Agarose Plate Research Articles

Multipad agarose plate: a rapid and high-throughput approach for antibiotic susceptibility testing.

We describe a phenotypic antibiotic susceptibility testing (AST) method that can provide an eightfold speed-up in turnaround time compared with the current clinical standard by leveraging advances in microscopy and single-cell imaging. A newly developed growth plate containing 96 agarose pads, termed the multipad agarose plate (MAP), can be assembled at low cost. Pads can be prepared with dilution series of antibiotics. Bacteria are seeded on the pads and automatically imaged using brightfield microscopy, with a fully automated segmentation pipeline quantifying microcolony formation and growth rate. Using a test set of nine antibiotics with very different targets, we demonstrate that accurate minimum inhibitory concentration (MIC) measurements can be performed based on the growth rate of microcolonies within 3 h of incubation with the antibiotic when started from exponential phase. Faster, reliable and high-throughput methods for AST, such as MAP, could improve patient care by expediting treatment initiation and alleviating the burden of antimicrobial resistance.

Enhanced glycolysis by ATPIF1 gene inactivation increased the anti-bacterial activities of neutrophils through induction of ROS and lactic acid

ATP synthase inhibitory factor 1 (ATPIF1) is a mitochondrial protein that regulates the activity of FoF1-ATP synthase. Mice lacking ATPIF1 throughout their bodies (Atpif1−/−) exhibit a reduction in the number of neutrophils. However, it remains unclear whether the inactivation of ATPIF1 impairs the antibacterial function of mice, this study aimed to evaluate it using a mouse peritonitis model. Mice were intraperitoneally injected with E. coli to induce peritonitis, and after 24 h, the colonies of E. coli were counted in agarose plates containing mice peritoneal lavage fluids (PLF) or extract from the liver. Neutrophils were analyzed for glucose metabolism in glycolysis following LPS stimulation. Reactive oxygen species (ROS) and lactic acid (LA) levels in neutrophils were measured using flow cytometry and Seahorse analysis, respectively. N-Acetylcysteine (NAC) and 2-Deoxy-d-glucose (2-DG) were employed to assess the role of ROS and LA in neutrophil bactericidal activity. RNA-seq analysis was conducted in neutrophils to investigate potential mechanisms. In ATPIF1−/− neutrophils, bactericidal activity was enhanced, accompanied by increased levels of ROS and LA compared to wildtype neutrophils. The augmented bactericidal activity of ATPIF1−/− neutrophils was reversed by pretreatment with NAC or 2-DG. RNA-seq analysis revealed downregulation of multiple genes involved in glutathione metabolism, pyruvate oxidation, and heme synthesis, along with increased expression of inflammatory and apoptotic genes. This study suggests that the inactivation of the Atpif1 gene enhances glucose metabolism in neutrophils, resulting in increased bactericidal activity mediated by elevated levels of ROS and LA. Inhibiting ATPIF1 may be a potential approach to enhance antibacterial immunity.

Screening of a Novel Fibrinolytic Enzyme-Producing Streptomyces from a Hyper-Arid Area and Optimization of Its Fibrinolytic Enzyme Production

Fibrinolytic enzymes are a kind of proteolytic enzymes that can hydrolyze fibrin and dissolve blood clots. They could be used as a therapeutic agent for treating thrombosis. It is important for the treatment of cardiovascular disease to find and develop new thrombolytic drugs. In order to explore new fibrinolytic enzymes, a strain named 214L-11 with protease and fibrinolytic enzyme activity, which was isolated from the Flaming Mountain of Xinjiang Province, was screened using the skimmed milk plate, the blood powder agarose plate and the fibrin plate methods. Phylogenetic analyses showed that strain 214L-11 shared the highest similarity with Streptomyces fumanus NBRC 13042T (98.88%), which indicated that it represented a potential novel species in the Streptomyces genus. The fibrinolytic enzyme produced by 214L-11 displayed thrombolytic and anticoagulant activities, and it could degrade a single specific protein in the thrombus, thereby destroying the thrombus structure. The fermentation medium optimized through response surface methodology was 15 g/L soluble starch, g/L KNO3 0.58, 0.43 g/L peptone, 0.01 g/L FeSO4·7H2O, 0.5 g/L MgSO4·7H2O, 0.2 g/L Mn2+, 0.5 g/L NaCl and 1 L distilled water, pH 8, and the maximum amount of fibrinolytic enzyme produced by strain 214L-11 in the optimal fermentation medium was 1255.3 FU/mL. Overall, the fibrinolytic enzyme-producing strain was screened from the Flaming Mountain of Xinjiang for the first time, which provided a basis for further research and the development of new efficient and safe hemolytic drugs.

A high-throughput sensory assay for parasitic and free-living nematodes.

Sensory pathways first elucidated in Caenorhabditis elegans are conserved across free-living and parasitic nematodes, even though each species responds to a diverse array of compounds. Most nematode sensory assays are performed by tallying observations of worm behavior on two-dimensional planes using agarose plates. These assays have been successful in the study of volatile sensation but are poorly suited for investigation of water-soluble gustation or parasitic nematodes without a free-living stage. In contrast, gustatory assays tend to be tedious, often limited to the manipulation of a single individual at a time. We have designed a nematode sensory assay using a microfluidics device that allows for the study of gustation in a 96-well, three-dimensional environment. This device is suited for free-living worms and parasitic worms that spend their lives in an aqueous environment, and we have used it to show that ivermectin inhibits the gustatory ability of vector-borne parasitic nematodes. Insight box Nematodes are powerful model organisms for understanding the sensory biology of multicellular eukaryotes, and many parasitic species cause disease in humans. Simple sensory assays performed on agarose plates have been the bedrock for establishing the neuronal, genetic, and developmental foundations for many sensory modalities in nematodes. However, these classical assays are poorly suited for translational movement of many parasitic nematodes and the sensation of water-soluble molecules (gustation). We have designed a device for high-throughput nematode sensory assays in a gel matrix. This 'gustatory microplate' is amenable to several species and reveals novel responses by free-living and parasitic nematodes to cues and drugs.

Automatically tracking feeding behavior in populations of foraging C. elegans.

Caenorhabditis elegans feeds on bacteria and other small microorganisms which it ingests using its pharynx, a neuromuscular pump. Currently, measuring feeding behavior requires tracking a single animal, indirectly estimating food intake from population-level metrics, or using restrained animals. To enable large throughput feeding measurements of unrestrained, crawling worms on agarose plates at a single worm resolution, we developed an imaging protocol and a complementary image analysis tool called PharaGlow. We image up to 50 unrestrained crawling worms simultaneously and extract locomotion and feeding behaviors. We demonstrate the tool's robustness and high-throughput capabilities by measuring feeding in different use-case scenarios, such as through development, with genetic and chemical perturbations that result in faster and slower pumping, and in the presence or absence of food. Finally, we demonstrate that our tool is capable of long-term imaging by showing behavioral dynamics of mating animals and worms with different genetic backgrounds. The low-resolution fluorescence microscopes required are readily available in C. elegans laboratories, and in combination with our python-based analysis workflow makes this methodology easily accessible. PharaGlow therefore enables the observation and analysis of the temporal dynamics of feeding and locomotory behaviors with high-throughput and precision in a user-friendly system.

Double-Gene Copromoting Expression Analysis in tPA/GH Transgenic Goat Mammary Epithelial Cells and Thrombolytic Activity of tPA In Vitro.

Human tissue-plasminogen activator (tPA) is a thrombolytic drug widely used in the treatment of stroke, pulmonary thrombosis, acute myocardial infarction, and other thrombotic diseases. The double genes cointegrated into the organisms and cells can produce a synergistic effect, which will improve the expression level of the target gene. However, the study of the integration of the GH and tPA genes to improve the expression level of tPA has not yet been reported. In order to elucidate this, we generated monoclonal goat mammary epithelial cell lines with tPA/GH double-gene integration and analyzed the tPA expression level in single- and double-gene integrated cells. We selected the mammary gland-specific expressing vectors BLC14/tPA and BLC14/GH with the β-lactoglobulin gene as a regulatory sequence in our previous research. The tPA and GH genes were electronically cotransfected into goat mammary epithelial cells. Resistant cell lines were screened by G418, and transgenic monoclonal cell lines were confirmed by PCR. The tPA expression was induced by prolactin and detected in the cell induction solution after 48 h by ELISA and Western blotting. We detected the tPA biological activity in vitro by fibrin agarose plate assay (FAPA). The results showed that a total of 207 resistant monoclonal cells were obtained, including 126 cell lines with tPA monogenic integration and 51 cell lines with tPA/GH double-gene integration. The rate of double-gene integration was 24.6% (51/207). A total of 48 cells expressed tPA, of which 25.3% (19/75) cells expressed single gene, and 56.9% (29/51) cells expressed double genes. The concentration of tPA in single-gene-expressing cells was 8.0-64.0 μg/mL, and the tPA level in double-gene-expressing cells was significantly higher (200-7200 μg/mL). In addition, the tPA had a relatively strong in vitro thrombolytic activity determined by FAPA. The results showed that goat mammary epithelial cell lines with tPA/GH gene integration were successfully established by electrotransfection, and the expression level of tPA in double-gene integrated cell lines was significantly increased. This study provided a new way for the preparation of a transgenic goat and other animal with high tPA expression by somatic cell nuclear transfer. The findings also laid a foundation for efficient production of pharmaceutical proteins in transgenic animal mammary gland bioreactors in the future.

Molecular Cloning of DARPins G3 in pET28b Expression Vector and Optimization of the Expression of This Protein in Escherichia Coli

Background: Human epidermal growth factor receptor 2 (HER2) is over-expressed in breast, ovarian, gastric, and prostate cancers and is used as a tumor marker in the diagnosis of cancer. Monoclonal antibodies have been used as a diagnostic and therapeutic tool against HER2. Because of the difficulties associated with the stability and complexity of the construct and the high cost of antibody production, we aimed to investigate, cloning, and expression of HER2- binding DARPins genes to identify, HER2-positive tumor markers, we aimed to investigate. Materials and Methods: After synthesis, the DARPins peptide gene was cloned into the M13 vector and sub-cloned into the TOP10 pet28b bacterial vector. After culturing the bacteria on an agarose plate containing antibiotics, the transfected bacteria expressing the DARPins gene were selected. To ensure gene cloning, we used enzymatic digestion and recombinant plasmid delivery for sequencing. Isopropyl β-d-1-thiogalactopyranosideIPTG was used for the induction of recombinant protein expression and the SDS-PAGE method and Western blot for expression confirmation. Results: The polymerase chain reaction (PCR) amplification product of DARPins was analyzed using agarose gel electrophoresis. Plasmid was purified from the positive clone by PCR cloning, sequenced and gene cloning was confirmed. After culturing from competent cells, protein expression was obtained from positive colonies. SDS- PAGE results showed the effect of different conditions including temperature, IPTG concentration, and time on the pET-DARPins expression. Conclusion: We were succeeded to express a new codon-optimized DARPins gene in Escherichia coli and HEK293t system.

Cyclic AMP signalling and glucose metabolism mediate pH taxis by African trypanosomes

The collective movement of African trypanosomes on semi-solid surfaces, known as social motility, is presumed to be due to migration factors and repellents released by the parasites. Here we show that procyclic (insect midgut) forms acidify their environment as a consequence of glucose metabolism, generating pH gradients by diffusion. Early and late procyclic forms exhibit self-organising properties on agarose plates. While early procyclic forms are repelled by acid and migrate outwards, late procyclic forms remain at the inoculation site. Furthermore, trypanosomes respond to exogenously formed pH gradients, with both early and late procyclic forms being attracted to alkali. pH taxis is mediated by multiple cyclic AMP effectors: deletion of one copy of adenylate cyclase ACP5, or both copies of the cyclic AMP response protein CARP3, abrogates the response to acid, while deletion of phosphodiesterase PDEB1 completely abolishes pH taxis. The ability to sense pH is biologically relevant as trypanosomes experience large changes as they migrate through their tsetse host. Supporting this, a CARP3 null mutant is severely compromised in its ability to establish infections in flies. Based on these findings, we propose that the expanded family of adenylate cyclases in trypanosomes might govern other chemotactic responses in their two hosts.

Retrieving sulfur in thiosulfate bio-oxidation: indigenous consortium vs. its dominant isolate Ochrobactrum sp.

Sulfur compounds such as thiosulfate are among contaminants with undesirable effects on ecosystems. Sulfur-oxidizing bacteria (SOB) can metabolize these compounds into less pernicious products such as elemental sulfur (S0). In this study, products distribution of thiosulfate bio-oxidation with emphasis on S0 was investigated in shake flasks (SFs) and 1 L stirred bioreactor (SB) using an indigenous SOB consortium. Enrichment of original cells taken from Atashkoh soil resulted in complete oxidation of 3000 ppm thiosulfate and 14% S0 formation as compared to 45% oxidation of original cells. The agarose plate method was selected to preserve this enriched-consortium due to the retrieval of more sulfur (30% in SF). Oxidation of 3000 ppm thiosulfate in SB resulted in 35.2% S0 formation and noticeably a higher thiosulfate depletion rate of 1 compared to 0.2 mmol S L−1 h−1 in SF. Complete oxidation of higher levels of thiosulfate (4000–6000 ppm) was achieved in SB and S0 was reclaimed by a maximum of 40% at 6000 ppm thiosulfate. The dominant strain was isolated and identified by 16S rRNA as Ochrobactrum sp. PN1. Although thiosulfate oxidation by this isolate was quite similar to the consortium, S0 formation was higher (195 mg S L−1) by the consortium compared to the isolated strain (138 mg S L−1). Our enriched SOB consortium was therefore well capable of partial oxidation of thiosulfate into S0, a less polluting intermediate product with versatile applications.

Recombinant Expression of Thrombolytic Agent Reteplase in Marine Microalga Tetraselmis subcordiformis (Chlorodendrales, Chlorophyta).

Tetraselmis subcordiformis, a unicellular marine green alga, is used widely in aquaculture as an initial feeding for fish, bivalve mollusks, penaeid shrimp larvae, and rotifers because of its rich content of amino acids and fatty acids. A stable nuclear transformation system using the herbicide phosphinothricin (PPT) as a selective reagent was established previously. In this research, the recombinant expression in T. subcordiformis was investigated by particle bombardment with the rt-PA gene that encodes the recombinant human tissue-type plasminogen activator (Reteplase), which is a thrombolytic agent for acute myocardial infarction treatment. Transgenic algal strains were selected by their resistance to PPT, and expression of rt-PA was validated by PCR, Southern blotting, and Western blotting, and bioactivity of rt-PA was confirmed by the fibrin agarose plate assay for bioactivity. The results showed that rt-PA was integrated into the genome of T. subcordiformis, and the expression product was bioactive, indicating proper post-transcriptional modification of rt-PA in T. subcordiformis. This report contributes to efforts that take advantage of marine microalgae as cell factories to prepare recombinant drugs and in establishing a characteristic pathway of oral administration in aquaculture.

Aerobic glycolysis supports hepatitis B virus protein synthesis through interaction between viral surface antigen and pyruvate kinase isoform M2.

As an intracellular pathogen, the reproduction of the hepatitis B virus (HBV) depends on the occupancy of host metabolism machinery. Here we test a hypothesis if HBV may govern intracellular biosynthesis to achieve a productive reproduction. To test this hypothesis, we set up an affinity purification screen for host factors that interact with large viral surface antigens (LHBS). This identified pyruvate kinase isoform M2 (PKM2), a key regulator of glucose metabolism, as a binding partner of viral surface antigens. We showed that the expression of viral LHBS affected oligomerization of PKM2 in hepatocytes, thereby increasing glucose consumption and lactate production, a phenomenon known as aerobic glycolysis. Reduction of PKM2 activity was also validated in several different models, including HBV-infected HepG2-NTCP-C4 cells, adenovirus mediated HBV gene transduction and transfection with a plasmid containing complete HBV genome on HuH-7 cells. We found the recovery of PKM2 activity in hepatocytes by chemical activators, TEPP-46 or DASA-58, reduced expressions of viral surface and core antigens. In addition, reduction of glycolysis by culturing in low-glucose condition or treatment with 2-deoxyglucose also decreased expressions of viral surface antigen, without affecting general host proteins. Finally, TEPP-46 largely suppressed proliferation of LHBS-positive cells on 3-dimensional agarose plates, but showed no effect on the traditional 2-dimensional cell culture. Taken together, these results indicate that HBV-induced metabolic switch may support its own translation in hepatocytes. In addition, aerobic glycolysis is likely essential for LHBS-mediated oncogenesis. Accordingly, restriction of glucose metabolism may be considered as a novel strategy to restrain viral protein synthesis and subsequent oncogenesis during chronic HBV infection.

A set of simple methods for detection and extraction of laminarinase

A carefully designed ammonium sulfate precipitation will simplify extraction of proteins and is considered to be a gold standard among various precipitation methods. Therefore, optimization of ammonium sulfate precipitation can be an important functional step in protein purification. The presence of high amounts of ammonium sulphate precludes direct detection of many enzymatically active proteins including reducing sugar assays (e.g. Nelson-Somogyi, Reissig and 3,5-dinitrosalicylic acid methods) for assessing carbohydrases (e.g. laminarinase (β (1–3)-glucanohydrolase), cellulases and chitinases). In this study, a simple method was developed using laminarin infused agarose plate for the direct analysis of the ammonium sulphate precipitates from Streptomyces rimosus AFM-1. The developed method is simple and convenient that can give accurate results even in presence of ammonium sulfate in the crude precipitates. Laminarin is a translucent substrate requiring the use of a stain to visualize the zones of hydrolysis in a plate assay. A very low-cost and locally available fluorescent optical fabric brightener Tinopal CBS-X has been used as a stain to detect the zones of hydrolysis. We also report simple methods to prepare colloidal chitin and cell free supernatant in this manuscript.

Simple Methods for the Preparation of Colloidal Chitin, Cell Free Supernatant and Estimation of Laminarinase.

Colloidal chitin (CC) is a common substrate used in research work involving chitin-active enzymes (chitinases). Cell free supernatant (CFS) is prepared from fermented broth. Preparation of CC and CFS usually involve large amounts of liquid, which must be separated from the solids. This necessitates the use of a large volume centrifugation facility, which may not be accessible to everyone. Filtration is a viable alternative to centrifugation, and several filter elements are described in the literature. Each of those elements has its own set of disadvantages like non-availability, high cost, fragility, and non-reusability. Here we describe the use of lab coat clothing material (LCCM) for the preparation of CC and CFS. For filtration purposes, the LCCM was found to be functional, rugged, reusable, and cost-effective. Also described here is a new method for the estimation of laminarinase using a laminarin infused agarose gel plate. An easily available optical fabric brightener (OFB) was used as a stain for the agarose plate. The laminarin infused agarose plate assay is simple, inexpensive, and was found to be impervious to high amounts of ammonium sulfate (AS) in enzyme precipitates.

Extracellular phosphoesterases of invertebrate origin: underrated actors in phosphorus recycling?

Extracellular enzymes contribute substantially to the remineralisation of organic matter in aquatic systems. Marine invertebrates release endogenous enzymes through activities such as sloppy feeding or egestion, but the significance of such processes is widely unexplored. We compared functional key properties such as activity, stability, and apparent molecular masses of extracellular phosphatases from faeces of the marine crustaceans Idotea balthica, Palaemon varians, and Homarus gammarus with those of their digestive organs. A fluorescent substrate was used to examine enzyme activity qualitatively on agarose plates and quantitatively in microplate assays. Apparent molecular masses and enzyme stability were examined by native substrate gel electrophoresis (NSGE). Active extracellular phosphatase were present in the faeces of all tested species. NSGE activity band patterns were similar in digestive tissue extracts and faeces extracts. The initial enzyme activity retained for about 2 days, but thereafter rapidly decreased. Activity band patterns of digestive organs and faeces of I. balthica remained consistent for up to 72 h. Antibiotic treatment did not reduce phosphatase activity in the faeces of I. balthica and H. gammarus but in the faeces P. varians. Because of the comparatively high activities in the faeces, the electrophoretic similarity between faeces and digestive organs, and the limited durability of the enzymes in faeces extracts, we conclude that the studied crustaceans release predominantly endogenous, rather than bacterial phosphatases through faeces. These can substantially contribute to the pool of active extracellular phosphatases and the recycling of phosphorus in aquatic systems. The wider physiological and ecological context is discussed.

Abstract P6-04-18: The regulatory role of activating transcription factor-2 (ATF2) in modulating tamoxifen resistance in estrogen-receptor positive breast cancer

Abstract Introduction: Activating transcription factor-2 (ATF2), a member of the ATF and CREB group of bZIP transcription factors regulates gene transcription by forming homodimers or heterodimers with other AP-1 family members. We have previously shown that ATF2 is important for modulating the effect of tamoxifen, whereas Thr71-ATF2 phosphorylation predicts for improved outcome of ER-positive breast cancers receiving tamoxifen. In this study we further explore the role of ATF2 in modulating breast cancer endocrine therapy resistance. Methods: ATF2 silencing: The MCF7, TamR, LCC2, LCC9 breast cancer cell lines were maintained in their appropriate growth media. Cells were transfected using RNAiMAX (ThermoFisher Scientific) with two different siRNAs and a Silencer® negative control. Gene expression was assessed by quantitative real-time PCR (RT-qPCR) (TaqMan assays, ThermoFisher Scientific) and the protein levels were analysed by western blotting. Cell growth, invasion, migration: Sulforhodamine B assay was used for cell growth determination following manufactures’ protocol. Soft-agar colony formation was performed by seeding and incubating the cells in low-melting agarose plates. The resulting colonies were 5% MTT-stained and counted in a Gel Count™ (Oxford Optronix). Invasion/migration was performed using the transwell matrigel-coated (BD Biosciences) plates and the scratch wound healing assay. All the assays were performed in triplicates in three independent experiments. Microarray profiling: Human HT12_V4 microarray (Illumina) was performed to identify genes that were differentially regulated by ATF-2 silencing in TamR cells compared to MCF-7. Data analyses including log2 transformation and quantile normalisation were performed on R Bioconductor. Differentially-expressed genes were selected using FDR correction for multiple testing with p-value cut-off <0.05. Results: ATF-2 silencing significantly inhibited the proliferation (p<0.005), colony formation (p<0.005) and migratory ability (p<0.01) of TamR, LCC2 and LCC9 cells but had a no effect on the growth and migration of MCF-7 cells. Moreover, differential gene expression analysis in MCF7 versus TamR cells revealed a shift from the ER-enriched transcription factor network towards an ATF2-enriched and TGF2β signalling pathway (p=0.025) in TamR cells suggesting alternative hormone-independent growth mechanisms. We then considered differentially-expressed genes specific to the TamR cells (not differentially-expressed in MCF7) and found a reintroduction of ER and its signalling pathway upon ATF2 knockdown along with a reduction in MYC transcriptional activity (EnrichR). RT-qPCR and western blot confirmed the increased expression of ER and its targets TFF1, GREB1, NCOA3, in TamR cells after ATF2 silencing in both the gene and protein levels. Conclusions: In conclusion, for the first time, ATF-2 was identified as a novel transcription factor that can modulate endocrine resistance by regulating ER expression and activity. This data suggests that ATF2 silencing may overcome endocrine resistance and highlights further the dual role of this transcription factor that can mediate endocrine sensitivity and resistance by modulating ER expression and activity. Citation Format: Athina Giannoudis, Mohammed I Malki, Hisham Mohammed, Bharath Rudraraju, Suraj Menon, Simak Ali, Jason S Carroll, Carlo Palmieri. The regulatory role of activating transcription factor-2 (ATF2) in modulating tamoxifen resistance in estrogen-receptor positive breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-18.

Transient expression of bioactive recombinant human plasminogen activator in tobacco leaf

To assess the potential of transient expression of recombinant human plasminogen activator (rhPA) in plants as a cost-effective approach for recombinant rhPA production. Tobacco mosaic virus-based expression vector pTMV rhPA-NSK and plant binary expression vector pJ Zera-rhPA were constructed by in vitro sequence synthesis and subcloning. The two vectors were inoculated on either Nicotiana benthamiana or N. excelsiana leaves via agroinfiltration. The expression of recombinant rhPA in Nicotiana leaves was examined using Western blotting and ELISA, and the in vitro fibrinolysis activity of plant-produced rhPA was assessed by fibrin agarose plate assay (FAPA). Five to nine days after infiltration with an Agrobacterium inoculum containing pTMV rhPA-NSK, necrosis appeared in the infiltrated area on the leaves of both Nicotiana plants, but intact recombinant rhPA was still present in the necrotic leaf tissues. The accumulation level of recombinant rhPA in infiltrated N. benthamiana leaves was significantly higher than that in N. excelsiana leaves (P < 0.05). The yield of recombinant rhPA was up to 0.6% of the total soluble protein (or about 60.0 μg per gram) in the fresh leaf biomass at 7 days post-inoculation. The plant-derived rhPA was bioactive to convert inactive plasminogen to active plasmin. No necrosis occurred in pJ Zera-rhPA-infiltrated leaves. The Zera-rhPA protein was partially cleaved between the site of Zera tag and rhPA sequence in both Nicotiana leaves. We speculated that the formation of Zera tags-induced particles in the plant cells was a dynamic process of progressive aggregation in which some of the soluble polypeptides were encapsulated in these particles. Enzymatically active recombinant rhPA can be rapidly expressed in tobacco plants using the plant viral ampliconbased system, which offers a promising alternative for cost-effective production of recombinant rhPA.

3D культура меланоцитов как тест-система и клеточная модель для изучения патологии меланогенеза

Функциональная активность меланоцитов обусловливает защитные свойства кожи против воздействия ультрафиолета, вызывающего фотостарение. Однако изучение меланоцитов in vivo затруднено тем, что необходимо использовать животные модели и трудоемкие методы анализа, а культивирование клеток в монослойной культуре in vitro сопряжено с потерей тканеспецифичных маркеров клеток. Данная работа посвящена получению и изучению сфероидов из меланоцитов, так как 3D культивирование меланоцитов в виде сфероидов может сохранить их фенотип и функциональность. Исследование проводили на первичной культуре меланоцитов кожи человека. Клетки культивировали в монослое в полной ростовой среде до 4 пассажа. Далее клетки помещали на агарозные планшеты с микролунками в посевной плотности 3,3 х 10 кл./мл. Анализ полученных сфероидов производили с помощью фотометрии, иммуноцитохимии и ПЦР в реальном времени. Было показано, что, при культивировании в монослое, к 4 пассажу снижалось количество синтезируемого меланина. Тогда как в 3D условиях меланоциты формировали компактные сфероиды, внутри которых в процессе культивирования не только сохранялся синтез, но и накапливался меланин. Была выявлена экспрессия специфических генов TYR и MCR1, и увеличивался синтез белков, участвующих в меланогенезе - gp100 и MITF. Таким образом, в данном исследовании было показано, что меланоциты in vitro способны формировать длительно живущие, жизнеспособные 3D структуры - сфероиды, с сохранением фенотипа и синтеза тканеспецифичных маркеров. Поэтому данные сфероиды могут быть успешно использованы как тест-системы для оценки эффективности препаратов, направленных на регуляцию уровня пигментации кожи. The functional activity of melanocytes determines protective properties of the skin against the effect of ultraviolet radiation, which causes photo-aging. However, studying melanocytes in vivo and in an in vitro monolayer culture is difficult because animal models and laborious analytical methods are required, and the culturing is associated with loss of tissue-specific cell markers. Since 3D cultivation of melanocytes in the form of spheroids can preserve their phenotype and functionality, this work focused on obtaining and studying melanocyte spheroids. The study was conducted using a primary culture of human skin melanocytes. Cells were cultured in a monolayer to the fourth passage. Then these cells were placed in agarose plates with microwells at the cell suspension concentration of 3.3 х 10 in vitro . These features make the melanocyte spheroids a convenient test-system for studying toxicity and efficiency of drugs targeted at regulation of skin pigmentation.

Process optimization for production and purification of novel fibrinolytic enzyme from Stenotrophomonas sp. KG-16-3

Intravascular thrombosis is a major cardiovascular complication responsible for high mortality worldwide. Existing thrombolytic agents are expensive and have various side effects. As a consequence, researchers continue to search for better thrombolytic agents. Fibrinolytic proteases especially those of microbial origin are considered as potential therapeutic candidates for thrombosis. The current study reports fibrinolytic protease from a bacterial isolate Stenotrophomonas sp. KG-16-3, as it exhibits high fibrinolytic activity on fibrin agarose plate. Studies on fibrinolytic protease from Stenotrophomonas sp. are lacking. So, a detailed study was conducted for the production and purification of fibrinolytic protease. Optimizing process parameters using the Design of Experiments method enhanced the yield by 1.5-fold. The fibrinolytic enzyme was purified by ammonium sulfate precipitation, ion-exchange and gel-filtration chromatography resulting in 7.1-fold purification and 16.7% yield with specific activity of 383.8 U/mg. The purified enzyme exhibited higher fibrinolytic activity than plasmin and had a molecular weight of 39 kDa. Optimal activity of the enzyme was observed at 50 °C and pH 10. The enzyme exhibited stability up to 60 °C, over pH 7–10 and in the presence of different metal ions and solvents. The activity of the enzyme was significantly reduced in the presence of phenylmethyl sulfonyl fluoride, iodoacetic acid and 1,10-phenanthroline, suggesting that the enzyme belonged to the serine–cysteine metalloprotease category. The present study is the first ever report on the Design of Experiments based optimization of fermentation conditions for the production of fibrinolytic protease from Stenotrophomonas sp.

Efficient increase of the novel recombinant human plasminogen activator expression level and stability through the use of homozygote transgenic rabbits.

Expression efficacy of recombinant protein in current expression systems is generally low. Therefore, the expression levels of recombinant proteins in the breast milk of transgenic animals are typically low. In view of this, the present study aimed to construct homozygous transgenic rabbits with a high expression level of recombinant human plasminogen activator (rhPA) during the entire lactation period. Homozygous transgenic rabbits were obtained using an effective rhPA mammary‑specific expression vector PCL25/rhPA. The expression level and thrombolytic ability of rhPA in the milk of both homozygous and hemizygous rabbits were detected by enzyme‑linked immunosorbent and fibrin agarose plate assays. It was observed that the expression of rhPA was constant during the entire lactation period in homozygous rabbits, while the expression of rhPA declined slowly in hemizygote rhPA transgenic rabbits during the lactation period. In addition, the expression of rhPA in homozygous transgenic rabbit was ~950µg/ml, which was markedly higher in comparison with that in hemizygote rabbits. Furthermore, increased gene copy number was observed to increase the expression level of rhPA at the same integration vector.

A Repeating Sulfated Galactan Motif Resuscitates Dormant Micrococcus luteus Bacteria.

Only a small fraction of bacteria can autonomously initiate growth on agar plates. Nongrowing bacteria typically enter a metabolically inactive dormant state and require specific chemical trigger factors or signals to exit this state and to resume growth. Micrococcus luteus has become a model organism for this important yet poorly understood phenomenon. Only a few resuscitation signals have been described to date, and all of them are produced endogenously by bacterial species. We report the discovery of a novel type of resuscitation signal that allows M. luteus to grow on agar but not agarose plates. Fractionation of the agar polysaccharide complex and sulfation of agarose allowed us to identify the signal as highly sulfated saccharides found in agar or carrageenans. Purification of hydrolyzed κ-carrageenan ultimately led to the identification of the signal as a small fragment of a large linear polysaccharide, i.e., an oligosaccharide of five or more sugars with a repeating disaccharide motif containing d-galactose-4-sulfate (G4S) 1,4-linked to 3,6-anhydro-α-d-galactose (DA), G4S-(DA-G4S) n≥2IMPORTANCE Most environmental bacteria cannot initiate growth on agar plates, but they can flourish on the same plates once growth is initiated. While there are a number of names for and manifestations of this phenomenon, the underlying cause appears to be the requirement for a molecular signal indicating safe growing conditions. Micrococcus luteus has become a model organism for studying this growth initiation process, often called resuscitation, because of its apparent connection with the persistent or dormant form of Mycobacterium tuberculosis, an important human pathogen. In this report, we identify a highly sulfated saccharide from agar or carrageenans that robustly resuscitates dormant M. luteus on agarose plates. We identified and characterized the signal as a small repeating disaccharide motif. Our results indicate that signals inherent in or absent from the polysaccharide composition of solid growth media can have major effects on bacterial growth.