Principles and approach to developing mammalian cell culture media for high cell density perfusion process leveraging established fed-batch media.

Perfusion medium was successfully developed based on our fed-batch platform basal and feed media. A systematic development approach was undertaken by first optimizing the ratios of fed-batch basal and feed media followed by targeted removal of unnecessary and redundant components. With this reduction in components, the medium could then be further concentrated by 2× to increase medium depth. The medium osmolality was also optimized where we found ∼360 mOsm/kg was desirable resulting in a residual culture osmolality of ∼300 mOsm/kg for our cell lines. Further building on this, the amino acids Q, E, N, and D were rebalanced to reduce lactate and ammonium levels, and increase the cell-specific productivity without compromising on cell viability while leaving viable cell density largely unaffected. Further modifications were also made by increasing certain important vitamin and lipid concentrations, while eliminating other unnecessary vitamins. Overall, an effective perfusion medium was developed with all components remaining in the formulation understood to be important and their concentrations increased to improve medium depth. The critical cell-specific perfusion rate using this medium was then established for a cell line of interest to be 0.075 nL/cell-day yielding 1.2 g/L-day at steady state. This perfusion process was then successfully scaled up to a 100 L single-use bioreactor with an ATF6 demonstrating similar performance as a 2 L bioreactor with an ATF2. Large volume handling challenges in our fed-batch facility were overcome by developing a liquid medium version of the powder medium product contained in custom totes for plug-and-play use with the bioreactor. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:891-901, 2017.

Mesoionic pyrido[1,2-a]pyrimidinones: Discovery of dicloromezotiaz as a lepidoptera insecticide acting on nicotinic acetylcholine receptors1,2.

A novel class of mesoionic pyrido[1,2-a]pyrimidinones has been discovered with exceptional insecticidal activity controlling a number of insect species. In this communication, we report the part of the optimization program that led to the identification of dicloromezotiaz as a potent insecticide to control a broad range of lepidoptera. Our efforts in discovery, synthesis, structure-activity relationship elucidation, and biological activity evaluation are also presented.

Mesoionic pyrido[1,2-a]pyrimidinones: Discovery of triflumezopyrim as a potent hopper insecticide1.

A novel class of mesoionic pyrido[1,2-a]pyrimidinones has been discovered with exceptional insecticidal activity controlling a number of insect species. In this communication, we report the part of the optimization program which led to the discovery of triflumezopyrim as a highly potent insecticide controlling various hopper species. Our efforts in discovery, synthesis, structure-activity relationship elucidation, and biological activity evaluation are also presented.

Mesoionic insecticides: a novel class of insecticides that modulate nicotinic acetylcholine receptors.

BACKGROUND: As the world population grows towards 9 billion by 2050, it is projected that food production will need to increase by 60%. A critical part of this growth includes the safe and effective use of insecticides to reduce the estimated 20-49% loss of global crop yields owing to pests. The development of new insecticides will help to sustain this protection and overcome insecticide resistance. RESULTS: A novel class of mesoionic compounds has been discovered, with exceptional insecticidal activity on a range of Hemiptera and Lepidoptera. These compounds bind to the orthosteric site of the nicotinic acetylcholine receptor and result in a highly potent inhibitory action at the receptor with minimal agonism. The synthesis, biological activity, optimization and mode of action will be discussed. CONCLUSION: Triflumezopyrim insect control will provide a powerful tool for control of hopper species in rice throughout Asia. Dicloromezotiaz can provide a useful control tool for lepidopteran pests, with an underexploited mode of action among these pests. © 2016 Society of Chemical Industry.

Mesoionic pyrido[1,2-a]pyrimidinones: A novel class of insecticides inhibiting nicotinic acetylcholine receptors.

A novel class of mesoionic pyrido[1,2-a]pyrimidinones has been discovered with exceptional insecticidal activity controlling a number of insect species, particularly hemiptera and lepidoptera. Mode-of-action studies showed that they act on nicotinic acetylcholine receptors (nAChRs) primarily as inhibitors. Here we report the discovery, evolution, and preparation of this class of chemistry. Our efforts in structure-activity relationship elucidation and biological activity evaluation are also presented.

Mode of action of triflumezopyrim: A novel mesoionic insecticide which inhibits the nicotinic acetylcholine receptor.

Triflumezopyrim, a newly commercialized molecule from DuPont Crop Protection, belongs to the novel class of mesoionic insecticides. This study characterizes the biochemical and physiological action of this novel insecticide. Using membranes from the aphid, Myzus persicae, triflumezopyrim was found to displace (3)H-imidacloprid with a Ki value of 43 nM with competitive binding results indicating that triflumezopyrim binds to the orthosteric site of the nicotinic acetylcholine receptor (nAChR). In voltage clamp studies using dissociated Periplaneta americana neurons, triflumezopyrim inhibits nAChR currents with an IC50 of 0.6 nM. Activation of nAChR currents was minimal and required concentrations ≥100 µM. Xenopus oocytes expressing chimeric nAChRs (Drosophila α2/chick ß2) showed similar inhibitory effects from triflumezopyrim. In P. americana neurons, co-application experiments with acetylcholine reveal the inhibitory action of triflumezopyrim to be rapid and prolonged in nature. Such physiological action is distinct from other insecticides in IRAC Group 4 in which the toxicological mode of action is attributed to nAChR agonism. Mesoionic insecticides act via inhibition of the orthosteric binding site of the nAChR despite previous beliefs that such action would translate to poor insect control. Triflumezopyrim is the first commercialized insecticide from this class and provides outstanding control of hoppers, including the brown planthopper, Nilaparvata lugens, which is already displaying strong resistance to neonicotinoids such as imidacloprid.

CD4+ T Cell Help Selectively Enhances High-Avidity Tumor Antigen-Specific CD8+ T Cells.

Maintaining antitumor immunity remains a persistent impediment to cancer immunotherapy. We and others have previously reported that high-avidity CD8(+) T cells are more susceptible to tolerance induction in the tumor microenvironment. In the present study, we used a novel model where T cells derived from two independent TCR transgenic mouse lines recognize the same melanoma antigenic epitope but differ in their avidity. We tested whether providing CD4(+) T cell help would improve T cell responsiveness as a function of effector T cell avidity. Interestingly, delivery of CD4(+) T cell help during in vitro priming of CD8(+) T cells improved cytokine secretion and lytic capacity of high-avidity T cells, but not low-avidity T cells. Consistent with this observation, copriming with CD4(+) T cells improved antitumor immunity mediated by higher avidity, melanoma-specific CD8(+) T cells, but not T cells with similar specificity but lower avidity. Enhanced tumor immunity was associated with improved CD8(+) T cell expansion and reduced tolerization, and it was dependent on presentation of both CD4(+) and CD8(+) T cell epitopes by the same dendritic cell population. Our findings demonstrate that CD4(+) T cell help preferentially augments high-avidity CD8(+) T cells and provide important insight for understanding the requirements to elicit and maintain durable tumor immunity.

Movement of cyantraniliprole in plants after foliar applications and its impact on the control of sucking and chewing insects.

BACKGROUND: Given the physical properties of insecticides, there is often some movement of these compounds within crop plants following foliar application. In this context, movement of two formulations of cyantraniliprole, an anthranilic diamide, was characterized for translocation to new growth, distribution within a leaf and penetration through the leaf cuticle. RESULTS: Upward movement of cyantraniliprole to new plant growth via the xylem was confirmed using (14) C-radiolabeled cyantraniliprole and from Helicoverpa zea mortality on tomato leaves that had not been directly treated. Within a leaf there was significant acropetal movement (base to apex) of cyantraniliprole, but no significant basipetal movement (apex to base). Translaminar movement, the ability of a compound to penetrate the leaf cuticle, was demonstrated in a variety of plants, both with and without the use of adjuvants, by treating only the adaxial surface of the leaf and measuring control of diamondback moth (Plutella xylostella), green peach aphid (Myzus persicae) and sweetpotato whitefly (Bemisia tabaci) exposed in clip cages to the untreated abaxial surface. CONCLUSION: The plant mobility and plant protection of cyantraniliprole is discussed with implications for use in insect resistance management and integrated pest management programs.

Interleukin-1 and interferon-γ orchestrate ß-glucan-activated human dendritic cell programming via IκB-ζ modulation.

Recognition of microbial components via innate receptors including the C-type lectin receptor Dectin-1, together with the inflammatory environment, programs dendritic cells (DCs) to orchestrate the magnitude and type of adaptive immune responses. The exposure to ß-glucan, a known Dectin-1 agonist and component of fungi, yeasts, and certain immune support supplements, activates DCs to induce T helper (Th)17 cells that are essential against fungal pathogens and extracellular bacteria but may trigger inflammatory pathology or autoimmune diseases. However, the exact mechanisms of DC programming by ß-glucan have not yet been fully elucidated. Using a gene expression/perturbation approach, we demonstrate that in human DCs ß-glucan transcriptionally activates via an interleukin (IL)-1- and inflammasome-mediated positive feedback late-induced genes that bridge innate and adaptive immunity. We report that in addition to its known ability to directly prime T cells toward the Th17 lineage, IL-1 by promoting the transcriptional cofactor inhibitor of κB-ζ (IκB-ζ) also programs ß-glucan-exposed DCs to express cell adhesion and migration mediators, antimicrobial molecules, and Th17-polarizing factors. Interferon (IFN)-γ interferes with the IL-1/IκB-ζ axis in ß-glucan-activated DCs and promotes T cell-mediated immune responses with increased release of IFN-γ and IL-22, and diminished production of IL-17. Thus, our results identify IL-1 and IFN-γ as regulators of DC programming by ß-glucan. These molecular networks provide new insights into the regulation of the Th17 response as well as new targets for the modulation of immune responses to ß-glucan-containing microorganisms.

Genome-wide DNA methylation patterns in LSH mutant reveals de-repression of repeat elements and redundant epigenetic silencing pathways.

Cytosine methylation is critical in mammalian development and plays a role in diverse biologic processes such as genomic imprinting, X chromosome inactivation, and silencing of repeat elements. Several factors regulate DNA methylation in early embryogenesis, but their precise role in the establishment of DNA methylation at a given site remains unclear. We have generated a comprehensive methylation map in fibroblasts derived from the murine DNA methylation mutant Hells(-/-) (helicase, lymphoid specific, also known as LSH). It has been previously shown that HELLS can influence de novo methylation of retroviral sequences and endogenous genes. Here, we describe that HELLS controls cytosine methylation in a nuclear compartment that is in part defined by lamin B1 attachment regions. Despite widespread loss of cytosine methylation at regulatory sequences, including promoter regions of protein-coding genes and noncoding RNA genes, overall relative transcript abundance levels in the absence of HELLS are similar to those in wild-type cells. A subset of promoter regions shows increases of the histone modification H3K27me3, suggesting redundancy of epigenetic silencing mechanisms. Furthermore, HELLS modulates CG methylation at all classes of repeat elements and is critical for repression of a subset of repeat elements. Overall, we provide a detailed analysis of gene expression changes in relation to DNA methylation alterations, which contributes to our understanding of the biological role of cytosine methylation.

Endogenous retrovirus insertion in the KIT oncogene determines white and white spotting in domestic cats.

The Dominant White locus (W) in the domestic cat demonstrates pleiotropic effects exhibiting complete penetrance for absence of coat pigmentation and incomplete penetrance for deafness and iris hypopigmentation. We performed linkage analysis using a pedigree segregating White to identify KIT (Chr. B1) as the feline W locus. Segregation and sequence analysis of the KIT gene in two pedigrees (P1 and P2) revealed the remarkable retrotransposition and evolution of a feline endogenous retrovirus (FERV1) as responsible for two distinct phenotypes of the W locus, Dominant White, and white spotting. A full-length (7125 bp) FERV1 element is associated with white spotting, whereas a FERV1 long terminal repeat (LTR) is associated with all Dominant White individuals. For purposes of statistical analysis, the alternatives of wild-type sequence, FERV1 element, and LTR-only define a triallelic marker. Taking into account pedigree relationships, deafness is genetically linked and associated with this marker; estimated P values for association are in the range of 0.007 to 0.10. The retrotransposition interrupts a DNAase I hypersensitive site in KIT intron 1 that is highly conserved across mammals and was previously demonstrated to regulate temporal and tissue-specific expression of KIT in murine hematopoietic and melanocytic cells. A large-population genetic survey of cats (n = 270), representing 30 cat breeds, supports our findings and demonstrates statistical significance of the FERV1 LTR and full-length element with Dominant White/blue iris (P < 0.0001) and white spotting (P < 0.0001), respectively.

The effect of temperature on germination of chlamydospores of Phytophthora ramorum.

Mycelium-free chlamydospores of 12 isolates of P. ramorum representing three clonal lineages were produced with a method involving incubation in nonsterile sand at 20 C in darkness for 30 d. Chlamydospores were incubated on selective agar medium at 5, 10, 15, 20, 25 and 30 C and germination assessed after 1, 2, 4, 6 and 8 d incubation. The optimal temperature for germination based on 8 d incubation was 20 C for all three clonal lineages tested (NA1, NA2, EU1). Mean germination rates were 2, 21, 44, 67, 32 and 0 percent at 5, 10, 15, 20, 25 and 30 C respectively for all isolates combined. The highest mean germination rate was scored by isolates of the EU1 clonal lineage at 20 C (85%) after 8 d incubation However, substantial variation was observed among isolates within each clonal lineage. Overall temperatures and days of incubation on which germination was assessed isolates of the NA1 clonal lineage had the lowest mean germination, even though one isolate had the highest germination of any isolate in any lineage. The results indicate that 20 C is the optimal germination temperature for P. ramorum chlamydospores and that a great disparity in germination percentage can exist within isolates, even within a single clonal lineage.

CG hypomethylation in Lsh-/- mouse embryonic fibroblasts is associated with de novo H3K4me1 formation and altered cellular plasticity.

DNA methylation patterns are established in early embryogenesis and are critical for cellular differentiation. To investigate the role of CG methylation in potential enhancer formation, we assessed H3K4me1 modification in murine embryonic fibroblasts (MEFs) derived from the DNA methylation mutant Lsh(-/-) mice. We report here de novo formation of putative enhancer elements at CG hypomethylated sites that can be dynamically altered. We found a subset of differentially enriched H3K4me1 regions clustered at neuronal lineage genes and overlapping with known cis-regulatory elements present in brain tissue. Reprogramming of Lsh(-/-) MEFs into induced pluripotent stem (iPS) cells leads to increased neuronal lineage gene expression of premarked genes and enhanced differentiation potential of Lsh(-/-) iPS cells toward the neuronal lineage pathway compared with WT iPS cells in vitro and in vivo. The state of CG hypomethylation and H3K4me1 enrichment is partially maintained in Lsh(-/-) iPS cells. The acquisition of H3K27ac and activity of subcloned fragments in an enhancer reporter assay indicate functional activity of several of de novo H3K4me1-marked sequences. Our results suggest a functional link of H3K4me1 enrichment at CG hypomethylated sites, enhancer formation, and cellular plasticity.

An analysis of select emerging executive skills in perinatally HIV-1-infected children.

This study examined the effect of perinatal HIV-1 infection on emerging executive skills in children (n = 161) ages 8 to 12 years. HIV-positive (n = 76) and HIV-negative (n = 85) children were eligible to participate. The HIV-positive children included those who had experienced a CDC Class C event (greater severity, n = 22) and those who were HIV-positive but who had not experienced a CDC Class C event (less severity, n = 54). Measures of emerging executive functions completed by the children included subtests from the Developmental Neuropsychological Assessment (NEPSY), the Trail-Making Test-Part B, and a subtest from the Woodcock-Johnson Battery-Revised. Ratings of executive functions were obtained from caretakers using the Behavior Rating Inventory of Executive Functions. Generalized estimating equations methods, discriminate analyses, and global deficit score analyses were performed to determine whether differences emerged between the three clinical groups while using strict controls. The present results revealed significant group differences in unadjusted mean scores measuring executive functioning. However, such differences did not remain statistically significant when moderating variables were taken into consideration in the models. The apparent deficit in executive functioning for the HIV-positive children was found to be largely due to differential psychosocial and environmental factors rather than HIV disease and its severity, and in this cohort, the effects of HIV-1 infection on emerging executive functions appeared to be negligible when controlling for treatment and moderating psychosocial variables.

Interferon-dependent IL-10 production by Tregs limits tumor Th17 inflammation.

The capacity of IL-10 and Tregs in the inflammatory tumor microenvironment to impair anticancer Th1 immunity makes them attractive targets for cancer immunotherapy. IL-10 and Tregs also suppress Th17 activity, which is associated with poor prognosis in several cancers. However, previous studies have overlooked their potential contribution to the regulation of pathogenic cancer-associated inflammation. In this study, we investigated the origin and function of IL-10­producing cells in the tumor microenvironment using transplantable tumor models in mice. The majority of tumor-associated IL-10 was produced by an activated Treg population. IL-10 production by Tregs was required to restrain Th17-type inflammation. Accumulation of activated IL-10+ Tregs in the tumor required type I IFN signaling but not inflammatory signaling pathways that depend on TLR adapter protein MyD88 or IL-12 family cytokines. IL-10 production limited Th17 cell numbers in both spleen and tumor. However, type I IFN was required to limit Th17 cells specifically in the tumor microenvironment, reflecting selective control of tumor-associated Tregs by type I IFN. Thus, the interplay of type I IFN, Tregs, and IL-10 is required to negatively regulate Th17 inflammation in the tumor microenvironment. Therapeutic interference of this network could therefore have the undesirable consequence of promoting Th17 inflammation and cancer growth.

COMPLETE-MFA: complementary parallel labeling experiments technique for metabolic flux analysis.

We have developed a novel approach for measuring highly accurate and precise metabolic fluxes in living cells, termed COMPLETE-MFA, short for complementary parallel labeling experiments technique for metabolic flux analysis. The COMPLETE-MFA method is based on combined analysis of multiple isotopic labeling experiments, where the synergy of using complementary tracers greatly improves the precision of estimated fluxes. In this work, we demonstrate the COMPLETE-MFA approach using all singly labeled glucose tracers, [1-(13)C], [2-(13)C], [3-(13)C], [4-(13)C], [5-(13)C], and [6-(13)C]glucose to determine precise metabolic fluxes for wild-type Escherichia coli. Cells were grown in six parallel cultures on defined medium with glucose as the only carbon source. Mass isotopomers of biomass amino acids were measured by gas chromatography-mass spectrometry (GC-MS). The data from all six experiments were then fitted simultaneously to a single flux model to determine accurate intracellular fluxes. We obtained a statistically acceptable fit with more than 300 redundant measurements. The estimated flux map is the most precise flux result obtained thus far for E. coli cells. To our knowledge, this is the first time that six isotopic labeling experiments have been successfully integrated for high-resolution (13)C-flux analysis.

Parallel labeling experiments with [U-13C]glucose validate E. coli metabolic network model for 13C metabolic flux analysis.

(13)C-metabolic flux analysis (MFA) is a widely used method for measuring intracellular metabolic fluxes in living cells. (13)C MFA relies on several key assumptions: (1) the assumed metabolic network model is complete, in that it accounts for all significant enzymatic and transport reactions; (2) (13)C-labeling measurements are accurate and precise; and (3) enzymes and transporters do not discriminate between (12)C- and (13)C-labeled metabolites. In this study, we tested these inherent assumptions of (13)C MFA for wild-type E. coli by parallel labeling experiments with [U-(13)C]glucose as tracer. Cells were grown in six parallel cultures in custom-constructed mini-bioreactors, starting from the same inoculum, on medium containing different mixtures of natural glucose and fully labeled [U-(13)C]glucose, ranging from 0% to 100% [U-(13)C]glucose. Macroscopic growth characteristics of E. coli showed no observable kinetic isotope effect. The cells grew equally well on natural glucose, 100% [U-(13)C]glucose, and mixtures thereof. (13)C MFA was then used to determine intracellular metabolic fluxes for several metabolic network models: an initial network model from literature; and extended network models that accounted for potential dilution effects of isotopic labeling. The initial network model did not give statistically acceptable fits and produced inconsistent flux results for the parallel labeling experiments. In contrast, an extended network model that accounted for dilution of intracellular CO(2) by exchange with extracellular CO(2) produced statistically acceptable fits, and the estimated metabolic fluxes were consistent for the parallel cultures. This study illustrates the importance of model validation for (13)C MFA. We show that an incomplete network model can produce statistically unacceptable fits, as determined by a chi-square test for goodness-of-fit, and return biased metabolic fluxes. The validated metabolic network model for E. coli from this study can be used in future investigations for unbiased metabolic flux measurements.

Molecular and organismal changes in offspring of male mice treated with chemical stressors.

Both gene methylation changes and genetic instability have been noted in offspring of male rodents exposed to radiation or chemicals, but few specific gene targets have been established. Previously, we identified the gene for ribosomal RNA, rDNA, as showing methylation change in sperm of mice treated with the preconceptional carcinogen, chromium(III) chloride. rDNA is a critical cell growth regulator. Here, we investigated the effects of paternal treatments on rDNA in offspring tissue. A total of 93 litters and 758 offspring were obtained, permitting rigorous mixed-effects models statistical analysis of the results. We show that the offspring of male mice treated with Cr(III) presented increased methylation in a promoter sequence of the rDNA gene, specifically in lung. Furthermore polymorphic variants of the multi-copy rDNA genes displayed altered frequencies indicative of structural changes, as a function of both tissue type and paternal treatments. Organismal effects also occurred: some groups of offspring of male mice treated with either Cr(III) or its vehicle, acidic saline, compared with those of untreated mice, had altered average body and liver weights and levels of serum glucose and leptin. Males treated directly with Cr(III) or acidic saline presented serum hormone changes consistent with a stress response. These results establish for the first time epigenetic and genetic instability effects in a gene of central physiological importance, in offspring of male mice exposed preconceptionally to chemicals, possibly related to a stress response in these males.

Sequence analysis of Kaposi sarcoma-associated herpesvirus (KSHV) microRNAs in patients with multicentric Castleman disease and KSHV-associated inflammatory cytokine syndrome.

BACKGROUND: Kaposi sarcoma-associated herpesvirus (KSHV) encodes 12 pre-microRNAs that yield 25 mature microRNAs. We previously reported phylogenetic analysis of the microRNA-coding region of KSHV from Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD) patients. We observed a high level of conservation for most sequences but also a divergent cluster of 5 KSHV sequences, including 2 from MCD patients. METHODS: KSHV microRNA sequences from 23 MCD patients and 7 patients with a newly described KSHV-associated inflammatory cytokine syndrome (KICS) were examined by amplification, cloning, and sequencing of a 646-bp fragment of K12/T0.7 encoding microRNA-K12-10 and microRNA-K12-12 and a 2.8-kbp fragment containing the remaining 10 pre-microRNAs. RESULTS: Phylogenetic analysis showed a distinct variant cluster consisting exclusively of MCD and KICS patients in all trees. Pearson χ(2) analysis revealed that 40 single-nucleotide polymorphisms (SNPs) at various loci were significantly associated with MCD and KICS risk. Cluster analysis of these SNPs generated several combinations of 3 SNPs as putative indicators of MCD and KICS risk. CONCLUSIONS: These findings show that MCD and KICS patients frequently have unusual KSHV microRNA sequences and suggest an association between the observed sequence variation and risk of MCD and KICS.

Dynamic metabolic flux analysis (DMFA): a framework for determining fluxes at metabolic non-steady state.

Metabolic flux analysis (MFA) is a key tool for measuring in vivo metabolic fluxes in systems at metabolic steady state. Here, we present a new method for dynamic metabolic flux analysis (DMFA) of systems that are not at metabolic steady state. The advantages of our DMFA method are: (1) time-series of metabolite concentration data can be applied directly for estimating dynamic fluxes, making data smoothing and estimation of average extracellular rates unnecessary; (2) flux estimation is achieved without integration of ODEs, or iterations; (3) characteristic metabolic phases in the fermentation data are identified automatically by the algorithm, rather than selected manually/arbitrarily. We demonstrate the application of the new DMFA framework in three example systems. First, we evaluated the performance of DMFA in a simple three-reaction model in terms of accuracy, precision and flux observability. Next, we analyzed a commercial glucose-limited fed-batch process for 1,3-propanediol production. The DMFA method accurately captured the dynamic behavior of the fed-batch fermentation and identified characteristic metabolic phases. Lastly, we demonstrate that DMFA can be used without any assumed metabolic network model for data reconciliation and detection of gross measurement errors using carbon and electron balances as constraints.