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The induction of viable but nonculturable (VBNC) bacteria with cellular integrity and low metabolic activity by chemical disinfection causes a significant underestimation of potential microbiological risks in drinking water. Herein, a physical Co3O4 nanowire-assisted electroporation (NW-EP) was developed to induce cell damage via the locally enhanced electric field over nanowire tips, potentially achieving effective inhibition of VBNC cells as compared with chemical chlorination (Cl2). NW-EP enabled over 5-log removal of culturable cell for various G+/G- bacteria under voltage of 1.0 V and hydraulic retention time of 180 s, and with ∼3-6 times lower energy consumption than Cl2. NW-EP also achieved much higher removals (∼84.6 % and 89.5 %) of viable Bacillus cereus (G+) and Acinetobacter schindleri (G-) via generating unrecoverable pores on cell wall and reversible/irreversible pores on cell membrane than Cl2 (∼28.6 % and 41.1 %) with insignificant cell damage. The residual VBNC bacteria with cell wall damage and membrane pore resealing exhibited gradual inactivation by osmotic stress, leading to ∼99.8 % cell inactivation after 24 h storage (∼59.4 % for Cl2). Characterizations of cell membrane integrity and cell morphology revealed that osmotic stress promoted cell membrane damage for the gradual inactivation of VBNC cells during storage. The excellent adaptability of NW-EP for controlling VBNC cells in DI, tap and lake waters suggested its promising application potentials for drinking water, such as design of an external device on household taps.

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Despite the diligent efforts of libraries, archives, and similar institutions to preserve cultural monuments, biodeterioration continues to pose a significant threat to these objects. One of the main sources of microorganisms responsible for the biodeterioration process is the presence of airborne microorganisms. Therefore, this research aims to monitor and compare outcomes of both culture-dependent (utilising various cultivation strategies) and culture-independent approaches (RNA-based sequencing) to identifying metabolically active airborne microorganisms in archives in the Czech Republic. Through this study, several species that have the potential to pose risks to both cultural heritage objects and the health of institution employees were found. Additionally, the efficacy of different cultivation media was demonstrated to be varied across archive rooms, highlighting the necessity of employing multiple cultivation media for comprehensive analyses. Of noteworthy importance, the resuscitating-promoting factor (Rpf) proved to be a pivotal tool, increasing bacterial culturability by up to 30% when synergistically employed Reasoner's 2A agar (R2A) and R2A + Rpf media. Next, the study emphasises the importance of integrating both culture-dependent and culture-independent approaches. The overlap between genera identified by the culture-dependent approach and those identified also by the culture-independent approach varied from 33% to surpassing 94%, with the maximum alignment exceeding 94% in only one case. Our results highlight the importance of actively monitoring and assessing levels of microbial air contamination in archives to prevent further deterioration of cultural heritage objects and to promote improved conditions for employees in archives and similar institutions.

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Compared to japonica, the lower genetic transformation efficiency of indica is a technical bottleneck for rice molecular breeding. Specifically, callus browning frequently occurs during the culture of the elite indica variety 93-11, leading to poor culturability and lower genetic transformation efficiency. Here, 67 QTLs related to culturability were detected using 97 introgression lines (designated as 9DILs) derived from Dongxiang common wild rice (DXCWR, Oryza rufipogon Griff.) with 93-11 genetic background, explaining 4% ~12% of the phenotypic variations. The QTL qCBT9 on chromosome 9 was a primary QTL for reducing callus browning derived from DXCWR. Five 9DILs with light callus browning and high differentiation were screened. We evaluated the callus browning index (CBI) of 100 F2 population crossed of 93-11 and 9DIL71 and the recombinant plants screened from 3270 individuals. The qCBT9 was delimited to a ~148kb region between the markers X16 and X23. RNA-seq analysis of DEGs between 9DIL71 and 93-11 showed three upregulated DEGs (Os09g0526500, Os09g0527900, Os09g0528200,) and three downregulated DEGs (Os09g0526700, Os09g0526800, Os09g0527700) were located in the candidate region of qCBT9. Furthermore, callus browning may be involved in cell senescence and death caused by oxidative stress. The differentiation of indica and japonica in this region suggested that qCBT9 was possibly a vital QTL contributed to better culturability of japonica. Our results laid a foundation for further cloning of the gene for reduced callus browning in O. rufipogon, and also provided a new genetic resource and material basis for improving the culturability and genetic transformation efficiency of cultivated rice. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01470-z.

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Callus browning during tissue culture of indica rice is genotype dependent, thus limiting the application of genetic transformation for editing-assisted breeding and elucidation of gene function. Here, using 124 introgression lines (HCLs) derived from a cross between the indica rice 9311 and Chaling common wild rice and 2059 SNPs for single-point and interval analysis, we identified two major QTLs, qCBT7 on chromosome 7 and qCBT10 on chromosome 10, related to callus browning, explaining 8-13% of callus browning. Moreover, we performed RNA-seq of two introgression lines with low callus browning, HCL183 and HCL232, with Oryza. rufipogon introgression fragments on chromosomes 10 and 7, respectively. Three candidate genes (Os07g0620700, Os10g0361000, and Os10g0456800) with upregulation were identified by combining interval mapping and weighted gene coexpression network analysis using the DEGs. The qRT-PCR results of the three candidate genes were consistent with those of RNA-seq. The differentiation of indica and japonica subspecies Oryza. sativa and Oryza. rufipogon suggests that these candidate genes are possibly unique in Oryza. rufipogon. GO analyses of hub genes revealed that callus browning may be mainly associated with ethylene and hormone signaling pathways. The results lay a foundation for future cloning of qCBT7 or qCBT10 and will improve genetic transformation efficiency in rice.

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Mycobacterium abscessus has recently emerged as the cause of an increasing number of human infections worldwide. Unfortunately, it is highly resistant to existing drugs, and new specific agents to combat M. abscessus have not yet been found. The discovery of antibiotics that are effective not only against replicating but also against dormant and often recalcitrant cells is a daunting challenge. In this study, we developed a model of non-replicating M. abscessus, which represents a valuable screening tool for antibacterial agents. Thus, we demonstrated that, under a deficiency of potassium ions in the growth media and prolonged incubation, M. abscessus entered a 'non-culturable' state with a significant loss of colony-forming ability, but it retained viability, as confirmed using the most-probable-number (MPN) assay. The 'non-culturable' mycobacteria possessed decelerated cellular metabolism and noticeable differences in cell morphology from actively growing mycobacteria. 'Non-culturable' cells were used in a comprehensive screening of the efficacy of antibiotics, along with actively growing cells. Both CFU and MPN tests confirmed the prominent bactericidal effect of moxifloxacin on actively growing and 'non-culturable' M. abscessus, as proven by less than 0.01% of cells surviving after antibiotic treatment and prolonged storage. Bedaquiline exhibited a comparable bactericidal effect only on metabolically inactive non-culturable cells aged for 44 days. There were reductions ranging from 1000 to 10,000-fold in CFU and MPN, but it was not so efficient with respect to active cells, resulting in a bacteriostatic effect. The demonstrated specificity of bedaquiline in relation to inert non-replicating M. abscessus offers a new and unexpected result. Based on the findings of this research, moxifloxacin and bedaquiline can be regarded as potential treatments for infections caused by M. abscessus. In addition, a key outcome is the proposal to include the combination of viability assays for comprehensive testing of drug candidates. Relying on CFU-based assays alone resulted in overestimates of antibacterial efficacy, as demonstrated in our experiments.

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IMPORTANCE: Microorganisms are a repository of interesting metabolites and functions. Therefore, accessing them is an important exercise for advancing not only basic questions about their physiology but also to advance technological applications. In this sense, increasing the culturability of environmental microorganisms remains an important endeavor for modern microbiology. Because microorganisms do not live in isolation in their environments, molecules can be added to the cultivation strategies to "inform them" that they are present in growth-permissive environmental conditions. Signaling molecules such as acyl-homoserine lactones and 3',5'-cyclic adenosine monophosphate belong to the plethora of molecules used by bacteria to communicate with each other in a phenomenon called quorum sensing. Therefore, including quorum sensing molecules can be an incentive for microorganisms, specifically soil bacteria, to increase their numbers on solid media.

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Fecal microbiota transplantation (FMT) is under investigation for several indications, including ulcerative colitis (UC). The clinical success of FMT depends partly on the engraftment of viable bacteria. Because the vast majority of human gut microbiota consists of anaerobes, the currently used aerobic processing protocols of donor stool may diminish the bacterial viability of transplanted material. This study assessed the effect of four processing techniques for donor stool (i.e., anaerobic and aerobic, both direct processing and after temporary cool storage) on bacterial viability. By combining anaerobic culturing on customized media for anaerobes with 16S rRNA sequencing, we could successfully culture and identify the majority of the bacteria present in raw fecal suspensions. We show that direct anaerobic processing of donor stool is superior to aerobic processing conditions for preserving the bacterial viability of obligate anaerobes and butyrate-producing bacteria related to the clinical response to FMT in ulcerative colitis patients, including Faecalibacterium, Eubacterium hallii, and Blautia. The effect of oxygen exposure during stool processing decreased when the samples were stored long-term. Our results confirm the importance of sample conditioning to preserve the bacterial viability of oxygen-sensitive gut bacteria. Anaerobic processing of donor stool may lead to increased clinical success of FMT, which should further be investigated in clinical trials.

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Isolation of microorganisms is a useful approach to gathering knowledge about their genomic properties, physiology, and ecology, in addition to allowing the characterization of novel taxa. We performed an extensive isolation effort on samples from seawater manipulation experiments that were carried out during the four astronomical seasons in a coastal site of the northwest Mediterranean to evaluate the impact of grazing, viral mortality, resource competition reduction, and light presence/absence on bacterioplankton growth. Isolates were retrieved using two growth media, and their full 16S rRNA gene was sequenced to assess their identity and calculate their culturability across seasons and experimental conditions. A total of 1,643 isolates were obtained, mainly affiliated to the classes Gammaproteobacteria (44%), Alphaproteobacteria (26%), and Bacteroidia (17%). Isolates pertaining to class Gammaproteobacteria were the most abundant in all experiments, while Bacteroidia were preferentially enriched in the treatments with reduced grazing. Sixty-one isolates had a similarity below 97% to cultured taxa and are thus putatively novel. Comparison of isolate sequences with 16S rRNA gene amplicon sequences from the same samples showed that the percentage of reads corresponding to isolates was 21.4% within the whole data set, with dramatic increases in the summer virus-reduced (71%) and diluted (47%) treatments. In fact, we were able to isolate the top 10 abundant taxa in several experiments and from the whole data set. We also show that top-down and bottom-up controls differentially affect taxa in terms of culturability. Our results indicate that culturing marine bacteria using agar plates can be successful in certain ecological situations. IMPORTANCE Bottom-up and top-down controls greatly influence marine microbial community composition and dynamics, which in turn have effects on their culturability. We isolated a high amount of heterotrophic bacterial strains from experiments where seawater environmental conditions had been manipulated and found that decreasing grazing and viral pressure as well as rising nutrient availability are key factors increasing the success in culturing marine bacteria. Our data hint at factors influencing culturability and underpin bacterial cultures as a powerful way to discover new taxa.

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Podaxis pistillaris is neutraceutically, cosmoceutically and medicinally recognised macrofungus. During this research work, this edible mushroom was systematically characterised. Its culturability, laccase production, and dye decolorisation potential were evaluated and optimised. Among the different media tested, PDA proved as most efficient medium for culturability of P. pistillaris. Conditions for laccase production were optimised in submerged state fermentation. Maximum laccase secretion was noted after 14th day of Incubation at 35 °C with 130 rpm and 5 pH of medium. Fructose and ammonium-phosphate was found as best carbon and nitrogen source, while wheat straw revealed as good ligno-cellulosic source for strengthening laccase production. Congo-red dye decolorisation capability by crude laccase enzyme was evaluated and found maximum decolorisation potential (92.2%) after 288h of incubation. From this pilot study, it was confirmed that this edible macrofungus has culturability, laccase production and dye decolorisation attributes that will further contribute in delignification, biosorption and bioremediation.

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The Podoscypha is a recognized therapeutically significant genus of mushrooms. A Podoscypha species under a Morus sp. Was found from the largest man-made forest Changa-Manga, Kasur during the exploration of fungal flora of Pakistan. Detailed morpho-anatomical and phylogenetic analysis identified it as P. petalodes, a part of common native flora of many regions of the world like Pakistan. The culturabilty and cultivation potential of this fungus was assessed for the first time using different media and substrates. Maximum cultural growth was observed on the Compost Extract Agar (CEA) medium at 28 °C. Mycelium of cultured strains on CEA medium was used for the spawn production on wheat, sorghum and barley grains. Cultivation potential in the form of spawn running period, harvesting time duration and yield was investigated on variety of substrates. A mixed substrate of sawdust and tea waste at 28 °C showed the optimum yield. Tea-waste was used as the casing material in all substrates and proved very effective. These results depicted that Podoscypha petalodes possesses the cultivation potential. Its cultivation on large scale can solve the major health concerns of the growing population. It would provide the people easy accessibility of economical, effective and natural medicine throughout the year that restrict in case of only natural production at specific time of the year.

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As a unique microbial response to adverse circumstances, the viable but nonculturable (VBNC) state is characterized by the loss of culturability of microbial cells on/in nutrient media that normally support their growth, while maintaining metabolic activity. These cells can resuscitate to a culturable state under suitable conditions. Given the intrinsic importance of the VBNC state and recent debates surrounding it, there is a need to redefine and standardize the term, and to address essential questions such as 'How to differentiate VBNC from other similar terms?' and 'How can VBNC cells be standardly and accurately determined?'. This opinion piece aims at contributing to an improved understanding of the VBNC state and promoting its proper handling as an underestimated and controversial microbial survival strategy.

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Over the last 30 years, the description of microbial diversity has been mainly based on culture-independent approaches (metabarcoding and metagenomics) allowing an in-depth analysis of microbial diversity that no other approach allows. Bearing in mind that culture-dependent approaches cannot replace culture-independent approaches, we have improved an original method for isolating strains consisting of "culturing" grains of sand directly on Petri dishes (grain-by-grain method). This method allowed to cultivate up to 10% of the bacteria counted on the surface of grains of the three sites studied in the Great Western Erg in Algeria (Timoudi, Béni Abbès, and Taghit), knowing that on average about 10 bacterial cells colonize each grain. The diversity of culturable bacteria (collection of 290 strains) predicted by 16S rRNA gene sequencing revealed that Arthrobacter subterraneus, Arthrobacter tecti, Pseudarthrobacter phenanthrenivorans, Pseudarthrobacter psychrotolerans, and Massilia agri are the dominant species. The comparison of the culture-dependent and -independent (16S rRNA gene metabarcoding) approaches at the Timoudi site revealed 18 bacterial genera common to both approaches with a relative overestimation of the genera Arthrobacter/Pseudarthrobacter and Kocuria, and a relative underestimation of the genera Blastococcus and Domibacillus by the bacterial culturing approach. The bacterial isolates will allow further study on the mechanisms of tolerance to desiccation, especially in Pseudomonadota (Proteobacteria).

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Both latent and active TB infections are caused by a heterogeneous population of mycobacteria, which includes actively replicating and dormant bacilli in different proportions. Dormancy substantially affects M. tuberculosis drug tolerance and TB clinical management due to a significant decrease in the metabolic activity of bacilli, which leads to the complexity of both the diagnosis and the eradication of bacilli. Most diagnostic approaches to latent infection deal with a subpopulation of active M. tuberculosis, underestimating the contribution of dormant bacilli and leading to limited success in the fight against latent TB. Moreover, active TB appears not only as a primary form of infection but can also develop from latent TB, when resuscitation from dormancy is followed by bacterial multiplication, leading to disease progression. To win against latent infection, the identification of the Achilles' heel of dormant M. tuberculosis is urgently needed. Regulatory mechanisms and metabolic adaptation to growth arrest should be studied using in vitro and in vivo models that adequately imitate latent TB infection in macroorganisms. Understanding the mechanisms underlying M. tuberculosis dormancy and resuscitation may provide clues to help control latent infection, reduce disease severity in patients, and prevent pathogen transmission in the population.

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Viability assessment is a critical step in evaluating bacterial pathogens to determine infectious risks to public health. Based on three accepted viable criteria (culturability, metabolic activity, and membrane integrity), current viability assessments are categorized into three main strategies. The first strategy relies on the culturability of bacteria. The major limitation of this strategy is that it cannot detect viable but nonculturable (VBNC) bacteria. As the second strategy, based on the metabolic activity of bacteria, VBNC bacteria can be detected. However, VBNC bacteria sometimes can enter a dormant state that allows them to silence reproduction and metabolism; therefore, they cannot be detected based on culturability and metabolic activity. In order to overcome this drawback, viability assessments based on membrane integrity (third strategy) have been developed. However, these techniques generally require multiple steps, bulky machines, and laboratory technicians to conduct the tests, making them less attractive and popular applications. With significant advances in microfluidic technology, these limitations of current technologies for viability assessment can be improved. This review summarized and discussed the advances, challenges, and future perspectives of current methods for the viability assessment of bacterial pathogens.

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The effect of oxygen on the germination and culturability of aerobic Bacillus atrophaeus spores was investigated in this study. Under oxic or anoxic conditions, various nutritional and non-nutritional germinants were utilized to induce germination. Tb3+-dipicolinic acid fluorescence assay and phase-contrast microscopy were used to track the germination process. The final germination level, germination half time, and germination speed were used to define germination kinetics. Colony-forming unit enumeration was used to assess the culturability of germinated spores germinated with or without oxygen. The results show that in the absence of oxygen, the final germination level was unaffected, germination half time decreased by up to 35.0%, germination speed increased by up to 27.4%, and culturability decreased by up to 95.1%. It is suggested that oxygen affects some germinant receptor-dependent germination pathways, implying that biomolecules engaged in these pathways may be oxygen-sensitive. Furthermore, spores that have completed the germination process in either anoxic or oxic conditions may have different culturability. This research contributed to a better understanding of the fundamental mechanism of germination.(AU)

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[This corrects the article DOI: 10.3389/fmicb.2021.784796.].

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Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant opportunistic pathogen with a great ability to form biofilms. Herein, the antimicrobial potential of Thymbra capitata essential oil (EO) against MRSA biofilms was investigated. The determination of the minimum inhibitory concentration (MIC) and the minimum lethal concentration (MLC) of the T. capitata EO was first investigated on a group of clinical isolates from septicaemias, diabetic foot ulcers and osteomyelitis. Biofilms were incubated with the EO at the MLC and its anti-biofilm potential was investigated. A strong antimicrobial activity was observed, with MIC and MLC values between 0·32 and 0·64 mg l-1 . However, the concentration of EO necessary for the eradication of planktonic cells was insufficient to significantly reduce the biofilm biomass of some isolates. Nevertheless, cell culturability and overall cellular metabolism was strongly reduced in all biofilms tested, only when the EO was tested. Contrary to the tested antibiotics, T. capitata EO showed a significant antimicrobial activity against MRSA biofilms, by reducing cellular metabolism and cellular culturability.

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The effect of oxygen on the germination and culturability of aerobic Bacillus atrophaeus spores was investigated in this study. Under oxic or anoxic conditions, various nutritional and non-nutritional germinants were utilized to induce germination. Tb3+-dipicolinic acid fluorescence assay and phase-contrast microscopy were used to track the germination process. The final germination level, germination half time, and germination speed were used to define germination kinetics. Colony-forming unit enumeration was used to assess the culturability of germinated spores germinated with or without oxygen. The results show that in the absence of oxygen, the final germination level was unaffected, germination half time decreased by up to 35.0%, germination speed increased by up to 27.4%, and culturability decreased by up to 95.1%. It is suggested that oxygen affects some germinant receptor-dependent germination pathways, implying that biomolecules engaged in these pathways may be oxygen-sensitive. Furthermore, spores that have completed the germination process in either anoxic or oxic conditions may have different culturability. This research contributed to a better understanding of the fundamental mechanism of germination.

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The contamination of fungi in water supply systems poses great risks to environment and human health. In this work, UV light-emitting diodes (UV-LEDs)-based advanced disinfection processes (ADPs) including UV-LEDs/hydrogen peroxide (H2O2), UV-LEDs/persulfate (PS) and UV-LEDs/peroxymonosulfate (PMS), were adopted for waterborne fungal spores inactivation. Overall comparisons of the UV-LEDs-based ADPs with respect to the control efficiency of photoreactivation and energy consumption were also evaluated. Results showed that culturability reduction of the fungal spores treated by UV-LEDs was not enhanced with the addition H2O2, PMS, and PS according to the results of heterotrophic plate counts and reaction rate constants; A. niger was expected to have higher UV resistance followed by T. harzianum and P. polonicum. However, UV-LEDs-ADPs inactivation, especially at the wavelengths of 280 and 265/280 nm, could accelerate the permeabilization of fungal spores as characterized by flow cytometry. Take P. polonicum for example, the percentage of membrane permeabilized spores was 98.0%, 98.7%, 97.6% and 82.6% after treatment by UV280/H2O2, UV280/PS, UV280/PMS and UV280 alone, respectively at the fluence of 100 mJ/cm2. The direct attack of free radicals in the processes of UV-LEDs-ADPs further enhanced the membrane damage and lowered the photoreactivation level, thus improved the inactivation efficiency. UV-LEDs/H2O2 was considered as an effective process in the disinfection of fungal spores with the advantages of enhancing the damage of membrane, inhibiting photoreactivation and comparable energy consumption compared with UV-LEDs alone.

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In this work, sequential applications of light-emitting diodes (UV-LEDs) with two wavelengths and chlorine (Cl2) were performed for fungal spores disinfection: UV-Cl2, Cl2-UV, UV/Cl2-UV, UV-UV/Cl2, Cl2-UV/Cl2-Cl2. Overall comparisons of the sequential processes with respect to the inhibitory effect on photoreactivation were also evaluated. According to the evaluation of culturability and membrane permeability, inactivation of fungal spores by UV was not enhanced by prior or post exposure to Cl2, but in the UV/Cl2 process with pre or post UV treatment, the inactivation efficiency was greatly enhanced. Take P. polonicum for example, pre-treatments by UV265 and UV280 (40 mJ/cm2) caused the log count reduction (LCR) of 1.05 log and 0.95 log, then the followed UV265/Cl2 and UV280/Cl2 at the same UV fluence caused additional LCR of 1.80 log and 2.00 log. The permeabilization of P. polonicum was also accelerated in the processes of UV/Cl2-UV and UV-UV/Cl2, especially at the wavelength of 280 nm. In the sequential processes, especially those containing UV/Cl2 or at the wavelength of 280 nm, could promote the formation of intracellular reactive oxygen species (ROS), thus leading to more severe damage to the spores as reflected in the culturability reduction, membrane permeability and inhibition of photoreactivation.

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