Single-cell transcriptomic analysis identifies an immune-prone population in erythroid precursors during human ontogenesis.

Nonimmune cells can have immunomodulatory roles that contribute to healthy development. However, the molecular and cellular mechanisms underlying the immunomodulatory functions of erythroid cells during human ontogenesis remain elusive. Here, integrated, single-cell transcriptomic studies of erythroid cells from the human yolk sac, fetal liver, preterm umbilical cord blood (UCB), term UCB and adult bone marrow (BM) identified classical and immune subsets of erythroid precursors with divergent differentiation trajectories. Immune-erythroid cells were present from the yolk sac to the adult BM throughout human ontogenesis but failed to be generated in vitro from human embryonic stem cells. Compared with classical-erythroid precursors, these immune-erythroid cells possessed dual erythroid and immune regulatory networks, showed immunomodulatory functions and interacted more frequently with various innate and adaptive immune cells. Our findings provide important insights into the nature of immune-erythroid cells and their roles during development and diseases.

Efficacy of venetoclax combined with decitabine conditioning regimen for allogeneic hematopoietic stem cell transplantation in high-risk and elderly patients with myeloid neoplasms.

This prospective clinical investigation focused on the addition of venetoclax and decitabine to myeloablative conditioning regimens, targeting high-risk and elderly individuals undergoing allogeneic hematopoietic stem cell transplantation. In total, 19 patients were enrolled in the trial between December 2021 and February 2023, and their progress was monitored for a median follow-up period of 258 days, ranging from 35 to 544 days. In the initial regimen (n=11), venetoclax was administered at a dosage of 400 mg per day from day -14 to day -1, while in the modified regimen (n=8), it was administered from day -14 to day -5. Decitabine was orally administered at a dosage of 20mg/m2/day from day -7 to day -3. Grade 3/4 adverse events observed included hematological events, hypertension, infections, allergy, and increased amylase. In the entire cohort, the overall survival (OS) and relapse-free survival (RFS) rates at 6 months were 63% (95% CI, 45-89) and 63% (95% CI, 45-89), respectively. The non-relapse mortality (NRM) rate at 6 months was 37% (95% CI, 16-58), while the cumulative incidence of relapse (CIR) was 0. However, the incidence of grade II-IV acute graft-versus-host disease (aGVHD) and grade III-IV aGVHD within 100 days was found to be 31% (95% CI, 12-53) and 26% (95% CI, 9-47), respectively. These rates indicate a relatively high occurrence, making it less suitable to administer the regimen to elderly patients. Therefore, further high-quality studies are required to enhance the conditioning regimen specifically for high-risk and elderly patients diagnosed with myeloid neoplasms. Clinical trial registration: ChiCTR2100050272.

Heterologous expression and characterization of Bacillus velezensis SW5 serine protease involved in the hydrolysis of anchovy protein.

BACKGROUND: Bacillus velezensis SW5, with good enzyme production ability, was isolated and identified in our laboratory from fermented fish sauce. Its galactosidase has been expressed in Escherichia coli, which could hydrolyze lactose in milk. The present study aims to express a novel serine protease gene (SPr-SW5) of this strain by Bacillus subtilis WB800N, and applies the expressed enzyme in hydrolysis of anchovy to prepare antioxidant substances, aiming to alleviate the waste of low-value fish resources. RESULTS: SPr-SW5 with the open reading frame of 1353 bp encodes a serine protease (SPr-SW5) with 450 amino acids. The theoretical molecular weight and isoelectric point are 47.2 kDa and 5.22, respectively. The successful expression of SPr-SW5 in B. subtilis WB800N was confirmed by a skim milk plate test. Its optimal temperature and pH were 50 °C and 8.0, respectively. SPr-SW5 activity was increased by Ca2+ and Zn2+ , but inhibited by Fe3+ . Furthermore, SPr-SW5 was tolerant to 1% Tween-40 and Tween-80; however, its activity was strongly inhibited by 10 mm phenylmethylsulfonyl fluoride. Additionally, SPr-SW5 could be capable of hydrolyzing anchovy, the hydrolysate (AHP) at 10 g L-1 , with 2,2-diphenyl-1-picrylhydrazyl and hydroxyl (·OH) scavenging rates of 73.21% and 79.71%, displaying good antioxidant activity. CONCLUSION: The novel SPr-SW5 was successfully expressed in B. subtilis WB800N. It exhibited excellent temperature stability and good tolerance to several metal ions. In addition, the anchovy hydrolyzed by expressed SPr-SW5 has good antioxidant ability. Overall, this research lays a good foundation for SPr-SW5 with respect to exploration and application in the food industry as enzyme preparation. © 2023 Society of Chemical Industry.

Peptide hydrogels: Synthesis, properties, and applications in food science.

Due to the unique and excellent biological, physical, and chemical properties of peptide hydrogels, their application in the biomedical field is extremely wide. The applications of peptide hydrogels are closely related to their unique responsiveness and excellent properties. However, its defects in mechanical properties, stability, and toxicity limit its application in the food field. In this review, we focus on the fabrication methods of peptide hydrogels through the physical, chemical, and biological stimulations. In addition, the functional design of peptide hydrogels by the incorporation with materials is discussed. Meanwhile, the excellent properties of peptide hydrogels such as the stimulus responsiveness, biocompatibility, antimicrobial properties, rheology, and stability are reviewed. Finally, the application of peptide hydrogel in the food field is summarized and prospected.

Ruania suaedae sp. nov. and Ruania halotolerans sp. nov., two actinobacteria isolated from saline soil, and reclassification of Haloactinobacterium kanbiaonis as Occultella kanbiaonis comb. nov.

Two Gram-stain-positive, non-motile, strictly aerobic, yellow-coloured, rod-shaped bacterial strains, designated LR1S40T and M4N3S171T, were isolated from rhizosphere and bulk saline soil of Suaeda salsa collected in Inner Mongolia, China. Phylogenetic trees based on 16S rRNA gene and whole genome sequences showed that the two strains clustered tightly with strains of the genus Ruania. Strains LR1S40T and M4N3S171T had 95.5% 16S rRNA gene similarity to each other, and strain LR1S40T had 98.8, 98.7, 97.4 and <97.0% similarity to Ruania alkalisoli RN3S43T, Ruania rhizosphaerae LNNU 22110T, Ruania alba YIM 93306T and all other current type strains, while strain M4N3S171T had 98.6 and <97.0% similarity to R. alba YIM 93306T, and all other current type strains, respectively. The average nucleotide identity based on blast (ANIb) and digital DNA-DNA hybridization (dDDH) values of LR1S40T and M4N3S171T with each other and to the other type strains of Ruania were well below the threshold values (95% for ANIb, 70% for dDDH) for differentiating a species. Diphosphatidylglycerol and phosphatidylglycerol were the major polar lipids in both strains. The predominant menaquinone in both strains was both MK-8. The genome of strain LR1S40T consisted of a 3557440 bp circular chromosome, with a G+C content of 71.1 mol%, while the genome of strain M4N3S171T consisted of 4270413 bp, with a G+C content of 67.6 mol%. The phylogenetic, physiological and phenotypic characteristics allowed discrimination of the two strains from their relatives. The names Ruania suaedae sp. nov. [type strain LR1S40T (=CGMCC 1.19028T=KCTC 49726T)] and Ruania halotolerans sp. nov. [type strain M4N3S171T (=CGMCC 1. 19142T=KCTC 49727T)] are therefore proposed. During the publication of Haloactinobacterium kanbiaonis, Haloactinobacterium glacieicola (type strain T3246-1T), which was selected as the reference strain for the identification of H. kanbiaonis, was reclassified as Occultella glacieicola. The two phylogenetic trees showed that H. kanbiaonis HY164T tightly clustered with Occultella aeris F300T, and had the highest 16S rRNA gene similarity (99.8%) to O. aeris F300T. Based on the phylogenetic analysis and the publication record, Haloactinobacterium kanbiaonis should be reclassified as Occultella kanbiaonis comb. nov.

Description and characterization of three endophytic Bacillaceae from the halophyte Suaeda salsa: Paenalkalicoccus suaedae gen. nov., sp. nov., Cytobacillus suaedae sp. nov., and Bacillus suaedae sp. nov.

Three strains of members of the family Bacillaceae, which can inhibit the growth of some Gram-stain-positive strains, designated M4U3P1T, HD4P25T and RD4P76T, were isolated from Suaeda salsa halophytes in Baotou, Inner Mongolia, PR China. A phylogenetic analysis based on the 16S rRNA gene and the whole genome sequences revealed that HD4P25T clustered with Cytobacillus luteolus YIM 93174T with a similarity of 98.4 %, and RD4P76T shared the highest similarity of 16S rRNA gene with Bacillus mesophilus SA4T (97.5 %). M4U3P1T clustered with strains of genera Salipaludibacillus and Alkalicoccus based on whole-genome sequence analyses, but its 16S rRNA gene had the highest similarity to 'Evansella tamaricis' EGI 80668 (96.1 %). The average nucleotide's identity by blast (ANIb) and digital DNA-DNA hybridization (dDDH) values of the three isolated strains to their close relatives were well below the threshold value for identifying a novel species.On the basis of the phylogenetic, physiological and phenotypic results, Paenalkalicoccus suaedae gen. nov., sp. nov. [type strain M4U3P1T (=CGMCC 1.17076T=JCM 33851T)], Cytobacillus suaedae sp. nov. [type strain HD4P25T (=CGMCC 1.18651T =JCM 34524T)], and Bacillus suaedae sp. nov. [type strain RD4P76T (=CGMCC 1.18659T=JCM 34525T)] were proposed, respectively. All three species are ubiquitous in the bulk saline-alkaline soils, but only the species represented by strain RD4P76T was widely distributed in the rhizosphere soil, the above-ground part and the roots of S. salsa. The species represented by M4U3P1T can be detected in the roots of S. salsa, and rarely detected in the above-ground parts of S. salsa. The species represented by HD4P25T was rarely detected in the interior of S. salsa. The three strains could inhibit some of the Gram-stain-positive bacteria (i.e. members of the genera Planococcus, Zhihengliuella and Sanguibacter) in the saline-alkali soil. A genomic analysis of these three strains revealed that they can synthesize different antagonistic compounds, such as aminobenzoate and bacitracin or subtilisin.

Potassium Phosphite Enhances the Antagonistic Capability of Bacillus amyloliquefaciens to Manage Tomato Bacterial Wilt.

Bacterial wilt caused by Ralstonia solanacearum is a distributed and worldwide soilborne disease. The application of biocontrol microbes or agricultural chemicals has been widely used to manage tomato bacterial wilt. However, whether and how agricultural chemicals affect the antagonistic ability of biocontrol microbes is still unknown. Here, we combined potassium phosphite (K-Phite), an environmentally friendly agricultural chemical, and the biocontrol agent Bacillus amyloliquefaciens QPF8 (strain F8) to manage tomato bacterial wilt disease. First, K-Phite at a concentration of 0.05% (wt/vol) could significantly inhibit the growth of R. solanacearum. Second, 0.05% K-Phite enhanced the antagonistic capability of B. amyloliquefaciens F8. Third, the greenhouse soil experiments showed that the control efficiency for tomato bacterial wilt in the combined treatment was significantly higher than that of the application of B. amyloliquefaciens F8 or K-Phite alone. Overall, our results highlighted a novel strategy for the control of tomato bacterial wilt disease via application and revealed a new integrated pattern depending on the enhancement of the antagonistic capability of biocontrol microbes by K-Phite.

Site-Specific Quantitation of Drug Conjugations on Antibody-Drug Conjugates (ADCs) Using a Protease-Assisted Drug Deconjugation and Linker-like Labeling (PADDLL) Method.

Antibody-drug conjugates (ADCs) are biopharmaceuticals for the targeted delivery of antitumor agents. ADCs can be highly heterogeneous with various drug-to-antibody ratio (DAR) species, conjugation sites, and occupancy levels. The conjugation site can modulate the ADC stability and efficacy and therefore can be considered to be a critical quality attribute (CQA) during development. Traditional mass spectrometry (MS)-based peptide mapping methods cannot accurately quantify site-specific conjugations due to a significant ionization discrepancy between unconjugated native peptides and conjugated peptides. Here, we developed a novel protease-assisted drug deconjugation and linker-like labeling (PADDLL) method to quantify the levels of linker payload at specific conjugation sites. We utilized optimized papain digestion to deconjugate the drug payload and labeled unoccupied conjugation sites with a linker-like structure to provide comparable ionization efficiency for MS-based quantitation. This method was successfully applied on two cysteine-linked, protease-cleavable ADCs, and the method demonstrated good linearity and reliability, reaching a limit of quantitation of below 1%. The calculated DARs were comparable with the results from intact mass analysis. The lot-to-lot variation in conjugation distribution and inferior conjugation stability at HC Cys225 to other interchain cysteines were observed. This method provides a valuable tool for ADC design and product development. To the best of our knowledge, this is the first analytical method developed to accurately quantify site-specific linker-drug payload conjugations for ADCs.

Soil fungal community affected by regional climate played an important role in the decomposition of organic compost.

A large amount of organic compost, produced with agricultural and breeding industry wastes by composting, is widely used in agriculture in China. The microbial decomposition of organic compost is a major flux in the nutrition cycle in sustainable agricultural soils. To explore the mechanism of organic compost mineralization in soil, in situ decomposition experiments of organic compost buried in soils were arranged in three different latitude regions located in Jilin, Jiangsu, and Yunnan in China. The results showed that organic compost had different decomposition rates at the three different sites, with the highest decomposition rate in Yunnan, followed by Jiangsu and Jilin. The decomposition rates of unsterilized organic compost were significantly greater than those of sterilized organic compost, indicating that the microorganisms in organic compost also made important contributions to the decomposition process. The soil microbial diversity and community structure among the three sites were significantly different. The fungal community, especially fungal richness, rather than the bacterial community in the soil, plays a major role in the decomposition of organic compost. The annual average temperature is an important environmental factor affecting fungal richness. This study will provide a reference for formulating agricultural fertilization models in different regions.

Dual-Sensitive Graphene Oxide Loaded with Proapoptotic Peptides and Anticancer Drugs for Cancer Synergetic Therapy.

A dual-sensitive drug delivery system (DDS) based on graphene oxide (GO) which is simultaneously loaded with proapoptotic peptides and anticancer drugs was rationally designed and fabricated for cancer synergetic therapy. Specifically, a kind of cell apoptosis peptide (KLAKLAK)2 (KLA) was anchored on the surface of GO via a disulfide bond to obtain GO-SS-KLA. Then, the aromatic anticancer drug doxorubicin (DOX) was loaded on GO through π-π conjugation and hydrogen bonding interactions. Finally, bovine serum albumin (BSA) was used to coat the GO carrier to obtain a biological medium-stable GO-based DDS, DOX@GO-SS-KLA/BSA. The results show that KLA and DOX can be released responding to the reductive and pH stimulus inside the cells, respectively, and achieve a synergetic therapy for cancer. Moreover, the results of stability studies show that DOX@GO-SS-KLA/BSA could be stably dispersed in water for more than 8 days and in 10% fetal bovine serum for at least 6 days. The constructed DOX@GO-SS-KLA/BSA exhibits great potential as a drug carrier for co-delivery of various therapeutic agents.

Influence of straw incorporation with and without straw decomposer on soil bacterial community structure and function in a rice-wheat cropping system.

To study the influence of straw incorporation with and without straw decomposer on bacterial community structure and biological traits, a 3-year field experiments, including four treatments: control without fertilizer (CK), chemical fertilizer (NPK), chemical fertilizer plus 7500 kg ha-1 straw incorporation (NPKS), and chemical fertilizer plus 7500 kg ha-1 straw incorporation and 300 kg ha-1 straw decomposer (NPKSD), were performed in a rice-wheat cropping system in Changshu (CS) and Jintan (JT) city, respectively. Soil samples were taken right after wheat (June) and rice (October) harvest in both sites, respectively. The NPKS and NPKSD treatments consistently increased crop yields, cellulase activity, and bacterial abundance in both sampling times and sites. Moreover, the NPKS and NPKSD treatments altered soil bacterial community structure, particularly in the wheat harvest soils in both sites, separating from the CK and NPK treatments. In the rice harvest soils, both NPKS and NPKSD treatments had no considerable impacts on bacterial communities in CS, whereas the NPKSD treatment significantly shaped bacterial communities compared to the other treatments in JT. These practices also significantly shifted the bacterial composition of unique operational taxonomic units (OTUs) rather than shared OTUs. The relative abundances of copiotrophic bacteria (Proteobacteria, Betaproteobacteria, and Actinobacteria) were positively correlated with soil total N, available N, and available P. Taken together, these results indicate that application of straw incorporation with and without straw decomposer could particularly stimulate the copiotrophic bacteria, enhance the soil biological activity, and thus, contribute to the soil productivity and sustainability in agro-ecosystems.

Origin of the enhanced visible-light photocatalytic activity of CNT modified g-C3N4 for H2 production.

Graphitic carbon nitride (g-C3N4) hybridized with a small number of multi-walled carbon nanotubes (CNT) was synthesized using cyanamide as precursor. The optimal CNT content is found to be ∼0.2 wt% in the composite, which displays a 2.4-fold enhancement in photocatalytic water splitting over pure g-C3N4. Characterizations by a series of joint techniques including Raman spectra, UV/vis diffuse reflectance spectra, steady and time-resolved fluorescence emission spectra, and photocurrent responses were carried out, aiming to reveal the determinative factor for the improved visible-light response. Our results indicate that the increased photoactivity originates from the enhanced charge-transfer effect due to the intimate interactions between g-C3N4 and conjugated CNT. The presence of CNT in the hybrids is beneficial for improving electron-hole separation on the excited g-C3N4 by prolonging the lifetimes of charge carriers and improving the population distribution of short-lived and long-lived charge carriers.

Isolation of Bacillus amyloliquefaciens S20 and its application in control of eggplant bacterial wilt.

Bacterial strain S20 was isolated and identified as Bacillus amyloliquefaciens based on physiological and biochemical characteristics and a 16S rRNA gene sequence analysis. Strain S20 inhibits the growth of Fusarium oxysporum and Ralstonia solanacearum. Some genes associated with the synthesis of some lipopeptides were detected in strain S20 by PCR. Iturins A were identified as the main antagonistic substrates by analysis with electrospray ionization mass spectrometry/collision-induced dissociation (ESI-MS/CID). Four homologues of iturin A (C13-C16) were identified. Pot experiments showed that the application of strain S20 alone could control eggplant wilt with an efficacy of 25.3% during a 40 day experiment. If strain S20 was used with organic fertilizer, the control efficacy against eggplant wilt reached as high as 70.7%. The application of organic fertilizer alone promotes the growth of R. solanacearum, resulting in a higher wilt incidence than that observed in control plants. The application of strain S20 effectively inhibits R. solanacearum in the rhizosphere soil of eggplant. The combined use of strain S20 and organic fertilizer more effectively controlled R. solanacearum in soil than the use of strain S20 alone. The soil count of strain S20 decreased gradually during the course of the experiment after inoculation. Organic fertilizer was beneficial for the survival of the antagonistic bacterial strain S20; a higher level of these bacteria could be maintained. The application of organic fertilizer with strain S20 increased bacterial diversity in rhizosphere soil.

Variations of culturable thermophilic microbe numbers and bacterial communities during the thermophilic phase of composting.

Composting is a process of stabilizing organic wastes through the degradation of biodegradable components by microbial communities under controlled conditions. In the present study, genera and species diversities, amylohydrolysis, protein and cellulose degradation abilities of culturable bacteria in the thermophilic phase of composting of cattle manure with plant ash and rice bran were investigated. The number of culturable thermophilic bacteria and actinomyces decreased with the increasing temperature. At the initiation and end of the thermophilic phase, genera and specie diversities and number of bacteria possessing degradation abilities were higher than during the middle phase. During the thermophilic composting phase, Bacillus, Geobacillus and Ureibacillus were the dominant genera, and Geobacillus thermodenitrificans was the dominant species. In later thermophilic phases, Geobacillus toebii and Ureibacillus terrenus were dominant. Bacillus, at the initiation, and Ureibacillus and Geobacillus, at the later phase, contributed the multiple degradation abilities. These data will facilitate the control of composting in the future.

Halomonas rhizosphaerae sp. nov. and Halomonas kalidii sp. nov., two novel moderate halophilic phenolic acid-degrading species isolated from saline soil.

Four vanillic acid-degrading bacterial strains, named LR5S13T, LR5S20, and M4R5S39T and LN1S58, were isolated from Kalidium cuspidatum rhizosphere and bulk soils, respectively. Phylogenetic analysis based on 16S rRNA gene as well as core genome revealed that LR5S13T and LR5S20 clustered closely with each other and with Halomonas ventosae Al12T, and that the two strains shared the highest similarities (both 99.3 %) with H. ventosae Al12T, in contrast, M4R5S39T and LN1S58 clustered together and with Halomonas heilongjiangensis 9-2T, and the two strains shared the highest similarities (99.4 and 99.2 %, respectively) with H. heilongjiangensis 9-2T. The average nucleotides identities based on BLAST (ANIb) and digital DNA-DNA hybridization (dDDH) values of strains LR5S13T to LR5S20, and M4R5S39T to LN1S58, were both higher than the threshold values for delineation of a species. The ANIb and dDDH values of the four strains to their closely relatives were lower than the threshold values. All four strains take phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol as the major polar lipids, Summed Feature 8, Summed Feature 3, and C16:0 as the major fatty acids. Based on the phylogenetic and phenotypic results, the four strains should be classified as two novel Halomonas species. Therefore, Halomonas rhizosphaerae sp. nov. (type strain LR5S13T = KCTC 8016T = CGMCC 1.62049T) and Halomonas kalidii (type strain M4R5S39T = KCTC 8015T = CGMCC 1.62047T) are proposed. The geographical distribution analysis based on 16S rRNA gene revealed that the two novel species are widely distributed across the globe, specifically in highly saline habits, especially in Central and Eastern Asia.

Rapid high-yield N-acylation of aminothiols: N-acetylglutathione and N-acetylhomocysteine and their thiol pK(a) values.

Methodology for the rapid N-acylation of aminothiols in aqueous solution using procedures commonly employed in biochemical studies is described here. Glutathione disulfide (GSSG) and homocystine were diN-acetylated in ~100% yield in 0.1 M aqueous NaHCO3 (pH 8.5) at room temperature by 2.5 equiv of the activated ester, N-hydroxysulfosuccinimidyl acetate, an efficient water-soluble acetylating reagent. Following acetone precipitation, diN-acetylGSSG was further purified and desalted on a strong anion-exchange (SAX) cartridge. DiN-acetylhomocystine was simultaneously purified and desalted on a C18 cartridge. The N-acetylated aminothiols were generated using gel-immobilized tris(2-carboxyethyl)phosphine as a reductant, which obviated the need for further purification. Alternatively, disulfide exchange with dissolved dithiothreitol yielded N-acetylglutathione, which was purified on the SAX cartridge. pH titrations of N-acetylglutathione (8.99) and N-acetylhomocysteine (9.66) as well as those of commercially available N-acetylcysteine (9.53) and N-acetylpenicillamine (10.21) yielded pK(a) (SH) values of importance for biological studies.

Promoter analysis and transcription regulation of fus gene cluster responsible for fusaricidin synthesis of Paenibacillus polymyxa SQR-21.

Fusaricidins produced by Paenibacillus polymyxa are lipopeptide antibiotics with outstanding antifungal activity. In this study, the whole gene cluster responsible for fusaricidin biosynthesis (fusA) was isolated and identified from the cDNA library of one biocontrol agent P. polymyxa SQR-21 (SQR-21). MALDI-TOF MS analysis confirmed that SQR-21 could produce four kinds of fusaricidins: A, B, C, and D. A central promoter that drove the transcription of fusGFEDCBA was revealed by mapping of the fus promoter region by 5' deletions. The disruption of fusA in SQR-21 led to the abolishment of fusaricidin production and antifungal activity. The direct interaction between a potential regulator, AbrB, and the promoter region of fus gene cluster was confirmed by electrophoretic mobility shift assays. One abrB disruption mutant showed significantly higher antifungal activity compared with the wild type. These results revealed a pathway for the transcriptional regulation of the fus gene cluster in P. polymyxa.

An optimized DNA extraction and purification method from dairy manure compost for genetic diversity analysis.

An unbiased DNA extraction protocol is necessary for analysis of genetic diversity, particularly, of genes in complex environmental samples by nucleic acid techniques. In the present study, three manual extraction methods and two commonly used commercial kits, which were accompanied by two DNA purification strategies, were compared based on cell lysis efficiency, DNA and humic acid yields, PCR amplification and denaturing gradient gel electrophoresis (DGGE) analysis. The results show that in spite of higher cell lysis efficiencies of the two commercial kits, the purified DNA yields were only one-third of that obtained by the two manual methods of FTSP (Freeze-thaw-SDS-Protein K) and FTSPP (Freeze-thaw-SDS-Protein K-Polyvinylpolypyrrolidone). The purified DNA from all five methods was pure enough for successful PCR and real-time PCR amplifications in the presence of 1 µg µL(-1) BSA. However, the FTSPP extraction method with DNA purification by a Wizard(®) kit yielded the largest number of 16S rRNA gene copies and ribotypes or bands in DGGE profiles, which indicated a superiority over the other four methods. The development of this optimized DNA extraction and purification method may provide a valuable tool for further molecular analysis of compost.