Pegaspargase Combined with Concurrent Radiotherapy for Early-Stage Extranodal Natural Killer/T-Cell Lymphoma, Nasal Type: A Two-Center Phase II Study.

BACKGROUND: Concurrent chemoradiotherapy (CCRT) is expected to improve local and systemic disease control and has been established as a standard therapy for several types of solid tumors. Considering the benefits of frontline radiation and pegaspargase in localized extranodal natural killer (NK)/T-cell lymphoma, nasal type (ENKTL), we conducted a phase II study on pegaspargase-based CCRT to explore an effective treatment. MATERIALS AND METHODS: In this study, 30 patients with newly diagnosed nasal ENKTL in stages IE to IIE received CCRT (radiation 50 Gy and two cycles of pegaspargase 2,500 unit/m2 every 3 weeks). Four courses of pegaspargase were performed after CCRT. RESULTS: The patients completed CCRT and four cycles of pegaspargase. The complete remission (CR) rate was 90%, with a 95% confidential interval (CI) of 73.5%-97.9% after CCRT. The CR rate was 100% (95% CI, 88.4%-100%) at the end of the treatment. The 2-year overall survival and progression-free survival rates were 90.9% (95% CI, 78.4%-100%) and 92.8% (95% CI, 83.2%-100%), respectively. The major adverse events were in grades 1-2. CONCLUSION: Preliminary data indicate that pegaspargase combined with concurrent radiotherapy for newly diagnosed patients with nasal ENKTL was efficacious and well tolerated. Registered at www.chictr.org. CLINICAL TRIAL REGISTRATION NUMBER: ChiCTR-OIC-15007662. IMPLICATIONS FOR PRACTICE: This clinical trial, evaluating the efficacy and toxicity of concurrent chemoradiotherapy by using single-drug pegaspargase for patients with extranodal natural killer/T-cell lymphoma, nasal type (ENKTL) in stage IE to IIE, showed pegaspargase combined with concurrent radiotherapy was efficacious and well tolerated. Pegaspargase has a long half-life and is easy to administer via intramuscular injection. Consequently, pegaspargase combined with concurrent radiotherapy for patients with ENKTL can be completed in the outpatient clinic.

An Extracytoplasmic Function Sigma Factor Controls Cellulose Utilization by Regulating the Expression of an Outer Membrane Protein in Cytophaga hutchinsonii.

The common soil cellulolytic bacterium known as Cytophaga hutchinsonii makes use of a unique but poorly understood strategy in order to utilize cellulose. While several genes have been identified as being an active part of the utilization of cellulose, the mechanism(s) by which C. hutchinsonii both (i) senses its environment and (ii) regulates the expression of those genes are not as yet known. In this study, we identified and characterized the gene CHU_3097 encoding an extracytoplasmic function (ECF) σ factor (σcel1), the disruption of which compromised C. hutchinsonii cellulose assimilation to a large degree. The σcel1 and its putative partner anti-σcel1, encoded by the CHU_3096 gene found immediately downstream from CHU_3097, copurified in vitro The σcel1 was discovered to be associated with inner membrane when cells were cultured on glucose and yet was partially released from the membrane in response to cellulose. This release was found to occur on glucose when the anti-σcel1 was absent. Transcriptome analyses found a σcel1-regulated, cellulose-responsive gene regulon, within which an outer membrane protein encoding the gene CHU_1276, essential for cellulose utilization, was discovered to be significantly downregulated by CHU_3097 disruption. The expression of CHU_1276 almost fully restored cellulose utilization to the CHU_3097 mutant, demonstrating that CHU_1276 represents a critical regulatory target of σcel1 In this way, our study provided insights into the role of an ECF σ factor in coordinating the cellulolytic response of C. hutchinsoniiIMPORTANCE The common cellulolytic bacterium Cytophaga hutchinsonii uses a unique but poorly understood strategy in order to make use of cellulose. Throughout the process of cellulosic biomass breakdown, outer membrane proteins are thought to play key roles; this is evidenced by CHU_1276, which is required for the utilization of cellulose. However, the regulatory mechanism of its expression is not yet known. We found and characterized an extracytoplasmic function σ factor that is involved in coordinating the cellulolytic response of C. hutchinsonii by directly regulating the expression of CHU_1276 This study makes a contribution to our understanding of the regulatory mechanism used by C. hutchinsonii in order to adjust its genetic programs and so deal with novel environmental cues.

Substrate binding interferes with active site conformational dynamics in endoglucanase Cel5A from Thermobifida fusca.

The role of protein dynamics in enzyme catalysis is one of the most active areas in current enzymological research. Here, using endoglucanase Cel5A from Thermobifida fusca (TfCel5A) as a model, we applied molecular dynamics simulations to explore the dynamic behavior of the enzyme upon substrate binding. The collective motions of the active site revealed that the mechanism of TfCel5A substrate binding can likely be described by the conformational-selection model; however, we observed that the conformations of active site residues changed differently along with substrate binding. Although most active site residues retained their native conformational ensemble, some (Tyr163 and Glu355) generated newly induced conformations, whereas others (Phe162 and Tyr189) exhibited shifts in the equilibration of their conformational distributions. These results showed that TfCel5A substrate binding relied on a hybrid mechanism involving induced fit and conformational selection. Interestingly, we found that TfCel5A active site could only partly rebalance its conformational dynamics upon substrate dissociation within the same simulation time, which implies that the conformational rebalance upon substrate dissociation is likely more difficult than the conformational selection upon substrate binding at least in the view of the time required. Our findings offer new insight into enzyme catalysis and potential applications for future protein engineering.

An Outer Membrane Protein Involved in the Uptake of Glucose Is Essential for Cytophaga hutchinsonii Cellulose Utilization.

Cytophaga hutchinsonii specializes in cellulose digestion by employing a collection of novel cell-associated proteins. Here, we identified a novel gene locus, CHU_1276, that is essential for C. hutchinsonii cellulose utilization. Disruption of CHU_1276 in C. hutchinsonii resulted in complete deficiency in cellulose degradation, as well as compromised assimilation of cellobiose or glucose at a low concentration. Further analysis showed that CHU_1276 was an outer membrane protein that could be induced by cellulose and low concentrations of glucose. Transcriptional profiling revealed that CHU_1276 exerted a profound effect on the genome-wide response to both glucose and Avicel and that the mutant lacking CHU_1276 displayed expression profiles very different from those of the wild-type strain under different culture conditions. Specifically, comparison of their transcriptional responses to cellulose led to the identification of a gene set potentially regulated by CHU_1276. These results suggest that CHU_1276 plays an essential role in cellulose utilization, probably by coordinating the extracellular hydrolysis of cellulose substrate with the intracellular uptake of the hydrolysis product in C. hutchinsonii.

A small periplasmic protein essential for Cytophaga hutchinsonii cellulose digestion.

Cytophaga hutchinsonii is a gliding cellulolytic bacterium that is ubiquitously distributed in soil. The mechanism by which C. hutchinsonii achieves cellulose digestion, however, is still largely unknown. In this study, we obtained a C. hutchinsonii mutant that was defective in utilizing filter paper or Avicel as the sole carbon source by transposon mutagenesis. The interrupted gene locus, CHU_2981, encodes a hypothetical protein with only 130 amino acids. Cell fractionation and western blot detection of CHU_2981 fused with a C-terminal green fluorescence protein (GFP) indicated that CHU_2981 is located in the periplasm. The CHU_2981-disrupted mutant cells exhibited a significant growth defect on Avicel but not on glucose and cellobiose. The absence of CHU_2981 also resulted in a significant defect in colony spreading and individual cell motility compared to wild-type cells. Further analysis demonstrated that the CHU_2981-disrupted mutant cells exhibited a different profile of cellulose-absorbed outer membrane proteins from that of wild-type cells, in which protein varieties and amounts were markedly decreased. Our results showed that CHU_2981, the periplasmic non-cellulolytic protein, plays an important role in both cellulose utilization and cell motility probably by being involved in the appropriate production of outer membrane proteins.

Identification and characterization of a novel locus in Cytophaga hutchinsonii involved in colony spreading and cellulose digestion.

Cytophaga hutchinsonii, an aerobic cellulolytic soil bacterium, is capable of degrading crystalline cellulose and gliding over surface rapidly. The involved mechanisms, however, are largely unknown. Here, we used the mariner-based transposon HimarEm1 to screen for C. hutchinsonii mutants deficient in utilizing filter paper as the sole carbon source. A novel gene locus, chu_1719, encoding a hypothetical protein was identified, whose inactivation resulted in a compromised growth of C. hutchinsonii on filter paper. Further analysis revealed that disruption of chu_1719 suppressed colony spreading but had no significant effect on Avicel degradation in liquid medium. Carboxymethylcellulase (CMCase) activity of the mutant membrane proteins was reduced by about 40% as compared with the wild-type strain. Moreover, profiles of cellulose-adsorbed outer membrane proteins were significantly different between the mutant and wild-type (WT) strains. These results suggest that chu_1719 plays an important role in controlling the spreading motility and cellulose utilization probably by affecting the appropriate production of membrane proteins in C. hutchinsonii.

Differential involvement of ß-glucosidases from Hypocrea jecorina in rapid induction of cellulase genes by cellulose and cellobiose.

Appropriate perception of cellulose outside the cell by transforming it into an intracellular signal ensures the rapid production of cellulases by cellulolytic Hypocrea jecorina. The major extracellular ß-glucosidase BglI (CEL3a) has been shown to contribute to the efficient induction of cellulase genes. Multiple ß-glucosidases belonging to glycosyl hydrolase (GH) family 3 and 1, however, exist in H. jecorina. Here we demonstrated that CEL1b, like CEL1a, was an intracellular ß-glucosidase displaying in vitro transglycosylation activity. We then found evidence that these two major intracellular ß-glucosidases were involved in the rapid induction of cellulase genes by insoluble cellulose. Deletion of cel1a and cel1b significantly compromised the efficient gene expression of the major cellulase gene, cbh1. Simultaneous absence of BglI, CEL1a, and CEL1b caused the induction of the cellulase gene by cellulose to further deteriorate. The induction defect, however, was not observed with cellobiose. The absence of the three ß-glucosidases, rather, facilitated the induced synthesis of cellulase on cellobiose. Furthermore, addition of cellobiose restored the productive induction on cellulose in the deletion strains. The results indicate that the three ß-glucosidases may not participate in transforming cellobiose beyond hydrolysis to provoke cellulase formation in H. jecorina. They may otherwise contribute to the accumulation of cellobiose from cellulose as inducing signals.

Purification and characterization of poly(L-lactic acid)-degrading enzymes from Amycolatopsis orientalis ssp. orientalis.

Polylactide or poly(l-lactic acid) (PLA) is a commercially promising material for use as a renewable and biodegradable plastic. Three novel PLA-degrading enzymes, named PLAase I, II and III, were purified to homogeneity from the culture supernatant of an effective PLA-degrading bacterium, Amycolatopsis orientalis ssp. orientalis. The molecular masses of these three PLAases as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 24.0, 19.5 and 18.0 kDa, with the pH optima being 9.5, 10.5 and 9.5, respectively. The optimal temperature for the enzyme activities was 50-60 degrees C. All the purified enzymes could degrade high-molecular-weight PLA film as well as casein, and the PLA-degrading activities were strongly inhibited by serine protease inhibitors such as phenylmethylsulfonyl fluoride and aprotinin, but were not susceptive to chymostatin and pepstatin. Taken together, these data demonstrated that A. orientalis ssp. orientalis produces multiple serine-like proteases to utilize extracellular polylactide as a sole carbon source.

[Progress on biodegradation of polylactic acid--a review].

Polylactic acid is a high molecular-weight polyester made from renewable resources such as corn or starch. It is a promising biodegradable plastic due to its mechanical properties, biocompatibility and biodegradability. To achieve natural recycling of polylactic acid, relative microorganisms and the underlying mechanisms in the biodegradation has become an important issue in biodegradable materials. Up to date, most isolated microbes capable of degrading polylactic acid belong to actinomycetes. Proteases secreted by these microorganisms are responsible for the degradation. However, subtle differences exist between these polylactic acid degrading enzymes and typical proteases with respect to substrate binding and catalysis. Amino acids relative to catalysis are postulated to be highly plastic allowing their catalytic hydrolysis of polylactic acid. In this paper we reviewed current studies on biodegradation of polylactic acid concerning its microbial, enzymatic reactions and the possible mechanisms. We also discussed the probability of biologically recycling PLA by applying highly efficient strains and enzymes.

Dynamic changes of the dominant functioning microbial community in the compost of a 90-m(3) aerobic solid state fermentor revealed by integrated meta-omics.

The dynamic changes in the composition and function of both bacterial and fungal communities over time and at various depths in the compost of a 90-m(3) industrial-scale fermentor were explored using integrated meta-omics. The microbial communities in the middle layer (1.2m) of the compost developed a stable and simple structure over time, which was mainly composed of Thermobifida, Bacillus, Thermomyces and Aspergillus. According to the metaproteomic results, the bacterial community was more focused on cellulose degradation, characterized by 44% of the cellulases that were secreted by Thermobifida, while the fungal community was more likely to degrade hemicellulose, mainly via Thermomyces and Aspergillus. The results revealed that, under artificial control of the temperature and oxygen concentration, the efficiency of organic waste degradation was greatly increased and the fermentation cycle was shortened to 11 days.

Wheat straw: An inefficient substrate for rapid natural lignocellulosic composting.

Composting is a promising method for the management of agricultural wastes. However, results for wheat straw composts with different carbon-to-nitrogen ratios revealed that wheat straw was only partly degraded after composting for 25days, with hemicellulose and cellulose content decreasing by 14% and 33%, respectively. No significant changes in community structure were found after composting according to 454-pyrosequencing. Bacterial communities were represented by Proteobacteria and Bacteroidetes throughout the composting process, including relatively high abundances of pathogenic microbes such as Pseudomonas and Flexibacter, suggesting that innocent treatment of the composts had not been achieved. Besides, the significant lignocellulose degrader Thermomyces was not the exclusively dominant fungus with relative abundance only accounting for 19% of fungal communities. These results indicated that comparing with maize straw, wheat straw was an inefficient substrate for rapid natural lignocellulose-based composting, which might be due to the recalcitrance of wheat straw.

Dynamic changes in xylanases and ß-1,4-endoglucanases secreted by Aspergillus niger An-76 in response to hydrolysates of lignocellulose polysaccharide.

Aspergillus niger is an effective secretor of glycoside hydrolases that facilitate the saprophytic lifestyle of the fungus by degrading plant cell wall polysaccharides. In the present study, a series of dynamic zymography assays were applied to quantify the secreted glycoside hydrolases of A. niger cultured in media containing different carbon sources. Differences in the diversity and concentrations of polysaccharide hydrolysates dynamically regulated the secretion of glycoside hydrolases. The secretion of ß-1,4-endoglucanase isozymes was observed to lag at least 24 h behind, rather than coincide with, the secretion of xylanase isozymes. Low concentrations of xylose could induce many endoxylanases (such as Xyn1/XynA, Xyn2, and Xyn3/XynB). High concentrations of xylose could sustain the induction of Xyn2 and Xyn3/XynB but repress Xyn1/XynA (GH10 endoxylanase), which has a broad substrate specificity, and also triggers the low-level secretion of Egl3/EglA, which also has a broad substrate specificity. Mixed polysaccharide hydrolysates sustained the induction of Egl1, whereas the other ß-1,4-endoglucanases were sustainably induced by the specific polysaccharide hydrolysates released during the hydrolysis process (such as Egl2 and Egl4). These results indicate that the secretion of glycoside hydrolases may be specifically regulated by the production of polysaccharide hydrolysates released during the process of biomass degradation.

Cellulose and cellodextrin utilization by the cellulolytic bacterium Cytophaga hutchisonii.

Cytophaga hutchinsonii is an abundant aerobic cellulolytic soil bacterium utilizing very few substrates as sole carbon and energy sources. In this study, growth of C. hutchinsonii on different substrates including crystalline cellulose, regenerated amorphous cellulose (RAC) as well as soluble sugars including cellodextrins was analyzed. Soluble sugars including glucose and cellodextrins were produced extracellularly when C. hutchinsonii was cultured on cellulose. Preferential use of cellulooligosaccharides as the carbon source by C. hutchinsonii was largely dependent on its inoculation status. Compared with glucose-grown cells, inoculation of cellobiose-grown cells led to a rapid assimilation of cellobiose or cellodextrins with longer-chain cellodextrins being hydrolyzed extracellularly to smaller oligomers during the culture. Further analysis of the distribution of cellulase activity revealed that, while the carboxymethylcellulase activity significantly induced by crystalline cellulose was highest in the outer membrane, the cellobiase activity was highest in the cytoplasmic membrane. These results suggest that membrane-bound cellulases may play an important role in cellulose solubilization by C. hutchinsonii and that metabolism of cello-oligosaccharides is a tightly coupled step in this process.

[Culture-independent digging of cellulases and genes from natural environments].

There is a great diversity for cellulolytic microbes in nature and the strategies they use to digest cellulose. In addition to the cultured cellulolytic microbes, there are still a great number of microbes being not readily culturable in natural environments, which may represent great potential for identifying novel cellulases and their encoding genes. The rise of metagenomics and metaproteomics provides essential technologic tools to dig up these resources and significant progress has been made so far. This review gives an insight into some relative results that have arisen from the meta-genomic or proteomic analysis of definitive uncultured microbe communities. Their potential role in elucidating the process and mechanisms of cellulose degradation in natural environment from the point of "community system microbiology" is also discussed.

Heterologous expression and site-directed mutagenesis of endoglucanase CelA from Clostridium thermocellum.

The endoglucanase CelA from Clostridium thermocellum was strongly expressed in Bacillus subtilis. The enzyme was purified by Ni(2+)-affinity chromatography. Site-directed substitution of D278 with an asparagine or an alanine residue surprisingly did not decrease the apparent k(cat) value. Further substitutions of two other potentially critical residues, Y215 and D152, resulted in a 2-fold decrease in apparent k(cat) value for Y215P and complete loss of activity for D152N.

Function of a low molecular weight peptide from Trichoderma pseudokoningii S38 during cellulose biodegradation.

The biochemical mechanism for cellulose decomposition by a low molecular weight peptide, named short fiber generating factor (SFGF), derived from the culture supernatant of a cellulolytic fungus Trichoderma pseudokoningii S-38, was determined. Sufficient information obtained by biochemical and biophysical studies and combined with observation with a scanning electron microscope provided further evidence for the earlier studies that the SFGF had a high capacity for chelating and reducing ferric ions, and could produce free radical by reduction of Fe(3+) to Fe(2+) in the presence of oxygen molecule. These studies suggested that the effect of SFGF on cellulose is directly related to an oxidative reaction and is different from the hydrolysis of cellulose by cellulases. The alcoholic hydroxyl groups in cellulose can be oxidized by SFGF, which leads to destruction of the hydrogen bond network in cellulose and cleavage of glycosidic linkages. Both effects led to the de-polymerization of cellulose and the formation of short fibers, and increase of reducing groups in residual cellulose, then the cellulose substrates became more susceptible for hydrolysis by cellulases.