Monosaccharide precursors for boosting chondroitin-like capsular polysaccharide production

Chondroitin sulfate is a well-known bioactive molecule, widely used as an anti-osteoarthritis drug, that is nowadays mainly produced by animal tissue sources with unsafe extraction procedures. Recent studies have explored an integrated biotechnological–chemical strategy to obtain a chondroitin sulfate precursor from Escherichia coli K4 capsular polysaccharide, demonstrating the influence of environmental and growth conditions on capsule synthesis. In this research work, the flexibility of the strain biosynthetic machinery was investigated to enhance the K4 capsular polysaccharide production by supplementing the growth medium with the monosaccharides (glucuronic acid, galactosamine and fructose) that constitute the chain. Shake flask experiments were performed by adding the sugars singularly or together, by testing monosaccharide different concentrations and times of addition and by observing the bacterial sugar consumption. A K4 capsular polysaccharide production enhancement, compared to the control, was observed in all cases of supplementation and, in particular, significant 68 and 57 % increases were observed when adding 0.385 mM glucuronic acid plus galactosamine or 0.385 mM fructose, respectively. Increased expression levels of the gene kfoC, coding for a K4 polymerase, evaluated in different growth conditions, confirmed the results at the molecular level. Furthermore, batch fermentations, performed in lab-scale reactors (2 L), allowed to double the K4 capsular polysaccharide production values obtained in shake flask conditions, by means of a strict control of the growth parameters.     

A monolithic silicon based integrated signal generation and detection system for monitoring DNA hybridisation

The aim of this work was to develop an integrated solution to DNA hybridisation monitoring for diagnostics on a monolithic silicon platform. A fabrication process was developed incorporating a gold initiation electrode patterned directly onto a PIN photodiode detector. Patterned interdigitated type electrodes exhibited the smallest reduction in photodiode sensitivity, therefore these were chosen as the ECL initiator design. A novel DNA hybridisation assay was developed based on the displacement of a partially mismatched complementary strand by a perfectly matched labelled complementary strand. Pre-hybridised thiolated oligonucleotide and unlabelled 25% mismatched oligonucleotide were assembled on the gold initiation electrode. On addition of the labelled perfectly complementary oligonucleotide, the mismatched strands were displaced and a signal was generated. The sensitivity of the photodiode to light emitted at 620 nm, the ruthenium emission wavelength, was determined and subsequently, the diode current response to light generated by flow addition of ruthenium solution was found to be measurable to a concentration of 10 fM. Pre-hybridised duplex DNA, consisting of thiolated oligonucleotide and ruthenium labelled complementary oligonucleotide, was assembled on the gold initiation electrode. The difference between the current measured during flow of buffer and the ECL co-reactant TPA was three orders of magnitude, indicating that DNA assembled on the surface comprised sufficient ruthenium to generate a measurable signal. Finally, the displacement of unlabelled partial mismatch oligonucleotide from the sensor surface was monitored on addition of the ruthenium labelled perfectly complementary oligonucleotide in TPA flow and the measured photodiode current response was up to 50 times greater.     

IgE recognition patterns of profilin, PR-10, and tropomyosin panallergens tested in 3,113 allergic patients by allergen microarray-based technology

Background

IgE recognition of panallergens having highly conserved sequence regions, structure, and function and shared by inhalant and food allergen sources is often observed.

Methods

We evaluated the IgE recognition profile of profilins (Bet v 2, Cyn d 12, Hel a 2, Hev b 8, Mer a 1, Ole e 2, Par j 3, Phl p 12, Pho d 2), PR-10 proteins (Aln g 1, Api g 1, Bet v 1.0101, Bet v 1.0401, Cor a 1, Dau c 1 and Mal d 1.0108) and tropomyosins (Ani s 3, Der p 10, Hel as 1, Pen i 1, Pen m 1, Per a 7) using the Immuno-Solid phase Allergen Chip (ISAC) microarray system. The three panallergen groups were well represented among the allergenic molecules immobilized on the ISAC. Moreover, they are distributed in several taxonomical allergenic sources, either close or distant, and have a route of exposure being either inhalation or ingestion.

Results

3,113 individuals (49.9% female) were selected on the basis of their reactivity to profilins, PR-10 or tropomyosins. 1,521 (48.8%) patients were reactive to profilins (77.6% Mer a 1 IgE⁺), 1,420 (45.6%) to PR-10 (92.5% Bet v 1 IgE⁺) and 632 (20.3%) to tropomyosins (68% Der p 10 IgE⁺). A significant direct relationship between different representative molecules within each group of panallergens was found. 2,688 patients (86.4%) recognized only one out of the three distinct groups of molecules as confirmed also by hierarchical clustering analysis.

Conclusions

Unless exposed to most of the allergens in the same or related allergenic sources, a preferential IgE response to distinct panallergens has been recorded. Allergen microarray IgE testing increases our knowledge of the IgE immune response and related epidemiological features within and between homologous molecules better describing the patients'' immunological phenotypes.     

Microtubule defects in mesenchymal stromal cells distinguish patients with Progressive Supranuclear Palsy

Progressive Supranuclear Palsy (PSP) is a rare neurodegenerative disease whose etiopathogenesis remains elusive. The intraneuronal accumulation of hyperphosphorylated Tau, a pivotal protein in regulating microtubules (MT), leads to include PSP into tauopathies. Pathological hallmarks are well known in neural cells but no word yet if PSP‐linked dysfunctions occur also in other cell types. We focused on bone marrow mesenchymal stromal cells (MSCs) that have recently gained attention for therapeutic interventions due to their anti‐inflammatory, antiapoptotic and trophic properties. Here, we aimed to investigate MSCs biology and to disclose if any disease‐linked defect occurs in this non‐neuronal compartment. First, we found that cells obtained from patients showed altered morphology and growth. Next, Western blotting analysis unravelled the imbalance in α‐tubulin post‐translational modifications and in MT stability. Interestingly, MT mass is significantly decreased in patient cells at baseline and differently changes overtime compared to controls, suggesting their inability to efficiently remodel MT cytoskeleton during ageing in culture. Thus, our results provide the first evidence that defects in MT regulation and stability occur and are detectable in a non‐neuronal compartment in patients with PSP. We suggest that MSCs could be a novel model system for unravelling cellular processes implicated in this neurodegenerative disorder.     

Assessing SSU rDNA Barcodes in Foraminifera: A Case Study using Bolivina quadrata

The Small Subunit Ribosomal RNA gene (SSU rDNA) is a widely used tool to reconstruct phylogenetic relationships among foraminiferal species. Recently, the highly variable regions of this gene have been proposed as DNA barcodes to identify foraminiferal species. However, the resolution of these barcodes has not been well established, yet. In this study, we evaluate four SSU rDNA hypervariable regions (37/f, 41/f, 43/e, and 45/e) as DNA barcodes to distinguish among species of the genus Bolivina, with particular emphasis on Bolivina quadrata for which ten new sequences ( KY468817 – KY468826 ) were obtained during this study. Our analyses show that a single SSU rDNA hypervariable sequence is insufficient to resolve all Bolivina species and that some regions (37/f and 41/f) are more useful than others (43/e and 45/e) to distinguish among closely related species. In addition, polymorphism analyses reveal a high degree of variability. In the context of barcoding studies, these results emphasize the need to assess the range of intraspecific variability of DNA barcodes prior to their application to identify foraminiferal species in environmental samples; our results also highlight the possibility that a longer SSU rDNA region might be required to distinguish among species belonging to the same taxonomic group (i.e. genus).     

Glucose compensation, serum lipids and thyroid hormones (rT3 and rT3/T3 ratio in non-insulin-dependent (type 2) diabetics: 2

In 45 type 2 diabetics it was unable to be found a relation between the plasma lipids and the fasting blood glucose (G), HbA1c, reverse T3 (rT3), rT3/T3 ratio, and relative body weight (R.B.W.). The conclusion was reached that the alteration of the lipoprotein metabolism and the thyroid hormones in type 2 diabetics could be primitive and independent from the availability of the insulin.     

Biochemical changes of rat brain membranes with aging

Modification of membrane composition and enzymatic activities both in total brain homogenate and purified synaptic plasma membrane of 3 and 24 month old rats has been investigated. Protein, cholesterol and phospholipid content and (Na+, K+)ATPase and 2',3' cyclic nucleotide phosphohydrolase activities were determined. The major changes occurred in the whole homogenate where a general increase in total protein and cholesterol content with age and a significant increase of the cholesterol/phospholipids molar ratio has been detected. In S.P.M. aging process induced a decrease of protein, cholesterol and phospholipids content associated with an increased membrane viscosity and a decrease of delta E. These data are consistent with a change in the structural organization and in the distribution pattern of different cell population in the aging brain. A possible artifactual effect of freezing on the reported parameter is also discussed.     

Environmental DNA metabarcoding as an effective and rapid tool for fish monitoring in canals

We focus on a case study along an English canal comparing environmental DNA (eDNA) metabarcoding with two types of electrofishing techniques (wade-and-reach and boom-boat). In addition to corroborating data obtained by electrofishing, eDNA provided a wider snapshot of fish assemblages. Given the semi-lotic nature of canals, we encourage the use of eDNA as a fast and cost-effective tool to detect and monitor whole fish communities.     

Sampling intraspecific variability in leaf functional traits: Practical suggestions to maximize collected information

The choice of the best sampling strategy to capture mean values of functional traits for a species/population, while maintaining information about traits’ variability and minimizing the sampling size and effort, is an open issue in functional trait ecology. Intraspecific variability (ITV) of functional traits strongly influences sampling size and effort. However, while adequate information is available about intraspecific variability between individuals (ITV_BI) and among populations (ITV_POP), relatively few studies have analyzed intraspecific variability within individuals (ITV_WI). Here, we provide an analysis of ITV_WI of two foliar traits, namely specific leaf area (SLA) and osmotic potential (π), in a population of Quercus ilex L. We assessed the baseline ITV_WI level of variation between the two traits and provided the minimum and optimal sampling size in order to take into account ITV_WI, comparing sampling optimization outputs with those previously proposed in the literature. Different factors accounted for different amount of variance of the two traits. SLA variance was mostly spread within individuals (43.4% of the total variance), while π variance was mainly spread between individuals (43.2%). Strategies that did not account for all the canopy strata produced mean values not representative of the sampled population. The minimum size to adequately capture the studied functional traits corresponded to 5 leaves taken randomly from 5 individuals, while the most accurate and feasible sampling size was 4 leaves taken randomly from 10 individuals. We demonstrate that the spatial structure of the canopy could significantly affect traits variability. Moreover, different strategies for different traits could be implemented during sampling surveys. We partially confirm sampling sizes previously proposed in the recent literature and encourage future analysis involving different traits.