A genome walking strategy for the identification of eukaryotic nucleotide sequences adjacent to known regions

Determination of nucleotide sequences adjacent to a known region is a recurring need in many genome scale studies. Various methods have been developed based on PCR techniques in order to fulfill th..

Phosphodiesterase Type 5 Inhibitors, Sport and Doping

Phosphodiesterase type 5 inhibitors (PDE5i) (e.g., sildenafil, tadalafil, vardenafil, and avanafil) are drugs commonly used to treat erectile dysfunction, pulmonary arterial hypertension, and benign prostatic hyperplasia. PDE5i are not prohibited by the World Anti-Doping Agency (WADA) but are alleged to be frequently misused by healthy athletes to improve sporting performance. In vitro and in vivo studies have reported various effects of PDE5i on cardiovascular, muscular, metabolic, and neuroendocrine systems and the potential, therefore, to enhance performance of healthy athletes during training and competition. This suggests well-controlled research studies to examine the ergogenic effects of PDE5i on performance during activities that simulate real sporting situations are warranted to determine if PDE5i should be included on the prohibited WADA list. In the meantime, there is concern that some otherwise healthy athletes will continue to misuse PDE5i to gain an unfair competitive advantage over their competitors

Iron Incorporation into Escherichia coli Dps Gives Rise to a Ferritin-like Microcrystalline Core

Abstract Escherichia coli Dps belongs to a family of bacterial stress-induced proteins to protect DNA from oxidative damage. It shares with Listeria innocua ferritin several structural features, such as the quaternary assemblage and the presence of an unusual ferroxidase center. Indeed, it was recently recognized to be able to oxidize and incorporate iron. Since ferritins are endowed with the unique capacity to direct iron deposition toward formation of a microcrystalline core, the structure of iron deposited in the E. coli Dps cavity was studied. Polarized single crystal absorption microspectrophotometry of iron-loaded Dps shows that iron ions are oriented. The spectral properties in the high spin 3d5 configuration point to a crystal form with tetrahedral symmetry where the tetrahedron center is occupied by iron ions and the vertices by oxygen. Crystals of iron-loaded Dps also show that, as in mammalian ferritins, iron does not remain bound to the site after oxidation has taken place. The kinetics of the iron reduction/release process induced by dithionite were measured in the crystal and in solution. The reaction appears to have two phases, witht of a few seconds and several minutes at neutral pH values, as in canonical ferritins. This behavior is attributed to a similar composition of the iron core

Plant Health and Rhizosphere Microbiome: Effects of the Bionematicide Aphanocladium album in Tomato Plants Infested by Meloidogyne javanica

The artificial introduction in the soil of antagonistic microorganisms can be a successful strategy, alternative to agrochemicals, for the control of the root-knot nematodes (Meloidogyne spp.) and for preserving plant health. On the other hand, plant roots and the associated rhizosphere constitute a complex system in which the contribution of microbial community is fundamental to plant health and development, since microbes may convert organic and inorganic substances into available plant nutrients. In the present study, the potential nematicidal activity of the biopesticide Aphanocladium album (A. album strain MX-95) against the root-knot nematode Meloidogyne javanica in infected tomato plants was investigated. Specifically, the effect of the A. album treatment on plant fitness was evaluated observing the plant morphological traits and also considering the nematode propagation parameters, the A. album MX-95 vitality and population density. In addition, the treatment effects on the rhizosphere microbiome were analysed by a metabarcoding procedure. Treatments with A. album isolate MX-95 significantly decreased root gall severity index and soil nematode population. The treatment also resulted in increased rhizosphere microbial populations. A. album MX-95 can be favourably considered as a new bionematicide to control M. javanica infestation

New insight into the genomic structure of dog T cell receptor beta (TRB) locus inferred from expression analysis

Here is an updated report on the genomic organization of T cell receptor beta (TRB) locus in the domestic dog (Canis lupus familiaris) as inferred from comparative genomics and expression analysis. The most interesting results we found were a second TRBD–J–C cluster, which is absent from the reference genome sequence, and the annotation of two additional TRBV genes. In dogs, TRB locus consists of a library of 37 TRBV genes positioned at the 50 end of two in tandem aligned D–J–C gene clusters, each composed of a single TRBD, 6 TRBJ and one TRBC genes, followed by a single TRBV gene with an inverted transcriptional orientation. The TRB genes are distributed in less than 300 kb, making the canine locus, one of the smaller mammalian TRB locus studied so far. The small size may be ascribed to reduced gene duplication occurrences and a lower density of total interspersed repeats compared to humans and mice. Despite the low TRBV gene content, a large and diversified beta chain repertoire is displayed in the dog peripheral blood. A full usage of TRBV and TRBJ genes, including pseudogenes, and a high level of allelic polymorphism contribute to generate diversity. Finally, this study suggests that the overall TRB locus organization is evolutionarily conserved supporting the dog as a highly suited model system for immune development and diseases

Identification of an Amylomaltase from the Halophilic Archaeon Haloquadratum walsbyi by Functional Metagenomics: Structural and Functional Insights

Amylomaltases are prokaryotic 4-α-glucanotransferases of the GH77 family. Thanks to the ability to modify starch, they constitute a group of enzymes of great interest for biotechnological applications. In this work we report the identification, by means of a functional metagenomics screening of the crystallization waters of the saltern of Margherita di Savoia (Italy), of an amylomaltase gene from the halophilic archaeon Haloquadratum walsbyi, and its expression in Escherichia coli cells. Sequence analysis indicated that the gene has specific insertions yet unknown in homologous genes in prokaryotes, and present only in amylomaltase genes identified in the genomes of other H. walsbyi strains. The gene is not part of any operon involved in the metabolism of maltooligosaccharides or glycogen, as it has been found in bacteria, making it impossible currently to assign a precise role to the encoded enzyme. Sequence analysis of the H. walsbyi amylomaltase and 3D modelling showed a common structure with homologous enzymes characterized in mesophilic and thermophilic bacteria. The recombinant H. walsbyi enzyme showed starch transglycosylation activity over a wide range of NaCl concentrations, with maltotriose as the best acceptor substrate compared to other maltooligosaccharides. This is the first study of an amylomaltase from a halophilic microorganism

Identification and characterization of the sucrose synthase 2 gene (Sus2) in durum wheat

Sucrose transport is the central system for the allocation of carbon resources in vascular plants. Sucrose synthase (SUS), which reversibly catalyzes sucrose synthesis and cleavage, represents a key enzyme in the control of the flow of carbon into starch biosynthesis. In the present study the genomic identification and characterization of the Sus2-2A and Sus2-2B genes coding for SUS in durum wheat (cultivars Ciccio and Svevo) is reported. The genes were analyzed for their expression in different tissues and at different seed maturation stages, in four tetraploid wheat genotypes (Svevo, Ciccio, Primadur, and 5-BIL42). The activity of the encoded proteins was evaluated by specific activity assays on endosperm extracts and their structure established by modeling approaches. The combined results of sucrose synthase 2 expression and activity levels were then considered in the light of their possible involvement in starch yield

Severe acute respiratory syndrome coronavirus 2 detection by real time polymerase chain reaction using pooling strategy of nasal samples

COVID-19 is a life-threatening multisistemic infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Infection control relies on timely identification and isolation of infected people who can alberg the virus for up to 14 days, providing important opportunities for undetected transmission. This note describes the application of rRT-PCR test for simpler, faster and less invasive monitoring of SARS-CoV-2 infection using pooling strategy of samples. Seventeen positive patients were provided with sterile dry swabs and asked to self-collected 2 nasal specimens (#NS1 and #NS2). The #NS1 was individually placed in a single tube and the #NS2 was placed in another tube together with 19 NSs collected from 19 negative patients. Both tubes were then tested with conventional molecular rRT-PCR and the strength of pooling nasal testing was compared with the molecular test performed on the single NS of each positive patient. The pooling strategy detected SARS-CoV-2 RNA to a similar extent to the single test, even when Ct value is on average high (Ct 37–38), confirming that test sensibility is not substantially affected even if the pool contains only one low viral load positive sample. Furthermore, the pooling strategy have benefits for SARS-CoV-2 routinary monitoring of groups in regions with a low SARS-CoV-2 prevalence

Polybrominated dibenzo-p-dioxins and furans (PBDD/Fs) in Italian food: Occurrence and dietary exposure

Human exposure to polychlorinated dioxins and furans (PCDD/Fs) through the dietary pathway is widely recognised and regulations in some regions of the world help to limit food contamination. Similar information on the analogous polybrominated dioxins and furans (PBDD/Fs) is scarce, partly due to the higher threshold to analytical access and unavailability of some standard materials. The analytical methodology developed here determined twelve planar PBDD/F congeners using 13Carbon labelled PBDD/F surrogates and high resolution mass spectrometric detection, and was extensively validated prior to the analysis of a range of commonly consumed Italian foods. The methodology also allowed simultaneous determination of PCDD/Fs and polychlorinated biphenyls (PCBs). The results show that PBDD/Fs occurred in different foods over a range of concentrations from <0.001 pg/g to 4.58 pg/g in fish. The dietary exposure (upper bound) of different Italian population groups, resulting from these occurrence levels was estimated using the toxic equivalency (TEQ) approach that is commonly used for dioxin-like contaminants and ranged from 0.17 to 0.42 pg TEQ/kg bodyweight/day (lower bound – 0.01 pg TEQ/kg bodyweight/day) depending on the population subgroup. Although precautionary, upper bound values may provide a more realistic estimate of toxicity as not all congeners and foods were measured. As expected, children were more highly exposed than adults due to lower body weight. These exposure levels were between a quarter and a third of that arising from the sum of PCDD/Fs and PCBs (0.61 to 1.38 pg WHO-TEQ/kg bodyweight/day), but they contribute to the dioxin-like toxicity. If this data is considered in view of the revised tolerable dioxin-like dietary intake published by EFSA in 2018, it is evident that the tolerable weekly intake of 2 pg/kg bodyweight/week would be exceeded by some of the assessed population sub-groups, or all sub-groups if the cumulative intake is considered

The oral and gut microbiota: beyond a short communication

Introduction. The current treatment and prevention of oral disorders, dental caries, periodontal and gum diseases, follow a very non-specific control of plaque as the main causative factor. The main therapeutically approach is carried out on the sole perspective to keep the levels of oral bacteria in an acceptable range compatible with one-way vision of oral-mouth health, as something completely separated from a systemic microbial homeostasis (dysbiosis) concomitant present in the gut. A sealed compartmental view which sees separate and incommunicable responses to a specific condition without considering the presence of interacting confounding factors can negatively influence the diagnosis a diseases and of course its progression. A general non-specific antimicrobial with more general antiplaque therapy based mainly on oral care products together with surgery interventions represent at the moment the only mechanical responses in treating oral diseases. Material and method. The present paper is a narrative review concening interractions between oral and gut microbiota, with a focus on the interdisciplinary approach in antimicrobial treatment. Pubmed, Cochrane Library database were used for searching engines. Key words used were as follows: "inflammatory bowel syndrome (IBS)", "ulcerative colitis", "oral dysbiosis", "gut dysbiosis", "probiotics", "periodontitis". Results and discussions. Literature research showed that there are few issues to be discussed the ever increasing resistance to antibiotics, the high consumption of industrial food and sugars and their negatively effect on gut and oral microbiota. There is a need to highlight and develop a novel philosophical approach in the treatments for oral diseases that will necessarily involve non-conventional antimicrobial solutions. Such approaches should preferably reduce the consumption of both intestinal and oral microbiota, that are intimately connected and host approximately well over 1000 different species of bacteria at 108–109 bacteria per mL of mucous and saliva. Preventive approaches based upon the restoration of the microbial ecological balance, rather than elimination of the disease associated species, have been proposed. Conclusions. Having both oral-gut microbiota screened is an essential moment that influence the healthy immune modulatory and regenerative capacity of the body and, the new proposed formula integrates a wider screen on the patients where oral condition is strictly evaluated together with gut screen; therefore any proposed treatment will be inevitably sustained by the use of prebiotics and probiotics to promote health-associated bacterial growth. Keywords: inflammatory bowel syndrome (IBS), ulcerative colitis, oral dysbiosis, gut dysbiosis, probiotics, periodontitis