ALBERT

Description: The protein 3D8 single-chain variable fragment (3D8 scFv) has potential anti-viral activity due to its ability to penetrate into cells and hydrolyze nucleic acids. Probiotic Lactobacillus paracasei engineered to secrete 3D8 scFv for oral administration was used to test the anti-viral effects of 3D8 scFv against gastrointestinal virus infections. We found that injection of 3D8 scFv into the intestinal lumen resulted in the penetration of 3D8 scFv into the intestinal villi and lamina propria. 3D8 scFv secreted from engineered L. paracasei retained its cell-penetrating and nucleic acid-hydrolyzing activities, which were previously shown with 3D8 scFv expressed in Escherichia coli . Pretreatment of RAW264.7 cells with 3D8 scFv purified from L. paracasei prevented apoptosis induction by murine norovirus infection and decreased messenger RNA (mRNA) expression of the viral capsid protein VP1. In a mouse model, oral administration of the engineered L. paracasei prior to murine norovirus infection reduced the expression level of mRNA encoding viral polymerase. Taken together, these results suggest that L. paracasei secreting 3D8 scFv provides a basis for the development of ingestible anti-viral probiotics active against gastrointestinal viral infection.

Description: Patients with a decrease in limb perfusion with a potential threat to limb viability manifested by ischemic rest pain, ischemic ulcers, and/or gangrene are considered to have critical limb ischemia (CLI). Because of this generally poor outcome, there is a strong need for attempting any procedure to save the affected limb. The aim of this work is to evaluate the possibility to use stem cell therapy as a treatment option for patients with chronic critical lower limb ischemia with no distal run off. This study includes 20 patients with chronic critical lower limb ischemia with no distal run off who are unsuitable for vascular or endovascular option. These patients underwent stem cell therapy (SCT) by autologous transplantation of bone marrow derived mononuclear cells. 55 % of patients treated with SCT showed improvement of the rest pain after the first month, 60 % continued improvement of the rest pain after 6 months, 75 % after 1 year and 80 % after 2 years and continued without any deterioration till the third year. Limb salvage rate after STC was 80 % after the first year till the end of the second and third years. SCT can result in angiogenesis in patients with no-option CLI, providing a foundation for the application of this therapy to leg ischemia.

Description: Bacterial contamination and biomass harvesting are still challenges associated with coupling of microalgae and wastewater treatment technology. This study investigated aggregation, bacterial growth, lipid production, and pollutant removal during bacteria contaminated Chlorella regularis cultivation under nutrient starvation stress, by supposing the C/N/P ratios of the medium to 14/1.4/1 (MB 2.5 ) and 44/1.4/1 (MB 4.0 ), respectively. Granules of 500–650 μm were formed in the bacteria contaminated inoculum; however, purified C. regularis were generally suspended freely in the medium, indicating that bacterial presence was a prerequisite for granulation. Extracellular polymeric substance (EPS) analysis showed that polysaccharides were dominant in granules, while protein mainly distributed in the outer layer. Denaturing gradient gel electrophoresis (DGGE) results revealed Sphingobacteriales bacterium and Sphingobacterium sp . are vital organisms involved in the flocculation of microalgae, and nitrifiers ( Stenotrophomonas maltophilia ) could co-exist in the granular. Both EPS and DGGE results further supported that bacteria played key roles in granulation. C. regularis was always dominant and determined the total biomass concentration during co-cultivation, but bacterial growth was limited owing to nutrient deficiency. Starvation strategy also contributed to enhancement of lipid accumulation, as lipid content in MB 4.0 with a greater C/N/P led to the greatest increase in the starvation period, and the maximum lipid productivity reached 0.057 g/(L·day). Chemical oxygen demand and nitrogen removal in MB 4.0 reached 92 and 96 %, respectively, after 3 days of cultivation. Thus, cultivation of microalgae in high C/N/P wastewater enabled simultaneous realization of biomass granulation, bacterial overgrowth limitation, enhanced lipid accumulation, and wastewater purification.

Description: Understanding the impact of polycyclic aromatic hydrocarbons (PAHs) on soil environments is of increasingly important concern. Therefore, the microbial degradation of PAHs in soils has drawn considerable attention, but little is known about the PAH degradation genes in urban soils. In this study, we examined the diversity and abundance of the PAH degradation bacteria and evaluated whether the specific bacteria can reflect PAH contents in the soils from urban roadsides directly receiving traffic emission. The results of phylogenetic analysis indicated that low PAH degradation bacterial diversity occurred in the urban roadside soils, only including Mycobacterium sp., Terrabacter sp., and one novel cluster. The community composition diversity of PAH degradation bacteria did not show a significant difference across the sampling sites. The abundance of PAH degradation genes ranged from 5.70 × 10 6 to 6.44 × 10 7  gene copies g −1 dry soil, with an average abundance of 1.43 × 10 7  gene copies g −1 dry soil, and their spatial variations were related significantly to PAH contents in the soils. The Mycobacterium sp. was the most widely detected and estimated to occupy 65.9–100 % of the total PAH degradation bacteria at most of the soil samples, implying that the Mycobacterium sp. might play a primary role in degrading PAHs in the contaminated urban soil environments.

Description: The ability of acetic acid bacteria (AAB) to produce cellulose has gained much industrial interest due to the physical and chemical characteristics of bacterial cellulose. The production of cellulose occurs in the presence of oxygen and in a glucose-containing medium, but it can also occur during vinegar elaboration by the traditional method. The vinegar biofilm produced by AAB on the air-liquid interface is primarily composed of cellulose and maintains the cells in close contact with oxygen. In this study, we screened for the ability of AAB to produce cellulose using different carbon sources in the presence or absence of ethanol. The presence of cellulose in biofilms was confirmed using the fluorochrome Calcofluor by microscopy. Moreover, the process of biofilm formation was monitored under epifluorescence microscopy using the Live/Dead BacLight Kit. A total of 77 AAB strains belonging to 35 species of Acetobacter , Komagataeibacter , Gluconacetobacter , and Gluconobacter were analysed, and 30 strains were able to produce a cellulose biofilm in at least one condition. This cellulose production was correlated with the PCR amplification of the bcsA gene that encodes cellulose synthase. A total of eight degenerated primers were designed, resulting in one primer pair that was able to detect the presence of this gene in 27 AAB strains, 26 of which formed cellulose.

Description: Pseudomonas aeruginosa is an opportunistic pathogen that localizes to and colonizes mucosal tissue. Thus, vaccines that elicit a strong mucosal response against P. aeruginosa should be superior to other vaccination strategies. In this study, to stimulate rapid and enhanced mucosal immune responses, mannose-modified chitosan microspheres loaded with the recombinant outer membrane protein OprF 190–342 -OprI 21–83 (FI) (FI-MCS-MPs) of P. aeruginosa were developed as a potent subunit vaccine for mucosal delivery. FI-MCS-MPs were successfully obtained via the tripolyphosphate ionic crosslinking method. Confocal and immunohistochemical analyses indicated that FI-MCS-MPs exhibited the ability to bind the macrophage mannose receptor (MMR, CD206) in vitro and in vivo. After intranasal immunization of mice with FI-MCS-MPs, FI-specific humoral immune responses were detected, measured as local IgM antibody titers in lung tissue slurry; IgA antibody titers in nasal washes, bronchoalveolar lavage (BAL), and intestinal lavage; and systemic IgA and IgG antibody titers in serum. FI-MCS-MPs induced early and high mucosal and systemic humoral antibody responses comparable to those in the group vaccinated with unmodified mannose. High levels of IFN-γ and IL-4 in addition to T lymphocyte subsets induced a mixed Th1/Th2 response in mice immunized with FI-MCS-MPs, resulting in the establishment of cellular immunity. Additionally, when immunized mice were challenged with P. aeruginosa via the nasal cavity, FI-MCS-MPs demonstrated 75 % protective efficacy. Together, these data indicate that mannose-modified chitosan microspheres are a promising subunit delivery system for vaccines against P. aeruginosa infection.

Description: The methylotrophic yeast Pichia pastoris is an attractive expression system due to its ability to secrete large amounts of recombinant protein, with the potential for glycosylation. Advances in glycoengineering of P. pastoris have successfully demonstrated the humanization of both the N- and O-linked glycosylation pathways in this organism. However, in certain cases, the presence of O-linked glycans on a therapeutic protein may not be desirable. Recently, we have reported the in vitro utility of jack bean α-1,2/3/6-mannosidase to remove O-linked mannose from intact undenatured glycoproteins produced in glycoengineered P. pastoris . However, one caveat of this strategy is that jack bean mannosidase has yet to be cloned and as such is only available as crude cellular extracts. This raises several concerns for using this reagent to treat large preparations of therapeutic proteins generated in P. pastoris . Therefore, we postulated that lysosomal mannosidases which have been cloned and demonstrated to have similar activities to jack bean mannosidase on N-linked glycans would also process O-linked glycans in a similar fashion. To this end, we screened a panel of recombinant lysosomal mannosidases from different organisms and identified several which cannot only reduce extended O-linked mannose chains but which can also hydrolyze the Man-α- O -Ser/Thr glycosidic bond on intact glycoproteins. As such, not only do we show for the first time the utility of lysosomal mannosidase for O-linked mannose processing, but since this is a recombinant enzyme, it has several benefits over the use of crude jack bean mannosidase extracts.

Description: The unfolded protein response (UPR) represents a mechanism to preserve endoplasmic reticulum (ER) homeostasis that is conserved in eukaryotes. ER stress caused by the accumulation of potentially toxic un- or misfolded proteins in the ER triggers UPR activation and the induction of genes important for protein folding in the ER, ER expansion, and transport from and to the ER. Along with this adaptation, the overall capacity for protein secretion is markedly increased by the UPR. In filamentous fungi, various approaches to employ the UPR for improved production of homologous and heterologous proteins have been investigated. As the effects on protein production were strongly dependent on the expressed protein, generally applicable strategies have to be developed. A combination of transcriptomic approaches monitoring secretion stress and basic research on the UPR mechanism provided novel and important insight into the complex regulatory cross-connections between UPR signalling, cellular physiology, and developmental processes. It will be discussed how this increasing knowledge on the UPR might stimulate the development of novel strategies for using the UPR as a tool in biotechnology.

Description: So far, the contribution of ammonia-oxidizing archaea (AOA) to ammonia oxidation in wastewater treatment processes has not been well understood. In this study, two soil aquifer treatment (SATs) systems were built up to treat synthetic domestic wastewater (column 1) and secondary effluent (column 4), accomplishing an average of 95 % ammonia removal during over 550 days of operation. Except at day 322, archaeal amoA genes always outnumbered bacterial amoA genes in both SATs as determined by using quantitative polymerase chain reaction (q-PCR). The ratios of archaeal amoA to 16S rRNA gene averaged at 0.70 ± 0.56 and 0.82 ± 0.62 in column 1 and column 4, respectively, indicating that all the archaea could be AOA carrying amoA gene in the SATs. The results of MiSeq-pyrosequencing targeting on archaeal and bacterial 16S rRNA genes with the primer pair of modified 515R/806R indicated that Nitrososphaera cluster affiliated with thaumarchaeal group I.1b was the dominant AOA species, while Nitrosospira cluster was the dominant ammonia-oxidizing bacteria (AOB). The statistical analysis showed significant relationship between AOA abundance (compared to AOB abundance) and inorganic and total nitrogen concentrations. Based on the mathematical model calculation for microbial growth, AOA had much greater capacity of ammonia oxidation as compared to the specific influent ammonia loading for AOA in the SATs, implying that a small fraction of the total AOA would actively work to oxidize ammonia chemoautotrophically whereas most of AOA would exhibit some level of functional redundancy. These results all pointed that AOA involved in microbial ammonia oxidation in the SATs.

Description: Selective capture of transcribed sequences (SCOTS) is an effective method to identify bacterial genes differentially expressed during different biological processes, including pathogenic interactions with a host species. The method can be used to elucidate molecular mechanisms driving and maintaining such interactions. The method is a powerful genetic tool that overcomes limitations found in other methods, by working with small amounts of mRNA and allowing for the separation of bacterial cDNA from host cDNA. It has been increasingly used in the discovery of genes involved in the bacterium-host interaction. In this review, we briefly introduce the SCOTS method, outline the technical advances offered in the method, and focus on the method’s applications in several microbial pathogens.

Description: A maltotriose-producing α-amylase, AmyA, from a newly isolated bacterial strain Microbulbifer thermotolerans DAU221 was purified and characterized in the heterologous host, Escherichia coli , using the pCold I vector. The amyA gene encoded a 761-residue protein composed of a 33 amino acid secretion signal peptide. The purified α-amylase with a molecular mass of 80 kDa, approximately, shared a sequence motif characteristic of the glycoside hydrolase family 13. The enzyme was optimally active, at 50 °C in sodium phosphate buffer (pH 6.0), by the traditional one factor-at-a-time method. But the optimal conditions of time, temperature, and pH for production of maltotriose from soluble starch were 1.76 h, 44.95 °C, and pH 6.35 by response surface methodology, respectively. Maltotriose, as the major enzyme reaction product, was analyzed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The enzyme was found to be inhibited by the addition of 10 mM Cu 2+ , Fe 3+ , Hg 2+ , Zn 2+ , and EDTA, but exhibited extreme stability toward hexane. The K m and V max values for the hydrolysis of soluble starch were 1.08 mg/mL and 1.736 mmol maltotriose/mg protein/min, respectively.

Description: The single-copy genes encoding putative polyphosphate–glucose phosphotransferases (PPGK, EC 2.7.1.63) from two nitrogen-fixing Cyanobacteria , Nostoc sp. PCC7120 and Nostoc punctiforme PCC73102, were cloned and functionally characterized. In contrast to their actinobacterial counterparts, the cyanobacterial PPGKs have shown the ability to phosphorylate glucose using strictly inorganic polyphosphates (polyP) as phosphoryl donors. This has proven to be an economically attractive reagent in contrast to the more costly ATP. Cyanobacterial PPGKs had a higher affinity for medium–long-sized polyP (greater than ten phosphoryl residues). Thus, longer polyP resulted in higher catalytic efficiency. Also in contrast to most their homologs in Actinobacteria , both cyanobacterial PPGKs exhibited a modest but significant polyP-mannokinase activity as well. Specific activities were in the range of 180–230 and 2–3 μmol min −1  mg −1 with glucose and mannose as substrates, respectively. No polyP-fructokinase activity was detected. Cyanobacterial PPGKs required a divalent metal cofactor and exhibited alkaline pH optima (approx. 9.0) and a remarkable thermostability (optimum temperature, 45 °C). The preference for Mg 2+ was noted with an affinity constant of 1.3 mM. Both recombinant PPGKs are homodimers with a subunit molecular mass of ca. 27 kDa. Based on database searches and experimental data from Southern blots and activity assays, closely related PPGK homologs appear to be widespread among unicellular and filamentous mostly nitrogen-fixing Cyanobacteria . Overall, these findings indicate that polyP may be metabolized in these photosynthetic prokaryotes to yield glucose (or mannose) 6-phosphate. They also provide evidence for a novel group-specific subfamily of strictly polyP-dependent gluco(manno)kinases with ancestral features and high biotechnological potential, capable of efficiently using polyP as an alternative and cheap source of energy-rich phosphate instead of costly ATP. Finally, these results could shed new light on the evolutionary origin of sugar kinases.

Description: Estuarine and tidal wetlands with high primary productivity and biological activity play a crucial role in coastal nutrient dynamics. Here, to better reveal the effects of extracellular enzymes and microbial community on carbon (C) and nitrogen (N) mineralization, the incubation experiments with different C and N addition patterns to the tidal sediments of the Yangtze Estuary (China) were conducted. The results suggested a significant increase in cumulative CO 2 effluxes in the C and CN treatment experiments, while no significant difference in cumulative CO 2 effluxes between the N treatment and control (CK) experiments was observed. In addition, the nutrient addition patterns had a great influence on dissolve organic C and N levels, but a small effect on microbial biomass C and N. Microbial community composition and microbial activity were found to be positively correlated with organic C (OC) and the molar ratio of C to N (C/N). Partial correlation analysis, controlling for C/N, supported direct effects of OC on the activity of carbon-cycling extracellular enzymes (cellulase and polyphenol oxidase), while C/N exhibited negatively correlations with urease and Gram-positive bacteria to Gram-negative bacteria (G+/G−). Strong relationships were found between CO 2 efflux and mineral nitrogen with the activity of specific enzymes (sucrase, cellulase, and polyphenol oxidase) and abundances of Gram-negative bacteria, arbuscular mycorrhizal fungi, and fungi, suggesting the significant influences of microbial community and enzyme activity on C and N mineralization in the estuarine and tidal wetlands. Furthermore, this study could highlight the need to explore effects of nutrient supply on microbial communities and enzyme activity changes associated with the C and N mineralization in these wetlands induced by the climate change.

Description: In this study, the process of pyrite colonization and leaching by three iron-oxidizing Acidithiobacillus species was investigated by fluorescence microscopy, bacterial attachment, and leaching assays. Within the first 4–5 days, only the biofilm subpopulation was responsible for pyrite dissolution. Pyrite-grown cells, in contrast to iron-grown cells, were able to oxidize iron(II) ions or pyrite after 24 h iron starvation and incubation with 1 mM H 2 O 2 , indicating that these cells were adapted to the presence of enhanced levels of reactive oxygen species (ROS), which are generated on metal sulfide surfaces. Acidithiobacillus ferrivorans SS3 and Acidithiobacillus ferrooxidans R1 showed enhanced pyrite colonization and biofilm formation compared to A. ferrooxidans T . A broad range of factors influencing the biofilm formation on pyrite were also identified, some of them were strain-specific. Cultivation at non-optimum growth temperatures or increased ionic strength led to a decreased colonization of pyrite. The presence of iron(III) ions increased pyrite colonization, especially when pyrite-grown cells were used, while the addition of 20 mM copper(II) ions resulted in reduced biofilm formation on pyrite. This observation correlated with a different extracellular polymeric substance (EPS) composition of copper-exposed cells. Interestingly, the addition of 1 mM sodium glucuronate in combination with iron(III) ions led to a 5-fold and 7-fold increased cell attachment after 1 and 8 days of incubation, respectively, in A. ferrooxidans T . In addition, sodium glucuronate addition enhanced pyrite dissolution by 25 %.

Description: Background Monoacetylated xylosyl residues of the main hardwood hemicellulose acetylglucuronoxylan undergo acetyl group migration between positions 2 and 3, and predominantly to position 4 of the non-reducing end xylopyranosyl (NRE-Xyl p ) residues which are amplified by saccharifying enzymes. On monoacetylated non-reducing end xylopyranosyl (NRE-Xyl p ) residues of xylooligosaccharides the acetyl group migrates predominantly to position 4 and hinders their hydrolysis by β-xylosidase. Methods Acetyl migration on the NRE-Xyl p residues and their enzymatic deacetylation by various xylan deacetylases was followed by 1 H-NMR spectroscopy and TLC. Results A 5-min heat treatment of 4-nitrophenyl 3- O -acetyl-β-D-xylopyranoside was sufficient to establish equilibrium between monoacetate derivatives acetylated at positions 2, 3 and 4. Rapid acetyl migration along the NRE-Xyl p ring at elevated temperature was confirmed in derivatives of methyl β-1,4-xylotrioside (Xyl 3 Me) monoacetylated solely on the NRE-Xyl p residue, the analogues of naturally occurring acetylated xylooligosaccharides. The Xyl 3 Me monoacetates were used as substrates to study regioselectivity of the NRE-Xyl p residue deacetylation by various acetylxylan esterases (AcXEs) of distinct carbohydrate esterase (CE) families. CE1, CE4 and CE6 AcXEs hydrolyzed considerably faster the 2″- O -acetyl derivative than the 3″- O -acetyl derivative. In contrast, the CE16 acetyl esterase preferred to attack the ester bond at position 3 followed by position 4. Conclusions Redistribution of acetyl group on the NRE-Xyl p residues is extremely rapid at elevated temperature and includes the formation of 4-acetate. Regioselectivity of AcXEs and CE16 acetyl esterase on these monoacetates is complementary. General significance The formation of all isomers of acetylated xylosyl residues must be taken into account after a long-term incubation of acetylxylan and acetylated xylooligosaccharides solutions or upon their treatment at elevated temperatures. This phenomenon emphasizes requirement of both types of xylan deacetylases to enable a rapid saccharification of xylooligosaccharides by glycoside hydrolases.

Description: Foot-and-mouth disease (FMD) remains a major threat to livestock worldwide, especially in developing countries. To improve the efficacy of vaccination against FMD, various types of vaccines have been developed, including synthetic peptide vaccines. We designed three synthetic peptide vaccines, 59 to 87 aa in size, based on immunogenic epitopes in the VP1, 3A, and 3D proteins of the A/HuBWH/CHA/2009 strain of the foot-and-mouth disease virus (FMDV), corresponding to amino acid positions 129 to 169 of VP1, 21 to 35 of 3A, and 346 to 370 of 3D. The efficacies of the vaccines were evaluated in cattle and guinea pigs challenged with serotype-A FMDV. All of the vaccines elicited the production of virus-neutralizing antibodies. The PB peptide, which contained sequences corresponding to positions 129 to 169 of V P1 and 346 to 370 of 3D, demonstrated the highest levels of immunogenicity and immunoprotection against FMDV. Two doses of 50 μg of the synthetic PB peptide vaccine provided 100 % protection against FMDV infection in guinea pigs, and a single dose of 100 μg provided 60 % protection in cattle. These findings provide empirical data for facilitating the development of synthetic peptide vaccines against FMD.

Description: Corynebacterium glutamicum can consume glucose to excrete glycerol under oxygen deprivation. Although glycerol synthesis from 1,3-dihydroxyacetone (DHA) has been speculated, no direct evidence has yet been provided in C. glutamicum . Enzymatic and genetic investigations here indicate that the glycerol is largely produced from DHA and, unexpectedly, the reaction is catalyzed by ( S , S )-butanediol dehydrogenase (ButA) that inherently catalyzes the interconversion between S -acetoin and ( S , S )-2,3-butanediol. Consequently, the following pathway for glycerol biosynthesis in the bacterium emerges: dihydroxyacetone phosphate is dephosphorylated by HdpA to DHA, which is subsequently reduced to glycerol by ButA. This study emphasizes the importance of promiscuous activity of the enzyme in vivo.

Description: Cofactor is especially important for biotransformation catalyzed by oxidoreductases. Many attempts in enhancing performance of the reactions by improving cofactor utilization have been reported. In this study, efficiency of cofactor-requiring biocatalysis was enhanced by improving cofactor recycling via spatially programmed assembling glycerol dehydrogenase (GlyDH, Escherichia coli MG1655) and glutamate dehydrogenase (GluDH, Bacillus subtilis str168), with the aid of single-stranded DNA (ssDNA). The two enzymes were first independently expressed as molecules fused with a phage protein A* that could covalently link ssDNA with certain features. After an enzymatic cross-linking reaction taking place under mild conditions, the conjugate of fused enzyme and ssDNA was assembled into desired structures through base pairing enabled by the ssDNA. Results showed that, to some extent, the fusion with protein A* could improve the specific activity of the enzymes (GlyDH-A*/GlyDH = 116.0 %; GluDH-A*/GluDH = 105.2 %). Additionally, in the coupled reaction system with glycerol and α-ketoglutaric acid as substrates, regarding the production of glutamic acid based on HPLC analysis, the efficiency of cofactor utilization was significantly enhanced (by 23.8- to 41.9-folds), indicating the existence of a substrate-channeling mechanism for cofactor utilization in the assembled reaction system due to the proximity effects. The degree of substrate channeling was calculated as from 1.65 to 1.73. Furthermore, the efficiency of cofactor utilization was influenced in an architecture-dependent manner when complexes with different stoichiometry of GlyDH and GluDH were utilized in biotransformation. This study demonstrated a novel strategy of cofactor recycling for enhanced performance of coupled oxidoreductive reactions.

Description: New 3-(hydroxyphenylphosphinyl)-propanoic acid (3-HPP) esters of cellulose were synthesized in N, N-dimethylacetamide/LiCl homogeneously by the method of in situ activation with p-toluenesulfonyl chloride. Chemical structure and thermal properties of the cellulose esters were investigated by FTIR, 13 C-NMR, TGA, RT-IR and Py–GC/MS, and their flame retardancy was studied by limiting oxygen index (LOI) test and vertical flammability test. It was found that the degree of substitution (DS) of cellulose esters, in the range from 0.62 to 1.42, had an obvious effect on solubility of cellulose esters. According to the FT-IR and Py–GC/MS results, flame retardant 3-HPP reacting with cellulose could accelerate dehydration action and decrease flammable released products. Besides, ESEM observation also confirmed that flame retardant cellulose (FRC) fibers with 3 wt% cellulose acetate prepared by dry-wet spinning technique possessed good flame resistance.

Description: In this study cotton fabric was coated with 5,5-dimethylhydantoin (DMH), followed by chlorination with sodium hypochlorite to impart antimicrobial properties and functions. An orthogonal array testing strategy was employed for obtaining the optimum treatment condition. After coating and chlorination, cotton fabrics were characterized with different methods. Ultraviolet spectroscopy, Scanning Electron Microscope and Fourier Transform Infrared Spectroscopy were used to observe the properties of cotton fabric after finishing, such as concentration of chlorine on cotton fabric, morphological properties of the surface of cotton fabric and functional groups on the cotton fabric. The results showed that cotton fabric coated with DMH followed with chlorination has antimicrobial properties that are able to resist S. aureus and this property is rechargeable.

Description: Stem cells (SCs) are known as undifferentiated cells with self-renewal and differentiation capacities. Regeneration is a phenomenon that occurs in a limited number of animals after injury, during which blastema tissue is formed. It has been hypothesized that upon injury, the dedifferentiation of surrounding tissues leads into the appearance of cells with SC characteristics. In present study, stem-like cells (SLCs) were obtained from regenerating tissue of New Zealand white rabbit’s pinna and their stemness properties were examined by their capacity to differentiate toward insulin producing cells (IPCs), as well as neural and osteogenic lineages. Differentiation was induced by culture of SLCs in defined medium, and cell fates were monitored by specific staining, RT-PCR and flow cytometry assays. Our results revealed that dithizone positive cells, which represent IPCs, and islet-like structures appeared 1 week after induction of SLCs, and this observation was confirmed by the elevated expression of Ins , Pax6 and Glut4 at mRNA level. Furthermore, SLCs were able to express neural markers as early as 1 week after retinoic acid treatment. Finally, SLCs were able to differentiate into osteogenic lineage, as confirmed by Alizarin Red S staining and RT-PCR studies. In conclusion, SLCs, which could successfully differentiate into cells derived from all three germ layers, can be considered as a valuable model to study developmental biology and regenerative medicine.

Description: Advancements in cell cultures are occurring at a rapid pace, an important direction is culturing cells in 3D conditions. We demonstrate the usefulness of agarose hydrogels in obtaining 3 dimensional aggregates of three cell lines, A549, MCF-7 and Sp2/0. The differences in culture phases, susceptibility to cisplatin-induced cytotoxicity are studied. Also, the 3D aggregates of the three cell lines were reverted into 2D cultures and the protein profile differences among the 2D, 3D and revert cultures were studied. The analysis of protein profile differences using UniProt data base further augment the usefulness of agarose hydrogels for obtaining 3D cell cultures.

Description: The catalytic and non-catalytic pyrolysis of microcrystalline cellulose and phosphoric acid pre-treated cellulose was investigated. The thermal processes were carried out applying two different methodologies: conventional fast pyrolysis and microwave-induced pyrolysis. For the catalytic experiments different catalysts were evaluated: CeO 2 , Nb 2 O 5 , SiO 2 , high surface area SiO 2 , Si-MCM-48 and Al-Fe-MCM-48. In all cases the liquid fraction was evaluated by quantifying the yields of anhydrosugars (mainly levoglucosan, levoglucosenone and 1,4:3,6-dianhydro-α- d -glucopyranose) and aromatic hydrocarbons. In the reaction of microcrystalline cellulose levoglucosan was the main product, while levoglucosenone was predominant in the pyrolysis of phosphoric acid pre-treated cellulose. Catalysts improved the fraction of bio-oil and the product distribution depended on the nature of catalytic materials as well as the starting cellulose. On the other hand, the microwave induced pyrolysis favored the formation of char at expenses of liquid fraction. In this case levoglucosenone and other anhydrosugars in conjunction with furan compounds were the main products.

Description: CuCl supported on molecular sieves has attracted increasing attention in catalyzing oxidative carbonylation of ethanol to diethyl carbonate. Mesoporous MCM-41 has been widely used as catalyst support due to its large surface area and well defined mesoporous structure. Considering its intrinsic weak acidity, MCM-41 was modified by a simple impregnation method to incorporate Al. The incorporation of Al components resulted in the high dispersion of Cu species and the increase of acid sites without changing the mesoporous structure of MCM-41, and thus enhanceed the catalytic activity of CuCl/MCM-41 for diethyl carbonate synthesis.

Description: In recent years, “orientationless technology” based on a fundamentally new approach to directional wellbore drilling, which consists in proper selection of the drill string bottom-hole assembly, is gaining popularity in oil and gas fields of Kazakhstan. The proposed approach rests on the use of the key parameter, namely, the stabilizing length of the bottom part of the drill string, which determines the direction of the wellbore deviation.

Description: It is established that carbonate dissolution kinetics have diffusion and kinetic control, i.e., chemical reaction rate determines the transfer of calcium and carbonate ions into solution and diffusion processes determine the removal of ions into the depth of solution. Kinetics of calcite and siderite dissolution in hydrochloric acid solution in relation to medium pH are studied. Orders of dissolution reaction rate with respect to hydrogen ions n (H + ) = 0.6±0.1 are calculated.

Description: Carbon-carbon composite materials are tested in order to evaluate corrosion resistance in a hydrogen sulfide atmosphere and under conditions of primary oil refining in order to determine the possibility of their use under petrochemical production conditions.

Description: Effective and inexpensive sorbing materials can be manufactured from secondary (recycled) polymers, which will help solve problems of water cleaning and waste recycling. A technology of sorbing material manufacture from polyethylene terephthalate wastes for waste and surface water cleaning is described, and the physicochemical properties of the sorbent and the efficiency of water cleaning from oil products are studied.

Description: Herein, the microwave-assisted grafting method was employed for the development of a novel pH-responsive graft copolymer derived from polyacrylamide-modified hydroxypropyl methyl cellulose [g-HPMC (M)]. The synthesised copolymer has been used for in vitro sustained release of ornidazole. Various characterizations confirm the formation of graft copolymer. Swelling studies indicate the pH-dependent swelling behaviour, while deswelling studies suggest that g-HPMC (M) shows faster deswelling in response to change in pH and/temperature. The cell viability study signifies that g-HPMC (M) is cytocompatible. The in-vitro release study demonstrates that g-HPMC (M) delivers ornidazole specifically in the colon pH, without release of the drug in the acidic environment, ensuring g-HPMC (M) as an ideal candidate for orally administered colonic drug carriers. The kinetics and mechanism of drug release suggest that it follows a non-Fickian release mechanism.

Description: The effect of the fibrillation process through a twin-screw extruder (TSE) on properties of pulp fibers was studied, considering the degree of both fibrillation and degradation of the fibers. Never-dried refined bleached kraft pulp (NBKP) was passed through a TSE several times at a high concentration of 28 wt%. The output of fibrillated fibers had a solid content up to ca. 50 wt%, and the material was in powder form. Characterizations of the morphology, dewatering speed, sedimentation, laser light scattering, scanning electron microscopy of cellulose suspensions, and light transmittance of resin-impregnated films showed that the fibrillation degree of the pulp was enhanced with a higher number of passes. However, the results from thermogravimetry, intrinsic viscosity, and X-ray diffraction analyses indicated that some degradation occurred during the fibrillation process in the TSE. In addition, the mechanical properties of the fibrillated pulp sheets reflected the effects of treatment on the fibrillation and degradation of the cellulose. For never-dried refined NBKP pulp, the best compromise in terms of fibrillation and degradation degree is between 3 and 14 passes, depending on the envisaged properties and applications. The possibility of nanocellulose production at the reported high solid contents is of great interest for industry.

Description: Recent ultrahigh-density tiling array and large-scale transcriptome analysis have revealed that large numbers of long non-coding RNAs (lncRNAs) are transcribed in mammals. Several lncRNAs have been implicated in transcriptional regulation, organization of nuclear structure, and post-transcriptional processing. However, the regulation of expression of lncRNAs is less well understood. Here, we show that the exogenous and endogenous expression of an oncogenic form of small GTPase Ras (called oncogenic Ras) decrease the expression of lncRNA ANRIL (antisense non-coding RNA in the INK4 locus), which is involved in the regulation of cellular senescence. We also show that forced expression of oncogenic Ras increases the expression of lncRNA PANDA (p21 associated ncRNA DNA damage activated), which is involved in the regulation of apoptosis. Microarray analysis demonstrated that expression of multiple lncRNAs fluctuated by forced expression of oncogenic Ras. These findings indicate that oncogenic Ras regulates the expression of a large number of lncRNAs including functional lncRNAs, such as ANRIL and PANDA .

Description: The efficiency of two cell types, namely adult fibroblasts, and amniotic fluid stem (AFS) cells as nuclear donor cells for somatic cell nuclear transfer by hand-made cloning in buffalo ( Bubalus bubalis ) was compared. The in vitro expanded buffalo adult fibroblast cells showed a typical “S” shape growth curve with a doubling time of 40.8 h and stained positive for vimentin. The in vitro cultured undifferentiated AFS cells showed a doubling time of 33.2 h and stained positive for alkaline phosphatase, these cells were also found positive for undifferentiated embryonic stem cell markers like OCT-4, NANOG and SOX-2, which accentuate their pluripotent property. Further, when AFS cells were exposed to corresponding induction conditions, these cells differentiated into osteogenic, adipogenic and chondrogenic lineages which was confirmed through alizaran, oil red O and alcian blue staining, respectively. Cultured adult fibroblasts and AFS cells of passages 10–15 and 8–12, respectively, were used as nuclear donors. A total of 94 embryos were reconstructed using adult fibroblast as donor cells with cleavage and blastocyst production rate of 62.8 ± 1.8 and 19.1 ± 1.5, respectively. An overall cleavage and blastocyst formation rate of 71.1 ± 1.2 and 29.9 ± 2.2 was obtained when 97 embryos were reconstructed using AFS cells as donor cells. There were no significant differences ( P  〉 0.05) in reconstructed efficiency between the cloned embryos derived from two donor cells, whereas the results showed that there were significant differences ( P  〈 0.05) in cleavage and blastocyst rates between the cloned embryos derived from two donor cell groups. Average total cell numbers for blastocyst generated using AFS cells (172.4 ± 5.8) was significantly ( P  〈 0.05) higher than from adult fibroblasts (148.2 ± 6.1). This study suggests that the in vitro developmental potential of the cloned embryos derived from AFS cells were higher than that of the cloned embryos derived from adult fibroblasts in buffalo hand-made cloning.

Description: The use of food additives has increased enormously in modern food technology but they have adverse effects in human healthy. The aim of this study was to investigate the DNA damage of some food additives such as citric acid (CA), benzoic acid (BA), brilliant blue (BB) and sunset yellow (SY) which were investigated in human male germ cells using comet assay. The sperm cells were incubated with different concentrations of these food additives (50, 100, 200 and 500 μg/mL) for 1 h at 32 °C. The results showed for CA, BA, BB and SY a dose dependent increase in tail DNA%, tail length and tail moment in human sperm when compared to control group. When control values were compared in the studied parameters in the treatment concentrations, SY was found to exhibit the highest level of DNA damage followed by BB 〉 BA 〉 CA. However, none of the food additives affected the tail DNA%, tail length and tail moment at 50 and 100 μg/mL. At 200 μg/mL of SY, the tail DNA% and tail length of sperm were 95.80 ± 0.28 and 42.56  ±  4.66, for BB the values were 95.06 ± 2.30 and 39.56 ± 3.78, whereas for BA the values were 89.05 ± 2.78 and 31.50 ± 0.71, for CA the values were 88.59 ± 6.45 and 13.59 ± 2.74, respectively. However, only the highest concentration of the used food additives significantly affected the studied parameters of sperm DNA. The present results indicate that SY and BB are more harmful than BA and CA to human sperm in vitro.

Description: In the present study, the use of Rhodococcus erythropolis mutant strain RG9 expressing the cytochrome P450 BM3 mutant M02 enzyme has been evaluated for whole-cell biotransformation of a 17-ketosteroid, norandrostenedione, as a model substrate. Purified P450 BM3 mutant M02 enzyme hydroxylated the steroid with 〉95 % regioselectivity to form 16-β-OH norandrostenedione, as confirmed by NMR analysis. Whole cells of R. erythropolis RG9 expressing P450 BM3 M02 enzyme also converted norandrostenedione into the 16-β-hydroxylated product, resulting in the formation of about 0.35 g/L. Whole cells of strain RG9 itself did not convert norandrostenedione, indicating that metabolite formation is P450 BM3 M02 enzyme mediated. This study shows that R. erythropolis is a novel and interesting host for the heterologous expression of highly selective and active P450 BM3 M02 enzyme variants able to perform steroid bioconversions.

Description: 2,4-Dichlorophenol (2,4-DCP) is considered as an important pollutant because of its high toxicity and wide distribution in wastewaters. Innocuous remediation technologies have been studied for the removal of this pollutant. This study investigated the feasibility of using garlic roots as a plant system for the removal of 2,4-DCP. The optimal conditions for its removal were established based on orthogonal experiments (OA 25 matrix). Significant factors that affect removal efficiency, arranged from high to low importance, include pH, reaction time, 2,4-DCP concentration, and H 2 O 2 concentration. In addition, garlic roots could be re-used for as much as three consecutive cycles. The decrease in pH and the increase of Cl − ion content in the post-removal solutions indicated that 2,4-DCP dehalogenation occurred during transformation. Changes in the deposition pattern of lignin in roots exposed to 2,4-DCP suggested that several of the products deposited were lignin-type polymers. The acute toxicity test revealed that the post-removal solutions were less toxic than the parent solutions. Therefore, garlic roots have considerable potential to effectively and safely remove 2,4-DCP from wastewater.

Description: Earlier findings on the nutritional benefits of diacylglycerols (DAGs) have attracted much attention on the synthesis of DAGs. In this study, we reported an improved method for the lipase-catalyzed synthesis of 1,3-diolein by the irreversible glycerolysis of vinyl oleate with glycerol. The effects of reaction system, lipase loading, molar ratio of vinyl oleate to glycerol, reaction temperature and time on 1,3-diolein content in crude reaction mixture were investigated. When the reaction was conducted in a solvent-free system at 30 °C for 8 h by reacting 2 mmol vinyl oleate with 1 mmol glycerol with 8 % (w/w, relative to total reactants) Lipozyme RM IM (Novozymes, Beijing, China) as catalyst, there were 90.5 ± 2.9 % (area/area) 1,3-diolein and (3.3 ± 0.3) % 1,2-diolein produced. After purification, 1,3-diolein was obtained at 81.4 % yield with 98.2 % purity. The lipase-catalyzed synthesis of 1,3-diolein using vinyl oleate as acyl donor by glycerolysis was also conducted using a medium with 50 mmol of glycerol and 100 mmol vinyl oleate. Compared to enzymatic esterification in a solvent, enzymatic glycerolysis for the synthesis 1,3-diolein is more effective due to the irreversible reaction, mild due to the low reaction temperature, and environmentally benign due to the use of solvent-free reaction system.

Description: Yro2 and its paralogous protein Mrh1 of Saccharomyces cerevisiae have seven predicted transmembrane domains and predominantly localize to the plasma membrane. Their physiological functions and regulation of gene expression have not yet been elucidated in detail. We herein demonstrated that MRH1 was constitutively expressed, whereas the expression of YRO2 was induced by acetic acid stress and entering the stationary phase. Fluorescence microscopic analysis revealed that Mrh1 and Yro2 were distributed as small foci in the plasma membrane under acetic acid stress conditions. The null mutants of these genes ( mrh1 ∆, yro2 ∆, and mrh1 ∆ yro2 ∆) showed delayed growth and a decrease in the productivity of ethanol in the presence of acetic acid, indicating that Yro2 and Mrh1 are involved in tolerance to acetic acid stress.

Description: The use of the food-grade bacterium Lactococcus lactis as a vehicle for the oral delivery of DNA vaccine plasmids constitutes a promising strategy for vaccination. The delivery of DNA plasmids into eukaryotic cells is of critical importance for subsequent DNA expression and effectiveness of the vaccine. In this context, the use of the recombinant invasive L. lactis FnBPA+ (fibronectin-binding protein A) strain for the oral delivery of the eukaryotic expression vector vaccination using lactic acid bacteria (pValac), coding for the 6-kDa early secreted antigenic target (ESAT-6) gene of Mycobacterium tuberculosis , could represent a new DNA vaccine strategy against tuberculosis. To this end, the ESAT-6 sequence was cloned into the pValac vector; the L. lactis fibronectin-binding protein A (FnBPA)+ (pValac: ESAT -6) strain was obtained, and its immunological profile was checked in BALB/c mice. This strain was able to significantly increase interferon gamma (IFN-γ) production in spleen cells, showing a systemic T helper 1 (Th1) cell response. The mice also showed a significant increase in specific secretory immunoglobulin A (sIgA) production in colon tissue and fecal extracts. Thus, this is the first time that L. lactis has been used to deliver a plasmid DNA harboring a gene that encodes an antigen against tuberculosis through mucous membranes.

Description: Both environmental agents and spontaneous cellular events cause serious DNA damage, threatening the integrity of the genome. In response to replication stress or genotoxic agents triggered DNA damage, degradation of p12 subunit of DNA polymerase delta (Pol δ) results in an inter-conversion between heterotetramer (Pol δ4) and heterotrimer (Pol δ3) forms and plays a significant role in DNA damage response in eukaryotic cells. In this work, we used mass spectrometry-based proteomic approach to identify those cellular stress response protein changes corresponding to the degradation of p12 in DNA-damaged HeLa cells by the treatment with hydroxyurea (HU). A total of 736 ± 13 proteins in non-treated control group and 741 ± 19 protein spots in HU-treated cells were detected, of which 34 proteins (17 up-regulated and 17 down-regulated) exhibited significantly altered protein expression levels. Their physiological roles are mainly associated with cellular components, molecular functions, and biological processes by gene ontology analysis, among which 21 proteins were mapped to KEGG pathways. They are involved in 5 primary pathways with the subsets involving 16 secondary pathways by further KEGG analysis. More interestingly, the up-regulation of translationally controlled tumor protein was further identified to be associated with p12 degradation by Western blot analysis. Our works may enlarge and broaden our view for deeply understanding how global cellular stress responds to DNA damage, which could contribute to the etiology of human cancer or other diseases that can result from loss of genomic stability.

Description: A series of unsupported MoS 2 catalysts with or without Al 2 O 3 modification was prepared using a modified thermal decomposition approach. The catalysts were tested for the methanation of carbon monoxide and the optimum one has 25.6 wt-% Al 2 O 3 content. The catalysts were characterized by nitrogen adsorption measurement, X-ray diffraction and transmission electron microscopy. The results show that adding appropriate amount of Al 2 O 3 increases the dispersion of MoS 2 , and the increased interaction force between MoS 2 and Al 2 O 3 can inhibit the sintering of active MoS 2 to some extent.

Description: Canolol-enriched extracts obtained from the extraction of fluidized bed treated canola meal with supercritical carbon dioxide were added to high-oleic canola oil in different concentrations (200, 500 and 750 mg/kg). After 30 h of deep-fat frying, oils fortified with canolol-enriched extracts showed a two to three times better frying performance in comparison to the commonly used antioxidants (TBHQ, 200 mg/kg; rosemary extract, 40 and 200 mg/kg) and a control without antioxidants with regards to the formation of di- and polymer triacylglycerols, total polar compounds, secondary degradation products (anisidine value) and the iodine value. The canolol-enriched extracts were also able to slow down the degradation of α- and γ-tocopherol during frying resulting in significant amounts of tocopherols after 30 h of frying in comparison to the other oils. The influence of the canolol-enriched extracts indicated strongly concentration-dependent performance. With increasing concentration of the extract, the thermal stability of the fortified oil was improved. The only disadvantage of the addition of the extracts was an increase in the initial acid value, but within the frying time, only oil fortified with 750 mg canolol-enriched extract/kg reached the limit given in different countries.

Description: Diacylglycerol acyltransferase (DGAT) catalyzes the acyl-CoA-dependent acylation of sn -1,2-diacylglycerol to produce triacylglycerol (TAG). This enzyme, which is critical to numerous facets of oilseed development, has been highlighted as a genetic engineering target to increase storage lipid production in microorganisms designed for biofuel applications. Here, four transcriptionally active DGAT1 genes were identified and characterized from the oil crop Brassica napus . Overexpression of each BnaDGAT1 in Saccharomyces cerevisiae increased TAG biosynthesis. Further studies showed that adding an N-terminal tag could mask the deleterious influence of the DGATs’ native N-terminal sequences, resulting in increased in vivo accumulation of the polypeptides and an increase of up to about 150-fold in in vitro enzyme activity. The levels of TAG and total lipid fatty acids in S. cerevisiae producing the N-terminally tagged BnaDGAT1.b at 72 h were 53 and 28 % higher than those in cultures producing untagged BnaA.DGAT1.b, respectively. These modified DGATs catalyzed the synthesis of up to 453 mg fatty acid/L by this time point. The results will be of benefit in the biochemical analysis of recombinant DGAT1 produced through heterologous expression in yeast and offer a new approach to increase storage lipid content in yeast for industrial applications.

Description: Naphtho-γ-pyrones (NGPs) are secondary metabolites mainly produced by filamentous fungi ( Fusarium sp., Aspergillus sp.) that should be considered by industrials. Indeed, these natural biomolecules show various biological activities: anti-oxidant, anti-microbial, anti-cancer, anti-HIV, anti-hyperuricuric, anti-tubercular, or mammalian triacylglycerol synthesis inhibition which could be useful for pharmaceutical, cosmetic, and/or food industries. In this review, we draw an overview on the interest in studying fungal NGPs by presenting their biological activities and their potential values for industrials, their biochemical properties, and what is currently known on their biosynthetic pathway. Finally, we will present what remains to be discovered about NGPs.

Description: This article presents results of an experimental study of the drying kinetics of silica gel in a microwave electromagnetic field. The influence exerted by various process parameters on the drying kinetics under a microwave energy supply is ascertained on the basis of the experimental data. The substantial impact of the initial moisture content of the silica gel and the power supplied on the time required for moisture desorption is demonstrated. Use of a proposed energy criterion has made it possible to generalize experimental results obtained for different regime parameters.

Description: Anode-mechanical treatment of surfaces with dielectric inclusions is described. Basic theoretical laws of anode-mechanical treatment of metallic surfaces are given.

Description: Mesenchymal stem cells (MSCs) offer promise as therapeutic aid in the repair of tendon and ligament injuries in race horses. Fetal adnexa is considered as an ideal source of MSCs due to many advantages, including non-invasive nature of isolation procedures and availability of large tissue mass for harvesting the cells. However, MSCs isolated from equine fetal adnexa have not been fully characterized due to lack of species-specific markers. Therefore, this study was carried out to isolate MSCs from equine umbilical cord blood (UCB) and characterize them using cross-reactive markers. The plastic-adherent cells could be isolated from 13 out of 20 (65 %) UCB samples. The UCB derived cells proliferated till passage 20 with average cell doubling time of 46.40 ± 2.86 h. These cells expressed mesenchymal surface markers but did not express haematopoietic/leucocytic markers by RT-PCR and immunocytochemistry. The phenotypic expression of CD29, CD44, CD73 and CD90 was shown by 96.36 ± 1.28, 93.40 ± 0.70, 73.23 ± 1.29 and 46.75 ± 3.95 % cells, respectively in flow cytometry, whereas, reactivity against the haematopoietic antigens CD34 and CD45 was observed only in 2.4 ± 0.20 and 0.1 ± 0.0 % of cells, respectively. Osteogenic and chondrogenic differentiation could be achieved using established methods, whereas the optimum adipogenic differentiation was achieved after supplementing media with 15 % rabbit serum and 20 ng/ml of recombinant human insulin. In this study, we optimized methodology for isolation, cultural characterization, differentiation and immunophenotyping of MSCs from equine UCB. Protocols and markers used in this study can be employed for unequivocal characterization of equine MSCs.

Description: Chronic inflammation is an important factor in colorectal carcinogenesis. However, evidence on the effect of pro-inflammatory and anti-inflammatory foods and nutrients is scarce. Moreover, there are few studies focusing on diet–gene interactions on inflammation and colorectal cancer (CRC). This study was designed to investigate the association between the novel dietary inflammatory index (DII) and CRC and its potential interaction with polymorphisms in inflammatory genes. Data from the Bellvitge Colorectal Cancer Study, a case–control study (424 cases with incident colorectal cancer and 401 hospital-based controls), were used. The DII score for each participant was obtained by multiplying intakes of dietary components from a validated dietary history questionnaire by literature-based dietary inflammatory weights that reflected the inflammatory potential of components. Data from four important single nucleotide polymorphisms located in genes thought to be important in inflammation-associated CRC: i.e., interleukin ( IL )- 4 , IL-6 , IL-8 , and peroxisome proliferator-activated receptor-γ ( PPARG ) were analyzed. A direct association was observed between DII score and CRC risk (OR Q4 vs. Q1 1.65, 95 % CI 1.05–2.60, and P trend 0.011). A stronger association was found with colon cancer risk (OR Q4 vs. Q1 2.24, 95 % CI 1.33–3.77, and P trend 0.002) than rectal cancer risk (OR Q4 vs. Q1 1.12, 95 % CI 0.61–2.06, and P trend 0.37). DII score was inversely correlated with SNP rs2243250 in IL-4 among controls, and an interaction was observed with CRC risk. Neither correlation nor interaction was detected for other inflammatory genes. Overall, high-DII diets are associated with increased risk of CRC, particularly for colon cancer, suggesting that dietary-mediated inflammation plays an important role in colorectal carcinogenesis.

Description: Cottonseed meal (CSM), a common agricultural by-product, was used as a nutrient source for the production of eicosapentaenoic acid (EPA) by Pythium irregulare . CSM can support good cell growth performance, as can yeast extract (YE). In terms of the maximum EPA content and EPA yield, CSM is superior to YE. Low concentrations of CSM are beneficial to lipid synthesis, and high concentrations favor the EPA content. Utilizing response surface methodology (RSM) analysis, the optimum contents of glucose and CSM in the fermentation medium were determined to be 40.2 and 16.1 g/l, respectively. After 6 days of fermentation at 25 °C and optimal conditions, the EPA yield and productivity were 245.3 and 40.9 mg/l day, respectively. Particle size of CSM was found to affect the EPA production, and a finely ground CSM (100 mesh) was determined to be best for EPA production. The variation in the fatty acid content of total fatty acid (TFA) indicates that EPA was synthesized through the n-6 route in P. irregulare and Δ12 desaturase was the key enzyme for EPA biosynthesis. Sodium carbonate was determined to be notably good at removing free gossypol attached to biomass. After fungal biomass from each flask had been harvested from Na 2 CO 3 -supplemented medium, 1 % (w/v) Na 2 CO 3 solution was used to wash the mycelia three times; free gossypol (FG) was not detected (detection limit 0.0018 %). This work provides a new approach using cottonseed meal to produce EPA through fungal fermentation.

Description: In Bacillus subtilis , natural genetic competence is subject to complex genetic regulation and quorum sensing dependent. Upon extracellular accumulation of the peptide-pheromone ComX, the membrane-bound sensor histidine kinase ComP initiates diverse signaling pathways by activating—among others—DegQ and ComS. While DegQ favors the expression of extracellular enzymes rather than competence development, ComS is crucial for competence development as it prevents proteolytic degradation of ComK, the key transcriptional activator of all genes required for the uptake and integration of DNA. In Bacillus licheniformis , ComX/ComP sensed cell density negatively influences competence development, suggesting differences from the quorum-sensing-dependent control mechanism in Bacillus subtilis . Here, we show that each of six investigated strains possesses both of two different, recently identified putative comS genes. When expressed from an inducible promoter, none of the comS candidate genes displayed an impact on competence development neither in B. subtilis nor in B. licheniformis . Moreover, disruption of the genes did not reduce transformation efficiency. While the putative comS homologs do not contribute to competence development, we provide evidence that the degQ gene as for B. subtilis negatively influences genetic competency in B. licheniformis .

Description: The adult heart contains a population of cardiac progenitor cells (CPCs). Growing and collecting an adequate number of CPCs demands complex culture media containing growth factors. Since activated macrophages secrete many growth factors, we investigated if activated isolated heart cells seeded on a feeder layer of activated peritoneal macrophages (PM) could result in CPCs and if these, in turn, could exert cardioprotection in rats with myocardial infarction (MI). Heart cells of inbred Wistar rats were isolated by collagenase digestion and cultured on PM obtained 72 h after intraperitoneal injection of 12 ml thioglycollate. Cells (1 × 10 6 ) exhibiting CPC phenotype (immunohistochemistry) were injected in the periphery of rat MI 10 min after coronary artery occlusion. Control rats received vehicle. Three weeks later, left ventricular (LV) function (echocardiogram) was assessed, animals were euthanized and the hearts removed for histological studies. Five to six days after seeding heart cells on PM, spherical clusters composed of small bright and spherical cells expressing mostly c-Kit and Sca-1 antigens were apparent. After explant, those clusters developed cobblestone-like monolayers that expressed smooth muscle actin and sarcomeric actin and were successfully transferred for more than ten passages. When injected in the MI periphery, many of them survived at 21 days after coronary ligature, improved LV ejection fraction and decreased scar size as compared with control rats. CPC-derived cells with cardiocyte and smooth muscle phenotypes can be successfully grown on a feeder layer of activated syngeneic PM. These cells decreased scar size and improved heart function in rats with MI.

Description: To better understand the quantitative relationships between messenger RNA (mRNA) and protein biomarkers relevant to bioremediation, we quantified and compared respiration-associated gene products in an anaerobic syntrophic community. Respiration biomarkers for Dehalococcoides , an organohalide reducer, and Methanospirillum , a hydrogenotrophic methanogen, were quantified via qRT-PCR for mRNA and multiple reaction monitoring (MRM) of proteotypic peptides for protein. mRNA transcripts of the Dehalococcoides reductive dehalogenases PceA, TceA, and DMC1545, and hydrogenase HupL, as well as the Methanospirillum oxidoreductases MvrD and FrcA were shown to be similarly regulated with respect to their temporal responses to substrate addition. However, MvrD was two orders of magnitude lower in mRNA abundance. Per cell, Dehalococcoides protein biomarkers quantified were more abundant than Methanospirillum proteins. Comparing mRNA with protein abundance, poor correlations were observed between mRNA transcript levels and the net protein produced. For example, Dehalococcoides HupL and TceA transcripts were similarly abundant though TceA was far more abundant at the protein level (167 ± 121 vs. 1095 ± 337 proteins per cell, respectively). In Methanospirillum , MvrD maintained comparable per-cell protein abundance to FrcA (42 ± 14 vs. 60 ± 1 proteins per cell, respectively) despite the significantly lower transcript levels. Though no variability in protein decay rates was observed, the mRNA translation rate quantified for TceA was greater than the other Dehalococcoides targets monitored. These data suggest that there is considerable variation in the relationship between mRNA abundance and protein production both across transcripts within an organism and across organisms. This highlights the importance of empirically based studies for interpreting biomarker levels in environmentally relevant organisms.

Description: Pyroligneous acid (PA) is a complex highly oxygenated aqueous liquid fraction obtained by the condensation of pyrolysis vapors, which result from the thermochemical breakdown or pyrolysis of plant biomass components such as cellulose, hemicellulose, and lignin. PA produced by the slow pyrolysis of plant biomass is a yellowish brown or dark brown liquid with acidic pH and usually comprises a complex mixture of guaiacols, catechols, syringols, phenols, vanillins, furans, pyrans, carboxaldehydes, hydroxyketones, sugars, alkyl aryl ethers, nitrogenated derivatives, alcohols, acetic acid, and other carboxylic acids. The phenolic components, namely guaiacol, alkyl guaiacols, syringol, and alkyl syringols, contribute to the smoky odor of PA. PA finds application in diverse areas, as antioxidant, antimicrobial, antiinflammatory, plant growth stimulator, coagulant for natural rubber, and termiticidal and pesticidal agent; is a source for valuable chemicals; and imparts a smoky flavor for food.

Description: Two-stage process based on photofermentation of dark fermentation effluents is widely recognized as the most effective method for biological production of hydrogen from organic substrates. Recently, it was described an alternative mechanism, named capnophilic lactic fermentation, for sugar fermentation by the hyperthermophilic bacterium Thermotoga neapolitana in CO 2 -rich atmosphere. Here, we report the first application of this novel process to two-stage biological production of hydrogen. The microbial system based on T. neapolitana DSM 4359 T and Rhodopseudomonas palustris 42OL gave 9.4 mol of hydrogen per mole of glucose consumed during the anaerobic process, which is the best production yield so far reported for conventional two-stage batch cultivations. The improvement of hydrogen yield correlates with the increase in lactic production during capnophilic lactic fermentation and takes also advantage of the introduction of original conditions for culturing both microorganisms in minimal media based on diluted sea water. The use of CO 2 during the first step of the combined process establishes a novel strategy for biohydrogen technology. Moreover, this study opens the way to cost reduction and use of salt-rich waste as feedstock.

Description: 1-Butyl-3-methylimidazolium chloride ([BMIM]Cl) was selected as co-solvent to dissolve cellulose and silk fibroin and the cellulose/silk fibroin blend fibers were fabricated with dry-jet wet spinning technology. The phase morphology of cellulose and silk fibroin in the blend fibers was studied by scanning electron microcopy and laser scanning confocal microscope. It is shown that the cellulose is in the continuous phase and silk fibroin exists as “fibril-like” in cellulose, in which the radial dimension of silk fibroin phase is 0.5–1.0 μm. The phase size of silk fibroin along the fiber axis increased with the increase of silk fibroin content and draw ratio. From the wide-angle X-ray scattering, it is found that the total crystallinity of the blend fibers decreased with increasing silk fibroin content. The hydrogen bond between cellulose and silk fibroin was observed from Fourier transform infrared spectra. Although the tensile strength and initial modulus of blend fibers decreased with increasing silk fibroin content, the tensile strength of blend fibers contain 35 wt% silk fibroin was up to 191 MPa.

Description: Mycoinsecticides application within Integral Pest Management requires high quantities of conidia, with the proper quality and resistance against environmental conditions. Metarhizium anisopliae var. lepidiotum conidia were produced in normal atmospheric conditions (21 % O 2 ) and different concentrations of oxygen pulses (16, 26, 30, and 40 %); conidia obtained under hypoxic conditions showed significantly lower viability, hydrophobicity, and virulence against Tenebrio molitor larvae or mealworm, compared with those obtained under normal atmospheric conditions. Higher concentrations of oxygen (26 and 30 %) improved conidial production. However, when a 30 % oxygen concentration was applied, maximal conidial yields were obtained at earlier times (132 h) relative to 26 % oxygen pulses (156 h); additionally, with 30 % oxygen pulses, conidia thermotolerance was improved, maintaining viability, hydrophobicity, and virulence. Although conidial production was not affected when 40 % oxygen pulses were applied, viability and virulence were diminished in those conidia. In order to find a critical time for mycelia competence to respond to these oxidant conditions, oxygen pulses were first applied either at 36, 48, 60, and 72 h. A critical time of 60 h was determined to be the best time for the M. anisopliae var. lepidiotum mycelia to respond to oxygen pulses in order to increase conidial production and also to maintain the quality features. Therefore, oxygen-enriched (30 %) pulses starting at 60 h are recommended for a high production without the impairment of quality of M. anisopliae var. lepidiotum conidia.

Description: Butenoic acid is a C 4 short-chain unsaturated fatty acid mainly used in the preparation of resins, pharmaceuticals, and fine chemicals. However, butenoic acid derived from petroleum is costly and unfriendly to the environment. Here, we report a novel biosynthetic strategy to produce butenoic acid by utilizing the intermediate of fatty acid biosynthesis pathway in engineered Escherichia coli . A thioesterase gene ( B. thetaiotaomicron thioesterase ( bTE )) from Bacteroides thetaiotaomicron was heterologously expressed in E. coli to specifically convert butenoyl-acyl carrier protein (ACP), a fatty acid biosynthesis intermediate, to butenoic acid. The titer of butenoic acid ranged from 0.07 to 11.4 mg/L in four different E. coli strains with varied expressing vectors. Deletion of endogenous fadD gene (encoding acyl-CoA synthetase) to block fatty acid oxidation improved the butenoic acid production in all strains to some extent. The highest butenoic acid accumulation of 18.7 mg/L was obtained in strain XP-2 (BL21-∆ fadD /pET28a- bTE ). Moreover, partially inhibiting the enoyl-ACP reductase (FabI) of strain XP-2 by triclosan increased butenoic acid production by threefold, and the butenoic acid titer was further increased to 161.4 mg/L by supplying glucose and tryptone in the M9 medium. Fed-batch fermentation of this strain further enhanced butenoic acid production to 4.0 g/L within 48 h. The butenoic acid tolerance assay revealed that this strain could tolerate 15–20 g/L of butenoic acid.

Description: Bacillus amyloliquefaciens strains FZBREP and FZBSPA were derived from the wild-type FZB42 by replacement of the native bacilysin operon promoter with constitutive promoters P repB and P spac from plasmids pMK3 and pLOSS, respectively. These strains contained two antibiotic resistance genes, and markerless strains were constructed by deleting the chloramphenicol resistance cassette and promoter region bordered by two lox sites ( lox 71 and lox 66) using Cre recombinase expressed from the temperature-sensitive vector pLOSS-cre. The vector-encoded spectinomycin resistance gene was removed by high temperature (50 °C) treatment. RT-PCR and qRT-PCR results indicated that P repB and especially P spac significantly increased expression of the bac operon, and FZBREP and FZBSPA strains produced up to 170.4 and 315.6 % more bacilysin than wild type, respectively. Bacilysin overproduction was accompanied by enhancement of the antagonistic activities against Staphylococcus aureus (an indicator of bacilysin) and Clavibacter michiganense subsp. sepedonicum (the causative agent of potato ring rot). Both the size and degree of ring rot-associated necrotic tubers were decreased compared with the wild-type strain, which confirmed the protective effects and biocontrol potential of these genetically engineered strains.

Description: Highly specific and fast multiplex detection methods are essential to conduct reasonable DNA-based diagnostics and are especially important to characterise infectious diseases. More than 1000 genetic targets such as antibiotic resistance genes, virulence factors and phylogenetic markers have to be identified as fast as possible to facilitate the correct treatment of a patient. In the present work, we developed a novel ligation-based DNA probe concept that was combined with the microarray technology and used it for the detection of bacterial pathogens. The novel linear chain (LNC) probes identified all tested species correctly within 1 h based on their 16S rRNA gene in a 25-multiplex reaction. Genomic DNA was used directly as template in the ligation reaction identifying as little as 10 7 cells without any pre-amplification. The high specificity was further demonstrated characterising a single nucleotide polymorphism leading to no false positive fluorescence signals of the untargeted single nucleotide polymorphism (SNP) variants. In comparison to conventional microarray probes, the sensitivity of the novel LNC3 probes was higher by a factor of 10 or more. In summary, we present a fast, simple, highly specific and sensitive multiplex detection method adaptable for a wide range of applications.

Description: Bacterial cellulose (BC) is used in different fields as a biological material due to its unique properties. Despite there being many BC applications, there still remain many problems associated with bioprocess technology, such as increasing productivity and decreasing production cost. New technologies that use waste from the food industry as raw materials for culture media promote economic advantages because they reduce environmental pollution and stimulate new research for science sustainability. For this reason, BC production requires optimized conditions to increase its application. The main objective of this study was to evaluate BC production by Gluconacetobacter xylinus using industry waste, namely, rotten fruits and milk whey, as culture media. Furthermore, the structure of BC produced at different conditions was also determined. The culture media employed in this study were composed of rotten fruit collected from the disposal of free markets, milk whey from a local industrial disposal, and their combination, and Hestrin and Schramm media was used as standard culture media. Although all culture media studied produced BC, the highest BC yield—60 mg/mL—was achieved with the rotten fruit culture. Thus, the results showed that rotten fruit can be used for BC production. This culture media can be considered as a profitable alternative to generate high-value products. In addition, it combines environmental concern with sustainable processes that can promote also the reduction of production cost.

Description: A biogas production plant operating with main and secondary digesters (MD, SD) was analysed for the diversity of bacteria from Clostridium cluster I and its pathogenic members. The plant was run in two parallel lines, both receiving silages, and one, in addition, cattle manure (CM). Quantitative PCR of 16S rRNA genes from directly extracted DNA indicated that cluster I represented 0.2 to 5.6 % of the total bacterial communities. Its prevalence was particularly low in CM and also in SD compared to MD, indicating its decline during fermentation. In contrast, another highly abundant clostridial group, i.e. the “faecal” cluster XIVa, remained quantitatively unaffected during fermentation. A total of 85.1 % of 581,934 rRNA gene sequences gathered by group-specific PCR from the silages, CM and digesters could be assigned to cluster I. All remaining sequences fell into other clostridial groups. The three most dominant operational taxonomic units (OTUs) introduced with CM were from cluster I, and they declined during fermentation. Fermentation with CM significantly increased OTUs of clostridia outside of cluster I but not within. The only OTUs related to pathogens were detected for Clostridium botulinum with 0.18 % of all cluster I sequences in maize silage and less than 0.01 % in the other substrates and digester materials. These OTUs could be assigned to all four established C. botulinum groups, thus, potentially covering all seven neurotoxins. Mouse lethality tests of samples with suspected presence of C. botulinum , however, indicated no toxigenic potential and, thus, no risk associated with the rare occurrence of these OTUs.

Description: The phenomenon of the cell density effect is not readily explained by an obvious nutrient limitation, and a recent study has suggested that for recombinant Autographa californica multiple nucleopolyhedrovirus (rAcMNPV)-infected Sf9 cells, a drop in messenger RNA (mRNA) levels may be sufficient to explain the cell density effect for this system. The current study aims to investigate the response in cell-specific yields (viral DNA (vDNA), LacZ mRNA and β-galactosidase (β-Gal) protein) with increasing infection cell density (ICD) for rAcMNPV-infected Hi5 cells, where the rAcMNPV expresses the β-Gal gene under control of the polyhedral promoter. Hi5 cells in suspension culture of Express Five® medium were synchronously infected with a rAcMNPV at multiple ICDs between 0.5 and 6 × 10 6 cells/mL and a multiplicity of infection of 10 plaque-forming units (PFU)/cell either in the original or fresh medium conditions. There were negative correlations between the three key virus infection indicators (vDNA, mRNA and β-Gal) and the peak cell density (PCD). However, unlike infected Sf9 cells, the yield decline started at the lowest PCD investigated (0.6 × 10 6 cells/mL). Generally, the yield decline with increasing PCD was most pronounced for β-Gal followed by mRNA and was more moderate for vDNA. The decline was significantly reduced but not totally arrested when fresh medium replacement was used. The results suggest that the reduction in recombinant protein-specific yields at high PCDs is associated with limitations during the up-stream processes of replication and transcription rather than entirely caused by limitations during translation. In addition, low production rates at late infection stages of moderate to high ICDs are a probable cause of the cell density effect.

Description: Steryl glucosides (SG) are common contaminants in biodiesel that form precipitates, which form and cause problems due to fouling during transport and storage. Therefore, their quantification is necessary to assess the quality of this fuel. The methods currently available for SG analysis require expensive instrumentation, need a previous concentration step by solid-phase extraction (SPE) or are of limited use for the quantitative assessment. We developed an enzymatic method for SG quantification in biodiesel samples based on the hydrolysis of the glucoside catalyzed by a broadly specific beta glucosidase and the subsequent determination of the glucose released by the reaction. The method is non-expensive, sensitive and was adapted to 96-well format fluorescence plate reader, making it useful for the parallel assay of multiple samples. The enzymatic assay presented here represent a valuable tool for both quality control and the development of improved biodiesel production and purification procedures.

Description: Di-hydroxylated soybean oil (DSO), a biobased polyol synthesized from epoxidized soybean oil (ESO) could be used to formulate resins for adhesives; however, current DSO synthesis requires harsh reaction conditions that significantly increase both cost and waste generation. In this paper, we investigate the kinetics of oxirane cleavage in ESO to DSO by water and elucidate the role of different process parameters in the reaction rate and optimization of reaction conditions. Our kinetic study showed that ESO oxirane cleavage was a first-order reaction and that the ESO oxirane cleavage rate was greatly influenced by tetrahydrofuran (THF)/ESO ratio, H 2 O/ESO ratio, catalyst content, and temperature. Optimized reaction parameters were THF/ESO of 0.5, H 2 O/ESO of 0.25, catalyst content of 1.5 %, and reaction time of 3 h at 25 °C. DSO with hydroxyl value of 242 mg KOH/g was obtained under these conditions. We also characterized the structure, thermal properties, adhesion performance, and viscoelasticity of UV-polymerized resins based on this DSO. The resin tape exhibited peel adhesion strength of 3.6 N/in., which is comparable to some commercial tapes measured under similar conditions.