Soil properties of Alhagi sparsifolia community in saline-sodic badlands in west China

A field experiment was conducted to assess the influences of soil chemical, physical, and biological properties of Alhagi sparsifolia community in Linze, Gaotai, and Guazhou County, Gansu province, China. Results showed that soils sampled were generally infertile with low levels of organic matter, available nitrogen, phosphorus, copper, manganese, and zinc with bacteria dominant microbial communities supporting A. sparsifolia. Available potassium and iron were sufficient in the study sites. With increasing soil layer depth, the contents of organic matter, available nitrogen, phosphorus, potassium, manganese, urease, dehydrogenase, bacteria, and actinomyces in the soil decreased significantly (P < 0.05), whereas the concentrations of moisture, available iron, and zinc in the soil increased significantly (P < 0.05). The contents of organic matter, available nitrogen, phosphorus, potassium, iron, manganese, zinc, copper, urease, dehydrogenase, bacteria, and actinomyces showed strong seasonal variations (P < 0.05). All these variables except dehydrogenase, bacteria, and actinomyces were the highest in summer and the lowest in spring. The comprehensive score of soil qualities was the greatest in Linze, medium in Guazhou, and lowest in Gaotai.     

Design,synthesis, molecular docking,anti-Proteus mirabilis and urease inhibition of new fluoroquinolone carboxylic acid derivatives

New hydroxamic acid, hydrazide and amide derivatives of ciprofloxacin in addition to their analogues of levofloxacin were prepared and identified by different spectroscopic techniques. Some of the prepared compounds revealed good activity against the urease splitting bacteria, Proteus mirabilis. The urease inhibitory activity was investigated using indophenol method. Most of the tested compounds showed better activity than the reference acetohydroxamic acid (AHA). The ciprofloxacin hydrazide derivative 3a and levofloxacin hydroxamic acid 7 experienced the highest activity (IC₅₀ = 1.22 μM and 2.20 μM, respectively). Molecular docking study revealed high spontaneous binding ability of the tested compounds to the active site of urease.     

Epidemiological characterization of serotype group B Streptococci neonatal infections associated with interleukin-6 level as a sensitive parameter for the early diagnosis

Group B streptococcal infection (Streptococcus agalactiae) is one of the leading causes of life-threatening disease in the early neonatal period, resulting in sepsis, pneumonia, and meningitis. During invasive infections, an excessive release of pro-inflammatory cytokine, such as interleukin-6 (IL-6), thus IL-6 gene is significant, as a diagnostic marker of systemic infection of the newborns. The present study aimed to describe the epidemiology diagnostic of GBS disease in neonatal by phenotypic and genotypic methods. Nine hundred and ninety-six samples were taken at Maternity and Children Hospital, Jeddah, Saudi Arabia for a period of one year (2011–2012). Results indicated that out of 217 infected samples, twenty (9.23.0%) were positive for group B Streptococci bacteria. This study also shows that female infants are more susceptible than males. The level of IL-6 was higher in mothers above 30 years. Twenty positive Streptococci group B isolates showed bands with the cylE gene primers in the border between 228 bp, 267 bp and 50 bp. Molecular detection by Real time polymerase chain reaction was also done to detect the target (Sip gene) encoding the Sip surface immunogenic protein. Specific primers and TaqMan probe were chosen for this purpose. A Real-time PCR method targeting the sip gene of GBS in neonates after delivery has been evaluated.     

Immobilization of urease on vapour phase stain etched porous silicon

Porous silicon layers fabricated by the reaction-induced vapor phase stain etch method were coated with 5% polyethylenimine. Urease from Canavalia brasiliensis beans was immobilized on this support through covalent linking with 2.5% glutaraldehyde. The pH and temperature profile of the immobilized and free urease exhibited higher activity at pH 6.5 and 37 °C. After being stored for 30 days at 4 °C, the immobilized enzyme had 75% of the initial activity. The maximum apparent Michaelis constant for free urease (K_m) was 94.33 mM whereas for immobilized urease was 53.04 mM. The maximum reaction velocity (V_max) for free urease was 3.51 mmol/min and for immobilized urease was 1.57 mmol/min.     

Degradation of 2,4-dichlorophenol by Bacillus sp. isolated from an aeration pond in the Baikalsk pulp and paper mill (Russia)

A pure culture of 2,4-dichlorophenol (2,4-DCP)-degrading bacteria was isolated from a natural enrichment that had been adapted to chlorophenols in the aeration pond of the Baikalsk pulp and paper mill (Russia). The bacteria were identified by 16S rDNA intergenic region analysis, using PCR with universal primers. Comparative analysis of the 16S rDNA sequence (1545 bp) in the GenBank database revealed that these bacteria are related to Bacillus cereus GN1. Degradation of 2,4-DCP was studied using this culture in liquid medium under aerobic conditions, at initial concentrations of 20–560 μM 2,4-DCP. The 2,4-DCP degradation rates by B. cereus GN1 could be determined at concentrations up to 400 μM. However, higher concentrations of 2,4-DCP (560 μM) were inhibitory to cell growth.     

In vitro biocontrol activity of halotolerant Streptomyces aureofaciens K20: A potent antagonist against Macrophomina phaseolina (Tassi) Goid

A halotolerant actinobacterial strain isolated from salinity affected soil of Eastern Indo-Gangetic plains (IGP), Uttar Pradesh, India, was characterised for its antagonistic potential against Macrophomina phaseolina by dual-culture assay. It was shown to effectively inhibit the growth of M. phaseolina with an inhibition zone of 27 ± 1.33 mm. Further the actinobacterial strain was evaluated for its plant growth promoting (PGP) properties and its ability to produce biocontrol related extracellular enzymes viz. amylase, protease, cellulase, chitinase, gelatinase and urease. The results revealed that the actinobacterial strain had PGP potential along with positive assay for amylase, chitinase and urease. The interaction study between antagonist strain and fungal pathogen, performed by scanning electron microscopy technique revealed that the actinobacterium was able to damage fungal mycelia may be due to chitinase, establishing its role as a potential antagonist against M. phaseolina. The actinobacterial isolate was characterised by 16S rDNA gene sequencing, and was identified as Streptomyces genera. The identified gene sequence was deposited to NCBI GenBank with an accession number KP331758.     

Distribution of anammox bacteria in domestic WWTPs and their enrichments evaluated by real-time quantitative PCR

Enrichment of anaerobic ammonium oxidation (anammox) bacteria using five activated sludges in three domestic wastewater treatment plants (WWTPs) were processed in a short term of 70 days and evaluated by real-time quantitative PCR (RTQ-PCR). Before the enrichment, building phylogenetic trees of Planctomycetes phylum in four reactors of sequencing batch reactor (SBR), anoxic and oxic reactors of anaerobic–anoxic–oxic (A2O) process, and rotating biological contactor (RBC) revealed six groups of distantly relative genera of Planctomyces, Pirellula, Gemmata, Isophaera, Candidatus and putative anammox bacteria. All clones of Candidatus sp. were affiliated with anammox bacteria and the majority of anammox clones were related to Planctomycete KSU-1 (AB057453). The discovery of anammox bacteria in raw activated sludges provided a partial rationale for the utilization of activated sludge as a seeding source of the anammox process. To verify the activity of anammox bacteria in the activated sludges, enrichment cultivations were conducted using SBRs. The enrichment of anammox bacteria resulted in the significant anammox activity of three samples. Quantification of 16S rRNA gene of anammox bacteria using RTQ-PCR showed the highest concentration of anammox bacteria of 2.48 ± 0.22 × 10⁹ copies of 16S rRNA gene/mg-volatile suspended solids (VSS), which was the same order of magnitude as that of the referential granular anammox sludge, 6.23 ± 0.59 × 10⁹ copies of 16S rRNA gene/mg-VSS, taken from an anammox upflow anaerobic sludge blanket (UASB) reactor. The doubling time of anammox bacteria enriched in this study was 1.18 days. The growth yield of anammox bacteria enriched in this study was 4.75 ± 0.57 × 10⁶ copies of 16S rRNA gene/mg of ammonium- and nitrite-nitrogen, which was similar to 4.50 ± 0.61 × 10⁶ copies of 16S rRNA gene/mg of ammonium- and nitrite-nitrogen for the referential anammox sludge. Substrate uptake rates of three successful enrichments at the end of the enrichment were comparable to those of granular and suspended anammox sludges. Rapid enrichment of anammox bacteria using activated sludge could offer an alternative method for obtaining a large volume of seeding anammox sludge.     

Synthesis,biological evaluation and molecular docking of N-phenyl thiosemicarbazones as urease inhibitors

Urease is an important enzyme which breaks urea into ammonia and carbon dioxide during metabolic processes. However, an elevated activity of urease causes various complications of clinical importance. The inhibition of urease activity with small molecules as inhibitors is an effective strategy for therapeutic intervention. Herein, we have synthesized a series of 19 benzofurane linked N-phenyl semithiocarbazones (3a–3s). All the compounds were screened for enzyme inhibitor activity against Jack bean urease. The synthesized N-phenyl thiosemicarbazones had varying activity levels with IC₅₀ values between 0.077 ± 0.001 and 24.04 ± 0.14 μM compared to standard inhibitor, thiourea (IC₅₀ = 21 ± 0.11 μM). The activities of these compounds may be due to their close resemblance of thiourea. A docking study with Jack bean urease (PDB ID: 4H9M) revealed possible binding modes of N-phenyl thiosemicarbazones.     

Lacipirellula parvula gen. nov., sp. nov., representing a lineage of planctomycetes widespread in low-oxygen habitats,description of the family Lacipirellulaceae fam. nov. and proposal of the orders Pirellulales ord. nov., Gemmatales ord. nov. and Isosphaerales ord. nov.

Pirellula-like planctomycetes are ubiquitous aquatic bacteria, which are often detected in anoxic or micro-oxic habitats. By contrast, the taxonomically described representatives of these bacteria, with very few exceptions, are strict aerobes. Here, we report the isolation and characterization of the facultatively anaerobic planctomycete, strain PX69^T, which was isolated from a boreal lake. Its 16S rRNA gene sequence is affiliated with the Pirellula-related Pir4 clade, which is dominated by environmental sequences retrieved from a variety of low-oxygen habitats. Strain PX69^T was represented by ellipsoidal cells that multiplied by budding and grew on sugars, some polysaccharides and glycerol. Anaerobic growth occurred by means of fermentation. Strain PX69^T grew at pH 5.5–7.5 and at temperatures between 10 and 30 °C. The major fatty acids were C18:1ω9c, C16:0 and C16:1ω7c; the major intact polar lipid was dimethylphosphatidylethanolamine. The complete genome of strain PX69^T was 6.92 Mb in size; DNA G + C content was 61.7 mol%. Among characterized planctomycetes, the highest 16S rRNA gene similarity (90.4%) was observed with ‘Bythopirellula goksoyri’ Pr1d, a planctomycete from deep-sea sediments. We propose to classify PX69^T as a novel genus and species, Lacipirellula parvula gen. nov., sp. nov.; the type strain is strain PX69^T (=KCTC 72398^T = CECT 9826^T = VKM B-3335^T). This genus is placed in a novel family, Lacipirellulaceae fam. nov., which belongs to the order Pirellulales ord. nov. Based on the results of comparative genome analysis, we also suggest establishment of the orders Gemmatales ord. nov. and Isosphaerales ord. nov. as well as an emendation of the order Planctomycetales.     

Effect of long-term exposure to mobile phone radiation on alpha-Int1 gene sequence of Candida albicans

Over the last decade, communication industries have witnessed a tremendous expansion, while, the biological effects of electromagnetic waves have not been fully elucidated. Current study aimed at evaluating the mutagenic effect of long-term exposure to 900-MHz radiation on alpha-Int1 gene sequences of Candida albicans. A standard 900 MHz radiation generator was used for radiation. 10 ml volumes from a stock suspension of C. albicans were transferred into 10 polystyrene tubes. Five tubes were exposed at 4 °C to a fixed magnitude of radiation with different time periods of 10, 70, 210, 350 and 490 h. The other 5 tubes were kept far enough from radiation. The samples underwent genomic DNA extraction. PCR amplification of alpha-Int1 gene sequence was done using one set of primers. PCR products were resolved using agarose gel electrophoresis and the nucleotide sequences were determined. All samples showed a clear electrophoretic band around 441 bp and further sequencing revealed the amplified DNA segments are related to alpha-Int1 gene of the yeast. No mutations in the gene were seen in radiation exposed samples. Long-term exposure of the yeast to mobile phone radiation under the above mentioned conditions had no mutagenic effect on alpha-Int1 gene sequence.     

3-Arylpropionylhydroxamic acid derivatives as Helicobacter pylori urease inhibitors: Synthesis,molecular docking and biological evaluation

Helicobacter pylori urease is involved in several physiologic responses such as stomach and duodenal ulcers, adenocarcinomas and stomach lymphomas. Thus, inhibition of urease is taken for a good chance to treat H. pylori-caused infections, we have therefore focused our efforts on seeking novel urease inhibitors. Here, a series of arylpropionylhydroxamic acids were synthesized and evaluated for urease inhibition. Out of these compounds, 3-(2-benzyloxy-5-chlorophenyl)-3-hydroxypropionylhydroxamic acid (d24) was the most active inhibitor with IC₅₀ of 0.15 ± 0.05 μM, showing a mixed inhibition with both competitive and uncompetitive aspects. Non-linear fitting of kinetic data gives kinetics parameters of 0.13 and 0.12 μg·mL⁻¹ for K_i and K_i′, respectively. The plasma protein binding assays suggested that d24 exhibited moderate binding to human and rabbit plasma proteins.     

Opisthorchis viverrini and Haplorchis taichui: Development of a multiplex PCR assay for their detection and differentiation using specific primers derived from HAT-RAPD

Specific primers for the detection of Opisthorchis viverrini and Haplorchis taichui were investigated by using the HAT-RAPD PCR method. Fourteen arbitrary primers (Operon Technologies) were performed for the generation of polymorphic DNA profiles. The results showed that a 319 bp fragment generated from the OPA-04 primer was expected to be O. viverrini-specific while a 256 bp fragment generated from the OPP-11 primer was considered to be H. taichui-specific. Based on each sequence data, two pairs of specific primers were designed and sequences of each primer were as follows; H. taichui; Hapt_F5′-GGCCAACGCAATCGTCATCC-3′and Hapt_R1 5′-CTCTCGACCTCCTCTAGAAT-3′ which yielded a 170 bp PCR product. For O. viverrini, OpV-1F: 5′-AATCGGGCTGCATATTGACCGAT-3′ and OpV-1R: 5′-CGGTGTTGCTTATTTTGCAGACAA-3′ which generated a 319 bp PCR product. These specific primers were tested for efficacy and specific detection for all parasites DNA samples. The results showed that 170 and 319 bp specific PCR products were generated as equivalent to positive result in H. taichui and O. viverrini, respectively by having no cross-reaction with any parasites tested. PCR conditions are recommended at 68 °C annealing temperature and with 0.5 mM magnesium chloride (Mg Cl₂). Additionally, specific primers developed in this study were effective to determine the presence of both parasites in fish and snail intermediate hosts, which the DNA of O. viverrini was artificially spiked since it is rarely found in northern Thailand.The H. taichui and O. viverrini-specific primers successfully developed in this study can be use for epidemiological monitoring, preventing management and control programs.     

Bacteria in bovine semen can increase sperm DNA fragmentation rates: a kinetic experimental approach

Cryopreserved straws of semen (n = 228) from Holstein bulls (n = 47) were examined for bacterial presence and sperm DNA fragmentation (SDF) dynamics. Commercial semen doses (representing six ejaculates per individual) were randomly selected from a bull stud in Spain. The dynamics of SDF were assessed after thawing (T0) and at 4, 24, 48, 72 and 96 h of incubation at 37 °C, using the commercial variant of the sperm chromatin dispersion test for Bovine (Halomax^®). One group of bulls showed a bacterial presence in semen samples between 0 and 96 h of incubation (n = 23, group A) while the other did not (n = 24, group B). Immediate post-thaw differences in SDF were not observed when both groups were compared. However, the rate of increase in SDF (rSDF) over time, considered as an estimate of the kinetic behaviour of sperm DNA survival, was significantly higher (P < 0.05) in semen samples from group A (0.7% per hour) versus group B (0.05% per hour). Polymerase Chain Reaction (PCR) assay was used for DNA amplification using primers designed for specific regions of the bacterial gene that codifies for 16S rRNA. Different species within the phyla Bacteroidetes, Firmicutes, Proteobacteria, Cyanobacteria, Fusobacteria and Actinobacteria were identified. The results show that (1) SDF at baseline (T0) may not be affected by the presence of bacteria but the rSDF can increase due to bacterial growth during incubation, (2) the increase in the rSDF is characteristic of some bulls but not for others, and (3) certain bacterial strains are repeatedly found in separate ejaculates from the same bull.     

RAPD-PCR and 16S rDNA phylogenetic analysis of alkaline protease producing bacteria isolated from soil of India: Identification and detection of genetic variability

178 bacterial strains were isolated from the soil samples collected from different regions of India out of which, 20 bacterial isolates were selected for alkaline protease production. The alkaline protease production efficiency of organisms was monitored at regular intervals (24 h) upto 7 days at 37 °C, pH 10. The 16S rDNA sequencing and RAPD-PCR based technique were used to identify the genetic variability among the 20 isolates of alkaline protease producing bacteria. The phylogenetic analysis indicated that the isolates can be separated into two clusters which could be further subdivided into five groups. Group 1 and 5 represented the family Bacillaceae, Groups 2 represented the Micrococcaceae family while Group 3 included the Arthrobacter bacterial group (family Micrococcaceae) from different geographical locations, respectively. Group 4 was identified as Pseudomonadaceae which was gram (−) bacteria. 21 different oligonucleotide primers were used to amplify approximately 261 fragments from each DNA sample. The bands were scored on the basis of their presence and absence and similarity between DNA samples was checked using Jaccard’s coefficient. Isolates were distinguished into distinct groups based on RAPD profiles from different geographical locations, morphological features and enzyme production efficiency. For cluster analysis the dendrogram was constructed using the unweighted pair group method with arithmetic averages (UPGMA). The results indicated that 16S rDNA and RAPD-PCR are suitable methods for rapid identification and differentiation of alkaline protease producing bacteria.     

Synthesis and antimicrobial activity of polyhalo isophthalonitrile derivatives

A series of polyhalo isophthalonitrile derivatives (3 and 4) that incorporate a variety of substituents at the 2-, 4-, 5- and/or 6-positions of the isophthalonitrile moieties have been designed and synthesized. These derivatives were evaluated for their antimicrobial activity against Staphylococcus aureus, Bacillus cereus (Gram-positive bacteria), Escherichia coli, Pseudomonas aeruginosa (Gram-negative bacteria); and Candida albicans (Fungi). Compounds 3 and 4 showed stronger inhibition of Gram-positive bacteria and fungi growth, and the antimicrobial ability of compound 3j (a 4-(benzylamino)-5-chloro-2,6-difluoro analog, MIC[SA] = 0.5 μg/mL; MIC[BC] = 0.4 μg/mL; MIC[CA] = 0.5 μg/mL) were close to nofloxacin and fluconazole and identified as the most potent antimicrobial agents in the series. The preliminary analysis of structure–activity relationships is also discussed.     

Synthesis and molecular docking study of some 5,6-dichloro-2-cyclopropyl-1H-benzimidazole derivatives bearing triazole,oxadiazole, and imine functionalities as potent inhibitors of urease

A new series of benzimidazole compounds including hydrazinecarbothioamide, 1,2,4-triazole, 1,3,4-oxadiazole and imine function were synthesized starting from 5,6-dichloro-2-cyclopropyl-1H-benzimidazole. All of the benzimidazole derivatives exhibited good urease inhibitor activity. Compound 6a proved to be the most potent showing an enzyme inhibitory activity with an IC₅₀ = 0.06 µM. Molecular docking studies were also conducted on enzyme extracted from Jack bean urease to identify the binding mode of the newly synthesized compounds.     

Rapid detection of Trichinella spiralis larvae in muscles by loop-mediated isothermal amplification

Trichinella spiralis is a tissue-dwelling nematode parasite. A loop-mediated isothermal amplification (LAMP) assay was developed and validated for the sensitive and rapid detection of T. spiralis larvae in muscle samples. Sixteen sets of primers were designed to recognise distinct sequences of a conserved gene, a 1.6 kb repetitive element of the Trichinella genome. One set of primers was selected as the most appropriate for rapid detection. The specificity and sensitivity of the primers in LAMP reactions for T. spiralis larvae and muscle samples of mice infected with T. spiralis were determined. Another 10 heterologous parasites were selected for specificity assays. The results showed that target DNA was amplified and visualised by monitoring turbidity and adding calcein detection methods within 70 min at an isothermal temperature of 63 °C. The sensitivity of LAMP with the detection limit of 362 fg/μl was >10 times higher than that for PCR. The designed primers had a good specificity. No cross-reactivity was found with the DNA of any other parasites. The assay was able to detect T. spiralis in all mouse muscle samples infected with 10 T. spiralis larvae on day 20 p.i. We believe this is the first report regarding the application of the LAMP assay for detection of T. spiralis larvae in muscle samples from experimentally infected mice. This method demonstrates a potentially valuable means for the direct detection of T. spiralis larvae in meat inspection.     

Barcoding old Korean lepidopteran specimens using newly designed specific primer pairs

Currently, DNA barcodes are often required to be analyzed using old museum specimens when they are the only available specimens for rare or endangered species, or even type series. In this study, using eight universal primers and newly designed 315 species-specific primers, we tried to recover full-length barcode sequences from 45 dried specimens of 36 butterfly species collected between 1959 and 1980 in Korea. The eight universal primers failed entirely in the PCR amplification and sequencing of all the specimens. On the other hand, 284 primer pairs consisting of the 315 primers, targeting fragments of 71–417 bp, amplified various lengths of barcode sequences from all specimens. The fragments were successfully combined to generate the barcode sequences ranging from 444 bp to 658 bp. Notably, of the 284 primer pairs, 26 primer pairs designed for Limenitis camilla, Argynnis niobe, and Brenthis daphne successfully amplified the barcode sequences of congeneric species, Limenitis doerriesi, Argynnis nerippe, and Brenthis ino, suggesting that the species-specific primers can be available for analyzing barcode sequences of closely related species. Our study reveals that the newly designed species-specific primers will be effective in acquiring COI sequences from old butterfly specimens.