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We describe herein a method of recharging used commercial spin columns or assembling homemade spin columns using filter paper as binding material for cost-effective, low throughput nucleic acid purification. The efficiency of filter paper-based spin columns was evaluated for purification of nucleic acids from various sources. Following protocols of commercial kits, we found filter paper to be a useful binding material for purification of nucleic acids, including plant genomic DNA, plant total RNA, PCR products, and DNA from agarose gels. However, filter paper has a weak binding affinity to plasmid DNA in tested miniprep protocols. Protocols for the use of filter paper recharged spin columns or homemade spin columns for low throughput purification of plant genomic DNA and total RNA with unused commercial kit buffers or less expensive homemade buffers are presented.

Amphimeriasis, a fish-borne zoonotic disease caused by the liver fluke Amphimerus spp., has recently been reported as an emerging disease affecting an indigenous Ameridian group, the Chachi, living in Ecuador. The only method for diagnosing amphimeriasis was the microscopic detection of eggs from the parasite in patients' stool samples with very low sensitivity. Our group developed an ELISA technique for detection of anti-Amphimerus IgG in human sera and a molecular method based on LAMP technology (named LAMPhimerus) for specific and sensitive parasite DNA detection. The LAMPhimerus method showed to be much more sensitive than classical parasitological methods for amphimeriasis diagnosis using human stool samples for analysis. The objective of this work is to demonstrate the feasibility of using dried stool samples on filter paper as source of DNA in combination with the effectiveness of our previously designed LAMPhimerus assay for successfully Amphimerus sp. detection in clinical stool samples. A total of 102 untreated and undiluted stool samples collected from Chachi population were spread as thin layer onto common filter paper for easily transportation to our laboratory and stored at room temperature for one year until DNA extraction. When LAMPhimerus method was applied for Amphimerus sp. DNA detection, a higher number of positive results was detected (61/102; 59.80%) in comparison to parasitological methods (38/102; 37.25%), including 28/61 (45.90%) microscopy-confirmed Amphimerus sp. infections. The diagnostic parameters for the sensitivity and specificity werecalculated for our LAMPhimerus assay, which were 79.17% and 65.98%, respectively. We demonstrate, for the first time, that common filter paper is useful for easy collection and long-term storage of human stool samples for later DNA extraction and molecular analysis of human-parasitic trematode eggs. This simple, economic and easily handling method combined with the specific and sensible LAMPhimerus assay has the potential to beused as an effective molecular large-scale screening test for amphimeriasis-endemic areas.

Humans introduced paper mulberry (Broussonetia papyrifera) from Taiwan into the Pacific over 5000 years ago as a fiber source to make barkcloth textiles that were, and still are, important cultural artifacts throughout the Pacific. We have used B. papyrifera, a species closely associated to humans, as a proxy to understand the human settlement of the Pacific Islands. We report the first genetic analysis of paper mulberry textiles from historical and archaeological contexts (200 to 50 years before present) and compare our results with genetic data obtained from contemporary and herbarium paper mulberry samples. Following stringent ancient DNA protocols, we extracted DNA from 13 barkcloth textiles. We confirmed that the fiber source is paper mulberry in nine of the 13 textiles studied using the nuclear ITS-1 marker and by statistical estimates. We detected high genetic diversity in historical Pacific paper mulberry barkcloth with a set of ten microsatellites, showing new alleles and specific genetic patterns. These genetic signatures allow tracing connections to plants from the Asian homeland, Near and Remote Oceania, establishing links not observed previously (using the same genetic tools) in extant plants or herbaria samples. These results show that historic barkcloth textiles are cultural materials amenable to genetic analysis to reveal human history and that these artifacts may harbor evidence of greater genetic diversity in Pacific B. papyrifera in the past.

Rocky desertification is a bottleneck that reduces ecological and environmental security in karst areas. Paper mulberry, a unique deciduous tree, shows good performance in rocky desertification areas. Its resistance mechanisms are therefore of high interest. In this study, a lysine acetylation proteomics analysis of paper mulberry seedling leaves was conducted in combination with the purification of acetylated protein by high-precision nano LC-MS/MS. We identified a total of 7130 acetylation sites in 3179 proteins. Analysis of the modified sites showed a predominance of nine motifs. Six positively charged residues: lysine (K), arginine (R), and histidine (H), serine (S), threonine (T), and tyrosine (Y) occurred most frequently at the +1 position, phenylalanine (F) was both detected both upstream and downstream of the acetylated lysines; and the sequence logos showed a strong preference for lysine and arginine around acetylated lysines. Functional annotation revealed that the identified enzymes were mainly involved in translation, transcription, ribosomal structure and biological processes, showing that lysine acetylation can regulate various aspects of primary carbon and nitrogen metabolism and secondary metabolism. Acetylated proteins were enriched in the chloroplast, cytoplasm, and nucleus, and many stress response-related proteins were also discovered to be acetylated, including PAL, HSP70, and ERF. HSP70, an important protein involved in plant abiotic and disease stress responses, was identified in paper mulberry, although it is rarely found in woody plants. This may be further examined in research in other plants and could explain the good adaptation of paper mulberry to the karst environment. However, these hypotheses require further verification. Our data can provide a new starting point for the further analysis of the acetylation function in paper mulberry and other plants.

India has the highest burden of Tuberculosis (TB) and multidrug-resistant TB (MDR-TB) worldwide. Innovative technology is the need of the hour to identify these cases that remain either undiagnosed or inadequately diagnosed due to the unavailability of appropriate tools at primary healthcare settings. We developed and evaluated 3 kits, namely ‘TB Detect’ (containing BioFM-Filter device), ‘TB Concentration and Transport’ (containing Trans-Filter device) and ‘TB DNA Extraction’ kits. These kits enable bio-safe equipment-free concentration of sputum on filters and improved fluorescence microscopy at primary healthcare centres, ambient temperature transport of dried inactivated sputum filters to central laboratories and molecular detection of drug resistance by PCR and DNA sequencing (Mol-DST). In a 2-site evaluation (n = 1190 sputum specimens) on presumptive TB patients, BioFM-Filter smear exhibited a significant increase in positivity of 7% and 4% over ZN smear and LED-FM smear (p

Biosensors have emerged as a valuable tool with high specificity and sensitivity for fast and reliable detection of hazardous substances in drinking water. Numerous substances have been addressed using synthetic biology approaches. However, many proposed biosensors are based on living, genetically modified organisms and are therefore limited in shelf life, usability and biosafety. We addressed these issues by the construction of an extensible, cell-free biosensor. Storage is possible through freeze drying on paper. Following the addition of an aqueous sample, a highly efficient cell-free protein synthesis (CFPS) reaction is initiated. Specific allosteric transcription factors modulate the expression of 'superfolder' green fluorescent protein (sfGFP) depending on the presence of the substance of interest. The resulting fluorescence intensities are analyzed with a conventional smartphone accompanied by simple and cheap light filters. An ordinary differential equitation (ODE) model of the biosensors was developed, which enabled prediction and optimization of performance. With an optimized cell-free biosensor based on the Shigella flexneri MerR transcriptional activator, detection of 6 mug/L Hg(II) ions in water was achieved. Furthermore, a completely new biosensor for the detection of gamma-hydroxybutyrate (GHB), a substance used as date-rape drug, was established by employing the naturally occurring transcriptional repressor BlcR from Agrobacterium tumefaciens.

Type 1 diabetes (T1D) is increasing in incidence and predictable with measurement of serum islet autoantibodies (iAb) years prior to clinical disease onset. Identifying iAb positive individuals reduces diabetic ketoacidosis and identifies individuals for T1D prevention trials. However, large scale screening for iAb remains challenging as assays have varying sensitivities and specificities, insulin autoantibodies remain difficult to measure and venipuncture is generally required to obtain serum. We developed an approach to reliably measure all four major iAb, including insulin autoantibodies, from dried blood spots (DBS) on filter-paper. By spiking iAb positive serum into iAb negative whole blood in a dose titration, we optimized the conditions for autoantibody elution from filter paper as measured by fluid phase radioimmunoassays. After assessing stability of measuring iAb from DBS over time, we then screened iAb from DBS and the corresponding serum in new-onset T1D (n = 52), and controls (n = 72) which included first-degree relatives of T1D patients. iAb measured from eluted DBS in new-onset T1D strongly correlated with serum measurements (R2 = 0.96 for mIAA, GADA = 0.94, IA-2A = 0.85, ZnT8A = 0.82, p

In this paper, we present a facile and cost-effective method to obtain superhydrophobic filter paper and demonstrate its application for efficient water/oil separation. By coupling structurally distinct organosilane precursors (e.g., octadecyltrichlorosilane and methyltrichlorosilane) to paper fibers under controlled reaction conditions, we have formulated a simple, inexpensive, and efficient protocol to achieve a desirable superhydrophobic and superoleophilic surface on conventional filter paper. The silanized superhydrophobic filter paper showed nanostructured morphology and demonstrated great separation efficiency (up to 99.4%) for water/oil mixtures. The modified filter paper is stable in both aqueous solutions and organic solvents, and can be reused multiple times. The present study shows that our newly developed binary silanization is a promising method of modifying cellulose-based materials for practical applications, in particular the treatment of industrial waste water and ecosystem recovery.

In vitro 3D culture is an important model for tissues in vivo. Cells in different locations of 3D tissues are physiologically different, because they are exposed to different concentrations of oxygen, nutrients, and signaling molecules, and to other environmental factors (temperature, mechanical stress, etc). The majority of high-throughput assays based on 3D cultures, however, can only detect the average behavior of cells in the whole 3D construct. Isolation of cells from specific regions of 3D cultures is possible, but relies on low-throughput techniques such as tissue sectioning and micromanipulation. Based on a procedure reported previously (‘‘cells-in-gels-in-paper’’ or CiGiP), this paper describes a simple method for culture of arrays of thin planar sections of tissues, either alone or stacked to create more complex 3D tissue structures. This procedure starts with sheets of paper patterned with hydrophobic regions that form 96 hydrophilic zones. Serial spotting of cells suspended in extracellular matrix (ECM) gel onto the patterned paper creates an array of 200 micron-thick slabs of ECM gel (supported mechanically by cellulose fibers) containing cells. Stacking the sheets with zones aligned on top of one another assembles 96 3D multilayer constructs. De-stacking the layers of the 3D culture, by peeling apart the sheets of paper, ‘‘sections’’ all 96 cultures at once. It is, thus, simple to isolate 200-micron-thick cell-containing slabs from each 3D culture in the 96-zone array. Because the 3D cultures are assembled from multiple layers, the number of cells plated initially in each layer determines the spatial distribution of cells in the stacked 3D cultures. This capability made it possible to compare the growth of 3D tumor models of different spatial composition, and to examine the migration of cells in these structures.

Made available in DSpace on 2018-11-26T16:33:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-05-11 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Recent studies have reported incipient morphological caste dimorphism in the Van der Vecht organ size of some temperate Polistes paper wasps. Whether species other than the temperate ones show a similar pattern remains elusive. Here, we have studied some Neotropical Polistes species. By comparing females collected through the year, we showed caste related differences in the size of the Van der Vecht organ in P. ferreri (body size corrected Van der Vech organ size of queens = 0.45 +/- 0.06, workers = 0.38 +/- 0.07 mm(2), p = 0.0021), P. versicolor (body size corrected Van der Vech organ size of queens = 0.54 +/- 0.11, workers = 0.46 +/- 0.09 mm(2), p = 0.010), but not P. simillimus (body size corrected Van der Vech organ size of queens = 0.52 +/- 0.05, workers = 0.49 +/- 0.06 mm(2), p = 0.238). Therefore, it seems that queens and workers of some Neotropical Polistes have diverged in their ontogenic trajectory of the Van der Vecht organ size, providing clear evidence for incipient morphological caste dimorphism. As Polistes are distributed mostly in the tropics, we propose that physical caste differences may be widespread in the genus. Also, we highlight that morphological divergence in the queen-worker phenotypes may have started through differential selection of body structures, like the Van der Vecht organ. Univ Fed Vicosa, Dept Entomol, BR-36570000 Vicosa, MG, Brazil Univ Florence, Dipartimento Biol Evoluzionist Leo Pardi, Via Romana 17, I-50125 Florence, Italy Univ Fed Vicosa, Dept Biol Geral, BR-36570000 Vicosa, MG, Brazil Univ Estadual Paulista, Inst Biociencias Letras & Ciencias Exatas, Dept Zool & Bot, BR-15054000 Sao Paulo, Brazil Univ Estadual Paulista, Inst Biociencias Letras & Ciencias Exatas, Dept Zool & Bot, BR-15054000 Sao Paulo, Brazil CNPq: 143246/2011-9

Many results have been obtained when studying scientific papers citations databases in a network perspective. Articles can be ranked according to their current in-degree and their future popularity or citation counts can even be predicted. The dynamical properties of such networks and the observation of the time evolution of their nodes started more recently. This work adopts an evolutionary perspective and proposes an original algorithm for the construction of genealogical trees of scientific papers on the basis of their citation count evolution in time. The fitness of a paper now amounts to its in-degree growing trend and a "dying" paper will suddenly see this trend declining in time. It will give birth and be taken over by some of its most prevalent citing "offspring". Practically, this might be used to trace the successive published milestones of a research field., SCOPUS: ar.j, info:eu-repo/semantics/published

In newborn screening, samples suspected for congenital adrenal hyperplasia (CAH), a potentially lethal inborn error of steroid biosynthesis, need to be confirmed using liquid chromatography–tandem mass spectrometry. Daily quality controls (QCs) for the 2nd-tier CAH assay are not commercially available and are therefore generally prepared within the laboratory. For the first time, we aimed to compare five different QC preparation approaches used in routine diagnostics for CAH on the concentrations of cortisol, 21-deoxycortisol, 11-deoxycortisol, 4-androstenedione and 17-hydroxyprogesterone in dried blood spots. The techniques from Prep1 to Prep5 were tested at two analyte concentrations by spiking aliquots of a steroid-depleted blood, derived from washed erythrocyte suspension and steroid-depleted serum. The preparation processes differed in the sequence of the preparation steps and whether freeze-thaw cycles were used to facilitate blood homogeneity. The five types of dried blood spot QCs were assayed and quantitated in duplicate on five different days using a single calibration row per day. Inter-assay variations less than 15% and concentrations within ±15% of the nominal values were considered acceptable. Results obtained by means of the four dried blood spot QC preparation techniques (Prep1, Prep2, Prep4 and Prep5) were statistically similar and remained within the ±15% ranges in terms of both reproducibility and nominal values. However, concentration results for Prep3 (spiking prior to three freeze-thaw cycles) were significantly lower than the nominal values in this setting, with differences exceeding the ±15% range in many cases despite acceptable inter-assay variations. These findings have implications for the in-house preparation of QC samples in laboratory developed tests for CAH, including 2nd-tier assays in newborn screening.

Passiflora quadrangularis L. belongs to the family Passifloraceae which bears larger fruit with edible juicy mesocarp and pulp known as a good source of phytochemicals. Cultivation and plant management practices are known to influence the phytochemical compositions of agricultural produce. This study aimed to examine the influence of the cultivation practices on the antioxidant activities and secondary metabolites of the organically and conventionally grown P. quadrangularis. Findings revealed organically treated P. quadrangularis plants showed enhancement in their antioxidant properties and secondary metabolites profiles. Among the plant parts, leaves of P. quadrangularis grown organically possessed higher antioxidant activities compared to the conventional in all assays evaluated. The antioxidant activities in the edible parts of the P. quadrangularis fruit have also been enhanced through organic cultivation with significantly higher total phenolic content and DPPH in mesocarp, and the pulp showed higher total flavonoid content, DPPH and FRAP. This observation is supported by a higher level of vitamins and secondary metabolites in the samples. The secondary metabolites profile showed mesocarps were phenolic rich, the pulps were flavonoids rich while leaves showed good composition of phenolics, flavonoids and terpenoids with outstanding antioxidant activities. The common secondary metabolites for organically produced P. quadrangularis in different plant parts include 2-isopropyl-3-methoxycinnamic acid (mesocarp and pulp), myricetin isomers (pulp and leaves), and malvidin-3-O-arabinoside isomers (pulp and leaves). This study confirmed that organic cultivated P. quadrangularis possessed higher antioxidant activities contributed by its vitamins and secondary metabolites.

Globally, the economic losses due to hard ticks infestation and the control of the associated diseases have been calculated at USD $13.9–18.7 billion per year. The economic impact is related to its direct damage to the skins, blood loss, anemia, severe immunological reactions and indirect losses that related to the effects of hemoparasites, cost of treatment for clinical cases and expenses incurred in the control of ticks. The current study evaluated the acaricidal activities of fennel Foeniculum vulgare essential oil and its main components; trans-anethole and fenchone; against R. annulatus. GC–MS analysis revealed that this oil contained 16 components representing 99.9% of the total identified compounds with E-anethole being the predominant component(64.29%), followed by fenchone (9.94%). The fennel oil and trans-anethole showed significant acaricidal activities. The LC50 of the fennel oil was attained at concentrations of 12.96% for adult ticks and 1.75% for tick larvae meanwhile the LC50 of trans-anethole was reached at concentrations of 2.36% for adult tick and 0.56% for tick larvae. On the contrary, fenchone showed no any significant adulticidal activities and its LC50 attained at a concentration of 9.11% for tick larvae. Regarding repellence activities, trans-anethole achieved 100% repellency at the concentration of 10% while fennel showed 86% repellency at the same concentration. Fenchone showed no repellency effect. Treatment of larvae with fennel, trans-anethole, and fenchone LC50 concentrations significantly inhibited the acetylcholinesterase activity. Meanwhile, glutathione s-transferase activity was significantly decreased in fennel treated larvae but no significant effect was found in the larvae of trans-anethole and fenchone groups. These results indicate that the acaricide effect of fennel oil may attributed to its high content of trans-anethole. This was supported by potent adulticidal, larvicidal, and repellency effects of trans-anethole against Rhipeciphalus annulatus tick and therefore it could be included in the list of acaricide of plant origin.

Annually, agricultural activity produces an enormous amount of plant biomass by-product. Many studies have reported the biomethane potential of agro-industrial wastes, but only a few studies have investigated applying the substrates in both batch and continuous mode. Tomato is one of the most popular vegetables globally; its processing releases a substantial amount of by-product, such as stems and leaves. This study examined the BMP of tomato plant (Solanum lycopersicum Mill. L. cv. Alfred) waste. A comparative test revealed that the BMPs of corn stover, tomato waste,and their combination were approximately the same, around 280 mL methane/g Volatile Solid. In contrast, the relative biogas production decreased in the presence of tomato waste in a continuous mesophilic anaerobic digestion system; the daily biogas productions were 860 ± 80, 290 ± 50, and 570 ± 70 mL biogas/gVolatile Solid/day in the case of corn stover, tomato waste, and their mixture, respectively. The methane content of biogas was around 46–48%. The fermentation parameters of the continuous AD experiments were optimal in all cases; thus, TW might have an inhibitory effect on the microbial community. Tomato plant materials contain e.g. flavonoids, glycoalkaloids (such as tomatine and tomatidine), etc. known as antimicrobial and antifungal agents. The negative effect of tomatine on the biogas yield was confirmed in batch fermentation experiments. Metagenomic analysis revealed that the tomato plant waste caused significant rearrangements in the microbial communities in the continuously operated reactors. The results demonstrated that tomato waste could be a good mono-substrate in batch fermentations or a co-substrate with corn stover in a proper ratio in continuous anaerobic fermentations for biogas production. These results also point to the importance of running long-term continuous fermentations to test the suitability of a novel biomass substrate for industrial biogas production.

Phytoseiids are predatory mites that prey on other mites and small arthropods, and several species are used in commercial agriculture for biological control of pests. To optimize phytoseiid mites' use in biocontrol, an efficient method for oral delivery of test compounds is required to assess their sensitivities to pesticides, RNAi for gene functional analysis and artificial diets. Here we developed four methods for oral delivery of a solution of xenobiotics to different life stages of the commercially available generalist predatory mite Neoseiulus californicus: (i) soaking mites in the solution, or allowing them to feed on (ii) spider mites soaked in the solution, (iii) a solution droplet, or (iv) solution-saturated filter paper. As measured by ingestion of a tracer dye, the droplet-based feeding system was most efficient; the dye was observed in the alimentary canal of >90% test mites of all life stages, with no mortality. The droplet-based feeding system was also effective for the commercially available specialist predatory mite Phytoseiulus persimilis, with >80% delivery efficiency. This study paves the way for development of methods for high-throughput RNAi and for toxicological or nutritional assays in phytoseiid mites.

Bacterial expansin-like proteins have synergistically increased cellulose hydrolysis by cellulolytic enzymes during the initial stages of biofuel production, but they have not been tested on livestock feeds. The objectives of this study were to: isolate and express an expansin-like protein (BsEXLX1), to verify its disruptive activity (expansion) on cotton fibers by immunodetection (Experiment 1), and to determine the effect of dose, pH and temperature for BsEXLX1 and cellulase to synergistically hydrolyze filter paper (FP) and carboxymethyl cellulose (CMC) under laboratory (Experiment 2) and simulated ruminal (Experiment 3) conditions. In addition, we determined the ability of BsEXLX1 to synergistically increase hydrolysis of corn and bermudagrass silages by an exogenous fibrolytic enzyme (EFE) (Experiment 4) and how different doses of BsEXLX1 and EFE affect the gas production (GP), in vitro digestibility and fermentation of a diet for dairy cows (Experiment 5). In Experiment 1, immunofluorescence-based examination of cotton microfiber treated without or with recombinant expansin-like protein expressed from Bacillus subtilis (BsEXLX1) increased the surface area by > 100% compared to the untreated control. In Experiment 2, adding BsEXLX1 (100 μg/g FP) to cellulase (0.0148 FPU) increased release of reducing sugars compared to cellulase alone by more than 40% (P < 0.01) at optimal pH (4.0) and temperature (50°C) after 24 h. In Experiment 3 and 4, adding BsEXLX1 to cellulase or EFE, synergistically increased release of reducing sugars from FP, corn and bermudagrass silages under simulated ruminal conditions (pH 6.0, 39°C). In Experiment 5, increasing the concentration of BsEXLX1 linearly increased (P < 0.01) GP from fermentation of a diet for dairy cows by up to 17.8%. Synergistic effects between BsEXLX1 and EFE increased in vitro NDF digestibility of the diet by 23.3% compared to the control. In vitro digestibility of hemicellulose and butyrate concentration were linearly increased by BsEXLX1 compared to the control. This study demonstrated that BsEXLX1 can improve the efficacy of cellulase and EFE at hydrolyzing pure substrates and dairy cow feeds, respectively.

Determining the distribution of the Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis, YFP) in the Yangtze River has to date relied on traditional visual and counting methods, but such field surveys are time-consuming and expensive. Analyses using environmental DNA (eDNA) to investigate the presence and range of endangered aquatic species have proven to be more economical and effective detection methods, and are a non-invasive approach to sampling. A challenge of relying on eDNA for YFP monitoring is that the Yangtze River is characterized by high turbidity and a strong current. Here, we used an eDNA-based approach to estimate the presence of YFP at 18 sites in the Yangtze River in August 2017 and at an additional 11 sites in January 2018. At each sampling site, we filtered six 1 L water samples with 5 µm pore size filter paper and quantified the amount of YFP eDNA in each water sample using quantitative real-time polymerase chain reaction (qPCR). In addition, YFP eDNA was successfully detected in locations where we visually observed YFP, as well as in locations where YFP were not observed directly. We found that our eDNA-based method had higher detection rates than traditional field survey methods. Although YFP was visually observed in the Yangtze River in winter, water samples collected during the summer contained significantly higher YFP eDNA than winter water samples. Our results demonstrate the potential effectiveness of eDNA detection methods in determining the distribution of YFP in the Yangtze River.

Due to the inefficient reproduction of microorganisms in oxygen-deprived environments of the reservoir, the applications of microbial enhanced oil recovery (MEOR) are restricted. To overcome this problem, a new type of air-assisted MEOR process was investigated. Three compounding oil degradation strains were screened using biochemical experiments. Their performances in bacterial suspensions with different amounts of dissolved oxygen were evaluated. Water flooding, microbial flooding and air-assisted microbial flooding core flow experiments were carried out. Carbon distribution curve of biodegraded oil with different oxygen concentration was determined by chromatographic analysis. The long-chain alkanes are degraded by microorganisms. A simulation model was established to take into account the change in oxygen concentration in the reservoir. The results showed that the optimal dissolved oxygen concentration for microbial growth was 4.5~5.5mg/L. The main oxygen consumption in the reservoir happened in the stationary and declining phases of the microbial growth systems. In order to reduce the oxygen concentration to a safe level, the minimum radius of oxygen consumption was found to be about 145m. These results demonstrate that the air-assisted MEOR process can overcome the shortcomings of traditional microbial flooding techniques. The findings of this study can help for better understanding of microbial enhanced oil recovery and improving the efficiency of microbial oil displacement.

Intelligent biorefinery design that addresses both the composition of the biomass feedstock as well as fermentation microorganisms could benefit from dedicated tools for computational simulation and computer-assisted optimization. Here we present the BioLego Vn2.0 framework, based on Microsoft Azure Cloud, which supports large-scale simulations of biomass serial fermentation processes by two different organisms. BioLego enables the simultaneous analysis of multiple fermentation scenarios and the comparison of fermentation potential of multiple feedstock compositions. Thanks to the effective use of cloud computing it further allows resource intensive analysis and exploration of media and organism modifications. We use BioLego to obtain biological and validation results, including (1) exploratory search for the optimal utilization of corn biomasses-corn cobs, corn fiber and corn stover-in fermentation biorefineries; (2) analysis of the possible effects of changes in the composition of K. alvarezi biomass on the ethanol production yield in an anaerobic two-step process (S. cerevisiae followed by E. coli); (3) analysis of the impact, on the estimated ethanol production yield, of knocking out single organism reactions either in one or in both organisms in an anaerobic two-step fermentation process of Ulva sp. into ethanol (S. cerevisiae followed by E. coli); and (4) comparison of several experimentally measured ethanol fermentation rates with the predictions of BioLego.

The pre-aerobic process of coking wastewater treatment has strong capacity of decarbonization and detoxification, which contribute to the subsequent dinitrogen of non-carbon source/heterotrophic denitrification. The COD removal rate can reach > 90% in the first aerobic bioreactor of the novel O/H/O coking wastewater treatment system during long-term operation. The physico-chemical characteristics of influent and effluent coking wastewater in the first aerobic bioreactor were analyzed to examine how they correlated with bacterial communities. The diversity of the activated sludge microbial community was investigated using a culture-independent molecular approach. The microbial community functional profiling and detailed pathways were predicted from the 16S rRNA gene-sequencing data by the PICRUSt software and the KEGG database. High-throughput MiSeq sequencing results revealed a distinct microbial composition in the activated sludge of the first aerobic bioreactor of the O/H/O system. Proteobacteria, Bacteroidetes, and Chlorobi were the decarbonization and detoxification dominant phyla with the relative abundance of 84.07 ± 5.45, 10.89 ± 6.31, and 2.96 ± 1.12%, respectively. Thiobacillus, Rhodoplanes, Lysobacter, and Leucobacter were the potential major genera involved in the crucial functional pathways related to the degradation of phenols, cyanide, benzoate, and naphthalene. These results indicated that the comprehensive understanding of the structure and function diversity of the microbial community in the bioreactor will be conducive to the optimal coking wastewater treatment.

Starch based gluten-free bread (formulations containing mixture of corn and potato starch with hydrocolloids) are deficient in nutrients and do not contain health promoting compounds. Therefore they could be supplemented with raw materials rich in such components, especially antioxidants. Among them pseudo-cereals, seeds, fruits and vegetables are often applied to this purpose. Potato pulp produced by processing red fleshed (Magenta Love) and purple fleshed (Violetta) varieties could become a new innovative substrate for gluten-free bread enrichment, because of high levels of endogenous polyphenols, namely flavonoids, flavonols, phenolic acids and especially anthocyanins with high antioxidant potential, as well as dietary fiber. Study material consisted of gluten-free bread enriched in the pulp. Dietary fiber, acrylamide content and antioxidant and antiradical potential of the bread were determined. Sensory evaluation included crumb elasticity, porosity and other characteristics, taste and smell. Among all analyzed gluten-free breads, the sample containing 7.5% share of freeze-dried red potato pulp Magenta Love was characterized by high content of phenolic compounds and dietary fiber, pronounced antioxidant activity, low levels of potentially dangerous acrylamide and good physical and sensory characteristics. Therefore such an addition (7.5% Magenta Love) could be recommended for industrial production of gluten-free bread.

Waste biomass from the palm oil industry is currently burned as a means of disposal and solutions are required to reduce the environmental impact. Whilst some waste biomass can be recycled to provide green energy such as biogas, this investigation aimed to optimise experimental conditions for recycling palm waste into substrate for insects, farmed as a sustainable high-protein animal feed. NMR spectroscopy and LC-HRMS were used to analyse the composition of palm empty fruit bunches (EFB) under experimental conditions optimised to produce nutritious substrate rather than biogas. Statistical pattern recognition techniques were used to investigate differences in composition for various combinations of pre-processing and anaerobic digestion (AD) methods. Pre-processing methods included steaming, pressure cooking, composting, microwaving, and breaking down the EFB using ionic liquids. AD conditions which were modified in combination with pre-processing methods were ratios of EFB:digestate and pH. Results show that the selection of pre-processing method affects the breakdown of the palm waste and subsequently the substrate composition and biogas production. Although large-scale insect feeding trials will be required to determine nutritional content, we found that conditions can be optimised to recycle palm waste for the production of substrate for insect rearing. Pre-processing EFB using ionic liquid before AD at pH6 with a 2:1 digestate:EFB ratio were found to be the best combination of experimental conditions.

Samples of bleached kraft pine cellulosic pulp, either treated with an enzyme preparation (a Thermomyces lanuginosus xylanase, an Aspergillus sp. cellulase, and a multienzyme preparation NS-22086 containing both these activities) or untreated, were refined in a laboratory PFI mill. The treatment with cellulases contained in the last two preparations significantly improved the pulp’s susceptibility to refining (the target freeness value of 30°SR was achieved in a significantly shorter time), increased water retention value (WRV) and fines contents while the weighted average fiber length was significantly reduced. These changes of pulp parameters caused deterioration of paper strength properties. The treatment with the xylanase, which partially hydrolyzed xylan, small amounts of which are associated with cellulose fibers, only slightly loosened the structure of fibers. These subtle changes positively affected the susceptibility of the pulp to refining (refining energy was significantly reduced) and improved the static strength properties of paper. Thus, the treatment of kraft pulps with xylanases may lead to substantial savings of refining energy without negative effects on paper characteristics.

Congenital cytomegalovirus (CMV) infection is a major cause of birth defects ranging from developmental disorders to stillbirth. Most newborns affected by CMV do not present with symptoms at birth but are at risk of sequelae at later stages of their childhood. Stored dried blood spots (DBS) taken at birth can be used for retrospective diagnosis of hereditary diseases, but detection of pathogens is challenged by potentially low pathogen concentrations in the small blood volume available in a DBS. Here we test four different extraction methods for optimal recovery of CMV DNA from DBS at low to high CMV titers. The recovery efficiencies varied widely between the different extractions (from 3% to 100%) with the most efficient method extracting up to 113-fold more CMV DNA than the least efficient and 8-fold more than the reference protocol. Furthermore, we amplified four immunomodulatory CMV genes from the extracted DNA: the UL40 and UL111A genes which occur as functional knockouts in some circulating CMV strains, and the highly variable UL146 and US28 genes. The PCRs specifically amplified the CMV genes at all tested titers with sufficient quality for sequencing and genotyping. In summary, we here report an extraction method for optimal recovery of CMV DNA from DBSs that can be used for both detection of CMV and for genotyping of polymorphic CMV genes in congenital CMV infection.

An electrochemically activated solution (ECAS), generated by electrolysis of a dilute sodium chloride solution in a four-chamber electrolytic cell (Ecas4), was tested as a sanitising aerosol in eliminating bacteria from the environment of a weaning room vacated 24-48h earlier, at a continuous flow pig farm. An ultrasonic humidifier was used to fill the environment with a fog (droplets with diameters of 1–5 μm) containing 0.25 ppm of hypochlorous acid. The weaning room was fogged for 3 min at 30 min intervals during five hours of aerosol disinfection. An innovative sample treatment with propidium monoazide dye in conjunction with cyclonic air sampling was optimised and adapted for discerning live/dead bacteria in subsequent molecular quantification steps. Without fogging, total bacterial load ranged from 5.06 ± 0.04 to 5.75 ± 0.04 Log10 CFU/m3. After the first hour of fogging, a 78% total bacterial reduction was observed, which further increased to > 97% after the second hour, > 99.4% after the third and 99.8% after the fourth hour, finally resulting in a 99.99% reduction from the farm environment over five hours. Unlike the current formaldehyde spray disinfection protocol, which requires a long empty period because of its hazardous properties, this economically viable and environmentally friendly disinfection protocol may significantly lower downtime. Moreover, ECAS fogging can be easily adapted to a variety of applications, including the elimination of pathogens from livestock farm air environment for disease prevention, as well as decontamination after disease outbreaks. Refereed/Peer-reviewed

Engineering faster cellulose deconstruction is difficult because it is a complex, cooperative, multi-enzyme process. Here we use experimental evolution to select for populations of Trichoderma citrinoviride that deconstruct up to five-fold more cellulose. Ten replicate populations of T. citrinoviride were selected for growth on filter paper by serial culture. After 125 periods of growth and transfer to fresh media, the filter paper deconstruction increased an average of 2.5 fold. Two populations were examined in more detail. The activity of the secreted cellulase mixtures increased more than two-fold relative to the ancestor and the largest increase was in the extracellular β-glucosidase activity. qPCR showed at least 16-fold more transcribed RNA for egl4 (endoglucanase IV gene), cbh1 (cellobiohydrolase I gene) and bgl1 (extracellular β-glucosidase I gene) in selected populations as compared to the ancestor, and earlier peak expressions of these genes. Deep sequencing shows that the regulatory strategies used to alter cellulase secretion differ in the two strains. The improvements in cellulose deconstruction come from earlier expression of all cellulases and increased relative amount of β-glucosidase, but with small increases in the total secreted protein and therefore little increase in metabolic cost.

BACKGROUND: The development in the deinking process has made recycled fiber a major part of the raw material for pulp and paper industry. Enzymes have revolutionized the deinking process obtaining brightness levels surpassing conventional deinking processes. This study explores the deinking efficiencies of bacterial alkalophilic laccase (L) and xylanase (X) enzymes along with physical deinking methods of microwaving (MW) and sonication (S) for recycling of old newsprint (ONP). METHODS AND RESULTS: The operational parameters viz. enzyme dose, pH and treatment time for X and L deinking were optimized statistically using Response Surface Methodology. Laccase did not require any mediator supplementation for deinking. Deinking of ONP pulp with a combination of xylanase and laccase enzymes was investigated, and fiber surface composition and morphological changes were studied using X-ray diffraction, fourier transform infrared spectroscopy and scanning electron microscopy. Compared to the pulp deinked with xylanase (47.9%) or laccase (62.2%) individually, the percentage reduction of effective residual ink concentration (ERIC) was higher for the combined xylanase/laccase-deinked pulp (65.8%). An increase in brightness (21.6%), breaking length (16.5%), burst factor (4.2%) tear factor (6.9%), viscosity (13%) and cellulose crystallinity (10.3%) along with decrease in kappa number (22%) and chemical consumption (50%) were also observed. Surface appeared more fibrillar along with changes in surface functional groups. A combination of physical and enzymatic processes (S-MW-XL) for deinking further improved brightness (28.8%) and decreased ERIC (73.9%) substantially. CONCLUSION: This is the first report on deinking of ONP with laccase without any mediator supplementation. XL pretreatment resulted in marked improvement in paper quality and a new sequence being reported for deinking (S-MW-XL) will contribute further in decreasing chemical consumption and making the process commercially feasible.

The current technologies of algal biofuels production and wastewater treatment (e.g., aerobic) process are still in question, due to the significant amount of fresh water and nutrients requirements for microalgae cultivation, and negative energy balance in both processes, especially when considered in the context of developing counties around the world. In this research, a simplistic sustainable approach of algal biofuels production from wastewater was proposed using a Hybrid Anaerobic Baffled Reactor (HABR) and Photobioreactor (PBR) system. The study suggests that the HABR was capable of removing most of the organic and solid (>90% COD and TSS removal) from wastewater, and produced a healthy feedstock (high N: P = 3:1) for microalgae cultivation in PBRs for biofuels production. A co-culture of Chlorella vulgaris, Chlorella sorokiniana, and Scenedesmus simris002 showed high lipid content up to 44.1%; and the dominant FAMEs composition (C16-C18) of 87.9% in produced biofuels. Perhaps, this proposed low-cost technological approach (e.g., HABR-PBR system) would connect the currently broken link of sustainable bioenergy generation and wastewater treatment pathway for developing countries.

A bioflocculant (MBFA18) was produced by Aspergillus niger (A18) using potato starch wastewater (PSW) as nutrients. The cultivation processes and flocculating treatment for PSW purification were systematically studied. The flocculating rate of the MBFA 18 achieved 90.06% (kaolin clay) under the optimal cultivation condition (PSW with 5950 mg/L COD, 20 g/L glucose, 0.2 g/L urea and without phosphorus source addition and pH adjustment). Furthermore, effects of flocculant dosage, initial pH, coagulant aid (CaCl2) addition and sedimentation time on the PSW treatment were discussed and studied in detail. The optimum flocculation treatment conditions were determined according to the treatment efficiency, cost and flocculation conditions. During the PSW treatment, 2 mL/L bioflocculant (1.89 g/L) dosage and 0.5 mol/L coagulant aid addition were applied without pH adjustment and 91.15% COD and 60.22% turbidity removal rate could be achieved within 20 min. The comparative study between the bioflocculant and conventional chemical flocculants showed excellent flocculating efficiency of MBFA 18 with lower cost (4.7 yuan/t), which indicated that the bioflocculant MBFA 18 produced in PSW substrate has a great potential to be an alternative flocculant in PSW treatment.

Proteins, metabolites, and 16S rRNA measurements were used to examine the community structure and functional relationships within a cellulose degrading anaerobic bioreactor. The bioreactor was seeded with bovine rumen fluid and operated with a 4 day hydraulic retention time on cellulose (avicel) as sole carbon and energy source. The reactor performance and microbial community structure was monitored during the establishment of the cellulose-degrading community. After stable operation was established in the bioreactor, the mixing intensity was increased in order to investigate the effect of a physical disruption of the microbial community structure. Finally, the original conditions were re-established to understand the stability of the microbial community after a perturbation. All factors measured were found to be inter-correlated during these three distinct phases of operation (establishment, perturbation and re-establishment). In particular, the return of community structure and function to pre-perturbed conditions suggests that propionate fermentation and acetate utilization were the explanatory factors for community establishment and re-establishment.

Efficient conversion of cellulosic sugars in cellulosic hydrolysates is important for economically viable production of biofuels from lignocellulosic biomass, but the goal remains a critical challenge. The present study reports a new approach for simultaneous fermentation of cellobiose and xylose by using the co-culture consisting of recombinant Saccharomyces cerevisiae specialist strains. The co-culture system can provide competitive advantage of modularity compared to the single culture system and can be tuned to deal with fluctuations in feedstock composition to achieve robust and cost-effective biofuel production. This study characterized fermentation kinetics of the recombinant cellobiose-consuming S. cerevisiae strain EJ2, xylose-consuming S. cerevisiae strain SR8, and their co-culture. The motivation for kinetic modeling was to provide guidance and prediction of using the co-culture system for simultaneous fermentation of mixed sugars with adjustable biomass of each specialist strain under different substrate concentrations. The kinetic model for the co-culture system was developed based on the pure culture models and incorporated the effects of product inhibition, initial substrate concentration and inoculum size. The model simulations were validated by results from independent fermentation experiments under different substrate conditions, and good agreement was found between model predictions and experimental data from batch fermentation of cellobiose, xylose and their mixtures. Additionally, with the guidance of model prediction, simultaneous co-fermentation of 60 g/L cellobiose and 20 g/L xylose was achieved with the initial cell densities of 0.45 g dry cell weight /L for EJ2 and 0.9 g dry cell weight /L SR8. The results demonstrated that the kinetic modeling could be used to guide the design and optimization of yeast co-culture conditions for achieving simultaneous fermentation of cellobiose and xylose with improved ethanol productivity, which is critically important for robust and efficient renewable biofuel production from lignocellulosic biomass.

Ricin is a highly-toxic compound derived from castor plant beans. Several incidents involving contamination of residences and buildings due to ricin production or dissemination have occurred in recent years. The goal of this study was to determine whether ricin bioactivity could be attenuated in reasonable time via simple modifications of the indoor environment. Attenuation was assessed on six different materials as a function of temperature, relative humidity (RH), and contact time, using both a pure and crude preparation of the toxin. Ricin bioactivity was quantified via a cytotoxicity assay, and attenuation determined as the difference in ricin recovered from test and positive controls. The results showed that pure ricin could be attenuated successfully, while the crude ricin was generally more persistent and results more variable. We found no significant attenuation in crude ricin after two weeks at typical indoor environmental conditions, except on steel. Attenuation mostly improved with increasing temperature, but the effect of RH varied. For pure ricin, heat treatments at 40°C for 5 days or 50°C for 2–3 days achieved greater than 96% attenuation on steel. In contrast, appreciable recovery of the crude ricin preparation still occurred at 40°C after two weeks.

The increasing water crisis makes fresh water a valuable resource, which must be used wisely. However, with growing population and inefficient waste treatment systems, the amount of wastewater dispelled in rivers is increasing abominably. Utilizing this freely available waste-water along with biodiesel industry waste- crude glycerol for bio-hydrogen production is being reported here. The bacterial cultures of Bacillus thuringiensis strain EGU45 and Bacillus amyloliquefaciens strain CD16 produced2.4–3.0 L H2/day/L feed during a 60 days continuous culture system at hydraulic retention time of 2 days. An average H2 yield of 100–120 L/L CG was reported by the two strains. Recycling of the effluent by up to 25% resulted in up to 94% H2 production compared to control.