STUDY OF LOW PHYTIC ACID 1 LOCUS IN MAIZE

Phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate; InsP6) is ubiquitous in eukaryotic cells and constitutes the major storage form of phosphate in plant seeds (from 60% to 80%). During maturation it is accumulated in the protein storage vacuole in inclusions called globoids; the phosphate groups present in phytic acid (PA) are able to form phytate salts (phytin) binding important mineral cations. In mature maize kernels, 80% of PA is localized in the scutellum and the remaining 20% in the aleurone layer. The phosphorus stored as PA is remobilized during germination by phytase enzymes: these are also found in many microorganisms. Regarding the involvement of P in agricultural production and its sustainability, it has been estimated that nearly 50% of elemental P used yearly in global agricultural activities is accumulated in the PA. PA forming mixed salts with mineral cations is mainly excreted by monogastric animals and humans because they do not have phytase activity in their digestive systems. Considering that seeds are an important component of animal feed and human food, the limitations of phosphorus and micronutrients bioavailability imply a decrease in their nutritional value. Furthermore the undigested phosphorous contained in excreted phytin can contribute to water pollution (eutrophication). These negative effects have led to breeding programmes which have the aim of reducing the PA content in the seeds of several cultivated plants. The main way to reach this result by conventional breeding is the isolation of low phytic acid (lpa) mutations, capable of restraining the biosynthesis or the storage of PA in the seed; the increased P and mineral cation bioavailability in lpa seeds is confirmed by nutritional trials. In maize three low phytic acid mutants have been isolated: lpa1 and lpa2 by chemical mutagenesis, lpa3 by transposon tagging. Compared to the other mutations in maize, lpa1 exhibited the major reduction of PA in the seed, this comes with a proportional increase of free P without changing the total P content. Taking advantage of this property, lpa mutants can be recognized by the HIP (high inorganic phosphate) phenotype of the seeds. The Lpa1 gene encodes for ZmMRP4 (accession number EF586878) a multidrug-associated-protein (MRP) belonging to the subfamily of ATP-binding cassette (ABC) transmembrane transporters. MRP proteins are implicated in different roles like the transport of organic ions and anthocyanins, detoxification of xenobiotic compounds, transpiration control, and tolerance to oxidative stress. The role of this MRP protein is not completely understood but it is fundamental for phytic acid accumulation and viability of seeds. low phytic acid mutants isolated in rice and soybean are related to defects in homologues of the maize ABC transporter. It was observed that lpa mutations found in several crops usually bring pleiotropic effects on plant and seed performance, such as reduced germination and emergence rate, lower seed filling, weakening in stress resistance. The presence of pleiotropic effects shows that lpa mutations influence not only the seed but also the whole plant and its production. This can reflect the relevance of inositol phosphates as multifunctioning molecules, and their involvement in fundamental signaling and developmental pathways, like DNA repair, RNA editing, chromatin remodeling and control of gene expression. Furthermore phytic acid exhibits, by its ability to chelate iron, a potent antioxidant activity, avoiding the formation of reactive oxygen species. With the aim to isolate new maize low phytic acid mutants mutagenesis treatment were performed with EMS (ethyl-methanesulfonate). Since wild type mature maize seeds contain high amount of phytic phosphate and low free phosphate content, we screened the mutagenized population looking for seeds containing high levels of free phosphate (HIP phenotype), a typical feature of lpa. In previous studies a single recessive lpa mutation (originally named lpa241 and obtained by EMS pollen-tratment mutagenesis) was isolated and described, it was allelic to the lpa1-1 mutant, and was consequently renamed lpa1-241. A first evidence of non-Mendelian inheritance of lpa1 trait came from the appearance of unexpected free phosphate phenotypes in Lpa1/lpa1-241. When heterozygous families were selfed, we observed an overall increase of the mutant phenotype ratio due to the appearance of weak and intermediate phenotype, not consistent with a monogenic recessive mutation. This phenomenon can be explained with a partial Lpa1 allele silencing caused by trans interaction with the paramutagenic lpa1-241 allele. We performed genetic and molecular analyses of the lpa1-241 mutation that indicate an epigenetic origin of this trait, that is, a paramutagenic interaction that results in meiotically heritable changes in ZmMRP4 gene expression, causing a strong pleiotropic effect on the whole plant. To our knowledge, this is the first report of a paramutagenic activity not involving flavonoid biosynthesis in maize, but regarding a key enzyme of an important metabolic pathway in plants. We isolate a new maize (Zea mays L.) low phytic acid 1 mutant allele obtained by chemical EMS seed mutagenesis. We performed the allelism test with two other lpa1 mutants: lpa1-1 and lpa1-241, our mutant failed to complement these mutants. This mutant, named lpa1-7, exhibits a monogenic recessive inheritance and lethality as homozygous. We demonstrate that in vitro cultivation can overcome lethality allowing the growth of adult plants and we report data regarding embryo and leaf abnormalities and other defects caused by negative pleiotropic effects of this mutation. We conducted two experiments to ascertain the nature of lpa1-7 and. we also performed physiological analysis, histological observations and considerations regarding the effects of the lpa1 mutations on the plant. Pigmented maize contains anthocyanins and phenolic compounds which are phytochemicals synthesized in the plant by secondary metabolism; although these compounds are considered as non-nutritive, in these years the interest in antioxidant and bioactive properties has increased due to their health benefits. Anthocyanins are water soluble secondary metabolites belonging to the class of flavonoids and they play important roles in several aspect of plant biology. The anthocyanins are present in the vacuole in a glycosilated form and their colour is influenced in part by the pH of this compart. In maize they are synthesized by a complex pathway made up of more than 20 genes, and regulated by two classes of transcription factors: r1/b1 bHLH genes and c1/pl1/p1 MYB gene families. Our aim is the constitution of maize inbred lines carrying low phytic acid mutations together with regulatory genes pushing the anthocyanin accumulation in the kernels and seedlings, so they can compensate the leak in antioxidant activity induced by the low phytic acid mutation. We found that the lpa1-241 line is able to alter the accumulation of anthocyanin in kernel tissues. The anthocyanins, are present in the vacuole where their colour is dependent on the pH. In maize the anthocyanins are cytoplasmically synthesized molecules probably transported in the vacuole by ZmMRP3 gene activity. We observed an interaction between the accumulation of anthocyanin pigments in the kernel and the lpa mutations. In fact the lpa1-241 mutant accumulates a higher level of anthocyanins as compared to wild type either in the embryo or in the aleurone layer in a genotype able to accumulate anthocyanin. Furthermore, we demonstrate that these pigments are mislocalised in the cytoplasm, conferring a blue pigmentation of the scutellum, because of the neutral/basic pH of this cellular compartment; expression analysis showed a reduction of ZmMRP3 anthocyanins\u2019 transporter gene expression. On the whole, these data strongly suggest a possible interaction between the lpa mutation and anthocyanin accumulation and compartmentalization in the kernel

Enhanced B-cell differentiation and reduced proliferative capacity in chronic hepatitis C and chronic hepatitis B virus infections

BACKGROUND & AIMS: Chronic microial infections aare frequently associated with B-cell activation and polyclonal proliferation, potentially leading to autoimmunity and lymphoproliferative disorders. We assessed B-cell phenotype and function in chronic hepatitis B (HBV) and chronic hepatitis C (HCV) virus infection. METHODS: We studied 70 patients with chronic HCV infection, 34 with chronic HBV infection and 54 healthy controls, B-cell phenotype was assessed by flow cytometry using monoclonal antibodies specific for CD27, the CD69, CD71, and CD86 activation markers and the chemokine receptor CXCR3. Differentiation into immunoglobulin-producing cells (IPC) was analysed by ELISpot upon stimulation and with CD40 ligand+IL-10 as surrogate bystander T-cell help or CpG oligodeoxynucleotide+IL-2, as innate immunity signal. Proliferation was examined by cytometry using carboxyfluorescein diacetate succinimidyl ester (CFSE) after stimulation with CpG. RESULTS: A significantly higher proportion of B cells from both HCV-and HBV-infected patients expressed activation markers compared with controls and a positive correlation was found between CXCR3(+) B cells and HCV RNA values. Memory B cells from patients with chronic HCV and HBV infections showed enhanced differentiation into IPC compared with controls, although this was restricted to IgG and at a lower level in HCV-compared with HBV-infected patients. Moreover, patients' activated B cells displayed significantly lower proliferative ability compared to healthy donors despite low expression of the FcRL4 exhaustin marker. CONCLUSIONS: B-cell activation, but not exhaustion, is common in chronic viral hepatitis. However, enhanced B-cell differentiation and deficient proliferative capacity were not associated with commitment to terminal differentiation

Multidisciplinary Design and Optimization of Regional Jet Retrofitting Activity

A retrofit analysis on a 90 passengers regional jet aircraft is performed through a multidisciplinary collaborative aircraft design and optimization highlighting the impact on costs and performance. Two different activities are accounted for selecting the best aircraft retrofit solution: a re-engining operation that allows to substitute a conventional power-plant platform with advanced geared turbofan and an on-board-systems architecture modernization, considering different levels of electrification. Besides the variables that are directly dependent from these activities, also scenario variables are considered during the optimization such as the fuel price, the fleet size and the years of utilization of the upgraded systems. The optimization is led by impacts of the retrofitting process on emissions, capital costs and saving costs, computed at industrial level. Overall aircraft design competences (aerodynamics, masses, performance, noise, and emissions) have been computed increasing the level of fidelity and reliability. The whole process is implemented in the framework of the AGILE 4.0 research project in a collaborative remote multidisciplinary approach. Results show that the engine retrofitting can be a profitable solution for both manufacturers and airliners. Conversely, the on-board-system electrification seems to be not convenient in a retrofitting process due to the high capital costs. Depending on the operative scenario, involved stakeholders can properly orient their decision on a retrofitting strategy

Unexpected silicon localization in calcium carbonate exoskeleton of cultured and fossil coccolithophores

Coccolithophores, marine calcifying phytoplankton, are important primary producers impacting the global carbon cycle at different timescales. Their biomineral structures, the calcite containing coccoliths, are among the most elaborate hard parts of any organism. Understanding the morphogenesis of coccoliths is not only relevant in the context of coccolithophore eco-physiology but will also inform biomineralization and crystal design research more generally. The recent discovery of a silicon (Si) requirement for crystal shaping in some coccolithophores has opened up a new avenue of biomineralization research. In order to develop a mechanistic understanding of the role of Si, the presence and localization of this chemical element in coccoliths needs to be known. Here, we document for the first time the uneven Si distribution in Helicosphaera carteri coccoliths through three synchrotron-based techniques employing X-ray Fluorescence and Infrared Spectromicroscopy. The enrichment of Si in specific areas of the coccoliths point to a targeted role of this element in the coccolith formation. Our findings mark a key step in biomineralization research because it opens the door for a detailed mechanistic understanding of the role Si plays in shaping coccolith crystals

Thermal damage study of beryllium windows used as vacuum barriers in synchrotron radiation beamlines

An experimental study to investigate thermal-induced damage to SSRL-designed beryllium foil windows was performed at LLNL's Laser Welding Research Facility. The primary goal of this study was to determine the threshold at which thermal-stress-induced damage occurs in these commonly used vacuum barriers. An Nd:Yag pulsed laser with cylindrical optics and a carefully designed test cell provided a test environment that closely resembles the actual beamline conditions at SSRL. Tests performed on two beryllium window geometries, with different vertical aperture dimensions but equal foil thicknesses of 0.254 mm, resulted in two focused total-power thresholds at which incipient damage was determined. For a beam spot size similar to that of the Beamline-X Wiggler Line, onset of surface damage for a 5-mm by 25-mm aperture window was observed at 170 W after 174,000 laser pulses (1.2-ms pulse at 100 pps). A second window with double the vertical aperture dimension (10 mm by 25 mm) was observed to have surface cracking after 180,000 laser pulses with 85 W impinging its front surface. It failed after approximately 1,000,000 pulses. Another window of the same type (10 mm by 25 mm) received 2,160,000 laser pulses at 74.4 W, and subsequent metallographic sectioning revealed no signs of through-thickness damage. Comparison of windows with equal foil thicknesses and aperture dimensions has effectively identified the heat flux limit for incipient failure. The data show that halving the aperture's vertical dimension allows doubling the total incident power for equivalent onsets of thermal-induced damage

Long-chain polyphosphates impair SARS-CoV-2 infection and replication

Inorganic polyphosphates (polyPs) are linear polymers composed of repeated phosphate (PO43−) units linked together by multiple high-energy phosphoanhydride bonds. In addition to being a source of energy, polyPs have cytoprotective and antiviral activities. Here, we investigated the antiviral activities of long-chain polyPs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In molecular docking analyses, polyPs interacted with several conserved amino acid residues in angiotensin-converting enzyme 2 (ACE2), the host receptor that facilitates virus entry, and in viral RNA-dependent RNA polymerase (RdRp). ELISA and limited proteolysis assays using nano– LC-MS/MS mapped polyP120 binding to ACE2, and site-directed mutagenesis confirmed interactions between ACE2 and SARS-CoV-2 RdRp and identified the specific amino acid residues involved. PolyP120 enhanced the proteasomal degradation of both ACE2 and RdRp, thus impairing replication of the British B.1.1.7 SARS-CoV-2 variant. We thus tested polyPs for functional interactions with the virus in SARS-CoV-2–infected Vero E6 and Caco2 cells and in primary human nasal epithelial cells. Delivery of a nebulized form of polyP120 reduced the amounts of viral positive-sense genomic and subgenomic RNAs, of RNA transcripts encoding proinflammatory cytokines, and of viral structural proteins, thereby presenting SARS-CoV-2 infection in cells in vitro

Hepatitis C Virus Antigenic Convergence

Vaccine development against hepatitis C virus (HCV) is hindered by poor understanding of factors defining cross-immunoreactivity among heterogeneous epitopes. Using synthetic peptides and mouse immunization as a model, we conducted a quantitative analysis of cross-immunoreactivity among variants of the HCV hypervariable region 1 (HVR1). Analysis of 26,883 immunological reactions among pairs of peptides showed that the distribution of cross-immunoreactivity among HVR1 variants was skewed, with antibodies against a few variants reacting with all tested peptides. The HVR1 cross-immunoreactivity was accurately modeled based on amino acid sequence alone. The tested peptides were mapped in the HVR1 sequence space, which was visualized as a network of 11,319 sequences. The HVR1 variants with a greater network centrality showed a broader cross-immunoreactivity. The entire sequence space is explored by each HCV genotype and subtype. These findings indicate that HVR1 antigenic diversity is extensively convergent and effectively limited, suggesting significant implications for vaccine development

Acute diverticulitis management: evolving trends among Italian surgeons. A survey of the Italian Society of Colorectal Surgery (SICCR)

Acute diverticulitis (AD) is associated with relevant morbidity/mortality and is increasing worldwide, thus becoming a major issue for national health systems. AD may be challenging, as clinical relevance varies widely, ranging from asymptomatic picture to life-threatening conditions, with continuously evolving diagnostic tools, classifications, and management. A 33-item-questionnaire was administered to residents and surgeons to analyze the actual clinical practice and to verify the real spread of recent recommendations, also by stratifying surgeons by experience. CT-scan remains the mainstay of AD assessment, including cases presenting with recurrent mild episodes or women of child-bearing age. Outpatient management of mild AD is slowly gaining acceptance. A conservative management is preferred in non-severe cases with extradigestive air or small/non-radiologically drainable abscesses. In severe cases, a laparoscopic approach is preferred, with a non-negligible number of surgeons confident in performing emergency complex procedures. Surgeons are seemingly aware of several options during emergency surgery for AD, since the rate of Hartmann procedures does not exceed 50% in most environments and damage control surgery is spreading in life-threatening cases. Quality of life and history of complicated AD are the main indications for delayed colectomy, which is mostly performed avoiding the proximal vessel ligation, mobilizing the splenic flexure and performing a colorectal anastomosis. ICG is spreading to check anastomotic stumps’ vascularization. Differences between the two experience groups were found about the type of investigation to exclude colon cancer (considering the experience only in terms of number of colectomies performed), the size of the peritoneal abscess to be drained, practice of damage control surgery and the attitude towards colovesical fistula

The Biodiversity of the Mediterranean Sea: Estimates, Patterns, and Threats

The Mediterranean Sea is a marine biodiversity hot spot. Here we combined an extensive literature analysis with expert opinions to update publicly available estimates of major taxa in this marine ecosystem and to revise and update several species lists. We also assessed overall spatial and temporal patterns of species diversity and identified major changes and threats. Our results listed approximately 17,000 marine species occurring in the Mediterranean Sea. However, our estimates of marine diversity are still incomplete as yet—undescribed species will be added in the future. Diversity for microbes is substantially underestimated, and the deep-sea areas and portions of the southern and eastern region are still poorly known. In addition, the invasion of alien species is a crucial factor that will continue to change the biodiversity of the Mediterranean, mainly in its eastern basin that can spread rapidly northwards and westwards due to the warming of the Mediterranean Sea. Spatial patterns showed a general decrease in biodiversity from northwestern to southeastern regions following a gradient of production, with some exceptions and caution due to gaps in our knowledge of the biota along the southern and eastern rims. Biodiversity was also generally higher in coastal areas and continental shelves, and decreases with depth. Temporal trends indicated that overexploitation and habitat loss have been the main human drivers of historical changes in biodiversity. At present, habitat loss and degradation, followed by fishing impacts, pollution, climate change, eutrophication, and the establishment of alien species are the most important threats and affect the greatest number of taxonomic groups. All these impacts are expected to grow in importance in the future, especially climate change and habitat degradation. The spatial identification of hot spots highlighted the ecological importance of most of the western Mediterranean shelves (and in particular, the Strait of Gibraltar and the adjacent Alboran Sea), western African coast, the Adriatic, and the Aegean Sea, which show high concentrations of endangered, threatened, or vulnerable species. The Levantine Basin, severely impacted by the invasion of species, is endangered as well