Filter Paper Assay Research Articles

Comparative susceptibility of adult house fly (Diptera: Muscidae) and an important predator, Carcinops pumilio (Coleoptera: Histeridae), to Beauveria bassiana (Hypocreales: Cordycipitaceae).

House flies (Musca domestica L.) (Diptera: Muscidae) are challenging pests to control. Biological control using Carcinops pumilio beetles may help to reduce house fly populations. However, it is unknown if C. pumilio beetles are compatible with Beauveria bassiana, another house fly biological control option. Five strains of commercially available (GHA, HF23, and L90) and newly discovered (NFH10 and PSU1) strains of B. bassiana were used to test the comparative susceptibility of adult house flies and adult C. pumilio using different laboratory exposure methods. Adult house flies were susceptible to B. bassiana in contact filter paper assays (89%-98% mortality) and immersion assays (100% mortality) at the same 108 conidia suspension using 0.1% CapSil as an aqueous surfactant. Carcinops pumilio were less susceptible than flies to B. bassiana infection using the contact and immersion assays at the same 108 conidial concentration, with 4.4%-12.2% and 8.3%-24.6% mortality, respectively. Immersion in an aqueous conidial suspension resulted in higher mortality compared to contact with treated filter papers at the same 108 concentration with house flies and beetles. We conclude that C. pumilio can safely be used as a biological control agent for house flies with B. bassiana in animal production systems.

Effect of medium supplementations and extraction conditions on cellulase production through solid state fermentation of oil palm empty fruit bunches

In this study, cellulase enzyme was produced through solid state fermentation (SSF), employing oil palm empty fruit bunches (EFB) as the primary substrate. Two key aspects were explored to enhance crude enzyme yield, which are medium supplementation effects during SSF and cellulase recovery. During medium supplementation, glucose and/or Tween 80 were added alongside EFB substrate and other nutrients. Enzyme yield was determined using a filter paper assay and expressed as enzyme activity. The initial addition of glucose during fermentation led to increased crude enzyme activity, as measured by the filter paper assay. The peak crude enzyme activity was observed with the addition of 3 mg of glucose, with higher amounts showing no further increase in activity. Conversely, the addition of Tween 80 did not yield any significant increase in crude enzyme activity across all concentrations tested. The extraction conditions were varied to study cellulase recovery, specifically by adjusting the solid‐to‐solvent ratio and the number of extraction stages. Higher enzyme activity was achieved with lower solid‐to‐liquid ratios, as the increased solvent volume facilitated greater enzyme extraction. However, increasing the number of extraction steps did not significantly affect the resulting cellulase activity. Overall, this research underscores the need for further process optimization for cellulase production via SSF, utilizing the widely available EFB in Indonesia.

Development and evaluation of agro-waste biodegradation formulation

In the study, twenty strains of cellulose degrading microbes (Pseudomonas spp., Bacillus spp., Aspergillus spp. etc.) were isolated from the gut of termites and earthworms collected from different locations. The cellulolytic activity of the isolates of Aspergillus spp. were determined based on the formation of clear zone on CMC (Carboxymethyl cellulose) media. The strain C18 of Aspergillus flavus isolated from the gut of termite produced the largest zone with maximum cellulolytic index of 7.64. All the purified strains were subjected to in vitro cellulase activity through DNS (3,5-dinitrosalicylic acid) method which showed that strain C18 of Aspergillus flavus produced the maximum amount of glucose (0.507 mg ML ) and the -1 cellulase enzyme ( IU ML ) was recorded as 0.187 IU ML . Out of twenty microbial strains, ten best -1 -1 microbial strains were selected and subjected to in vitro FPA (filter paper assay). Aspergillus flavus strain C18 was found most effective with maximum glucose (1.059 mg ml ) and cellulase enzyme (0.196 -1 IUML ) production after 9 days of inoculation and therefore the consortium was made from two most -1 effective Aspergillus flavus strains (C12 and C18) which were found best effective in degradation of wheat straw substrate i.e., 61 per cent degradation of initial substrate weight. After evaluation of each strain, effective strains (C12 and C18) were used to prepare consortium formulations for commercial use in the field. These cellulolytic microbes have significant role in agro-waste management by degradation without harming the land and environment.. KEYWORDS :Agro-waste, Aspergillus flavus, biodegradation, cellulolytic microbes, formulation

Phytotoxic phenols from the needles of Cedrus deodara

During the course of screening for anti-seed germination phytochemicals, the methanol fraction of the Cedrus deodara fresh needles showed potent activity. Bioactivity-guided fractionation led to the isolation of thirty-eight phenolic compounds. Four ones were identified as previously undescribed including (7S,8S)-3-methoxy-9′-acetoxy-3′,7-epoxy-8,4′-oxyneoligna-4,9-diol (7), (7S,8R)-dihydro-3′-hydroxy-8-acetoxymethyl-7-(4-hydroxy-3-methoxy-phenyl)-1′-benzofuranpropanol (10), (8S)-4,9,9′-trihydroxy-3,3′-dimethoxy-8,4′-oxyneolignan (11) and (7S,8S)-4,7,9′-trihydroxy-3,3′-dimethoxy-9-acetoxy-8,4′-oxyneolignan (16), respectively. The potential phytotoxic effects of these compounds on the seed germination and root elongation of Arabidopsis thaliana were evaluated by the filter paper assay developed in our laboratory. Bioassay results indicated that caffeic acid (36) displayed most significant inhibitory activities against the seed germination and root elongation of A. thaliana, stronger than those of the commercial herbicides acetochlor and glyphosate at the same concentration of 200 μg/mL. Ditetrahydrofuran lignan (1), dihydrochalcone (25), and eight simple phenols (28, 29, 31, 33–35, 37 and 38) completely inhibited the seed germination of A. thaliana at the concentration of 400 μg/mL, which were as active as acetochlor. Dihydroflavone (21) and the simple phenols 32–34 displayed stronger inhibitory effects on the root elongation of A. thaliana than that of glyphosate. The inhibitory effects of these active compounds on the seed germination and root elongation of Amaranthus tricolor and Lactuca sativa were evaluated as well. The phytotoxic activity of 11, 16, 22, 25, 31, 34, 37 and 38 were detected for the first time. In addition, the structure-activity relationships of the same class of these phytochemicals were discussed.

Screening, enzyme activity and genomic analysis of Paenibacillus silvae CH2

Mangrove soil is a reliable source for screening cellulose-degrading bacteria due to the high diversity of microbes. To effectively utilize crop straw resources, a cellulolytic bacterium, Paenibacillus silvae strain CH2 was isolated from mangrove soil. We determined the carboxymethyl cellulose (CMC) and filter paper assay (FPA) activities of CH2 at different incubation times, NaCl concentrations, pH and temperatures, estimated the degradation efficiencies of rice and maize straw by CH2, sequenced and analyzed the whole genome of CH2. The results showed that along with the increases of incubation time, NaCl concentration, pH and temperature, the CMC and FPA activities increased first and then decreased . The highest CMC and FPA activities were observed at incubation time of 72-84 h, NaCl concentration of 6.0 g·L-1, pH of 7 and temperature of 36 ℃. Degradation of straw assays revealed that CH2 could effectively degrade rice and maize straw. At 0 g·L-1 NaCl (the control), the 10-day degradation rates of rice and maize straw were 30.4% and 47.0%, respectively. In the presence of 15 g·L-1 NaCl, the degradation rates were not significantly different from the control, indicating that CH2 had a high tolerance to salts. The whole genome of P. silvae CH2 was 6797325 bp, containing 6312 coding genes. P. silvae CH2 contained multiple genes encoding cellulose and hemicellulose degrading enzymes. These enzymes mainly belonged to the GH family, including endo-1,4-β-xylanase, Xylan 1,4-β-xylosidase, β-glucosidase, and endoglucanase. The results indicated that the bacterium had the potential to be used in crop straw degradation.

Screening and genome-wide analysis of lignocellulose-degrading bacteria from humic soil.

Crop straw contains huge amounts of exploitable energy, and efficient biomass degradation measures have attracted worldwide attention. Mining strains with high yields of cellulose-degrading enzymes is of great significance for developing clean energy and industrial production of related enzymes. In this study, we reported a high-quality genome sequence of Bacillus velezensis SSF6 strain using high-throughput sequencing technology (Illumina PE150 and PacBio) and assessed its lignocellulose degradation potential. The results demonstrated that the genome of B. velezensis SSF6 was 3.89 Mb and contained 4,015 genes, of which 2,972, 3,831 and 158 genes were annotated in the COGs (Clusters of Orthologous Groups), KEGG (Kyoto Encyclopedia of Genes and Genomes) and CAZyme (Carbohydrate-Active enZymes) databases, respectively, and contained a large number of genes related to carbohydrate metabolism. Furthermore, B. velezensis SSF6 has a high cellulose degradation capacity, with a filter paper assay (FPA) and an exoglucanase activity of 64.48 ± 0.28 and 78.59 ± 0.42 U/mL, respectively. Comparative genomic analysis depicted that B. velezensis SSF6 was richer in carbohydrate hydrolase gene. In conclusion, the cellulose-degrading ability of B. velezensis SSF6 was revealed by genome sequencing and the determination of cellulase activity, which laid a foundation for further cellulose degradation and bioconversion.

772 SCREENING FOR ANDERSON-FABRY DISEASE IN RELATIVELY YOUNG PATIENTS WITH UNEXPLAINED CONDUCTION DISORDERS REQUIRING PACEMAKER IMPLANTATION

Abstract Background Anderson-Fabry Disease (AFD, OMIM 301500) is a rare X-linked lysosomal storage disorder caused by GLA gene mutation resulting in a deficit or absent activity of the α-galactosidase A enzyme (α-Gal A). This deficiency involves the impossibility of cleavage of glycophospholipids, resulting in an intralisosomal accumulation of them in different tissues. Due to an incidence of 1 in 80000, AFD is considered the second most common glycosphingolipid storage disorder after Gaucher disease. Cardiac manifestations include left ventricular hypertrophy (LVH) and arrhythmias. The rate of pacemaker implantation (PMI) in AFD has been described to be 25 times higher than in the general population, and the requirement of PMI has been reported to be as high as 8% in some AFD series. In this context, the presence of conduction abnormalities in young patients may suggest an undiagnosed AFD.1 Therefore, early diagnosis is important in AFD because appropriate therapies seem to be more effective when initiated promptly. Purpose Our aim is to detect AFD among relatively patients with unexplained conduction disturbances requiring PMI, not submitted to newborn screening. Methods Among 650 patients afferent to our ambulatory for routinary pacemaker follow-up, we considered a selected population with diagnosis of sinus node dysfunction or atrioventricular block (confirmed by atrial pacing rate ≥ 60% or ventricular pacing percentage ≥ 80%) and an age, at the time of PMI, ranging between ≥40 and ≤70 years old. The exclusion criteria were: patients with previous myocardial infarction; patient whit known cardiac disease (such as hypertrophic cardiomyopathy); patients who underwent cardiac surgery and patients with extracardiac disease with cardiac involvement such as autoimmune disorders. For this cohort of 26 adult patients (13 males; 13 females; mean age 63 ± 7 years) a prospective screening study for AFD was performed. After clinical evaluation, transthoracic echocardiography (analyzing signs of left ventricular hypertrophy) and pacemaker check, a dried blood spot sampled in filter paper was analyzed. This filter paper assay was performed in male patients in order to evaluate the α-Galactosidase A enzyme activity through the detection of Fabry disease biomarkers; only in the case of abnormal values, genetic investigation was performed. In female patients, the analysis was exclusively genetic. Results The analyses revealed 58% (15/26) of patients affected by mild LVH (IVS diameter ranging from 11 to 15 mm). No patient had severe LVH (IVS diameter ≥15 mm) or moderate-severe renal dysfunction (more than stage 3B, GFR below than 30-44 mL/min). In the restrict cohort considered, we found one 69 yo female patient with heterozygosis GLA pathogenic mutation, NM_000169.2:c.638A>C p.(Lys213Thr). She had normal value of liso-Gb3 1,1 ng/ml (n.v. ≤ 1,8 ng/ml). She had mild LVH (IVS diameter 12 mm) and no renal dysfunction. Familiar screening was programmed. Conclusion In a highly selected sample of relatively young patients with conduction disturbances requiring pacemaker implantation, a female patient with genetic mutation causing AFD has been identified. Therefore, it seems that screening efforts should be increased in this patient population. 1 Hemelsoet D, De Keyser J, Van Heuverswyn F et al. Screening for Fabry Disease in Male Patients With Arrhythmia Requiring a Pacemaker or an Implantable Cardioverter-Defibrillator. Circulation. 2021 Feb 23;143(8):872-874. doi: 10.1161/CIRCULATIONAHA.120.051400.

Viral infection and allergy status impact severity of asthma symptoms in children with asthma exacerbations

BackgroundAlthough viral infection is known to be associated with asthma exacerbations, prior research has not identified reliable predictors of acute symptom severity in virus-related asthma exacerbations (VRAEs). ObjectiveTo determine the effect of asthma control and viral infection on the severity of current illness and evaluate biomarkers related to acute symptoms during asthma exacerbations. MethodsWe prospectively enrolled 120 children with physician-diagnosed asthma and current wheezing who presented to Arkansas Children's Hospital emergency department. The asthma control test (ACT) stratified controlled (ACT > 19) and uncontrolled (ACT ≤ 19) asthma, whereas pediatric respiratory symptom scores evaluated symptoms. Nasopharyngeal swabs were obtained for viral analysis, and inflammatory mediators were evaluated by nasal filter paper and Luminex assays. ResultsThere were 33 children with controlled asthma and 87 children with uncontrolled asthma. In those with uncontrolled asthma, 77% were infected with viruses during VRAE compared with 58% of those with controlled asthma. Uncontrolled subjects with VRAE had more acute symptoms compared with the controlled subjects with VRAE or uncontrolled subjects without a virus. The uncontrolled subjects with VRAE and allergy had the highest acute symptom scores (3.363 point pediatric respiratory symptom; P = .04). Children with asthma with higher symptom scores had more periostin (P = .02). ConclusionDetection of respiratory viruses is frequent in those with uncontrolled asthma. Uncontrolled subjects with viruses have more acute symptoms during exacerbations, especially in those with allergy. Periostin was highest in subjects with the most acute symptoms, regardless of control status. Taken together, these data imply synergy between viral infection and allergy in subjects with uncontrolled asthma when considering acute asthma symptoms and nasal inflammation during an exacerbation of asthma.

Lignocellulosic Ezymes of Basidial Fungi-Isolated from Different Ecological Niches of Georgia

Great interest in basidiomycetes for targeted technological treatment of agro-industrial plant substrates are conditioned by their ability to produce lignocellulosic enzymes. Consisting of 29 soil-climatic zones, Georgia (South Caucasus area) represents a unique place for the isolation of microorganisms. In order to study the biochemistry and physiology of wood-degrading basidiomycetes, samples of wood-degrading basidiomycetes were collected from different taxonomic niches of Georgia. 45 strains were obtained as pure cultures and 32 ones – identified. Producers of lignocellulosic enzymes were revealed among test fungi under solid-state and submerged cultivation conditions. Pleurous ostreatus GV10, Pleurotus drynus IN 11 and Fomes fomentarius GK33 were found to be the best producers of cellulosic enzymes using orange and mandarin peels, wheat and Potato straw, wheat bran as substrates, and Ganoderma lucidum GB03 – the best producer of laccase during cultivation on orange and mandarin peels waste. The influence of lignocellulose on the accumulation of the enzymes laccase, xylanase and Filter paper assay was studied.

Lignocellulosic Ezymes of Basidial Fungi-Isolated from Different Ecological Niches of Georgia

Great interest in basidiomycetes for targeted technological treatment of agro-industrial plant substrates are conditioned by their ability to produce lignocellulosic enzymes. Consisting of 29 soil-climatic zones, Georgia (South Caucasus area) represents a unique place for the isolation of microorganisms. In order to study the biochemistry and physiology of wood-degrading basidiomycetes, samples of wood-degrading basidiomycetes were collected from different taxonomic niches of Georgia. 45 strains were obtained as pure cultures and 32 ones – identified. Producers of lignocellulosic enzymes were revealed among test fungi under solid-state and submerged cultivation conditions. Pleurous ostreatus GV10, Pleurotus drynus IN 11 and Fomes fomentarius GK33 were found to be the best producers of cellulosic enzymes using orange and mandarin peels, wheat and Potato straw, wheat bran as substrates, and Ganoderma lucidum GB03 – the best producer of laccase during cultivation on orange and mandarin peels waste. The influence of lignocellulose on the accumulation of the enzymes laccase, xylanase and Filter paper assay was studied.

Tamarind extract pretreatment: Valorization of sugarcane bagasse for cellulase production by Aspergillus flavus

Effective pretreatment is crucial for cellulase production from sugarcane bagasse. Pretreatment with tamarind extract could reduce the hazardous effect associated with chemical pretreatment. The present work investigated tamarind (Tamarindus indica) extract in combination with H2SO4 and thermal pretreatment of sugarcane bagasse for cellulase production by Aspergillus flavus. The sugarcane bagasse was pretreated with tamarind extract pH 2 and pH 4, followed by 1% H2SO4 and thermal treatment at 121°C for 15 min. The pretreatment slurry was analysed for reducing sugar while solid bagasse was analysed for weight loss. Aspergillus flavus grew on sugarcane bagasse under solid state fermentation and the Carboxy Methyl Cellulase (CMCase) and Filter Paper Assay (FPA) activities were compared on the various pretreatments. The pretreatments changed the visible morphology of the sugarcane bagasse observed by the swelling, fibrous appearance and colour change. Pretreatment slurry yielded highest soluble reducing sugar at 60.01 mg/ml in tamarind extract (pH 4/1% H2SO4 ) and highest weight loss of solids at 73.70% in tamarind extract (pH 2/1% H2SO4 /thermal 121°C). Aspergillus flavus performed better on tamarind extract (pH 2/1% H2SO4 ) by producing optimal CMCase and FPA activities at 0.100 U/ml and 0.409 U/ml respectively after 3 days of fermentation. Cellulase was maximally active at temperature of 50 °C. The tamarind extract pretreatment successfully proved to be an alternative organo-chemical pretreatment of sugarcane bagasse as evidenced by the physical properties, soluble reducing sugars and cellulase activities.
 Keywords: Aspergillus flavus, Cellulase, Pretreatment, Sugarcane bagasse, Tamarind extract

Engineering the endoplasmic reticulum secretory pathway in Trichoderma reesei for improved cellulase production

The filamentous fungus Trichoderma reesei is an extraordinarily efficient cell factory of industrial cellulase for production of biofuels and other bio-based products because of its excellent potential to secrete cellulolytic enzymes. Engineering the protein secretory pathway may be a powerful means for efficient protein production. However, it is uncertain whether this engineering approach could improve cellulase production in T. reesei. Herein, the endoplasmic reticulum (ER) secretory pathway was engineered for the production of cellulolytic enzymes by multiple strategies, including: (I) overexpression of the key components of protein folding (Pdi1, Ero1 and BiP); (II) overexpression of the glycosylation-related elements (Gpt1 and Gls2); (III) knockout of the ER mannosidase I (Mns1) encoding gene mns1. By utilizing these ER engineering strategies, the secretion of β-glucosidase was remarkably elevated in the engineered strains, ranging from 29.2 % to 112.5 %. Furthermore, it was found that engineering these components also regulated the ER stress resistance. More importantly, the total cellulase production was increased with varying degrees, which reached a maximum of 149.4 %, using the filter paper assay (FPA) as a characterization method. These results demonstrated that engineering the ER secretory pathway can enhance protein secretion, particularly for cellulase production, which shed light for the development of high-efficient cellulolytic enzymes for economically feasible bioethanol production from lignocellulosic biomass.

Isolation and characterization of anaerobic bacteria with fiber degradation potential from faeces of Boselaphus tragocamelus grazing on semi arid Indian conditions.

Hindgut of wild ruminants harbours diversified anaerobic bacteria with promising fiber degrading ability. Fibrolytic enzyme activity is strongly influenced by diet and host species which till date remains unexplored for harnessing their optimum benefits. The present study was conducted to isolate and characterize fiber degrading anaerobic bacteria from faeces of wild blue bull (Boselaphus tragocamelus) inhabiting in semiarid regions. A total of 167 isolates were obtained from 85 faecal samples using M-10 medium, on the basis of clear zones formed on Congo red plates 20 isolates were selected and designated as NLG1-20 for microscopic and biochemical characterization. Further, molecular confirmation was done by PCR analysis with universal 16S rDNA primers. All isolates were obligatory anaerobes except, NLG4, NLG19 and NLG20. Majority of the sugars tested were utilized by most of the isolates except arabinose. Fibrolytic enzyme activities revealed that NLG1 had highest endoglucanase activity, NLG13 had highest exoglucanase activity while NLG8 showed maximum xylanase activity. In case of FPase assay, highest and lowest values were observed in isolate NLG11 (8.96 U/mL) and NLG8 (5.58 U/mL), respectively. Phylogenetic analysis of the isolates revealed a highly diverse group of microbes mainly belonging to the family Paenibacillaceae which have not been previously characterized in ruminants for fiber degradation. Therefore, results obtained in the present study indicated that the screened isolates showed promising fiber degrading potential in terms of filter paper assay and fibrolytic enzyme activity which can be explored further for improving lignocellulose digestibility in ruminants as an additive.

Isolation of alkali-thermotolerant cellulolytic bacteria from spent mushroom substrate

In this study, the isolation of new alkali-thermophilic bacteria with enhanced cellulase producing ability from the spent mushroom substrate was conducted. Repeated streaking was performed on the Microcrystalline-cellulose (MCC) to obtain a pure culture. Two thermophiles bacteria isolates were used for subsequent experiment. Morphological characteristic by gram staining and endospore staining also genetic identification of 16S rDNA was performed and the growth profile had been generated using Polymath Software. Filter paper assay (determination of enzyme activity), Bradford protein concentration were used in plotting the growth associated product curve. Based on the 16S rDNA sequencing results, the bacterium strains of Anoxybacillus geothermalis, incubated at 50˚C, pH 7 and Aeribacillus pallidus, incubated at 60˚C, pH 8 was successfully isolated. Both strains are gram-positive and endospore-forming bacteria. Based on the growth curve, the doubling time for Anoxybacillus geothermalis was calculated to be 49.5 minutes. Whereas, doubling time of Aeribacillus pallidus was 56.7 minutes. Both strains represent the growth assosiated product curve in which, when product formation is growth associated the specific rate of the product formation increases with specific growth rate and vice versa.

Biocatalyst: Cellulase Production in Solid State Fermentation (SSF) Using Rice Bran as Substrate

The study was aimed to analyze the biological transformation of cellulose in rice bran by Aspergillus flavus SB04 in SSF for 28 days. The culture conditions such as pH, temperature, moisture content were optimized for the effective production of the enzyme in SSF. Effect of carbon and nitrogen sources on cellulase production was further estimated in SMF and were quantified for 24hrs intervals for 7 days Maximum cellulase production for rice bran was observed to be high in glucose (carbon source) and yeast extract (nitrogen source) at initial moisture 75ml, pH 6, temperature 33°C and fermentation period was 14th day that was optimized using response surface methodology. The enzyme production was analyzed individually by dinitrosalicylic acid (DNS) method, Lowry protein estimation, and filter paper assay. The lignocellulosic degradation was observed and confirmed by FTIR and SEM. The degradation of cellulose periodically increases after 7 days, which influences the yield of cellulase enzyme.

Substrate Characteristics That Influence the Filter Paper Assay’s Ability to Predict the Hydrolytic Potential of Cellulase Mixtures

Currently, filter paper assay (FPA) is used to measure the hydrolytic potential of cellulase enzyme mixtures that are used to hydrolyze a range of cellulosic substrates. However, some of the Whatman No. 1 filter paper substrate characteristics, such as its high cellulose content, cellulose accessibility, dried nature, etc., compromise the ability of the FPA to predict how well a complex cellulase cocktail will hydrolyze pretreated lignocellulosic substrates. The influence of factors such as drying, papermaking, and the presence of lignin and hemicellulose on the FPA was assessed by producing and using “model/paper sheets” as a substitute for the Whatman No. 1 filter paper. Drying was shown to have a major, negative influence resulting in a 60% reduction in the assessed cellulase activity. Similarly, a 53% decrease in cellulase activity resulted when an organosolv-pretreated hardwood containing lignin and hemicellulose was used instead of the Whatman No. 1 filter paper. It was apparent that the FPA was a poor predictor of the hydrolytic potential of an enzyme mixture on cellulosic/lignocellulosic substrates. However, other than assessing the ease of hydrolysis of a specific substrate under a defined set of conditions, it is likely that the FPA will continue to be used to indicate the broad, hydrolytic potential of an enzyme mixture in terms of the amount of protein required to hydrolyze the cellulosic component over a defined set of temperature, time, and pH conditions.

MP48-16 COMPARATIVE EVALUATION OF WHITE MATTER DEMYELINATION BY MAGNETIC RESONANCE IMAGING AND NEUROGENIC VOIDING DYSFUNCTION IN A VIRAL MURINE MODEL OF MULTIPLE SCLEROSIS

You have accessJournal of UrologyUrodynamics/Lower Urinary Tract Dysfunction/Female Pelvic Medicine: Neurogenic Voiding Dysfunction (MP48)1 Apr 2020MP48-16 COMPARATIVE EVALUATION OF WHITE MATTER DEMYELINATION BY MAGNETIC RESONANCE IMAGING AND NEUROGENIC VOIDING DYSFUNCTION IN A VIRAL MURINE MODEL OF MULTIPLE SCLEROSIS Li Lu, Alison Xie, Natalie Serkova, and Anna Malykhina* Li LuLi Lu More articles by this author , Alison XieAlison Xie More articles by this author , Natalie SerkovaNatalie Serkova More articles by this author , and Anna Malykhina*Anna Malykhina* More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000903.016AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Lower urinary tract symptoms (LUTS) are present in 70–80% of patients with multiple sclerosis (MS), having a negative effect on the quality of the individual’s life. In 10–15% of patients, neurogenic LUTS are present at the onset of MS when there may be only few lesions identified in the CNS. In this study, we aimed to establish if early white matter demyelination could be detected by MRI, and whether it could be associated with the first signs of neurogenic LUTS. METHODS: Adult mice were inoculated with A59 strain of mouse hepatitis virus (coronavirus-induced encephalomyelitis model, CIE). The level of neurodegeneration was assessed by the Clinical Symptom Score (CSS). MRI experiments were performed on a 9.4 Tesla/ 20-cm BioSpec MRI scanner. Bladder function was evaluated in vivo by a filter paper assay, and in vitro by detrusor contractility studies. RESULTS: The spines from control and CIE mice (CSS≤1-2, 10 wks) were subjected to a FLASH MRI ultra-high resolution sequence, and DTI-EPI protocols. The mean diffusivity (D) and fractional anisotropy (FA) parameters were used to assess the integrity and organization of the white matter. The representative T2 MRI and DTI images, and FA maps are included in Fig. 1. The D values were significantly reduced in CIE mice (5.36±0.21x10-4 mm2/s) in comparison to control (6.98±0.37x10-4 mm2/s, p≤0.01). The FA parameters were also significantly lower in CIE mice (0.39±0.02 vs 0.56±0.02 in control, p≤0.002). Micturition patterns in CIE mice were not significantly changed, but the differences were detected in the contractile responses of the detrusor to various stimuli. CONCLUSIONS: The early changes in D and FA parameters reflect the ongoing changes in white matter structural integrity of the spinal cord. These early signs of demyelination are associated with modified detrusor contractile responses, which likely reflect the early stage of neurogenic LUTS development. Source of Funding: The study was supported by the NIH/NIDDK grant DK121506 and Department of Surgery AEF grant. © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e700-e701 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Li Lu More articles by this author Alison Xie More articles by this author Natalie Serkova More articles by this author Anna Malykhina* More articles by this author Expand All Advertisement PDF downloadLoading ...

Production of cellulase from Aspergillus niger and Trichoderma reesei mixed culture in carboxymethylcellulose medium as sole carbon

Abstract. Septiani D, Suryadi H, Mun’im A, Mangunwardoyo W. 2019. Production of cellulase from Aspergillus niger and Trichoderma reesei mixed culture in carboxymethylcellulose medium as sole carbon. Biodiversitas 20: 3539-3544. Cellulase is one of hydrolytic enzymes that breakdown cellulose into glucose. Cellulases are promising to be applied in natural products which may improve the yield of bioactive in plant extract through cellulose depolymerization. Cellulases from mixed culture of Aspergillus niger and Trichoderma reesei can produce a high cellulase activity because of the synergism activity among endoglucanase, exoglucanase, and also β-glucoside. Cellulase production and partial purification of monoculture and mixed culture (1:1) of these fungi on carboxymethylcellulose media were investigated in this study. Total cellulase activity was measured by filter paper assay followed by protein estimation with Bradford method. The crude extract of Aspergillus niger monoculture has the highest cellulase activity (0.131 U/mL, P<005) followed by mixed culture (0.109 U/mL) and Trichoderma reesei (0.106 U/mL). The cellulase activity of partially purified cellulase from mixed culture significantly increased (0.335, 0.348, 0.374 U/mL, P<0.05) compared to crude extract along with stepwise addition of ammonium sulfate. Cellulase activity of mixed culture at 80% ammonium sulfate increase up to 2.238-fold and showed highest value (P<0.05) compared to monocultures. In conclusion, combination of Aspergillus niger and Trichoderma reesei fungi in carboxymethyl cellulose media followed by 80% ammonium sulfate precipitation can be a promising cellulase production with high cellulase activity.

Comparative Susceptibility of Western Corn Rootworm (Coleoptera: Chrysomelidae) Neonates to Selected Insecticides and Bt Proteins in the Presence and Absence of Feeding Stimulants.

The susceptibility of western corn rootworm, Diabrotica virgifera virgifera LeConte, larvae to nine insecticides from five different classes and to Bt proteins eCry3.1Ab and mCry3A in the presence or absence of feeding stimulants, was estimated in filter paper and diet toxicity assays, respectively. The use of a synthetic feeding stimulant blend of the sugars glucose, sucrose, and fructose plus linoleic acid at a ratio of 30:4:4:0.3 mg/ml of distilled water was evaluated to determine whether they increase the efficacy of insecticides and Bt proteins. The efficacy of thiamethoxam diluted in solutions with feeding stimulants was significantly increased when compared to thiamethoxam dilutions in water (>60-fold). Differences in the efficacy of the other insecticide classes when diluted in feeding stimulant solutions were no greater than fivefold when compared to the insecticides diluted in water. The presence of corn root juice as a natural feeding stimulant diminished toxicity of the insecticides, except for thiamethoxam, even though larval fresh weight was higher when fed on root juice compared to feeding stimulant or water. The use of feeding stimulants in diet toxicity assays did not enhance efficacy of eCry3.1Ab nor mCry3A proteins. Feeding stimulants can be recommended in combination with thiamethoxam to increase larval mortality. These results are discussed in terms of applicability of feeding stimulants to improve susceptibility of western corn rootworm larvae to pesticides in general.

Solid State Fermentation Parameters Effect on Cellulase Production from Empty Fruit Bunch

In this study, agriculture waste palm empty fruit bunch (EFB) was used as carbon/cellulose source in solid state fermentation for cheaper cellulase production. Fermentation operation parameters, such as: solid to liquid ratio, temperature, and pH, were varied to study the effect of those parameters towards crude cellulase activity. Two different fungi organisms, Trichoderma viride and Trichoderma reesei were used as the producers. Extracellular cellulase enzyme was extracted using simple contact method using citrate buffer. Assessment of the extracted cellulase activity by filter paper assay showed that Trichoderma viride is the superior organism capable of producing higher cellulase amount compared to Trichoderma reesei at the same fermentation condition. The optimum cellulase activity of 0.79 FPU/g dry substrate was obtained when solid to liquid ratio used for the fermentation was 1:1, while the optimum fermentation temperature and pH were found to be 30 °C and 5.5, respectively. The result obtained in this research showed the potential of EFB utilization for enzyme production.