Growth Medium Research Articles

Characteristics of Aqueous Chemical Species Generation in Plasma-Facing Liquid Systems Using Helium Jet Plasma.

Plasma-facing liquids (PFLs) facilitate the storage of reactive O and N species (RONS), including H2O2 and NO2 -, which remain in the PFL after plasma treatment, and they can continuously influence the target immersed in the liquid. However, their behaviors and levels of generation and extinction depend strongly on the plasma characteristics and liquid condition. Therefore, understanding the effects of the liquid type on the plasma discharge characteristics and the RONS generated via plasma discharge is necessary. We compared the RONS generation and storage trends of deionized H2O and a high-conductivity PFL, RPMI 1640, which is a well-known cell culture medium commonly used to culture mammalian cells. RPMI 1640 acted as an electrode and enhanced the plasma discharge power by supplying abundant radicals and RONS. The production of gaseous hydroxyl radicals and NO markedly increased, which facilitated H2O2 and NO2 - production in the PFL for the first 200 s, and then the increase in the RONS concentration stagnated. With respect to storage, as the components within RMPI 1640 exhibited high reaction constants for their reactions with H2O2, H2O2 elimination was completed in <30 min. Unlike H2O2, the concentration of NO2 - in the PFL was unchanged.

In vitro mass propagation of Dendrocalamus asper (Giant bamboo) through direct organogenesis

Dendrocalamus asper (giant bamboo) is a clumping type of bamboo belonging to the Poaceae family. Due to its economic and environmental value, demand for this species has increased tremendously. Conventional propagation methods have limitations due to low seed viability and the lack of healthy clumps. Therefore, an in vitro mass propagation protocol was developed to provide healthy plants for large-scale plantations. Seeds were used as the explant and they were surface sterilized and cultured on MS medium free of growth regulators. Nodal segments taken from in vitro germinated seedlings were used for shoot initiation. The best medium for shoot induction (MS medium supplemented with 0.0–2.5 mg/L BAP), best medium for multiple shoot induction (MS medium supplemented with 0.0–5.0 mg/L BAP), effect of shoot cluster size (shoot clusters containing 1–4 shoots) and effect of physical state of the medium (semisolid and liquid media) on multiple shoot induction were determined using shoots per node, mean shoot length and mean number of leaves per shoot after 6 weeks of incubation. Elongated shoots were transferred into a rooting medium and the best medium for root induction (MS medium supplemented with 0.0–5.0 mg/L IBA and IAA) and effect of cluster size (shoot clusters containing 1–4 shoots) on rooting were determined using the number of roots and root length after 6 weeks of incubation. All the cultures were maintained under a 16-hour photoperiod. Well-developed plantlets were transferred to coir pellets and after four weeks transferred into different potting mixtures containing different combinations of sand, compost and coir dust. Unless otherwise mentioned there were at least twenty replicates in all treatments. The highest mean number of shoots per node (16.87±0.52), mean shoot length (4.12±0.27 cm) and mean number of leaves per shoot (4.80±0.33) were observed in the MS medium supplemented with 1.0 mg/L BAP. The MS medium supplemented with 2.0 mg/L BAP was the best for multiple shoot induction, shoot cluster with 3 shoots was the best cluster size and the liquid medium had a better effect on shoot multiplication. The half-strength MS medium supplemented with 2.0 mg/L IBA was the best for in vitro root induction with the highest mean number of roots (7.15±0.77) and a mean root length of 10.79±1.11 cm. Shoot clusters with 3 shoots was the best cluster size for root induction. A sand: compost: coir dust (1:1:1) mixture was the best potting mixture giving 100% survival. These findings provide a reliable micropropagation protocol for D. asper, which holds great promise for meeting the growing demand for bamboo resources and promoting sustainable bamboo cultivation.

Lack of cytotoxic and genotoxic effects of mPEG-silane coated iron(III) oxide nanoparticles doped with magnesium despite cellular uptake in cancerous and noncancerous lung cells

Cytotoxic and genotoxic effects of novel mPEG-silane coated iron(III) oxide nanoparticles doped with magnesium (Mg0.1-γ-Fe2O3(mPEG-silane)0.5) have been investigated on human adenocarcinomic alveolar basal epithelial (A549) and human normal bronchial epithelial (BEAS-2B) cells. In the studies several molecular and cellular targets addressing to cell membrane, cytoplasm organelles and nucleus components were served as toxicological endpoints. The as-synthesized nanoparticles were found to be stable in the cell culture media and were examined for different concentration and exposure times. No cytotoxicity of the tested nanoparticles was found although these nanoparticles slightly increased reactive oxygen species in both cell types studied. Mg0.1-γ-Fe2O3(mPEG-silane)0.5 nanoparticles did not produce any DNA strand breaks and oxidative DNA damages in A549 and BEAS-2B cells. Different concentration of Mg0.1-γ-Fe2O3(mPEG-silane)0.5 nanoparticles and different incubation time did not affect cell migration. The lung cancer cells' uptake of the nanoparticles was more effective than in normal lung cells. Altogether, the results evidence that mPEG-silane coated iron(III) oxide nanoparticles doped with magnesium do not elucidate any deleterious effects on human normal and cancerous lung cells despite cellular uptake of these nanoparticles. Therefore, it seems reasonable to conclude that these novel biocompatible nanoparticles are promising candidates for further development towards medical applications.

Культуральные признаки и рост на сложных жидких питательных средах перспективного грибного штамма антагониста М-24 Penicillium sp. возбудителя фомоза подсолнечника

A microbiological method decreasing harmfulness of Phoma rot on sunflower, which is one of the prevailing disease in all regions of crop cultivation, is developed at the V.S. Pustovoit All-Russian Research Institute of Oil Crops. The cultural traits of antagonist fungus of phoma pathogen on sunflower М-24 Penicillium sp. on three agar media and strain growth on four complicated liquid nutrient media were studied at surface cultivation of a strainproducer to develop elements of technological regimen for production of microbiopreparations in a preparative form wettable powder. On the tenth days of cultivation at +25 °С temperature, strong growth of the fungus with abundant conidial sporulation and maximal diameter of a colony was noted on the agar medium by Rudakov. The medium by Rudakov is stated as optimal among complicated liquid nutrient media. In ten days of the surface cultivation, a thick folded mycelial film with an abundant sporulation was formed on this medium as well as maximal weight of a dry mycelium – 1.44 g per 100 ml of nutrient medium vs. 0.20–1.39 g on the other media.

Физиологические свойства перспективного грибного штамма антагониста возбудителя сухой гнили подсолнечника Т-5 Trichoderma sp.

Dry rot is one of the most dangerous and wide spreading disease on sunflower in last years. Due to the growing demand for environmentally friendly production of agricultural products, the demand for competitive microbiological products is increasing. Since 2020, there are studied and developed environmentally friendly microbiological preparations of a multifunctional type of action against the dry rot pathogen on sunflower Rhizopus oryzae Went &amp; Prins. Geerl. at the V.S. Pustovoit All-Russian Research Institute of Oil Crops. The physiological qualities of a promising antagonist strain of the dry rot pathogen T-5 Trichoderma sp. isolated as a result of stepwise screening were studied during stationary cultivation on liquid nutrient media in order to develop elements of a technological regimen for the production of a microbiological preparation in a preparative form wettable powder. Optimal conditions for surface cultivation of the fungus T-5 Trichoderma sp. on a liquid nutrient medium were determined as following: temperature +25 °C; acidic reaction of the medium (pH 3); glucose and starch as carbon source; source of nitrogen nutrition is corn extract. The media by Rudakova and No. 1 were identified as the best complicated liquid nutrient media.

Quality Control of L-Ascorbic Acid 2-Phosphate Magnesium Using Reversed-Phase Liquid Chromatography.

L-Ascorbic acid 2-phosphate magnesium (AP-Mg) salt is a Vitamin C derivative frequently used as a raw material in cell culture media for research purposes as well as for Good Manufacturing Practice (GMP)-manufacturing of cell and tissue advanced therapy medicinal products. A selective reversed-phase HPLC (RP-LC) method was developed and validated. Commercially available AP-Mg products from different suppliers were analyzed. Various new impurities were found using this newly developed RP-LC method. Using quantitative nuclear magnetic resonance spectroscopy, a mass balance of roughly 99.9% was obtained; the total numbers of impurities detected in both methods are also identical. The values of the relative ultraviolet (UV) response factors at λ = 210nm of the impurities in this RP-LC method were discussed. When equaling the overall mean relative response factor of the impurities to 0.6 (estimated central value), the mass balance in the RP-LC method was nearly 100%. The structures of the new impurities are proposed as ethylation derivatives of open-ring AP-Mg products as well as phosphorylated derivatives of ascorbic acid.

A new hiPSC-based viral expression system to study virus host-cell interaction in hiPSC-derived cardiomyocytes

Abstract Coxsackievirus B3 (CVB3) belongs to the Picornaviridae family and is a member of the enterovirus genus. CVB3 is the most frequently detected pathogen in viral myocarditis, which leads to acute heart infections in 20-40% of cases and to dilated cardiomyopathy (DCM) in cases of prolonged viral persistence. Complex arrhythmias and sudden deaths due to CVB infections have also been reported, particularly in newborns and children. Observed arrhythmias include bradycardia, AV block and junctional ectopic tachycardia, suggesting that in addition to the functioning myocardium, the pacemaker cells and cardiac conduction system may also be impaired by CVB3 infection. The symptomatic phenotype is highly variable and depends on unclear host factors. It ranges from inconspicuous inflammation and rapid healing to acute congestive heart failure or viral persistence with progressive chronic disease. Despite the increasing and profound knowledge about viral myocarditis, it is still a challenge to identify further host factors that contribute to acute and chronic myocarditis, as science lacks sensitive and controllable disease models. The presented project shows the generation of a controlled, inducible virus-expressing hiPSC line that provides a solution for the beforementioned problems. The new human induced pluripotent stem cell line (hiPSC) i can be used to carry out virologic experiments in a controlled manner and without risk to the user. For this purpose, the modified and non-infectious genome of the enterovirus Coxsackievirus B3 (CVB3) is stably integrated into the genome of a wild-type hiPSC line. The CVB3 genome to be stably integrated was modified to prevent the formation of functional capsids by a DVP0 mutation. In addition, the 3'UTR and 5'UTR regions of the CVB3 genome were removed, which prevents uncontrolled self-replication of viral RNA. As a result, work with this CVB3DVP0-UTR genome is classified as S1 compliant. The expression of the CVB3DP0-UTR virus genome can be controlled by adding doxycycline to the cell culture medium by a TeT-On System. The transfected cells are then cultivated as clones and tested for virus expression. The dose-dependent CVB3DVP0 expression is verified using qPCR and FACS after 1 day of doxycycline application in the cell culture medium. Clones isolated and positively tested for CVB3 expression are used to carry out initial experiments on the interaction of the expressed viral proteins with the cell. Particular focus is placed on the induction of cell death, remodelling of the cytoskeleton and increase of reactive oxygen species (ROS) in mitochondria after CVB3DVP0-UTR induction in order to validate the cell system for application. Differentiation of the CVB3-expressing hiPSC-line into cardiomyocytes with subsequent virus induction results in the elevation of mitochondrial ROS, degradation of the cytoskeleton and arrhythmic behaviour of the cells.

Discovery, characterization, and engineering of an advantageous Streptomyces host for heterologous expression of natural product biosynthetic gene clusters

BackgroundStreptomyces is renowned for its robust biosynthetic capacity in producing medically relevant natural products. However, the majority of natural products biosynthetic gene clusters (BGCs) either yield low amounts of natural products or remain cryptic under standard laboratory conditions. Various heterologous production hosts have been engineered to address these challenges, and yet the successful activation of BGCs has still been limited. In our search for a valuable addition to the heterologous host panel, we identified the strain Streptomyces sp. A4420, which exhibited rapid initial growth and a high metabolic capacity, prompting further exploration of its potential.ResultsWe engineered a polyketide-focused chassis strain based on Streptomyces sp. A4420 (CH strain) by deleting 9 native polyketide BGCs. The resulting metabolically simplified organism exhibited consistent sporulation and growth, surpassing the performance of most existing Streptomyces based chassis strains in standard liquid growth media. Four distinct polyketide BGCs were chosen and expressed in various heterologous hosts, including the Streptomyces sp. A4420 wild-type and CH strains, alongside Streptomyces coelicolor M1152, Streptomyces lividans TK24, Streptomyces albus J1074, and Streptomyces venezuelae NRRL B-65442. Remarkably, only the Streptomyces sp. A4420 CH strain demonstrated the capability to produce all metabolites under every condition outperforming its parental strain and other tested organisms. To enhance visualization and comparison of the tested strains, we developed a matrix-like analysis involving 15 parameters. This comprehensive analysis unequivocally illustrated the significant potential of the new strain to become a popular heterologous host.ConclusionOur engineered Streptomyces sp. A4420 CH strain exhibits promising attributes for the heterologous expression of natural products with a focus on polyketides, offering an alternative choice in the arsenal of heterologous production strains. As genomics and cloning strategies progress, establishment of a diverse panel of heterologous production hosts will be crucial for expediting the discovery and production of medically relevant natural products derived from Streptomyces.

Rice Weevil (Sitophilus oryzae L.) Gut Bacteria Inhibit Growth of Aspergillus flavus and Degrade Aflatoxin B1.

In this study, bacteria residing in the gut of the rice weevils (Sitophilus oryzae L.) (Coleoptera: Curculionidae) feeding on aflatoxin-contaminated corn kernels were isolated and evaluated for their ability to suppress Aspergillus flavus and to remove/degrade aflatoxin B1 (AFB1). Four morphologically distinct S. oryzae gut-associated bacterial isolates were isolated and identified as Bacillus subtilis (RWGB1), Bacillus oceanisediminis (RWGB2), Bacillus firmus (RWGB3), and Pseudomonas aeruginosa (RWGB4) based on 16S rRNA gene sequence analysis. These bacterial isolates inhibited A. flavus growth in the dual culture assay and induced morphological deformities in the fungal hyphae, as confirmed by scanning electron microscopy. All four bacterial isolates were capable of removing AFB1 from the nutrient broth medium. In addition, culture supernatants of these bacterial isolates degraded AFB1, and the degradation of toxin molecules was confirmed by liquid chromatography-mass spectrometry. The bacterial isolates, B. subtilis RWGB1, B. oceanisediminis RWGB2, and P. aeruginosa RWGB4, were capable of producing antifungal volatile organic compounds that inhibited A. flavus growth. These results suggest that the bacterial isolates from S. oryzae gut have the potential to bind and/or degrade AFB1. Further research on their application in the food and feed industries could enhance the safety of food and feed production.

Antimicrobial metabolites from the marine-derived fungus Aspergillus sp. ZZ1861

Fungi from the genus Aspergillus are important resources for the discovery of bioactive agents. This investigation characterized the isolation, structural elucidation, and antimicrobial evaluation of 46 metabolites produced by the marine-derived fungus Aspergillus sp. ZZ1861 in rice solid and potato dextrose broth liquid media. The structures of these isolated compounds were determined based on their HRESIMS data, NMR spectral analyses, and data from ECD, NMR, and optical rotation calculations. Emericelactones F and G, 20R,25S-preshamixanthone, 20R,25R-preshamixanthone, phthalimidinic acid A, phthalimidinic acid B, aspergilol G, and 2-hydroxyemodic amide are eight previously undescribed compounds and (S)-2-(5-hydroxymethyl-2-formylpyrrol-1-yl) propionic acid lactone is reported from a natural resource for the first time. It is also the first report of the configurations of 25S–O-methylarugosin A, 25R-O-methylarugosin A, 5R-(+)-9-hydroxymicroperfuranone, and 5R-(+)-microperfuranone. Phthalimidinic acid A, phthalimidinic acid B, aspergilol G, and 2-hydroxyemodic amide have antifungal activity against Candida albicans with MIC values of 1.56, 3.12, 1.56, and 12.5 μg/mL, respectively, 20R,25S-preshamixanthone (MIC 25 μg/mL) shows antibacterial activity against Escherichia coli, and 20R,25R-preshamixanthone exhibits antimicrobial activity against all three tested pathogens of methicillin-resistant Staphylococcus aureus, E. coli, and C. albicans with MIC values of 50, 25, 25 μg/mL, respectively.

Study of metal organic framework with siRNA for overcoming matrix barrierin breast cancer

Objective: This study aimed to develop a new delivery strategy that utilized metal organic framework (MOF) loaded with small-interfering RNA (siRNA) targeting ITGAV to overcome tumor matrix barrier, and thus enhance drug penetration and immune accessibility in breast cancer. Methods: MOF@siITGAV particles were constructed and characterized. The uptake of MOF@siITGAV in breast cancer cell line 4T1 was observed by the cellular uptake assay. The toxicity of MOF@siITGAV was detected by cell counting kit 8 (CCK-8). The blank control group, naked siITGAV group and MOF@siITGAV group were set. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) and Western blot were used to detect the expressions of ITGAV. The level of transforming growth factor β1 (TGF-β1) in the cell culture medium was detected by enzyme-linked immunosorbent assay (ELISA). The penetration of MOF@siITGAV in 4T1 cells was tested by constructing 3D spheroids. Mouse models of triple negative breast cancer were established. The effect of MOF@siITGAV on the growth of transplanted tumors and main organs was verified. Imminohistochemical (IHC) was used to test the expression of collagen and CD8. Results: MOF@siITGAV particles were constructed with sizes of (198.0±3.3) nm and zeta potential of -(20.2±0.4) mV. MOF@siITGAV could be engulfed by 4T1 cells and triggered to release siRNA. Compared to the blank control group, the expression of ITGAV in the MOF@siITGAV group [(46.5±11.3)%] and the naked siITGAV group [(109.9±19.0)%] was lower. TGF-β1 in the cell culture medium of the blank control group, naked siITGAV group, and MOF@siITGAV group was (474.5±34.4) pg/ml, (437.2±16.5) pg/ml, and (388.4±14.4) pg/ml, respectively. MOF@siITGAV could better penetrate into 4T1 spheroids and exhibit no obvious toxicity. The cell viability was (99.7±3.5)%, (98.2±5.2)%, (97.3±6.6)%, (92.1±8.1)%, and (92.4±4.1)%, respectively, after MOF@siITGAV treatment with the concentration of 0, 10, 20, 40, 80, and 160 μg/ml, respectively, for 24 h. The tumor growth in the MOF@siITGAV group was suppressed significantly. After 15-day treatment, the tumor volume of the MOF@siITGAV group was (135.3±41.9) mm3, smaller than that of the blank control group [(691.1±193.0) mm3] (P=0.025). The expression of collagen and the number of CD8 positive cells of the MOF@siITGAV group were lower than those of the other two groups. No significant abnormalities were observed in the main organs of mice. Conclusions: Targeting the integrinαv on the surface of cancer cells could destroy extracellular matrix, improve drug delivery, and increase immune infiltration.

#1261 A small-molecule inhibitor of Bruton's tyrosine kinase reduces production of galactose-deficient IgA1 in IgA nephropathy

Abstract Background and Aims IgA nephropathy (IgAN) is a chronic kidney disease characterized by glomerular immunodeposits that contain aberrantly glycosylated IgA1, i.e., IgA1 with some hinge-region O-glycans deficient in galactose (galactose-deficient IgA1; Gd-IgA1). These immunodeposits originate from circulating immune complexes that contain Gd-IgA1 bound by IgG autoantibodies. Consistent with this hypothesis, serum levels of Gd-IgA1 and IgG autoantibodies independently predict disease progression in IgAN patients. Biochemical studies using IgA1-secreting cell lines derived from peripheral blood of IgAN patients and healthy controls have revealed that Gd-IgA1 production is driven by altered biosynthetic pathways of IgA1 O-glycans. Specifically, cell lines from IgAN patients have altered expression and activity of several glycosyltransferases, C1GalT1 and ST6GalNAc2, and a C1GalT1-specific chaperone, C1GalT1C1. Genetic studies have shown that serum levels of Gd-IgA1 are genetically co-determined and genome-wide association studies (GWAS) identified genetic variations that impact the expression C1GALT1 and C1GALT1C1 genes. Moreover, macroscopic hematuria during an upper-respiratory-tract infection is common at the clinical onset of IgAN and during episodes of accentuated disease activity, indicating a connection between IgAN and mucosal inflammation. Notably, a pro-inflammatory cytokine interleukin-6 (IL-6), enhances production of Gd-IgA1 exclusively in the cell lines derived from IgAN patients. Leukemia inhibitory factor (LIF) has similar effects. LIF-induced effects are mediated by abnormal STAT1 signaling and pathways that include Lyn, a Src-family kinase. The corresponding gene, LYN, was recently identified in one of the 16 new IgAN-associated GWAS loci. Lyn, together with spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (Btk), is involved in B-cell-receptor (BCR)-signaling pathways that include activation and inhibitory signals. The aim of this study was to assess if Btk may be involved in Gd-IgA1 production in IgAN. Method IgA1-producing cell lines were cloned by limiting dilution of Epstein-Barr-virus-immortalized peripheral-blood B cells from patients with IgAN (n = 5) and healthy controls (n = 5). Cell signaling, induced by LIF or by BCR crosslinking with anti-IgA antibody, was assessed by flow cytometry or SDS-PAGE/immunoblotting using antibodies for specific phosphorylated sites of selected proteins. Data were normalized using antibodies for specific proteins. Total IgA and Gd-IgA1 were quantified in cell-culture medium using standard ELISA and lectin-ELISA, respectively. Gd-IgA1, normalized to IgA, was expressed in Units (U) of standard Gd-IgA1 protein. Results IgA1-producing cell lines from patients with IgAN and healthy controls secreted similar amounts of total IgA (17.2 ± 9.3 ug/ml vs. 14.6 ± 9.5 ug/ml), but the cells from IgAN patients secreted more Gd-IgA1 (24.2 ± 7.5 U vs. 15.5 ± 2.7 U; p &amp;lt; 0.05). Baseline phosphorylation of Btk (Y223), but not Syk (Y551), was higher in the cells from patients with IgAN than in those from healthy controls. We used an IgA1-secreting cell line from an IgAN patient to confirm that BCR crosslinking with F(ab’)2 anti-IgA antibody in IgA + CD19+ cells lines induced phosphorylation of Btk (Y223). Conversely, anti-IgM antibody, used as a negative control, did not activate Btk. LIF stimulation did not affect phosphorylation of Btk or Syk. A Btk inhibitor, PCI-32765 (ibrutinib), reduced Gd-IgA1 production in a dose-dependent fashion exclusively in the IgAN-derived cells. Notably, 1 uM PCI-32765 reduced Gd-IgA1 production to that for healthy controls (25.7 ± 1.2 U to 17.3 ± 1.8 U; p &amp;lt; 0.01). Conclusion Inhibition of Btk with a small-molecule inhibitor normalized Gd-IgA1 production in IgA1-secreting cells from patients with IgAN, revealing a previously unknown role of Btk in aberrant O-glycosylation of IgA1. Btk may thus represent a potential new target for disease-specific therapy of IgAN.

Cartilage-Specific Gene Expression and Extracellular Matrix Deposition in the Course of Mesenchymal Stromal Cell Chondrogenic Differentiation in 3D Spheroid Culture.

Articular cartilage damage still remains a major problem in orthopedical surgery. The development of tissue engineering techniques such as autologous chondrocyte implantation is a promising way to improve clinical outcomes. On the other hand, the clinical application of autologous chondrocytes has considerable limitations. Mesenchymal stromal cells (MSCs) from various tissues have been shown to possess chondrogenic differentiation potential, although to different degrees. In the present study, we assessed the alterations in chondrogenesis-related gene transcription rates and extracellular matrix deposition levels before and after the chondrogenic differentiation of MSCs in a 3D spheroid culture. MSCs were obtained from three different tissues: umbilical cord Wharton's jelly (WJMSC-Wharton's jelly mesenchymal stromal cells), adipose tissue (ATMSC-adipose tissue mesenchymal stromal cells), and the dental pulp of deciduous teeth (SHEDs-stem cells from human exfoliated deciduous teeth). Monolayer MSC cultures served as baseline controls. Newly formed 3D spheroids composed of MSCs previously grown in 2D cultures were precultured for 2 days in growth medium, and then, chondrogenic differentiation was induced by maintaining them in the TGF-β1-containing medium for 21 days. Among the MSC types studied, WJMSCs showed the most similarities with primary chondrocytes in terms of the upregulation of cartilage-specific gene expression. Interestingly, such upregulation occurred to some extent in all 3D spheroids, even prior to the addition of TGF-β1. These results confirm that the potential of Wharton's jelly is on par with adipose tissue as a valuable cell source for cartilage engineering applications as well as for the treatment of osteoarthritis. The 3D spheroid environment on its own acts as a trigger for the chondrogenic differentiation of MSCs.

#2716 Selenoprotein-P1 (SEPP-1) in human proximal tubule cells after ischemic-reperfusion injury: an in-vitro model

Abstract Background and Aims Novel biomarkers, anticipating serum creatinine rise, are eagerly needed to have a speedy diagnosis of acute kidney injury (AKI) and thus to mitigate and to preserve kidney function with well-timed therapeutic plans. Recently, in a pilot study on patients undergoing elective major cardiac surgery with cardiopulmonary bypass (CPB), selenoprotein-p1 (SEPP-1) has emerged as a promising candidate plasmatic biomarker for an early AKI risk stratification. SEPP-1 increased particularly between 4th to 8th hour after CPB and with a strict relation with ischemia duration (Bolignano et Al. Rev. Cardiovasc. Med. 2022). SEPP1 is primarily secreted from liver and acts as a selenium transporter supplying tissues and organs with this trace mineral which elicits the activity of specific glutathione peroxidase selenoenzymes. Additionally, renal tubular cells are able to synthetize this protein but remain unknown if, during AKI, SEPP1 increases as defeat answer to renal cells damage or if it's hepatic SEPP1 that accumulates for a reduced renal glomerular filtration rate. The primary aim of this study was to investigate the release of SEPP-1 by renal tubular cells in an experimental model of AKI induced by ischemia-reperfusion and secondarily evaluate the effect of selenium supplying on renal cells injury. Method Human proximal tubular cells (PTC), HK2 cells and human embryonic kidney 293 cells (HEK293) were cultured in Keratinocyte-SFM a complete serum-free medium supplemented with human recombinant Epidermal Growth Factor (rEGF) and Bovine Pituitary Extract (BPE) (LGC Standards s.r.l., Milan, Italy) and in Eagle's Minimum Essential Medium (E-MEM) (LGC Standards s.r.l., Milan, Italy) containing 10% fetal bovine serum, respectively. The cells were pre-incubated with medium containing Sodium Selenite 100 nM for 24 h and then were treated for 24 hours with CoCl2 (Sigma Aldrich S.r.l., Milan, Italy) a chemical hypoxia inducer (500 µM for HK-2 and 250 µM for HEK-293). After that cell viability was evaluated by MTT and trypan bleu dye exclusion assay, while, protein expression of HIF (hypoxia marker) and SEPP1 was analyzed by western blot analysis. Results Ischemia-reperfusion was simulated by the exposure of HK-2 and HEK-293 to CoCl2 and confirmed by HIF expression. The expression of SEPP-1 increased significantly in HK-2 and HEK-293 after hypoxia stress (Fig. 1). Furthermore, the treatment with CoCl2 determined a decrease of cell viability (about 50% less) and Sodium selenite alone increased cell proliferation (about 15% more) in both cell lines. Interestingly cell growth after hypoxia augmented significantly if cell lines were pre-treated with sodium-selenite, highly in HK-2 cells (p &amp;lt; 0.001). Conclusion These preliminary data evidenced, in vitro, that AKI is able to induce renal tubular expression of SEPP-1 and probably SEPP-1 release in human after renal injury is not only a result of a reduced filtration of the hepatic released protein. Furthermore, SEPP-1 showed a possible protective role in AKI. In fact, AKI induced by ischemia-reperfusion, in our in vitro cellular model, was able to induce an increased expression of SEPP-1 in renal tubular cells suggesting a shielding mechanism against injury related to selenium transport. This hypothesis was supported by an exalted SEPP-1 expression after Sodium-Selenite adding to cells’ culture. Supplementary experiments, including functional molecular assays in vitro and in vivo studies will allow to better characterize the role of SEPP-1 in AKI.

A non-spherical nanoparticles system for the delivery of peptides using polymer grafted nanocellulose

AbstractPeptide drugs are increasingly used to treat a variety of diseases ranging from cancer, and infections to cardiovascular diseases. However, peptides can suffer from low stability in the bloodstream. Entrapment of peptides into nano-sized carriers of various types has widely been explored, but all of them have spherical shapes. Nanocellulose can in contrast serve as a non-spherical nanoparticle with a high aspect ratio. After the isolation of nanocellulose by TEMPO-mediated oxidation, the material needs to be modified with polymers to generate nanoparticles with high water-solubility that can also favourably interact with peptide drugs. We have here chosen insulin as the model drug, which can strongly interact with cationic polymers. As it is known that cationic polymer may retain charged drugs too tightly, we have selected poly(2-(dimethylamino)ethyl acrylate) PDMAEA as a degradable polymer that undergoes self-hydrolysis to poly(acrylic acid) in water. This polymer was compared to poly(N-(3-(dimethylamino)propyl) acrylamide) PDMAPAA, which is a stable cationic polymer. The cationic polymer was co-grafted with poly(2-hydroxyethyl acrylate) PHEA as a water-soluble neutral polymer using the three-component Passerini reaction. A combination of fluorescence and UV-Vis techniques were used to quantify the amount of polymer that was conjugated to the surface. The polymer-coated nanocellulose was labelled with the fluorescent cyanine dye Cy5 while insulin was labelled with Cy3 creating a FRET system that allows monitoring of the interaction between insulin and polymer in cell growth media. We observed that despite the self-hydrolysis of PDMAEA into a negatively charged polymer, the negatively charged insulin was not released in buffer solution according to the FRET studies. Only the addition of serum-supplemented cell growth media led to insulin release. The limited release was explained with the fact that insulin, as well as other peptides, have a mixture of negative and positive charges, with the pH value and the isoelectric point determining the balance between both. Negative-charged polymers can therefore still interact favourably with negatively charged peptides by interacting with cationic amino acids. Graphical Abstract

Метафорические образы перемещения в жидкой среде в диалекте английского языка Scots

The paper attempts to study the semantics of verbs denoting movement in a liquid medium in the Scots dialect. The aim of the study is to determine the ways of figurative interpretation of the semantics of movement in a liquid medium in Scottish lexical metaphor. The scientific novelty lies in the fact that for the first time in the Scots dialect, the semantics of verbs of movement in a liquid medium have been studied and described as a basis for creating a system of metaphorical images. The results showed that such a class of verbs is small and poorly differentiated. In the original meaning of the verbal lexemes of this class, such semantic components as “medium”, “speed” and “sound” turned out to be important. These components of the semantics in dialectal verbs served as the basis for a figurative assimilation of processes and phenomena of various spheres of human activity. The metaphorical evaluation of verbs in which the main semantic component of the original (nominative) meaning is movement in a “liquid dirty medium” is most often negative. Movement in such a medium served as a source sphere for the figurative comprehension of a number of deviant social actions, deeds, and human behavior.

Hypoxia‑regulated exosomal miR‑185 inhibits esophageal squamous cell carcinoma progression and predicts prognosis.

Despite advances in treatment and diagnosis, the prognosis of patients with esophageal squamous cell carcinoma (ESCC) remains poor. MicroRNAs (miRNAs/miRs) are associated with prognosis in esophageal cancer, indicating that they may help guide treatment decisions. The aim of the present study was to explore exosomal miR-185 as a candidate prognostic biomarker and therapeutic target in ESCC, to investigate its biological function and clinical significance, and to ascertain the applicability of circulating exosomal miR-185 for the development of targeted drugs for ESCC treatment. A GeneChip miRNA array was used to compare exosomal miRNA expression in ESCC cell lines under hypoxia with those under normoxia. Exosomal miR-185 expression was then confirmed by reverse transcription-quantitative PCR. Patient background and prognosis were compared between high and low miR-185 expression groups. Functional analyses were performed to evaluate the antitumor effects of miR-185 in ESCC cells. Global Gene Set Enrichment Analysis of The Cancer Genome Atlas data was also performed, and differentially expressed exosomal miRNAs under hypoxia were identified compared to those under normoxia. Hypoxia markedly decreased the expression of exosomal miR-185 in KYSE-960 and T.Tn cell culture media. Overexpression of miR-185 suppressed the migration, invasion and colony-forming abilities of ESCC lines, and also suppressed cell cycle progression and promoted apoptosis after cisplatin treatment. Notably, high miR-185 expression was associated with signaling pathways related to cell death, DNA damage and p53. Furthermore, circulating exosomal miR-185 levels were associated with cN and cStage, and could predict progression-free survival and disease-specific survival of patients with ESCC after initial treatment. In conclusion, miR-185 holds potential as a prognostic biomarker and therapeutic target in ESCC.

Extractive Mass Transfer of Zr(IV) into a Hydrophobic Ionic Liquid Medium Containing Diglycolamide Extractant: Solvent Extraction and Spectroscopic Analysis.

Solvent extraction of Zr(IV) in ionic liquid (IL) medium is less known and Zr(IV) - IL chemistry indeed needs exploration to realize the coordination approach of Zr(IV) in IL phase. In view of this, in the present work, a strongly hydrophobic imidazolium based IL: 1-methyl-3-octylimidazolium bis(trifluoromethylsulfonyl)imide ([C8mim][NTf2]) as the medium containing a diglycolamide (DGA) extractant: N,N,N',N'-tetra-n-octyldiglycolamide (TODGA) was opted to understand the extraction behavior of Zr(IV) from nitric acid medium. Different experimental parameters such as the concentration of initial nitric acid, initial feed metal concentration, equilibration time and ligand concentration were tuned to unravel the extraction efficiency of the proposed IL phase towards Zr(IV). The extraction scenario was completely dependent on the hydrophobicity of IL diluent and increasing extraction trend with an increase in feed acidity along with the coordination of two TODGA molecules ensured Zr(IV) - complex formation in a neutral solvation mechanistic pathway. The extraction trend of Zr(IV) was compared with molecular diluent (n-dodecane (n-DD)) containing TODGA and a phase modifier: N,N-dihexyloctanamide (DHOA). The coordination aspect of Zr(IV) with ligand in IL phase was ascertained spectroscopically to validate Zr(IV) loading in IL phase. Thermodynamics of Zr(IV) extraction revealed the nature of the extraction process and the complex formation.

An Efficient Propagation Approach to Forcing Softwood Shoots from Epicormic Buds and Subsequent Rooting of Paulownia elongata S. Y. Hu.

The current research describes the multiplication of Paulownia elongata S. Y. Hu, a timber plant, through the forcing of softwood shoots from epicormic buds under glasshouse conditions in spring and fall seasons. Different growth media were used to compare their effect on the forcing potential of epicormic buds. For this, 25-30-cm-long and 1.2-2-cm-diameter stem segments taken from the lower juvenile portion of a mother plant were placed horizontally in flat trays containing media, i.e., sterilized well-moistened sand, peat moss, perlite, and vermiculite individually. Furthermore, 4-6-cm-long forced softwood shoots were detached and treated with various concentrations of IBA (indole-3-butyric acid) and NAA (α-naphthyl acetic acid) either individually or in combinations for subsequent rooting. The response of shoot forcing was better in spring as compared to fall in terms of shoot length (cm), and number of shoots or leaves; however, an earlier bud break was observed during fall after 30 days of the initial experiment. The use of peat moss and vermiculite proved to be equally suitable for early bud break in both seasons, whereas in terms of shoot and leaf number as well as the shoot length (cm), the best outcome was observed in sand. Best rooting was observed at 3 gL-1 IBA + 3 gL-1 NAA in terms of root number per shoot, root length (cm), and days to root initiation while using sand as the growth medium after 50 days of the rooting experiment. The successfully established plantlets were further shifted to soil at Botanical Garden, University of the Punjab, Lahore, Pakistan, exhibiting an 87.5% survival rate. On the basis of the results obtained, it may be concluded that reasonable softwood shoot forcing in P. elongata may further be exploited for its mass scale nursery propagation as well as use in future in vitro studies.

Glyphosate resistance and biodegradation by Burkholderia cenocepacia CEIB S5-2.

Glyphosate is a broad spectrum and non-selective herbicide employed to control different weeds in agricultural and urban zones and to facilitate the harvest of various crops. Currently, glyphosate-based formulations are the most employed herbicides in agriculture worldwide. Extensive use of glyphosate has been related to environmental pollution events and adverse effects on non-target organisms, including humans. Reducing the presence of glyphosate in the environment and its potential adverse effects requires the development of remediation and treatment alternatives. Bioremediation with microorganisms has been proposed as a feasible alternative for treating glyphosate pollution. The present study reports the glyphosate resistance profile and degradation capacity of the bacterial strain Burkholderia cenocepacia CEIB S5-2, isolated from an agricultural field in Morelos-México. According to the agar plates and the liquid media inhibition assays, the bacterial strain can resist glyphosate exposure at high concentrations, 2000mg·L-1. In the degradation assays, the bacterial strain was capable of fast degrading glyphosate (50mg·L-1) and the primary degradation metabolite aminomethylphosphonic acid (AMPA) in just eight hours. The analysis of the genomic data of B. cenocepacia CEIB S5-2 revealed the presence of genes that encode enzymes implicated in glyphosate biodegradation through the two metabolic pathways reported, sarcosine and AMPA. This investigation provides novel information about the potential of species of the genus Burkholderia in the degradation of the herbicide glyphosate and its main degradation metabolite (AMPA). Furthermore, the analysis of genomic information allowed us to propose for the first time a metabolic route related to the degradation of glyphosate in this bacterial group. According to the findings of this study, B. cenocepacia CEIB S5-2 displays a great glyphosate biodegradation capability and has the potential to be implemented in glyphosate bioremediation approaches.