Suspended Cultures Research Articles

Comparative analysis of whole-genome sequences of African swine fever virus (Asfarviridae: Asfivirus) isolates сollected on the territory of the left bank of the Dnieper River in 2023

Introduction. The lack of data on the whole-genome sequences of African swine fever virus (ASFV) variants circulating on the territory of the left bank of the Dnieper River complicates the understanding of the molecular evolution of the virus and the character of the epidemic process development in Russia and Ukraine. Understanding the genetic divergence and phylogenetic relatedness of isolates can largely adjust the strategy of general and specific prevention of the disease. The aim of the study – search and description of unique mutations (deletions/insertions/substitutions) in isolates collected from domestic pigs in Donetsk, Luhansk and Zaporozhye regions in 2023; determination of relatedness and level of homology with reference strains of ASFV genotype II; sub-genotyping and clustering of isolates based on whole-genome analysis. Materials and methods. The samples used were a culture suspension of porcine bone marrow (PBM) cells containing ASFV isolates obtained from pathologic material from domestic pig carcasses. Genomic DNA was prepared by purification and concentration of virus followed by phenol-chloroform extraction of total nucleic acid. The high-throughput sequencing process was performed using MGI technology. Consensus sequences were assembled by mapping reads to the reference genome of strain Georgia 2007/1. Results. All isolates are assigned to genotype II, have a monophyletic origin, are phylogenetically close to the clusters «Europe» (4/5) and «Bryansk 2021» (1/5), and are divergent from the original parental genetic variants that make up the enlarged clades. In addition, numerous substitutions in the loci of the multigene family MGF 110, 505, and 360, encoding virulence proteins, were detected in 4 isolates from Donetsk and Zaporozhye regions. Conclusion. The phylogeny of the genotype II ASFV, which originated from the reference strain Georgia 2007/1, is shown to be sufficient for isolate differentiation. The presented data are of theoretical and practical importance for domestic and international ASFV surveillance.

Establishment, Multiplication, and Biochemical Analysis of Embryogenic Lines of the Amazonian Palm Euterpe precatoria Mart. under Suspension Culture

The palm Euterpe precatoria holds great social, cultural, and environmental importance. The heart of palm and the fruit are the main products used for industrialization due to their energetic properties. Thus, the aim of this study was to establish a suspension cultivation protocol for the species using different explant sources. For this, eight lineages of E. precatoria embryogenic calluses were tested, with five in liquid medium Murashige and Skoog (MS) with 5 μM Picloram and three for comparison in semisolid medium MS with 20 μM Picloram and 5 μM 2iP. The growth curve was obtained by weighing the calli from 60 to 180 days of cultivation. The Gompertz model was applied, and growth kinetics were evaluated. At 100 days, the contents of total soluble sugars (TSSs) and total soluble proteins (TSPs) were determined. Principal components (PCA) were measured. According to the analysis of the data, the cultivation of E. precatoria lineages in liquid medium was successfully carried out, and the establishment was achieved. The model can be considered adequate since the R2 values found describe more than 90% of the growth kinetics of the lineages. In the liquid system, lineages L1 (from leaf explants and multiplied in semisolid medium—SM), L2 (from leaf explants and multiplied in SM), and L6 (from zygotic embryo explants and multiplied in liquid medium—LM) showed the shortest time to double the biomass accumulation. Multivariate analysis reveals a significant increase in masses in liquid cultures, represented by lineages L6 and L2. There was statistical difference in the amount of TSSs extracted; the highest TSS levels were observed in lineages cultivated in LM. The protein content found was very low, showing statistical differences among the lineages. In this work, the establishment and multiplication of embryogenic calli of E. precatoria are described for the first time, and they emerge as viable alternatives for the vegetative propagation of the species.

Improving transient gene expression and agroinfiltration‐based transformation effectiveness in Indonesian orchid Phalaenopsis amabilis (L.) Blume

Transient gene expression is an approach used to study transient genes across various species, with infiltration by Agrobacterium tumefaciens (agroinfiltration) being a commonly used method. Agroinfiltration offers a simple and effective means of delivering transgenes into the plant genome. An alternative method for enhancing the quality and productivity of orchids as ornamental plants is genetic modification through agroinfiltration. Although Agrobacterium‐mediated genetic transformation by immersion has been used on the Phalaenopsis amabilis (L.) Blume species of orchid, transformation efficiency using the immersion technique remains relatively low and the method itself is challenging due to its requirement for aseptic handling. The application of agroinfiltration in P. amabilis has not previously been reported. This study investigates the impact of the injection site, acetosyringone concentration, bacterial density (OD600), and injection volume to determine the optimum conditions for agroinfiltration on P. amabilis. The results demonstrated that injection site had a noticeably distinct impact on transformation effectiveness, with the abaxial position of the leaf being the optimal site for Agrobacterium culture suspension injection. While adjustments in acetosyringone concentration, bacterial density (OD600), and injection volume did not significantly affect transformation efficiency, they did influence the peak time of GFP fluorescence. Acetosyringone at a concentration of 200 µM, an OD600 of 1.0 for Agrobacterium culture, and an injection volume of 500 µL effectively accelerated GFP expression duration.

Streptomyces avermitilis MICNEMA2022: a new biorational strain for producing abamectin as an integrated nematode management agent

BackgroundAbamectin (ABA) is considered a powerful insecticidal and anthelmintic agent. It is an intracellular product of Streptomyces avermitilis; is synthesized through complicated pathways and can then be extracted from mycelial by methanol extraction. ABA serves as a biological control substance against the root-knot nematode Meloidogyne incognita. This investigation is intended to reach a new strain of S. avermitilis capable of producing ABA effectively.ResultsAmong the sixty actinobacterial isolates, Streptomyces St.53 isolate was chosen for its superior nematicidal effectiveness. The mycelial-methanol extract of isolate St.53 exhibited a maximum in vitro mortality of 100% in one day. In the greenhouse experiment, the mycelial-methanol extract demonstrated, for the second-stage juveniles (J2s), 75.69% nematode reduction and 0.84 reproduction rate (Rr) while for the second-stage juveniles (J2s), the culture suspension demonstrated 75.38% nematode reduction and 0.80 reproduction rate (Rr). Molecular identification for St.53 was performed using 16 S rRNA gene analysis and recorded in NCBI Genbank as S. avermitilis MICNEMA2022 with accession number (OP108264.1). LC-MS was utilized to detect and identify abamectin in extracts while HPLC analysis was carried out for quantitative determination. Both abamectin B1a and abamectin B1b were produced and detected at retention times of 4.572 and 3.890 min respectively.ConclusionStreptomyces avermitilis MICNEMA2022 proved to be an effective source for producing abamectin as a biorational agent for integrated nematode management.

First Report of Colletotrichum gigasporum causing Anthracnose of chili pepper in South Korea.

Anthracnose, a destructive fungal disease, poses a significant threat to chili pepper (Capsicum annuum L.) production worldwide (de Silva et al. 2019). In South Korea, anthracnose outbreaks have traditionally been attributed to several Colletotrichum species such as C. gloeosporioides and C. acutatum. About 10% of the yield (chili production) is lost annually in South Korea due to chili anthracnose (Oo et al. 2020). During field surveys conducted in August 2017, symptomatic lesions resembling anthracnose were observed on chili pepper in two farmer's fields (Gochang and Cheongyang) in South Korea. Affected fruits exhibited characteristic symptoms, including circular sunken lesions with dark margins and abundant orange spore masses on the surface. About 20% of chili pepper fruit were affected in each field with an area of about 0.2 ha. Five putative Colletotrichum spp. isolates were obtained from six affected fruits (three from each field) following the procedure described by Cai et el. (2009). Three isolates (C01049, C01111, and C01115), representing each location, were selected to identify at the species level. Colonies on potato dextrose agar (incubated at 25°C in the dark for 7 days) were cottony with entire margins, white aerial mycelium and dark gray in the center. Conidia were hyaline, aseptate, cylindrical with bothnds round, and 17.8 - 30.5 × 6.0 -10.0 µm (mean 23.8 ×7.9 μm, n = 30). Appressoria were dark brown, irregular but mostly ovoid with smooth walls. These morphological features align with those of Colletotrichum spp. within the Colletotrichum gigasporum species (Liu et al. 2014). The identity of the pathogen was further confirmed through multi-locus phylogenetic analysis. The target genes including ITS, ACT, CHS-1, GAPDH, TUB2, and GS were amplified and sequenced using the primer sets ITS1/ITS4, ACT 512F/ ACT-783R, CHS-79F/ CHS-345R, GDF/GDR, T1/Bt2b, and GSF1/GSR1, respectively (Weir et al. 2012; Liu et al. 2014). The resulting sequences were deposited in GenBank (accession no: ITS: MT605261, MT605262, LC823714; ACT: MT612991, MT612992, LC823718; CHS-1: MT612993, MT612994, LC823717; GAPDH: LC811375, LC811376, LC823716; TUB2: MT612997, MT612998, LC823715; GS: LC811377, LC811378, LC823719). The constructed Bayesian and maximum likelihood tree based on combined sequences of ITS, ACT, CHS-1, GAPDH, TUB2, and GS confirmed the identification of the isolates (C01049, C01111, C01115) as C. gigasporum. Pathogenicity tests were conducted by inoculating healthy chili fruit with 70 µL of a conidial suspension (1×106 conidia /mL) of pure cultures of the isolates. The conidial suspension was applied on 10 wounded or 10 non-wounded fruit. The same number of fruit were treated with sterile distilled water as controls. Within 5 days of inoculation, symptoms consistent with anthracnose developed on the inoculated wounded fruit, whereas non-wounded and control fruit remained asymptomatic. This experiment was repeated twice. Colletotrichum gigasporum was re-isolated from diseased tissue of inoculated fruit. Colletotrichum gigasporum has been identified as the cause of anthracnose on Dalbergia odorifera, Carica papaya in China, and Brassica oleracea in India (Wan et al., 2018; Saini et al. 2022; He et al. 2023). To the best of our knowledge, this report marks the first documented instance of C. gigasporum causing anthracnose of chili pepper in South Korea. These results indicate that various species of Colletotrichum can be the fungi causing chili pepper anthracnose. The findings of this study emphasize the need for effective disease management strategies to mitigate impact of C. gigasporum on chili pepper cultivation in the region.

Light footprint of microalgae cultivation system addressed by modified Cornet model

The cultivation of microalgae is significantly influenced by light intensity and utilization efficiency. This study developed a modified Cornet (M−Cornet) model to assess the distribution of light intensity and flux in microalgae cultivation systems. Algal biofilm cultivation represents a more concentrated approach of algal suspension cultivation. Both follow the M−Cornet model and exhibit the same growth rates when cultured under identical conditions. Algal pigments and morphologies greatly impact the light absorption and scattering, resulting in light attenuation in intensity, penetration distance and light flux distribution. Algae varieties exhibit diverse light flux characteristics. 37% − 90% of the incident light is absorbed, of which, 80% to 90% is dissipated as heat. 10% to 63% of the incident light is scattered off the photobioreactor. The overall light utilization efficiency ranges 6% to 13%. The light footprint using the M−Cornet model offers valuable insights for photobioreactors designing and cultivation operating.

Rapid susceptibility of Carbapenem resistant Pseudomonas aeruginosa and its resistance gene to non-thermal plasma treatment in a batch reactor

The critically ranked carbapenem-resistant Pseudomonas aeruginosa has been observed to infect immunocompromised patients that consume polluted waters, leading to critical infections and more hospital costs. To save lives and unburden the public health sectors of preventable costs, non-thermal plasma (NTP) technology was investigated as an alternative disinfection step that could be applied in wastewater treatment plants (WWTPs) to inactivate this bacterium and its prominent carbapenem resistance gene (blaNDM-1). Culture and molecular-based techniques were employed to confirm carbapenem resistance in P. aeruginosa (27853). Culture suspensions of carbapenem-resistant ATCC P. aeruginosa (16 h culture) were prepared from confirmed isolates and subjected to plasma treatment at varying time intervals (3 min, 6 min, 9 min, 12 min and 15 min) in triplicates. The plasma treated samples were evaluated for re-growth and the presence of blaNDM-1. The treatment resulted in a 0.68 log reduction after 3 min and the highest log reduction of ≥8 after 12 min, suggesting that plasma disinfection has a great potential to be an efficient tertiary treatment step for WWTPs. Moreover, the gel image showed that band intensity of blaNDM-1 reduced with treatment time, thereby suggesting a probable reduction of amplified genes. Notwithstanding, longer treatment time, a grounded electrode with a larger surface (≥ 40 mm diameter) and/or oxygen-containing feeding gas is warranted to completely inactivate its antibiotic resistance gene (ARG), which might be bound by biofilms as they seem to protect P. aeruginosa from the action of non-thermal plasma (NTP) disinfection.

Isolation and Characterization of a (Surfactin-Like Molecule) Produced by Bacillus subtilis: Antagonistic Impact on Root-Knot Nematodes

Plant-parasitic nematodes are severe soil-borne pathogens that cause significant damage to agricultural products each year, resulting in substantial financial losses globally. Thus, there is an urgent need to identify novel biological control agents or nematicides. The nematicidal potential of Bacillus subtilis-derived lipopeptides against Meloidogyne incognita was investigated at various concentrations (35 ppm, 25 ppm, 15 ppm, 5 ppm) under in vitro conditions. Egg hatching inhibition and mortality of second-stage juveniles (J2s) of M. incognita were analyzed after exposure for 6, 12, 24, 48, and 96 hours. Data showed that with the increase in concentration and exposure period, egg hatching inhibition and percent mortality increases. Maximum percent mortality of J2s was reported at 35ppm i.e., 45%, 55%, 67.75%, 77% and 85% at 6, 12, 24, 48 and 96 hrs, respectively. The maximum ovicidal activity was reported at 35ppm concentration, with 84.61% of eggs hatching inhibition on 96 hrs of the exposure period. The bacterial culture suspension of Bacillus subtilis and Pseudomonas putida at1.2x108cfu/ml, and the crude lipopeptide (35ppm) was also investigated as a biological control agent against M. incognita on tomato in a pot experiment under glasshouse condition. Combinational treatment of P. putida and B. subtilis culture, prior to inoculation of M. incognita on tomato plant caused a significant increase in plant growth attributes and in biochemical parameters over the inoculated control. In the same treatment, the maximum reduction in nematode population and root galling was recorded. However, in the crude lipopeptide experiment study, root dip and inoculation of crude lipopeptide in tomato after the introduction of M. incognita caused a major augment in all the parameters over the inoculated control. MALDI-TOF MS analysis of crude lipopeptide shows surfactin like molecules at m/z 1058 [M+Na]+. It is concluded that crude lipopeptide or combinational treatment of B. subtilis and P. putida culture suspension can be employed as a biocontrol agent against M. incognita and may act as a source of a novel nematicidal agent of bacterial origin.

Methane-Driven Perchlorate Reduction by a Microbial Consortium.

The phenomenon of methane oxidation linked to perchlorate reduction has been reported in multiple studies; yet, the underlying microbial mechanisms remain unclear. Here, we enriched suspended cultures by performing methane-driven perchlorate reduction under oxygen-limiting conditions in a membrane bioreactor (MBR). Batch test results proved that perchlorate reduction was coupled to methane oxidation, in which acetate was predicted as the potential intermediate and oxygen played an essential role in activating methane. By combining DNA-based stable isotope probing incubation and high-throughput sequencing analyses of 16S rRNA gene and functional genes (pmoA, pcrA, and narG), we found that synergistic interactions between aerobic methanotrophs (Methylococcus and Methylocystis) and perchlorate-reducing bacteria (PRB; Denitratisoma and Dechloromonas) played active roles in mediating methane-driven perchlorate reduction. This partnership was further demonstrated by coculture experiments in which the aerobic methanotroph could produce acetate to support PRB to complete perchlorate reduction. Our findings advance the understanding of the methane-driven perchlorate reduction process and have implications for similar microbial consortia linking methane and chlorine biogeochemical cycles in natural environments.

Biocontrol of Fusarium head blight in rice using Bacillus velezensis JCK-7158.

Fusarium head blight (FHB) is a destructive disease caused by several species of Fusarium, such as Fusarium graminearum and F. asiaticum. FHB affects cereal crops, including wheat, barley, and rice, worldwide. Fusarium-infected kernels not only cause reduced yields but also cause quality loss by producing mycotoxins, such as trichothecenes and zearalenone, which are toxic to animals and humans. For decades, chemical fungicides have been used to control FHB because of their convenience and high control efficacy. However, the prolonged use of chemical fungicides has caused adverse effects, including the emergence of drug resistance to pathogens and environmental pollution. Biological control is considered one of the most promising alternatives to chemicals and can be used for integrated management of FHB due to the rare possibility of environment pollution and reduced health risks. In this study, Bacillus velezensis JCK-7158 isolated from rice was selected as an ecofriendly alternative to chemical fungicides for the management of FHB. JCK-7158 produced the extracellular enzymes protease, chitinase, gelatinase, and cellulase; the plant growth hormone indole-3-acetic acid; and the 2,3-butanediol precursor acetoin. Moreover, JCK-7158 exhibited broad antagonistic activity against various phytopathogenic fungi and produced iturin A, surfactin, and volatile substances as active antifungal compounds. It also enhanced the expression of PR1, a known induced resistance marker gene, in transgenic Arabidopsis plants expressing β-glucuronidase (GUS) fused with the PR1 promoter. Under greenhouse conditions, treatments with the culture broth and suspension concentrate formulation of JCK-7158 at a 1,000-fold dilution inhibited the development of FHB by 50 and 66%, respectively. In a field experiment, treatment with the suspension concentrate formulation of JCK-7158 at a 1,000-fold dilution effectively controlled the development of FHB with a control value of 55% and reduced the production of the mycotoxin nivalenol by 40%. Interestingly, treatment with JCK-7158 enhanced the expression of plant defense-related genes in salicylic acid, jasmonic acid, ethylene, and reactive oxygen species (ROS) signaling pathways before and after FHB pathogen inoculation. Taken together, our findings support that JCK-7158 has the potential to serve as a new biocontrol agent for the management of FHB.

Efficacy of entomopathogenic fungi against Philaenus spumarius, the vector of Xylella fastidosa.

Xylella fastidiosa is an important causative agent of Olive Quick Decline Syndrome in the Apulia region of Italy. The current study evaluated the bioefficacy of three entomopathogenic fungal strains: Beauveria bassiana SGB7004, Metarhizium robertsii SGB1K, and Akanthomyces lecanii SGB4711 against Philaenus spumarius the main vector of this pathogen, under laboratory conditions. Pathogenicity bioassays were performed by dipping nymphs and adults of P. spumarius in an aqueous suspension of powdered fungal culture (PFC) or conidial suspension (CS) of the three fungal strains. Both B. bassiana SGB7004 and M. robertsii SGB1K affected the viability of nymphs, resulting in more than 80% mortality at 48 h post treatment, while the effect of A. lecanii SGB4711 was not statistically significant. On adults, all three biocontrol strains were effective in a time- and concentration-dependent manner. The PFCs of B. bassiana SGB7004, M. robertsii SGB1K, and A. lecanii SGB4711 at the highest concentration tested (120 mg mL-1) resulted in 97%, 83% and 27% mortality at the trial endpoint (120 h), respectively. Mycelial growth was observed on 38.5%, 37.0% and 61.5% of dead insects treated with B. bassiana SGB7004 (2.3 × 108 CFU mL-1), M. robertsii SGB1K (3.8 × 106 CFU mL-1) and A. lecanii SGB4711 (5.4 × 108 CFU mL-1), respectively. None of the PFCs of the tested strains was pathogenic when injected into nymph spittle. Beauveria bassiana SGB7004 and M. robertsii SGB1K significantly affected the survival of P. spumarius nymphs and adults, while A. lecanii SGB4711 was not effective on nymphs and only slightly effective against adults. © 2024 Society of Chemical Industry.

Capsaicin attenuates Porphyromonas gingivalis-suppressed osteogenesis of periodontal ligament stem cells via regulating mitochondrial function and activating PI3K/AKT/mTOR pathway.

Prevention of periodontal bone resorption triggered by Porphyromonas gingivalis (P. gingivalis) is crucial for dental stability. Capsaicin, known as the pungent ingredient of chili peppers, can activate key signaling molecules involved in osteogenic process. However, the effect of capsaicin on osteogenesis of periodontal ligament stem cells (PDLSCs) under inflammation remains elusive. P. gingivalis culture suspension was added to mimic the inflammatory status after capsaicin pretreatment. The effects of capsaicin on the osteogenesis of PDLSCs, as well as mitochondrial morphology, Ca2+ level, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and osteogenesis-regulated protein expression levels were analyzed. Furthermore, a mouse experimental periodontitis model was established to evaluate the effect of capsaicin on alveolar bone resorption and the expression of osteogenesis-related proteins. Under P. gingivalis stimulation, capsaicin increased osteogenesis of PDLSCs. Not surprisingly, capsaicin rescued the damage to mitochondrial morphology, decreased the concentration of intracellular Ca2+ and ROS, enhanced MMP and activated phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway. The invivo results showed that capsaicin significantly attenuated alveolar bone loss and augmented the expression of bone associated proteins. Capsaicin increases osteogenesis of PDLSCs under inflammation and reduces alveolar bone resorption in mouse experimental periodontitis.

Experimental Susceptibility of Nyssomyia antunesi and Lutzomyia longipalpis (Psychodidae: Phlebotominae) to Leishmania (Viannia) lainsoni and L. (V.) lindenbergi (Trypanosomatidae: Leishmaniinae).

The present work assessed the experimental susceptibility of Nyssomyia antunesi and Lutzomyia longipalpis to Leishmania (Viannia) lainsoni and L. (V.) lindenbergi. A L. (Leishmania) chagasi-Lu. longipalpis combination was used as a susceptible control. Wild-caught Ny. antunesi and laboratory-bred Lu. longipalpis were membrane-fed on blood with a 5 × 106/mL log-phase promastigote culture suspension and dissected on days 2 and 8 post-blood meal (pbm) for analysis focused on the assessment of parasitoses, as well as placement and promastigote morphotyping. Survival curves were constructed. In all combinations, promastigotes were observed on day 8 pbm. For both Leishmania species, in Lu. longipalpis, the presence of parasites was observed up to the stomodeal valve, while in Ny. antunesi, the presence of parasites was observed up to the cardia. There were no significant differences in parasitosis between L. (V.) lainsoni and L. (V.) lindenbergi in either Ny. antunesi or Lu. longipalpis. Six morphological promastigote forms were distinguished in Giemsa-stained gut smears. The survival curves of all combinations decreased and were affected differently by several Lu. longipalpis-parasite combinations, as well with Lu. longipalpis-uninfected blood. These findings stress Lu. longipalpis as experimentally susceptible to Leishmania spp. and suggest the putative susceptibility of Ny. antunesi to L. (V.) lainsoni and L. (V.) lindenbergi.

Effect of drugs regulating acute immune responses on the liver in septic processes

Objective. To study the effect of drugs regulating acute immune responses on liver in septic processes. Materials and methods. The study was conducted on 39 male white rats weighing 250-400 g. The animals were initiated septic process development by intraperitoneal injection of Klebsiella pneumoniae culture suspension with simultaneous intravenous injection of polyvinylpyrrolidone (PVP) and gadolinium in the volume of 0.6 ml. On the 14th day the animals were removed from the experiment by decapitation under light ether anesthesia, blood and liver were taken for biochemical and histological studies. Results. When analyzing the morphometric parameters of liver preparations, an increase in the number of nuclei and a slight increase in the area of nuclei in three experimental groups was established by 1,2 times (p < 0,05) compared to the control group, which reflects the ongoing processes of hepatocyte regeneration due to possible mechanisms, namely hypertrophy nuclei and proliferation with both PVP and gadolinium in systemic inflammatory response and sepsis. Moreover, the use of PVP and gadolinium led to a decrease in the likelihood of perinuclear edema, protein degeneration and large droplet vacuolization (p < 0,05). When using gadolinium, the lumen diameter of the sinusoids was the largest and amounted to 4,47; 3,22-5,63 µm (p<0,05), and in septic shock it did not differ from the group where PVP was used (p > 0,05). Of the laboratory parameters of surviving individuals, the lowest ALaT level was noted in the gadolinium experimental group – 53,7; 51,8-55,1 U/L (p<0,05), while the lowest urea level was observed when using PVP (5,0; 4,99-5,15 mmol/l) (p < 0,05) as part of a systemic inflammatory response. Conclusion. Gadolinium and PVP have a positive effect on the detoxification function of liver. Moreover, the effect of PVP on the morphology and function of the liver differs at the stages of septic process.

Получение и применение иодированного бактерицида для дезинфекции воды в локальных системах очистки

The paper presents the results of studies on obtaining a bactericide based on iodized anion exchange resin АВ-17-8. The obtained compound bactericidal efficiency was assessed on Escherichia сoli culture suspensions at 103–109 CFU/mL. All experiments gave positive results and demonstrated the possibility of their use for natural water treatment in local water treatment systems. The obtained bactericidal filter can be used for quantitative determination of bacteriological contamination of the studied waters. Water samples contaminated with E. coli at 500, 1000, 5000 and 10000 CFU/mL were prepared to quantify the bactericidal activity of the iodine-containing compound obtained on an anion-exchange resin. Then samples were sequentially passed through a 100 cm3 bactericidal filter. By the amount of iodine released, the degree of microbial contamination can be determined. There were no microbial cells in the filtrate at zero concentrations of released iodine in the solution. The degree of microbiological contamination of water Q was 10,000 CFU/mL at 0.00391 g/L of iodine. A linear dependence between bactericidal activity and iodine release from the anion-exchange resin was established. This allows us to assume that the positive iodine radical is released “on a signal” – at the appearance of living microorganisms carrying excessive electrostatic charge. The released by the bactericide positive iodine radical (I*+) interacts only with the electrostatic charge of microorganisms, and not with the functional groups of their shells. The found quantitative dependence makes it possible to determine microbiological contamination based on the iodine concentration in the treated solution. Based on the test results, a flowchart of the technological process of surface water treatment was proposed. The presence of a bactericidal filter allows the use of a reverse osmosis filter, sensitive to biological water contaminants, as part of the installation.

Abstract 1705: 3D growth modulates the competition between STAT3 and STAT5 in breast cancer

Abstract Approximately 13% of women are diagnosed with invasive breast cancer. Signal Transducer and Activator of Transcription 3 (STAT3) is a transcription factor that is often inappropriately activated in breast cancer and is frequently associated with Triple Negative Breast Cancer (TNBC). STAT5 can also be activated and inappropriate activation of STAT5 alone or with STAT3 is generally correlated with a more favorable prognosis, a lower grade tumor, and a better response to therapies compared to inappropriate activation of STAT3 alone. Therefore, knowing the activation status of STAT5 and understanding how STAT5 activation can attenuate STAT3-driven breast cancers could help guide therapeutic choices and patient treatment. STAT3 and STAT5 can regulate a set of overlapping target genes. For example, STAT3 and STAT5 can regulate BCL6 expression, where STAT5 represses expression and STAT3 enhances it. Importantly, STAT5 outcompetes and preferentially binds in the presence of activated STAT3. This suggests that the overlapping target genes could play an important role in understanding how activation of STAT5 can lead to a more favorable breast cancer compared to activation of STAT3 alone. This previous work was conducted in a 2D cell culture model; however, 3D models can better mimic clinical settings and the use of 3D models can help bridge the gap. Therefore, understanding the relationship between STAT3 and STAT5 in 3D is important. SK-BR-3 cells (HER2+ cells with low basal STAT3 activity) and MDA-MB-231 cells (TNBC cells with constitutive STAT3 activity) are being compared between 2D cell culture and 3D media suspension. We have shown using chromatin immunoprecipitation that STAT3 DNA-binding is enhanced in 3D at certain binding sites, whereas STAT5 is not. STAT5 has reduced dominance and preferential binding over STAT3 for BCL6 in 3D compared to 2D. In other cases, STAT3 binding is decreased upon STAT5 activation in 3D but not 2D. To begin to understand these differences, we analyzed phosphorylation of these STATs in 2D and 3D. Phospho-STAT3 levels were similar between 2D and 3D in SK-BR-3 cells when activated with LIF stimulation but were higher in 3D in the MDA-MB-231 cells which contain constitutive STAT3 activity. STAT5 activation by prolactin stimulation did not directly affect STAT3 phosphorylation in 2D or 3D but does affect STAT3 binding in 3D growing cells. However, prolactin induced phospho-STAT5 seems to be reduced in 3D in both cell lines. We are currently analyzing additional binding sites to better define the differences between STAT3 and STAT5 mediated gene regulation in 2D and 3D since STAT phosphorylation does not fully address it. Overall, our data suggests that differences between 2D and 3D breast cancer models will be important for translation to patient tumors and understanding the relationship between STAT3 and STAT5 will help elucidate how STAT5 activation status can be used when treating patients with breast cancer. Citation Format: Alexandra Elizabeth Temple, Sarah R. Walker. 3D growth modulates the competition between STAT3 and STAT5 in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1705.

Arthrospira platensis growth control system in semi-industrial environment

The paper proposes the design of a device based on the Arduino platform for obtaining data on the biomass of micro-algae, the temperature of the cultural suspension and the irradiation of the pool surface during semi-industrial cultivation of A. platensis. The task was to offer the simplest possible design, consisting of widely available components. In the course of the work, sensor designs were developed, calibration curves were taken, and correction factors were calculated that are necessary for using this device. The device includes a temperature, irradiance and optical density sensor connected to the Arduino Mega platform. The data from the sensors are displayed on the LCD screen and recorded on the Micro SD card.

Small-Scale Cultivation and Transfection of ExpiCHO and HEK293E Cells in Single-Use Orbitally Shaken Bioreactors.

Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK293) cells are the two most important mammalian hosts for the production of recombinant proteins. In this chapter, the suspension cultivation and transfection of these cells in small-scale, single-use orbitally shaken bioreactors, TubeSpin™ bioreactor 50 [orbitally shaken reactor 50 (OSR50)], and TubeSpin™ bioreactor 600 [orbitally shaken reactor 600 (OSR600)] are described. These are conical centrifuge tubes with nominal volumes of 50mL and 600mL, respectively, that have been redesigned with a ventilated cap for the cultivation of animal cells in suspension at working volumes up to 20mL and 400mL, respectively.

Large-Scale Production of Specialized Metabolites In Vitro Cultures.

For centuries plants have been intensively utilized as reliable sources of food, flavoring, and pharmaceutical ingredients. However, plant natural habitats are being rapidly lost due to the climate change and agriculture. Plant biotechnology offers a sustainable approach for the bioproduction of specialized plant metabolites. The unique structural features of plant-derived specialized metabolites, such as their safety profile and multi-target spectrum, have led to the establishment of many plant-derived drugs. However, there are still many challenges to overcome regarding the production of these metabolites from plant in vitro systems and establish a sustainable large-scale biotechnological process. These challenges are due to the peculiarities of plant cell metabolism, the complexity of plant specialized metabolite pathways, and the correct selection of bioreactor systems and bioprocess optimization. In this book chapter, we attempted to focus on the advantages of plant in vitro systems and in particular plant cell suspensions for their cultivation as a source of plant-derived specialized metabolites. A state-of-the-art technological platform for plant cell suspension cultivation from callus induction to lab-scale cultivation, extraction, and purification is presented. Possibilities for bioreactor cultivation of plant cell suspensions in benchtop and large-scale volumes are highlighted, including several examples and patents for industrial production of specialized metabolites.

Untargeted metabolomic in basil cell cultures - a case study of Ocimum basilicum L. var. minimum Alef.

Due to the lack of experimental databases, together with the chemical complexity and the dynamic nature of plants' metabolome, most of the metabolites in complex biological materials (like plant in vitro tissue cultures) are not-annotated, unidentified metabolites. In this study, a method for further metabolite characterization and classification based on the UPLC-HESI-HRMS/MS approach for small-leaved basil (Ocimum basilicum L. var. minimum Alef.) callus and cell suspension culture is presented. A total of 2168 metabolic features were detected, out of which the database for exact mass metabolic profiling for 1949 metabolites is presented here since there is no available database dedicated to O. basilicum. We further focused on secondary metabolites (particularly phenolic compounds). The presence of 60 different phenolic compounds belonging mainly to the groups of flavonoids, glycosides, terpenoids, and phenolic acids is confirmed. By comparing relative abundances of phenolic compounds from callus culture and cell suspension culture, both grown on two types of media, via svd-PCА, univariate analysis, post-hoc tests, and heatmapping of metabolites, we provided a practical example of how resources presented here can be further applied in tissue culture-based basil metabolomics studies. This study represents the first approach toward routine targeted investigation of secondary metabolites in basil in vitro cultures and provides various opportunities for new-generation analyses.