Differential expression of bio-active metabolites produced by chitosan polymers-based Bacillus amyloliquefaciens fermentation.

Bacillus amyloliquefaciens strain PPL shows a potential for the control of phytopathogenic fungi. In the present study, upon growing the strain PPL on various forms of chitosan (0.5 % powder, 0.1 % soluble, and 0.15 % colloidal) as the carbon source, the antifungal activity on tomato Fusarium wilt correlated with the activity of chitosanase and ß-1,3-glucanase. The colloidal substrate-based strain PPL fermentation displayed the highest degree of spore germination inhibition (79.5 %) and biocontrol efficiency (76.0 %) in tomato by increased biofilm formation. The colloidal culture upregulated the expression of chitosanase gene (5.9-fold), and the powder attributed to the expression of cyclic lipopeptides-genes (2.5-5.7 fold). Moreover, the three chitosan cultures induced the morphological changes of Fusarium oxysporum. These results suggest that the choice of growth substrate synergistically affects the production of secondary metabolites by PPL strain, and consequently its antifungal activity.

Venetoclax with Azacitidine Induced Tumor Lysis Syndrome in an Elderly Patient with Acute Myeloid Leukemia: A Case Report.

Combination treatment with hypomethylating agents (HMAs) and venetoclax is being used increasingly in elderly patients with acute myeloid leukemia (AML). Venetoclax with HMAs has been reported to be associated with tumor lysis syndrome (TLS) in AML patients with high leukemic burden. We present a case of an elderly AML patient with low leukemic burden who developed TLS while receiving venetoclax and azacitidine (AZA). A 74-year-old man with newly diagnosed AML with NPM1 mutation received combination therapy with venetoclax and AZA in an outpatient clinic. Within 12 hours after starting venetoclax and AZA, the patient was admitted to the emergency room with fever, general weakness, and laboratory findings consistent with TLS. Based on our results, we recommend monitoring at the start of the treatment with venetoclax and HMAs to prevent and control TLS regardless of the leukemic burden and favorable genetic risk.

Antibacterial activity of various chitosan forms against Xanthomonas axonopodis pv. glycines.

In this study, the antibacterial activities of colloidal chitosan, chitosan solution, and chitooligosaccharide solution were evaluated against Xanthomonas axonopodis pv. glycines grown in peptone sucrose broth (PSB) medium. Treatment with colloidal chitosan (0.01, 0.025, and 0.05%) inhibited X. axonopodis pv. glycines growth only until 36 h. Thin-layer chromatography analysis detected some metabolites, consistently with the cell growth pattern. Two chitooligosaccharides (1-3 kDa and 5-10 kDa) dissolved in distilled water and acetic acid did not exhibit antibacterial activity against X. axonopodis pv. glycines at all tested concentrations (0, 0.001, 0.005, 0.01, 0.015, 0.02, 0.025, and 0.05%). Compared to the control, the chitosan solution decreased X. axonopodis pv. glycines cell growth by 58.7% and 99.0% at concentrations of 0.015% and 0.02%, respectively, after 3 d of incubation. The chitosan solution exhibited the highest antibacterial activity at pH 6.5. However, the antibacterial activity of chitosan decreased in the presence of NaCl and MgCl2.

Characterization and antifungal activity of chitosanase produced by Pedobacter sp. PR-M6.

To investigate the temperature requirements of chitosanase activity, as well as the degradation patterns generated by enzyme-induced chitosan oligomer hydrolysis, Pedobacter sp. PR-M6 was inoculated onto 0.5% colloidal chitosan medium agar plates. Cell growth was higher at 30 °C than at 20 °C during the initial 2 days of incubation. The protein content rapidly increased on day 1 at both temperatures and then it slowly increased at 20 °C and slowly decreased at 30 °C during the following 5 days of incubation. In order to characterize the electrophoretic pattern, Pedobacter sp. PR-M6 was cultured in 1% powder chitosan medium at 20 °C and 30 °C for 5 days after incubation and analyzed by SDS-PAGE. Four bands were visible, corresponding to ct1 (25 kDa), ct2 (17 kDa), ct3 (15 kDa), and ct4 (14 kDa), at both 20 °C and 30 °C. The optimal conditions for the activity of chitosanase produced from Pedobacter sp. PR-M6 were 60 °C and 1.81 enzyme units/mg protein. Two major isozyme bands (ct3 and ct4) exhibited their strongest chitosanase activity at 50 °C in SDS-PAGE gel. The reaction products generated from (GlcN)2-(GlcN)5 substrates at 60 °C after a 1 h incubation were investigated by thin-layer chromatography. Low-molecular weight chitosan and oligochitosan (LCOC) and soluble chitosan showed antifungal activity against A. brassicicola, B. cinerea, F. solani, and R. solani. LCOC exhibited higher antifungal activity than soluble chitosan. Moreover, LCOC treatments (500 ppm and 1000 ppm) inhibited conidia germination in A. brassicicola.

Expression and degradation patterns of chitinase purified from Xuehuali (Pyrus bretschneiderilia) pollen.

The present study investigated the expression pattern of chitinase in Xuehuali (Pyrus bretschneiderilia) pollen, as well as its subsequent degradation. The chitinase was purified and collected using chitin affinity column chromatography with regenerated chitin. After purification, four additional chitinase isozymes (chiA, chiB, chiC, and chiD) and chitinase (Chi II) were clearly expressed on SDS-PAGE gels that contained 0.01% glycol chitin. The chitinase reaction products were examined using GlcNAc, (GlcNAc)2, (GlcNAc)3, (GlcNAc)4, (GlcNAc)5, and (GlcNAc)6 as substrates at 2 and 24h after reaction via TLC and HPLC. The (GlcNAc)4 oligosaccharide was slightly degraded to (GlcNAc)2 after 24h of reaction with Xuehuali pollen chitinase on TLC. Meanwhile, (GlcNAc)5 was degraded to (GlcNAc)2-4, and 2300ppm (GlcNAc)6 was degraded to 246ppm (GlcNAc)2, 208ppm (GlcNAc)3, 572ppm (GlcNAc)4, and 336ppm (GlcNAc)5 on HPLC. With regard to temperature, the strongest Xuehuali pollen chitinase activity (0.69 unit/mL) was observed at 37°C after 3h of incubation, and with regard to pH, the strongest activity (0.72unit/mL) was observed at pH 3 after 3h of incubation. The main chitin oligomers degraded from (GlcNAc)6 were (GlcNAc)2 and (GlcNAc)4.

Antifungal activity and patterns of N-acetyl-chitooligosaccharide degradation via chitinase produced from Serratia marcescens PRNK-1.

Serratia marcescens PRNK-1, which has strong chitinolytic activity, was isolated from cockroaches (Periplaneta americana L.). The chitinase from S. marcescens PRNK-1 was characterized after incubation in a 0.5% colloidal chitin medium at 30 °C for 3 days. The molecular weights of three bands after staining for chitinase activity were approximately 34, 41, and 48 kDa on an SDS-PAGE gel. S. marcescens PRNK-1 strain strongly inhibited hyphal growth of Rhizoctonia solani and Fusarium oxysporum. Thin-layer chromatography (TLC) and high performance liquid chromatograph (HPLC) analyses were conducted to investigate the degradation patterns of N-acetyl-chitooligosaccharides by PRNK-1 chitinase. The N-acetyl-chitooligosaccharides: N-acetyl-chitin dimer (GlcNAc)2, N-acetyl-chitin trimer (GlcNAc)3, and N-acetyl-chitin tetramer (GlcNAc)4 were degraded to (GlcNAc)1-3 on a TLC plate. In an additional experiment, (GlcNAc)6 was degraded to (GlcNAc)1-4 on a TLC plate. The optimal temperature for chitinase activity of the PRNK-1 was 50 °C, producing 32.8 units/mL. As seen via TLC, the highest degradation of (GlcNAc)4 by PRNK-1 chitinase occurred with 50 °C incubation. The optimal pH for chitinase activity of PRNK-1 was pH 5.5, producing 24.6 units/mL. As seen via TLC, the highest degradation of (GlcNAc)4 by PRNK-1 chitinase occurred at pH 5.0-6.0. These results indicate that chitinase produced from S. marcescens PRNK-1 strain showed strong antifungal activity and potential of production of N-acetyl-chitooligosaccharides.

Enzyme activity and expression pattern of intra- and extracellular chitinase and ß-1,3-glucanase of Wickerhamomyces anomalus EG2 using glycol chitin and glucan-containing high polymer complex.

We investigated cell growth and activity of intra- and extracellular chitinase, ß-1,3-glucanase, and chitin deacetylase with SDS-PAGE by incubating W. anomalus EG2 in PDB and YPD media for 24h in presence of different concentrations (0%, 0.1%, 0.3%, and 0.5%) of colloidal chitin. Maximum cell growth was observed in both PDB and YPD media without colloidal chitin. In the absence of colloidal chitin, maximum extracellular ß-1,3-glucanase activity of 32.96 and 47.28 units/mL was reported at 18h in PDB medium and 6h in YPD medium, respectively. In addition, extracellular chitinase was unaffected by various concentrations of carboxymethyl chitin in both PDB and YPD media. In the absence of colloidal chitin, maximum intracellular chitinase activity was indicated to be 9.82 and 9.86 units/mg protein in PDB and YPD media, respectively. Maximum intracellular ß-1,3-glucanase activity reported was 17.34 units/mg protein in PDB medium containing 0.5% colloidal chitin and 15.0 units/mg protein in YPD medium containing 0.3% colloidal chitin. Five major isozymes, GN1, GN2, GN3, GN4, and GN5, of intracellular ß-1,3-glucanase were detected with glucan-containing high polymer complex as a substrate with or without colloidal chitin.

Antifungal activity and expression patterns of extracellular chitinase and ß-1,3-glucanase in Wickerhamomyces anomalus EG2 treated with chitin and glucan.

In this study, the expression patterns of extracellular chitinase and ß-1,3-glucanase from cultured Wickerhamomyces anomalus EG2 treated with chitin, glucan, and chemical chitinase inhibitors (kinetin, caffeine, and acetazolamide) were investigated using SDS-PAGE. Relationship between enzyme expression and antifungal activity from yeast plays a very important role for biocontrol of phytopathoges. To determine antifungal activity against phytopathogens, W. anomalus EG2 was shown to strongly inhibit hyphal growth of Fusarium oxysporum KACC 40032 and Rhizoctonia solani KACC 40111. Slight chitinase activity was observed 12 h after incubation in both PDB and YPD medium without colloidal chitin. The molecular weight of chitinase was approximately 124 kDa ß-1,3-Glucanase isoenzyme (GN1 and GN2) was observed distinctly on SDS-PAGE gels when laminarin was used as a substrate. ß-1,3-Glucanase isoenzyme was not observed when using glucan-containing high polymer complex (GHPC) as a substrate. Production of chitinase from W. anomalus EG2 was inhibited slightly by acetazolamide. Abnormal and cluster-shaped cells of W. anomalus EG2 were observed in both PDB and YPD medium treated with colloidal chitin. These results indicated that W. anomalus EG2 could be applied commercially as a biological control agent of phytopathogens and as a bioinhibitor of yeast cell growth.

Identification, purification, and expression patterns of chitinase from psychrotolerant Pedobacter sp. PR-M6 and antifungal activity in vitro.

In this study, a novel psychrotolerant chitinolytic bacterium Pedobacter sp. PR-M6 that displayed strong chitinolytic activity on 0.5% colloidal chitin was isolated from the soil of a decayed mushroom. Chitinase activity of PR-M6 at 25 °C (C25) after 6 days of incubation with colloidal chitin increased rapidly to a maximum level (31.3 U/mg proteins). Three chitinase isozymes (chiII, chiIII, and chiIV) from the crude enzyme at 25 °C (C25) incubation were expressed on SDS-PAGE gels at 25 °C. After purification by chitin-affinity chromatography, six chitinase isozymes (chiI, chiII, chiIII, chiIV, chiV, and chiVI) from C25-fractions were expressed on SDS-PAGE gels at 25 °C. Major bands of chitinase isozymes (chiI, chiII, and chiIII) from C4-fractions were strongly expressed on SDS-PAGE gels at 25 °C. Pedobacter sp. PR-M6 showed high inhibition rate of 60.9% and 57.5% against Rhizoctonia solani and Botrytis cinerea, respectively. These results indicated that psychrotolerant Pedobacter sp. PR-M6 could be applied widely as a microorganism agent for the biocontrol of agricultural phytopathogens at low temperatures.

Enzymatic properties of chitinase-producing antagonistic bacterium Paenibacillus chitinolyticus with various substrates.

Various chitin substrates were used to investigate the properties of enzymes produced from the chitinase-producing bacterium Paenibacillus chitinolyticus MP-306 against phytopathogens. The MP-306 bacterium was incubated in nine culture media [crab shell powder chitin (CRS), chitin-protein complex powder (CPC), carboxymethyl-chitin powder (CMC), yeast extract only (YE), LB (Trypton, NaCl, and yeast extract), GT (Trypton, NaCl, and glucose), crab shell colloidal chitin (CSC), squid pen powder chitin (SPC), and cicada slough powder chitin (CSP)] at 30 °C for 3 days. Chitinase isozymes in CPC medium were expressed strongly as CN1, CN2, CN3, CN4, CN5, and CN6 bands on native-PAGE gels. Chitinase isozymes in CPC and CMC medium were expressed as 13 bands (CS1-CS13) on SDS-PAGE gels. Chitinase isozymes were expressed strongly on SDS-PAGE gels as two bands (CS6 and CS8) on YE and LB medium and 13 bands (CS1-CS13) on SPC medium. In crude enzyme, chitinase isozymes at pH 7 and pH 9 in chitin media appeared strongly on SDS-PAGE gels. Partial purified enzyme indicated high stability of enzyme activity at various temperatures and pHs in chitin medium, while these enzymes indicated low activity staining of enzyme on electrophoresis gels at various temperatures and pHs condition of chitin medium.

Enhanced production of poly-γ-glutamic acid by a newly-isolated Bacillus subtilis.

Application of poly-gamma-glutamic acid (γ-PGA), an unusual macromolecular anionic polypeptide, is limited due to the high cost associated with its low productivity. Screening bacterial strains to find a more efficient producer is one approach to overcome this limitation. Strain MJ80 was isolated as a γ-PGA producer among 1,500 bacterial colonies obtained from soil samples. It was identified as Bacillus subtilis, based on the biochemical and morphological properties and 16S rDNA gene sequencing. It produced γ-PGA from both glutamic acid and soybean powder, identifying it as a facultative glutamic acid-metabolizing bacterium. After optimization of its culture conditions, B. subtilis MJ80 showed γ-PGA productivity of 75.5 and 68.7 g/l in 3 and 300 l jar fermenters for 3 days cultivation, respectively, the highest productivity reported to date, suggesting MJ80 to be a promising strain for γ-PGA production.

Expression patterns of chitinase and chitosanase produced from Bacillus cereus in suppression of phytopathogen.

Bacillus cereus MP-310 was incubated on various culture media substrates as LB, colloidal chitin, chitosan powder, and chitosan beads to investigate the concurrent expression patterns of chitinase and chitosanase isozymes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Chitinase activity increased rapidly with a maximum level after 6 days of incubation in CM-chitin medium. Major bands of chitinase isozymes were strongly expressed on SDS-PAGE in LB medium (four bands) and in colloidal chitin medium (five bands) after 6 days after incubation, and in chitosan powder medium (one band) and in chitosan beads medium (five bands) after 12 days after incubation. A major band of chitosanase isozymes was strongly expressed on SDS-PAGE in chitosan powder medium (one band) and in chitosan beads medium (one band) after 12 days of incubation.

Molecular and immunohistochemical characterization of the chitinase gene from Pieris rapae granulovirus.

The chitinase gene of baculoviruses is expressed in the late phase of virus replication in insects and possesses high exo- and endochitinase activity, which can hydrolyze chitin in the body of the insect, thus promoting terminal host liquefaction. Alphabaculovirus viral chitinases (vChitA) have been well analyzed, but information regarding viral chitinases from betabaculoviruses is limited. Whole-genome sequencing of a Korean isolate of Pieris rapae GV (PiraGV-K) predicted a putative chitinase gene corresponding to ORF10. The PiraGV-K chitinase gene had a coding sequence of 1,761 bp, encoding a protein of 586 amino acid (aa) residues, including an 18-aa putative signal peptide. Time course induction pattern observed by SDS-PAGE and subsequent Western blot with anti-PiraGV-K chitinase antibody revealed the cleavage of the signal peptide from the intact chitinase. Edman sequencing analysis was further conducted to confirm the exact nature of the mature chitinase, and the N-terminal amino acid sequence (KPGAP) exactly matched the sequence following the signal peptide sequence. The transcriptomics of PiraGV-K chitinase in infected P. rapae larvae, examined by real-time PCR, revealed a significant 75-fold increase after four days of feeding with PiraGV-K-treated leaves, with a subsequent decline at the later stages of infection. Confocal microscopic analysis showed that PiraGV-K chitinase possibly exists as a secreted protein, with strong chitinase-specific signals in fat body cells and integument at four days postinfection. Furthermore, immunogold labeling and electron microscopy studies localized the PiraGV-K chitinase in the cytoplasm and sparsely within vacuolar structures in the fat body apart from the extensive aggregation in the cuticular lining of the integument.

Detection of chitinase ChiA produced by Serratia marcescens PRC-5, using anti-PrGV-chitinase.

In this study, a bacterium Serratia marcescens PRC-5 that displayed strong chitinolytic activity on 0.5% colloidal chitin-containing agar medium was isolated from soil. The chitinase activity increased rapidly with a maximum level (6.14 U/mL) on 4 days of incubation with swollen chitin (pH 5.0). Three active bands of chitinase isozymes were observed (53, 44, and 34 kDa) on SDS-PAGE gel and there pI values ranged from pI 5.4 to 5.8 on 2D gels. The chitinase from the PRC-5 strain was also able to produce GlcNAc monomers on TLC plates. The chitinase of PRC-5 inhibited the mycelial growth of Rhizoctonia solani KACC40111, which indicates that it could be used as a biocontrol agent for phytophathogens. The chitinase isozyme N1, which had a molecular weight of 62 kDa, was transferred from a native and SDS-PAGE gel onto an immunoblot and was probed using an anti-PrGV-chitinase.

Expression patterns of chitinase produced from Paenibacillus chitinolyticus with different two culture media.

To investigate the expression patterns of chitinase isozymes on native-PAGE and SDS-PAGE gels Paenibacillus chitinolyticus MP-306 was cultured on culture media with and without chitin substrate. P. chitinolyticus MP-306 had a strong chitinolytic activity on colloidal chitin medium. Chitinase isozymes of MP-306 were expressed as six bands (CN1-CN6) on native-PAGE gels and thirteen bands (CS1-CS13) on SDS-PAGE gels after incubation in chitin medium. Three bands (CN1, CN2, and CN3) of chitinase isozymes of MP-306 on native-PAGE gels were expressed as nine bands (CS1, CS2, CS3, CS4, CS5, CS6, CS8, CS10, and CS13) of chitinase isozymes on SDS-PAGE gels. Three bands (CN4, CN5, and CN6) of chitinase isozymes of MP-306 were strongly inhibited by metal ions on native-PAGE and SDS-PAGE gels.

Induction of defense response against Rhizoctonia solani in cucumber plants by endophytic bacterium Bacillus thuringiensis GS1.

An endophytic bacterium, Bacillus thuringiensis GS1, was isolated from bracken (Pteridium aquilinum) and found to have maximal production of chitinase (4.3 units/ml) at 5 days after culture. This study investigated the ability of B. thuringiensis GS1 to induce resistance to Rhizoctonia solani KACC 40111 (RS) in cucumber plants. Chitinase activity was greatest in RS-treated plants at 4 days. beta-1,3- Glucanase activity was highest in GS1-treated plants at 5 days. Guaiacol peroxidase (GPOD) activity increased continuously in all treated plants for 5 days. Ascorbate peroxidase (APX) activity in RS-treated plants was increased 1.5-fold compared with the control at 4 days. Polyphenol oxidase (PPO) activity in RS-treated plants was increased 1.5-fold compared with the control at 3 days. At 5 days after treatment, activity staining revealed three bands with chitinase activity (Ch1, Ch2, and Ch3) on SDSPAGE of cucumber plants treated with GS1+RS, whereas only one band was observed for RS-treated plants (Ch2). One GPOD isozyme (Gp1) was also observed in response to treatment with RS and GS1+RS at 4 days. One APX band (Ap2) was present on the native-PAGE gel of the control, and GS1- and GS1+RS-treated plants at 1 day. PPO bands (Po1 and Po2) from RS- and GS1+RS-treated plants were stronger than in the control and GS1-treated plants upon native-PAGE at 5 days. Taken together, these results indicate that the induction of PR proteins and defense-related enzymes by B. thuringiensis GS1 might have suppressed the damping-off caused by R. solani KACC 40111 in cucumber plants.

Molecular cloning and characterization of novel Morus alba germin-like protein gene which encodes for a silkworm gut digestion-resistant antimicrobial protein.

BACKGROUND: Silkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances) and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens. METHODOLOGY/PRINCIPAL FINDINGS: Silkworm fecal matter extracts prepared in 0.02 M phosphate buffer saline (pH 7.4), at a temperature of 60°C was subjected to 40% saturated ammonium sulphate precipitation and purified by gel-filtration chromatography (GFC). SDS-PAGE under denaturing conditions showed a single band at about 21.5 kDa. The peak fraction, thus obtained by GFC wastested for homogeneityusing C18reverse-phase high performance liquid chromatography (HPLC). The activity of the purified protein was tested against selected Gram +/- bacteria and phytopathogenic Fusarium species with concentration-dependent inhibitionrelationship. The purified bioactive protein was subjected to matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS) and N-terminal sequencing by Edman degradation towards its identification. The N-terminal first 18 amino acid sequence following the predicted signal peptide showed homology to plant germin-like proteins (Glp). In order to characterize the full-length gene sequence in detail, the partial cDNA was cloned and sequenced using degenerate primers, followed by 5'- and 3'-rapid amplification of cDNA ends (RACE-PCR). The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps) involved in plant development and defense. CONCLUSIONS/SIGNIFICANCE: The study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter. The N-terminal amino acid sequence of the purified protein was found similar to the deduced amino acid sequence (without the transit peptide sequence) of the full length cDNA from M. alba.

Caudal epidural anesthesia administered intraoperatively provides for effective postoperative analgesia after total hip arthroplasty.

STUDY OBJECTIVE: To investigate the use of caudal epidural anesthesia for postoperative pain after total hip arthroplasty. DESIGN: Prospective study. SETTING: University-affiliated hospital. PATIENTS: 32 (4 men and 28 women) patients, aged 49 to 89 years, scheduled for total hip arthroplasty for osteoarthritis of the hip. INTERVENTIONS AND MEASUREMENTS: Patients were allocated to three groups: lumbar epidural anesthesia (EA group; n = 16) or caudal epidural anesthesia (CA group; n = 16) groups, which were case-matched according to patient demographics. Nine patients received general anesthesia only (GA group). We evaluated the level of postoperative pain using a 100-mm Visual Analog Scale (VAS) recorded at 3, 6, 9, 12, and 24 hours after surgery. MAIN RESULTS: Total requirement of diclofenac sodium suppositories was significantly larger in the GA group than in the EA or CA groups (444 +/- 302 vs 188 +/- 124 and 145 +/- 130 mg). The number of days requiring analgesics was significantly prolonged in the GA group compared with the EA or CA groups (14 +/- 9 vs 4 +/- 3 and 4 +/- 4 days). These items were similar between the EA group and the CA group. All VAS values for pain, rest, and movement in the postsurgical period over 24 hours were significantly higher in the GA group than in either the EA or CA groups. CONCLUSIONS: Caudal epidural anesthesia provides effective postoperative analgesia similar to lumbar epidural anesthesia.