In silico prediction and in vivo testing of promoters targeting GABAergic inhibitory neurons.

Impairment of GABAergic inhibitory neuronal function is linked to epilepsy and other neurological and psychiatric disorders. Recombinant adeno-associated virus (rAAV)-based gene therapy targeting GABAergic neurons is a promising treatment for GABA-associated disorders. However, there is a need to develop rAAV-compatible gene-regulatory elements capable of selectively driving expression in GABAergic neurons throughout the brain. Here, we designed several novel GABAergic gene promoters. In silico analyses, including evolutionarily conserved DNA sequence alignments and transcription factor binding site searches among GABAergic neuronal genes, were carried out to reveal novel sequences for use as rAAV-compatible promoters. rAAVs (serotype 9) were injected into the CSF of neonatal mice and into the brain parenchyma of adult mice to assess promoter specificity. In mice injected neonatally, transgene expression was detected in multiple brain regions with very high neuronal specificity and moderate-to-high GABAergic neuronal selectivity. The GABA promoters differed greatly in their levels of expression and, in some brain regions, showed strikingly different patterns of GABAergic neuron transduction. This study is the first report of rAAV vectors that are functional in multiple brain regions using promoters designed by in silico analyses from multiple GABAergic genes. These novel GABA-targeting vectors may be useful tools to advance gene therapy for GABA-associated disorders.

GABAergic Gene Regulatory Elements Used in Adeno-Associated Viral Vectors.

Several neurological and psychiatric disorders have been associated with impairments in GABAergic inhibitory neurons in the brain. Thus, in the current era of accelerated development of molecular medicine and biologically-based drugs, there is a need to identify gene regulatory sequences that can be utilized for selectively manipulating the expression of nucleic acids and proteins in GABAergic neurons. This is particularly important for the use of viral vectors in gene therapy. In this Mini Review, we discuss the use of various gene regulatory elements for targeting GABAergic neurons, with an emphasis on adeno-associated viral vectors, the most widely used class of viral vectors for treating brain diseases.

Targeting DNA Damage Response and Repair to Enhance Therapeutic Index in Cisplatin-Based Cancer Treatment.

Platinum-based chemotherapies, such as cisplatin, play a large role in cancer treatment. The development of resistance and treatment toxicity creates substantial barriers to disease control, yet. To enhance the therapeutic index of cisplatin-based chemotherapy, it is imperative to circumvent resistance and toxicity while optimizing tumor sensitization. One of the primary mechanisms by which cancer cells develop resistance to cisplatin is through upregulation of DNA repair pathways. In this review, we discuss the DNA damage response in the context of cisplatin-induced DNA damage. We describe the proteins involved in the pathways and their roles in resistance development. Common biomarkers for cisplatin resistance and their utilization to improve patient risk stratification and treatment personalization are addressed. Finally, we discuss some of the current treatments and future strategies to circumvent the development of cisplatin resistance.

Impact of periprocedural morphine use on mortality in STEMI patients treated with primary PCI.

BACKGROUND: Intravenous morphine (MO) decreases the effect of all oral platelet P2Y12 receptor inhibitors in vitro and observational reports suggest that its use may be associated with larger infarct size. Yet, there are limited data available about the impact of this interaction on clinical outcomes. We studied the effect of MO on mortality in ST-segment elevation myocardial infarction (STEMI) patients treated with primary PCI using a prospective registry. METHODS: Of the 1255 patients who underwent primary PCI, 397 received MO based on physician's judgment. Clopidogrel was used as P2Y12 receptor antagonist in all cases. Median follow-up time was 7.5 years with 457 deaths. To adjust for confounding, two propensity score-based procedures were performed: 1 to 1 matching (PSM, 728 cases), and inverse probability of treatment weighting (IPTW) retaining data from all patients. Primary outcome measure was time to all-cause death, whereas predischarge left ventricular ejection fraction (LVEF) was used as secondary end point. RESULTS: An adequate balance on baseline covariates was achieved by both methods. We found no difference in survival as the HR (MO/no MO) was 0.98 (95% confidence interval [CI]: 0.76-1.26), p = 0.86 using PSM and 1.01 (95% CI: 0.84-1.23), p = 0.88 with IPTW. Likewise, distributions of LVEFs were similar using either methods: with PSM, median LVEFs were 50.0% (interquartile range [IQR]: 43.0%-55.3%) vs 50.0% (IQR: 42.0%-55.0%) in the no MO and MO groups, respectively (p = 0.76), whereas using IPTW, they were 50.0% (IQR: 42.5%-55.0%) vs 50.0% (IQR: 41.0%-55.0%), respectively (p = 0.86). CONCLUSIONS: Our data suggest that morphine use may have no impact on long-term mortality and on predischarge ejection fraction in STEMI patients treated with primary PCI.

The Phylum Bryozoa: From Biology to Biomedical Potential.

Less than one percent of marine natural products characterized since 1963 have been obtained from the phylum Bryozoa which, therefore, still represents a huge reservoir for the discovery of bioactive metabolites with its ~6000 described species. The current review is designed to highlight how bryozoans use sophisticated chemical defenses against their numerous predators and competitors, and which can be harbored for medicinal uses. This review collates all currently available chemoecological data about bryozoans and lists potential applications/benefits for human health. The core of the current review relates to the potential of bryozoan metabolites in human diseases with particular attention to viral, brain, and parasitic diseases. It additionally weighs the pros and cons of total syntheses of some bryozoan metabolites versus the synthesis of non-natural analogues, and explores the hopes put into the development of biotechnological approaches to provide sustainable amounts of bryozoan metabolites without harming the natural environment.

Proprotein convertase 7 (PCSK7) reduces apoA-V levels.

The locus of the human proprotein convertase subtilisin-kexin type-7 (PC7) gene (PCSK7) is on chromosome 11q23.3 close to the gene cluster APOA5/APOA4/APOC3/APOA1, a region implicated in the regulation of lipoprotein metabolism. A GWAS reported the association of PCSK7 SNPs with plasma triglyceride (TG), and exome sequencing of African Americans revealed the association of a low-frequency coding variant of PC7 (R504H; SNP rs142953140) with a ~ 30% TG reduction. Another PCSK7 SNP rs508487 is in linkage disequilibrium with a promoter variant of the liver-derived apolipoprotein A-V (apoA-V), an indirect activator of the lipoprotein lipase (LpL), and is associated with elevated TG levels. We thus hypothesized that PC7 regulates the levels/activity of apoA-V. Studies in the human hepatic cell line HuH7 revealed that wild-type (WT) PC7 and its endoplasmic reticulum (ER)-retained forms bind to and enhance the degradation of human apoA-V in acidic lysosomes in a nonenzymatic fashion. PC7-induced degradation of apoA-V is inhibited by bafilomycin A1 and the alkalinizing agents: chloroquine and NH4 Cl. Thus, the PC7-induced apoA-V degradation implicates an ER-lysosomal communication inhibited by bafilomycin A1. In vitro, the natural R504H mutant enhances PC7 Ser505 phosphorylation at the structurally exposed Ser-X-Glu507 motif recognized by the secretory kinase Fam20C. Co-expression of the phosphomimetic PC7-S505E with apoA-V resulted in lower degradation compared to WT, suggesting that Ser505 phosphorylation of PC7 lowers TG levels via reduced apoA-V degradation. In agreement, in Pcsk7-/- mice fed high-fat diet, plasma apoA-V levels and adipocyte LpL activity are increased, providing an in vivo mechanistic link for a role of liver PC7 in enhanced TG storage in adipocytes.

Long-term ticagrelor for secondary prevention in patients with prior myocardial infarction and no history of coronary stenting: insights from PEGASUS-TIMI 54.

AIMS: PEGASUS-TIMI 54 demonstrated that long-term dual antiplatelet therapy (DAPT) with aspirin and ticagrelor reduced the risk of major adverse cardiovascular events (MACE), with an acceptable increase in bleeding, in patients with prior myocardial infarction (MI). While much of the discussion around prolonged DAPT has been focused on stented patients, patients with prior MI without prior coronary stenting comprise a clinically important subgroup. METHODS AND RESULTS: This was a pre-specified analysis from PEGASUS-TIMI 54, which randomized 21 162 patients with prior MI (1-3 years) and additional high-risk features to ticagrelor 60 mg, 90 mg, or placebo twice daily in addition to aspirin. A total of 4199 patients had no history of coronary stenting at baseline. The primary efficacy outcome (MACE) was the composite of cardiovascular death, MI, or stroke. Patients without history of coronary stenting had higher baseline risk of MACE [13.2% vs. 8.0%, adjusted hazard ratio (HR) 1.41, 95% confidence interval (CI) 1.15-1.73, in the placebo arm]. The relative risk reduction in MACE with ticagrelor (pooled doses) was similar in patients without (HR 0.82, 95% CI 0.68-0.99) and with prior stenting (HR 0.85, 95% CI 0.75-0.96; P for interaction = 0.76). CONCLUSION: Long-term ticagrelor reduces thrombotic events in patients with prior MI regardless of whether they had prior coronary stenting. These data highlight the benefits of DAPT in prevention of spontaneous atherothrombotic events and indicate that long-term ticagrelor may be considered in high-risk patients with prior MI even if they have not been treated with stenting. CLINICALTRIALS.GOV IDENTIFIER: NCT01225562.

Renal Function and Outcomes With Dabigatran Dual Antithrombotic Therapy in Atrial Fibrillation Patients After PCI.

OBJECTIVES: The study sought to evaluate the effect of dabigatran dual therapy versus warfarin triple therapy across categories of renal function in the RE-DUAL PCI (Randomized Evaluation of Dual Antithrombotic Therapy with Dabigatran versus Triple Therapy with Warfarin in Patients with Nonvalvular Atrial Fibrillation Undergoing Percutaneous Coronary Intervention) trial. BACKGROUND: The RE-DUAL PCI (NCT02164864) trial of patients with atrial fibrillation undergoing percutaneous coronary intervention reported that dabigatran dual therapy (110 or 150 mg twice daily, plus clopidogrel or ticagrelor) reduced the primary endpoint of major bleeding events (MBE) or clinically relevant nonmajor bleeding events (CRNMBE) compared with warfarin triple therapy, with noninferiority in overall thromboembolic events. METHODS: Risk of a first MBE or CRNMBE and the composite of death or thromboembolic event (DTE) or unplanned revascularization were evaluated in 2,725 patients according to baseline creatinine clearance (CrCl) categories: 30 to <50, 50 to <80, and ≥80 ml/min. RESULTS: Compared with warfarin, dabigatran 110 mg dual therapy reduced risk of MBE or CRNMBE across all categories of CrCl (p for interaction = 0.19). Dabigatran 150 mg dual therapy reduced risk of MBE or CRNMBE regardless of the CrCl category (p for interaction = 0.31). Risk of DTE or unplanned revascularization was similar to warfarin triple therapy for dabigatran 110 mg dual therapy across all CrCl categories. Dabigatran 150 mg dual therapy versus warfarin triple therapy had similar risk for DTE or unplanned revascularization in patients with CrCl 30 to <80 ml/min and lower risk at CrCl ≥80 ml/min (p for interaction = 0.02). CONCLUSIONS: In the RE-DUAL PCI trial, dabigatran dual therapy reduced bleeding events versus warfarin triple therapy irrespective of renal function, with overall similar risks of thromboembolic events but lower risks with dabigatran 150 mg in patients with normal CrCl.

Efficacy and safety with ticagrelor in patients with prior myocardial infarction in the approved European label: insights from PEGASUS-TIMI 54.

AIMS: In PEGASUS-TIMI 54, ticagrelor significantly reduced the risk of the composite of major adverse cardiovascular (CV) events by 15-16% in stable patients with a prior myocardial infarction (MI) 1-3 years earlier. We report the efficacy and safety in the subpopulation recommended for treatment in the European (EU) label, i.e. treatment with 60 mg b.i.d. initiated up to 2 years from the MI, or within 1 year after stopping previous adenosine diphosphate receptor inhibitor treatment. METHODS AND RESULTS: Of the 21 162 patients enrolled in PEGASUS-TIMI 54, 10 779 patients were included in the primary analysis for this study, randomized to ticagrelor 60 mg (n = 5388) or matching placebo (n = 5391). The cumulative proportions of patients with events at 36 months were calculated by the Kaplan-Meier (KM) method. The composite of CV death, MI, or stroke occurred less frequently in the ticagrelor group (7.9% KM rate vs. 9.6%), hazard ratio (HR) 0.80 [95% confidence interval (CI) 0.70-0.91; P = 0.001]. Ticagrelor also reduced the risk of all-cause mortality, HR 0.80 (0.67-0.96; P = 0.018). Thrombolysis in myocardial infarction major bleeding was more frequent in the ticagrelor group 2.5% vs. 1.1%; HR 2.36 (1.65-3.39; P < 0.001). The corresponding HR for fatal or intracranial bleeding was 1.17 (0.68-2.01; P = 0.58). CONCLUSION: In PEGASUS-TIMI 54, treatment with ticagrelor 60 mg as recommended in the EU label, was associated with a relative risk reduction of 20% in CV death, MI, or stroke. Thrombolysis in myocardial infarction major bleeding was increased, but fatal or intracranial bleeding was similar to placebo. There appears to be a favourable benefit-risk ratio for long-term ticagrelor 60 mg in this population. CLINICAL TRIAL REGISTRATION: http://www.clinicaltrials.gov NCT01225562.

Halogenated Diterpenes with In Vitro Antitumor Activity from the Red Alga Sphaerococcus coronopifolius.

Eight new (1-8) structurally diverse diterpenes featuring five different carbocycles were isolated from the organic extracts of the red alga Sphaerococcus coronopifolius collected from the coastline of the Ionian Sea in Greece. The structures of the new natural products, seven of which were halogenated, and the relative configuration of their stereocenters were determined on the basis of comprehensive spectroscopic analyses, including NMR and HRMS data. Compounds 5 and 8 were found to possess in vitro antitumor activity against one murine and five human cancer cell lines with mean IC50 values 15 and 16 µM, respectively.

Algae metabolites: from in vitro growth inhibitory effects to promising anticancer activity.

Covering: 1957 to 2017 Algae constitute a heterogeneous group of eukaryotic photosynthetic organisms, mainly found in the marine environment. Algae produce numerous metabolites that help them cope with the harsh conditions of the marine environment. Because of their structural diversity and uniqueness, these molecules have recently gained a lot of interest for the identification of medicinally useful agents, including those with potential anticancer activities. In the current review, which is not a catalogue-based one, we first highlight the major biological events that lead to various types of cancer, including metastatic ones, to chemoresistance, thus to any types of current anticancer treatment relating to the use of chemotherapeutics. We then review algal metabolites for which scientific literature reports anticancer activity. Lastly, we focus on algal metabolites with promising anticancer activity based on their ability to target biological characteristics of cancer cells responsible for poor treatment outcomes. Thus, we highlight compounds that have, among others, one or more of the following characteristics: selectivity in reducing the proliferation of cancer cells over normal ones, potential for killing cancer cells through non-apoptotic signaling pathways, ability to circumvent MDR-related efflux pumps, and activity in vivo in relevant pre-clinical models.

Glioblastoma quo vadis: Will migration and invasiveness reemerge as therapeutic targets?

PURPOSE: The purpose of the current review is to highlight, on one hand, the fact that the migratory pattern of glioma cells is the major obstacle to combat them with chemotherapy, and on the other one, the new treatment strategies to overcome this obstacle. METHODS: This review surveys several membrane and extracellular molecules involved in glioma cell migration, invasiveness and resistance to apoptosis. RESULTS: This review focuses on signaling pathways implicated in the positive regulation of glioblastoma cell migration, including glutamate and ion channel networks, microtubes and membrane-derived extracellular vesicles (EV) containing microRNAs. Glioma cells release glutamate to the extracellular matrix, inducing neuronal cell death, which may facilitate glioma growth and invasion. Glioma cell migration and invasion are further facilitated through ion channels and transporters that modify cellular volume. Microtubes and EV promote connections and communication among glioma cells and with the microenvironment and are associated with progression and resistance to therapy. Potential therapies linked to these pathways for glioblastoma are being developed. CONCLUSION: Our view is evolving from an intracellular view of the complex intracellular signaling pathways to one of orchestral machinery, including connections between heterogeneous tumoral and nontumoral cells and with the microenvironment through channels, microtubes, and extracellular miRNA, generating different signals at different times. All of these elements give rise to a new perspective for the treatment of glioblastoma.

Effect of cell culture medium additives on color and acidic charge variants of a monoclonal antibody.

This manuscript summarizes the effect of certain cell culture medium additives on antibody drug substance coloration and acidic charge variants. It has been shown previously that B-vitamins and iron in the cell culture medium could significantly impact color intensity. In this manuscript, we detail the effect of several other cell culture components that have been shown to impact coloration. It is shown that if cystine is used instead of cysteine in the cell culture medium, coloration was reduced. Hydrocortisone has been shown to reduce coloration and boost specific productivity. The effect of a peptone/hydrolysate on coloration was investigated in cell culture experiments, which showed its use can lead to reduced coloration. Mechanisms by which these compounds influence coloration will be briefly discussed. Since it has been previously shown that antibody oxidation could potentially lead to coloration, the current effort was focused on screening for specific antioxidant additives to the culture medium to reduce coloration. An in-vitro incubation model was used to screen antioxidant compounds, several of which were found to significantly reduce antibody color, while some led to significantly increased color. Hypotaurine and carboxymethylcysteine, which had the most significant color reducing effect in the incubation study, were further tested in small-scale bioreactor cell culture experiments. These studies demonstrated that these compounds lead to reduced coloration in cell culture without affecting cell growth and titer. Hypotaurine, hydrocortisone, peptone, and cystine were also shown to reduce the acidic charge variant levels, which was previously shown to correlate with color. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018 © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1298-1307, 2018.

An Introduction to "Recent Trends in the Biotechnology Industry: Development and Manufacturing of Recombinant Antibodies and Proteins".

The production of the first therapeutic proteins in the early 1980s heralded the launch of the biopharmaceuticals industry. The number of approved products has grown year on year over the past three decades to now represent a significant share of the entire pharmaceuticals market. More than 200 therapeutic proteins have been approved, approximately a quarter of which are represented by monoclonal antibodies and their derivatives. In 2016, the list of the top 15 best-selling drugs included more than eight biologics and in 2020 the trend will continue, with more than 50% of the top 20 best-selling drugs predicted to be biologics. From 1986 to 2014 several first-in-class, advance-in-class, and breakthrough designated therapeutic options were approved, with advanced therapies such as immuno-oncology and cell-based therapies being approved for several indications.

Effect of polygodial and its direct derivatives on the mammalian Na+/K+-ATPase activity.

The sesquiterpene polygodial is an agonist of the transient receptor potential vanilloid 1 (TRPV1). Our group recently reported the synthesis and anticancer effects of polygodial and its derivatives, and showed that these compounds retain activity against apoptosis- and multidrug-resistant cancer cells. Herein, we tested the inhibitory effect of these compounds on the activity of the enzyme Na+/K+-ATPase (NKA) from kidney (α1 isoform) and brain (α2 and α3 isoforms) guinea pig extracts. Polygodial (1) displayed a dose-dependent inhibition of both kidney and brain purified NKA preparations, with higher sensitivity for the cerebral isoforms. Polygo-11,12-diol (2) and C11,C12-pyridazine derivative (3) proved to be poor inhibitors. Unsaturated ester (4) and 9-epipolygodial (5) inhibited NKA preparations from brain and kidney, with the same inhibitory potency. Nevertheless, they did not achieve maximum inhibition even at higher concentration. Comparing the inhibitory potency in crude homogenates and purified preparations of NKA, compounds 4 and 5 revealed a degree of selectivity toward the renal enzyme. Kinetic studies showed a non-competitive inhibition for Na+ and K+ by compounds 1, 4 and 5 and for ATP by 1 and 4. However, compound 5 presented a competitive inhibition type. Furthermore, K+-activated p-nitrophenylphosphatase activity of these purified preparations was not inhibited by 1, 4 and 5, suggesting that these compounds acted in the initial phase of the enzyme's catalytic cycle. These findings suggest that the antitumor action of polygodial and its analogues may be linked to their NKA inhibitory properties and reinforce that NKA may be an important target for cancer therapy.

Comparison of Platelet Function Guided Versus Unguided Treatment With P2Y12 Inhibitors in Patients With Acute Myocardial Infarction (from the Hungarian Myocardial Infarction Registry).

Evidence is conflicting regarding the clinical benefits of selecting P2Y12 inhibitors based on platelet function testing (PFT). Between March 1, 2013 and March 1, 2014, we collected clinical characteristics and platelet function data in a nationwide acute myocardial infarction (AMI) registry from 15 interventional cardiology centers in Hungary. The risk of all-cause mortality at 1 year were compared after propensity score (PS) matching between patients receiving PFT-guided and unguided P2Y12-inhibitor therapies. High platelet reactivity on clopidogrel (HPRoC) was uniformly defined with the Multiplate assay. A total of 5,583 patients with AMI and coronary intervention were registered. After exclusion of cases with contraindication to prasugrel, propensity matching resulted in a sample of 2,104 patients with well-adjusted characteristics. Clopidogrel was the dominant P2Y12 inhibitor in both groups (unguided: 96% vs PFT guided: 85%, p <0.001). In the PFT-guided group, 19% of patients had HPRoC and 77% of them were switched to prasugrel. According to the adjusted analysis, all-cause mortality at 1 year was significantly lower in the PFT-guided compared with the unguided group (hazard ratio 0.57 [95% confidence interval 0.43 to 0.77], p <0.001). Although prasugrel treatment was not associated with lower all-cause mortality in the overall cohort, patients with HPRoC who switched to prasugrel had significantly lower mortality when compared with those continuing clopidogrel (hazard ratio 0.33 [95% confidence interval 0.12 to 0.92], p <0.05). In conclusion, in patients with AMI, PFT-guided treatment with a high rate of switchover to prasugrel was associated with a lower risk of mortality. Prasugrel was a predictor of lower mortality in patients with HPRoC but not in the overall cohort of AMI.

[Steroid 21-hydroxylase deficiency, the most frequent cause of congenital adrenal hyperplasia]. / Szteroid-21-hidroxiláz-deficientia, a congenitalis adrenalis hyperplasia leggyakoribb oka.

Congenital adrenal hyperplasia is a group of genetic diseases due to the disablement of 7 genes; one of them is steroid 21-hydroxylase deficiency. The genes of congenital adrenal hyperplasia encode enzymes taking part in the steroidogenesis of adrenal gland. Steroid 21-hydroxylase deficiency is an autosomal recessive disorder caused by mutations of the steroid 21-hydroxylase gene. The mutations of steroid 21-hydroxylase gene cause 95% of the congenital adrenal hyperplasia cases. Although the non-classic steroid 21-hydroxylase deficiency with mild symptoms is seldom diagnosed, the classic steroid 21-hydroxylase deficiency may lead to life-threatening salt-wasting and adrenal crises due to the insufficient aldosterone and cortisol serum levels. The classic type requires life-long steroid replacement which may result in cushingoid side effects, and typical comorbidities may be also developed. The patients' quality of life is decreased, and their mortality is much higher than that of the population without steroid 21-hydroxylase deficiency. The diagnosis, consequences and the patients' life-long clinical care require a multidisciplinary approach: the specialists in pediatrics, internal medicine, endocrinology, laboratory medicine, genetic diagnostics, surgery, obstetrics-gynecology and psychology need to work together. Orv Hetil. 2018; 159(7): 269-277.

The Fungal Metabolite Eurochevalierine, a Sequiterpene Alkaloid, Displays Anti-Cancer Properties through Selective Sirtuin 1/2 Inhibition.

NAD⁺-dependent histone deacetylases (sirtuins) are implicated in cellular processes such as proliferation, DNA repair, and apoptosis by regulating gene expression and the functions of numerous proteins. Due to their key role in cells, the discovery of small molecule sirtuin modulators has been of significant interest for diverse therapeutic applications. In particular, it has been shown that inhibition of sirtuin 1 and 2 activities is beneficial for cancer treatment. Here, we demonstrate that the fungal metabolite eurochevalierine from the fungus Neosartorya pseudofischeri inhibits sirtuin 1 and 2 activities (IC50 about 10 µM) without affecting sirtuin 3 activity. The binding modes of the eurochevalierine for sirtuin 1 and 2 have been identified through computational docking analyses. Accordingly, this sequiterpene alkaloid induces histone H4 and α-tubulin acetylation in various cancer cell models in which it induces strong cytostatic effects without affecting significantly the viability of healthy PBMCs. Importantly, eurochevalierine targets preferentially cancer cell proliferation (selectivity factor ≫ 7), as normal human primary CD34⁺ stem/progenitor cells were less affected by the treatment. Finally, eurochevalierine displays suitable drug-likeness parameters and therefore represent a promising scaffold for lead molecule optimization to study the mechanism and biological roles of sirtuins and potentially a basis for development into therapeutics.

Risk Mitigation in Preventing Adventitious Agent Contamination of Mammalian Cell Cultures.

Industrial-scale mammalian cell culture processes have been contaminated by viruses during the culturing phase. Although the historical frequency of such events has been quite low, the impact of contamination can be significant for the manufacturing company and for the supply of the product to patients. This chapter discusses sources of adventitious agent contamination risk in a cell culture process, provides a semiquantitative assessment of such risks, and describes potential process barriers that can be used to reduce contamination risk. High-temperature, short-time (HTST) heat treatment is recommended as the process barrier of choice, when compatible with the process. A case study assessing the compatibility of HTST heat treatment with a cell culture medium is presented, and lessons learned are shared from our experiences over many years of developing and implementing virus barriers in mammalian cell culture processes. Graphical Abstract.