Enhancing the functionality of a microscale bioreactor system as an industrial process development tool for mammalian perfusion culture

Without a scale-down model for perfusion, high resource demand makes cell line screening or process development challenging, therefore, potentially successful cell lines or perfusion processes are unrealized and their ability untapped. We present here the refunctioning of a high-capacity microscale system that is typically used in fed-batch process development to allow perfusion operation utilizing in situ gravity settling and automated sampling. In this low resource setting, which involved routine perturbations in mixing, pH and dissolved oxygen concentrations, the specific productivity and the maximum cell concentration were higher than 3.0 × 10⁶ mg/cell/day and 7 × 10 ⁷ cells/ml, respectively, across replicate microscale perfusion runs conducted at one vessel volume exchange per day. A comparative analysis was conducted at bench scale with vessels operated in perfusion mode utilizing a cell retention device. Neither specific productivity nor product quality indicated by product aggregation (6%) was significantly different across scales 19 days after inoculation, thus demonstrating this setup to be a suitable and reliable platform for evaluating the performance of cell lines and the effect of process parameters, relevant to perfusion mode of culturing.     

Cellular Expression of Cyclooxygenase,Aromatase, Adipokines,Inflammation and Cell Proliferation Markers in Breast Cancer Specimen

Current evidences suggest that expression of Ki67, cyclooxygenase (COX), aromatase, adipokines, prostaglandins, free radicals, β-catenin and α-SMA might be involved in breast cancer pathogenesis. The main objective of this study was to compare expression/localization of these potential compounds in breast cancer tissues with tissues collected adjacent to the tumor using immunohistochemistry and correlated with clinical pathology. The breast cancer specimens were collected from 30 women aged between 49 and 89 years who underwent breast surgery following cancer diagnosis. Expression levels of molecules by different stainings were graded as a score on a scale based upon staining intensity and proportion of positive cells/area or individually. AdipoR1, adiponectin, Ob-R, leptin, COX-1, COX-2, aromatase, PGF_2α, F₂-isoprostanes and α-SMA were localised on higher levels in the breast tissues adjacent to the tumor compared to tumor specimens when considering either score or staining area whereas COX-2 and AdipoR2 were found to be higher considering staining intensity and Ki67 on score level in the tumor tissue. There was no significant difference observed on β-catenin either on score nor on staining area and intensity between tissues adjacent to the tumor and tumor tissues. A positive correlation was found between COX-1 and COX-2 in the tumor tissues. In conclusion, these suggest that Ki67, COXs, aromatase, prostaglandin, free radicals, adipokines, β-catenin and α-SMA are involved in breast cancer. These further focus the need of examination of tissues adjacent to tumor, tumor itself and compare them with normal or benign breast tissues for a better understanding of breast cancer pathology and future evaluation of therapeutic benefit.     

Optic Disc - Fovea Distance,Axial Length and Parapapillary Zones. The Beijing Eye Study 2011

Purpose

To measure the distance between the optic disc center and the fovea (DFD) and to assess its associations.

Methods

The population-based cross-sectional Beijing Eye Study 2011 included 3468 individuals aged 50+ years. The DFD was measured on fundus photographs.

Results

Readable fundus photographs were available for 2836 (81.8%) individuals. Mean DFD was 4.76 ± 0.34mm (median: 4.74 mm; range: 3.76–6.53mm). In multivariate analysis, longer DFD was associated with longer axial length (P<0.001; standardized correlation coefficient beta: 0.62), higher prevalence of axially high myopia (P<0.001; beta:0.06), shallower anterior chamber depth (P<0.001; beta:-0.18), thinner lens thickness (P = 0.004; beta: -0.06), smaller optic disc-fovea angle (P = 0.02; beta: -0.04), larger parapapillary alpha zone (P = 0.008; beta: 0.05), larger parapapillary beta/gamma zone (P<0.001; beta: 0.11), larger optic disc area (P<0.001; beta: 0.08), lower degree of cortical cataract (P = 0.002; beta: -0.08), and lower prevalence of age-related macular degeneration (P = 0.001; beta: -0.06). Bruch´s membrane opening-fovea distance (DFD minus disc radius minus parapapillary beta/gamma zone width) in non-glaucomatous eyes was not significantly (P = 0.60) related with axial length in emmetropic or axially myopic eyes (axial length ≥23.5 mm), while it increased significantly (P<0.001; r: 0.32) with longer axial length in eyes with an axial length of <23.5mm. Ratio of mean DFD to disc diameter was 2.65 ± 0.30. If the ratio of disc-fovea distance to disc diameter was considered constant and if the individual disc diameter was calculated as the individual disc-fovea distance divided by the constant factor of 2.65, the resulting calculated disc diameter differed from the directly measured disc diameter by 0.16 ±0.13 mm (median: 0.13 mm, range: 0.00–0.89 mm) or 8.9 ± 7.3% (median: 7.4%; range: 0.00–70%) of the measured disc diameter.

Conclusions

DFD (mean: 4.76mm) increases with longer axial length, larger parapapillary alpha zone and parapapillary beta/gamma zone, and larger disc area. The axial elongation associated increase in DFD was due to an enlargement of parapapillary beta/gamma zone while the Bruch’s membrane opening-fovea distance did not enlarge with longer axial length. This finding may be of interest for the process of emmetropization and myopization. Due to its variability, the disc-fovea distance has only limited clinical value as a relative size unit for structures at the posterior pole.     

Apoptosis of human breast carcinoma cells in the presence of disialosyl gangliosides: II. Treatment of SKBR3 cells with GD3 and GD1b gangliosides

Apoptosis, or programmed cell death, plays an important role in many physiological and diseased conditions. Induction of apoptosis in cancer cells has been monitored during the cells' progression to apoptosis by anti-cancer drugs and inhibitors of the cell surface glycolipids, gangliosides and SA-Le(x) biosyntheses [Basu, S (1991) Glycobiology, 1, 469-475; and ibid, 427-435] in animal tissues and human carcinoma cells, respectively. Induction of apoptosis in cancer cells by cell surface glycolipids in the human breast cancer (SKBR3) cells is the aim in this study. We have employed the disialosyl gangliosides (GD3 and GD1b) to initiate apoptosis in SKBR3 cells grown in culture in the presence of (14)C-L-Serine. At lower concentrations (0-20 microM) of exogenously added non-radioactive GD3, GD1b, or bovine ganglioside mixture (GM1:GD1a:GD1b:GT1a 2:4:4:2), the incorporation of radioactivity in both (14)C-sphingolipid and (14)C-ceramide was higher. However, at higher concentrations (20-100 microM), wherein apoptosis occurred in high frequency, the (14)C-incorporation decreased in both GSLs and ceramide. Apoptosis induction was monitored by the concomitant appearance of caspase-3 activation and the binding of a fluorescent dye PSS-380 to the outer leaflet of phosphatidyl-serine. These results indicated that, in addition to many unknown cell surface glycoconjugates GD3 or GD1b (disialosyl ganglioside) could play an important role in the regulation of breast carcinoma cell death.     

Catalase-peroxidases (KatG) exhibit NADH oxidase activity

Catalase-peroxidases (KatG) produced by Burkholderia pseudomallei, Escherichia coli, and Mycobacterium tuberculosis catalyze the oxidation of NADH to form NAD+ and either H2O2 or superoxide radical depending on pH. The NADH oxidase reaction requires molecular oxygen, does not require hydrogen peroxide, is not inhibited by superoxide dismutase or catalase, and has a pH optimum of 8.75, clearly differentiating it from the peroxidase and catalase reactions with pH optima of 5.5 and 6.5, respectively, and from the NADH peroxidase-oxidase reaction of horseradish peroxidase. B. pseudomallei KatG has a relatively high affinity for NADH (Km=12 microm), but the oxidase reaction is slow (kcat=0.54 min(-1)) compared with the peroxidase and catalase reactions. The catalase-peroxidases also catalyze the hydrazinolysis of isonicotinic acid hydrazide (INH) in an oxygen- and H2O2-independent reaction, and KatG-dependent radical generation from a mixture of NADH and INH is two to three times faster than the combined rates of separate reactions with NADH and INH alone. The major products from the coupled reaction, identified by high pressure liquid chromatography fractionation and mass spectrometry, are NAD+ and isonicotinoyl-NAD, the activated form of isoniazid that inhibits mycolic acid synthesis in M. tuberculosis. Isonicotinoyl-NAD synthesis from a mixture of NAD+ and INH is KatG-dependent and is activated by manganese ion. M. tuberculosis KatG catalyzes isonicotinoyl-NAD formation from NAD+ and INH more efficiently than B. pseudomallei KatG.     

The Binding of Indole-3-acetic Acid and 3-Methyleneoxindole to Plant Macromolecules

Homogenates of pea (Pisum sativum L., var. Alaska) seedlings exposed to ¹⁴C-indole-3-acetic acid or ¹⁴C-3-methyleneoxindole, an oxidation product of indole-3-acetic acid, were extracted with phenol. In both cases 90% of the bound radioactivity was found associated with the protein fraction and 10% with the water-soluble, ethanol-insoluble fraction. The binding of radioactivity from ¹⁴C-indole-3-acetic acid is greatly reduced by the addition of unlabeled 3-methyleneoxindole as well as by chlorogenic acid, an inhibitor of the oxidation of indole-3-acetic acid to 3-methyleneoxindole. Chlorogenic acid does not inhibit the binding of ¹⁴C-3-methyleneoxindole. The labeled protein and water-soluble, ethanol-insoluble fractions of the phenol extract were treated with an excess of 2-mercaptoethanol. Independently of whether the seedlings had been exposed to ¹⁴C-indole-3-acetic acid or ¹⁴C-3-methyleneoxindole, the radioactivity was recovered from both fractions in the form of a 2-mercaptoethanol-3-methyleneoxindole adduct. These findings indicate that 3-methyleneoxindole is an intermediate in the binding of indole-3-acetic acid to macromolecules.     

Membrane active chelators as novel anti-African trypanosome and anti-malarial drugs

Malaria (Plasmodium spp.) and human African trypanosomiasis (Trypanosoma brucei spp.) are vector borne, deadly parasitic diseases. While chemotherapeutic agents for both diseases are available, difficulty in disease eradication and development of drug resistance require that new therapies targeting unexplored pathways or exploiting novel modes of action be developed. Intracellular Plasmodium and extracellular Trypanosoma brucei may have unique and essential requirements for divalent metal ions, beyond that deemed physiological for the host. Membrane Active Chelators (MACs), biologically active only in a hydrophobic lipid environment, are able to bind metal ions at elevated non-physiological concentrations in the vicinity of cell membranes. A dose–response relationship study using validated viability assays revealed that two MAC drugs, DP-b99 and DP-460, were cytotoxic for these parasites in vitro. The 50% effective concentration (EC₅₀) values for DP-b99 and DP-460 were 87 μM and 39 μM for Trypanosoma brucei brucei and 21 μM and 28 μM for erythrocytic Plasmodium falciparum, respectively. Furthermore, drug potency was maintained for at least 24 h in serum containing medium at 37 °C. While the exact mechanism of action of MACs against intracellular malaria and extracellular African trypanosome parasites has yet to be determined, their potential as antiparasitic agents warrants further investigation.     

Pneumocytes are distinguished by highly elevated expression of the ER stress biomarker GRP78, a co-receptor for SARS-CoV-2, in COVID-19 autopsies

Vaccinations are widely credited with reducing death rates from COVID-19, but the underlying host-viral mechanisms/interactions for morbidity and mortality of SARS-CoV-2 infection remain poorly understood. Acute respiratory distress syndrome (ARDS) describes the severe lung injury, which is pathologically associated with alveolar damage, inflammation, non-cardiogenic edema, and hyaline membrane formation. Because proteostatic pathways play central roles in cellular protection, immune modulation, protein degradation, and tissue repair, we examined the pathological features for the unfolded protein response (UPR) using the surrogate biomarker glucose-regulated protein 78 (GRP78) and co-receptor for SARS-CoV-2. At autopsy, immunostaining of COVID-19 lungs showed highly elevated expression of GRP78 in both pneumocytes and macrophages compared with that of non-COVID control lungs. GRP78 expression was detected in both SARS-CoV-2-infected and un-infected pneumocytes as determined by multiplexed immunostaining for nucleocapsid protein. In macrophages, immunohistochemical staining for GRP78 from deceased COVID-19 patients was increased but overlapped with GRP78 expression taken from surgical resections of non-COVID-19 controls. In contrast, the robust in situ GRP78 immunostaining of pneumocytes from COVID-19 autopsies exhibited no overlap and was independent of age, race/ethnicity, and gender compared with that from non-COVID-19 controls. Our findings bring new insights for stress-response pathways involving the proteostatic network implicated for host resilience and suggest that targeting of GRP78 expression with existing therapeutics might afford an alternative therapeutic strategy to modulate host-viral interactions during SARS-CoV-2 infections.     

Metabolic oligosaccharide engineering with N-Acyl functionalized ManNAc analogs: cytotoxicity, metabolic flux, and glycan-display considerations

Metabolic oligosaccharide engineering (MOE) is a maturing technology capable of modifying cell surface sugars in living cells and animals through the biosynthetic installation of non‐natural monosaccharides into the glycocalyx. A particularly robust area of investigation involves the incorporation of azide functional groups onto the cell surface, which can then be further derivatized using “click chemistry.” While considerable effort has gone into optimizing the reagents used for the azide ligation reactions, less optimization of the monosaccharide analogs used in the preceding metabolic incorporation steps has been done. This study fills this void by reporting novel butanoylated ManNAc analogs that are used by cells with greater efficiency and less cytotoxicity than the current “gold standard,” which are peracetylated compounds such as Ac₄ManNAz. In particular, tributanoylated, N‐acetyl, N‐azido, and N‐levulinoyl ManNAc analogs with the high flux 1,3,4‐O‐hydroxyl pattern of butanoylation were compared with their counterparts having the pro‐apoptotic 3,4,6‐O‐butanoylation pattern. The results reveal that the ketone‐bearing N‐levulinoyl analog 3,4,6‐O‐Bu₃ManNLev is highly apoptotic, and thus is a promising anti‐cancer drug candidate. By contrast, the azide‐bearing analog 1,3,4‐O‐Bu₃ManNAz effectively labeled cellular sialoglycans at concentrations ～3‐ to 5‐fold lower (e.g., at 12.5–25 µM) than Ac₄ManNAz (50–150 µM) and exhibited no indications of apoptosis even at concentrations up to 400 µM. In summary, this work extends emerging structure activity relationships that predict the effects of short chain fatty acid modified monosaccharides on mammalian cells and also provides a tangible advance in efforts to make MOE a practical technology for the medical and biotechnology communities. Biotechnol. Bioeng. 2012; 109:992–1006. © 2011 Wiley Periodicals, Inc.