BioTech最新文献

{"title":"Efficient Purification of Polyhistidine-Tagged Recombinant Proteins Using Functionalized Corundum Particles.","authors":"Jule L Völzke,&nbsp;Sarah Smatty,&nbsp;Sarah Döring,&nbsp;Shireen Ewald,&nbsp;Marcus Oelze,&nbsp;Franziska Fratzke,&nbsp;Sabine Flemig,&nbsp;Zoltán Konthur,&nbsp;Michael G Weller","doi":"10.3390/biotech12020031","DOIUrl":"https://doi.org/10.3390/biotech12020031","url":null,"abstract":"<p><p>Immobilized metal affinity chromatography (IMAC) is a popular and valuable method for the affinity purification of polyhistidine-tagged recombinant proteins. However, it often shows practical limitations, which might require cumbersome optimizations, additional polishing, and enrichment steps. Here, we present functionalized corundum particles for the efficient, economical, and fast purification of recombinant proteins in a column-free format. The corundum surface is first derivatized with the amino silane APTES, then EDTA dianhydride, and subsequently loaded with nickel ions. The Kaiser test, well known in solid-phase peptide synthesis, was used to monitor amino silanization and the reaction with EDTA dianhydride. In addition, ICP-MS was performed to quantify the metal-binding capacity. His-tagged protein A/G (PAG), mixed with bovine serum albumin (BSA), was used as a test system. The PAG binding capacity was around 3 mg protein per gram of corundum or 2.4 mg per 1 mL of corundum suspension. Cytoplasm obtained from different <i>E. coli</i> strains was examined as examples of a complex matrix. The imidazole concentration was varied in the loading and washing buffers. As expected, higher imidazole concentrations during loading are usually beneficial when higher purities are desired. Even when higher sample volumes, such as one liter, were used, recombinant protein down to a concentration of 1 µg/mL could be isolated selectively. Comparing the corundum material with standard Ni-NTA agarose beads indicated higher purities of proteins isolated using corundum. His6-MBP-mSA2, a fusion protein consisting of monomeric streptavidin and maltose-binding protein in the cytoplasm of <i>E. coli</i>, was purified successfully. To show that this method is also suitable for mammalian cell culture supernatants, purification of the SARS-CoV-2-S-RBD-His8 expressed in human Expi293F cells was performed. The material cost of the nickel-loaded corundum material (without regeneration) is estimated to be less than 30 cents for 1 g of functionalized support or 10 cents per milligram of isolated protein. Another advantage of the novel system is the corundum particles' extremely high physical and chemical stability. The new material should be applicable in small laboratories and large-scale industrial applications. In summary, we could show that this new material is an efficient, robust, and cost-effective purification platform for the purification of His-tagged proteins, even in challenging, complex matrices and large sample volumes of low product concentration.</p>","PeriodicalId":34490,"journal":{"name":"BioTech","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10204482/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9508957","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Impact of Biomass Drying Process on the Extraction Efficiency of C-Phycoerythrin.","authors":"Ariadna H Vergel-Suarez,&nbsp;Janet B García-Martínez,&nbsp;Germán L López-Barrera,&nbsp;Andrés F Barajas-Solano,&nbsp;Antonio Zuorro","doi":"10.3390/biotech12020030","DOIUrl":"https://doi.org/10.3390/biotech12020030","url":null,"abstract":"<p><p>Drying the biomass produced is one of the critical steps to avoid cell degradation; however, its high energy cost is a significant technological barrier to improving this type of bioprocess's technical and economic feasibility. This work explores the impact of the biomass drying method of a strain of <i>Potamosiphon</i> sp. on the extraction efficiency of a phycoerythrin-rich protein extract. To achieve the above, the effect of time (12-24 h), temperature (40-70 °C), and drying method (convection oven and dehydrator) were determined using an I-best design with a response surface. According to the statistical results, the factors that most influence the extraction and purity of phycoerythrin are temperature and moisture removal by dehydration. The latter demonstrates that gentle drying of the biomass allows removing the most significant amount of moisture from the biomass without affecting the concentration or quality of temperature-sensitive proteins.</p>","PeriodicalId":34490,"journal":{"name":"BioTech","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10204359/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9515219","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cyanidiales-Based Bioremediation of Heavy Metals.","authors":"Hari Lal Kharel, Ina Shrestha, Melissa Tan, Mohammad Nikookar, Negar Saraei, Thinesh Selvaratnam","doi":"10.3390/biotech12020029","DOIUrl":"10.3390/biotech12020029","url":null,"abstract":"<p><p>With growing urbanization and ongoing development activities, the consumption of heavy metals has been increasing globally. Although heavy metals are vital for the survival of living beings, they can become hazardous when they surpass the permissible limit. The effect of heavy metals varies from normal to acute depending on the individual, so it is necessary to treat the heavy metals before releasing them into the environment. Various conventional treatment technologies have been used based on physical, chemical, and biological methods. However, due to technical and economic constraints and poor sustainability towards the environment, the use of these technologies has been limited. Microalgal-based heavy metal removal has been explored for the past few decades and has been seen as an effective, environment-friendly, and inexpensive method compared to conventional treatment technology. Cyanidiales that belong to red algae have the potential for remediation of heavy metals as they can withstand and tolerate extreme stresses of heat, acid salts, and heavy metals. Cyanidiales are the only photosynthetic organisms that can survive and thrive in acidic mine drainage, where heavy metal contamination is often prevalent. This review focuses on the algal species belonging to three genera of Cyanidiales: <i>Cyanidioschyzon</i>, <i>Cyanidium</i>, and <i>Galdieria.</i> Papers published after 2015 were considered in order to examine these species' efficiency in heavy metal removal. The result is summarized as maximum removal efficiency at the optimum experimental conditions and based on the parameters affecting the metal ion removal efficiency. This study finds that pH, initial metal concentration, initial algal biomass concentration, algal strains, and growth temperature are the major parameters that affect the heavy metal removal efficiency of Cyanidiales.</p>","PeriodicalId":34490,"journal":{"name":"BioTech","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2023-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10123701/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9388827","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Protein Delivery to Insect Epithelial Cells In Vivo: Potential Application to Functional Molecular Analysis of Proteins in Butterfly Wing Development.","authors":"Yugo Nakazato,&nbsp;Joji M Otaki","doi":"10.3390/biotech12020028","DOIUrl":"https://doi.org/10.3390/biotech12020028","url":null,"abstract":"<p><p>Protein delivery to cells in vivo has great potential for the functional analysis of proteins in nonmodel organisms. In this study, using the butterfly wing system, we investigated a method of protein delivery to insect epithelial cells that allows for easy access, treatment, and observation in real time in vivo. Topical and systemic applications (called the sandwich and injection methods, respectively) were tested. In both methods, green/orange fluorescent proteins (GFP/OFP) were naturally incorporated into intracellular vesicles and occasionally into the cytosol from the apical surface without any delivery reagent. However, the antibodies were not delivered by the sandwich method at all, and were delivered only into vesicles by the injection method. A membrane-lytic peptide, L17E, appeared to slightly improve the delivery of GFP/OFP and antibodies. A novel peptide reagent, ProteoCarry, successfully promoted the delivery of both GFP/OFP and antibodies into the cytosol via both the sandwich and injection methods. These protein delivery results will provide opportunities for the functional molecular analysis of proteins in butterfly wing development, and may offer a new way to deliver proteins into target cells in vivo in nonmodel organisms.</p>","PeriodicalId":34490,"journal":{"name":"BioTech","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10123617/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9389213","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Molecular Characterization of Dehydrin in Azraq Saltbush among Related <i>Atriplex</i> Species.","authors":"Anas Musallam,&nbsp;Saeid Abu-Romman,&nbsp;Monther T Sadder","doi":"10.3390/biotech12020027","DOIUrl":"https://doi.org/10.3390/biotech12020027","url":null,"abstract":"<p><p><i>Atriplex</i> spp. (saltbush) is known to survive extremely harsh environmental stresses such as salinity and drought. It mitigates such conditions based on specialized physiological and biochemical characteristics. Dehydrin genes (<i>DHNs</i>) are considered major players in this adaptation. In this study, a novel <i>DHN</i> gene from Azrak (Jordan) saltbush was characterized along with other <i>Atriplex</i> species from diverse habitats. Intronless <i>DHN</i>-expressed sequence tags (495-761 bp) were successfully cloned and sequenced. Saltbush dehydrins contain one S-segment followed by three K-segments: an arrangement called SK3-type. Two substantial insertions were detected including three copies of the K2-segemnet in <i>A. canescens</i>. New motif variants other than the six-serine standard were evident in the S-segment. AhaDHN1 (<i>A. halimus</i>) has a cysteine residue (SSCSSS), while AgaDHN1 (<i>A. gardneri var. utahensis</i>) has an isoleucine residue (SISSSS). In contrast to the conserved K1-segment, both the K2- and K3-segment showed several substitutions, particularly in AnuDHN1 (<i>A. nummularia</i>). In addition, a parsimony phylogenetic tree based on homologs from related genera was constructed. The phylogenetic tree resolved DHNs for all of the investigated <i>Atriplex</i> species in a superclade with an 85% bootstrap value. Nonetheless, the DHN isolated from Azraq saltbush was uniquely subclustred with a related genera <i>Halimione portulacoides</i>. The characterized DHNs revealed tremendous diversification among the <i>Atriplex</i> species, which opens a new venue for their functional analysis.</p>","PeriodicalId":34490,"journal":{"name":"BioTech","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10123722/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9382421","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biomolecular Liquid-Liquid Phase Separation for Biotechnology.","authors":"Sumit Shil,&nbsp;Mitsuki Tsuruta,&nbsp;Keiko Kawauchi,&nbsp;Daisuke Miyoshi","doi":"10.3390/biotech12020026","DOIUrl":"https://doi.org/10.3390/biotech12020026","url":null,"abstract":"<p><p>The liquid-liquid phase separation (LLPS) of biomolecules induces condensed assemblies called liquid droplets or membrane-less organelles. In contrast to organelles with lipid membrane barriers, the liquid droplets induced by LLPS do not have distinct barriers (lipid bilayer). Biomolecular LLPS in cells has attracted considerable attention in broad research fields from cellular biology to soft matter physics. The physical and chemical properties of LLPS exert a variety of functions in living cells: activating and deactivating biomolecules involving enzymes; controlling the localization, condensation, and concentration of biomolecules; the filtration and purification of biomolecules; and sensing environmental factors for fast, adaptive, and reversible responses. The versatility of LLPS plays an essential role in various biological processes, such as controlling the central dogma and the onset mechanism of pathological diseases. Moreover, biomolecular LLPS could be critical for developing new biotechnologies such as the condensation, purification, and activation of a series of biomolecules. In this review article, we introduce some fundamental aspects and recent progress of biomolecular LLPS in living cells and test tubes. Then, we discuss applications of biomolecular LLPS toward biotechnologies.</p>","PeriodicalId":34490,"journal":{"name":"BioTech","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10123627/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10349283","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Computational Screening of Approved Drugs for Inhibition of the Antibiotic Resistance Gene <i>mecA</i> in Methicillin-Resistant <i>Staphylococcus aureus</i> (MRSA) Strains.","authors":"Benson Otarigho,&nbsp;Mofolusho O Falade","doi":"10.3390/biotech12020025","DOIUrl":"https://doi.org/10.3390/biotech12020025","url":null,"abstract":"<p><p>Antibiotic resistance is a critical problem that results in a high morbidity and mortality rate. The process of discovering new chemotherapy and antibiotics is challenging, expensive, and time-consuming, with only a few getting approved for clinical use. Therefore, screening already-approved drugs to combat pathogens such as bacteria that cause serious infections in humans and animals is highly encouraged. In this work, we aim to identify approved antibiotics that can inhibit the <i>mecA</i> antibiotic resistance gene found in methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) strains. The MecA protein sequence was utilized to perform a BLAST search against a drug database containing 4302 approved drugs. The results revealed that 50 medications, including known antibiotics for other bacterial strains, targeted the <i>mecA</i> antibiotic resistance gene. In addition, a structural similarity approach was employed to identify existing antibiotics for <i>S. aureus</i>, followed by molecular docking. The results of the docking experiment indicated that six drugs had a high binding affinity to the <i>mecA</i> antibiotic resistance gene. Furthermore, using the structural similarity strategy, it was discovered that afamelanotide, an approved drug with unclear antibiotic activity, had a strong binding affinity to the MRSA-MecA protein. These findings suggest that certain already-approved drugs have potential in chemotherapy against drug-resistant pathogenic bacteria, such as MRSA.</p>","PeriodicalId":34490,"journal":{"name":"BioTech","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-03-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10123713/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9382418","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimization of Melanin Production by <i>Streptomyces antibioticus</i> NRRL B-1701 Using <i>Arthrospira (Spirulina) platensis</i> Residues Hydrolysates as Low-Cost L-tyrosine Supplement.","authors":"Oranit Kraseasintra,&nbsp;Sritip Sensupa,&nbsp;Kanjana Mahanil,&nbsp;Sada Yoosathaporn,&nbsp;Jeeraporn Pekkoh,&nbsp;Sirasit Srinuanpan,&nbsp;Wasu Pathom-Aree,&nbsp;Chayakorn Pumas","doi":"10.3390/biotech12010024","DOIUrl":"https://doi.org/10.3390/biotech12010024","url":null,"abstract":"<p><p>Melanin is a functional pigment that is used in various products. It can be produced by <i>Streptomyces antibioticus</i> NRRL B-1701 when supplemented with L-tyrosine. <i>Arthrospira (Spirulina) platensis</i> is a cyanobacterium with high protein content, including the protein phycocyanin (PC). During PC's extraction, biomass residues are generated, and these residues still contain various amino acids, especially L-tyrosine, which can be used as a low-cost supplement for melanin production. Thus, this study employed a hydrolysate of <i>A. platensis</i> biomass residue for L-tyrosine substitution. The effects of two drying methods, namely, lyophilization and dying via a hot air oven, on the proximate composition and content of L-tyrosine in the biomass residue were evaluated. The highest L-tyrosine (0.268 g L-tyrosine/100 g dried biomass) concentration was obtained from a hot-air-oven-dried biomass residue hydrolysate (HAO-DBRH). The HAO-DBRH was then used as a low-cost L-tyrosine supplement for maximizing melanin production, which was optimized by the response surface methodology (RSM) through central composite design (CCD). Using the RSM-CCD, the maximum level of melanin production achieved was 0.24 g/L, which is approximately four times higher than it was before optimization. This result suggests that <i>A. platensis</i> residue hydrolysate could be an economically feasible and low-cost alternative source of L-tyrosine for the production of melanin.</p>","PeriodicalId":34490,"journal":{"name":"BioTech","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10046677/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9209306","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Thermostable Lipase Isolated from <i>Brevibacillus thermoruber</i> Strain 7 Degrades Ɛ-Polycaprolactone.","authors":"Nikolina Atanasova,&nbsp;Tsvetelina Paunova-Krasteva,&nbsp;Margarita Kambourova,&nbsp;Ivanka Boyadzhieva","doi":"10.3390/biotech12010023","DOIUrl":"https://doi.org/10.3390/biotech12010023","url":null,"abstract":"<p><p>The tremendous problem with plastic waste accumulation has determined an interest in biodegradation by effective degraders and their enzymes, such as thermophilic enzymes, which are characterized by high catalytic rates, thermostability, and optimum temperatures close to the melting points of some plastics. In the present work, we report on the ability of a thermophilic lipase, by <i>Brevibacillus thermoruber</i> strain 7, to degrade Ɛ-polycaprolactone (PCL), as well as the enzyme purification, the characterization of its physicochemical properties, the product degradation, and its disruptive effect on the PCL surface. The pure enzyme showed the highest reported optimum temperature at 55 °C and a pH of 7.5, while its half-life at 60 °C was more than five hours. Its substrate specificity referred the enzyme to the subgroup of lipases in the esterase group. A strong inhibitory effect was observed by detergents, inhibitors, and Fe³⁺ while Ca²⁺ enhanced its activity. The monomer Ɛ-caprolactone was a main product of the enzyme degradation. Similar elution profiles of the products received after treatment with ultra-concentrate and pure enzyme were observed. The significant changes in PCL appearance comprising the formation of shallower or deeper in-folds were observed after a week of incubation. The valuable enzyme properties of the lipase from <i>Brevibacillus thermoruber</i> strain 7, which caused a comparatively quick degradation of PCL, suggests further possible exploration of the enzyme for effective and environment-friendly degradation of PCL wastes in the area of thermal basins, or in thermophilic remediation processes.</p>","PeriodicalId":34490,"journal":{"name":"BioTech","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-03-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10046884/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9202484","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Large-Scale Production of Isocitric Acid Using <i>Yarrowia lipolytica</i> Yeast with Further Down-Stream Purification.","authors":"Svetlana V Kamzolova,&nbsp;Vladimir A Samoilenko,&nbsp;Julia N Lunina,&nbsp;Igor G Morgunov","doi":"10.3390/biotech12010022","DOIUrl":"https://doi.org/10.3390/biotech12010022","url":null,"abstract":"<p><p>Isocitric acid (ICA) refers to a group of promising regulators of energy metabolism which has antistress, antihypoxic, and antioxidant activities. In this paper, we reported a process of ICA production from rapeseed oil using yeast <i>Yarrowia lipolytica</i> VKM Y-2373 in a 500-L fermentor. The producer synthesized 64.1 g/L ICA with a product yield of 0.72 g/g and a productivity 0.54 g/L·h. We also developed an effective purification method, including a cell separation, clarification, concentration, acidification, and crystallization process, which resulted in the formation of the crystals of monopotassium salt of ICA with a purity of 99.0-99.9%. To the best of our knowledge, this is the first report on an ICA production process at an upscaled bioreactor level.</p>","PeriodicalId":34490,"journal":{"name":"BioTech","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10046092/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9202481","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}