Prevalence of Histidine Decarboxylase Genes of Gram-Positive Bacteria in Surströmming as Revealed by qPCR

Histamine is a degradation product of the bacterial decarboxylation of the amino acid histidine; such activity is determined by histidine decarboxylase encoded by a gene cluster, carried by some Gram-positive bacteria, that includes the hdcA gene. In this study, the presence of the hdcA gene in ready-to-eat surströmming samples collected from three producers based in Sweden was directly assessed via qPCR analysis for the very first time. Samples from producer A showed hdcA average gene abundance of 6.67 ± 0.13 Log cells/gene copies g⁻¹; in samples from producer B the average value attested at 5.56 ± 0.06 Log cells/gene copies g⁻¹, whereas for samples of producer C hdcA average gene abundance attested at 5.30 ± 0.08 Log cells/gene copies g⁻¹. ANOVA showed a significantly higher average hdcA gene copy number in samples from producer A, whereas no significant differences were seen between average values of hdcA gene copy numbers detected in samples from producer B and C. The hdcA gene copies detected in the present study could give an estimation of the load of potential histamine-producing bacteria in surströmming.     

Wildland fire as an atmospheric source of viable microbial aerosols and biological ice nucleating particles

The environmental sources of microbial aerosols and processes by which they are emitted into the atmosphere are not well characterized. In this study we analyzed microbial cells and biological ice nucleating particles (INPs) in smoke emitted from eight prescribed wildland fires in North Florida. When compared to air sampled prior to ignition, samples of the air–smoke mixtures contained fivefold higher concentrations of microbial cells (6.7 ± 1.3 × 10⁴ cells m⁻³) and biological INPs (2.4 ± 0.91 × 10³ INPs m⁻³ active at temperatures ≥ −15 °C), and these data significantly positively correlated with PM₁₀. Various bacteria could be cultured from the smoke samples, and the nearest neighbors of many of the isolates are plant epi- and endophytes, suggesting vegetation was a source. Controlled laboratory combustion experiments indicated that smoke emitted from dead vegetation contained significantly higher numbers of cells, INPs, and culturable bacteria relative to the green shrubs tested. Microbial viability of smoke aerosols based on formazan production and epifluorescent microscopy revealed no significant difference in the viable fraction (~80%) when compared to samples of ambient air. From these data, we estimate each fire aerosolized an average of 7 ± 4 × 10⁹ cells and 2 ± 1 × 10⁸ biological INPs per m² burned and conclude that emissions from wildland fire are sources of viable microbial aerosols to the atmosphere.Subject terms: Air microbiology, Microbial ecology, Microbial ecology, Forest ecology     

Glycoside hydrolase from the GH76 family indicates that marine Salegentibacter sp. Hel_I_6 consumes alpha-mannan from fungi

Microbial glycan degradation is essential to global carbon cycling. The marine bacterium Salegentibacter sp. Hel_I_6 (Bacteroidota) isolated from seawater off Helgoland island (North Sea) contains an α-mannan inducible gene cluster with a GH76 family endo-α-1,6-mannanase (ShGH76). This cluster is related to genetic loci employed by human gut bacteria to digest fungal α-mannan. Metagenomes from the Hel_I_6 isolation site revealed increasing GH76 gene frequencies in free-living bacteria during microalgae blooms, suggesting degradation of α-1,6-mannans from fungi. Recombinant ShGH76 protein activity assays with yeast α-mannan and synthetic oligomannans showed endo-α-1,6-mannanase activity. Resolved structures of apo-ShGH76 (2.0 Å) and of mutants co-crystalized with fungal mannan-mimicking α-1,6-mannotetrose (1.90 Å) and α-1,6-mannotriose (1.47 Å) retained the canonical (α/α)₆ fold, despite low identities with sequences of known GH76 structures (GH76s from gut bacteria: <27%). The apo-form active site differed from those known from gut bacteria, and co-crystallizations revealed a kinked oligomannan conformation. Co-crystallizations also revealed precise molecular-scale interactions of ShGH76 with fungal mannan-mimicking oligomannans, indicating adaptation to this particular type of substrate. Our data hence suggest presence of yet unknown fungal α-1,6-mannans in marine ecosystems, in particular during microalgal blooms.Subject terms: Metagenomics, Microbial ecology, Structural biology, Fungal ecology, Molecular ecology     

Phycosphere pH of unicellular nano- and micro- phytoplankton cells and consequences for iron speciation

Surface ocean pH is declining due to anthropogenic atmospheric CO₂ uptake with a global decline of ~0.3 possible by 2100. Extracellular pH influences a range of biological processes, including nutrient uptake, calcification and silicification. However, there are poor constraints on how pH levels in the extracellular microenvironment surrounding phytoplankton cells (the phycosphere) differ from bulk seawater. This adds uncertainty to biological impacts of environmental change. Furthermore, previous modelling work suggests that phycosphere pH of small cells is close to bulk seawater, and this has not been experimentally verified. Here we observe under 140 μmol photons·m⁻²·s⁻¹ the phycosphere pH of Chlamydomonas concordia (5 µm diameter), Emiliania huxleyi (5 µm), Coscinodiscus radiatus (50 µm) and C. wailesii (100 µm) are 0.11 ± 0.07, 0.20 ± 0.09, 0.41 ± 0.04 and 0.15 ± 0.20 (mean ± SD) higher than bulk seawater (pH 8.00), respectively. Thickness of the pH boundary layer of C. wailesii increases from 18 ± 4 to 122 ± 17 µm when bulk seawater pH decreases from 8.00 to 7.78. Phycosphere pH is regulated by photosynthesis and extracellular enzymatic transformation of bicarbonate, as well as being influenced by light intensity and seawater pH and buffering capacity. The pH change alters Fe speciation in the phycosphere, and hence Fe availability to phytoplankton is likely better predicted by the phycosphere, rather than bulk seawater. Overall, the precise quantification of chemical conditions in the phycosphere is crucial for assessing the sensitivity of marine phytoplankton to ongoing ocean acidification and Fe limitation in surface oceans.Subject terms: Water microbiology, Microbial biooceanography, Biogeochemistry     

Small phytoplankton contribute greatly to CO2-fixation after the diatom bloom in the Southern Ocean

Phytoplankton is composed of a broad-sized spectrum of phylogenetically diverse microorganisms. Assessing CO₂-fixation intra- and inter-group variability is crucial in understanding how the carbon pump functions, as each group of phytoplankton may be characterized by diverse efficiencies in carbon fixation and export to the deep ocean. We measured the CO₂-fixation of different groups of phytoplankton at the single-cell level around the naturally iron-fertilized Kerguelen plateau (Southern Ocean), known for intense diatoms blooms suspected to enhance CO₂ sequestration. After the bloom, small cells (<20 µm) composed of phylogenetically distant taxa (prymnesiophytes, prasinophytes, and small diatoms) were growing faster (0.37 ± 0.13 and 0.22 ± 0.09 division d⁻¹ on- and off-plateau, respectively) than larger diatoms (0.11 ± 0.14 and 0.09 ± 0.11 division d⁻¹ on- and off-plateau, respectively), which showed heterogeneous growth and a large proportion of inactive cells (19 ± 13%). As a result, small phytoplankton contributed to a large proportion of the CO₂ fixation (41–70%). The analysis of pigment vertical distribution indicated that grazing may be an important pathway of small phytoplankton export. Overall, this study highlights the need to further explore the role of small cells in CO₂-fixation and export in the Southern Ocean.Subject terms: Biogeochemistry, Biogeochemistry, Stable isotope analysis, Microbial ecology     

Active predation,phylogenetic diversity,and global prevalence of myxobacteria in wastewater treatment plants

The operation of modern wastewater treatment plants (WWTPs) is driven by activated sludge microbiota, a complex assemblage of trophically interacting microorganisms. Microbial predation is crucial to fundamental understanding of how biological interactions drive microbiome structuring and functioning of WWTPs. However, predatory bacteria have received little attention regarding their diversity, activity, and ecological function in activated sludge, limiting the exploitation of food web interactions for wastewater microbiome engineering. Here, by using rRNA-stable isotope probing of activated sludge microbiota with ¹³C-labeled prey bacteria, we uncovered diverse as-yet-uncultivated putative predatory bacteria that actively incorporated ¹³C-biomass. Myxobacteria, especially Haliangium and the mle1-27 clade, were found as the dominant active predators, refreshing conventional views based on a few predatory isolates of Bdellovibrionota from WWTPs. The identified predatory bacteria showed more selective predation on prey compared with the protists dominated by ciliates, providing in situ evidence for inter-domain predation behavior divergence in activated sludge. Putative predatory bacteria were tracked over a two-year microbiome monitoring effort at a local WWTP, revealing the predominance of Myxococcota (6.5 ± 1.3%) over Bdellovibrionota (1.0 ± 0.2%) lineages. Phylogenetic analysis unveiled highly diverse myxobacteria inhabiting activated sludge and suggested a habitat filtering effect in global WWTPs. Further mining of a global activated sludge microbiome dataset revealed the prevalence of Myxococcota (5.4 ± 0.1%) species and potential impacts of myxobacterial predation on process performance. Collectively, our findings provided unique insights into the predating activity, diversity, and prevalence of Myxococcota species in activated sludge, highlighting their links with wastewater treatment processes via trophic regulation of enteric and functional bacteria.Subject terms: Microbial ecology, Biodiversity, Environmental microbiology, Microbial ecology, Environmental sciences     

An all-taxon microbial inventory of the Moorea coral reef ecosystem

The Moorea Coral Reef Long Term Ecological Research (LTER) Site (17.50°S, 149.83°W) comprises the fringe of coral reefs and lagoons surrounding the volcanic island of Moorea in the Society Islands of French Polynesia. As part of our Microbial Inventory Research Across Diverse Aquatic LTERS biodiversity inventory project, we characterized microbial community composition across all three domains of life using amplicon pyrosequencing of the V6 (bacterial and archaeal) and V9 (eukaryotic) hypervariable regions of small-subunit ribosomal RNA genes. Our survey spanned eight locations along a 130-km transect from the reef lagoon to the open ocean to examine changes in communities along inshore to offshore gradients. Our results illustrate consistent community differentiation between inshore and offshore ecosystems across all three domains, with greater richness in all domains in the reef-associated habitats. Bacterial communities were more homogenous among open ocean sites spanning >100 km than among inshore sites separated by <1 km, whereas eukaryotic communities varied more offshore than inshore, and archaea showed more equal levels of dissimilarity among subhabitats. We identified signature communities representative of specific geographic and geochemical milieu, and characterized co-occurrence patterns of specific microbial taxa within the inshore ecosystem including several bacterial groups that persist in geographical niches across time. Bacterial and archaeal communities were dominated by few abundant taxa but spatial patterning was consistent through time and space in both rare and abundant communities. This is the first in-depth inventory analysis of biogeographic variation of all three microbial domains within a coral reef ecosystem.     

Allergic airway inflammation induces the migration of dendritic cells into airway sensory ganglia

Background

A neuroimmune crosstalk between dendritic cells (DCs) and airway nerves in the lung has recently been reported. However, the presence of DCs in airway sensory ganglia under normal and allergic conditions has not been explored so far. Therefore, this study aims to investigate the localisation, distribution and proliferation of DCs in airway sensory ganglia under allergic airway inflammation.

Methods

Using the house dust mite (HDM) model for allergic airway inflammation BALB/c mice were exposed to HDM extract intranasally (25 μg/50 μl) for 5 consecutive days a week over 7 weeks. With the help of the immunohistochemistry, vagal jugular-nodose ganglia complex (JNC) sections were analysed regarding their expression of DC-markers (MHC II, CD11c, CD103), the neuronal marker PGP 9.5 and the neuropeptide calcitonin gene-related peptide (CGRP) and glutamine synthetase (GS) as a marker for satellite glia cells (SGCs). To address the original source of DCs in sensory ganglia, a proliferation experiment was also carried in this study.

Results

Immune cells with characteristic DC-phenotype were found to be closely located to SGCs and vagal sensory neurons under physiological conditions. The percentage of DCs in relation to neurons was significantly increased by allergic airway inflammation in comparison to the controls (HDM 51.38 ± 2.38% vs. control 28.16 ± 2.86%, p < 0.001). The present study also demonstrated that DCs were shown to proliferate in jugular-nodose ganglia, however, the proliferation rate of DCs is not significantly changed in the two treated animal groups (proliferating DCs/ total DCs: HDM 0.89 ± 0.38%, vs. control 1.19 ± 0.54%, p = 0.68). Also, increased number of CGRP-positive neurons was found in JNC after allergic sensitisation and challenge (HDM 31.16 ± 5.41% vs. control 7.16 ± 1.53%, p < 0.001).

Conclusion

The present findings suggest that DCs may migrate from outside into the ganglia to interact with sensory neurons enhancing or protecting the allergic airway inflammation. The increase of DCs as well as CGRP-positive neurons in airway ganglia by allergic airway inflammation indicate that intraganglionic DCs and neurons expressing CGRP may contribute to the pathogenesis of bronchial asthma. To understand this neuroimmune interaction in allergic airway inflammation further functional experiments should be carried out in future studies.     

Mitochondrial DNA alteration in obstructive sleep apnea

Background

Obstructive Sleep Apnea (OSAS) is a disease associated with the increase of cardiovascular risk and it is characterized by repeated episodes of Intermittent Hypoxia (IH) which inducing oxidative stress and systemic inflammation. Mitochondria are cell organelles involved in the respiratory that have their own DNA (MtDNA). The aim of this study was to investigate if the increase of oxidative stress in OSAS patients can induce also MtDNA alterations.

Methods

46 OSAS patients (age 59.27 ± 11.38; BMI 30.84 ± 3.64; AHI 36.63 ± 24.18) were compared with 36 control subjects (age 54.42 ± 6.63; BMI 29.06 ± 4.7; AHI 3.8 ± 1.10). In blood cells Content of MtDNA and nuclear DNA (nDNA) was measured in OSAS patients by Real Time PCR. The ratio between MtDNA/nDNA was then calculated. Presence of oxidative stress was evaluated by levels of Reactive Oxygen Metabolites (ROMs), measured by diacron reactive oxygen metabolite test (d-ROM test).

Results

MtDNA/nDNA was higher in patients with OSAS than in the control group (150.94 ± 49.14 vs 128.96 ± 45.8; p = 0.04), the levels of ROMs were also higher in OSAS subjects (329.71 ± 70.17 vs 226 ± 36.76; p = 0.04) and they were positively correlated with MtDNA/nDNA (R = 0.5, p < 0.01).

Conclusions

In OSAS patients there is a Mitochondrial DNA damage induced by the increase of oxidative stress. Intermittent hypoxia seems to be the main mechanism which leads to this process.     

In vitro and in silico studies of bis (indol-3-yl) methane derivatives as potential α-glucosidase and α-amylase inhibitors

In this paper, bis (indol-3-yl) methanes (BIMs) were synthesised and evaluated for their inhibitory activity against α-glucosidase and α-amylase. All synthesised compounds showed potential α-glucosidase and α-amylase inhibitory activities. Compounds 5 g (IC₅₀: 7.54 ± 1.10 μM), 5e (IC₅₀: 9.00 ± 0.97 μM), and 5 h (IC₅₀: 9.57 ± 0.62 μM) presented strongest inhibitory activities against α-glucosidase, that were ∼ 30 times stronger than acarbose. Compounds 5 g (IC₅₀: 32.18 ± 1.66 µM), 5 h (IC₅₀: 31.47 ± 1.42 µM), and 5 s (IC₅₀: 30.91 ± 0.86 µM) showed strongest inhibitory activities towards α-amylase, ∼ 2.5 times stronger than acarbose. The mechanisms and docking simulation of the compounds were also studied. Compounds 5 g and 5 h exhibited bifunctional inhibitory activity against these two enzymes. Furthermore, compounds showed no toxicity against 3T3-L1 cells and HepG2 cells.

Highlights

1. A series of bis (indol-3-yl) methanes (BIMs) were synthesised and evaluated inhibitory activities against α-glucosidase and α-amylase.
2. Compound 5g exhibited promising activity (IC₅₀ = 7.54 ± 1.10 μM) against α-glucosidase.
3. Compound 5s exhibited promising activity (IC₅₀ = 30.91 ± 0.86 μM) against α-amylase.
4. In silico studies were performed to confirm the binding interactions of synthetic compounds with the enzyme active site.

     

Exploring the interaction of myricetin with human alpha-2-macroglobulin: biophysical and in-silico analysis

Myricetin (MYR) is a bioactive secondary metabolite found in plants that is recognized for its nutraceutical value and is an essential constituent of various foods and beverages. It is reported to exhibit a plethora of activities, including antioxidant, antimicrobial, antidiabetic, anticancer, and anti-inflammatory. Alpha-2-macroglobulin (α2M) is a major plasma anti-proteinase that can inhibit proteinases of both human and non-human origin, regardless of their specificity and catalytic mechanism. Here, we explored the interaction of MYR-α2M using various biochemical and biophysical techniques. It was found that the interaction of MYR brings subtle change in its anti-proteolytic potential and thereby alters its structure and function, as can be seen from absorbance and fluorescence spectroscopy. UV spectroscopy of α2M in presence of MYR indicated the occurrence of hyperchromism, suggesting complex formation. Fluorescence spectroscopy reveals that MYR reduces the fluorescence intensity of native α2M with a shift in the wavelength maxima. At 318.15 K, MYR binds to α2M with a binding constant of 2.4 × 10³ M⁻¹, which indicates significant binding. The ΔG value was found to be − 7.56 kcal mol⁻¹ at 298.15 K, suggesting the interaction to be spontaneous and thermodynamically favorable. The secondary structure of α2M does not involve any major change as was confirmed by CD analysis. The molecular docking indicates that Asp-146, Ser-172, Glu-174, and Tyr-180 were the key residues involved in α2M-MYR complex formation. This study contributes to our understanding of the function and mechanism of protein and flavonoid binding by providing a molecular basis of the interaction between MYR and α2M.     

Engineering defensin α‐helix to produce high‐affinity SARS‐CoV‐2 spike protein binding ligands

The binding of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike protein to the angiotensin‐converting enzyme 2 (ACE2) receptor expressed on the host cells is a critical initial step for viral infection. This interaction is blocked through competitive inhibition by soluble ACE2 protein. Therefore, developing high‐affinity and cost‐effective ACE2 mimetic ligands that disrupt this protein–protein interaction is a promising strategy for viral diagnostics and therapy. We employed human and plant defensins, a class of small (2–5 kDa) and highly stable proteins containing solvent‐exposed alpha‐helix, conformationally constrained by two disulfide bonds. Therefore, we engineered the amino acid residues on the constrained alpha‐helix of defensins to mimic the critical residues on the ACE2 helix 1 that interact with the SARS‐CoV‐2 spike protein. The engineered proteins (h‐deface2, p‐deface2, and p‐deface2‐MUT) were soluble and purified to homogeneity with a high yield from a bacterial expression system. The proteins demonstrated exceptional thermostability (Tm 70.7°C), high‐affinity binding to the spike protein with apparent K _d values of 54.4 ± 11.3, 33.5 ± 8.2, and 14.4 ± 3.5 nM for h‐deface2, p‐deface2, and p‐deface2‐MUT, respectively, and were used in a diagnostic assay that detected SARS‐CoV‐2 neutralizing antibodies. This work addresses the challenge of developing helical ACE2 mimetics by demonstrating that defensins provide promising scaffolds to engineer alpha‐helices in a constrained form for designing of high‐affinity ligands.     

The effects of acute oral glutamine supplementation on exercise-induced gastrointestinal permeability and heat shock protein expression in peripheral blood mononuclear cells

Chronic glutamine supplementation reduces exercise-induced intestinal permeability and inhibits the NF-κB pro-inflammatory pathway in human peripheral blood mononuclear cells. These effects were correlated with activation of HSP70. The purpose of this paper is to test if an acute dose of oral glutamine prior to exercise reduces intestinal permeability along with activation of the heat shock response leading to inhibition of pro-inflammatory markers. Physically active subjects (N = 7) completed baseline and exercise intestinal permeability tests, determined by the percent ratio of urinary lactulose (5 g) to rhamnose (2 g). Exercise included two 60-min treadmill runs at 70 % of VO₂max at 30 °C after ingestion of glutamine (Gln) or placebo (Pla). Plasma levels of endotoxin and TNF-α, along with peripheral blood mononuclear cell (PBMC) protein expression of HSP70 and IκBα, were measured pre- and post-exercise and 2 and 4 h post-exercise. Permeability increased in the Pla trial compared to that at rest (0.06 ± 0.01 vs. 0.02 ± 0.018) and did not increase in the Gln trial. Plasma endotoxin was lower at the 4-h time point in the Gln vs. 4 h in the Pla (6.715 ± 0.046 pg/ml vs. 7.952 ± 1.11 pg/ml). TNF-α was lower 4 h post-exercise in the Gln vs. Pla (1.64 ± 0.09 pg/ml vs. 1.87 ± 0.12 pg/ml). PBMC expression of IkBα was higher 4 h post-exercise in the Gln vs. 4 h in the Pla (1.29 ± 0.43 vs. 0.8892 ± 0.040). HSP70 was higher pre-exercise and 2 h post-exercise in the Gln vs. Pla (1.35 ± 0.21 vs. 1.000 ± 0.000 and 1.65 ± 0.21 vs. 1.27 ± 0.40). Acute oral glutamine supplementation prevents an exercise-induced rise in intestinal permeability and suppresses NF-κB activation in peripheral blood mononuclear cells.     

Genomic and kinetic analysis of novel Nitrospinae enriched by cell sorting

Chemolithoautotrophic nitrite-oxidizing bacteria (NOB) are key players in global nitrogen and carbon cycling. Members of the phylum Nitrospinae are the most abundant, known NOB in the oceans. To date, only two closely affiliated Nitrospinae species have been isolated, which are only distantly related to the environmentally abundant uncultured Nitrospinae clades. Here, we applied live cell sorting, activity screening, and subcultivation on marine nitrite-oxidizing enrichments to obtain novel marine Nitrospinae. Two binary cultures were obtained, each containing one Nitrospinae strain and one alphaproteobacterial heterotroph. The Nitrospinae strains represent two new genera, and one strain is more closely related to environmentally abundant Nitrospinae than previously cultured NOB. With an apparent half-saturation constant of 8.7 ± 2.5 µM, this strain has the highest affinity for nitrite among characterized marine NOB, while the other strain (16.2 ± 1.6 µM) and Nitrospina gracilis (20.1 ± 2.1 µM) displayed slightly lower nitrite affinities. The new strains and N. gracilis share core metabolic pathways for nitrite oxidation and CO₂ fixation but differ remarkably in their genomic repertoires of terminal oxidases, use of organic N sources, alternative energy metabolisms, osmotic stress and phage defense. The new strains, tentatively named “Candidatus Nitrohelix vancouverensis” and “Candidatus Nitronauta litoralis”, shed light on the niche differentiation and potential ecological roles of Nitrospinae.Subject terms: Water microbiology, Microbial ecology, Biogeochemistry     

Protistan microbial observatory in the Cariaco Basin,Caribbean. I. Pyrosequencing vs Sanger insights into species richness

Microbial diversity and distribution are topics of intensive research. In two companion papers in this issue, we describe the results of the Cariaco Microbial Observatory (Caribbean Sea, Venezuela). The Basin contains the largest body of marine anoxic water, and presents an opportunity to study protistan communities across biogeochemical gradients. In the first paper, we survey 18S ribosomal RNA (rRNA) gene sequence diversity using both Sanger- and pyrosequencing-based approaches, employing multiple PCR primers, and state-of-the-art statistical analyses to estimate microbial richness missed by the survey. Sampling the Basin at three stations, in two seasons, and at four depths with distinct biogeochemical regimes, we obtained the largest, and arguably the least biased collection of over 6000 nearly full-length protistan rRNA gene sequences from a given oceanographic regime to date, and over 80 000 pyrosequencing tags. These represent all major and many minor protistan taxa, at frequencies globally similar between the two sequence collections. This large data set provided, via the recently developed parametric modeling, the first statistically sound prediction of the total size of protistan richness in a large and varied environment, such as the Cariaco Basin: over 36 000 species, defined as almost full-length 18S rRNA gene sequence clusters sharing over 99% sequence homology. This richness is a small fraction of the grand total of known protists (over 100 000–500 000 species), suggesting a degree of protistan endemism.     

Tuning the activity of iminosugars: novel N-alkylated deoxynojirimycin derivatives as strong BuChE inhibitors

We have designed unprecedented cholinesterase inhibitors based on 1-deoxynojirimycin as potential anti-Alzheimer’s agents. Compounds are comprised of three key structural motifs: the iminosugar, for interaction with cholinesterase catalytic anionic site (CAS); a hydrocarbon tether with variable lengths, and a fragment derived from 2-phenylethanol for promoting interactions with peripheral anionic site (PAS). Title compounds exhibited good selectivity towards BuChE, strongly depending on the substitution pattern and the length of the tether. The lead compounds were found to be strong mixed inhibitors of BuChE (IC₅₀ = 1.8 and 1.9 µM). The presumptive binding mode of the lead compound was analysed using molecular docking simulations, revealing H-bond interactions with the catalytic subsite (His438) and CAS (Trp82 and Glu197) and van der Waals interactions with PAS (Thr284, Pro285, Asn289). They also lacked significant antiproliferative activity against tumour and non-tumour cells at 100 µM, making them promising new agents for tackling Alzheimer’s disease through the cholinergic approach.     

Genetic distance as an alternative to physical distance for definition of gene units in association studies

Background

Some association studies, as the implemented in VEGAS, ALIGATOR, i-GSEA4GWAS, GSA-SNP and other software tools, use genes as the unit of analysis. These genes include the coding sequence plus flanking sequences. Polymorphisms in the flanking sequences are of interest because they involve cis-regulatory elements or they inform on untyped genetic variants trough linkage disequilibrium. Gene extensions have customarily been defined as ± 50 Kb. This approach is not fully satisfactory because genetic relationships between neighbouring sequences are a function of genetic distances, which are only poorly replaced by physical distances.

Results

Standardized recombination rates (SRR) from the deCODE recombination map were used as units of genetic distances. We searched for a SRR producing flanking sequences near the ± 50 Kb offset that has been common in previous studies. A SRR ≥ 2 was selected because it led to gene extensions with median length = 45.3 Kb and the simplicity of an integer value. As expected, boundaries of the genes defined with the ± 50 Kb and with the SRR ≥2 rules were rarely concordant. The impact of these differences was illustrated with the interpretation of top association signals from two large studies including many hits and their detailed analysis based in different criteria. The definition based in genetic distance was more concordant with the results of these studies than the based in physical distance. In the analysis of 18 top disease associated loci form the first study, the SRR ≥2 genes led to a fully concordant interpretation in 17 loci; the ± 50 Kb genes only in 6. Interpretation of the 43 putative functional genes of the second study based in the SRR ≥2 definition only missed 4 of the genes, whereas the based in the ± 50 Kb definition missed 10 genes.

Conclusions

A gene definition based on genetic distance led to results more concordant with expert detailed analyses than the commonly used based in physical distance. The genome coordinates for each gene are provided to maintain a simple use of the new definitions.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-408) contains supplementary material, which is available to authorized users.     

Development of Acid-Resistant Alginate/Trimethyl Chitosan Nanoparticles Containing Cationic β-Cyclodextrin Polymers for Insulin Oral Delivery

In this study, the use of trimethylchitosan (TMC), by higher solubility in comparison with chitosan, in alginate/chitosan nanoparticles containing cationic β-cyclodextrin polymers (CPβCDs) has been studied, with the aim of increasing insulin uptake by nanoparticles. Firstly, TMCs were synthesized by iodomethane, and CPβCDs were synthesized within a one-step polycondensation reaction using choline chloride (CC) and epichlorohydrine (EP). Insulin–CβCDPs complex was prepared by mixing 1:1 portion of insulin and CPβCDs solutions. Then, nanoparticles prepared in a three-step procedure based on the iono-tropic pregelation method. Nanoparticles screened using experimental design and Placket Burman methodology to obtain minimum size and polydispercity index (pdI) and the highest entrapment efficiency (EE). CPβCDs and TMC solution concentration and pH and alginate and calcium chloride solution concentrations are found as the significant parameters on size, PdI, and EE. The nanoparticles with proper physicochemical properties were obtained; the size, PdI, and EE% of optimized nanoparticles were reported as 150.82 ± 21 nm, 0.362 ± 0.036, and 93.2% ± 4.1, respectively. The cumulative insulin release in intestinal condition achieved was 50.2% during 6 h. By SEM imaging, separate, spherical, and nonaggregated nanoparticles were found. In the cytotoxicity studies on Caco-2 cell culture, no significant cytotoxicity was observed in 5 h of incubation, but after 24 h of incubation, viability was decreased to 50% in 0.5 mμ of TMC concentration. Permeability studies across Caco-2 cells had been carried out, and permeability achieved in 240 min was 8.41 ± 0.39%, which shows noticeable increase in comparison with chitosan nanoparticles. Thus, according to the results, the optimized nanoparticles can be used as a new insulin oral delivery system.KEY WORDS: alginate, cationic β-cyclodextrin, insulin nanoparticle, oral delivery, trimethyl chitosan     

Design and green synthesis of novel quinolinone derivatives of potential anti-breast cancer activity against MCF-7 cell line targeting multi-receptor tyrosine kinases

A new set of 4,6,7,8-tetrahydroquinolin-5(1H)-ones were designed as cytotoxic agents against breast cancer cell line (MCF-7) and synthesised under ultrasonic irradiation using chitosan decorated copper nanoparticles (CS/CuNPs) catalyst. The new compounds 4b, 4j, 4k, and 4e exhibited the most potent cytotoxic activity of IC₅₀ values (0.002 − 0.004 µM) comparing to Staurosporine of IC₅₀; 0.005 μM. The latter derivatives exhibited a promising safety profile against the normal human WI38 cells of IC₅₀ range 0.0149 − 0.048 µM. Furthermore, the most promising cytotoxic compounds 4b, 4j were evaluated as multi-targeting agents against the RTK protein kinases; EGFR, HER-2, PDGFR-β, and VEGFR-2. Compound 4j showed promising inhibitory activity against HER-2 and PDGFR-β of IC₅₀ values 0.17 × 10⁻³, 0.07 × 10⁻³ µM in comparison with the reference drug sorafenib of IC₅₀; 0.28 × 10⁻³, 0.13 × 10⁻³ µM, respectively. In addition, 4j induced apoptotic effect and cell cycle arrest at G2/M phase preventing the mitotic cycle in MCF-7 cells.