Isolation and characterization of bacterial cellulose produced from soybean whey and soybean hydrolyzate.

Soybean whey and soybean hydrolyzate can be used for the biotechnological production of high-value products. Herein, we isolate soybean whey (SW)-and soybean hydrolyzate (SH)-derived bacterial cellulose (BC, produced by kombucha) and characterize it by a range of instrumental techniques to reveal differences in micromorphology, crystallinity, and themal behavior. Studies have shown that the amounts of wet state BC produced from HS, SW and SH was 181 g/L, 47 g/L and 83 g/L, respectively. The instrumental analysis of BC, included SEM, AFM, FT-IR, XRD and TGA. It is shown that the FT-IR spectra of BC have a similar character, but we found differences in the micromorphology,crystallinity and thermal temperature of BC. The minimum average widths of the fibers produced from SH medium was 100 ± 29 nm. The CrI values of BC produced from SH medium was 61.8%. The maximum thermal degradation rate temperature of BC produced from SW medium was 355.73 °C. The combined results demonstrate that soybean industrial waste can be used as a cost-effective raw material for BC production.

Development of a Simple Extraction Procedure and an HPLC Method for the Determination of Delmopinol in a Challenging Rubbery Dental Chew.

BACKGROUND: Delmopinol hydrochloride (delmopinol) is widely used in oral hygiene products such as dental chews for preventing dental plaque buildup and gingivitis. OBJECTIVE: This study aimed to develop a simple and inexpensive extraction method, followed by a stability-indicating reversed-phase HPLC (RP-HPLC) method for the determination of delmopinol from rubbery dental chews. METHODS: The extraction method was optimized in terms of pH, temperature, solvents, repeatability, and reproducibility. Delmopinol was extracted from the rubbery dental chews using 6 N NaOH-MeOH (1 + 1, v/v) at 65°C. The delmopinol peak was eluted in about 7 min by gradient elution on a pH-stable (alkaline) Phenomenex Gemini-NX-C18 column (50 × 4.6 mm id, 110 Å, 3 µm particle size) maintained at 50°C. Mobile phase A was composed of aqueous 10 mM ammonium hydroxide and mobile phase B was acetonitrile. The analyte was monitored by UV detection at 220 nm with a flow rate of 2.0 mL/min. RESULTS: The method was successfully validated as per the current International Conference on Harmonization guidelines for delmopinol with respect to specificity, linearity (R2 >0.999), accuracy, precision, LOQ (2 µg/mL), LOD (0.6 µg/mL), and robustness. The reliability of the method was also demonstrated by batch analysis of 132 dental chews. CONCLUSION: A simple extraction method and RP-HPLC method were successfully developed and validated for the determination of delmopinol from rubbery dental chews. HIGHLIGHTS: The overall method was demonstrated to be accurate, robust, specific, and stability-indicating. Therefore, the developed method is suitable and highly desirable for routine analysis of delmopinol in its finished drug products.

PPARγ-AGPAT6 signaling mediates acetate-induced mTORC1 activation and milk fat synthesis in mammary epithelial cells of dairy cows.

This research communication investigated the role and the underlying mechanism of sn-1-acylglycerol-3-phosphate O-acyltransferase 6 (AGPAT6) in acetate-induced mTORC1 signaling activation and milk fat synthesis in dairy cow mammary epithelial cells. The data showed AGPAT6 knockdown significantly decreased acetate-induced phosphorylation of mTORC1 signaling molecules and intracellular triacylglycerol (TAG) content, whereas this inhibition effect was reversed after the addition of 16:0,18:1 phosphatidic acid (PA), suggesting that AGPAT6 could generate PA in response to acetate simulation, that in turn activates mTORC1 signaling. PPARγ is the upstream regulator of AGPAT6 upon acetate stimulation. Luciferase assay with clones containing various deletions and mutation in AGPAT6 promoter showed that there is a RXRα binding sequence located at -96 bp of AGPAT6 promoter. Acetate stimulation significantly increased the interaction between PPARγ and AGPAT6 via this RXRα binding site. Taken together, our data indicated that AGPAT6 could activate mTORC1 signaling by producing PA during acetate-induced milk fat synthesis, and PPARγ acts as a transcription factor to mediate the effect of acetate on AGPAT6 via RXRα.

Pore Structure Characteristics and Affecting Factors of Shale in the First Member of the Qingshankou Formation in the Gulong Sag, Songliao Basin.

A major historical breakthrough has been made in the exploration of the GK (the first member of the Qingshankou Formation (K2qn1), Gulong Sag) shale oil of the Songliao Basin. However, few reports have been reported on the pore structure characteristics of this large-scale lacustrine medium-high maturity shale. In addition, the difference between the pore structure characteristics of the GK shale and medium-low maturity marine/continental shale is unknown, and the affecting factors of pore development are still unclear. Therefore, in order to clarify the pore structure characteristics of the GK shale and its affecting factors, this study characterized them experimentally and revealed the law of pore evolution using the mineral composition and geochemical analysis, microscopic observations, and pore quantification techniques. Results indicate that (1) the pore system of GK shale reservoirs is divided into micropores (pore diameter < 10 nm), mesopores (10 nm < pore diameter < 50 nm), and macropores (pore diameter > 50 nm); (2) the pore structure of the GK shale is mainly affected by the clay content, siliceous mineral content, and thermal maturity; and (3) when the content of clay minerals and siliceous minerals in the GK shale reservoir is high, and ∼0.8% < R o < ∼1.4%, the storage capacity and oil content of the GK shale show high values, and it can be considered as a strong candidate for further exploration and development. This research can push the shale oil revolution to a new height and is significant to promote the development of the petroleum industry.

Pore types, genesis, and evolution model of lacustrine oil-prone shale: a case study of the Cretaceous Qingshankou Formation, Songliao Basin, NE China.

A comprehensive characterisation of the pore structure in shale oil reservoirs is essential for forecasting oil production and exploration risks. This study forecasted these risks in the oil-rich Songliao Basin using combination of high-resolution field emission scanning electron microscopy and quantitative X-ray diffraction to analyze the pore genesis and evolution mode within the first member of the Cretaceous Qingshankou Formation (K2qn1). The results showed the dominance of inorganic pores over organic pores, wherein diagenetic processes, such as compaction, pressure solution, and cementation, were responsible for the destruction of pore structure in the formation. Notably, the pores formed by dissolution and shrinkage cracks resulting from clay mineral transformation improved the oil storage space. Furthermore, according to the geochemical data and clay composition, the K2qn1 shale is in the middle diagenetic stage A, which can be further subdivided into A1 and A2 stages from top to bottom. The porosity slowly decreased in both sub-stages A1 and A2, wherein the decrease was stable in the latter. The diagenetic observations in this study are significant for the exploration of unconventional shale oil in petroliferous basins worldwide.

A DNA microarray assay for authenticating five important marine mammal species in food and feed.

Material identification in processed and unprocessed food and feed is crucial for ensuring the safety and hygiene of food and feed products. Therefore, to identify possible marine mammal components in feed, we study developed a DNA microarray with species-specific oligonucleotide probes that enable the rapid identification of five important marine mammal species (dolphins, seals, sea lions, white whales, and finless porpoises). The assay was tested using five target marine mammal species, and the probe patterns were compared with those of three fish meals (for feed) to see if they contained traces of marine mammals. All five marine mammal species could be distinguished by the microarray, and no marine mammal-derived ingredients were detected in the three fish meals. This study indicates that DNA microarray-based detection is relatively easy and effective for identification of non-compliant marine mammal ingredients in seafood or feed.

Acute cardiac injury in adult hospitalized COVID-19 patients in Zhuhai, China.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has already became a public health emergency of international concern. COVID-19 related cardiac injury remains largely unclear. METHODS: We retrospectively analyzed demographic, clinical, laboratory and cardiovascular imaging data of all consecutively admitted adult COVID-19 patients in Zhuhai, China from January 17th, 2020 to February 18th, 2020. RESULTS: A total of 93 patients were included in the study. Acute cardiac injury was found in 9 (9.7%) COVID-19 patients with median level of hypersensitive cardiac troponin I (hs-cTnI) to be 0.085 µg/L (IQR 0.027-0.560 µg/L). Compared with patients without cardiac injury, the median age of patients with cardiac injury was significantly older (65.0 vs. 44.0, P<0.05), hypertension was significantly more common (44.4% vs. 14.3%, P<0.05), and the proportion of severe-critical cases were greater (77.8% vs. 17.9%, P<0.05). Patients with cardiac injury were more likely have elevation of N-terminal proBNP (NT-proBNP) in comparison (66.7% vs. 10.0%, P<0.05). There was no significant difference in echocardiographic parameters between patients with and without cardiac injury. Multivariable logistic regression analysis indicated that older age (OR: 1.093, 95% CI: 1.011-1.182) and increased NT-proBNP (OR: 10.979, 95% CI: 2.024-59.555) were independent risk factors for cardiac injury. Cardiac magnetic resonance (CMR) imaging performed on three patients at around one month after they underwent significant hs-cTnI elevation showed that they had underlying cardiovascular comorbidities. CONCLUSIONS: Acute cardiac injury was seen in the minority of hospitalized COVID-19 patients in Zhuhai, China. Older age and increased NT-proBNP were associated with acute cardiac injury. REGISTRATION NUMBER: ChiCTR2000030952.

Long-term cardiac-specific mortality among 44,292 acute myeloid leukemia patients treated with chemotherapy: a population-based analysis.

Background: Acute myeloid leukemia (AML) is a common hematological malignancy treated with regimens containing anthracycline, an agent with cardiotoxicity. However, the cardiac-specific mortality in AML patients receiving chemotherapy remains unknown. Methods: In this population-based study, patients diagnosed with AML between 1973 and 2015 were identified in the Surveillance, Epidemiology, and End Results database. Cumulative mortality by cause of death was calculated. To quantify the excessive cardiac-specific death compared with the general population, standardized mortality ratios (SMRs) were calculated. Multivariate Cox regression analyses were performed to identify risk factors associated with cardiac-specific death and AML-specific death. Results: A total of 64,679 AML patients were identified between 1973 and 2015; 68.48% of patients (44,292) received chemotherapy. Among all possible competing causes of death, AML was associated with the highest cumulative mortality. The AML patients who received chemotherapy showed excessive cardiac-specific mortality compared with the general population, with an SMR of 6.35 (95% CI: 5.89-6.82). Age, year of diagnosis, sex, and marital status were independently associated with patient prognosis. Conclusion: Cardiac-specific mortality in AML patients receiving chemotherapy is higher than that in the general population.

Lysosomes are involved in induction of steroidogenic acute regulatory protein (StAR) gene expression and progesterone synthesis through low-density lipoprotein in cultured bovine granulosa cells.

Progesterone is an important steroid hormone in the regulation of the bovine estrous cycle. The steroidogenic acute regulatory protein (StAR) is an indispensable component for transporting cholesterol to the inner mitochondrial membrane, which is one of the rate-limiting steps for progesterone synthesis. Low-density lipoprotein (LDL) supplies cholesterol precursors for progesterone formation, and the lysosomal degradation pathway of LDL is essential for progesterone biosynthesis in granulosa cells after ovulation. However, it is currently unknown how LDL and lysosomes coordinate the expression of the StAR gene and progesterone production in bovine granulosa cells. Here, we investigated the role of lysosomes in LDL-treated bovine granulosa cells. Our results reported that LDL induced expression of StAR messenger RNA and protein as well as expression of cholesterol side-chain cleavage cytochrome P-450 (CYP11A1) messenger RNA and progesterone production in cultured bovine granulosa cells. The number of lysosomes in the granulosa cells was also significantly increased by LDL; whereas the lysosomal inhibitor, chloroquine, strikingly abolished these LDL-induced effects. Our results indicate that LDL promotes StAR expression, synthesis of progesterone, and formation of lysosomes in bovine granulosa cells, and lysosomes participate in the process by releasing free cholesterol from hydrolyzed LDL.

Purification and characterization of a dehalogenase from Pseudomonas stutzeri DEH130 isolated from the marine sponge Hymeniacidon perlevis.

2-haloacid dehalogenases are enzymes that are capable of degrading 2-haloacid compounds. These enzymes are produced by bacteria, but so far they have only been purified and characterized from terrestrial bacteria. The present study describes the purification and characterization of 2-haloacid dehalogenase from the marine bacterium Pseudomonas stutzeri DEH130. P. Stutzeri DEH130 contained two kinds of 2-haloacid dehalogenase (designated as Dehalogenase I and Dehalogenase II) as detected in the crude cell extract after ammonium sulfate fractionation. Both enzymes appeared to exhibit stereo-specificity with respect to substrate. Dehalogenase I was a 109.9-kDa enzyme that preferentially utilized D-2-chloropropropionate and had optimum activity at pH 7.5. Dehalogenase II, which preferentially utilized L-2-chloropropionate, was further purified by ion-exchange chromatography and gel filtration. Purified Dehalogenase II appeared to be a dimeric enzyme with a subunit of 26.0-kDa. It had maximum activity at pH 10.0 and a temperature of 40 °C. Its activity was not inhibited by DTT and EDTA, but strongly inhibited by Cu²âº, Zn²âº, and Co²âº. The K(m) and V(max) for L-2-chloropropionate were 0.3 mM and 23.8 µmol/min/mg, respectively. Its substrate specificity was limited to short chain mono-substituted 2-halocarboxylic acids, with no activity detected toward fluoropropionate and monoiodoacetate. This is the first report on the purification and characterization of 2-haloacid dehalogenase from a marine bacterium.

Novel micro-bioreactor high throughput technology for cell culture process development: Reproducibility and scalability assessment of fed-batch CHO cultures.

With increasing timeline pressures to get therapeutic and vaccine candidates into the clinic, resource intensive approaches such as the use of shake flasks and bench-top bioreactors may limit the design space for experimentation to yield highly productive processes. The need to conduct large numbers of experiments has resulted in the use of miniaturized high-throughput (HT) technology for process development. One such high-throughput system is the SimCell platform, a robotically driven, cell culture bioreactor system developed by BioProcessors Corp. This study describes the use of the SimCell micro-bioreactor technology for fed-batch cultivation of a GS-CHO transfectant expressing a model IgG4 monoclonal antibody. Cultivations were conducted in gas-permeable chambers based on a micro-fluidic design, with six micro-bioreactors (MBs) per micro-bioreactor array (MBA). Online, non-invasive measurement of total cell density, pH and dissolved oxygen (DO) was performed. One hundred fourteen parallel MBs (19 MBAs) were employed to examine process reproducibility and scalability at shake flask, 3- and 100-L bioreactor scales. The results of the study demonstrate that the SimCell platform operated under fed-batch conditions could support viable cell concentrations up to least 12 x 10(6) cells/mL. In addition, both intra-MB (MB to MB) as well as intra-MBA (MBA to MBA) culture performance was found to be highly reproducible. The intra-MB and -MBA variability was calculated for each measurement as the coefficient of variation defined as CV (%) = (standard deviation/mean) x 100. The % CV values for most intra-MB and intra-MBA measurements were generally under 10% and the intra-MBA values were slightly lower than those for intra-MB. Cell growth, process parameters, metabolic and protein titer profiles were also compared to those from shake flask, bench-top, and pilot scale bioreactor cultivations and found to be within +/-20% of the historical averages.

[Purification and characterization of a bromoperoxidase from Gracilaria lemaneiformis].

A bromoperoxidase from Gracilaria lemaneiformis was purified to homogeneity using a multi-step process of ammonium sulfate precipitation (AS), dialysis, and DEAE-cellulose 52 anion exchange chromatography. The bromoperoxidase activity was unstable or undetectable in crude extract solution. However, it became stable with electrophoretic purity after this multiple purification process. The anion exchange chromatography purification was a critical step in the purification process, which effectively eliminated the phycobiliprotein and smucilaginous polysaccharides. The purified bromoperoxidase was a monomeric enzyme with the relative molecular masses of 66 kD as determined by denaturing and native gradient gel electrophoresis. The optimal pH for bromoination was 6.0 and bromoperoxidase activity was stable as stored at a broad pH range of 3.0-9.0. Of a range of compounds tested, only vanadium enhanced bromoperoxidase activity. Kinetic studies for the bromination of monochlorodimedone (MCD) showed that the Km values of Br- and H2O2 are 53.5 micromol/L, 38 micromol/L respectively.

Cholesterol delivery to NS0 cells: challenges and solutions in disposable linear low-density polyethylene-based bioreactors.

We report the successful cultivation of cholesterol dependent NS0 cells in linear low-density polyethylene (LLDPE) Wave Bioreactors when employing a low ratio of cyclodextrin to cholesterol additive mixture. While cultivation of NS0 cells in Wave Bioreactors was successful when using a culture medium supplemented with fetal bovine serum (FBS), cultivation with the same culture medium supplemented with cholesterol-lipid concentrate (CLC), which contains lipids and synthetic cholesterol coupled with the carrier methyl-beta-cyclodextrin (mbetaCD), proved to be problematic. However, it was possible to cultivate NS0 cells in the medium supplemented with CLC when using conventional cultivation vessels such as disposable polycarbonate shake-flasks and glass bioreactors. A series of experiments investigating the effect of the physical conditions in Wave Bioreactors (e.g., rocking rate/angle, gas delivery mode) ruled out their likely influence, while the exposure of the cells to small squares of Wave Bioreactor film resulted in a lack of growth as in the Wave Bioreactor, suggesting an interaction between the cells, the CLC, and the LLDPE contact surface. Further experiments with both cholesterol-independent and cholesterol-dependent NS0 cells established that the concurrent presence of mbetaCD in the culture medium and the LLDPE film was sufficient to inhibit growth for both cell types. By reducing the excess mbetaCD added to the culture medium, it was possible to successfully cultivate cholesterol-dependent NS0 cells in Wave Bioreactors using a cholesterol-mbetaCD complex as the sole source of exogenous cholesterol. We propose that the mechanism of growth inhibition involves the extraction of cholesterol from cell membranes by the excess mbetaCD in the medium, followed with the irreversible adsorption or entrapment of the cholesterol-mbetaCD complexes to the LLDPE surface of the Wave Bioreactor. Controlling and mitigating these negative interactions enabled the routine utilization of disposable bioreactors for the cultivation of cholesterol-dependent NS0 cell lines in conjunction with an animal component-free cultivation medium.

A novel function for selenium in biological system: selenite as a highly effective iron carrier for Chinese hamster ovary cell growth and monoclonal antibody production.

As the market for biopharmaceuticals especially monoclonal antibodies (MAbs) rapidly grows, their manufacturing methods are coming under increasing regulatory scrutiny, particularly due to concerns about the potential introduction of adventitious agents from animal-sourced components in the media used for their production in mammalian cell culture. Chinese hamster ovary (CHO) cells are by far the most commonly used production vehicles for these recombinant glycoproteins. In developing animal-component free media for CHO and other mammalian cell lines, the iron-transporter function of serum or human/bovine transferrin is usually replaced by certain organic or inorganic chelators capable of delivering iron for cell respiration and metabolism, but few of them are sufficiently effective. Selenium is a well-known essential trace element (TE) for cell growth and development, and its positive role in biological system includes detoxification of free radicals by activating glutathione peroxidase. In cell culture, selenium in the form of selenite can help cells to detoxify the medium thus protect them from oxidative damage. In this presentation, we describe the discovery and application of a novel function of selenite, that is, as a highly effective carrier to deliver iron for cell growth and function. In our in-house-developed animal protein-free (APF) medium for CHO cells, using an iron-selenite compound to replace the well-established tropolone delivery system for iron led to comparable or better cell growth and antibody production. A high cell density of >10 x 10(6) viable cells/mL and excellent antibody titer of approximately 3 g/L were achieved in 14-day fed-batch cultures in shake flasks, followed by successful scale-up to stirred bioreactors. The preparation of the commercially unavailable iron-selenite compound from respective ions, and its effectiveness in cell-culture performance, were dependent on reaction time, substrates, and other conditions.

Development of Animal-free, Protein-Free and Chemically-Defined Media for NS0 Cell Culture.

There has been a recent boom of monoclonal antibodies on the market, and a significant portion of them were produced by NS0 cell lines. As regulations become more stringent in ensuring production processes are free of potential contamination by adventitious agents, it is highly desirable to further develop serum-free media into ones that do not contain any components of animal origin, or 'animal-free media'. Using a shake-flask batch culture system, recombinant proteins (human albumin and human insulin) and synthetic compounds (tropolone and ferric ammonium citrate) were identified to be capable of replacing the animal-sourced proteins commonly found in serum-free media for NS0 cell culture, namely bovine albumin, insulin and transferrin. The cholesterol requirement of NS0 cells was satisfied by the use of a commercially available non-proteinaceous, non-animal sourced cholesterol/fatty acid mix in place of bovine lipoproteins, which in effect also eliminated the need for recombinant albumin. In the animal-free medium thus formulated, NS0 cell lines, either the host or recombinant constructs, were all able to grow in batch culture to 1~ 3x10(6) viable cells/ml for multiple passages, with no requirement for gradual adaptation even when seeded from 10% serum-containing cultures. It was surprising to observe that the recombinant insulin was essentially ineffective as sodium salt compared to its zinc salt. Studies showed that the zinc deficiency in the former resulted in a rapid decline of cell viabilities. Supplementation of zinc ions greatly improved growth, and even led to the total replacement of recombinant insulin and hence the formulation of a protein-free medium. When the cell lines were adapted to cholesterol-independent growth which eliminated the need for any lipid source, a completely chemically-defined animal-free medium was formulated. In all cases, antibody production by various GS-NS0 constructs in animal-free media was stable for multiple passages and at least similar to the original serum-free medium containing the animal-sourced proteins. The medium also served well for cryopreservation of NS0 cells in the absence of serum.

Toward consistent and productive complex media for industrial fermentations: studies on yeast extract for a recombinant yeast fermentation process.

Yeast extract (YE) is commonly used as a key component in the complex media for industrial fermentations. However, the lot-to-lot variation of this raw material frequently requires extensive "use testing" of many lots to identify only the few that support desired fermentation performance. Through extensive fermentation studies and chemical analyses, we have identified adenine and two metabolizable carbon sources, trehalose and lactate, as the principle components in YE that affect the production of a recombinant protein antigen by a yeast strain. Adenine is required for culture growth and the relationship between biomass and measured adenine can be expressed by a Michaelis-Menten model, while the slowly metabolized trehalose serves to maintain the energy supply to the continued antigen synthesis. The rapidly utilized lactate exerts an indirect positive effect by sparing some of the accumulated ethanol from being consumed for growth to being utilized in the product formation. The effects of these YE components are mutually dependent. Based on the database generated from 40 lots at laboratory scale, a relatively high level of carbon sources in YE (trehalose plus lactate, >9.5% w/w) and an intermediate level of adenine (0.14-0.24% w/w) appear to be the minimal requirement of a good lot for this recombinant yeast fermentation. Many poor lots were improved in lab fermenters by rational supplementation of trehalose, lactate, or adenine to compensate for their insufficiencies. At the large production scale, predictions based on adenine and trehalose/lactate contents in various YE lots used correlated reasonably well with culture growth and antigen yield, illustrating the feasibility of such a simple chemical/biochemical analysis as a rapid and reliable initial screening tool. Without incurring any compositional change to an established manufacturing medium, this study demonstrates an effective approach to achieve consistency in fermentations employing complex nutrients and to improve fermentation productivities supported by suboptimal lots of raw material.

Use of glucose feeding to produce concentrated yeast cells.

A defined medium and fed-batch feeding process for the production of a yeast biocatalyst, developed at the 23-L scale, was scaled up to the 600-L pilot scale. Presterilized 100-L-vol plastic bags were implemented for the pilot-scale nutrient feeding. Medium of increased concentration Oqs implemented at the pilot scale, and equivalent dry cell weights were reached with a medium 80% more concentrated than that used at the laboratory scale. The higher medium concentration was believed to be necessary at the pilot scale owing to the additional heat stresses on key components (e.g., complexing of magnesium sulfate with phosphate), increased dilution during sterilization, lower evaporation rate owing to the lower vessel volume per minute air flow rate, and increased dilution owing to nutrient feeding or shot additions. Peak cell density was found to be somewhat insensitive to variations in residual glucose levels. These results suggest that defined medium developed at the laboratory scale may need to be further optimized at the pilot scale for equivalent performance.