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The Ediacaran-Cambrian transition documents a critical stage in the diversification of animals. The global fossil record documents the appearance of cloudinomorphs and other shelled tubular organisms followed by non-biomineralized small carbonaceous fossils and by the highly diversified small shelly fossils between ~ 550 and 530 Ma. Here, we report diverse microfossils in thin sections and hand samples from the Ediacaran Bocaina Formation, Brazil, separated into five descriptive categories: elongate solid structures (ES); elongate filled structures (EF); two types of equidimensional structures (EQ 1 and 2) and elongate hollow structures with coiled ends (CE). These specimens, interpreted as diversified candidate metazoans, predate the latest Ediacaran biomineralized index macrofossils of the Cloudina-Corumbella-Namacalathus biozone in the overlying Tamengo Formation. Our new carbonate U-Pb ages for the Bocaina Formation, position this novel fossil record at 571 ± 9 Ma (weighted mean age). Thus, our data point to diversification of metazoans, including biomineralized specimens reminiscent of sections of cloudinids, protoconodonts, anabaritids, and hyolithids, in addition to organo-phosphatic surficial coverings of animals, demonstrably earlier than the record of the earliest known skeletonized metazoan fossils., (© 2024. The Author(s).)

Over the last 20 years, both severe acute respiratory syndrome coronavirus-1 and severe acute respiratory syndrome coronavirus-2 have transmitted from animal hosts to humans causing zoonotic outbreaks of severe disease. Both viruses originate from a group of betacoronaviruses known as subgroup 2b. The emergence of two dangerous human pathogens from this group along with previous studies illustrating the potential of other subgroup 2b members to transmit to humans has underscored the need for antiviral development against them. Coronaviruses modify the host innate immune response in part through the reversal of ubiquitination and ISGylation with their papain-like protease (PLpro). To identify unique or overarching subgroup 2b structural features or enzymatic biases, the PLpro from a subgroup 2b bat coronavirus, BtSCoV-Rf1.2004, was biochemically and structurally evaluated. This evaluation revealed that PLpros from subgroup 2b coronaviruses have narrow substrate specificity for K48 polyubiquitin and ISG15 originating from certain species. The PLpro of BtSCoV-Rf1.2004 was used as a tool alongside PLpro of CoV-1 and CoV-2 to design 30 novel noncovalent drug-like pan subgroup 2b PLpro inhibitors that included determining the effects of using previously unexplored core linkers within these compounds. Two crystal structures of BtSCoV-Rf1.2004 PLpro bound to these inhibitors aided in compound design as well as shared structural features among subgroup 2b proteases. Screening of these three subgroup 2b PLpros against this novel set of inhibitors along with cytotoxicity studies provide new directions for pan-coronavirus subgroup 2b antiviral development of PLpro inhibitors.

The stimulating and performance-enhancing properties of caffeine are often explored in one the most consumed types of supplements: the pre-workout supplements (PWS). However, despite the popularity of PWS, previous studies have reported incompatibilities between what is described in their labels and their actual caffeine content. This study aimed to develop, to optimize, and to validate a gas-chromatography coupled to nitrogen-phosphorus detector (GC-NPD) method to quantify caffeine in PWS and to analyze commercial PWS marketed in Brazil to estimate the caffeine daily intake. For this purpose, three different extraction procedures were evaluated: agitation in vortex, shaker, and sonication. Sonication yielded the best extraction results. Next, the parameters' temperature and time were optimized by using central composite rotatable design (CCRD) and response surface methodology, which revealed the optimal values of 70°C and 10 min. The method was validated and applied to quantify caffeine in 52 PWS. From the 36 PWS labels that specified the caffeine amount, seven (19%) presented more than 120% of the declared quantity, whereas 15 (42%) contained less than 80% of the labeled caffeine. Additionally, six products presented undeclared caffeine. Considering the label stated doses, five supplements exceeded the safe caffeine daily intake (400 mg). On the basis of these findings, supplement quality control remains an issue that deserves more attention from consumers, manufacturers, and regulatory agencies. Finally, we suggest that PWS consumers be careful of the habit of ingesting caffeine through other sources and avoid ingesting two or more different PWS products in the same day., (© 2021 John Wiley & Sons, Ltd.)

Post-translational modification of host and viral proteins by ubiquitin and ubiquitin-like proteins plays a key role in a host's ability to mount an effective immune response. Avian species lack a ubiquitin-like protein found in mammals and other non-avian reptiles; interferon stimulated gene product 15 (ISG15). ISG15 serves as a messenger molecule and can be conjugated to both host and viral proteins leading them to be stabilized, degraded, or sequestered. Structurally, ISG15 is comprised of a tandem ubiquitin-like domain (Ubl), which serves as the motif for post-translational modification. The 2'-5' oligoadenylate synthetase-like proteins (OASL) also encode two Ubl domains in series near its C-terminus which binds OASL to retinoic acid inducible gene-I (RIG-I). This protein-protein interaction increases the sensitivity of RIG-I and results in an enhanced production of type 1 interferons and a robust immune response. Unlike human and other mammalian OASL homologues, avian OASLs terminate their tandem Ubl domains with the same LRLRGG motif found in ubiquitin and ISG15, a motif required for their conjugation to proteins. Chickens, however, lack RIG-I, raising the question of structural and functional characteristics of chicken OASL (chOASL). By investigating chOASL, the evolutionary history of viruses with deubiquitinases can be explored and drivers of species specificity for these viruses may be uncovered. Here we show that the chOASL tandem Ubl domains shares structural characteristics with mammalian ISG15, and that chOASL can oligomerize and conjugate to itself. In addition, the ISG15-like features of avian OASLs and how they impact interactions with viral deubiquitinases and deISGylases are explored., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shepard, Freitas, Rodriguez, Scholte, Baker, Hutchison, Longo, Miller, O’Boyle, Tandon, Zhao, Grimsey, Wells, Bergeron and Pegan.)

Objective: Examine the driving impairment effects of alcohol alone and of alcohol combined with texting., Methods: Fifteen drivers (nine male, six female; mean age: 31.1 ± 6.9 years, range: 23 to 43 years) with similar drinking habit (i.e., social drinkers) completed a lap in a closed-course section in six different situations: (I) sober; (II) sober and while texting; (III) 30 minutes after ingesting a moderate dose of ethanol (0.50 g/kg); (IV) 30 minutes after drinking and while texting; (V) 60 minutes after drinking, (VI) 60 minutes after drinking and while texting. Driving performance was analyzed by means of maximum and mean speed, braking time and braking distance; and ability to control the car (i.e., evaluating if the drivers hit a traffic cone or exceeded the boundaries of the course). P values of < 0.05 were considered significant., Results: Pre and post-alcohol consumption results show a significant increase concerning the drivers' mean and maximum speed after drinking (p < 3.2x10 -8 ). However, neither alcohol nor texting had significant effects on braking parameters (p > 0.05). Traffic cones were knocked down only in texting experiments. In addition, when using the cell phone drivers tended to reduce the speed, and to accelerate abruptly right after they finish texting., Conclusion: Our findings strengthen the hypothesis that even moderate alcohol doses may significantly impair the driving performance. Additionally, alcohol and texting have complementary effects on driving impairment, and their combination represents a significant risk factor for crashes.

Viral proteases are highly specific and recognize conserved cleavage site sequences of ∼6-8 amino acids. Short stretches of homologous host-pathogen sequences (SSHHPS) can be found spanning the viral protease cleavage sites. We hypothesized that these sequences corresponded to specific host protein targets since >40 host proteins have been shown to be cleaved by Group IV viral proteases and one Group VI viral protease. Using PHI-BLAST and the viral protease cleavage site sequences, we searched the human proteome for host targets and analyzed the hit results. Although the polyprotein and host proteins related to the suppression of the innate immune responses may be the primary targets of these viral proteases, we identified other cleavable host proteins. These proteins appear to be related to the virus-induced phenotype associated with Group IV viruses, suggesting that information about viral pathogenesis may be extractable directly from the viral genome sequence. Here we identify sequences cleaved by the SARS-CoV-2 papain-like protease (PLpro) in vitro within human MYH7 and MYH6 (two cardiac myosins linked to several cardiomyopathies), FOXP3 (an X-linked T reg cell transcription factor), ErbB4 (HER4), and vitamin-K-dependent plasma protein S (PROS1), an anticoagulation protein that prevents blood clots. Zinc inhibited the cleavage of these host sequences in vitro . Other patterns emerged from multispecies sequence alignments of the cleavage sites, which may have implications for the selection of animal models and zoonosis. SSHHPS/nsP is an example of a sequence-specific post-translational silencing mechanism.

The interest in alternative biological matrices (e.g., hair and saliva) for forensic toxicology analysis has increased, and recent developments in sample preparation have targeted rapid, cheap, efficient and eco-friendly methods, including microextraction techniques. For this review, we have gathered information about these two hot topics. We discuss the composition, incorporation of analytes and advantages and disadvantages of different biological matrices, and also present the operation principles of the most reported microextraction procedures and their application in forensic toxicology. The outcome of this review may encourage future forensic researches into alternative samples and microextraction techniques.

Nairoviruses are arthropod-borne viruses with a nearly global geographical distribution. Several are known causative agents of human disease, including Crimean-Congo hemorrhagic fever virus (CCHFV), which has a case fatality rate that can exceed 30%. Nairoviruses encode an ovarian tumour domain protease (OTU) that can suppress the innate immune response by reversing post-translational modifications by ubiquitin (Ub) and/or interferon-stimulated gene product 15 (ISG15). As a result, the OTU has been identified as a potential target for the development of CCHFV therapeutics. Despite sharing the same general fold, nairoviral OTUs show structural and enzymatic diversity. The CCHFV OTU, for example, possesses activity towards both Ub and ISG15, while the Hazara virus (HAZV) OTU interacts exclusively with Ub. Virology studies focused on the OTU have mostly been restricted to CCHFV, which requires BSL-4 containment facilities. Although HAZV has been proposed as a BSL-2 alternative, differences in the engagement of substrates by CCHFV and HAZV OTUs may present complicating factors when trying to model one using the other. To understand the molecular underpinnings of the differences in activity, a 2.78 Å resolution crystal structure of HAZV OTU bound to Ub was solved. Using structure-guided site-directed mutagenesis, HAZV OTUs were engineered with altered or eliminated deubiquitinase activity, including one with an exclusive activity for ISG15. Additionally, analysis of the structure yielded insights into the difference in inhibition observed between CCHFV and HAZV OTUs with a Ub-based inhibitor. These new insights present opportunities to utilize HAZV as a model system to better understand the role of the OTU in the context of infection.

Interferon (IFN)-stimulated gene product 15 (ISG15) is a ubiquitin-like protein critical for the control of microbial infections. ISG15 appears to serve a wide variety of functions, which regulate multiple cellular responses contributing to the development of an antiviral state. ISG15 is a versatile molecule directly modulating both host and virus protein function which regulate many signaling pathways, including its own synthesis. Here we review the various roles ISG15 plays in the antiviral immune response, and examine the mechanisms by which viruses attempt to mitigate or exploit ISG15 activity., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for COVID-19, is a novel human betacoronavirus that is rapidly spreading worldwide. The outbreak currently includes over 3.7 million cases and 260,000 fatalities. As a betacoronavirus, SARS-CoV-2 encodes for a papain-like protease (PLpro) that is likely responsible for cleavage of the coronavirus (CoV) viral polypeptide. The PLpro is also responsible for suppression of host innate immune responses by virtue of its ability to reverse host ubiquitination and ISGylation events. Here, the biochemical activity of SARS-CoV-2 PLpro against ubiquitin (Ub) and interferon-stimulated gene product 15 (ISG15) substrates is evaluated, revealing that the protease has a marked reduction in its ability to process K48 linked Ub substrates compared to its counterpart in SARS-CoV. Additionally, its substrate activity more closely mirrors that of the PLpro from the Middle East respiratory syndrome coronavirus and prefers ISG15s from certain species including humans. Additionally, naphthalene based PLpro inhibitors are shown to be effective at halting SARS-CoV-2 PLpro activity as well as SARS-CoV-2 replication.

Tick-borne nairoviruses (order Bunyavirales) encode an ovarian tumor domain protease (OTU) that suppresses the innate immune response by reversing the post-translational modification of proteins by ubiquitin (Ub) and interferon-stimulated gene product 15 (ISG15). Ub is highly conserved across eukaryotes, whereas ISG15 is only present in vertebrates and shows substantial sequence diversity. Prior attempts to address the effect of ISG15 diversity on viral protein-ISG15 interactions have focused on only a single species' ISG15 or a limited selection of nairovirus OTUs. To gain a more complete perspective of OTU-ISG15 interactions, we biochemically assessed the relative activities of 14 diverse nairovirus OTUs for 12 species' ISG15 and found that ISG15 activity is predominantly restricted to particular nairovirus lineages reflecting, in general, known virus-host associations. To uncover the underlying molecular factors driving OTUs affinity for ISG15, X-ray crystal structures of Kupe virus and Ganjam virus OTUs bound to sheep ISG15 were solved and compared to complexes of Crimean-Congo hemorrhagic fever virus and Erve virus OTUs bound to human and mouse ISG15, respectively. Through mutational and structural analysis seven residues in ISG15 were identified that predominantly influence ISG15 species specificity among nairovirus OTUs. Additionally, OTU residues were identified that influence ISG15 preference, suggesting the potential for viral OTUs to adapt to different host ISG15s. These findings provide a foundation to further develop research methods to trace nairovirus-host relationships and delineate the full impact of ISG15 diversity on nairovirus infection., Competing Interests: The authors have declared that no competing interests exist.

Platymiscium floribundum lectin (PFL), a mannose/N-acetyl-D-glucosamine-specific lectin, was isolated from P. floribundum seeds using Sepharose-mannose affinity media chromatography. PFL is a glycoprotein that is a potent agglutinin for rabbit erythrocytes. In addition, PFL is highly stable because it is able to maintain its hemagglutinating activity after exposure to temperatures of up to 60 °C for 1 h and exposure to a wide pH range. The PFL purification process was monitored using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the results showed that the purified lectin consists of a single band with a molecular mass of approximately 29 kDa in either the presence or the absence of a reducing agent. The analysis of purified PFL by electrospray ionization-mass spectrometry showed that most ions had a molecular weight of 27,053 ± 2 Da, and other less abundant ions had similar molecular weights. Gel filtration shows that the lectin exists as a dimer in solution with mass at approximately 65 kDa. Sixteen peptides were sequenced, and as a result, a total of 130 amino acids were identified and resulted in a coverage of approximately 65% of the PFL sequence. The partial sequence of PFL was aligned with sequences of other lectins from evolutionarily related species, and PFL showed considerable similarity to the other lectins., (Copyright © 2012 John Wiley & Sons, Ltd.)

Background: Lectins are mainly described as simple carbohydrate-binding proteins. Previous studies have tried to identify other binding sites, which possible recognize plant hormones, secondary metabolites, and isolated amino acid residues. We report the crystal structure of a lectin isolated from Canavalia gladiata seeds (CGL), describing a new binding pocket, which may be related to pathogen resistance activity in ConA-like lectins; a site where a non-protein amino-acid, alpha-aminobutyric acid (Abu), is bound., Results: The overall structure of native CGL and complexed with alpha-methyl-mannoside and Abu have been refined at 2.3 A and 2.31 A resolution, respectively. Analysis of the electron density maps of the CGL structure shows clearly the presence of Abu, which was confirmed by mass spectrometry., Conclusion: The presence of Abu in a plant lectin structure strongly indicates the ability of lectins on carrying secondary metabolites. Comparison of the amino acids composing the site with other legume lectins revealed that this site is conserved, providing an evidence of the biological relevance of this site. This new action of lectins strengthens their role in defense mechanisms in plants.

Here, we report the crystallographic study of a lectin from Canavalia maritima seeds (ConM) and its relaxant activity on vascular smooth muscle, to provide new insights into the understanding of structure/function relationships of this class of proteins. ConM was crystallized and its structure determined by standard molecular replacement techniques. The amino acid residues, previously suggested incorrectly by manual sequencing, have now been determined as I17, I53, S129, S134, G144, S164, P165, S187, V190, S169, T196, and S202. Analysis of the structure indicated a dimer in the asymmetric unit, two metal binding sites per monomer, and loops involved in the molecular oligomerization. These confer 98% similarity between ConM and other previously described lectins, derived from Canavalia ensiformis and Canavalia brasiliensis. Our functional data indicate that ConM exerts a concentration-dependent relaxant action on isolated aortic rings that probably occurs via an interaction with a specific lectin-binding site on the endothelium, resulting in a release of nitric oxide.

A lectin from Canavalia maritima seeds (ConM) was purified and submitted to crystallization experiments. The best crystals were obtained using the vapour-diffusion method at a constant temperature of 293 K and grew in 7 d. A complete structural data set was collected to 2.1 A resolution using a synchrotron-radiation source. The ConM crystal belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 67.15, b = 70.90, c = 97.37 A. A molecular-replacement search found a solution with a correlation coefficient of 69.2% and an R factor of 42.5%. Crystallographic refinement is under way.

The seed lectin from Canavalia gladiata was purified and crystallized. Orthorhombic crystals belonging to space group C222(1) grew within three weeks at 293 K using the hanging-drop vapour-diffusion method. Using synchrotron X-ray radiation, a complete structural data set was collected at 2.3 A resolution. The preliminary crystal structure of the lectin, determined by molecular replacement, had a correlation coefficient of 0.569 and an R factor of 0.412.

A glucose/mannose-binding lectin was isolated from seeds of Parkia discolor (Mimosoideae) using affinity chromatography on Sephadex G-100 gel. The protein presented a unique component in SDS-PAGE corresponding to a molecular mass of 58,000 Da, which is very similar to that of a closely related lectin from Parkia platycephala. Among the simple sugars tested, mannose was the best inhibitor, but biantennary glycans, containing the trimannoside core, present in N-glycoproteins, also seem to be powerful inhibitors of the haemagglutinating activity induced by the purified lectin. The protein was characterised by high content of glycine and proline and absence of cysteine. Rabbit antibodies, anti-P. platycephala seed lectin, recognised the P. discolor lectin. However, no cross-reaction was observed when a set of other legume lectins from sub-family Papilionoideae and others from families Moraceae and Euphorbiaceae were assayed with the Parkia lectins. This suggests that Parkia lectins comprise a new group of legume lectins exhibiting distinct characteristics.

The primary structure of a lectin isolated from the red alga Bryothamnion triquetrum was established by combination of Edman degradation of sets of overlapping peptides and mass spectrometry. It contains 91 amino acids and two disulphide bonds. The primary structure of the B. triquetrum lectin does not show amino acid sequence similarity with known plant and animal lectin structures. Hence, this protein may be the paradigm of a novel lectin family.