cGMP Regulation Research Articles

Downregulation of miR-214 promotes dilated Cardiomyopathy Progression through PDE5A-Mediated cGMP regulation.

Dilated cardiomyopathy (DCM) is a myocardial disorder resulting in a substantial decline in cardiac function and potentially leading to heart failure. This research combines bioinformatics analysis with empirical validation to explore the roles and mechanisms of miR-214 in DCM. Using the DEseq2 R package, a total of 125 differentially expressed circulating miRNAs (DE c-miRNAs) and 784 DE genes (DEGs) were identified. Cross-analysis between target genes of DE c-miRNAs and DEGs identified 124 common genes, and protein-protein interaction analysis of common genes identified 11 hub genes. Twelve DE c-miRNAs were further verified by quantifying their levels in the serum of DCM patients and healthy individuals. miR-214 levels were significantly decreased in serum from DCM patients, positively correlated with left ventricular ejection fraction and left ventricular fractional shortening. Further analysis showed that miR-214 directly targets and negatively regulates phosphodiesterase 5A (PDE5A). Elevated PDE5A expression reduced cGMP levels; however, using sildenafil, a PDE5A inhibitor, reversed this effect, substantiating the regulatory mechanism of miR-214 on cGMP via PDE5A. These results provide new potential targets for the diagnosis and treatment of DCM.

Scalable bioprocess for high-yield production of SARS-CoV-2 trimeric spike protein-based immunogen (IMT-CVAX) using suspension CHO cells

The COVID-19 pandemic has brought global attention towards the readiness of biotechnology platforms for rapid development of immunogens to be used as vaccine antigens, in diagnostics and in fundamental research. In the present study we provide a bioprocess that is both robust and industry-compatible for high level expression, efficient purification, and scale-up of IMT-CVAX, a prefusion-stabilized trimeric spike protein of SARS-CoV-2. The recombinant IMT-CVAX protein was produced using stable cell pool of Chinese Hamster Ovary (CHO-S) cells that were banked in compliance with cGMP (Current Good Manufacturing Practices) regulations. High quantity expression of IMT-CVAX was achieved through optimization of the shake flask-based process with regards to media, nutrient feeding regimen, incubation temperature, viable cell density and cell viability. We employed refined expression procedures to scale up IMT-CVAX using bioreactor, resulting in a significantly higher yield of around 500 mg/L. Subsequently, a straightforward and simple purification process was developed to produce high-quality IMT-CVAX protein. This procedure consisted of centrifugation, tangential flow filtration (TFF), and either one- or two-step liquid chromatography. A robust anti-spike IgG response was observed in mice after immunization with purified adjuvanted IMT-CVAX. In pseudoviral neutralization assay, mice-generated anti-IMT-CVAX sera could neutralize various SARS-CoV-2 variants. The human convalescent sera from COVID-19-recovered patients recognized IMT-CVAX effectively, which confirms the conformational integrity of IMT-CVAX epitopes. The bioprocess demonstrated here is able to produce sufficient quantities of biopharmaceutical-quality spike protein, which can be used as immunogen against emerging SARS-CoV-2 variants, and can aid in the rapid response to any other pandemic-potential pathogens.

The Combination of Oolonghomobisflavan B and Diallyl Disulfide Induces Apoptotic Cell Death via 67-kDa Laminin Receptor/Cyclic Guanosine Monophosphate in Acute Myeloid Leukemia Cells.

Diallyl disulfide (DADS) is a well-known principal functional component derived from garlic (Allium sativum) that has various health benefits. Previously, we identified a 67-kDa laminin receptor, a receptor for oolong tea polyphenol oolonghomobisflavan B (OHBFB). However, its molecular mechanisms still remain to be elucidated. Here, we show that DADS synergistically enhanced the effect of the oolong tea polyphenol oolonghomobisflavan B (OHBFB), which induces apoptosis in acute myeloid leukemia (AML) cancer cells without affecting normal human peripheral blood mononuclear cells (PBMCs). The underlying mechanism of OHBFB-induced anti-AML effects involves the upregulation of the 67-kDa laminin receptor/endothelial nitric oxide synthase/cyclic guanosine monophosphate (cGMP)/protein kinase c delta (PKCδ)/acid sphingomyelinase (ASM)/cleaved caspase-3 signaling pathway. In conclusion, we show that the combination of OHBFB and DADS synergistically induced apoptotic cell death in AML cells through activation of 67LR/cGMP/PKCδ/ASM signaling pathway. Moreover, in this mechanism, we demonstrate DADS may reduce the enzyme activity of phosphodiesterase, which is a negative regulator of cGMP that potentiates OHBFB-induced AML apoptotic cell death without affecting normal PBMCs.

Analysis of cGMP Signaling in Dictyostelium.

Biochemical assays are described to analyze signal transduction by the second messenger cGMP in Dictyostelium. The methods include enzyme assays to measure the activity and regulation of cGMP synthesizing guanylyl cyclases and cGMP-degrading phosphodiesterases. In addition, several methods are described to quantify cGMP levels. The target of cGMP in Dictyostelium is the large protein GbpC that has multiple domains including a Roc domain, a kinase domain, and a cGMP-stimulated Ras-GEF domain. A cGMP-binding assay is described to detect and quantify GbpC.

Signaling Pathway Gene Set Enrichment Analysis in Whole Exome Sequencing Data from Discordant Identical Twins Unveils Relevant Pathways As a Clue to Understand Somatic Mutation Acquisition and Clonal Evolution in Myeloproliferative Neoplasms

Although the role of classic driver mutations (i.e., JAK2, MPL, and CALR) in myeloproliferative neoplasm (MPN) development is well characterized, additional somatic mutations likely modulate the phenotype. Recurrent somatic mutations, such as those in TET2, DNMT3A, ASXL1 genes are commonly identified in MPN using targeted myeloid panels, however identification of somatic mutations using an unbiased approach may unveil novel pathways involved in MPN pathogenesis. We performed whole exome sequencing of blood samples of three sets of discordant identical twins in which one twin has MPN and the other twin is unaffected. The twin sets included all MPN subtypes (1 polycythemia vera, 1 essential thrombocythemia, 1 primary myelofibrosis). We also performed whole exome sequencing on additional family members, including the mother and father of the PV twin and the brother of the ET twin. Sequencing information from the discordant identical twins is a unique opportunity to identify somatic mutations in the affected twin that may contribute to MPN pathogenesis, and also to potentially identify clonal hematopoiesis in the unaffected twin. We mapped all the identified genetic variants to the affected genes, and we filtered all the genes mapped to those variants depending on whether they were present either in the MPN-affected twin or in the unaffected counterpart for further gene set enrichment pathway analysis. The gene set enriched signaling pathways from identified genetic variants for each condition-exclusive (either MPN or unaffected twin) allows us to identify and prioritize key pathways in MPN development in terms of somatic mutations acquisition. Furthermore, besides performing the analysis within each single MPN set, we analyzed the gene sets resulting from combining the different families allowing us not only to identify pathways at each single MPN type level but also among the different MPN diseases (PV, ET, and MF). We divided all the enrichment analyses into coding and silent variants to obtain a higher degree of pathway refinement. In single discordant twin sets we identified several pathways that may play an important role within MPN pathogenesis such as defective mismatch DNA repair, facilitated mitotic progression, extracellular matrix degradation, MAPK signaling, FGFR signaling, O-glycosylation of proteins, collagen metabolism and laminin interactions, neutrophil degranulation, interleukin-33 signaling, Rho GTPases and cGMP regulation, and nuclear pore complex (NPC) assembly. Interestingly, only when we analyze all the MPN-types together all the statistically significant pathways resulting from coding variants correspond only to DNA repair, specifically defective mismatch repair (PMS2, MLH1, MutSα, MutSβ, and TP53). Our findings reveal that DNA repair genes might occupy a central role within MPN acquisition and development, whereas at the same time each single MPN clinical entity might possess its own distinct mechanisms differentiating each one from the others.

Current Good Manufacturing Practice (cGMP): An Overview for the Analytical Chemist

Good Manufacturing Practice (GMP) is the most significant regulation impacting the pharmaceutical industry and requires substantial investment in resources and time commitments. This paper provides a high-level overview of Current Good Manufacturing Practice (cGMP) regulations and related public quality standards. It discusses GMP compliance requirements and practices, emphasizing those relevant to the analytical chemist in development and quality control.

Promises and challenges of a decentralized CAR T-cell manufacturing model.

Autologous chimeric antigen receptor-modified T-cell (CAR T) products have demonstrated un-precedent efficacy in treating many relapsed/refractory B-cell and plasma cell malignancies, leading to multiple commercial products now in routine clinical use. These positive responses to CAR T therapy have spurred biotech and big pharma companies to evaluate innovative production methods to increase patient access while maintaining adequate quality control and profitability. Autologous cellular therapies are, by definition, manufactured as single patient batches, and demand has soared for manufacturing facilities compliant with current Good Manufacturing Practice (cGMP) regulations. The use of a centralized production model is straining finite resources even in developed countries in North America and the European Union, and patient access is not feasible for most of the developing world. The idea of having a more uniform availability of these cell therapy products promoted the concept of point-of-care (POC) manufacturing or decentralized in-house production. While this strategy can potentially decrease the cost of manufacturing, the challenge comes in maintaining the same quality as currently available centrally manufactured products due to the lack of standardized manufacturing techniques amongst institutions. However, academic medical institutions and biotech companies alike have forged ahead innovating and adopting new technologies to launch clinical trials of CAR T products produced exclusively in-house. Here we discuss POC production of CAR T products.

Phosphodiesterase type 9 opposes relaxation of mouse pulmonary artery but does not influence chronic hypoxia-induced pulmonary hypertension in mouse

PDE9 is a phosphodiesterase which has recently emerged as a regulator of cGMP in cardiac and vascular myocytes. Its contribution in vasculature is, however, unclear. We sought to investigate PDE9 expression and function in mouse pulmonary artery by comparing wild type (WT) and PDE9-deficient, Pde9a(-/-), mice. We also addressed the hypothesis that PDE9 could represent a new therapeutic target to treat pulmonary arterial hypertension, a severe, deadly disease. PDE9 expression was examined in mouse tissues and human pulmonary artery from subjects with or without pulmonary arterial hypertension using RT-PCR and immunoblots. Relaxant responses were studied in mouse pulmonary arteries mounted on a myograph and contracted with phenylephrine. A challenge with 21-day, 10% O2-hypoxia was used as a model of pulmonary hypertension in mice and was compared to normoxia. Right ventricle and pulmonary artery morphology and function were determined by echocardiography, right ventricle catheterization and gravimetric analysis. PDE9 protein and Pde9a transcript were detected in human and mouse pulmonary arteries, respectively. Pulmonary arteries from Pde9a(-/-) mice displayed more potent relaxant responses to acetylcholine, to the NO donor diethylamine NONOate and to atrial natriuretic peptide than vessels from WT animals. Following hypoxia, Pde9a expression was increased in lung and right ventricle from WT mice. PDE9 deficiency failed to ameliorate pulmonary hypertension hemodynamic criteria, namely higher right ventricular systolic pressure and lower echocardiographic pulmonary arterial acceleration time. Right ventricle hypertrophy, reflected by increased Fulton index, right ventricle thickness and fetal genes expression, were also similar in Pde9a(-/-) and WT groups. PDE9 is expressed in pulmonary artery and takes part in the relaxant responses mediated by the cGMP pathway. The ablation of Pde9a in mouse, however, does not prevent the development of pulmonary hypertension in a 21-day hypoxia model, nor does it attenuate right ventricle deleterious remodeling.

A Brief Review On Cleaning Validation-Regulatory Guidelines For Pharmaceuticals

Cleaning validation is a process used by pharmaceutical manufacturers to ensure that their equipment and facilities are thoroughly cleaned and free of any residual contaminants that could potentially compromise the quality of their products. This process is guided by regulatory guidelines set forth by FDA and European Medicines Agency (EMA) are two examples of such agencies.
 Cleaning validation protocols are typically required by these guidelines, which outline cleaning procedures, acceptance criteria for evaluating the effectiveness, as well as testing and documenting the results. The protocol should also include a risk assessment to determine which equipment and areas of the facility are most critical to the product's quality and safety.
 Manufacturers are also expected to periodically review and update their cleaning validation protocols as necessary, and to keep detailed records of all cleaning validation activities. A manufacturer's cleaning validation protocol and the presence of contaminants may also be verified by FDA or EMA inspections. To ensure consistently high quality, pharmaceutical manufacturers must control cross-contamination. There must be a comprehensive cleaning regime in place to demonstrate that the methods employed in a facility consistently control the carryover of product (including intermediates and impurities), cleaning agents, and extraneous material into subsequent products. An effective cleaning regime provides documented evidence that these aspects are consistently controlled within a facility. As a pharmaceutical manufacturer, it is your responsibility to ensure that your cleaning process complies with cGMP regulations, first and foremost.

ROS and cGMP signaling modulate persistent escape from hypoxia in Caenorhabditis elegans.

The ability to detect and respond to acute oxygen (O2) shortages is indispensable to aerobic life. The molecular mechanisms and circuits underlying this capacity are poorly understood. Here, we characterize the behavioral responses of feeding Caenorhabditis elegans to approximately 1% O2. Acute hypoxia triggers a bout of turning maneuvers followed by a persistent switch to rapid forward movement as animals seek to avoid and escape hypoxia. While the behavioral responses to 1% O2 closely resemble those evoked by 21% O2, they have distinct molecular and circuit underpinnings. Disrupting phosphodiesterases (PDEs), specific G proteins, or BBSome function inhibits escape from 1% O2 due to increased cGMP signaling. A primary source of cGMP is GCY-28, the ortholog of the atrial natriuretic peptide (ANP) receptor. cGMP activates the protein kinase G EGL-4 and enhances neuroendocrine secretion to inhibit acute responses to 1% O2. Triggering a rise in cGMP optogenetically in multiple neurons, including AIA interneurons, rapidly and reversibly inhibits escape from 1% O2. Ca2+ imaging reveals that a 7% to 1% O2 stimulus evokes a Ca2+ decrease in several neurons. Defects in mitochondrial complex I (MCI) and mitochondrial complex I (MCIII), which lead to persistently high reactive oxygen species (ROS), abrogate acute hypoxia responses. In particular, repressing the expression of isp-1, which encodes the iron sulfur protein of MCIII, inhibits escape from 1% O2 without affecting responses to 21% O2. Both genetic and pharmacological up-regulation of mitochondrial ROS increase cGMP levels, which contribute to the reduced hypoxia responses. Our results implicate ROS and precise regulation of intracellular cGMP in the modulation of acute responses to hypoxia by C. elegans.

Structural Modifications of Nimodipine Lead to Novel PDE1 Inhibitors with Anti-pulmonary Fibrosis Effects.

Our previous research demonstrated that phosphodiesterase-1 (PDE1) could work as a potential target against idiopathic pulmonary fibrosis. Nimodipine, a calcium antagonist commonly used to improve hypertension, was reported to have inhibition against PDE1. Herein, a series of nimodipine analogues were discovered as novel selective and potent PDE1 inhibitors after structural modifications. Compound 2g exhibited excellent inhibitory activity against PDE1C (IC50 = 10 nM), high selectivity over other PDEs except for PDE4, and weak calcium channel antagonistic activity. Administration of compound 2g exhibited remarkable therapeutic effects in a rat model of pulmonary fibrosis induced by bleomycin and prevented myofibroblast differentiation induced by TGF-β1. The expressions of PDE1B and PDE1C were found to be increased and concentrated in the focus of fibrosis. Compound 2g increased the levels of 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) in the lungs of rats with pulmonary fibrosis, supporting the fact that the anti-fibrosis effects of 2g were through the regulation of cAMP and cGMP.

A Review on Cleaning Validation-Regulatory Guidelines for The Pharmaceutical Industry

Manufacturing of Pharmaceutical products shall demonstrate a control to reproduce consistently the desired quality of product, wherein the control of cross-contamination plays an important role. An effective cleaning shall be in place to provide documented evidence that the cleaning methods employed within a facility consistently controls potential carryover of product (including intermediates and impurities), cleaning agents and extraneous material into subsequent product to a level which is below predetermined levels. Pharmaceutical manufacturers must validate their cleaning process to ensure compliance with cGMP regulations. So it is necessary to validate the cleaning procedures to ensure safety, efficacy, quality of the subsequent batches of drug product and regulatory requirements in Pharmaceutical product manufacture. In this article cleaning validation and cleaning validation program discussed in brief.

Validation of an automated cell counting method for cGMP manufacturing of human induced pluripotent stem cells

Human induced pluripotent stem cells (hiPSCs) must be manufactured as advanced therapy medicinal products (ATMPs) for innovative tissue replacement clinical applications. Yet, production of hiPSCs under current Good Manufacturing Practice (cGMP) presents many hurdles, such as the large-scale cell expansion needed to reach therapeutically-relevant hiPSC doses. For the monitoring of this phase, a fast and reliable cell counting method should be used. Conventional manual cell counting by the hemocytometer method is dependent on the operator's expertise and is time-consuming. Therefore, automation of sample preparation and analysis is needed to improve precision and rapidity of hiPSC cell counting. We investigated whether an automated cell counting method could be validated for use with hiPSCs, in comparison with a reference cell counting method included in the European Pharmacopeia, 10th edition. The proposed method was the fluorescence imaging-based NucleoCounter NC-100 system, whereas the reference method was manual cell counting using a Bürker hemocytometer. The validation strategy complied with EudraLex cGMP regulations for ATMP manufacturing and ICH Q2(R1) indications for validation of analytical methods. The use of the NucleoCounter NC-100 system for automated cell counting was validated, focusing on accuracy, specificity, intra- and inter-operator reproducibility, range and linearity, showing higher precision than the manual method. The automated method can be used more effectively than the manual one for hiPSC cell counting. Thus, this piece of work paves the way for all cGMP facilities that want to pursue hiPSC manufacturing for clinical use.

Evolutionary impacts of purine metabolism genes on mammalian oxidative stress adaptation.

Many mammals risk damage from oxidative stress stemming from frequent dives (i.e., cycles of ischemia/reperfusion and hypoxia/reoxygenation), high altitude and subterranean environments, or powered flight. Purine metabolism is an essential response to oxidative stress, and an imbalance between purine salvage and de novo biosynthesis pathways can generate damaging reactive oxygen species (ROS). Here, we examined the evolution of 117 purine metabolism-related genes to explore the accompanying molecular mechanisms of enhanced purine metabolism in mammals under high oxidative stress. We found that positively selected genes, convergent changes, and nonparallel amino acid substitutions are possibly associated with adaptation to oxidative stress in mammals. In particular, the evolution of convergent genes with cAMP and cGMP regulation roles may protect mammals from oxidative damage. Additionally, 32 genes were identified as under positive selection in cetaceans, including key purine salvage enzymes (i.e., HPRT1), suggesting improved re-utilization of non-recyclable purines avoid hypoxanthine accumulation and reduce oxidative stress. Most intriguingly, we found that six unique substitutions in cetacean xanthine dehydrogenase (XDH), an enzyme that regulates the generation of the ROS precursor xanthine oxidase (XO) during ischemic/hypoxic conditions, show enhanced enzyme activity and thermal stability and diminished XO conversion activity. These functional adaptations are likely beneficial for cetaceans by reducing radical oxygen species production during diving. In summary, our findings offer insights into the molecular and functional evolution of purine metabolism genes in mammalian oxidative stress adaptations.

The sGC-cGMP Signaling Pathway as a Potential Therapeutic Target in Doxorubicin-Induced Heart Failure: A Narrative Review.

The anti-cancer agent doxorubicin (DOX) has high cardiotoxicity that is linked to DOX-mediated increase in oxidative stress, mitochondrial iron overload, DNA damage, autophagy, necrosis, and apoptosis, all of which are also associated with secondary tumorigenicity. This limits the clinical application of DOX therapies. Previous studies have attributed DOX-mediated cardiotoxicity to mitochondrial iron accumulation and the production of reactive oxygen species (ROS), which seem to be independent of its anti-tumor DNA damaging effects. Chemo-sensitization of soluble guanylate cyclase (sGC) in the cyclic guanosine monophosphate (cGMP) pathway induces tumor cell death despite the cardiotoxicity associated with DOX treatment. However, sGC-cGMP signaling must be activated during heart failure to facilitate myocardial cell survival. The sGC pathway is dependent on nitric oxide and signal transduction via the nitric oxide-sGC-cGMP pathway and is attenuated in various cardiovascular diseases. Additionally, cGMP signaling is regulated by the action of certain phosphodiesterases (PDEs) that protect the heart by inhibiting PDE, an enzyme that hydrolyses cGMP to GMP activity. In this review, we discuss the studies describing the interactions between cGMP regulation and DOX-mediated cardiotoxicity and their application in improving DOX therapeutic outcomes. The results provide novel avenues for the reduction of DOX-induced secondary tumorigenicity and improve cellular autonomy during DOX-mediated cardiotoxicity.

Enhanced brain delivery and therapeutic activity of trastuzumab after blood-brain barrier opening by NEO100 in mouse models of brain-metastatic breast cancer.

The antitumor efficacy of human epidermal growth factor receptor 2 (HER2)-targeted therapies, such as humanized monoclonal antibody trastuzumab (Herceptin®, Roche), in patients with breast-to-brain cancer metastasis is hindered by the low permeability of the blood-brain barrier (BBB). NEO100 is a high-purity version of the natural monoterpene perillyl alcohol, produced under current good manufacturing practice (cGMP) regulations, that was shown previously to reversibly open the BBB in rodent models. Here we investigated whether NEO100 could enable brain entry of trastuzumab to achieve greater therapeutic activity. An in vitro BBB, consisting of human astrocytes and brain endothelial cells, was used to determine trastuzumab penetration in the presence or absence of NEO100. For in vivo studies, we administered intravenous (IV) trastuzumab or the trastuzumab-drug conjugate ado-trastuzumab emtansine (T-DM1; Kadcyla®, Roche), to mouse models harboring intracranial HER2+ breast cancer, with or without BBB opening via IA NEO100. Brain and tumor tissues were examined for the presence of trastuzumab and infiltration of immune cells. Therapeutic impact was evaluated based on overall survival. NEO100 greatly increased trastuzumab penetration across an in vitro BBB. In vivo, IA NEO100-mediated BBB opening resulted in brain tumor-selective accumulation of trastuzumab, without detectable presence in normal brain tissue, along with increased presence of immune cell populations. IV delivery of trastuzumab or T-DM1 achieved significantly greater overall survival of tumor-bearing mice when combined with IA NEO100. IA NEO100 facilitates brain tumor entry of trastuzumab and T-DM1 and significantly enhances their therapeutic efficacy, along with increased antibody-dependent immune cell recruitment.

Expansion of Human Pluripotent Stem Cells in Stirred Tank Bioreactors.

Bioreactor technolology enables the expansion of mammalian cells, which can be translated to processes compatible with Current Good Manufacturing Practice (cGMP) regulations. Cells are introduced to the bioreactor vessel, wherein key parameters such as temperature, pH, and oxygen levels are tightly controlled to facilitate growth over time. Here, we describe the microcarrier-based expansion of human pluripotent stem cells in a 3L stirred tank bioreactor.

Quality control unit-the science gatekeeper of CGMP products

The importance of the quality control unit to gatekeep pharmaceutical products to satisfy CGMP regulation is discussed. Scientific results and data are needed in most cases to answer PDA's questions. Laboratory's work functions, i.e. quality control (QC) , analytical method development and validation (AM) and stability (STB) have been included. The role of quality assurance (QA) has been discussed briefly. Analytical method validation, stability indicating method development and validation, specification and standard testing method preparations are critical tasks that may affect PDA's approval of the product and decision on whether a quality control unit of a comapny can ensure the products' good quality. Also mentioned are the "retest", "training/qualification" and "calibration of instruments" issues. A discussion is also made on the most recent trend in the US for considering using the first party or the third party to audit or inspect facilities to lessen the PDA's burden on frequent inspection.

Application of an Inclined Settler for Cell Culture-Based Influenza A Virus Production in Perfusion Mode.

Influenza viruses have been successfully propagated using a variety of animal cell lines in batch, fed-batch, and perfusion culture. For suspension cells, most studies reported on membrane-based cell retention devices typically leading to an accumulation of viruses in the bioreactor in perfusion mode. Aiming at continuous virus harvesting for improved productivities, an inclined settler was evaluated for influenza A virus (IAV) production using the avian suspension cell line AGE1.CR.pIX. Inclined settlers present many advantages as they are scalable, robust, and comply with cGMP regulations, e.g., for recombinant protein manufacturing. Perfusion rates up to 3000 L/day have been reported. In our study, successful growth of AGE1.CR.pIX cells up to 50 × 106 cells/mL and a cell retention efficiency exceeding 96% were obtained with the settler cooled to room temperature. No virus retention was observed. A total of 5.4–6.5 × 1013 virions were produced while a control experiment with an ATF system equaled to 1.9 × 1013 virions. For infection at 25 × 106 cells/mL, cell-specific virus yields up to 3474 virions/cell were obtained, about 5-fold higher than for an ATF based cultivation performed as a control (723 virions/cell). Trypsin activity was shown to have a large impact on cell growth dynamics after infection following the cell retention device, especially at a cell concentration of 50 × 106 cells/mL. Further control experiments performed with an acoustic settler showed that virus production was improved with a heat exchanger of the inclined settler operated at 27°C. In summary, cell culture-based production of viruses in perfusion mode with an inclined settler and continuous harvesting can drastically increase IAV yields and possibly the yield of other viruses. To our knowledge, this is the first report to show the potential of this device for viral vaccine production.

Myocardial Phosphodiesterases and Their Role in cGMP Regulation.

Cyclic nucleotide phosphodiesterases comprise an 11-member superfamily yielding near 100 isoform variants that hydrolyze cAMP or cGMP to their respective 5'-monophosphate form. Each plays a role in compartmentalized cyclic nucleotide signaling, with varying selectivity for each substrate, and conveying cell and intracellular-specific localized control. This review focuses on the 5 phosphodiesterases (PDEs) expressed in the cardiac myocyte capable of hydrolyzing cGMP and that have been shown to play a role in cardiac physiological and pathological processes. PDE1, PDE2, and PDE3 catabolize cAMP as well, whereas PDE5 and PDE9 are cGMP selective. PDE3 and PDE5 are already in clinical use, the former for heart failure, and PDE1, PDE9, and PDE5 are all being actively studied for this indication in patients. Research in just the past few years has revealed many novel cardiac influences of each isoform, expanding the therapeutic potential from their selective pharmacological blockade or in some instances, activation. PDE1C inhibition was found to confer cell survival protection and enhance cardiac contractility, whereas PDE2 inhibition or activation induces beneficial effects in hypertrophied or failing hearts, respectively. PDE3 inhibition is already clinically used to treat acute decompensated heart failure, although toxicity has precluded its long-term use. However, newer approaches including isoform-specific allosteric modulation may change this. Finally, inhibition of PDE5A and PDE9A counter pathological remodeling of the heart and are both being pursued in clinical trials. Here, we discuss recent research advances in each of these PDEs, their impact on the myocardium, and cardiac therapeutic potential.