Genetic Code Expansion in Shewanella oneidensis MR-1 Allows Site-Specific Incorporation of Bioorthogonal Functional Groups into a c-Type Cytochrome.

Genetic code expansion has enabled cellular synthesis of proteins containing unique chemical functional groups to allow the understanding and modulation of biological systems and engineer new biotechnology. Here, we report the development of efficient methods for site-specific incorporation of structurally diverse noncanonical amino acids (ncAAs) into proteins expressed in the electroactive bacterium Shewanella oneidensis MR-1. We demonstrate that the biosynthetic machinery for ncAA incorporation is compatible and orthogonal to the endogenous pathways of S. oneidensis MR-1 for protein synthesis, maturation of c-type cytochromes, and protein secretion. This allowed the efficient synthesis of a c-type cytochrome, MtrC, containing site-specifically incorporated ncAA in S. oneidensis MR-1 cells. We demonstrate that site-specific replacement of surface residues in MtrC with ncAAs does not influence its three-dimensional structure and redox properties. We also demonstrate that site-specifically incorporated bioorthogonal functional groups could be used for efficient site-selective labeling of MtrC with fluorophores. These synthetic biology developments pave the way to expand the chemical repertoire of designer proteins expressed in S. oneidensis MR-1.

The structure of a gilled stonefly larva from the mid-Cretaceous Kachin amber.

Perlidae stands as the most diverse family within Plecoptera, with evidence suggesting possible adaptation to warmer aquatic environments. Tracheal gills are hypothesized to have played a pivotal role in this radiation process. This study presents the description of a fossilized stonefly larva with gills, preserved as a fresh exuvia in mid-Cretaceous Kachin amber from northern Myanmar. The larva was classified within the family Perlidae based on distinctive morphological traits, including toothed lacinia and sharp-cusped mandibles, slender palps, glossae shorter than rounded paraglossae, and highly branched gills on the sides and ventral surface of thoracic segments. Additionally, the presence of a transverse, sparse, and irregular setal row on the occiput further indicates classification within the subfamily Acroneuriinae. Notably, the fossilized larva displays striking similarities in gill morphology and distribution to certain extant members within Perlidae, suggesting that these gill structures have an advantage in various aquatic habitats.

Cretaceous crown male ant reveals the rise of modern lineages.

Most described Mesozoic ants belong to stem groups that existed only during the Cretaceous period. Previously, the earliest known crown ants were dated to the Turonian (Late Cretaceous, ca. 94-90 million years ago (Ma)) deposits found in the USA, Kazakhstan, and Botswana. However, the recent discovery of an alate male ant in Kachin amber from the earliest Cenomanian (ca. 99 Ma), representing a new genus and species, Antiquiformica alata, revises the narrative on ant diversification. Antiquiformica can be distinctly differentiated from all known male stem ants by its geniculate antennae with elongated scape, extending far beyond the occipital margin of the head and half the length of the funiculus, as well as its partly reduced forewing venation. Furthermore, the combination of a one-segmented waist with a well-developed node, elongated scape extending beyond the occipital margin, and reduced forewing venation, particularly the completely reduced m-cu and rs-m crossveins and absence of rm and mcu closed cells, firmly places the fossil within the extant subfamily Formicinae. Fourier transform infrared spectroscopy (FTIR) confirmed that the amber containing Antiquiformica alata originated from the Kachin mines in Myanmar. This discovery significantly revises our understanding of the early evolution of Formicinae. The presence of Antiquiformica in Cenomanian amber indicates that the subfamily Formicinae emerged at least by the start of the Late Cretaceous, with crown ants likely originating earlier during the earliest Cretaceous or possibly the Late Jurassic, although paleontological evidence is lacking to support the latter hypothesis.

Interaction between spiders and ticks-ancient arthropod predatory behavior?

Ticks are ectoparasite vectors of pathogens affecting human and animal health worldwide. Rational integration of different control interventions including plant-derived repellents and acaricides, management of natural predators, and vaccines is required for innovative approaches to reduce the risks associated with ticks and tick-borne diseases. How tick populations are naturally controlled is always a question. Tick interactions with other arthropods including predators evolved from ancient times. In this study, Cretaceous (ca. 100 Mya) Burmese amber inclusions were identified as probably related to Compluriscutula vetulum (Acari: Ixodida: Ixodidae) tick larvae and spider silk. As illustrated in this study, ancient interactions between ticks and spiders may support arthropod predatory behavior as a natural control intervention. Rational integrative management of different tick control interventions including natural predators under a One Health perspective will contribute to effectively and sustainably reducing the risks associated with ticks and tick-borne diseases.

Unjustified use of amber necklaces for teething symptoms alleviation: Succinic acid release underperforms compared with natural skin bacteria production.

BACKGROUND: There is limited evidence of succinic acid release from amber necklace that justifies its biological plausibility. AIM: This study aimed to evaluate the release of succinic acid from Baltic amber beads in the presence of Staphylococcus epidermidis. DESIGN: The Baltic amber beads from the necklace were stratified according to their weight (average 0.05 g ± 0.067). Subsequently, the beads (n = 8) were submerged in 0.9% buffered saline (Control) or brain-heart infusion culture medium in the presence of a commercial strain of S. epidermidis, a resident skin bacterium incubated at 37°C for 24 h or 7 days. The samples were centrifuged, and the supernatants were analyzed by 1H Nuclear Magnetic Resonance. Multivariate analyses were adopted using the sparse partial least squares discriminant analysis method (p < .05). RESULTS: The group incubated with saline solution showed small release of succinic acid only after 7 days. In the groups with S. epidermidis, the release of succinic acid was observed in the both presence and absence of amber beads, indicating that succinic acid is a product released by bacteria. CONCLUSIONS: It was found that amber beads do not exhibit the ability to release expressive succinic acid, especially in a short period of time, which does not justify their use in infants. The most production of succinic acid is tributed to S. epidermidis.

In some cases more complicated approaches to refinement of macromolecular structures are not necessary.

The manuscript `Modeling a unit cell: crystallographic refinement procedure using the biomolecular MD simulation platform Amber' presents a novel protein structure refinement method claimed to offer improvements over traditional techniques like Refmac5 and Phenix. Our re-evaluation suggests that while the new method provides improvements, traditional methods achieve comparable results with less computational effort.

First fossil species of family Hyidae (Arachnida: Pseudoscorpiones) confirms 99 million years of ecological stasis in a Gondwanan lineage.

Burmese amber preserves a diverse assemblage of Cretaceous arachnids, and among pseudoscorpions (Arachnida: Pseudoscorpiones), ten species in five families have already been named. Here, we describe a new fossil species from Burmese amber in the pseudoscorpion family Hyidae, providing detailed measurements, photographs and 3D-models from synchrotron scanning. Based on morphology, the new fossil, Hya fynni sp. nov. is placed in the genus Hya, and is nearly identical to extant species in the genus, except for the position of trichobothrium est on the pedipalpal chela, thereby indicating extreme morphological stasis in this invertebrate lineage over the last 99 million years. Hya fynni represents the first described fossil species in Hyidae, and the third described Burmese fossil in the superfamily Neobisioidea. It also joins the garypinid, Amblyolpium burmiticum, in representing the oldest fossil records for extant pseudoscorpion genera. Considering proposed divergence dates, the newly described fossil species bolsters a Gondwanan origin for Hyidae, and provides evidence for the "Late Jurassic Rifting" hypothesis for the Burma Terrane, in which this landmass rifted from Gondwana in the Late Jurassic and collided with Eurasia by the Cretaceous/Eocene. Like Hya species today, H. fynni likely inhabited humicolous microhabitats in tropical forests on the Burma Terrane, supporting ecological niche stasis for this family since the Mesozoic.

Foveapeltis gen. nov., an unusual cleroid genus with large hypomeral cavities from mid-Cretaceous amber (Coleoptera: Cleroidea).

Beetles have a remote evolutionary history dating back to the Carboniferous, with Mesozoic fossils playing a pivotal role in elucidating the early evolution of extant families. Despite their exceptional preservation in amber, deciphering the systematic positions of Mesozoic trogossitid-like beetles remains challenging. Here, we describe and illustrate a new trogossitid-like lineage from mid-Cretaceous Kachin amber, Foveapeltis rutai Li, Kolibác, Liu & Cai, gen. et sp. nov. Foveapeltis stands out within the Cleroidea due to the presence of a significant large cavity on each hypomeron. While the exact phylogenetic placement of Foveapeltis remains uncertain, we offer a discussion on its potential affinity based on our constrained phylogenetic analyses.

Exploring genetic codon expansion for unnatural amino acid incorporation in filamentous fungus Aspergillus nidulans.

Genetic code expansion technology allows the incorporation of unnatural amino acids (UAAs) into proteins, which is useful in protein engineering, synthetic biology, and gene therapy. Despite its potential applications in various species, filamentous fungi remain unexplored. This study aims to address this gap by developing these techniques in Aspergillus nidulans. We introduced an amber stop codon into a specific sequence within the reporter gene expressed in A. nidulans and replaced the anticodon of the fungal tRNATyr with CUA. This resulted in the synthesis of the target protein, confirming the occurrence of amber suppression in the fungus. When exogenous E. coli tRNATyrCUA (Ec. tRNATyrCUA) and E. coli tyrosyl-tRNA (Ec.TyrRS) were introduced into A. nidulans, they successfully synthesized the target protein via amber suppression and were shown to be orthogonal to the fungal translation system. By replacing the wild-type Ec.TyrRS with a mutant with a higher affinity for the UAA O-methyl-L-tyrosine, the fungal system was able to initiate the synthesis of the UAA-labeled protein (UAA-protein). We further increased the expression level of the UAA-protein through several rational modifications. The successful development of a genetic code expansion technique for A. nidulans has introduced a potentially valuable approach to the study of fungal protein structure and function.

Patterns of morphological evolution in the raptorial appendages of praying mantises.

Mantodea (praying mantises) is a group of exclusively predatory insects, which, together with nonraptorial blattodeans (cockroaches and termites) and groups exclusively found in the fossil record, form the group Dictyoptera. A central characteristic of Mantodea is the specialization of their first pair of legs as raptorial grasping appendages, but the evolution from walking to raptorial legs is not yet fully understood. Here, we trace the evolution of the raptorial appendages in Dictyoptera through time using a morphometric (morphospaces) approach. We also describe two new mantodean nymphs preserved in amber from the Cretaceous and Eocene, which expand the scarce mantodean fossil record. Blattodean and mantodean appendages appear distinct in morphospace, but several appendages of fossil non-mantodeans can be considered raptorial, providing a potential transitional link between walking and raptorial morphotypes. Therefore, we discuss potential mantodean affinities for other predatory fossil dictyopterans. We examine changes across extant mantodeans, characterized by a straightening of the tibia especially associated with the rise of the diversification of the Mantidea and discuss whether a thickening of the femur could reflect an early adaptation to cursorial hunting.

A Practical Guide to All-Atom and Coarse-Grained Molecular Dynamics Simulations Using Amber and Gromacs: A Case Study of Disulfide-Bond Impact on the Intrinsically Disordered Amyloid Beta.

Intrinsically disordered proteins (IDPs) pose challenges to conventional experimental techniques due to their large-scale conformational fluctuations and transient structural elements. This work presents computational methods for studying IDPs at various resolutions using the Amber and Gromacs packages with both all-atom (Amber ff19SB with the OPC water model) and coarse-grained (Martini 3 and SIRAH) approaches. The effectiveness of these methodologies is demonstrated by examining the monomeric form of amyloid-ß (Aß42), an IDP, with and without disulfide bonds at different resolutions. Our results clearly show that the addition of a disulfide bond decreases the ß-content of Aß42; however, it increases the tendency of the monomeric Aß42 to form fibril-like conformations, explaining the various aggregation rates observed in experiments. Moreover, analysis of the monomeric Aß42 compactness, secondary structure content, and comparison between calculated and experimental chemical shifts demonstrates that all three methods provide a reasonable choice to study IDPs; however, coarse-grained approaches may lack some atomistic details, such as secondary structure recognition, due to the simplifications used. In general, this study not only explains the role of disulfide bonds in Aß42 but also provides a step-by-step protocol for setting up, conducting, and analyzing molecular dynamics (MD) simulations, which is adaptable for studying other biomacromolecules, including folded and disordered proteins and peptides.

A new fossil species of the extant genus Vicelva from mid-Cretaceous Kachin amber (Coleoptera: Staphylinidae).

A new species of the extant staphylinid genus Vicelva Moore & Legner, V. rasilis sp. nov., is reported from mid-Cretaceous Kachin amber of northern Myanmar. Vicelva rasilis is distinguishable from extant members of Vicelva by the smoother dorsal surface of head, pronotum and elytra, less prominent median projection of clypeus, unnotched mesal edge of mandibles, semiglabrous antennomere 6, and longer tarsomere 1. The pollen-containing coprolite attached to the beetle and the crystals within the beetle body provide valuable information about the biology and taphonomy of the fossil.

New Genera and Species of Trigonidiidae (Orthoptera: Grylloidea) from the Mid-Cretaceous of Myanmar with a Redescription of Birmaninemobius hirsutus.

The abundance of insects in Burmese amber illustrates a highly diverse insect community from the mid-Cretaceous period; yet, records of crickets (Grylloidea) are notably scarce. In this study, we describe two new genera with three new species, Palaeotrigonidium concavoculus gen. et sp. nov., Palaeotrigonidium defectivus sp. nov., and Tricalcaratus longilineus gen. et sp. nov., based on three specimens collected in north Myanmar. These new species can be placed within the Trigonidiidae (Orthoptera: Grylloidea) by their triangular head, compound eyes that protrude in dorsal view, and a body entirely covered with robust setae, particularly noticeable in the head and pronotum; however, subfamily assignments are not possible. Another known species, Birmaninemobius hirsutus, Xu et al., 2020, from Myanmar amber is redescribed based on a new specimen and a recheck of the holotype.

Lytic transglycosylase Slt of Pseudomonas aeruginosa as a periplasmic hub protein.

Peptidoglycan is a major constituent of the bacterial cell wall. Its integrity as a polymeric edifice is critical for bacterial survival and, as such, it is a preeminent target for antibiotics. The peptidoglycan is a dynamic crosslinked polymer that undergoes constant biosynthesis and turnover. The soluble lytic transglycosylase (Slt) of Pseudomonas aeruginosa is a periplasmic enzyme involved in this dynamic turnover. Using amber-codon-suppression methodology in live bacteria, we incorporated a fluorescent chromophore into the structure of Slt. Fluorescent microscopy shows that Slt populates the length of the periplasmic space and concentrates at the sites of septation in daughter cells. This concentration persists after separation of the cells. Amber-codon-suppression methodology was also used to incorporate a photoaffinity amino acid for the capture of partner proteins. Mass-spectrometry-based proteomics identified 12 partners for Slt in vivo. These proteomics experiments were complemented with in vitro pulldown analyses. Twenty additional partners were identified. We cloned the genes and purified to homogeneity 22 identified partners. Biophysical characterization confirmed all as bona fide Slt binders. The identities of the protein partners of Slt span disparate periplasmic protein families, inclusive of several proteins known to be present in the divisome. Notable periplasmic partners (KD < 0.5 µM) include PBPs (PBP1a, KD = 0.07 µM; PBP5 = 0.4 µM); other lytic transglycosylases (SltB2, KD = 0.09 µM; RlpA, KD = 0.4 µM); a type VI secretion system effector (Tse5, KD = 0.3 µM); and a regulatory protease for alginate biosynthesis (AlgO, KD < 0.4 µM). In light of the functional breadth of its interactome, Slt is conceptualized as a hub protein within the periplasm.

Deciphering the conformational dynamics of Myelin Oligodendrocyte glycoprotein in the myelin sheath.

Myelin Oligodendrocyte Glycoprotein (MOG) is a transmembrane protein in the myelin sheath. It acts as an auto-antigen under certain unknown conditions causing demyelination, thus resulting in Myelin Oligodendrocyte Glycoprotein Antibody-associated Disease (MOGAD). The significance of glycosylation in the conformational dynamics of the extracellular region (EC1) of the MOG were evident from the previous computational studies. Here, in this study, we performed the molecular dynamics simulation of the entire human MOG in the myelin sheath for 100 ns using the NAMD program. The results indicated that the EC1 and cytoplasmic region (CP) dominate the conformational rigidity of the protein, and enhance its interaction with lipids. This in turn helps in maintaining the myelin integrity in the presence of glycan. The transmembrane regions have reduced interaction with lipids in the glycosylated system. Moreover, the C-terminal extracellular region 2 (EC2) behaves exactly opposite to that of EC1 in the glycan presence. This may be attributed to the glycosylation site in the EC1 region. Hence, not only the region EC1 (having 3 crucial epitopes) but even the CP region were important for understanding the proper function of MOG in the glycan presence.Communicated by Ramaswamy H. Sarma.

Amber coloured plasma-Case for illustration.

INTRODUCTION: Abnormal colour of plasma is infrequently identified during processing of blood and blood components. Common reasons include haemolysis, medications or diet related. Sometimes, the aetiology is unknown. It is a dilemma for every transfusion specialist encountering this situation. Effort should be made to find the aetiology of discolouration of plasma, so that the blood donor can be suitably advised, and a decision can be made regarding the use of blood products. MATERIALS AND METHODS: We encountered two cases of orange coloured (amber coloured) plasma in our regular blood donors. All the common reasons for abnormal plasma discolouration were evaluated, including the donor's medication and diet. Spectrophotometry along with Liquid Chromatography-Mass Spectrometry (LC-MS) in both the positive and negative ion modes with literature search helped in arriving at a conclusion. RESULTS: Haemolysis was ruled out by estimation of plasma haemoglobin. Spectrophotometric analysis of the coloured plasma samples showed a peak, which was absent in normal coloured plasma. This was further investigated using Liquid Chromatography-Mass Spectrometry (LC-MS) in both the positive and negative ion modes. There was no significant difference between the coloured and normal samples in the positive ion mode. But in the negative ion mode, there was a peak observed at 110.5 and 191 m/z value in the profile of the coloured samples in comparison with the normal sample. Literature review shows the peak was corresponding to the presence of quinic acid residues-a substance found in coffee, and potentially excreted into the plasma of an individual with high coffee consumption. CONCLUSION: Reporting unusual causes associated with plasma discolouration is important. Present guidelines forbid issue of abnormal coloured blood and blood components for transfusion. Further such reports are necessary to confirm the safety of recipients receiving such units. This is the first case report to our knowledge of quinic acid discolouring blood products.

Development of orthogonal aminoacyl-tRNA synthetase mutant for incorporating a non-canonical amino acid.

Genetic code expansion involves introducing non-canonical amino acids (ncAAs) with unique functional groups into proteins to broaden their applications. Orthogonal aminoacyl tRNA synthetase (aaRS), essential for genetic code expansion, facilitates the charging of ncAAs to tRNA. In this study, we developed a new aaRS mutant from Methanosaeta concilii tyrosyl-tRNA synthetase (Mc TyrRS) to incorporate para-azido-L-phenylalanine (AzF). The development involved initial site-specific mutations in Mc TyrRS, followed by random mutagenesis. The new aaRS mutant with amber suppression was isolated through fluorescence-activated cell sorting. The M. concilii aaRS mutant structure was further analyzed to interpret the effect of mutations. This research provides a novel orthogonal aaRS evolution pipeline for highly efficient ncAA incorporation that will contribute to developing novel aaRS from various organisms.

Amber (Succinite) Extract Enhances Glucose Uptake through the Up-Regulation of ATP and Down-Regulation of ROS in Mouse C2C12 Cells.

Traditionally, amber (Succinite) has been used to alleviate all types of pain, skin allergies, and headaches. However, no studies have been conducted on its antidiabetic and antioxidant effects. In this study, differentiated skeletal muscle C2C12 cells were used to demonstrate the protective effects of amber (AMB) against H2O2-induced cell death. In addition, the effects of AMB on glucose uptake and ATP production were investigated. Our results showed that AMB at 10, 25, and 50 µg/mL suppressed the elevation of ROS production induced by H2O2 in a dose-dependent manner. Moreover, AMB enhanced glucose utilization in C2C12 cells through the improvement of ATP production and an increase in PGC-1α gene expression resulting in an amelioration of mitochondrial activity. On the other hand, AMB significantly increased the gene expression of glucose transporters GLUT4 and GLUT1. Our finding suggests that AMB can be used as a natural supplement for diabetes treatment and for the promotion of skeletal muscle function.

A New Genus of Praeaulacidae (Hymenoptera: Evanioidea) from Mid-Cretaceous Kachin Amber: Insights into a Putative New Praeaulacinae Subclade.

A new praeaulacid genus and species, Azygdellitha nova gen. et sp. nov., is described and illustrated based on a male specimen from mid-Cretaceous Kachin amber from Hukawng Valley, Myanmar. This newly discovered taxon increased the diversity of praeaulacid wasps during the Cretaceous period. While this new taxon shares similarities of wing venation with most species of the subfamily Praeaulacinae, it strongly differs from that of three genera previously described from mid-Cretaceous Kachin amber: Mesevania, Paleosyncrasis, and Praegastrinus. We explore the possibility that these genera constitute a distinct tribe within the Praeaulacinae, distinguished by their wing venation. We provide illustrations and emphasize the potentially diagnostic traits supporting this classification.

Dataset on vibrational spectra of amber and amber-like resins.

The article presents experimental data on vibrational spectroscopy of worldwide amber and amber-like resins. IR spectra were obtained using SHIMADZU IRPrestige-2 (Japan). Raman spectra were obtained using a Virsa Raman analyzer (UK). The data includes analyzed IR and Raman spectra of amber and amber-like resins, obtained to identify markers that determine spectral characteristics of natural resin and its deposits.