Importance of Sequencing HBA1, HBA2 and HBB Genes to Confirm the Diagnosis of High Oxygen Affinity Hemoglobin

High oxygen affinity hemoglobin (HOAH) is the main cause of constitutional erythrocytosis. Mutations in the genes coding the alpha and beta globin chains (HBA1, HBA2 and HBB) strengthen the binding of oxygen to hemoglobin (Hb), bringing about tissue hypoxia and a secondary erythrocytosis. The diagnosis of HOAH is based upon the identification of a mutation in HBA1, HBA2 or HBB in specialized laboratories. Phenotypic studies of Hb are also useful, but electrophoretic analysis can be normal in 1/3 of cases. The establishment of the dissociation curve of Hb can be used as another screening test, a shift to the left indicating an increased affinity for Hb. The direct measurement of venous P50 using a Hemox Analyzer is of great importance, but due to specific analytic conditions, it is only available in a few specialized laboratories. Alternatively, an estimated measurement of the P50 can be obtained in most of the blood gas analyzers on venous blood. The aim of our study was therefore to determine whether a normal venous P50 value could rule out HOAH. We sequenced the HBB, HBA1 and HBA2 genes of 75 patients with idiopathic erythrocytosis. Patients had previously undergone an exhaustive medical check-up after which the venous P50 value was defined as normal. Surprisingly, sequencing detected HOAH in three patients (Hb Olympia in two patients, and Hb St Nazaire in another). A careful retrospective examination of their medical files revealed that (i) one of the P50 samples was arterial; (ii) there was some air in another sample; and (iii) the P50 measurement was not actually done in one of the patients. Our study shows that in real life conditions, due to pre-analytical contingencies, a venous P50 value that is classified as being normal may not be sufficient to rule out a diagnosis of HOAH. Therefore, we recommend the systematic sequencing of the HBB, HBA1 and HBA2 genes in the exploration of idiopathic erythrocytosis.

Selected Quality Attributes of Freshwater Mussel Powder as a Promising Ingredient for Pet Food

The aim of this study was to describe the quality attributes of a freeze-dried preparation obtained from freshwater mussel Sinanodonta woodiana (SW) soft tissue in respect to its potential as a novel pet food ingredient. After ecotoxicological testing of the raw material with MARA (Microbial Assay for Risk Assessment), the basic physico-chemical properties of the powder, such as approximate composition, bulk density, color parameters, water activity, electrophoretic analysis (SDS-PAGE), solubility, gelling and emulsifying capacity, were analyzed. The powder with a water activity of 0.43 offers a toxically safe preparation that contains over 34% protein/100 g of dry matter (DM). The SDS-PAGE profile showed twelve protein bands with a molecular weight (MW) ranging from >250 to 10 kDa. Taurine content has been estimated at an essential amount above 150 mg/100 g of DM. The powder possessed desirable emulsifying properties with 230 mL per 1 g and demonstrated the ability to form a firmer gel with a strength of 152.9 g at a temperature above 80 °C with at least 10% protein content. The L*, a*, and b* values characterizing powder color were found to be 69.49, 16.33, and 3.86, respectively. The SW mussel powder seems to be a promising ingredient that can be added with other binding or gelling agents in order to improve both the taste and acceptance of the final pet food products.

First Report of Schlumbergera virus X in Dragon Fruit (Hylocereus spp.) in Spain

Dragon fruit (Hylocereus undatus) is a high-value fruit crop, introduced about a decade ago in the mainland of Spain. In 2021, chlorotic spots were observed on young cladodes in a commercial dragon fruit orchard in the province of Seville (southern Spain). Sap extracts from 4 symptomatic cladodes were used to mechanically inoculate indicator plants: no symptoms were produced in Datura stramonium plants, but Chenopodium amaranticolor reacted with chlorotic local lesions and prickly pear plants (Opuntia ficus-indica) showed irregular yellow ringspot symptoms on young cladodes at 30 days post inoculation. Total RNA was extracted from all 4 symptomatic cladodes as previously described (Pallas et al. 1987). Reverse transcription (RT)-PCR, which was carried out with M-MLV-RT and Go Taq Pol (Promega Biotech Ibérica, SL, Madrid, Spain) and tobamovirus primers (Dovas et al. 2004), failed to produce any amplicons. Electrophoretic analysis of dsRNA, extracted from symptomatic cladodes, yielded a banding pattern similar to the one reported for potexviruses (Valverde et al. 1986). Primers specific for Cactus virus X (Kim et al., 2016) failed to produce amplicons, whereas potexvirus group primers (Potex F5/Potex R2) (van der Vlugt and Berendsen 2002), amplified an expected 584-bp amplicon from RNA extracts of all 4 field-collected samples. The RT-PCR products from the four samples were Sanger-sequenced. All showed identical sequence results (GenBank Accession MZ614940) with a predicted amino acid identity of 99% with the corresponding RNA-dependent RNA polymerase amino acid sequence of Schlumbergera virus X (SchVX) (GenBank Accession No. ACD99908). SchVX-specific primers (431s, 5‘-TTTGAGGAGTTCGTCAGCAAGA-3‘ and 431As, 5‘-TCAAGAGCCCATTGAGAGAGTG-3‘) that were designed based on the new sequence, amplified the expected amplicon of around 430 nucleotides from the total RNA extracts of the four samples. The amplicons were Sanger-sequenced and the expected nucleotide sequence was obtained. This pair of primers were used in RT-PCR tests on subsequent surveys in 2 commercial dragon fruit greenhouses from the province of Seville, and in 1 experimental greenhouse in the province of Almeria. All samples from 25 symptomatic plants of H. undatus, H. hybridum, H. costaricensis, and H. purpusii in Seville and from 1 symptomatic H. undatus plant from Almeria tested positive for SchVX, while 15 asymptomatic plants tested negative. The results obtained in this investigation support that SchVX is present in the cladodes of dragon fruit plants expressing the symptoms. SchVX has been reported previously from H. undatus from Brasil (Duarte et al. 2008) and from prickly pear in Mexico (De La Torre-Almaráz et al. 2016), and to our knowledge, this is the first report of the virus in Spain. These findings suggest that SchVX has been introduced in dragon fruit farms from Spain and propagation of this emerging crop through planting of cuttings should include testing for this virus in order to prevent further spread.

Development and evaluation of potential functional food biscuits made from White Lupin

Potential functional food bakery products were developed and characterized based on White Lupin (Lupinus albus cv. Nelly) flour. Analytical properties of the seeds resemble to previously described Lupinus species, with significantly high protein content (45%). The high protein and dietetic fiber content of the seeds makes Lupin flour suitable to develop potential functional food products with high nutritional values. Results of the development of sweet biscuits and salty crackers enriched with Lupin flour are presented. Sensory evaluation of the bakery products was carried out by 15 panelists using the nine points hedonic scale. Heat stability of White Lupin proteins were investigated by gel-electrophoretic analysis, White Lupin proteins are quite stable at 140°C, after 35 min heating the biscuits still contain 69% of the original amount of proteins. Baking conditions were optimized also based on gel-electrophoretic experiments, the optimal baking time was 30 min at 140°C. Gluten-free Lupin-based biscuits and crackers were produced by completely omitting wheat flour from the recipes.

Application of Fish Collagen-Nanochitosan-Henna Extract Composites for the Control of Skin Pathogens and Accelerating Wound Healing

Skin is the largest protective organ that could be recurrently wounded and attacked by microorganisms. The wounded skin safeguarding and supporting were intended through natural derivatives. Fish collagen (Cg) type I, extracted from sea bream (Spondyliosoma cantharus), chitosan nanoparticles (NCht) from shrimp shells, and henna (Lawsonia inermis L.) leaves extract (He) were produced and physiochemically characterized. The antimicrobial potentialities of these compounds and their composites were assessed toward skin pathogens (Candida albicans and Staphylococcus aureus) using various assaying methods and microimaging techniques. The infrared and electrophoretic analysis of Cg validated its characteristics, and the IR-spectroscopic analysis of the compounds/composites indicated their physiochemical attributes and interrelations. The produced NCht particles had a diameter range of 64.6-308.8 nm, 104 nm mean diameter, and +31.3 mV zeta potentiality. Both NCht, He, and NCht/He composite exhibited significant antimicrobial potentiality toward skin pathogens; NCht/He was the strongest with inhibitory concentrations of 20.0 and 22.5 μg/mL and inhibition zones of 25.7 and 26.8 mm against S. aureus and C. albicans, respectively. The electron micrographs verified the synergistic microbicidal action of NCht/He, as they led to severe microbial lysis and deformations. The skin wounds’ treatment with NCht/He/Cg composite promoted the fastest and complete healing of wounded rats’ skin during 8 days of local treatment, with the absence of inflammation and infection signs; treated with NCht/He/Cg composite, the wound area vastly reduced from 63.6 mm2 to 15.9 and 9.1 mm2 after 2 and 4 days, respectively. The natural NCht/He/Cg composites are recommended as topical applications for optimum skin disinfection and regeneration.

Mineralogical and physico-chemical properties of halloysite-bearing slip surface material from a landslide during the 2018 Eastern Iburi earthquake, Hokkaido

Destructive landslides were triggered by the 6.7 Mw Eastern Iburi earthquake that struck southern Hokkaido, Japan, on 6 September 2018. Heavy rainfall on 4 September in addition to intermittent rainfall around the Iburi Tobu area saturated and weakened the slope-forming materials (mostly altered volcanoclastic soils), making them susceptible to failure because of the earthquake’s strong ground motion. Most of the shallow landslides exhibited long runouts along gentle hill slopes, with characteristic halloysite-bearing slip surface at the base of the volcanic soils. This study investigated the mineralogical and physico-chemical properties of the slip surface material with the aim of understanding weakening and post-failure behaviors during the landslides. Halloysite in the slip surface had irregular-to-hollow-spherical morphology with higher mesopore volumes than tubular halloysite, which is related to a high capacity for water retention after rainfall. To reproduce possible chemical changes in the slip surface during rainfall, the sample was immersed in varying amounts of rainwater; solution pH increased and ionic strength decreased with increasing water content. These findings, alongside electrophoretic analysis, suggest that rainwater infiltration could have increased the absolute zeta potential value of the slip surface material. It is suggested that rainfall before the earthquake enhanced the colloidal stability of halloysite particles within the slip surface, owing to an increase in electrostatic repulsion. This decreased the material’s cohesive strength, which might have led to destabilization of the slope during ground shaking generated by the earthquake, and subsequent high-mobility flow after failure.

Development of hull-less barley with ultra-low gluten content via target genes combination. I. Isolation of triple mutants and black grained genotypes

Aim. The purpose of the research, presented in this paper, is to develop (as the first one in Ukraine) hull-less barley breeding material with ultra-low gluten, combining three hordein-deficient mutations, derived from Risø56, Risø1508, and R118, with black grain color as an indication of bioactive pigments content, enhancing the functional status of grain as a food product. Methods. We used electrophoretic analysis of proteins in polyacrylamide gel, DNA isolation by the CTAB method, polymerase chain reaction with DNA-markers, restriction analysis and electrophoretic separa tion of DNA in agarose gel. Results. The article presents the results of the first stage of combining target hordeindefi cient mutations by binary crosses. The F2/3 and F3/4 populations were derived from binary crosses of Risø56 × Risø1508 (reciprocal Risø1508 × Risø56) and R118 × hull-less barley Achilles. The new PCR protocol was optimized. Markers B1hor, 3a5F and Dhor-m were selected to identify mutations in B, C and D hordeins. Conclusions. Several barley genotypes with one or two target hordein-deficient mutations and black grain color were isolated. The genotypes obtained will be used in a further backcrosses program, aimed at developing of a black hull-less barley variety with ultra-low gluten content.