Three-dimensional structure-guided evolution of a ribosome with tethered subunits.

RNA-based macromolecular machines, such as the ribosome, have functional parts reliant on structural interactions spanning sequence-distant regions. These features limit evolutionary exploration of mutant libraries and confound three-dimensional structure-guided design. To address these challenges, we describe Evolink (evolution and linkage), a method that enables high-throughput evolution of sequence-distant regions in large macromolecular machines, and library design guided by computational RNA modeling to enable exploration of structurally stable designs. Using Evolink, we evolved a tethered ribosome with a 58% increased activity in orthogonal protein translation and a 97% improvement in doubling times in SQ171 cells compared to a previously developed tethered ribosome, and reveal new permissible sequences in a pair of ribosomal helices with previously explored biological function. The Evolink approach may enable enhanced engineering of macromolecular machines for new and improved functions for synthetic biology.

Cell-Free Systems: Ideal Platforms for Accelerating the Discovery and Production of Peptide-Based Antibiotics.

Peptide-based antibiotics (PBAs), including antimicrobial peptides (AMPs) and their synthetic mimics, have received significant interest due to their diverse and unique bioactivities. The integration of high-throughput sequencing and bioinformatics tools has dramatically enhanced the discovery of enzymes, allowing researchers to identify specific genes and metabolic pathways responsible for producing novel PBAs more precisely. Cell-free systems (CFSs) that allow precise control over transcription and translation in vitro are being adapted, which accelerate the identification, characterization, selection, and production of novel PBAs. Furthermore, these platforms offer an ideal solution for overcoming the limitations of small-molecule antibiotics, which often lack efficacy against a broad spectrum of pathogens and contribute to the development of antibiotic resistance. In this review, we highlight recent examples of how CFSs streamline these processes while expanding our ability to access new antimicrobial agents that are effective against antibiotic-resistant infections.

Surgical vs nonsurgical treatments in patients with anterior open-bite have similar effects in occlusal function: A 2-year follow-up study.

INTRODUCTION: This study aimed to investigate changes in bite force (BF) and occlusal contact area after anterior open-bite (AOB) treatment and compare the changes in surgical vs nonsurgical treatment. METHODS: This retrospective study included patients with AOB compared with normal occlusion. AOB was corrected by either intrusion of the maxillary molars (intrusion group, n = 19) or orthognathic surgery (surgery group, n = 37). The control group (n = 35) had a normal overbite relationship. Records of lateral cephalograms, BF, and occlusal contact area taken before (T0), immediately after (T1), and 2 years after (T2) orthodontic treatment were compared within and among the 3 groups. RESULTS: The open-bite group, including intrusion and surgery groups, had a lower BF and less occlusal contact area than the control group at T0 and T1 (P <0.001). However, there were no significant differences among the 3 groups at T2 (P >0.05). The intrusion and surgery groups showed no significant differences throughout the observation period extending from T0 to T2 (P >0.05). Although BF and occlusal contact area decreased at T1 compared with T0, they increased during retention and showed higher values at T2 than at T0. CONCLUSIONS: Treatment of AOB improved BF and occlusal contact area 2 years posttreatment. Orthognathic surgery and molar intrusion using orthodontic miniscrews can improve occlusal function similarly. Orthodontists can select either method depending on malocclusion severity and patient demand.

Cell-free Biosynthesis of Peptidomimetics.

A wide variety of peptidomimetics (peptide analogs) possessing innovative biological functions have been brought forth as therapeutic candidates through cell-free protein synthesis (CFPS) systems. A key feature of these peptidomimetic drugs is the use of non-canonical amino acid building blocks with diverse biochemical properties that expand functional diversity. Here, we summarize recent technologies leveraging CFPS platforms to expand the reach of peptidomimetics drugs. We also offer perspectives on engineering the translational machinery that may open new opportunities for expanding genetically encoded chemistry to transform drug discovery practice beyond traditional boundaries.

A Medicinal Halophyte Ipomoea pes-caprae (Linn.) R. Br.: A Review of Its Botany, Traditional Uses, Phytochemistry, and Bioactivity.

Ipomoea pes-caprae (Linn.) R. Br. (Convolvulaceae) is a halophytic plant that favorably grows in tropical and subtropical countries in Asia, America, Africa, and Australia. Even though this plant is considered a pan-tropical plant, I. pes-caprae has been found to occur in inland habitats and coasts of wider areas, such as Spain, Anguilla, South Africa, and Marshall Island, either through a purposeful introduction, accidentally by dispersal, or by spreading due to climate change. The plant parts are used in traditional medicine for treating a wide range of diseases, such as inflammation, gastrointestinal disorders, pain, and hypertension. Previous phytochemical analyses of the plant have revealed pharmacologically active components, such as alkaloids, glycosides, steroids, terpenoids, and flavonoids. These phytoconstituents are responsible for the wide range of biological activities possessed by I. pes-caprae plant parts and extracts. This review arranges the previous reports on the botany, distribution, traditional uses, chemical constituents, and biological activities of I. pes-caprae to facilitate further studies that would lead to the discovery of novel bioactive natural products from this halophyte.

Design of a Scalable and Fast YOLO for Edge-Computing Devices.

With the increase in research cases of the application of a convolutional neural network (CNN)-based object detection technology, studies on the light-weight CNN models that can be performed in real time on the edge-computing devices are also increasing. This paper proposed scalable convolutional blocks that can be easily designed CNN networks of You Only Look Once (YOLO) detector which have the balanced processing speed and accuracy of the target edge-computing devices considering different performances by exchanging the proposed blocks simply. The maximum number of kernels of the convolutional layer was determined through simple but intuitive speed comparison tests for three edge-computing devices to be considered. The scalable convolutional blocks were designed in consideration of the limited maximum number of kernels to detect objects in real time on these edge-computing devices. Three scalable and fast YOLO detectors (SF-YOLO) which designed using the proposed scalable convolutional blocks compared the processing speed and accuracy with several conventional light-weight YOLO detectors on the edge-computing devices. When compared with YOLOv3-tiny, SF-YOLO was seen to be 2 times faster than the previous processing speed but with the same accuracy as YOLOv3-tiny, and also, a 48% improved processing speed than the YOLOv3-tiny-PRN which is the processing speed improvement model. Also, even in the large SF-YOLO model that focuses on the accuracy performance, it achieved a 10% faster processing speed with better accuracy of 40.4% mAP@0.5 in the MS COCO dataset than YOLOv4-tiny model.

Semisynthetic protein nanoreactor for single-molecule chemistry.

The covalent chemistry of individual reactants bound within a protein pore can be monitored by observing the ionic current flow through the pore, which acts as a nanoreactor responding to bond-making and bond-breaking events. In the present work, we incorporated an unnatural amino acid into the α-hemolysin (αHL) pore by using solid-phase peptide synthesis to make the central segment of the polypeptide chain, which forms the transmembrane ß-barrel of the assembled heptamer. The full-length αHL monomer was obtained by native chemical ligation of the central synthetic peptide to flanking recombinant polypeptides. αHL pores with one semisynthetic subunit were then used as nanoreactors for single-molecule chemistry. By introducing an amino acid with a terminal alkyne group, we were able to visualize click chemistry at the single-molecule level, which revealed a long-lived (4.5-s) reaction intermediate. Additional side chains might be introduced in a similar fashion, thereby greatly expanding the range of single-molecule covalent chemistry that can be investigated by the nanoreactor approach.

Three-dimensional evaluation of dentofacial transverse widths in adults with different sagittal facial patterns.

INTRODUCTION: The aim of this study was to evaluate the dentofacial transverse dimensions of subjects with different sagittal facial patterns using 3-dimensional cone-beam computed tomography images. METHODS: Cone-beam computed tomography images of 63 men and 80 women were divided into skeletal Class I, Class II, and Class III groups. Skeletal and dental evaluations were made on frontal views and coronal cross-sections of the images. Independent 2-sample t tests and 1-way analysis of variance followed by post hoc Tukey tests were used for sex and group differences. Pearson correlation analysis was used to identify factors related to changes in ANB angle. RESULTS: The Class II subjects did not show differences in maxillomandibular width and maxillary width compared with Class I subjects; however, their maxillary molars were more lingually tipped. The Class III subjects showed greater maxillomandibular width differences and smaller maxillary widths and maxillary buccolingual alveolar widths at midroot level compared with Class I subjects. The maxillary molars were buccally inclined, and the mandibular molars were lingually compensated in Class III subjects. The ANB angle showed positive correlations with jugal process width, maxillary width, and maxillary buccolingual alveolar width at midroot level as well as mandibular molar buccal inclination; negative correlations were found in maxillomandibular width difference, mandibular width at midroot level, and maxillary molar buccal inclination. CONCLUSIONS: A relative comparison of Class I, Class II, and Class III subjects showed that dental compensation had occurred to overcome the transverse skeletal discrepancies in the maxillary posterior segments of Class II and Class III subjects. This could escalate unidentified periodontal and functional problems in the long term. Future studies of transverse dentofacial dimensions, including periodontal evaluations and occlusal forces, would be useful for delivering proper orthodontic treatment for skeletal Class II and Class III subjects.

Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation.

The roles of 2-oxoglutarate (2OG)-dependent prolyl-hydroxylases in eukaryotes include collagen stabilization, hypoxia sensing, and translational regulation. The hypoxia-inducible factor (HIF) sensing system is conserved in animals, but not in other organisms. However, bioinformatics imply that 2OG-dependent prolyl-hydroxylases (PHDs) homologous to those acting as sensing components for the HIF system in animals occur in prokaryotes. We report cellular, biochemical, and crystallographic analyses revealing that Pseudomonas prolyl-hydroxylase domain containing protein (PPHD) contain a 2OG oxygenase related in structure and function to the animal PHDs. A Pseudomonas aeruginosa PPHD knockout mutant displays impaired growth in the presence of iron chelators and increased production of the virulence factor pyocyanin. We identify elongation factor Tu (EF-Tu) as a PPHD substrate, which undergoes prolyl-4-hydroxylation on its switch I loop. A crystal structure of PPHD reveals striking similarity to human PHD2 and a Chlamydomonas reinhardtii prolyl-4-hydroxylase. A crystal structure of PPHD complexed with intact EF-Tu reveals that major conformational changes occur in both PPHD and EF-Tu, including a >20-Å movement of the EF-Tu switch I loop. Comparison of the PPHD structures with those of HIF and collagen PHDs reveals conservation in substrate recognition despite diverse biological roles and origins. The observed changes will be useful in designing new types of 2OG oxygenase inhibitors based on various conformational states, rather than active site iron chelators, which make up most reported 2OG oxygenase inhibitors. Structurally informed phylogenetic analyses suggest that the role of prolyl-hydroxylation in human hypoxia sensing has ancient origins.

'Shotgun DNA synthesis' for the high-throughput construction of large DNA molecules.

We developed a highly scalable 'shotgun' DNA synthesis technology by utilizing microchip oligonucleotides, shotgun assembly and next-generation sequencing technology. A pool of microchip oligonucleotides targeting a penicillin biosynthetic gene cluster were assembled into numerous random fragments, and tagged with 20 bp degenerate barcode primer pairs. An optimal set of error-free fragments were identified by high-throughput DNA sequencing, selectively amplified using the barcode sequences, and successfully assembled into the target gene cluster.

A review of food contamination with nitrated and oxygenated polycyclic aromatic hydrocarbons: toxicity, analysis, occurrence, and risk assessment.

Prolonged exposure to polycyclic aromatic hydrocarbons (PAHs) and their derivatives, particularly nitrated polycyclic aromatic hydrocarbons (NPAHs) and oxygenated polycyclic aromatic hydrocarbons (OPAHs), can result in adverse health effects and may carry higher toxicity risks compared to PAHs alone. Various extraction methods have been utilized for PAHs derivatives from food samples. The analytes are then analyzed using gas chromatography/mass spectrometry and high-performance liquid chromatography techniques. PAHs derivatives are increasingly being detected in the environment, prompting scrutiny from numerous researchers. Similarly, their presence in food is becoming a significant concern. The elevated levels of PAH derivatives found in smoked food may result in detrimental dietary exposure and pose potential health hazards. Furthermore, investigating the level of exposure to these contaminants in food is imperative, as their consumption by humans carries inherent risks. Consequently, this review concentrates on the toxicity, analysis, occurrence, and risk evaluation of NPAHs and OPAHs present in food sources.

Plant proteins, peptides, and non-protein amino acids: Toxicity, sources, and analysis.

Plants have evolved various mechanisms to synthesize diverse range of substances that contribute to their survival against pests, pathogens, predators, and adverse environmental conditions. Although several plant metabolites possess therapeutic potential, some can be potentially harmful to human and animal health when consumed in large proportion. Proteins, peptides, and non-protein amino acids are products of plant biochemical pathways with proven beneficial and nutritional effects. Despite these benefits, the in vivo toxicities associated with certain plant-derived proteins, peptides, and non-protein amino acids pose a significant risk to humans and animals. Symptoms of poisoning include nausea, vomiting, diarrhea, hair and weight loss, goiter, cataracts, and infertility. Even though plant processing methods such as soaking and drying can reduce the amount of toxin contained in plants, complete riddance is often impossible. As such, food regulatory bodies need to prevent uncontrolled consumption of the listed and many other toxin-containing plant species to keep the public safe. For this purpose, this review collates crucial insights into the sources, and in vivo toxicity associated with certain plant-derived proteins, peptides, and non-protein amino acids that have the clear potential to adversely affect human health. Additionally, this review provides information on analytical methods suitable for the detection of these substances in plants.

Contamination of ultraviolet absorbers in food: toxicity, analytical methods, occurrence and risk assessments.

Ultraviolet (UV) absorbers are chemical substances that are widely used as defenses against the damaging effects of solar radiations. UV absorbers, despite their benefits, are categorized as emerging pollutants because they have been demonstrated to be mutagenic, toxic, pseudo-persistent, bio-accumulative, and to have strong estrogenic effects. Because of their common use in personal care products, they continue to enter the environment. Several food samples, particularly those derived from aquatic sources, have been found to be contaminated with these compounds. Toxic effects on aquatic life, such as metabolic imbalance and developmental toxicity, result from the continued presence of UV absorbers in aquatic bodies. In addition, the degree of exposure to these pollutants in foods should be examined because there are certain risks associated with their consumption by humans. Therefore, this review focuses on the toxicity, analytical techniques, occurrence, and risk assessments of UV absorbers found in food.

Determination of 57 pesticide residues in fishery products by GC tandem mass spectrometry combined with QuEChERS (quick, easy, cheap, effective, rugged, and safe) extraction.

An analytical method using GC-MS/MS combined with quick, easy, cheap, effective, rugged, and safe extraction was developed to determine 57 pesticides in fishery products. The limits of detection and quantification (LOD and LOQ, respectively) of the analytical method ranged between 0.91 and 2.12 ng/g wet mass and 3 and 7 ng/g wet mass, respectively. Moreover, the linearity of the calibration curves was acceptable (R2 > 0.99). The relative pesticide recoveries ranged between 53.87 and 127.2%, and reproducibility ranged between 0.25 and 10.87%. The pesticide residues in brown seaweed, eel, flatfish, shrimp, and abalone samples were analyzed using the developed analytical method, and the results indicate that most samples were not contaminated by the 57 target pesticides, except low levels (< 10 ng/g) of 1,1-dichloro-2,2-bis(4-chlorophenyl)ethene, alachlor, ametryn, isoprothiolane, and prometryn in several samples.

Effect of rice milling, washing, and cooking on reducing pesticide residues.

The effects of milling, washing, and cooking on etofenprox, flubendiamide, and tebufenozide levels in brown and polished rice were investigated by HPLC using a UV detector. The reduction rates of etofenprox, flubendiamide, and tebufenozide after milling were 68.74-93.16%, 64.49-90.25%, and 69.74-92.58%, respectively, 11.64-41.44%, 31.36-65.37%, and 31.61-73.79%, respectively, after washing brown rice, and 30.85-82.08%, 52.13-83.05%, and 43.04-83.89%, respectively, after washing polished rice. The residue levels of the three pesticides in brown rice decreased after electric and pressure cooking by 56.49 and 54.41%, 75.80 and 73.42%, and 70.01 and 71.27%, respectively, and the corresponding levels in polished rice decreased after electric and pressure cooking by 85.58 and 85.82%, 86.70 and 87.06%, and 89.89 and 89.68%, respectively. In conclusion, various processing methods decrease the residual levels of etofenprox, flubendiamide, and tebufenozide in rice.

Alkaloids in food: a review of toxicity, analytical methods, occurrence and risk assessments.

Alkaloids have been utilized by humans for years. They have diverse applications in pharmaceuticals. They have been proven to be effective in treating a number of diseases. They also form an important part of regular human diets, as they are present in food items, food supplements, diet ingredients and food contaminants. Despite their obvious importance, these alkaloids are toxic to humans. Their toxicity is dependent on a range of factors, such as specific dosage, exposure time and individual properties. Mild toxic effects include nausea, itching and vomiting while chronic effects include paralysis, teratogenicity and death. This review summarizes the published studies on the toxicity, analytical methods, occurrence and risk assessments of six major alkaloid groups that are present in food, namely, ergot, glycoalkaloids, purine, pyrrolizidine, quinolizidine and tropane alkaloids.

Programmable Synthesis of Biobased Materials Using Cell-Free Systems.

Motivated by the intricate mechanisms underlying biomolecule syntheses in cells that chemistry is currently unable to mimic, researchers have harnessed biological systems for manufacturing novel materials. Cell-free systems (CFSs) utilizing the bioactivity of transcriptional and translational machineries in vitro are excellent tools that allow supplementation of exogenous materials for production of innovative materials beyond the capability of natural biological systems. Herein, recent studies that have advanced the ability to expand the scope of biobased materials using CFS are summarized and approaches enabling the production of high-value materials, prototyping of genetic parts and modules, and biofunctionalization are discussed. By extending the reach of chemical and enzymatic reactions complementary to cellular materials, CFSs provide new opportunities at the interface of materials science and synthetic biology.

Comparison study of validation parameters and measurement uncertainty of rapid analytical methods for piperine in black pepper by ultraviolet spectroscopy and high-performance liquid chromatography.

Piperine, one of the main constituents of black pepper, is an important marker of black pepper quality because of its bio-activity and characteristic flavour. Ultraviolet spectroscopy and high-performance liquid chromatography-ultraviolet detection were used for rapid determination of piperine in black pepper. The performance parameters and measurement uncertainties of the methods were estimated to identify the most efficient method. Ultraviolet spectroscopy had good specificity and linearity. Its limit of detection was 0.65. Accuracy and precision ranged from 96.7 to 101.5% and from 0.59 to 2.12%, respectively. Measurement uncertainty was estimated as 4.29% for 49.481 g/kg (k = 2). High-performance liquid chromatography also provided good specificity and linearity. The limit of detection was 0.23. Accuracy and precision were 98.2-100.6% and 0.83-1.58%, separately. Measurement uncertainty was calculated as 2.47% for 34.819 g/kg (k = 2). High-performance liquid chromatography is more sensitive and accurate than ultraviolet spectroscopy.

Ribosome-mediated biosynthesis of pyridazinone oligomers in vitro.

The ribosome is a macromolecular machine that catalyzes the sequence-defined polymerization of L-α-amino acids into polypeptides. The catalysis of peptide bond formation between amino acid substrates is based on entropy trapping, wherein the adjacency of transfer RNA (tRNA)-coupled acyl bonds in the P-site and the α-amino groups in the A-site aligns the substrates for coupling. The plasticity of this catalytic mechanism has been observed in both remnants of the evolution of the genetic code and modern efforts to reprogram the genetic code (e.g., ribosomal incorporation of non-canonical amino acids, ribosomal ester formation). However, the limits of ribosome-mediated polymerization are underexplored. Here, rather than peptide bonds, we demonstrate ribosome-mediated polymerization of pyridazinone bonds via a cyclocondensation reaction between activated γ-keto and α-hydrazino ester monomers. In addition, we demonstrate the ribosome-catalyzed synthesis of peptide-hybrid oligomers composed of multiple sequence-defined alternating pyridazinone linkages. Our results highlight the plasticity of the ribosome's ancient bond-formation mechanism, expand the range of non-canonical polymeric backbones that can be synthesized by the ribosome, and open the door to new applications in synthetic biology.