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

Reasons for a Lower Rate of Epidural Anesthesia During Birth for Immigrant Women in the Eyes of Medical Staff: A Mixed-Methods Analysis

Various studies have shown that immigrant women in comparison to non-immigrant women of the same parity have lower rates of epidural anesthesia (EDA). Data from two studies on immigrant obstetric care in Berlin, Germany were analyzed to answer the following question: What reasons do the medical staff see for the lower rate of EDA in immigrant women? Between May and August 2017, 34 interviews with obstetricians and midwives in four obstetric clinics in Berlin were conducted on the topic of obstetric care for immigrant women. After anonymizing the more than 20 h of interview material, transcripts were coded with MaxQDa and analyzed according to the qualitative content analysis.The quantitative data is from an online survey conducted between May and October 2017, in all but one obstetric clinic in Berlin with obstetricians and midwives. Regarding the research question, 121 questionnaires could be analyzed. In the online survey, (multiple answers were possible), the top reason for a lower rate of EDA given was mostly fear on the part of the immigrant women (64%). A language barrier, which results in logistic and time constrictions, is mentioned as the second most frequent reason (50%). The explorative analysis of the interviews shows that doctors and midwives regard cultural aspects such as different expectations on the birth experience as a reason for a lower EDA rate. Furthermore, within the medical staff the impression persists that in some cases the companion decides on the behalf of the patient about the application of an EDA, which from time to time is against the wish of the immigrant woman giving birth. In the view of the medical staff, the reasons for a lower rate of EDA during birth for immigrant women were varied. On one side, this is attributed to the wishes of the respective women ("demand") but on the other side this can be attributed to the health care system ("supply"). In the case of a language barrier, the "supply" and the access of EDA for immigrant women is limited and can be then shifted to the German-speaking companion to make a decision regarding EDA ("structural deprivation of self-determination")

Optimized RNA isolation of FFPE uterine scar tissues for RNA expression analyses delineated by laser microdissection

ABSTRACT Samples for histological analyses are often formalin-fixed paraffin-embedded (FFPE) and slide-mounted, which complicates RNA extraction for many downstream molecular applications. Furthermore, when the region of interest is extremely small due to isolation with lasermicrodissection (LMD), extracting RNA of adequate quality and quantity is difficult. We describe an optimized protocol for maximizing RNA output from FFPE tissue devised to identify and analyze gene expression of humanmaternal uterine scar tissue obtained fromuterotomy scars resulting from prior cesarean deliveries. Gomori trichrome staining allowed for region identification for LMD. Successful RNA isolation, reverse transcription and, importantly, quantitative real-time PCR (qRT-PCR) were performed. This report provides an optimized step-by-step protocol yielding sufficient RNA for qRT-PCR analyses from challenging tissue/LMD-FFPE samples. METHOD SUMMARY FFPE sample sections weremounted on glass slides and stained with hematoxylin and eosin (HE). A corresponding slide was stained according to Gomori trichrome for orientation. Regions of interest in HE-stained samples weremarked using lasermicrodissection (LMD) and subsequently scratched off the slide with a sterile scalpel. RNA extraction and cDNA synthesis were carried out, and quantitative real-time PCR with TaqMan probes was performed. TWEETABLE ABSTRACT We describe a protocol for maximized RNA output from FFPE tissue delineated by laser microdissection devised to analyze gene expression of human maternal uterine scar tissue obtained from uterotomy scars of prior cesarean deliveries