Continuous Feeding Reduces the Generation of Metabolic Byproducts and Increases Antibodies Expression in Chinese Hamster Ovary-K1 Cells

Chinese hamster ovary (CHO) cells are the most important host system used for monoclonal antibody (mAb) expression. Moreover, the fed-batch culture mode is the most widely used method to increase mAb expression in CHO cells by increasing the amount of feed. However, a high amount of culture feed results in the production of metabolic byproducts. In this work, we used a continuous feeding strategy to reduce metabolic byproducts and improve mouse–human chimeric anti-epidermal growth factor receptor vIII (EGFRvIII) antibody C12 expression in Chinese hamster ovary-K1 cells. Moreover, the effects of the feeding strategy on the cell culture and monoclonal antibody production were evaluated in chemically defined suspension cultures of recombinant CHO-K1 cells. Compared with bolus feeding methods, the continuous feeding method did not have any advantages when the feeding amount was low, but with a high feeding amount, the continuous feeding method significantly reduced the concentrations of lactate and NH4+ in the later culture stage. At the end of the culture stage, compared with bolus feeding methods, the lactate and NH4+ concentrations under the continuous feeding mode were reduced by approximately 45% and 80%, respectively. In addition, the antibody C12 expression level was also increased by almost 10%. Compared to the bolus feeding method, the antibody C12 produced by the continuous feeding method had a lower content of high-mannose glycoforms. Further analysis found that the osmolality of the continuous feeding method was lower than that of the typical fed-batch bolus feeding method. Conclusively, these results indicate that the continuous feeding method is very useful for reducing metabolic byproducts and achieving higher levels of mAb production.

Chronic and Intermittent Hyperglycemia Modulates Expression of Key Molecules of PI3K/AKT Pathway in Differentiating Human Visceral Adipocytes

Background: Due to its prominence in the regulation of metabolism and inflammation, adipose tissue is a major target to investigate alterations in insulin action. This hormone activates PI3K/AKT pathway which is essential for glucose homeostasis, cell differentiation, and proliferation in insulin-sensitive tissues, like adipose tissue. The aim of this work was to evaluate the impact of chronic and intermittent high glucose on the expression of biomolecules of insulin signaling pathway during the differentiation and maturation of human visceral preadipocytes. Methods:Human visceral preadipocytes (HPA-V) cells were treated with high glucose (30mM)during the proliferation and/or differentiation and/or maturation stage. The level of mRNA (by Real-Time PCR) and protein (by Elisa tests) expression of IRS1, PI3K, PTEN, AKT2, and GLUT4 was examined after each culture stage. Furthermore, we investigated whether miR-29a-3p, miR-143-3p, miR-152-3p, miR-186-5p, miR-370-3p, and miR-374b-5p may affect the expression of biomolecules of the insulin signaling pathway. Results: Both chronic and intermittent hyperglycemia affects insulin signaling in visceral pre/adipocytes by upregulation of analyzed PI3K/AKT pathway molecules. Both mRNA and protein expression level is more dependent on stage-specific events than the length of the period of high glucose exposure. What is more, miRs expression changes seem to be involved in PI3K/AKT expression regulation in response to hyperglycemic stimulation.

Novel Osteogenic Behaviors around Hydrophilic and Radical-Free 4-META/MMA-TBB: Implications of an Osseointegrating Bone Cement

Poly(methyl methacrylate) (PMMA)-based bone cement, which is widely used to affix orthopedic metallic implants, is considered bio-tolerant but lacks osteoconductivity and is cytotoxic. Implant loosening and toxic complications are significant and recognized problems. Here we devised two strategies to improve PMMA-based bone cement: (1) adding 4-methacryloyloxylethyl trimellitate anhydride (4-META) to MMA monomer to render it hydrophilic; and (2) using tri-n-butyl borane (TBB) as a polymerization initiator instead of benzoyl peroxide (BPO) to reduce free radical production. Rat bone marrow-derived osteoblasts were cultured on PMMA-BPO, common bone cement ingredients, and 4-META/MMA-TBB, newly formulated ingredients. After 24 h of incubation, more cells survived on 4-META/MMA-TBB than on PMMA-BPO. The mineralized area was 20-times greater on 4-META/MMA-TBB than PMMA-BPO at the later culture stage and was accompanied by upregulated osteogenic gene expression. The strength of bone-to-cement integration in rat femurs was 4- and 7-times greater for 4-META/MMA-TBB than PMMA-BPO during early- and late-stage healing, respectively. MicroCT and histomorphometric analyses revealed contact osteogenesis exclusively around 4-META/MMA-TBB, with minimal soft tissue interposition. Hydrophilicity of 4-META/MMA-TBB was sustained for 24 h, particularly under wet conditions, whereas PMMA-BPO was hydrophobic immediately after mixing and was unaffected by time or condition. Electron spin resonance (ESR) spectroscopy revealed that the free radical production for 4-META/MMA-TBB was 1/10 to 1/20 that of PMMA-BPO within 24 h, and the substantial difference persisted for at least 10 days. The compromised ability of PMMA-BPO in recruiting cells was substantially alleviated by adding free radical-scavenging amino-acid N-acetyl cysteine (NAC) into the material, whereas adding NAC did not affect the ability of 4-META/MMA-TBB. These results suggest that 4-META/MMA-TBB shows significantly reduced cytotoxicity compared to PMMA-BPO and induces osteoconductivity due to uniquely created hydrophilic and radical-free interface. Further pre-clinical and clinical validations are warranted.

Strategies adopted by Aphanizomenon flos-aquae in response to phosphorus deficiency and their role on growth

Background The N2-fixing cyanobacterium, Aphanizomenon flos-aquae is a globally distributed bloom causing species that degrades water quality of fresh and marine water bodies. Overcoming phosphorus (P) deficiency is one of the ecological advantages for bloom forming cyanobacteria. It remains unclear to what extent can A. flos-aquae alleviate P deficiency by regulating P using strategies. Results Based on in situ observations of extracellular alkaline phosphatase (APase) in A. flos-aquae via enzyme-labeled fluorescence in freshwater bodies in China, Poland and Czechia, we further investigated responses of isolated A. flos-aquae to different P supplies (dissolved inorganic P (Pi) as +DIP, dissolved organic α-glycerophosphate and β-glycerophosphate as +DOPα and +DOPβ, P-free condition as P-depleted). The significantly negative relationships between percentage of APase producing cells and soluble reactive P concentration in both fields and cultures suggested that the excretion of APase in cyanobacterium was regulated by ambient Pi supply. Suffering from P deficiency in the P-depleted treatments, A. flos-aquae showed the highest APase activity but a vigorous growth at the early culture stage, which might also benefit from the formation of polyphosphate body (PPB) and the decrease of cell P quota. In the +DOP treatments, the coordination of dissolved DOP mineralization and continuous prompt utilization of PPB might contribute to a maintenance but not reproduction of A. flos-aquae when relying on DOP, since the specific growth rate kept around 0 cells L-1 day-1 at the second half culture period and the highest cell density reached only 13.38% of that in +DIP treatments while photochemical efficiency was comparable during the whole experiment. Luxury uptake of phosphate as PPB in the +DIP treatments was consistent to the canonical view of polyphosphate as P storage. Conclusion A. flos-aquae could achieve an instantaneous growth in response to P deficiency with the coordination of P utilization strategies, while it maintained a long-term sustainable growth but not reproduction under sole DOP supply. Persistent and active reproduction could only be achieved in high Pi supply, which implying that an effective consequence can be expected for combating the bloom of A. flos-aquae when controlling P supply.

Plant tissue culture environment as a switch-key of (epi)genetic changes

The in vitro tissue cultures are, beyond all difficulties, an essential tool in basic research as well as in commercial applications. Numerous works devoted to plant tissue cultures proved how important this part of the plant science is. Despite half a century of research on the issue of obtaining plants in in vitro cultures, many aspects remain unknown. The path associated with the reprogramming of explants in the fully functioning regenerants includes a series of processes that may result in the appearance of morphological, physiological, biochemical or, finally, genetic and epigenetic changes. All these changes occurring at the tissue culture stage and appearing in regenerants as tissue culture-induced variation and then inherited by generative progeny as somaclonal variation may be the result of oxidative stress, which works at the step of explant preparation, and in tissue culture as a result of nutrient components and environmental factors. In this review, we describe the current status of understanding the genetic and epigenetic changes that occur during tissue culture.

Effect of light specters (red and blue) on two-potato varieties tissue (spunta and Agria)

This study was carried out in plant tissue culture laboratory which belong to bio technology research center in order to test the effect of raid and blue spectral light colors and white lighting as a control and white fluorescent lamps as a second control on plant growth stages while two potato variety ( Spunta , Agria) were used in this experiment . potato plant samples were collected and cultured in special jars contain MS media after being sterilized by Clorox solution in order to avoid the any contamination risk , while the culture stage of the explant started in sterilized condition in sited the hood cabinet, wherever the culture stage completed the cultured plants were placed in the growth room under controlled condition with raid , blue and normal light condition. The obtained results showed that the wait normal color treatment was significantly higher than the other two treatments in plant length factor for the tow used potato variety, while for the number of leaves factor the results showed that the normal light color treatment was significantly higher than the other two treatments in plant number of leaves factor followed by raid color treatment in the two used variety Spunta and Agria while the Raid color treatment scored average of leaves (23.9, 23.3 ) leaf respectively.

Evolution of Western Ukrainian folk instrumental ensembles in the 20th – early 21st centuries

In the article the author traces the changes that have taken place with traditional instrumental ensembles of Western Ukraine during the 20th – beginning of the 21st centuries. Through this period the communicative capabilities of the environment increased rapidly, which significantly influenced the evolution of folk culture. The basis of the traditional ensembles of Western Ukrainian was the violin, to which was added, depending on the regions, a drum (tambourine), cymbals, bass, and a pipe instrument. Under the influence of various factors (interaction with representatives of other ethnographic regions or national minorities, writing culture, stage performances, remote communication, etc.), the instruments, repertoire, manner of performing, etc. in folk bands changed. During the 20th century the traditional instruments of ensembles were supplemented by others (clarinet, accordion, button accordion, trumpet, saxophone, trombone, etc.). Also, existing instruments were modified in some places (for example, the Hutsuls replaced small cymbals with large ones), or completely new ensembles were introduced to accompany traditional rituals or entertainments (brass bands or a variety of other popular ensembles). The repertoire of traditional ensembles is nowadays constantly replenished not only by the works of the local area in which musicians have an opportunity to perform, but also by the new-fangled samples of popular music. Under the influence of the written tradition, the manner of performing ensemble music is somewhat simplified. Currently, in most regions of Western Ukraine there is a setback and decline of the folk-instrumental ensemble tradition, with the exception of the Hutsul region (where young musicians still adopt this tradition orally) and partly Pokuttia, Zakarpattia, Podillia, Bukovyna, and Bessarabia, where for some occasions sometimes ―live‖ music of traditional or somewhat modified instruments is invited).

CONTACTS OF ENEOLITHIC CULTURES AND THEIR LOCAL GROUPS ON THE TERRITORY OF SUB-CARPATHIAN, WESTERN PODILLIA AND VOLHYNIA

According to data of analysis of materials from the settlement of Kozyna, process of Enolitization of Sub-Carpathian region began about 4500–4400 BC and it is associated with the arrival of bearers of Trypillia culture (Stage A) on these lands. Contacts with bearers of Malitska and Tiszapolgár cultures were confirmed. About 4400–4300 BC in Sub-Carpathians Trypillia culture with polychrome painting and in-depth ceramic ornament of Nezvysko II type (stage BI) existed. Trypillian population of this region had contacts with Tiszapolgár culture. At the period between 4200–4000 BC, this territory was densely populated by Trypillians of Zalischyky group (stage B1–BII). Its representatives contacted with Malitska and Liublin-Vohlynian cultures, as well as with Tiszapolgár culture. Approximately about 4000 BC in the area of modern Gorodenka district settlements from the phase I of Shypyntsi group of Nezvysko III type (stage BII) existed. They were not found in northern part of Sub-Carpathian region. Bearers of this group penetrated to the territory of Goryn’ (Bodaky), Podillia (Goloskiv) and further – to Middle Bug region (Voroshylivka). They had contacts with Liublin-Volhynian culture. Around 3900–3800 BC in southern part of Sub-Carpathian and Podillia regions settlements of Shypyntsi group of Tripillia culture (stage CI) existed. Representatives of this group had contacts with Bodrogkeresztúr culture. About 3800 BC expansion of Bădragii group of Trypillia culture began. Its “imports” were found in Bilche-Zolote–Verteba II and Bilyi Potik in Podillia. Around 3600 BC (beginning of CII stage), migration of Brînzeni group of Trypillia culture began. Under its influence Koshylivtsi one was formed. Bearers of Brînzeni traditions reached Volhynia. Close and distant contacts with the Funnelbeaker culture began. About 3300 BC (stage CII) bearers of Trypillia culture almost completely abandoned painted ceramic ware. It was replaced by corded and stamped ornament (Gordineşti-Kasperivtsi-Erbiceni group) and covering of the surface with red paint (Trojaniv group in Volhynia). Contacts with Funnelbeaker and Baden cultures are continued. Key words: Trypillia, Eneolithic, ceramics, “import”, ornamentation, contacts.

Optimasi sterilisasi permukaan eksplan stek mikro tanaman karet Optimization of surface sterilization on rubber microcutting explan

An increasing number of explants is necessary toobtain plantlets in large quantities, for mass propagationof rubber plants. However, high level of contamination atthe primary culture stage is still a major constraint in invitro microcutting of rubber. The aim of this study was tooptimize surface sterilization procedures to reduce micro-bial contamination at the primary culture. Sterilizationexperiment was conducted in two step., The first step wasto determine the effect of washing the explants withrunning water prior to sterilization and then using Deso-germe, ethanol or H 2 O 2 , while the second step was toidentify the suitable sterilization process on reducing thelevel of contamination. The results showed that the surfacesterilization with only one type of sterilization agent couldnot reduce contamination level caused either by bacteriaor fungi, while sterilization with three types of sterilizingagents increased the number of dead explants. The besttreatment for surface sterilization was the directsterilization of explants using 70% ethanol for one minuteand 17.6% H 2 O 2 for 20 minutes without washing with tapwater (A-CD treatment). The percentage of viable andaseptic explantsof this treatment was 76.7%, which wassignificantly higher than those of other treatments. Thistreatment reduced contamination level to 21.7%.AbstrakPeningkatan jumlah eksplan sangat diperlukan untukmemperoleh planlet dalam jumlah besar pada perbanyakanmassal tanaman karet secara in vitro. Namun, tingginyatingkat kontaminasi pada tahap kultur primer masih me-rupakan kendala utama dalam kultur stek mikro tanamankaret. Tujuan penelitian adalah mengoptimasi prosedursterilisasi permukaan eksplan untuk mengurangi jumlaheksplan yang terkontaminasi mikroba pada tahap kulturprimer. Percobaan sterilisasi dilaksanakan dalam duatahap, tahap pertama untuk mengetahui pengaruh pen-cucian eksplan dengan air mengalir pada awal sterilisasiserta penggunaan Desogerme, etanol dan H 2 O 2 , sedang-kan tahap kedua untuk mendapatkan proses sterilisasi yangpaling sesuai dalam menurunkan tingkat kontaminasi.Hasil penelitian menunjukkan bahwa perlakuan sterilisasipermukaan yang menggunakan satu jenis bahan sterilantidak dapat mengurangi kontaminasi, baik oleh bakterimaupun cendawan. Perlakuan sterilisasi eksplan dengantiga jenis bahan sterilan meningkatkan kematian eksplan.Perlakuan sterilisasi permukaan terbaik adalah sterilisasilangsung eksplan menggunakan etanol 70% selama satu menit dan H 2 O 2 17,6% selama 20 menit, tanpa pencuciandengan air mengalir (perlakuan A-CD). Persentase eksplansteril yang hidup sebesar 76,7%, berbeda nyata dibanding-kan dengan perlakuan lainnya. Perlakuan tersebut dapatmengurangi kontaminasi menjadi sebesar 21,7%.