Digitaria exilis Stapf) is an ancient African cereal that represents a rich source of carbohydrate, fat, fiber, vitamins, minerals, and sulfur-containing amino acids. Processing and utilization of fonio require adequate knowledge of its structural, chemical, and nutritional characteristics. The present work evaluates the structural, techno-functional, and gelling properties of fonio and compares them to other major gluten-free cereals . Fonio flour presented significantly higher water absorption index and swelling power, while it scored a lower water solubility index than the reference flours. The pasting viscosity profile of fonio was similar to that of rice, with equivalent peak viscosity but a breakdown viscosity 24% lower than rice, indicative of higher stability and resistance to shearing and heating. Rheological properties demonstrated that fonio generates gels with remarkably strong structures. At 15% concentration, fonio gel withstood stress 579% higher than those observed in the reference flours without breaking its structure. Fonio flour presented the highest gelatinization enthalpy (11.45 J/g) and a narrow gelatinization temperature range (9.96 °C), indicative of a better-packed starch structure than the other analyzed flours. The texture of the gels made with fonio showed higher firmness over the evaluated period. These combined results suggest that fonio is a suitable ingredient for gel-like food formulations.Fonio ( Over the last decade, the market for gluten-free (GF) products has grown considerably as a consequence of better diagnostic methods for identifying an increasing number of people suffering from celiac disease and other gluten-related disorders, and people who have eliminated gluten from their diet because they perceive it as a healthy improvement . The nutDigitaria exilis Stapf, also known as fonio or acha, is a naturally gluten-free African cereal suitable for use in the diet of celiac patients  were recorded. Endothermic transitions of the retrograded starches were determined with a second scan made after 7 days of sample storage at 4 °C, following the same procedure described for the gelatinization evaluation. Each experimental sample was measured at least in duplicate.Thermal transitions  were determined by differential scanning calorimetry (DSC) . Samples (~6 mg) were prepared at a 30:70  provided with the software “Texture expert exceed” version 2.63. The gels were prepared in 50 mL centrifuge tubes by heating 28 g of flour dispersion (at a concentration of 15 g/100 g) in a boiling water bath for 30 min, stirring the dispersion every 2–3 min until the boiling temperature was achieved. The tubes were cooled at room temperature and stored for 2, 6, 10, 24, 48, 96, and 192 h at 4 °C. Gels were left at room temperature for 15 min before the analysis of the texture. Measurements were done on gel cylinders of 2.7 cm diameter and 2 cm height. A “Texture Profile Analysis” (TPA) double compression test was performed using a 75 mm diameter aluminum probe (SMSP/75) to suppress 50% depth, at 1 mm/s speed test, with a 30 s delay between compressions. Firmness (N), springiness, cohesiveness, and gumminess (N) were calculated from the TPA graphs.p < 0.05) was used to establish significant differences among means.One-way ANOVA with parametric tests was used for the statistical analysis using Statgraphics Centurion XVIII software . The Levene test was used to check the homogeneity of variances. The normality of the studentized residuals was evaluated with the Kolmogorov–Smirnov test. Fisher’s Least Significant Difference test at 95% confidence intervals  followed an identical evolution as WAI, given that both parameters associate the weight of the formed gel with the number of insoluble compounds in the flour and the whole amount of flour, respectively. When the fonio flour gelatinizes, better interaction with water is exhibited, which could be due to the very small dimension of the starch granules, ~8 µm . Said smThe results obtained from viscometric tests are presented in In millet and sorghum, a second peak of viscosity appeared at the final cooling phase (50 °C) of the pasting curve. It is believed that this behavior could be due to the storage of the flours for a time equal to or greater than two months, given that a second peak during the holding stage was also found by Zhang and Hamaker  in sorghAt the end of the pasting profile, during the temperature holding at 50 °C, the final viscosity (FV) was recorded. This value showed the capacity of the material to form a viscous paste that reflects the retrogradation of amylose . The valmax) that samples could tolerate before the collapse of their structure. The effects of flour type and concentration on the value of maximum stress (τmax) were statistically significant (p < 0.05), following an increasing trend with increasing concentration in all the studied GF flours. The strain sweep assays also provided the stress at which the gels passed from a solid-like to a liquid-like behavior (the crossing point of the curves where G′ = G″ and tan(δ) = 1) .The viscoelastic properties of the gels were assessed by dynamic oscillatory tests at 25 °C see . The sam(δ) = 1) . Results1′, G1″, tan(δ)1 and a, b and c exponents were generated from fitting the frequency sweeps data in the range of 1–10 Hz to the power-law model. The high value of R2 (0.955–0.999) indicates to what extent the model adjusted to the studied systems. Elastic and viscous moduli significantly increased when the flour concentration increased. However, the rate of increase varied depending on the type of flour 1 < 1, in all the studied concentrations, indicating a solid-like behavior of the gels. This indicates that the gel structure was already formed at a concentration of 6%, regardless of the flour source. Except for the gels made from rice flour, tan(δ)1 decreased with the increase in flour concentration  than fonio. This denotes a higher increase in fonio gels’ viscoelastic moduli with flour concentration and a higher modulation capacity of the gel’s viscoelasticity by varying its concentration than rice gels. The exponents n and q of millet and sorghum were notably higher than that of fonio; however, their viscoelastic moduli at low concentrations (quantified by the m and p coefficients) were much lower than those obtained for fonio. The combination of a high consistency at low concentrations  and a high increase in consistency with increasing concentration (compared to rice) makes fonio an interesting ingredient for the production of GF products of gel-like nature.The high dependence of storage modulus on the concentration allows gathering information of the gelation efficiency and the structure of the particle network of the gel . Clark etructure  (Table 4odel see . Linear gel). Fonio and rice gels presented the highest values , while maize presented the lowest (5.1 J/g). Higher values of ΔHgel are indicative of a better-packed starch structure requiring more energy to fully gelatinize. Significant differences were also found among gelatinization temperatures. Ji et al. [Thermal properties of the gluten-free flours were evaluated using Differential Scanning Calorimetry (DSC). Gelatinization, retrogradation, and amylose-lipid complex data obtained from DSC are shown in i et al.  discoveri et al. . The disret) during the gel staling. All values determined for ΔHret were lower than their corresponding ΔHgel value, but showed the same trend, with fonio presenting the highest value. The width of this first peak was observed to be higher in fonio flour, while maize presented the lowest value. The second peak registered was related to the reversible amylose–lipid complex dissociation and appeared approximately at the same temperature as it did in the first scan. The enthalpies of the amylose–lipid complex were increased in the second scan with respect to the first scan. Eliasson [A second scan was performed to assess the retrogradation properties of the flours after storage of the gelatinized samples in their corresponding pans at 4 °C for 7 days . Two visEliasson  indicateThe texture properties of the gels made from the studied flours and their evolution during a period of 192 h of storage at 4 °C, depicted in p < 0.05) with storage time, except for rice gel which showed a very stable springiness value during the whole studied period. Fonio was the only sample to show some springiness recovery after 100 h, with the value corresponding to 192 h being higher than those determined at 48 and 96 h. In general, the cohesiveness of all gels showed a similar trend to that obtained for springiness, with decreasing values during the studied storage time.Springiness is a mechanical textural attribute related to the rapidity and degree of recovery from a deforming force . The iniThe value of gumminess increased after 6 h and decreased at longer times. The highest value was recorded in fonio and millet gels, but after 192 h, the fonio gel had only lost 21% of this maximum value, while millet lost 59%. This parameter is often used to characterize the necessary energy to disintegrate semisolid foods and is adequately correlated to sensory evaluation by a trained panel .Fonio flour demonstrated good performance when comparing its properties to other gluten-free sources, showing significant differences in techno-functional, pasting, rheological, and gelling properties. Fonio presented a higher ability to absorb water during gelatinization and swelling in excess water, resulting in the highest determined values of WAI and SP, believed to be influenced by its small starch granule size and high specific surface area. This greater ability to interact with water was also verified in the evaluated gel properties. Fonio had a higher pasting viscosity, as well as high shearing and heating stability and resistance, consistent with a better-packed starch structure than the other analyzed GF flours, as shown by the thermal analysis. Rheological properties revealed that fonio flour generated gels with a remarkably strong structure, particularly at high flour concentrations (12% and 15%). Fonio gels also exhibited notably higher elastic modulus and firmness values than the rest of the GF flours. These combined results lead to the conclusion that fonio has the potential to be used in food, especially in preparations such as porridge, where better gelling properties are appreciated. Therefore, it is suggested that fonio is a promising starch source to compete with other commercially important flours, such as rice, in industrial applications.


The paper presents the theoretical and experimental research conducted to date regarding the possibility of obtaining round bars from AZ31 magnesium alloy with a bimodal structure rolled in the radial shear rolling process (RSR) technology. There is no analysis of the impact of the deformation path  on the mechanical properties and the obtained bar structure. The feedstock, namely, AZ31 magnesium alloy round bars with a diameter of 30 mm, were rolled in RSR to the final diameter of 15 mm with different levels of deformation in successive passes, at a temperature of 400 °C. The bars obtained as a result of the RSR rolling process have different hardness on the cross-section as well as a characteristic gradient grain size distribution. Based on the conducted research, it can be concluded that the use of a larger number of passes with a smaller cross-section reduction will result in an improved formation of a bimodal structure consisting of a highly fragmented near-surface structure and in the half of the radius of the structure of fragmented grains at the boundaries of larger grains. The intensive development of technologies that has been observed for over a dozen years, facilitating the production of materials and finished products where the properties are shaped not only by changes in the chemical composition or heat treatment but primarily by means of unique methods of plastic processing, has led to the popularization of severe plastic deformation (SPD) methods ,2,3. TheThe radial shear rolling (RSR) process is used for rolling round bars made of various metallic materials, including steel ,18, titaNaizabekov et al.  showed tMagnesium alloys are some of the prospective light metals very popular in the industry. These alloys are widely used in research on obtaining materials with a UFG or bimodal structure by a variety of SPD methods, among which the RSR is widely used. Dobatkin et al. presents research results on structural changes, texture and mechanical properties of AZ41 alloy rolled on a multi-pass basis using RSR . The autIn the theoretical and experimental research conducted to date on obtaining bars with a bimodal structure from magnesium alloys, rolled in the RSR technology, no analysis is present of the impact of the deformation path  on the mechanical properties and structure of bars thus obtained. Thus, the goal of the paper was to determine the effect of the deformation path on the properties and structure of the obtained bars by applying various rolling patterns. For this purpose, numerical simulations and experimental tests were applied to the RSR process in bars with a diameter of 15 mm from a batch with an initial diameter of 30 mm made of AZ31 magnesium alloy. Three variants of deformation pattern tests were carried out: I: six passes-small deformation in a single pass, II: four passes-medium deformation in a single pass and III: three passes-large deformation in a single pass. Strength, microhardness and microstructure of bars and the material in a non-deformed state were tested, which allowed determining of the impact of the applied deformation pattern on the bimodality of the bars.In the RSR tests, the magnesium alloy AZ31 for plastic processing was used, with the chemical composition defined by the manufacturer specified in −1. Based on the results of the experimental studies, their approximation used extended the Hensel–Spittel [fσ dependence of strain T expressed as:The accuracy of numerical modeling depends largely on the accurate determination of the properties of materials used for simulations. A model of the deformed medium developed by the authors and determined from plastometric tests of compression tests performed using the Gleblee 3800 metallurgical process simulator  was used for numerical testing. The plastometric tests for the analyzed magnesium alloy were carried out for the temperatures 350, 400 and 450 °C and for the strain rates of 0.1, 1.0 and 10.0 s–Spittel  formula Example flow curves for the AZ31 magnesium alloy under the conditions mentioned above are presented in The plastometric tests showed that during plastic deformation of AZ31 magnesium alloy at low strain rates, recovery and recrystallization processes take place ,36,37. ATo increase the accuracy of numerical calculations, the properties of AZ31 alloy are presented in a table format, combining experimental test results (for a specific range) supplemented by the results calculated from Equation (1) using the factors from The tests involved using round feedstock with a diameter of 30 mm and a length of 200 mm, made of cast ingots 65 × 120 × 650 mm in size. Before making the feedstock for the rolling process, the ingots were subjected to the homogenization process  to homogenize the structure after casting. For the as-poured material and as-homogenized material, samples were taken for structural tests. Furthermore, for the post-homogenization material, standardized samples for tensile tests were prepared.17Al12 and Mg17Al12+ α at the boundaries at the dendrite boundaries of a solid solution of aluminum in magnesium [agnesium . The perTensile tests were performed with a Zwick Z100  testing device, using standard round samples with a diameter of 5 mm for the initial material and the length of the measuring base of 100 mm. However, for as-rolled bars, no samples were taken and the tearing was performed for three ready-made bars with the diameter of 15 mm rolled according to the assumed variants, which was to determine the influence of the obtained bimodal structure on their properties. Furthermore, both for the feedstock and for the samples taken from rolled rods, the microhardness measurement was carried out by the Vickers method using a semi-automatic Future-Tech FM700  microhardness tester. The microhardness tests were performed for samples taken from rolled bars in accordance with the assumed variant (a sample from each rolled bar), and for each of them, the measurement was taken on the longitudinal section, with the load of 50 g.ijS is the deviatoric stress tensor, ijε is the equivalent strain rate tensor, T is the temperature, K—consistence being dependent on the yield stress σp, n—factor characterizing hot metal deformation (0 < n < 1).Theoretical tests of the rolling process of AZ31 alloy bars were conducted using commercial software, i.e., Forge NxT 2.1 . The visco-plastic deformed metal model and three-dimensional state of strain were assumed for the numerical examination:jτ—friction stress, σ0—base stress, nσ—normal stress, μ—friction coefficient, m—friction factor.The friction conditions on the surface of metal-rolls contact are described with the Coulomb friction model and the Tresca friction model, in which appropriate coefficient values are taken:For the determination of the temperature field, a differential equation is used, which describes the temperature variations for a transient heat flow. This is a quasi-harmonic equation that can be expressed in the following form:xk, yk and zk are the functions of distribution of thermal conductivity coefficients in the directions x, y, z. The Ts is a function used to describe the temperature in the zone under consideration. The Q is the function of distribution of the deformation heat generation rate; pc is the function of distribution of metal specific heat; and ρ is the function of distribution of metal density. As the boundary conditions, the combined limiting conditions of the second and third kinds were adopted, which can be written in the form below:In the above equation, xl, yl and zl are the directional cosines of the normal to the band surface, q is the intensity of heat flow over the cooled zone surface and kα represents convective losses. Equation (5) and boundary condition (6) uniquely define the heat exchange during modeling of the rolling process.In the above equation, The die model was constructed from a triangle-based mesh, while the AZ31 feedstock model was constructed from tetrahedral elements. During deformation, the volumetric mesh is automatically remeshed, depending on the deformation value in order to avoid excessive deformation and improving the quality of the solutions. The AZ31 feedstock model was built from ca. 120 thousand elements (each element of average edge length was different for each rolling pass from 1.0 for the first pass to 0.6 in the last pass), and ca. 20 thousand elements for working rolls, each element having average edge length of 2 mm.2); coefficient of heat exchange between the material and the air αair—10 W/(K·m2), working rolls temperature—30 °C; ambient temperature—20 °C. To introduce a computer model, a model of the RSR SVP-08 laboratory rolling mill  was used, located at the Czestochowa University of Technology   . In the The pattern of deformations used in the tests for the analyzed variants of the RSR process on AZ31 alloy bars is presented in In order to assess the impact of changing the degree of the unit deformation on the possibility of a gradient structure, the sum of deformations for subsequent passes was analyzed.Before each rolling pass, the samples were heated in an electric chamber furnace LAC KC 120/14  to the assumed initial rolling temperature of 400 °C.Due to the nature of the deformation in the RSR rolling process, the parameter to identify the possibility of the formation of a bimodal structure in the rolled bar is the effective deformation, defined as the sum of deformations in subsequent passes in each analyzed rolling variant .ρzγ) in the axial zone. On the other hand, the use of a larger unit deformation in a single pass with a smaller number of passes will result in an increase in the proportion of shear strain in the direction of twisting ρzγ in the near-surface zone.By analyzing the computer simulations presented in The changes in the nature of the effective strain distributions for the analyzed variants result from the differences in the velocities and directions of metal flow  during tρzτ on the longitudinal section (deformation zone) of the rolled bar and on the cross-section (located in the exit of the deformation zone) for intermediate passes in each rolling variant .ρzτ; however, when analyzing the obtained distributions for cross-sections, the value of the stress tensor component ρzτ increases with the increase of the deformation in a single pass at the place of contact of the deformed metal with the working tools. Furthermore, in a larger volume of the deformed metal, the analyzed tensor component changes the orientation.When analyzing the data presented in ρzγ was performed for intermediate passes in each of the rolling variants (characterized by a different level of deformation), Additionally, an analysis of changes in the distribution of shear strain in the direction of torsion ρzγ on the cross-section is sequential  associated with the torsion and change in the length of the spiral line along which the deformed metal flows [ρzγ. Along with the increase of the level of deformation, a decrease in the longitudinal length of the range can be observed, where the analyzed component has a positive direction, from approx. 25.7 mm for the smallest deformation (ρzγ in the near-surface zones (from 0.2 for Variant I to 0.4 for Variant II). It can, therefore, be assumed that the use of a larger number of passes with a lower deformation in a single pass has a more beneficial effect on the homogenization of deformations between the surface and the intermediate and central zones  are a natural site of initiation of the formation of local shear bands. As previous research in this field has shown, there is significant fragmentation of the structure [ρzγ  for Variant I, pass 3  for Variant II and pass 2  for Variant III. The distribution of shear strain presented in al flows ,39. The ormation a to apprormation c. Increaal zones . On the tructure ,33, whicure [γρz .ρzγ for all analyzed cases does not exceed 0.1. Increasing the value of shear strains ρzγ is closely related to the reduction of the length of the zone and to the diameter at which they have a constant sign (positive or negative), which results from the reduction of the longitudinal length of twist line in the deformed medium. The obtained distributions and changes in the value of the component of shear strains ρzγ translates directly into the decomposition of the effective deformation, where in the variant with a greater number of passes with a relatively small unit deformation (Variant I), the effective deformation values in the bar axis are higher compared to the application for rolling of a smaller number of passes with a higher deformation in a single pass (Variant II).In the axis of the rolled bar, the value of the component of shear strain To determine the effect of the applied rolling variants on the mechanical properties of the finished AZ31 alloy bars, the mean values of the yield strength (YS) and the ultimate tensile strength (UTS) were determined in the tensile test. These results were compared to the value of the tested as-homogenized alloy in 0.05 = 37. On the other hand, the determined average microhardness  values determined for rolled bars on the longitudinal section as a function of the distance from the axis of the rolled bar are shown in Based on the tests of the microhardness of the studied as-homogenized AZ31 alloy, the average determined value was HVρzγ observed in subsequent passes, which for Variants II and III have larger local maxima that contribute to the fragmentation of the structure. Nevertheless, reducing the number of passes causes a higher heterogeneity in the structure in the final product. On the other hand, for Variant I, the greater number of passes  has a positive effect on the uniformity of the grain size. When analyzing the data showing the microstructure in surface zones, a strong fragmentation of the structure is observed for all passes, while in the case of Variants II and III, clusters of larger partially recrystallized grains can be observed, which are smaller for Variant I. This may result indirectly from the values of strain tensor component ρzγ observed in subsequent passes, which for Variants II and III have larger local maxima that contribute to the fragmentation of the structure.In the transition zones, an increased number share of larger grains with colonies of a finely divided structure can be observed for Variant I. The obtained structure evidences the formation of shear bands in the RSR process, in which grain refinement occurs in the surface zone through activating additional deformation mechanisms in the AZ31 magnesium alloy. For Variants II and III, a decrease in the number of small, finely divided grains is observed, while the maximum size of single magnesium grains increases. This tendency continues for the central zone in all analyzed variants. The structure becomes coarser with an increased amount of distinct twins and recrystallized grains. In the central zone, as shown in theoretical tests, the value of the set deformation in the subsequent passes is smaller than the intermediate and outer zones, which, at a given rolling temperature, favors recrystallization.The determined average grain sizes for all variants evidence that as a result of RSR process, it is possible to obtain an uneven distribution of magnesium grain sizes , which proves its bimodality. The results obtained for theoretical and experimental tests confirm the possibility of using the RSR process for the production of bars with a bimodal structure from magnesium alloys. Despite the possibility of generating relatively large deformations in subsequent single passes on the RSR rolling mill, the results of the tests carried out have evidenced that it is the most advantageous for the tested alloy to conduct the process with a greater number of passes with smaller single deformation than the use of larger unit deformations and reducing the number of passes. Moreover, due to the fact that for the central zones of the bar rolled in the RSR technology, the deformation is smaller than in the outer zones. It seems beneficial to conduct preliminary plastic working of the feedstock, which is characterized by high deformation in the central part of the processed bar. For this purpose, the process or rolling in modified stretching passes can be used, which provides the fragmentation of the structure in the central zone of the manufactured bars .The theoretical and experimental tests carried out have evidenced that the use of various deformation patterns affects the obtained structure and grain refinement. Based on the conducted research for the rolling process at 400 °C, it can be concluded that the use of a larger number of passes with a smaller cross-section reduction will positively impact the formation of a bimodal structure consisting of a highly fragmented near-surface structure and in the half of the radius of the structure of fragmented grains at the boundaries of larger grains. Strong grain fragmentation is the result of shear bands that constitute locations of nucleation and dynamic recrystallization of grains, which is beneficial to the fragmentation of the structure. When larger deformations are used during rolled bars, larger grains with distinct twins are visible. In the tested variants of rolling in the twinning areas, no formation of new embryos and no dynamic recrystallization were observed. The use of a smaller number of passes does not ensure complete fragmentation of the structure in the near-surface layers of the bar and results in a band structure consisting of larger grains surrounded by smaller grains. The grain refinement in the areas near the axial of the bar is characterized by a larger grain size due to the small impact of shear strain. Elongation of grains can be expected in areas near the axial zones due to the occurrence of only tensile stresses resulting from the elongation of the samples.


Methyltransferase enzymes have been associated with different processes within cells and viruses. Specifically, within viruses, methyltransferases are used to form the 5′cap-0 structure for optimal evasion of the host innate immune system. In this paper, we seek to discuss the various methyltransferases that exist within single-stranded RNA (ssRNA) viruses along with their respective inhibitors. Additionally, the importance of motifs such as the KDKE tetrad and glycine-rich motif in the catalytic activity of methyltransferases is discussed. The denotation of a protein as a methyltransferase is a general term for the enzymatic activity of a protein transferring a one-carbon group onto a select substrate. These substrates and the role that they play can vary based on the organism and the viral genome that codes for that particular methyltransferase. This review seeks to categorize a majority of the known methyltransferases within the viral world to explore their role and implications in drug design.Viruses can be further split into categories based on the genome that they possess . From thViruses can be classified using the Baltimore classification system . This clMethyltransferases are present within both Class 4 and Class 5 viruses . Once ClSARS-CoV, Berne virus, Zika virus, Rubella virus, Semliki virus, and Hepatitis E. In this method of replication, the virus enters the cell and releases its genomic material. Since the positive-sense strand is of the same nature as what mRNA would be, the ribosome of the host cell would be able to effortlessly translate the viral proteins to continue its life cycle. It can thus create its polymerase, coupled with cleavage of its genome, to develop additional proteins needed for its life cycle. Once maturation is achieved, the virus is packaged and released for infection of another cell.The following are positive-sense single-stranded RNA viruses that are further reviewed in this paper: Nishimuro ledantevirus, rabies virus, and Human Respiratory Syncytial virus A. In this type of replication, the virus releases its contents into the cell. Since this strand is not of the same nature as mRNA, the virus codes for the RNA-dependent RNA polymerase to convert the negative-sense RNA to a positive-sense mRNA for translation. The virus then follows similar subsequent steps as the positive-sense single-stranded RNA virus. This cycle begins anew with the release of the newly packaged viral material.The following are negative-sense single-stranded RNA viruses: Methyltransferases are mainly seen to be utilized within cap formation . Generalbacteria, restriction-modification systems are used to rid the cell of foreign DNA [In the realm of eign DNA . The sysThe restriction-modification system can be divided into four main types: Type 1, Type 2, Type 3, and Type 4. These are categorized according to subunit composition, cofactor requirements, and other properties.Methyltransferases are further split into multiple categories based on their structural features, such as the catalytic domain and structural motifs that comprise them. While not all methyltransferases can be confined to a single category, a majority subside under the larger umbrella of Class 1 methyltransferases. A generalized classification of methyltransferases and their corresponding names can be found in The following list encompasses methyltransferases that have their substrate as single-stranded RNA. Therefore, only methyltransferases involving Class 4, 5, and 6 of the Baltimore classification system will be discussed. In In SARS-CoV’s NSP14. This makes the replication and infection process much simpler for the virus if their genome is not large. The above image displays the available PDB files for the methyltransferases that have been discussed so far. The bright pink highlight seeks to partition the methyltransferase domain from the rest of the protein. While the methyltransferase domain is highlighted above, the domain can be further explored as there are specific motifs that are conserved amongst these proteins.Not all viruses have one protein dedicated solely to acting as a methyltransferase. Some methyltransferases have to make do with what they have and form bifunctional proteins such as that of The KDKE motif is comprised of four residues involved with the catalytic exchange of a methyl group onto the cap-acceptor site of the RNA strand. This motif is more common with the formation of cap-1 instead of cap-0, as most proteins responsible for sole cap-0 formation lack this motif. The residues involved are two lysines, one aspartic acid, and one glutamic acid. While this motif is very common, some variations can exist that are capable of catalyzing the same mechanism. In the above images, the KDKE motifs are displayed within their respective PDB files. In PDB 5M2X, the KDKE motif is K61, D146, K182, and E218. In PDB 3R24, the KDKE motif is K46, D130, K170, and E203. In PDB 6UEB, the KDKE motif is K1685, D1797, K1829, E1867. In PDB 5A22, the KDKE motif is K1651, D1762, K1795, and E1833. The aspartic acid (D) within the KDKE motif is responsible for the initiation of the cap structure formation. The two lysine residues have a possible role in stabilizing the incoming RNA molecule as it prepares to be methylated. This interaction involves the positively charged lysine residues and the negatively charged phosphate backbone of the RNA strand. Regarding the possible role of the glutamic acid residue, its role could be associated with maintaining the structural integrity of the RNA binding pocket by retaining the two lysine residues in a position where it can optimally interact with the RNA phosphate backbone. In SARS-CoV and NSP16, NSP16 has a strong preference for the Cap0-mRNA complex as a substrate rather than an mRNA substrate without the Cap0 structure. To understand the selectivity of this cap-dependent process, one must first compare the difference between normal guanine and the 7-methyl guanine. While the guanine is composed of a bicyclic delocalized ring, the 7-methyl guanine has the same components with the added benefit of a methyl which grants the 7mG group a positive charge. The type of bond between this positive charge and the aromatic residue seen in a majority of these RNA-binding domains can be described as a cation–pi interaction. The most common pi system involved in this type of interaction is typically tryptophan and is seen to be energetically favorable. Based on the characterization of the interaction of methylated N7 vs. non-methylated N7 within the EIF4E binding protein by the Brown lab, it was concluded that methylated N7 was calculated to have a tighter binding to the cap-0 structure vs. no cap at all [In the case of p at all . This exAnother common motif that is found within methyltransferase domains is the glycine-rich motif. This can come in the form of DxG, GxG, GxGxG, or some other variation. This motif usually will mark the area where SAM will dock within the methyltransferase domain. The methionine portion of the molecule will dock above this motif and stabilize via small hydrophobic interactions seen between the SAM molecule and the motif. The purpose of this conservation most likely serves as a designated docking location for SAM.With an introduction to the main motifs seen within methyltransferases, the different domains can now be presented via the different classes that they are categorized in. The composite diagram below, For the amarillovirales-like methyltransferase domain family, there appear to be conserved motifs such as the KDKE tetrad and the glycine-rich SAM-binding domain. For the alpha-like virus methyltransferase domain family, there appear to be conserved motifs such as a conserved histidine residue, DXXR SAM-binding motif, and conserved tryptophan residue. While the full mechanism is unclear, the conserved histidine and tryptophan residues are essential for methyltransferase activity and are shown to be associated with catalytic activity.SARS-CoV and NSP15 of Berne, but not in NSP14 of SARS-CoV. The KDKE tetrad is found within the NSP16 and NSP15, but not NSP14. A glycine-rich SAM-binding motif can be found conserved in NSP16 and NSP15. For the mononegavirales-like methyltransferase domain family, there appear to be conserved motifs such as KDKE tetrad, conserved aspartic acid residue, and glycine-rich SAM-binding motif. The conserved aspartic acid may play a role in interacting with the hydroxyl groups on the ribose of SAM to aid in retaining the molecule during methyl transfer. A more in-depth explanation of the motif composition is given in the virus’ respective subsections below.For the nidovirales-like methyltransferase domain family, we can see a conserved aspartic acid residue within NSP16 of Zika is a positive-sense ssRNA that enters the host cell via receptor-mediated endocytosis [Zika genome. After translation of the viral proteins, NS5 plays an essential role in the life cycle of Zika. NS5 is composed of two domains: an N-methyltransferase domain and an RNA-dependent RNA polymerase. In this reaction, SAM is used as a cofactor to catalyze the transfer of a methyl group to its final acceptor. In vitro studies have shown that while the MTase domain is associated with transferring a methyl group to the N7 of guanine, the domain has been seen to transfer a methyl group to the ribose-2′OH and act as a guanylyltransferase.ocytosis . Figure Based on different mutagenesis studies, the following sites can be seen as important for functionality: mRNA cap-binding sites, SAM binding sites, and general sites that are seen to be essential for methyltransferase activity . For mRNFor residues that are classified as being generally involved in methyltransferase activity, the following have been seen to exhibit this: K2581, D2666, K2702, and E2738. Additionally, a glycine-rich motif can be seen which can indicate the canonical SAM-binding domain. This can be defined as G2601/C2602/G2603/R2604/G2605.Semliki genome. Proteins are synthesized to create a complex that is capable of cap formation. The cap will form solely in the nucleus; however, this virus replicates exclusively in the cytoplasm. RNA TPase will remove the 5′ gamma-phosphate of the nascent RNA molecule. mRNA guanylyltransferase will donate a GMP to form a 5′-5′ TP linkage. NSP1 will bind to GMP to form an enzyme-GMP complex. The GMP is transferred to mRNA where SAM is used to transfer a methyl group to guanine-N7 to form the cap structure.Semliki virus is a positive-sense ssRNA virus that undergoes early translation once in the cell . Figure There have been residues reported that are important in this enzyme’s catalytic activity. Residue 38H is involved in creating the initial phosphoramide link between the enzyme and the mRNA structure. The following residues are seen to interact with the adenine base of the mRNA strand: A39, D153, Y155, Y249, and Y286. The following residues are seen to interact with the triphosphate portion of the mRNA strand: R42, S45, H46, K50, T70, Q152, and D153. The following residues are seen to interact with SAM within the binding pocket: G66, R71, R72, P84, E89, D90, R93, Q152, and D153.Additional site-directed mutagenesis studies further show how mutations in specific residues are needed for functionality. Specifically, within the methyltransferase domain, mutations in the following sites have shown decreased activity: L19, H38, D64, C81, V82, C83, D90, R93, C135, C142, K169, C214, Y249, and K317.Semliki virus is expected to have some key motifs that can be found within its sequence: an invariant histidine residue, a DXXR motif, and an invariant tyrosine residue. The histidine residue is found as H38, the DXXR motif as D90/P91/E92/R93, and the tyrosine residue is Y249.The methyltransferase domain of the Hepatitis E virus is located within the family Hepeviridae and genus Orthohepevirus [Hepatitis E Virus Genotype 1 genome. This is a single-stranded positive-sense ssRNA that is transmitted via the fecal–oral route. Once viral particles are ingested, the virus is absorbed via GI mucosa and into portal circulation to enter the liver. The following regions are present in ORF1 polyprotein: methyltransferase (60–240), Y-domain (239–439), putative papain-like cysteine protease (440–610), hinge (712–778), X-domain (785–942), NTPase/helicase (960–1204), RdRP (1207–1693) [The epevirus . Figure ge 712–77, X-domaiThe methyltransferase domain is expected to contain the following: an invariant histidine residue, a DXXR motif, and an invariant tyrosine residue . The invRubella is a positive-sense ssRNA virus where the virus initially binds to an unknown receptor of susceptible cells to catalyze receptor-mediated endocytosis and subsequent release of the genomic material into the cytoplasm of the host cell [Rubella genome. The genomic RNA is translated to produce the non-structural polyprotein which is comprised of gene products after further cleavage, P150 and P90 (RdRP). P150 has three subdomains that are capable of: methyltransferase functionality, a domain of an unknown function, and proteolytic function. While the specifics of the methyltransferase are not further discussed, the function can be assumed to be related to cap formation as this domain is found in the polymerase of the virus.ost cell . Figure Hepatitis E genome, Rubella will also contain some key motifs within the methyltransferase domain such as an invariant histidine residue, a DXXR motif, and an invariant tyrosine residue. An invariant histidine residue can be found as H65, the DXXR motif as D155/V156/P157/R158, and an invariant tyrosine residue at Y237.Similar to that of the ClustalO is a sequence alignment software that is utilized to discover potential conservation of residues amongst the methyltransferase domains within this classification of methyltransferases, alpha-like methyltransferase domain . Figure Semliki Forest Virus: H38, I65, P69, R93, A103, A125, V150, P160, P183, G191, and A216. The following is found to be conserved within the methyltransferase domain of PORF1 in the Hepatitis E virus: H65, I89, P93, R117, A127, A134, V161, P171, P194, G201, and A227. The following is found to be conserved within the methyltransferase domain of P150 in the Rubella virus: H66, I93, P97, R109, A119, A141, V164, P174, P196, G204, and A231.The following is found to be conserved within the methyltransferase domain of NSP1 in the Semliki) is compared to PORF1 (Hepatitis E), 15.68% identity is observed. When NSP1 (Semliki) is compared to P150 (Rubella), 18.85% identity is observed. When PORF1 (Hepatitis E) is compared to P150 (Rubella), 17.84% identity is observed.When comparing percent identity amongst the sequences, the values observed are less than 20%. When NSP1  motif is not similar to the GxG motif, it is a slight variation of the SAM-binding motif.Berne virus. This SAM-ribose binding residue can be defined as D6684. While this same residue seems to be conserved within NSP14, the residue is outside of the N7-methyltransferase domain and no further experimental data is available to confirm its participation in SAM-binding. It can be hypothesized that since the substrates of NSP14 are different from that of NSP14 and NSP15, this conserved aspartic acid residue may not play a role within the functionality of NSP14.With discussing NSP16, the following motifs are seen: GxG motif and KDKE tetrad. The GxG motif can be found at G6846/A6847/G6848 while the KDKE tetrad is found at K6821/D6905/K6945/E6978. When viewing PDB 3R24, a key SAM-binding residue (D6874), is essential for stabilizing and forming a sturdy hydrogen bond with the ribose hydroxyl groups to anchor the SAM molecule. Upon further inspection, this residue is conserved within close relative NSP15 from the 17 Since toroviruses are ancestrally similar to coronaviruses, there is some similar nomenclature amongst the gene names. Berne genome. The following genes are translated: ORF1ab is known as the replicase polyprotein, spike (S), membrane (M), hemagglutinin-esterase (HE), and nucleocapsid (N). The replicase polyprotein is cleaved into 14 chains: NSP 1–8, 10–15. NSP15 assumes the role of a methyltransferase. The domain is similar to that of other nidoviral-type SAM-dependent methyltransferases. This specifically will methylate the ribose-2′OH structure to form the cap-1 structure.The torovirus is a positive-sense ssRNA virus with a peplomer-bearing envelope.With discussing NSP15, the following motifs are seen: KDKE tetrad. The KDKE tetrad is found at K6633/D6709/K6737/E6771.ClustalO is a sequence-alignment software that was utilized to discover potential conservation of residues amongst the methyltransferase domains within this classification of methyltransferases, alpha-like methyltransferase domain . Figure The following is found to be conserved within the methyltransferase domain of NSP14 in SARS-CoV: L6079, K6098, Y6099, C6111, P6141, L6155, D6160, D6175, E6204, K6227, F6252, S6259, G6319. The following is found to be conserved within the methyltransferase domain of NSP16 in SARS-CoV: L6802, K6821, Y6822, C6826, P6855, L6869, D6874, D6889, E6922, K6945, F6968, S6975, G7031. The following is found to be conserved within the methyltransferase domain of NSP15 in the Berne virus: L6615, K6633, Y6634, C6638, P6666, L6679, D6684, D6693, E6719, K6737, F6761, S6768, G6824.When comparing percent identity amongst the sequences, the values observed ranged from 10% to 25%. When NSP14 (SARS-CoV) is compared to NSP16 (SARS-CoV), 12.75% identity is observed. When NSP14 (SARS-CoV) is compared to NSP15 (Berne), 10.57% identity is observed. When NSP16 (SARS-CoV) is compared to NSP15 (Berne), 25.28% identity is observed.HRSV is a negative-sense ssRNA that encodes for a total of 11 proteins. The following are coded: major nucleocapsid protein (N), nucleocapsid phosphoprotein (P), transcription processivity factor M2-1 and 2, L protein, large glycoprotein (G), fusion glycoprotein (F), small hydrophobic protein (SH), non-structural proteins 1 and 2, and non-glycosylated M protein. HRSV genome. The L protein assumes the role of viral polymerase to aid in the replication of the viral genome.HRSV-A virus, there is D1912 which is classified under this motif.There are key motifs present within mononegavirus-like methyltransferase domains that are seen to be pertinent to efficient catalytic activity: GxGxG and KDKE tetrad. The KDKE tetrad can be seen to catalyze the transfer of the methyl group from SAM to its substrate. The glycine-rich domain can be characteristic of the SAM-binding site. This motif usually sits somewhere close to the center of the SAM molecule. The GxGxG can be defined as G1853/E1854/G1855/A1856/G1857. This GxGxG motif can be referred to as Motif I. The following residues have been observed to be essential for methyltransferase activity: K1831, D1936, K1973, and E2004 otherwise known as KDKE tetrad. Motif III is usually a conserved aspartic acid or tryptophan. In the case of the N. ledantevirus is a negative-sense single-stranded RNA virus that contains a leader sequence followed by five structural protein genes that are separated by non-transcribed intergenic regions [Nishimuro Ledantevirus genome. The five structural proteins are N (nucleoprotein), P (phosphoprotein), M (matrix), G (transmembrane glycoprotein), and L (RNA-dependent RNA polymerase). regions . Figure Rabies virus, this protein is capable of catalyzing the necessary reactions responsible for cap formation of the mRNA. According to domain similarity, this enzyme has the following reported capabilities: GTP phosphohydrolase, PRNTase, RNA-directed RNA polymerase, replicase, transcriptase, guanine-N7-methyltransferase, and nucleoside-2′-O-methyltransferase.Similar to that of the L protein in the The first step in mRNA capping is a guanosine 5′-triphosphatase (GTPase) that can hydrolyze GTP into GDP and a side product, inorganic phosphate. The GDP acts as an acceptor for the subsequent enzymatic reaction via GDP polyribonucleotidyltransferase (PRNTase). The PRNT domain of the protein recognizes the 5′triphosphate end and forms an intermediate complex between L protein and the pRNA. The pRNA is then transferred to the GDP to form the 5′-terminal cap structure. Since the methyltransferase is non-specific, it can catalyze the formation of cap-0 and cap-1 structures.HRSV-A virus, N. ledantevirus has the characteristic GxGxG motif and KDKE tetrad to have a functional methyltransferase domain. The GxGxG motif is defined as G1668/D1669/G1670/S1671/G1672. This GxGxG motif can be referred to as Motif I. The KDKE tetrad is defined as K1649/D1760/K1793/E1831. Motif III tends to be a conserved aspartic acid or tryptophan. In the case of the N. ledantevirus, there is D1733 which is classified under this motif.Similar to the Rabies virus is a negative-sense single-stranded RNA virus that contains a leader sequence followed by five structural protein genes that are separated by intergenic regions that are not transcribed [Rabies genome. The five structural proteins are N (nucleoprotein), P (phosphoprotein), M (matrix), G (transmembrane glycoprotein), and L (RNA-dependent RNA polymerase).The nscribed ,20. FiguRABV genome in the order of the following proteins: N, P, M, G, and L. The L protein is heavily involved with cap formation.Based on the structural similarity to that of the vesicular stomatitis virus, it can be assumed that all enzymatic reactions related to viral RNA synthesis and processing can be linked to the L protein. The RNA-dependent RNA polymerase encoded with the rabies virus will initiate transcription at the 3′end of the The first step in mRNA capping is a guanosine 5′-triphosphatase (GTPase) that can hydrolyze GTP into GDP and a side product, inorganic phosphate. The GDP acts as an acceptor for the subsequent enzymatic reaction via GDP polyribonucleotidyltransferase (PRNTase). The PRNT domain of the L protein recognizes the specific mRNA viral sequence, 5′ppp-AACAG, and forms an intermediate complex between L protein and the pRNA. The pRNA is then transferred to the GDP to form the 5′-terminal cap structure.The methyltransferase domain is considered to be the motif VI within the L protein sequence. This can be labeled as a mononegavirus-type SAM-dependent methyltransferase. If looking at the full genomic sequence for Protein L, the methyltransferase domain spans residues 1674 to 1871. This methyltransferase can be seen to be utilized for both the N7 of the cap and the 2′OH of the nearby ribose sugar to catalyze the formation of both cap-0 and cap-1 structures.HRSV-A virus, the Rabies virus has the characteristic GxGxG motif and KDKE tetrad to have a functional methyltransferase domain. The GxGxG motif is defined as G1704/D1705/G1706/S1707/G1708. The KDKE tetrad is defined as K1685/D1796/K1829/E1867. This GxGxG motif can be referred to as Motif I. Motif III is usually a conserved aspartic acid or tryptophan. In the case of the Rabies virus, there is D1770 which is classified under this motif.Similar to the ClustalO was utilized to discover potential conservation of residues amongst the methyltransferase domains within this classification of methyltransferases, mononegavirales-like methyltransferase domain . Figure HRSV-A: T1828, G1829, I1837, L1841, C1848, G1853, G1855, G1857, S1877, L1878, P1887, P1909, D1912, W1919, L1932, D1936, E1938, I1948, K1973, I1987, L1990, 2003S, E2004, and Y2006. The following conserved residues are observed in N. ledantevirus: T1644, G1645, I1653, L1659, C1666, G1668, G1670, G1672, S1691, L1692, P1708, P1731, D1733, W1740, L1756, D1760, E1762, I1772, K1793, I1807, L1810, S1830, E1831, and Y1833. The following conserved residues are observed in the rabies virus: T1680, G1681, I1689, L1693, C1700, G1704, G1706, G1708, S1727, L1728, P1744, P1768, D1770, W1776, L1793, D1796, E1798, I1809, K1829, I1843, L1846, S1866, E1867, and Y1869.The following conserved residues are observed in N. ledantevirus) is compared to Protein L (rabies), 34.85% identity is observed. When GDP-PRNT (N. ledantevirus) is compared to Protein L , 20.63% identity is observed. When Protein L (rabies) is compared to Protein L , 23.28% identity is observed.When comparing percent identity amongst the sequences, the values observed ranged from 20 to 35%. When GDP-PRNT  is a protein present with the eukaryotic host cell that is responsible for recognizing mRNA and promoting the translation of that strand . More sp complex . The inhWhile the Cap-0 structure is methylation of the initial guanine nucleotide at the N7 position, Cap-1 is methylation on the 2′-hydroxyl group of ribose following the cap-0 nucleotide. Without the protection of cap structures, the RNA can be recognized by host processes such as the retinoic acid-inducible gene 1 (RIG-1) which leads to eventual induction of type-1 interferon (IFN). RIG-1 is defined as a cytosolic innate immune receptor that can differentiate cellular self-RNA from that of pathogens. In addition to RIG-1, IFN-induced protein with tetratricopeptide repeats (IFIT-1 and IFIT-5) also can bind exposed 5′-TP on viral mRNA which can induce a similar process .In the situation where a cap-0 has been placed but no 2′O-methylation has occurred (cap-1) then the unmethylated 2′O can be seen as a pathogen-associated molecular pattern (PAMP) where it can be recognized by melanoma differentiation-associated protein 5 (MDA5).Overall, the binding of this PAMP to a pattern recognition receptor (PRR) can induce a variety of signaling pathways (including IFN-1 production) to rid the cell of the foreign molecule.As mentioned above, mediators of this pathway include but are not limited to, IFIT, MDA5, and RIG-1 ,24,25. FRIG-1 and MDA5 are a part of the RIG-1 gene family that participates as PRR to recognize PAMP. IFIT1/5 will be induced by Type 1 IFN induction . The IFIFor mRNA to begin translation, the cap-binding protein eIF4E is needed. IFIT1 has been seen to compete for binding with CBP eIF4E and has a higher affinity for the cap-0 structure. Additionally, IFIT-1 shows low binding for Cap-1 so if the initial methylation of 2′O occurs, then it is less likely for IFIT-1 to complete its role. Thus, by inhibiting methyltransferase activity, the host cell can activate the RIG-1 system to rid the cell of this pathogenic virus.In the case of successful cap-inhibition, there are other possible processes in place to be cognizant of when approaching a virus. There are multiple alternative mechanisms to EIF4E that are present: Phosphorylation at S209, 4EBP regulation, alteration of EIF4E expression, EIF4E replacement, EIF4E regulation via binding alternative viral proteins .In adenoviruses, there is a mechanism involving EIF4E dephosphorylation to inhibit cap-dependent translation . Synthesherpes simplex virus (HSV), HSV can form ICP0 protein which promotes the degradation of 4EBP-1 via interaction with a proteasome. If there is less 4EBP-1 available, there is more EIF4E available for translation.In an alternative mechanism, 4EBP is used. 4EBP can also be known as EIF4E-binding protein which acts as translational repressors . In cellEpstein–Barr virus can induce EIF4E overexpression via latent membrane protein 1 (LMP1). LMP1 can induce over-expression of EIF4E via C-myc which can lead to the development of nasopharyngeal carcinoma.In an alternative mechanism, there is an alteration of EIF4E expression . For exaHIV-1, there is the binding of the DEAD motif to EIF4G and PABP. This induced displacement of EIF4E and allows for efficient translation.In an alternative mechanism, there is a displacement of EIF4E via viral proteins . In the sapovirus, its viral genome codes for the VPg protein that binds to EIF4E to stimulate its function, thus, increasing viral protein synthesis.In the final alternative mechanism, viral proteins can be induced to bind EIF4E to alter its function . In the Methyltransferases should be considered as desirable drug targets within antiviral treatments. The methyltransferases discussed play an essential role in forming cap structures needed for the avoidance of the host immune system to prolong the viral life cycle and infection into adjacent cells. By interacting with these methyltransferases and ceasing their activity, one would be able to expose areas of the viral genome that can be recognized by the host immune system, leading to its eventual demise. This section will be divided into experimental inhibitors within positive-sense and negative-sense ssRNA viruses.SARS-CoV with an ethyl linker connecting both groups with variations within the N atom substituent contained in the linker. The following substrates are based on the concept of creating a singular molecule with bifunctionality that can bind to both the mRNA binding pocket and SAM binding pocket . The desSARS-CoV [The following molecules are observed to act as inhibitors for the NSP16 belonging to SARS-CoV . For NSPThe following compounds are excluded from further testing/results due to the presence of a sulfonium ion that displayed false impressions of potential activity: 144802777, 25245977, and 140671722. This sulfonium ion was labeled as “PAINS” or “Pan-assay interference compounds”. This “PAINS” label refers to molecules that can give false-positive results within screening modalities.Berne virus has not been as extensively studied as that of the coronaviruses, however, Berne’s NSP15 is within the Toroviridae family which is under the umbrella of the Nidovirales order. Therefore, since coronaviridae and toroviridae are somewhat similar, it can be proposed that there is potential for cross-reactivity with these inhibitors in toroviridae viruses.The Dengue-3 virus (DENV-3) [Zika virus and dengue virus are within the same genus, flavivirus, so one can propose that there is a high similarity amongst the NS5 protein and domain. Similar to Berne and SARS-CoV, as mentioned above, there is potential for cross-reactivity with the Zika virus if the drugs were introduced to the virus. The following compounds are shown to have some inhibitory activity against the NS5 methyltransferase domain from the (DENV-3) ,30. BothThis group of viruses contains proteins with very similar methyltransferase domains that can be described to be alphavirus-like methyltransferases based on sequence and structure . While tThe following inhibitors, in RSV Polymerase. This group comprises ALS-8112, ALS-8176, and 2′F-4′CN C-adenosine. These work by halting the progression of replication by acting as bases. It can be proposed that halting this domain of the L protein can drastically affect the ability of the methyltransferase domain in the same protein.A subgroup of these inhibitors can be labeled as 2′-fluoronucleoside analogs with inhibition of the This group of inhibitors is comprised of BI-compound D, YM-53403, AZ-27, Compound-1, BRD9101, BRD3969.HRSV, there might be cross-reactivity with it due to high similarity amongst sequences and domains. The following inhibitors comprise this group: ERDRP-0519 and AS-136a.This group of inhibitors is labeled as molecules able to inhibit the L protein of the measles virus. While not directly related to that of Similar to that of the measles inhibitor section, Ebola has a high sequence similarity and domain structure to that of HRSV . The folWhile the variety of methyltransferase domains is immense, both in structure and specificity for different substrates, there is significant conservation of residues that are needed to maintain optimal functionality. Once a clear understanding of a virus’ methyltransferase is understood, one could develop new inhibitors or variations of existing inhibitors to target them. Vaccination efforts in recent years have exponentially increased, as we have seen with the recent SARS-CoV-2 pandemic. It is important to note that while vaccinations are extremely helpful in teaching the body how to develop an immune response against the spike proteins or proteins involved with entry into the host cell, they may not prove the same efficacy against variants. The outermost proteins of viruses may deviate amongst variations but key residues seem to be conserved when observing the main viral proteins, such as methyltransferases. Understanding these concepts is key to the future of drug design to help us in the case of a future pandemic.


In our study, we added a three-dimensional automated breast ultrasound (3D ABUS) to mammography to evaluate the performance and cancer detection rate of mammography alone or with the addition of 3D prone ABUS in women with dense breasts. Our prospective observational study was based on the screening of 1165 asymptomatic women with dense breasts who selected independent of risk factors. The results evaluated include the cancers detected between June 2017 and February 2019, and all surveys were subjected to a double reading. Mammography detected four cancers, while mammography combined with a prone Sofia system (3D ABUS) doubled the detection rate, with eight instances of cancer being found. The diagnostic yield difference was 3.4 per 1000. Mammography alone was subjected to a recall rate of 14.5 for 1000 women, while mammography combined with 3D prone ABUS resulted in a recall rate of 26.6 per 1000 women. We also observed an additional 12.1 recalls per 1000 women screened. Integrating full-field digital mammography (FFDM) with 3D prone ABUS in women with high breast density increases and improves breast cancer detection rates in a significant manner, including small and invasive cancers, and it has a tolerable impact on recall rate. Moreover, 3D prone ABUS performance results are comparable with the performance results of the supine 3D ABUS system. Numerous studies have proven that, in the last 30 years, cancer mortality has decreased; this is predominantly due to an increase in mammography screening programs. Given the diminishing number of late-stage cancers, it should be clear that mortality has also diminished, with statistics reporting a 45% decrease in death rates . ProgresStandard mammography breast cancer screening has helped to reduce breast cancer mortality . That saSome studies have reported a good correlation between 3D ABUS and MRI for lesion measurement, while others have evaluated the performances of 3D ABUS second look and HHUS second look in detecting additional suspicious lesions in patients undergoing preoperative breast MRI. Second look 3D ABUS detected more injuries than HHUS, while 10% of injuries detected by 3D ABUS were not evident in HHUS .Plentiful studies ,17,18,19The aim of our work is to compare the performance and detection rate of mammography alone versus double mammography—3D ABUS detection. Regarding the fact that the second-generation 3D automatic breast ultrasound system involved patients being in a prone rather than a supine position, our studio appears to be the first study to carry out this type of assessment.This study has been evaluated and approved by the Regional Ethics Review Board (registration number 189.20). This study is carried out in the manner prescribed by the Helsinki Statement. Study-specific informed consent was signed by each patient enrolled.All patients involved have signed their consent to use the data. Patients’ signed consent clearly states that the 3D ABUS was not a mammography substitute but an additional investigational analysis procedure. From June 2017 to February 2019, all women who came for routine mammograms underwent a selection process to be enrolled in our study. Follow-up of all enrolled patients covered a period of 24 months (February 2021). We chose only those women who fell into the following categories: women with breast imaging reporting and data system (BI-RADS) density 3/4; heterogeneously or extremely dense breasts; with no implants; not pregnant or breast feeding; with neither personal nor familial cancer history; within the age bracket of 40 to 75; not showing clear breast cancer symptoms; and those who had undergone surgery and percutaneous biopsy in the previous year .The density BI-RADS category was assessed during the screenings by radiographers who received specific training, or by the first radiologist reader if present at the screening. Based upon the above selection criteria, the eligible participants included in our study totaled 1165.The equipment used on all the patients who underwent mammography was FFDM Mammomat Inspiration (Siemens Erlangen Germany). The first mammography examination covered 3 different projections (six images): craniocaudal (CC), mediolateral oblique (MLO) and mediolateral (ML) views, while subsequent examinations were performed with only two images (MLO and CC).The automated breast ultrasound system equipment used was the prone system “Arietta/Sofia” . Two out of the three radiologist who took part in the trial were specifically trained by 3D ABUS professional technicians in order to be fully qualified to best utilize the system’s capacities, while the third radiologist was trained by the other two radiologists. Sofia is a 3D scan system device made of three different parts: a scan bed, a probe and a workstation. The scan bed table (on which the patient lies) has a 184 mm circular area in which a 92 mm long multifrequency (5–10 MHz) ultrasound transducer is embedded. The probe is sealed underneath a glass layer on which the ultrasound gel lotion is applied. A specific and dedicated workstation elaborates all of the scanned images as 3D images. The pre scan touch panel interface option allows the parameter to be modified A patient lies down prone with the bent contralateral leg positioned slightly rotated so that the breast flattens on the glass, with the nipple centered in the cone; the arm can be placed on the side . The opeThe original acquisitions were achieved in the transverse plane—the same as in supine ABUS systems—and they are then rebuilt in the sagittal and coronal planes.The process takes roughly 10 min per patient before the data are sent to the workstation. Three experienced breast radiologists (10 to 20 years’ experience) read and interpret the images; two of the three radiologists were fully trained by a Sofia ABUS technician, while the third one was trained by the other two radiologists.For each case, two of the three radiologists involved in the study sequentially interpreted both the mammogram and the 3D prone ABUS. In case of a reading of concern, none of the radiologists specified whether the concern was about full-field digital screening mammography (FFDSM) or 3D ABUS, nor did they discuss the position of the finding. This was executed to perform a double-blinded reading and to enable the double evaluation of BI-RADS results of the 3D ABUS prone exam and establish inter-observer variability.To evaluate possible malignant cases or possible recalls, each radiologist evaluated the FFDSM exams on the same workstation (5 Mega Pixel resolution), without access to the 3D prone ABUS images. Only after assigning the final Bi-RADS opinion to the screening mammography did the same radiologist interpret the 3D prone ABUS images and express a final opinion based on the assessment of mammography + 3D prone ABUS. All three radiologists evaluated 3D ABUS images on the same reconstruction workstation. Therefore, for each case, the mammography images and the three-dimensional ABUS images were evaluated in a double reading by two radiologists, with a determination for the final consensus.The radiologists interpreted the 3D ABUS findings  with respect to the following parameters: shape of mass, margins, orientation, echo pattern, lesion boundary and posterior acoustic features. Moreover, 3D prone ABUS System BI-RADS classification was assessed with the following criteria: 1 = negative; 2 = benign; 3 = probably benign; 4a = low suspicion; 4b = moderate suspicion; 4c = high suspicion; 5 = suggestive of malignancy. Readings of the BI-RADS images’ concordance were assigned by the K Fleiss statistic.We recalled all women with suspicious findings detected in either the 3D ABUS or FFDSM to perform detailed mammography and/or handheld ultrasonography exams. After the recall, all women for whom no signs of malignancy were found were informed, and they were asked to come back for their next screening following normal timings and procedures . In case of suspected malignancy, women were subjected to biopsy; the removed tissue was then analyzed by a pathologist to provide a final and confirmed diagnosis. For examinations evaluated with BI-RADS 1 or 3, an additional imaging investigation was carried out in order to determine a final and defined BI-RADS assessment. When the assigned BI-RADS was 4 or 5, a stereotactic or percutaneous guided ultrasound biopsy was performed. In case of benign biopsy results, no further investigations were required. If a malignancy was proven by biopsy results, the lesions were surgically removed.In each observation group (mammography versus mammography + 3D ABUS), deviations in cancer detection rate, recall rate, sensitivity, specificity, positive predictive value (PPV) and negative predicative value (NPV) were evaluated. We assigned the women into three groups: PPV1 , PPV2 (proportion of cancer detection among women recommended for biopsy), and PPV3 (proportion of cancer detection among women undergoing biopsy). All recommended biopsies were performed, and PPV2 and PPV3 were matched.McNemar’s test was utilized to compare diagnostic yield between mammography and automated Sofia ultrasound system. Results below 0.05 were classified as statically significant. Analyses were carried out using IBM Spss Statistics 23 software .t-test. Sensitivity and specificity calculations, against both mammography and the 3D prone ABUS system, were determined using a 2 × 2 table. McNemar’s SPSS was used for specificity testing and binomial testing was used to assess sensitivity. To evaluate PPV, we took into account only the recalled patients, and three different American College of Radiology (ACR) classification values were applied as follows: (a) Cancer percentage found at screening but where further investigation was needed (PPV1); (b) cancer percentage where biopsy was requested (PPV2); and (c) proportion of detected cancer after biopsy (PPV3). All requested biopsies were performed; therefore, the estimated PPV2 and PPV3 results were identical. No patient skipped the follow-up. Our study statistics results were compared to the BI-RADS classification using the Fleiss statistic K test with the Landis and Koch scale value. Diagnostic estimates interval of confidence applied was 95%; SPSS (IBM CORPORATION SPSS Statistics 23) was used for our analysis.Categorical and continuous variables were assessed using a Clopper–Pearson chi-square test and a Student’s The selected patients divided by age group and mammographic density are shown in Among the patients selected, twelve women were diagnosed with breast cancer, including four cancers detected with mammography alone, while another eight cancers were detected by adding 3D prone ABUS , Table 2p ˂ 0.001). However, the addition of interval cancer resulted in a sensitivity increase of 31.8% .By adding 3D prone ABUS to mammography, screening sensitivity saw of a 34.7% increase , while surgery increased by a rate of 3.4% . There were fewer biopsies (PPV2 and PPV3) requested and carried out after performing mammography in conjunction with 3D prone ABUS than there were compared to those requested and carried out after performing mammography alone: 41%  in relation to the two examinations versus 58.8%  for mammography. Furthermore, the inclusion of interval cancer in the NPV increased in mammography combined with 3D prone ABUS in comparison to mammography alone, though only by 0.2% (95% CI:0.12–3.1)  .p ˂ 0.001). while four cancers were detected with the mammographic screening alone  (p ˂ 0.001). Consequently. there was an increase of 50% in terms of cancer detection  . The difetection .p ˂ 0.001) and 26.6 per 1000 women for mammography combined with 3D prone ABUS ; this is a difference in recall of 12.1‰ . During mammography screening, four interval cancers (0.3%) were , and they were only recognized only after patients perceived some irregularity during self-palpation. Three of four interval cancers were found in patients classified as ACR, while the fourth one was ACR3. In retrospect, we hypothesize that two interval cancers (ACR4) might have been spotted due to an unstructured opacity (obscured view in part of the breast) that was placed where the cancer developed, but it showed a low cancer predictive value.In our study, the recall rate of mammography alone is 14.5 per 1000 women . Moreover. ABUS alone detected cancers that presented with a lower stage at diagnosis, suggesting positive prognostic implications. In this study, recall rates increased from 150.2 per 1000 women with mammography alone to 284.9 per 1000 women by adding ABUS, and specificity decreased by 13.4% for combined modalities  [Our study shows a high sensitivity of the prone ABUS system associated with mammography (93.5%). which is comparable to the sensitivity found in the more famous Somoinsight study (ABUS supine. 100%) . This fi3D ABUS) ,27. FurtIn other studies in the literature ,29,30, tOur study shows a specificity of 87% with the addition of 3D ABUS, though this is reduced compared to mammography alone and is in line with other studies in the literature: it should be noted, however, that Choi  showed aMoreover, an increase in cancer detection rates after the addition of prone ABUS to mammography (26.6 per 1000 women) is perfectly comparable with the results of other contributions in the literature that use the 3D ABUS supine system, though the number of patients enrolled should be taken into account ,16,17. OPossible future evolutions of the present study could concern comparisons between prone 3D ABUS and prone breast MRI, as well as comparisons between supine ABUS and supine breast MRI. As far as we know, there are no comparative studies of this type in the literature when analyzing large patient samples; a few of the existing studies focus on small samples of patients evaluated with supine ABUS and prone MRI for the assessment of density or in the search for residual disease in patients treated with neoadjuvant chemotherapy .As far as we know, there are no comparative studies between 3D ABUS and MRI in the supine position, and there are only studies that take into consideration single methods of analysis ,44.Another possible development of this study could be a comparison of the performance of CAD systems associated with mammography ,47,48,49It is also possible to carry out comparative evaluations between ABUS and MRI with associated computed aided detection (CAD) in the latter method that partially reduce the visual disturbance effect on reporting caused by background parenchymal enhancement ,51.Another technique with the potential to become a valuable complement to mammography screening is tomosynthesis .In this regard, however, it is important to consider that multi-centric prospective trials ASTOUND and ASTOUND2 by Tagliafico et al.which compare the addition of HHUS and tomosynthesis in patients with dense breasts and negative mammography at screening, report a better diagnostic increase for echography than tomosynthesis, with a percentage of false positive recall rate matching the first study (interim report). However, this percentage is significantly higher with the addition of echography in the second study ,51. In oTo conclude, we can assert that, for women with dense breasts, the cancer screening process is enhanced by combining 3D prone ABUS and FFDSM. Through this combination, cancer detection rates increase, but they also do so within an admissible recall rate.The above has been already stated in previous studies, and we need more studies and further investigations to better assess the combined methodologies’ advantages and benefits, as well as their feasibility in terms of costs in screening programs for women with dense breasts or very dense breasts. The results of the present contribution suggest that the total performances of the 3D prone ABUS system are substantially comparable to those of the 3D ABUS systems in which patients are supine. That said, to confirm this notion, further comparative studies with larger numbers of patients are needed.


Dioscorea spp.) into flour is aimed at optimizing techniques to obtain a material with high physicochemical and functional properties. The present study investigates the effect of the processing techniques on the levels of inulin, organic acids, total phenolics (TP), antioxidant capacity, and polyphenol oxidase (PPO) and peroxidase (POD) activities, as well as on the physicochemical properties of flour derived from two yam species—Dioscorea esculenta and Dioscorea bulbifera. All tubers were peeled and sliced, then subjected to different processing techniques through blanching, soaking, and drying. The results revealed that freeze-drying appears to be the best technique in achieving the highest whiteness index of yam flour. This coincided well with the low phenolics content and POD activity, which suggests a reduced enzymatic browning reaction in the freeze-dried yam flour. On the other hand, chemical analyses showed that D. esculenta and D. bulbifera flours have the highest levels of inulin  and succinic acid  in the samples subjected to direct oven-drying. Oven drying without pretreatment neither blanching nor water steeping maintained antioxidant activity in the flour derived from both D. esculenta and D. bulbifera.Research on the processing of yam ( Dioscorea spp.). Research has shown that yam tubers, particularly Dioscorea esculenta and Dioscorea bulbifera, are rich in inulin, organic acids, phenolics, and other antioxidants [Indonesia has a wide variety of carbohydrate-rich foods, among which is yam tuber  from Yogyakarta, Indonesia, were washed, peeled, and sliced (2 mm thickness). The lot was then portioned into four treatment groups as described in Two yam species  and sieved through a 60-mesh screen. The yam flour was sealed in polyethylene laminated bags to prevent moisture absorption and stored in a freezer (−20 °C) until used for further analysis.w/w) was calculated by weighing the yam flour, which was produced after drying. Color profiles  were measured using a chromameter . These values were used to calculate the whiteness index (WI), commonly used to indicate the color quality of flour, with the equation.Moisture contents of yam flour were measured by the air oven method of the Association of Official Analytical Chemists (AOAC) . The rep−1 DW). Yam flour (1 g) were mixed with 10 mL of 80% chilled ethanol  for 20 min with continuous shaking at 27 °C. The suspension was centrifuged and the supernatant was collected. The residue was re-extracted twice with 10 mL of 80% chilled ethanol and all supernatants were combined. The phenolic extracts were concentrated using a rotary evaporator  and then filled up with methanol to a final volume of 10 mL. The phenolic compounds were then stored at −20 °C until use.Total phenolics (TP) were measured according to previously described methods by Hung et al.  and Okarg and the absorbance of the clear supernatants was measured at 725 nm using a spectrophotometer . A standard calibration was prepared using gallic acid  and the content of total phenolics in each extract was calculated and expressed as milligrams of gallic acid equivalent (GAE) per gram of the sample.The phenolic contents in yam flour were determined using Folin-Ciocalteu’s method. The phenolic extracts were diluted to an appropriate concentration. The diluted solution (0.5 mL) was then oxidized with Folin-Ciocalteu’s reagent  and then neutralized with saturated sodium carbonate solution . The volume was adjusted to 10 mL with distilled water, then thoroughly mixed and allowed to stand for 45 min at ambient temperature. The solution was centrifuged for 5 min at 4000× −1 g−1 DW. Enzyme extracts were prepared from 5 g yam flour by homogenization of 25 mL cold extraction solution . The homogenate was held at 5 °C for 2 h, then centrifuge  at 32,300× g at 4 °C for 10 min, and filtered through a 0.45 µm filter . The filtrate was then kept in an ice bath. The PPO assay was performed using caffeic acid  as substrate. A total of 0.5 mL of substrate (1 mg/mL caffeic acid) was mixed with 0.6 mL of filtrate and the absorbance was measured at 450 (A450) using a spectrophotometer . The sample A450 was recorded at 1 min intervals for 7 min vs. a 1.1 mL blank containing no substrate. The net A450 was tabulated and plotted against time and the slope was calculated for the linear portion of the curve.Polyphenol oxidase (PPO) activities were determined spectrophotometrically as described previously by Bach et al.  with ming for 5 min. The substrate was prepared just before use by mixing 0.5 mL of 1% (p/v) aqueous pyrogallol  with 6 mL of 0.3% (v/v) aqueous H2O2 . The reaction was run in 1.5 mL UV-cuvette semi-micro containing 0.05 mL of peroxidase extract and 1.45 mL of substrate; the blank was obtained by replacing the peroxidase extract by pure buffer (pH 7.0). The maximum increase in absorbance at 460 nm was determined; one unit of peroxidase activity was defined as an increase in absorbance of 0.001 min−1.Peroxidase (POD) activity was determined with a procedure adapted from previously reported methods by Akissoe et al.  and MestThe scavenging of the stable DPPH radical was widely used to evaluate antioxidant activity of phenolic compounds extracted from tuber, cereal, and fruit ,21,23,27sequential arrangement and stored at −30 °C until use. Inulin was measured by HPLC  equipped with a Shim-pack SCR-101N column  and refractive index detector RID-10A . Column oven temperature was 80 °C, flow rate was 0.25 mL/min with a pump, and pure water was used as the carrier. Inulin  was used as standards.The inulin in yam flour was determined using an adjusted procedure as described previously by Takeuchi et al. . Soluble2SO4 as mobile phase, at a flow rate of 0.5 mL/min, the Diode Array Detector (DAD) operating at 214 nm and column oven temperature was 30 °C. For calibration of the HPLC system, a standard mixture of oxalic acid , citric acid , succinic acid , and glutaric acid  in 100 mL of 0.0125 M H2SO4 was used.The organic acids were determined by HPLC, using the extraction and analysis method, as described previously by Bhandari et al. . A totalThe experimental used completely randomized design with three replications. Normally distributed data with homogenous variance were subjected to analysis of variance (ANOVA) and DMRT test was used for comparison of the means, utilizing SAS software . All other data were subjected to the Kruskal–Wallis rank sum test using Minitab 14.D. esculenta and D. bulbifera ranged from 20.64–29.17% and 18.02–27.52%, respectively .D. bulbifera flour were between 74.84 and 92.24. In order to prevent the discoloration during processing, yam slices were pretreated with blanching and steeping steps before drying. The yam flour from both species that were water-steeped, blanched, and freeze-dried (SB-Freeze) had a lighter color as indicated by higher L-values.Color of yam flour prepared with different processing techniques are shown in D. esculenta and D. bulbifera flour.The water-steeped, blanched, and freeze-dried flour (SB-Freeze) from both species used showed the lowest redness  and lowest yellowness  compared to flour produced by other treatments. These low colors attributes reflected on the significant increase in whiteness index (WI) of this group. The SB-Freeze treatment had significant effect on increasing whiteness index of Color changes due to the drying process can affect the final food quality. Freeze-drying applied in this study prevents color degradation better than oven-drying. This method also produces a less damaging color than air-drying and spray-drying . Freeze-D. esculenta flour. −1 g−1 DW and 3.19 and 11.86 mg GAE/g, respectively. The loss of TP content was caused by leaching during the blanching. This phenomenon may be explained by the fact that phenolics are highly soluble in water [Enzymatic browning is linked to polyphenol oxidase (PPO) and peroxidase (POD) activities, and the production of polyphenols and their derivates ,35,36. Tin water ,41. The in water . The redin water ,43,44,45D. bulbifera flour  prior to analysis. However, this implicates a probably rapid color degradation of this flour during storage in the conditions commonly used by the industry [Contrary to the previous report , the resindustry .D. esculenta and D. bulbifera flour from treatment group of Oven (control) have the highest DPPH scavenging capacities due to the higher TP content. The lower TP content in samples groups of B-Oven, SB-Oven, and SB-Freeze was caused by leaching during steeping and blanching, consistent with the previous report of Xiao et al. [The percentage of DPPH scavenging of yam flour are between 5.00 and 18.24% . The reso et al. D. esculenta and D. bulbifera flour range from 4.99 to 27.80 g/100 g DW .For the different application, oven-drying can also be used as a pretreatment prior to extraction and spray drying process as developed in the previous study  to produD. esculenta and D. bulbifera flour ranged between 549.7 and 7957.7, and 447.8 and 7653.7 mg/100 g DW, respectively. Citric acid was determined to be the second most abundant organic acid in yam flour. Citric acid concentrations in D. esculenta and D. bulbifera flour ranged between 47.5 and 1739.2, and 30.2 and 1374.1 mg/100 g DW, respectively. Oxalic acid was determined in considerable amount in all yam species. The concentration of oxalic acid in D. esculenta and D. bulbifera flour were found to be 11.8 and 31.2, and 11.8 and 18.8 mg/100 g DW, respectively.Succinic acid appears to be the prominent organic acid in both species of yam. Succinic acid concentrations in D. esculenta and D. bulbifera. While, decreasing of oxalic acid is apparently affected by the blanching and drying steps in D. esculenta flour but not in D. bulbifera flour. Oven drying without pretreatment significantly maintained the amount of organic acid in all yam species. Blanching, steeping and freeze-drying significantly decreased the amount of succinic acid in the yam flour of both Organic acids as phenolic compounds have a preventive role against various diseases due to their antioxidant properties. Oxalic, citric, and succinic acids are able to chelate metals . Citric D. bulbifera flour. A previous study [D bulbifera flour increased with decreasing TP content and PPO activity.Correlations between TP content or PPO activities or POD activities and color  are given in us study  showed tD. bulbifera flour. These results show that browning index did not correlate with POD activity of D. bulbifera flour.In contrast, a positive correlation was seen between POD activities and color  in D. esculenta flour (r = 0.931 ****). Antioxidant activity of the yam flour increased with increasing its TP content. In food, polyphenols may contribute to the bitterness, astringency, color, flavor, odor, and oxidative stability [A strong positive correlation was shown between TP content and antioxidant activity in tability . Polyphetability .D. esculenta flour increased with increasing total phenolics content. Freeze-drying in the production of D. bulbifera flour either improved physical properties or reduced enzymatic browning. Enzymatic browning was due to the production of polyphenols and PPO activity, but not POD activity. Antioxidant activity in D. esculenta flour increased with increasing of TP content. With further research, these results may prove valuable in customizing the processing steps and conditions to achieve the desired functional characteristics of yam flour intended for the production of foods and nutrition with special dietary uses. Yam flour as an intermediate product has health-promoting characteristics that can be developed in many sectors, responding to the worldwide trend toward foods that have nutrition and health benefits.Functional properties of yam flour prepared by different processing techniques have been determined. Inulin, total phenolic content and succinic acid were highly preserved by direct oven drying without pretreatment. The addition of steeping and blanching steps as pretreatment to drying led to considerable losses of the functional constituents of yam. However, flour prepared without pretreatment displayed massive discoloration indicating the importance of optimization in the preparation processing of yam flour. Antioxidant activity in