Amino Acid Research Articles

Enhancement of N-Methyl Amino Acid Incorporation into Proteins and Peptides Using Modified Bacterial Ribosomes and Elongation Factor P.

N-Methylated amino acids are constituents of natural bioactive peptides and proteins. Nα-methylated amino acids appear abundantly in natural cyclic peptides, likely due to their constraint of peptide conformation and contribution to peptide stability. Peptides containing Nα-methylated amino acids have long been prepared by chemical synthesis. While such natural peptides are not produced ribosomally, recent ribosomal strategies have afforded Nα-methylated peptides. Presently, we define new strategies for the ribosomal incorporation of Nα-methylated amino acids into peptides and proteins. First, we identify modified ribosomes capable of facilitating the incorporation of six N-methylated amino acids into antibacterial scorpion peptide IsCT. Also synthesized analogously was a protein domain (RRM1) from hnRNP LL; improved yields were observed for nearly all tested N-methylated amino acids. Computational modeling of the ribosomal assembly illustrated how the distortion imposed by N-methylation could be compensated by altering the nucleotides in key 23S rRNA positions. Finally, it is known that incorporation of multiple prolines (an N-alkylated amino acid) ribosomally can be facilitated by bacterial elongation factor P. We report that supplementing endogenous EF-P during IsCT peptide and RRM1 protein synthesis gave improved yields for most of the N-methylated amino acids studied.

CSF amino acid profiles in ICV-streptozotocin-induced sporadic Alzheimer's disease in male Wistar rat: a metabolomics and systems biology perspective.

Alzheimer's disease (AD) is an increasingly important public health concern due to the increasing proportion of older individuals within the general population. The impairment of processes responsible for adequate brain energy supply primarily determines the early features of the aging process. Restricting brain energy supply results in brain hypometabolism prior to clinical symptoms and is anatomically and functionally associated with cognitive impairment. The present study investigated changes in metabolic profiles induced by intracerebroventricular-streptozotocin (ICV-STZ) in an AD-like animal model. To this end, male Wistar rats received a single injection of STZ (3 mg·kg-1) by ICV (2.5 μL into each ventricle for 5 min on each side). In the second week after receiving ICV-STZ, rats were tested for cognitive performance using the Morris Water Maze test and subsequently prepared for positron emission tomography (PET) to confirm AD-like symptoms. Tandem Mass Spectrometry (MS/MS) analysis was used to detect amino acid changes in cerebrospinal fluid (CFS) samples. Our metabolomics study revealed a reduction in the concentrations of various amino acids (alanine, arginine, aspartic acid, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophane, tyrosine, and valine) in CSF of ICV-STZ-treated animals as compared to controls rats. The results of the current study indicate amino acid levels could potentially be considered targets of nutritional and/or pharmacological interventions to interfere with AD progression.

Regional features of camel milk composition and properties in the Republic of Kazakhstan

Camel milk, renowned for its distinctive nutritional qualities, has captured the interest of scientific researchers due to its potential health benefits. This study aims to compare the biochemical composition of camel milk sourced from two distinct regions of Kazakhstan: Jetisu and Mangystou. Analytical methods were employed to achieve this objective, including gas chromatography for fatty acid analysis, chemical methods for physicochemical parameter determination, and assessment of amino acid, fatty acid, mineral, and vitamin content. In samples from the Jetisu region, protein content ranged from 3.61% to 3.70%, fat from 3.85% to 4.64%, and lactose from 4.80% to 4.85%. In comparison, samples from the Mangystou region exhibited protein content ranging from 3.65% to 3.81%, fat from 4.72% to 5.75%, and lactose from 4.21% to 4.28%. Regarding amino acid composition, Mangystou region samples contained more essential amino acids per 100 g of protein than Jetisu region samples: 41.29 g versus 38.20 g, respectively. Additionally, the Jetisu region sample contained 64.291% saturated fatty acids, while the Mangystou region sample had 62.135%, indicating differences in fatty acid composition based on geographical origin. In terms of mineral composition, camel milk from the Mangystou region exhibited higher calcium and zinc content compared to Jetisu region samples, with calcium and zinc content measured at 124.50 mg/100 g and 490.15 μg/100 g, respectively, for Mangystou samples, and 112.50 mg/100 g and 321.24 μg/100 g, respectively, for Jetisu samples. Overall, the study underscores regional variances in camel milk's biochemical composition, which can impact its nutritional and biological value. These findings provide dairy product producers in Kazakhstan with enhanced opportunities to create healthy, high-quality dairy products.

Metabuli: sensitive and specific metagenomic classification via joint analysis of amino acid and DNA.

Metagenomic taxonomic classifiers analyze either DNA or amino acid (AA) sequences. Metabuli ( https://metabuli.steineggerlab.com ), however, jointly analyzes both DNA and AA to leverage AA conservation for sensitive homology detection and DNA mutations for specific differentiation of closely related taxa. In the Critical Assessment of Metagenome Interpretation 2 plant-associated dataset, Metabuli covered 99% and 98% of classifications of state-of-the-art DNA- and AA-based classifiers, respectively.

Quality Evaluation of Polygonatum cyrtonema Hua Based on UPLC-Q-Exactive Orbitrap MS and Electronic Sensory Techniques with Different Numbers of Steaming Cycles

In this study, electronic sensory techniques were employed to comprehensively evaluate the organoleptic quality, chemical composition and content change rules for Polygonatum cyrtonema Hua (PCH) during the steaming process. The results were subjected to hierarchical cluster analysis (HCA), principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). These analyses revealed, from a sensory product perspective, overall differences in colour, odour and taste among the samples of PCH with different numbers of steaming cycles. Using the UPLC-Q-Exactive Orbitrap MS technique, 64 chemical components, including polysaccharides, organic acids, saponins and amino acids were detected in PCH before and after steaming. The sensory traits were then correlated with the chemical composition. From the perspectives of sensory traits, chemical composition, and multi-component index content, it was preliminarily deduced that carrying out five cycles of steaming and sun-drying was optimal, providing evidence for the quality evaluation of PCH during the steaming process.

Effect of Overripening on the Physico-Chemical and Sensory Characteristics of Boneless, Salt-Reduced Iberian Dry-Cured Ham

The aim of this study was to evaluate the effect of extended maturation and temperature increase on the physico-chemical, biochemical, instrumental color and texture, sensory, and acceptability parameters of cured and boneless Iberian hams. Given the limited knowledge in this area, our objective was to develop a ham with enhanced proteolysis, potentially leading to increased bioactive peptide generation and superior sensory characteristics compared to salt-reduced counterparts. To achieve this, a batch of hams cured up to 38% loss at 30 °C and two batches cured up to 42% loss at 30 °C and 36 °C were evaluated. Results showed that the increase in processing time and temperature significantly enhanced (p < 0.05) ham proteolysis and amino acid content without adversely affecting its texture. No significant differences were observed in instrumental texture parameters or sensory attributes as evaluated by consumers. These processing conditions also increased the content of free amino acids, improving the product quality. Overall, these processing modifications resulted in hams with excellent sensory acceptability and enhanced bioactive potential despite the salt reduction.

Single-Scan Heteronuclear 13C-15N J-Coupling NMR Observations Enhanced by Dissolution Dynamic Nuclear Polarization.

Heteronuclear 13C-15N couplings were measured in single-scan nuclear magnetic resonance (NMR) experiments for a variety of nitrogen-containing chemical compounds with varied structural characteristics, by using a one-dimensional (1D) 13C-15N multiple-quantum (MQ)-filtered experiment. Sensitivity limitations of the MQ filtering were overcome by the combined use of 15N labeling and dissolution dynamic nuclear polarization (dDNP), performed at cryogenic conditions and followed by quick and optimized sample melting and transfer procedures. Coupling information could thus be obtained from nucleotide bases, amino acids, urea, and aliphatic and aromatic amides, including the measurement of relatively small J-couplings directly from the 1D filtered spectra. This experiment could pave the way for NMR-based analytical applications that investigate structural and stereochemical insights into nitrogen-containing compounds, including dipeptides and proteins, while relying on heteronuclear couplings and nuclear hyperpolarization.

Immunoglobulin repertoire sequencing and de novo sequencing – Powerful tools for identifying free light chains from patients with light chain cast nephropathy

In patients with light chain cast nephropathy (LCCN), abundantly produced monoclonal immunoglobulin free light chains (FLCs) play a vital role in pathogenesis. Determining the precise sequences of patient-derived FLCs is therefore highly desirable. Although immunoglobulin repertoire sequencing (5′ RACE-seq) has been proven to be sensitive enough to provide full-length V(D)J region (variable, diversity and joining genes) of FLCs using bone marrow samples, an invasive and bone marrow independent method is still in demand. Here a de novo sequencing workflow based on the bottom-up proteomics for patient-derived FLCs was established. PEAKS software was used for the de novo sequencing of peptides that were further assembled into full-length FLC sequences. This de novo protein sequencing method can obtain the full-length amino acid sequences of FLCs, and had been shown to be as reliable as 5′ RACE-seq. The two LCCN sequences derived from above the two methods were identical, and they possessed more hydrophobic or nonpolar amino acids compared with the corresponding germline, which may be associated with the pathogenesis.

Nutritional values, antioxidant, and cytotoxic activities of selected edible marine macroalgae: a comparative study

AbstractThe objective of this study is to assess the chemical composition, nutritional values, and bioactivities of four macroalgae from the Egyptian Red Sea coasts using standard methods. Of these, three Rhodophyceae species, Digenea simplex (D. simplex), Laurencia papillosa (L. papillosa), and Galaxaura oblongata (G. oblongata), and one Phyaeophyceae species, Turbinaria decurrens (T. decurrens), were selected. The results of proximate and chemical composition analyses based on the algal dry weight (DW) showed that carbohydrate content was the highest, ranging from 32.47 ± 1.03% to 45.5 ± 1.23%. Other algal constituents, including ash, protein, moisture, sulfate, lipid, phenolics, and flavonoids, have contents that depend on the algal species. Besides, HPLC analysis revealed that each algal extract contained varying amounts of nine phenolic acids. Atomic absorption spectrometry, the HPLC-Pico-Tag method, the ion chromatography technique, and GC/MS analyses were used to determine the chemical profiles of the elemental, amino acid, halide, and fatty acid of each algal extract. Bioactivities revealed that both the •DPPH and ABTS assays showed that all the algae studied had a significant ability to scavenge free radicals in a dosage-dependent way. They also had strong selective cytotoxic activity against HEPG-2 and HCT-116 cell lines, but only weak activity against MCF-7 and A549 cell lines. Finally, our findings suggested that the selected algae might be efficiently used as nutraceuticals and functional foods, indicating an increase in their proliferation.

Rapid Synthesis of Peptides Mediated by PPh3−I2

AbstractWe report a mild, operationally simple, convenient, and rapid method of peptide bond formation using triphenylphosphine and iodine. The developed protocol was utilized to couple coded, non‐coded, and challenging amino acids in 64–92 % yield. Sterically hindered α,α‐disubstituted amino acids, which are important constituents of natural peptides, were also coupled in good yield. Mild reaction conditions in open air produce peptide bonds in a short time (30 min) without racemization.

Improving nitrofurantoin resistance prediction in Escherichia coli from whole-genome sequence by integrating NfsA/B enzyme assays.

Nitrofurantoin resistance in Escherichia coli is primarily caused by mutations damaging two enzymes, NfsA and NfsB. Studies based on small isolate collections with defined nitrofurantoin MICs have found significant random genetic drift in nfsA and nfsB, making it extremely difficult to predict nitrofurantoin resistance from whole-genome sequence (WGS) where both genes are not obviously disrupted by nonsense or frameshift mutations or insertional inactivation. Here, we report a WGS survey of 200 oqxAB-negative E. coli from community urine samples, of which 34 were nitrofurantoin resistant. We characterized individual non-synonymous mutations seen in nfsA and nfsB among this collection using complementation cloning and NfsA/B enzyme assays in cell extracts. We definitively identified R203C, H11Y, W212R, A112E, and A112T in NfsA and R121C, Q142H, F84S, P163H, W46R, K57E, and V191G in NfsB as amino acid substitutions that reduce enzyme activity sufficiently to cause resistance. In contrast, E58D, I117T, K141E, L157F, A172S, G187D, and A188V in NfsA and G66D, M75I, V93A, and A174E in NfsB are functionally silent in this context. We identified that 9/166 (5.4%) nitrofurantoin-susceptible isolates were "pre-resistant," defined as having loss of function mutations in nfsA or nfsB. Finally, using NfsA/B enzyme assays and proteomics, we demonstrated that 9/34 (26.5%) ribE wild-type nitrofurantoin-resistant isolates also carried functionally wild-type nfsB or nfsB/nfsA. In these cases, NfsA/B activity was reduced through downregulated gene expression. Our biological understanding of nitrofurantoin resistance is greatly improved by this analysis but is still insufficient to allow its reliable prediction from WGS data.

Proscan: a structure-based proline design web server.

The ability to control protein conformations and dynamics through structure-based design has been useful in various scenarios, including engineering of viral antigens for vaccines. One effective design strategy is the substitution of residues to proline amino acids, which due to its unique cyclic side chain can favor and rigidify key backbone conformations. To provide the community with a means to readily identify and explore proline designs for target proteins of interest, we developed the Proscan web server. Proscan provides assessment of backbone angles, energetic and deep learning-based favorability scores, and other parameters for proline substitutions at each position of an input structure, along with interactive visualization of backbone angles and candidate substitution sites on structures. It identifies known favorable proline substitutions for viral antigens, and was benchmarked against datasets of proline substitution stability effects from deep mutational scanning and thermodynamic measurements. This tool can enable researchers to identify and prioritize designs for prospective vaccine antigen targets, or other designs to favor stability of key protein conformations. Proscan is available at: https://proscan.ibbr.umd.edu.

Characterization of the genetic variation and evolutionary divergence of the CLEC18 family.

The C-type lectin family 18 (CLEC18) with lipid and glycan binding capabilities is important to metabolic regulation and innate immune responses against viral infection. However, human CLEC18 comprises three paralogous genes with highly similar sequences, making it challenging to distinguish genetic variations, expression patterns, and biological functions of individual CLEC18 paralogs. Additionally, the evolutionary relationship between human CLEC18 and its counterparts in other species remains unclear. To identify the sequence variation and evolutionary divergence of human CLEC18 paralogs, we conducted a comprehensive analysis using various resources, including human and non-human primate reference genome assemblies, human pangenome assemblies, and long-read-based whole-genome and -transcriptomesequencing datasets. We uncovered paralogous sequence variants (PSVs) and polymorphic variants (PVs) of human CLEC18 proteins, and identified distinct signatures specific to each CLEC18 paralog. Furthermore, we unveiled a novel segmental duplication for human CLEC18A gene. By comparing CLEC18 across human and non-human primates, our research showed that the CLEC18 paralogy probably occurred in the common ancestor of human and closely related non-human primates, and the lipid-binding CAP/SCP/TAPS domain of CLEC18 is more diverse than its glycan-binding CTLD. Moreover, we found that certain amino acids alterations at variant positions are exclusive to human CLEC18 paralogs. Our findings offer a comprehensive profiling of the intricate variations and evolutionary characteristics of human CLEC18.

Systematic-Narrative Hybrid Literature Review: Crosstalk between Gastrointestinal Renin–Angiotensin and Dopaminergic Systems in the Regulation of Intestinal Permeability by Tight Junctions

In the first part of this article, the role of intestinal epithelial tight junctions (TJs), together with gastrointestinal dopaminergic and renin–angiotensin systems, are narratively reviewed to provide sufficient background. In the second part, the current experimental data on the interplay between gastrointestinal (GI) dopaminergic and renin–angiotensin systems in the regulation of intestinal epithelial permeability are reviewed in a systematic manner using the PRISMA methodology. Experimental data confirmed the copresence of DOPA decarboxylase (DDC) and angiotensin converting enzyme 2 (ACE2) in human and rodent enterocytes. The intestinal barrier structure and integrity can be altered by angiotensin (1-7) and dopamine (DA). Both renin–angiotensin and dopaminergic systems influence intestinal Na+/K+-ATPase activity, thus maintaining electrolyte and nutritional homeostasis. The colocalization of B0AT1 and ACE2 indicates the direct role of the renin–angiotensin system in amino acid absorption. Yet, more studies are needed to thoroughly define the structural and functional interaction between TJ-associated proteins and GI renin–angiotensin and dopaminergic systems.

Unguarded liabilities: Borrelia burgdorferi’s complex amino acid dependence exposes unique avenues of inhibition

Recent reports from the Centers for Disease Control and Prevention approximate 500,000 cases of Lyme disease in the United States yearly, a significant economic burden on the healthcare system. The standard treatment for Lyme disease includes broad-spectrum antibiotics, which may be administered for extensive periods of time and result in significant impacts to the patient. Recently, we demonstrated that Borrelia burgdorferi, the causative agent of Lyme disease, is uniquely dependent upon peptide acquisition via an oligopeptide transport (Opp) system. This dependence appears unique to the spirochete; thus, the Opp system may constitute a novel and specific target for the inhibition of B. burgdorferi. For proof of concept, we conducted a pilot screen to determine if the Opp system constitutes a viable inhibitor target. OppA2 was utilized as our target protein as it is the most prolific peptide-binding protein throughout the enzootic cycle. We validated a thermal shift assay (TSA) to detect ligand binding against OppA2 and performed a high-throughput screen of 2,240 molecules from a diversity set library. The TSA results identified eight compounds (C1–8) demonstrating potential binding to OppA2, and growth assays identified C2 and C7 as inhibitors of B. burgdorferi growth. We confirmed by TSA that these two compounds interact with additional B. burgdorferi OppAs, potentially resulting in a cumulative inhibitory effect. Additionally, we showed that these compounds have no effect on Escherichia coli, a bacterium that encodes a dispensable Opp system which serves only as an ancillary nutrient transporter. These data demonstrate that the Opp system of B. burgdorferi acts as a viable drug target, with the potential for targeting multiple OppAs with a single compound. Moreover, the lack of inhibition against E. coli suggests that selective targeting of B. burgdorferi via the Opp system may be possible.

The Influence of Zinc Oxide Nanoparticles and Salt Stress on the Morphological and Some Biochemical Characteristics of Solanum lycopersicum L. Plants

Salinity reduces crop yields and quality, causing global economic losses. Zinc oxide nanoparticles (ZnO-NPs) improve plant physiological and metabolic processes and abiotic stress resistance. This study examined the effects of foliar ZnO-NPs at 75 and 150 mg/L on tomato Kecskeméti 549 plants to alleviate salt stress caused by 150 mM NaCl. The precipitation procedure produced ZnO-NPs that were characterized using UV-VIS, TEM, STEM, DLS, EDAX, Zeta potential, and FTIR. The study assessed TPCs, TFCs, total hydrolyzable sugars, total free amino acids, protein, proline, H2O2, and MDA along with plant height, stem width, leaf area, and SPAD values. The polyphenolic burden was also measured by HPLC. With salt stress, plant growth and chlorophyll content decreased significantly. The growth and development of tomato plants changed by applying the ZnO-NPs. Dosages of ZnO-NPs had a significant effect across treatments. ZnO-NPs also increased chlorophyll, reduced stress markers, and released phenolic chemicals and proteins in the leaves of tomatoes. ZnO-NPs reduce salt stress by promoting the uptake of minerals. ZnO-NPs had beneficial effects on tomato plants when subjected to salt stress, making them an alternate technique to boost resilience in saline soils or low-quality irrigation water. This study examined how foliar application of chemically synthesized ZnO-NPs to the leaves affected biochemistry, morphology, and phenolic compound synthesis with and without NaCl.

The pma-miR172 -PmAP2L3 module participates in the floral development of Masson pine (Pinus massoniana)

Masson pine (Pinus massoniana) is a core industrial tree species that is used for afforestation in southern China. Previous studies have shown that miR172 regulates plant growth and development, and the mechanism by which pma-miR172 affects flower development was explored in our study. In this research, we cloned and identified P. massoniana miR172 and its target gene PmAP2L3. The results showed that the 225 bp miR172 precursor was obtained, and the phylogenetic tree constructed based on the miR172 precursor sequence showed that P. massoniana miR172 has high homology with miR172 from Picea sitchensis and Picea glauca in gymnosperms. The target gene PmAP2L3 encodes a protein of 637 amino acids, and subcellular analysis revealed that the PmAP2L3 protein localised predominantly in the cytoplasm of P. massoniana.Compared with wild-type (WT) Arabidopsis, the miR172-overexpressing transgenic line blossomed more, while the 35 S::PmAP2L3 and 35 S::rAP2L3 transgenic lines were shorter. These results suggest that miR172 may promote flower development by inhibiting the target gene PmAP2L3.

Dietary Threonine Promoted the Growth and Ovarian Development of the Red Swamp Crayfish (Procambarus clarkii)

To explore the effects of dietary threonine on growth and ovarian development of red swamp crayfish (Procambarus clarkii), crayfish (5.48 ± 0.19 g) were fed six isoproteic and isoenergetic diets with varying levels of threonine (7.16 g/kg (control), 9.19, 12.74, 16.44, 20.83, and 23.78 g/kg) for 8 weeks. The results showed that weight gain rate, feed conversion ratio, protein efficiency rate, protein deposition rate, and essential amino acid deposition rates obtained the optimal values when the dietary threonine level was 12.74 or 16.44 g/kg. Compared to the control group, the 12.74 g/kg group exhibited enhanced nonspecific immunity and antioxidant properties. The 16.44 g/kg group demonstrated a significant increase in the frequency of B cells and R cells in the hepatopancreas, the length and width of intestinal villi, and the activities of protease and lipase. It also showed elevated ecdysterone hormone, gonadal index (GI), cAMP content, and the relative abundance of beneficial intestinal microflora. Compared to the control group, the mRNA expression of mTOR, S6K1, 4EBP1, EcR, RXR, chitinase, PKA, Vg, cdc2, and cyclin B was significantly upregulated, and the mRNA expression of MIH was significantly downregulated in the 16.44 g/kg group. Overall, optimal dietary threonine could improve intestinal health, regulate immune function, and enhance protein utilization, molting, and growth performance of red swamp crayfish. Additionally, it improved the synthesis of yolk substance and facilitated the development of ovarian cells of female crayfish. The optimal threonine level was 14.87–16.94 g/kg (dry matter), corresponding to 42.51–48.42 g/kg of dietary protein in red swamp crayfish.

Epistasis facilitates functional evolution in an ancient transcription factor.

A protein's genetic architecture - the set of causal rules by which its sequence produces its functions - also determines its possible evolutionary trajectories. Prior research has proposed that the genetic architecture of proteins is very complex, with pervasive epistatic interactions that constrain evolution and make function difficult to predict from sequence. Most of this work has analyzed only the direct paths between two proteins of interest - excluding the vast majority of possible genotypes and evolutionary trajectories - and has considered only a single protein function, leaving unaddressed the genetic architecture of functional specificity and its impact on the evolution of new functions. Here, we develop a new method based on ordinal logistic regression to directly characterize the global genetic determinants of multiple protein functions from 20-state combinatorial deep mutational scanning (DMS) experiments. We use it to dissect the genetic architecture and evolution of a transcription factor's specificity for DNA, using data from a combinatorial DMS of an ancient steroid hormone receptor's capacity to activate transcription from two biologically relevant DNA elements. We show that the genetic architecture of DNA recognition consists of a dense set of main and pairwise effects that involve virtually every possible amino acid state in the protein-DNA interface, but higher-order epistasis plays only a tiny role. Pairwise interactions enlarge the set of functional sequences and are the primary determinants of specificity for different DNA elements. They also massively expand the number of opportunities for single-residue mutations to switch specificity from one DNA target to another. By bringing variants with different functions close together in sequence space, pairwise epistasis therefore facilitates rather than constrains the evolution of new functions.

Structure-Activity Relationships, Deuteration, and Fluorination of Synthetic Cannabinoid Receptor Agonists Related to AKB48, 5F-AKB-48, and AFUBIATA.

Synthetic cannabinoid receptor agonists (SCRAs) are a growing class of new psychoactive substances (NPS) commonly derived from an N-alkylated indole, indazole, or 7-azaindole scaffold. Diversification of this core (at the 3-position) with amide-linked pendant amino acid groups and modular N-alkylation (of the indole/indazole/7-azaindole core) ensures that novel SCRAs continue to enter the illicit drug market rapidly. In response to the large number of SCRAs that have been detected, pharmacological evaluation of this NPS class has become increasingly common. Adamantane-derived SCRAs have consistently appeared throughout the market since 2011, and as such, a systematic set of these derivatives was synthesized and pharmacologically evaluated. Deuterated and fluorinated adamantane derivatives were prepared to evaluate typical hydrogen bioisosteres, as well as evaluation of the newly detected AFUBIATA.