Integrated Biologics Manufacturing in Stirred-Suspension Bioreactor: A Stem Cell Perspective

Stem cell therapy is garnering attention as several clinical trials have taken place in the recent years by using human pluripotent stem cells (hPSCs). Hundreds of biotechnological companies are investing to find a permanent cure for difficult-to-treat diseases like age-related macular degeneration, Parkinson’s disease, diabetes, etc. by using hPSCs. Therefore, clinical-grade cell manufacturing has become an important issue to make cell therapy products safe and effective. Current manufacturing practices are adopted from conventional antibody or protein production in the pharmaceutical industry where cells are used as a vector for producing the desired products. In cell therapy applications, cells are the products that are sensitive to physicochemical parameters and storage conditions anywhere between isolation to patient administration. Moreover, cell-based product manufacturing consists of multi-step processing, including isolation from patients, genetic modification, derivation, expansion, differentiation, purification, characterization, cryopreservation, etc. This can require long processing times and pose high risk of product contamination as well as high production cost. Herein, we discuss the current methods of biologics manufacturing and its limitations. We also review current practices for integrating and automating cell manufacturing facilities. Finally, we propose how to integrate multi-step cell processing in a single bioreactor to make the cell manufacturing practices more direct

Visceral Adipose Tissue Inflammatory Factors (TNF-Alpha, SOCS3) in Gestational Diabetes (GDM): Epigenetics as a Clue in GDM Pathophysiology

Gestational diabetes (GDM) is among the most challenging diseases in westernized countries, affecting mother and child, immediately and in later life. Obesity is a major risk factor for GDM. However, the impact visceral obesity and related epigenetics play for GDM etiopathogenesis have hardly been considered so far. Our recent findings within the prospective 'EaCH' cohort study of women with GDM or normal glucose tolerance (NGT), showed the role, critical factors of insulin resistance (i.e., adiponectin, insulin receptor) may have for GDM pathophysiology with epigenetically modified expression in subcutaneous (SAT) and visceral (VAT) adipose tissues. Here we investigated the expression and promoter methylation of key inflammatory candidates, tumor necrosis factor-alpha (TNF-α) and suppressor of cytokine signaling 3 (SOCS3) in maternal adipose tissues collected during caesarian section (GDM, n = 19; NGT, n = 22). The mRNA expression of TNF-α and SOCS3 was significantly increased in VAT, but not in SAT, of GDM patients vs. NGT, accompanied by specific alterations of respective promoter methylation patterns. In conclusion, we propose a critical role of VAT and visceral obesity for the pathogenesis of GDM, with epigenetic alterations of the expression of inflammatory factors as a potential factor

A novel non-Fermi-liquid state in the iron-pnictide FeCrAs

We report transport and thermodynamic properties of stoichiometric single crystals of the hexagonal iron-pnictide FeCrAs. The in-plane resistivity shows an unusual "non-metallic" dependence on temperature T, rising continuously with decreasing T from ~ 800 K to below 100 mK. The c-axis resistivity is similar, except for a sharp drop upon entry into an antiferromagnetic state at T_N 125 K. Below 10 K the resistivity follows a non-Fermi-liquid power law, rho(T) = rho_0 - AT^x with x<1, while the specific heat shows Fermi liquid behaviour with a large Sommerfeld coefficient, gamma ~ 30 mJ/mol K^2. The high temperature properties are reminiscent of those of the parent compounds of the new layered iron-pnictide superconductors, however the T -> 0 properties suggest a new class of non-Fermi liquid.Comment: 6 pages, 4 figure

Releases and first recovery of Lathrolestes ensator (Hymenoptera: Ichneumonidae) in North America, a parasitoid of Hoplocampa testudinea (Hymenoptera: Tenthredinidae)

The European apple sawfly, Hoplocampa testudinea (Klug), is a pest of apple, Malus pumila Bork. (Rosaceae), in orchards. Introduced from Europe onto Long Island, New York, in 1939, it gradually invaded the New England states (Pyenson 1943). Downes and Andison (1942) also reported the apple sawfly on Vancouver Island in 1940. In 1979, H. testudinea was discovered for the first time in southern Quebec (Paradis 1980), after which it spread throughout the apple-growing areas of this province (Vincent and Mailloux 1988). There are no published records on any natural enemies of H. testudinea in North Americ

DNA methylation and expression of proopiomelanocortin (POMC) gene in the hypothalamus of three-week-old chickens show sex-specific differences

Increased availability and improved sequence annotation of the chicken (Gallus gallus f. domestica) genome have sparked interest in the bird as a model system to investigate translational embryonic development and health/disease outcomes. However, the epigenetics of this bird genome remain unclear. The aim of this study was to determine the levels of gene expression and DNA methylation at the proopiomelanocortin (POMC) gene in the hypothalamus of 3-week-old chickens. POMC is a key player in the control of the stress response, food intake, and metabolism. DNA methylation of the promoter, CpG island, and gene body regions of POMC were measured. Our data illustrate the pattern, variability, and functionality of DNA methylation for POMC expression in the chicken. Our findings show correlation of methylation pattern and gene expression along with sex-specific differences in POMC. Overall, these novel data highlight the promising potential of the chicken as a model and also the need for breeders and researchers to consider sex ratios in their studies

Maternal but Not Paternal High-Fat Diet (HFD) Exposure at Conception Predisposes for 'Diabesity' in Offspring Generations

While environmental epigenetics mainly focuses on xenobiotic endocrine disruptors, dietary composition might be one of the most important environmental exposures for epigenetic modifications, perhaps even for offspring generations. We performed a large-scale rat study on key phenotypic consequences from parental (F0) high-caloric, high-fat diet (HFD) food intake, precisely and specifically at mating/conception, focusing on 'diabesity' risk in first- (F1) and second- (F2) generation offspring of both sexes. F0 rats (maternal or paternal, respectively) received HFD overfeeding, starting six weeks prior to mating with normally fed control rats. The maternal side F1 offspring of both sexes developed a 'diabesity' predisposition throughout life (obesity, hyperleptinemia, hyperglycemia, insulin resistance), while no respective alterations occurred in the paternal side F1 offspring, neither in males nor in females. Mating the maternal side F1 females with control males under standard feeding conditions led, again, to a 'diabesity' predisposition in the F2 generation, which, however, was less pronounced than in the F1 generation. Our observations speak in favor of the critical impact of maternal but not paternal metabolism around the time frame of reproduction for offspring metabolic health over generations. Such fundamental phenotypic observations should be carefully considered in front of detailed molecular epigenetic approaches on eventual mechanisms

Visceral adipose tissue alteration of PI3KR1 expression is associated with gestational diabetes but not promoter DNA methylation

Obesity and diabetes are at an epidemic rate, as well as growing incidences of gestational diabetes mellitus (GDM) which causes pregnancy risks, and harm in both maternal and child health. It remains unclear which molecular mechanisms are driving the functional differences between visceral and subcutaneous fat and how these types directly affect an individual's health outcome. Paired abdominal subcutaneous and omental visceral adipose tissue were collected from women with GDM (n = 20) and with normal glucose tolerance (NGT, n = 22) during planned caesarian section. Both groups had similar maternal age (average 32.5 years) and BMI at delivery (average 33.3 kg/m2). Adipose tissue mRNA expression analyses of insulin signalling genes: PI3KCA, PI3KR1, IRS1 and IRS2 showed significantly decreased PI3KR1 expression (-23%) in visceral fat in GDM with no association to promoter DNA methylation. Reduced visceral fat PI3KR1 expression appears to be a pathogenic factor in GDM but not through altered promoter methylation

SERCA2 Regulates Non-CF and CF Airway Epithelial Cell Response to Ozone

Calcium mobilization can regulate a wide range of essential functions of respiratory epithelium, including ion transport, ciliary beat frequency, and secretion of mucus, all of which are modified in cystic fibrosis (CF). SERCA2, an important controller of calcium signaling, is deficient in CF epithelium. We conducted this study to determine whether SERCA2 deficiency can modulate airway epithelial responses to environmental oxidants such as ozone. This could contribute to the pathogenesis of pulmonary exacerbations, which are important and frequent clinical events in CF. To address this, we used air-liquid interface (ALI) cultures of non-CF and CF cell lines, as well as differentiated cultures of cells derived from non-CF and CF patients. We found that ozone exposure caused enhanced membrane damage, mitochondrial dysfunction and apoptotic cell death in CF airway epithelial cell lines relative to non-CF. Ozone exposure caused increased proinflammatory cytokine production in CF airway epithelial cell lines. Elevated proinflammatory cytokine production also was observed in shRNA-mediated SERCA2 knockdown cells. Overexpression of SERCA2 reversed ozone-induced proinflammatory cytokine production. Ozone-induced proinflammatory cytokine production was NF-κB- dependent. In a stable NF-κB reporter cell line, SERCA2 inhibition and knockdown both upregulated cytomix-induced NF-κB activity, indicating importance of SERCA2 in modulating NF-κB activity. In this system, increased NF-κB activity was also accompanied by increased IL-8 production. Ozone also induced NF-κB activity and IL-8 release, an effect that was greater in SERCA2-silenced NF-κB-reporter cells. SERCA2 overexpression reversed cytomix-induced increased IL-8 release and total nuclear p65 in CFTR-deficient (16HBE-AS) cells. These studies suggest that SERCA2 is an important regulator of the proinflammatory response of airway epithelial cells and could be a potential therapeutic target