of 7.15 µg/ml and 20.86 µg/ml respectively against the Aedes aegypti mosquito larva after 1hr of exposure. The synthesised nanoparticles displayed scavenging activity of DPPH at all concentrations. High anticoagulant efficacy was displayed, while 45.61% clot lysis was observed. This study therefore demonstrated the effectiveness of biosynthesised Chasmanthera dependens stem extract silver nanoparticles as antimicrobial, antioxidant, anticoagulant, thrombolytic and larvicidal agents.