Micro-aggregation of a pristine grassland soil selects for bacterial and fungal communities and changes in nitrogen cycling potentials

Microbial analysis at the micro scale of soil is essential to the overall understanding of microbial organization and interactions, and necessary for a better understanding of soil ecosystem functioning. While bacterial communities have been extensively described, little is known about the organization of fungal communities as well as functional potentials at scales relevant to microbial interactions. Fungal and bacterial communities and changes in nitrogen cycling potentials in the pristine Rothamsted Park Grass soil (bulk soil) as well as in its particle size sub-fractions (PSFs; > 250 µm, 250-63 µm, 63-20 µm, 20-2 µm, < 2 µm and supernatant) were studied. The potential for nitrogen reduction was found elevated in bigger aggregates. The relative abundance of Basidiomycota deceased with decreasing particle size, Ascomycota showed an increase and Mucoromycota became more prominent in particles less than 20 µm. Bacterial community structures changed below 20 µm at the scale where microbes operate.Strikingly, only members of two bacterial and one fungal phyla (Proteobacteria, Bacteroidota and Ascomycota, respectively) were washed-off the soil during fractionation and accumulated in the supernatant fraction where most of the detected bacterial genera (e.g., Pseudomonas, Massilia, Mucilaginibacter, Edaphobaculum, Duganella, Janthinobacterium and Variovorax) were previously associated with exopolysaccharide production and biofilm formation.Overall, the applied method shows potential to study soil microbial communities at micro scales which might be useful in studies focusing on the role of specific fungal taxa in soil structure formation as well as research on how and by whom biofilm-like structures are distributed and organized in soil.

Influence of Chymosin on Physicochemical and Hydrolysis Characteristics of Casein Micelles and Individual Caseins

The effects of chymosin on the physicochemical and hydrolysis characteristics of casein micelles and individual caseins were investigated. Adding 0.03 units of chymosin/mL led to the casein micelles in skim milk coagulating after a 3 h incubation period at 30 °C. SDS–PAGE investigation showed that β-CN, κ-CN, αs-CN, and a portion of β-lactoglobulin (β-LG) in the milk supernatant fraction (MSF) were precipitated into the milk pellet fraction (MPF). The mean particle size of the MSF with chymosin decreased from 254.4 nm to 179.2 nm after a 3 h incubation period. Mass spectrometry and SDS–PAGE analysis suggested that chymosin hydrolyzed individual β-CN, κ-CN, and αs-CN, but not β-LG. Chymosin hydrolysis led to a decrease in the molecular weights of the hydrolyzed β-CN, κ-CN, and αs-CN. Particle size analysis indicated that there was no difference in the particle size distribution of hydrolyzed β-CN and αs-CN. Moreover, our outcomes demonstrated that the hydrolysis of κ-CN by chymosin occurs before that of β-CN and αs-CN.

Statins Accelerated Arthritic Incidence: Ex vivo Antioxidant and Histopathological Analysis

Statins are conventionally used medicaments for the treatment of hyperlipidemia and hypertension. Apart from the aforementioned pharmacological action, statin use was associated with increased risk for developing rheumatoid arthritis due to its immunomodulatory property. The present study focused to demonstrate whether statin use accelerated arthritic incidence. The Ex vivo antioxidant study was performed using Lipid peroxidation (LPO) and Superoxide dismutase (SOD) activity assay. Both the Antioxidant study was evaluated from tissue homogenates. Each homogenate was centrifuged, and the resulting supernatant fraction was used to determine the enzyme activity. The histopathological analysis of ankle joints was conducted by hematoxylin eosin staining technique. The result obtained from Lipid peroxidation study showed that both Pitavastatin and Lovastatin treated tissues exhibited significant increase (p<0.001) in enzyme activity due to the elevated level of malonaldehyde formation. The Superoxide dismutase activity assay of both test drugs on tissue homogenate was found that significant decrease (p<0.001) in SOD level contributed cartilage degradation due to the generation of free radicals. The histopathological analysis also revealed that scattered inflammatory cells destructs the normal articular structure in rat joint tissues showed the evidence of arthritic induction. The study confirmed that statins had a strong influence on arthritic onset by oxidative stress in patients with hyperlipidemia and hypertension.

Novel Indigenous Probiotic Lactobacillus reuteri Strain Produces Anti-biofilm Reuterin against Pathogenic Periodontal Bacteria

Objective The aim of this study was to evaluate the effect of reuterin produced by a novel probiotic strain of Lactobacillus reuteri against periodontal biofilms. Materials and Methods L. reuteri LC382415 (an indigenous Indonesian strain) was cultured in Man, Rogosa, and Sharpe (MRS) agar in anaerobic conditions for 24 hours. To isolate reuterin, L. reuteri was suspended in 300-mM glycerol in MRS broth and incubated under anaerobic conditions for 3 hours, and the supernatant fraction was filtered. The presence of reuterin was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and its concentration was determined. The effect of reuterin on Porphyromonas gingivalis ATCC 33277 and T. denticola ATCC 35405 biofilms was evaluated using biofilm assays. Biofilms were formed by incubating bacteria in 96-well microplates for 48 hours. A dose-dependent experiment was performed with reuterin concentrations of 12.5, 25, 50, and 100 μg/mL on biofilms. The inhibitory effect was measured at 1, 3, 6, and 24 hours. The biofilm masses were measured at 490 nm. Statistical analysis was using one-way ANOVA. Results The SDS-PAGE assay confirmed the presence of reuterin (52 kDa) in the culture supernatant of the L. reuteri strain. Reuterin in a concentration as low as 12.5 μg/mL significantly inhibited single- and mixed-species biofilms (p < 0.05). Conclusions This is the first study to demonstrate the promising effect of reuterin isolated from L. reuteri LC382415 against periodontal bacteria. Further studies are warranted to explore the mechanism of this active component.

Assessing the Adsorption of Bipyridinium Herbicides on Model Soil Granular Media

In this research work, the adsorption of two bipyridinium herbicides (i. e., Diquat and Paraquat) on natural soil and on model soil surfaces has been studied at different water chemistries commonly found in the environment (e.g., pH, supporting electrolyte, and presence of humic or fulvic acids). The experimental work was carried out in the laboratory, using experimental batches of clean quartz sand, silanized quartz sand and sandy soil as a model of agricultural topsoil where herbicides are commonly used and can be adsorbed. The concentrations reached at the equilibrium were analyzed by UV-Visible Spectroscopy for the supernatant fraction of the samples. The concentrations were fitted using adsorption isotherms to determine the adsorption mechanisms (i.e., chemisorption or physisorption) at the interface. In general terms, we have encountered that the nature of the soil matrix plays an important role on the study of pollutant adsorption. In experiments carried out on silica sand, the most abundant component of the natural soil matrix, no significant sorption was observed (&lt;1.5 mg/g) for any of the herbicides. Yet, in experiments carried out on the presence of clay and natural organic matter (i.e., fulvic and humic acids), the adsorption of both herbicides is much higher, likely due to the chemical structure of the molecules that might facilitate the complexation with both herbicides. This investigation improves our understanding of the role that soil granular components play on the absorption of two commonly used herbicides and adequately predict their fate in natural aquatic environments.

Accumulation of Cerebrospinal Fluid Glycerophospholipids and Sphingolipids in Cognitively Healthy Participants With Alzheimer’s Biomarkers Precedes Lipolysis in the Dementia Stage

Insight into lipids’ roles in Alzheimer’s disease (AD) pathophysiology is limited because brain membrane lipids have not been characterized in cognitively healthy (CH) individuals. Since age is a significant risk factor of AD, we hypothesize that aging renders the amyloid precursor protein (APP) more susceptible to abnormal processing because of deteriorating membrane lipids. To reflect brain membranes, we studied their lipid components in cerebrospinal fluid (CSF) and brain-derived CSF nanoparticle membranes. Based on CSF Aβ42/Tau levels established biomarkers of AD, we define a subset of CH participants with normal Aβ42/Tau (CH-NAT) and another group with abnormal or pathological Aβ42/Tau (CH-PAT). We report that glycerophospholipids are differentially metabolized in the CSF supernatant fluid and nanoparticle membrane fractions from CH-NAT, CH-PAT, and AD participants. Phosphatidylcholine molecular species from the supernatant fraction of CH-PAT were higher than in the CH-NAT and AD participants. Sphingomyelin levels in the supernatant fraction were lower in the CH-PAT and AD than in the CH-NAT group. The decrease in sphingomyelin corresponded with an increase in ceramide and dihydroceramide and an increase in the ceramide to sphingomyelin ratio in AD. In contrast to the supernatant fraction, sphingomyelin is higher in the nanoparticle fraction from the CH-PAT group, accompanied by lower ceramide and dihydroceramide and a decrease in the ratio of ceramide to sphingomyelin in CH-PAT compared with CH-NAT. On investigating the mechanism for the lipid changes in AD, we observed that phospholipase A2 (PLA2) activity was higher in the AD group than the CH groups. Paradoxically, acid and neutral sphingomyelinase (SMase) activities were lower in AD compared to the CH groups. Considering external influences on lipids, the clinical groups did not differ in their fasting blood lipids or dietary lipids, consistent with the CSF lipid changes originating from brain pathophysiology. The lipid accumulation in a prodromal AD biomarker positive stage identifies perturbation of lipid metabolism and disturbances in APP/Amyloid beta (Aβ) as early events in AD pathophysiology. Our results identify increased lipid turnover in CH participants with AD biomarkers, switching to a predominantly lipolytic state in dementia. This knowledge may be useful for targeting and testing new AD treatments.

The utility of sputum supernatant as an alternative liquid biopsy specimen for next-generation sequencing-based genomic profiling

Background Comprehensive mutation profiling has become a standard clinical practice in the management of advanced lung cancer. In addition to tissue and plasma, other body fluids are also being actively explored as alternative sources of tumor DNA. In this study, we investigated the potential of induced sputum obtained from patients with non-small cell lung cancer (NSCLC) for mutation profiling. Methods Capture-based targeted sequencing was performed on matched tumor, plasma, and induced sputum samples of 41 treatment-naïve patients with NSCLC using 168 gene panel. Results Comparative analysis on the mutation detection using matched tumor sample as reference revealed detection rates of 76.9% for plasma, 72.4% for sputum-supernatant, and 65.7% for sputum-sediment samples. Plasma, sputum-supernatant, and sputum-sediment achieved positive predictive values of 73.3%, 80.4%, and 55.6% and sensitivities of 50.0%, 36.9%, 31.3%, respectively, relative to tumor samples for 168 genes. Sputum-supernatants had significantly higher concordance rates relative to matched tumor samples (69.2% vs 37.8%; P = 0.031) and maximum allelic fraction (P < 0.001) than its matched sputum-sediments. Sputum-supernatants had comparable detection rates (71.4% vs. 67.9%; P = 1) but with significantly higher maximum allelic fraction than their matched plasma samples (P = 0.003). Furthermore, sputum-supernatant from smokers had a significantly higher maximum allelic fraction than sputum-supernatant from non-smokers (P = 0.021). Conclusions Our study demonstrated that supernatant fraction from induced sputum is a better sampling source than its sediment and has comparable performance as plasma samples. Induced sputum from NSCLC patients could serve as an alternative media for next-generation sequencing-based mutation profiling.

ML-06 Diagnostic value of liquid biopsy for CNS lymphoma by detection of specific gene mutations in the cerebrospinal fluid

Backgrounds & Purpose: Central nervous system lymphoma (CNSL) is the second most common primary malignant brain tumor. Brain biopsy is indispensable to confirm the diagnosis of CNSL, but has a potential risk of inducing hemorrhagic complications in the brain. Therefore, liquid biopsy using the cerebrospinal fluid (CSF) has attracted an attention as a less invasive diagnostic method. In this study, we established a digital PCR-based method to detect MYD88 mutations in CSF and evaluated its efficacy. Methods: Matched CSF and biopsy samples from CNSL patients collected before the start of chemotherapy were used. Cellular DNA and cell free DNA (cfDNA) of CSF were separately extracted from the pellet and the supernatant fraction of CSF, respectively. Presence of the MYD88 L265P mutation was examined in each fraction by the digital PCR. The mutational status obtained by liquid biopsy was compared with that of the matched biopsy specimen examined by pyrosequencing. Result: A total of 36 paired samples were used. When the cutoff value of Target/Total ratio was 0.25%, sensitivity, specificity, and area under the curve (AUC) of the digital PCR detection using cellular DNA were 92.9%, 100%, and 0.95, respectively, while they were 100%, 100%, and 1.00 using cfDNA. Conclusion: We showed that the digital PCR method was highly sensitive and specific in detecting MYD88 mutations in the CSF. We propose that CSF liquid biopsy may serve a clinically applicable surrogate to make a diagnosis of CNSL.

Novel non-specific lipid-Transfer Protein (TdLTP4) isolated from Durum wheat: Antimicrobial activities to control pathogen and spoilage bacteria and Anti-Inflammatory properties in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages

Background: Lipid transfer proteins (LTP) are members of the family of pathogenesis-related proteins (PR-14) that play a key role in plant defense mechanisms.Methods: In this study, a novel gene TdLTP4 encoding an antifungal protein from wheat (cv. Om Rabiaa) was subcloned, overexpressed in Escherichia coli BL-21 (DE3) and enriched using ammonium sulfate fractionation. The TdLTP4 fusion protein was then tested against a panel of pathogens, food-borne and spoilage bacteria and fungi in order to evaluate the antimicrobial properties. Our protein was applied to 0.5 µg/mL LPS-induced RAW 264.7 macrophages in vitro at different concentrations (5, 10, 20, 50 and 100 µg/ml). Levels of nitric oxide (NO), pro-inflammatory cytokines interleukin (IL)-1β (IL-1 β), interleukin (IL)-6 (IL-6), tumor necrosis factor (TNF-α) and anti-inflammatory cytokine IL-10 in the supernatant fraction were measured using enzyme-linked immunosorbent assay (ELISA). Expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were detected via Western blot. Results: The inhibition zones and minimal inhibitory concentration (MIC) values of bacterial strains were in the range of 14-26 mm and 62.5-250 µg/mL, respectively. Moreover, a remarkable activity against several fungal strains was revealed. TdLTP4 (5–100 µg/mL) decreased the production of NO (IC50= 4.32 µg/mL), IL-6 (IC50= 11.52 µg/mL), IL-1β (IC50= 7.87 µg/mL) and TNF-α (IC50 =8.66 µg/mL) by lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Our protein could modulate the macrophages inflammatory mode by causing reduction in iNOS and COX2.Conclusion: According to these findings, LTP fusion protein could be used as natural anti-inflammatory and antimicrobial agent in food preservation and human health.

Biological Control of Aedes albopictus: Obtained from the New Bacterial Candidates with Insecticidal Activity

Vector-borne deadly pathogens cause more than 700,000 deaths annually. They are transmitted by several vectors, among which the mosquito is the most important. Chemical compounds often have devastating side effects, leading to the abandonment of the majority of them. Biological control has been performed by using formulations of Bacillus sphaericus and Bacillus thuringiensis, but their intensive use has led to the emergence of resistance. Currently, the development of new alternative molecules is urgently needed, in order to use them in mosaics or in rotation with already known insecticides for the control of vectors, especially mosquitoes. Here, we attempted to identify bacterial species with potential anti-mosquito actions. Among bacterial strains isolated from dry sandy soil from Senegal, eleven strains from the Bacillales and Actinomycetales orders were chosen for the entomopathogenic activity experiments. Then, we tested their secondary metabolites, which were obtained from the supernatant fraction, and their cell wall and cytoplasmic compounds, which were found in the pellet fraction, in Aedes albopictus larvae, and compared the larval mortality rate with that obtained by using a commercial product. A total of 4/11 (36.36%) of the isolated species exhibited insecticidal activity. B. nealsonii, which is not a well-known bacterium, had the highest larvicidal effect with 70% of the larval mortality, which is highlighted for the first time. The Streptomyces species we isolated seem to be potential new species, and 3/5 (60%) of them exhibited insecticidal activity. Our study reports provide potential candidates for the identification of active molecules to be developed for strengthening the biological control of infectious diseases agents transmitted by mosquitoes.