Rewiring Metabolic Flux in Corynebacterium glutamicum Using a CRISPR/dCpf1-Based Bifunctional Regulation System.

Corynebacterium glutamicum, a microorganism classified as generally recognized as safe for use in the industrial production of food raw materials and additives, has encountered challenges in achieving widespread adoption and popularization as microbial cell factories. These obstacles arise from the intricate nature of manipulating metabolic flux through conventional methods, such as gene knockout and enzyme overexpression. To address this challenge, we developed a CRISPR/dCpf1-based bifunctional regulation system to bidirectionally regulate the expression of multiple genes in C. glutamicum. Specifically, through fusing various transcription factors to the C-terminus of dCpf1, the resulting dCpf1-SoxS exhibited both CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) capabilities in C. glutamicum by altering the binding sites of crRNAs. The bifunctional regulation system was used to fine-tune metabolic flux from shikimic acid (SA) and l-serine biosynthesis, resulting in 27-fold and 10-fold increases in SA and l-serine production, respectively, compared to the original strain. These findings highlight the potential of the CRISPR/dCpf1-based bifunctional regulation system in effectively enhancing the yield of target products in C. glutamicum.

An armoured marine reptile from the Early Triassic of South China and its phylogenetic and evolutionary implications.

Sauropterygia was a taxonomically and ecomorphologically diverse clade of Mesozoic marine reptiles spanning the Early Triassic to the Late Cretaceous. Sauropterygians are traditionally divided into two groups representing two markedly different body plans - the short-necked, durophagous Placodontia and the long-necked Eosauropterygia - whereas Saurosphargidae, a small clade of armoured marine reptiles, is generally considered as the sauropterygian sister-group. However, the early evolutionary history of sauropterygians and their phylogenetic relationships with other groups within Diapsida are still incompletely understood. Here, we report a new saurosphargid from the Early Triassic (Olenekian) of South China - Prosaurosphargis yingzishanensis gen. et sp. nov. - representing the earliest known occurrence of the clade. An updated phylogenetic analysis focussing on the interrelationships among diapsid reptiles recovers saurosphargids as nested within sauropterygians, forming a clade with eosauropterygians to the exclusion of placodonts. Furthermore, a clade comprising Eusaurosphargis and Palatodonta is recovered as the sauropterygian sister-group within Sauropterygomorpha tax. nov. The phylogenetic position of several Early and Middle Triassic sauropterygians of previously uncertain phylogenetic affinity, such as Atopodentatus, Hanosaurus, Majiashanosaurus, and Corosaurus, is also clarified, elucidating the early evolutionary assembly of the sauropterygian body plan. Finally, our phylogenetic analysis supports the placement of Testudines and Archosauromorpha within Archelosauria, a result strongly corroborated by molecular data, but only recently recovered in a phylogenetic analysis using a morphology-only dataset. Our study provides evidence for the rapid diversification of sauropterygians in the aftermath of the Permo-Triassic mass extinction event and emphasises the importance of broad taxonomic sampling in reconstructing phylogenetic relationships among extinct taxa.

Around 252 million years ago, just before the start of a period of time known as the Triassic, over 90% of animals, plants and other species on Earth went extinct in what was the worst mass extinction event in the planet's history. It is thought to have happened because of an increase in volcanic eruptions that led to global warming, acid rain and other catastrophic changes in the environment. The loss of so many species caused ecosystems to restructure as the surviving species evolved to fill niches left by those that had gone extinct. On land, reptiles diversified to give rise to dinosaurs, the flying pterosaurs, and the ancestors of modern crocodiles, lizards, snakes and turtles. Some of these land-based animals evolved to live in water, resulting in many species of marine reptiles emerging during the Triassic period. This included the saurosphargids, a group of marine reptiles that lived in the Middle Triassic around 247­237 million years ago. They were 'armoured' with a shield made of broadened ribs superficially similar to that of turtles, and a covering of bony plates. However, it is unclear how the saurosphargids evolved and how closely they are related to other marine reptiles. Here, Wolniewicz et al. studied a new species of saurosphargid named Prosaurosphargis yingzishanensis that was found fossilized in a quarry in South China. The animal was around 1.5 metres long and had a chest shield and armoured plates like other saurosphargids. The characteristics of the rock surrounding the fossil suggest that this individual lived in the Early Triassic, several million years before other saurosphargid species. The team used a phylogenetic approach to infer the evolutionary relationships between P. yingzishanensis and numerous other land-based and marine reptiles based on over 220 anatomical characteristics of the animals. The resulting evolutionary tree indicated that the saurosphargids represented an early stage in the evolution of a larger group of marine reptiles known as the sauropterygians. The analysis also identified the closest land-based relatives of sauropterygians. These findings provide evidence that marine reptiles rapidly diversified in the aftermath of the mass extinction event 252 million years ago. Furthermore, they contribute to our understanding of how ecosystems recover after a major environmental crisis.

A globally distributed durophagous marine reptile clade supports the rapid recovery of pelagic ecosystems after the Permo-Triassic mass extinction.

Marine ecosystem recovery after the Permo-Triassic mass extinction (PTME) has been extensively studied in the shallow sea, but little is known about the nature of this process in pelagic ecosystems. Omphalosauridae, an enigmatic clade of open-water durophagous marine reptiles, potentially played an important role in the recovery, but their fragmentary fossils and uncertain phylogenetic position have hindered our understanding of their role in the process. Here we report the large basal ichthyosauriform Sclerocormus from the Early Triassic of China that clearly demonstrates an omphalosaurid affinity, allowing for the synonymy of the recently erected Nasorostra with Omphalosauridae. The skull also reveals the anatomy of the unique feeding apparatus of omphalosaurids, likely an adaptation for feeding on hard-shelled pelagic invertebrates, especially ammonoids. Morphofunctional analysis of jaws shows that omphalosaurids occupy the morphospace of marine turtles. Our discovery adds another piece of evidence for an explosive radiation of marine reptiles into the ocean in the Early Triassic and the rapid recovery of pelagic ecosystems after the PTME.

Effect of Lactobacillus acidophilus fermentation on the composition of chlorogenic acids and anti-hyperuricemia activity of Artemisia selengensis Turcz.

Background: Lactobacillus fermentation has become a typical food processing method with the development of the modern food industry. Aims: This study aimed to assess the effects of Lactobacillus acidophilus NCUF202.2 fermentation on Artemisia selengensis Turcz (AST) in relieving hyperuricemia (HUA) and the mechanisms involved. Results: The fermented Artemisia selengensis Turcz extracts (FASTE) could significantly increase the content of free polyphenol and enhance the inhibitory effect on xanthine oxidase (XOD) in vitro. The result of ultrafiltration coupled with UPLC-MS/MS screened out the components that might be combined with XOD in the caffeoylquinic acids (CQAs) of Artemisia selengensis Turcz extracts (ASTE) as 5-CQA, 3-CQA, 4-CQA, 1-CQA, 3,4-diCQA, 3,5-diCQA, 1,5-diCQA and 4,5-diCQA as well as caffeic acid (CA) of FASTE except the above. Our results also showed that ASTE and FASTE could alleviate HUA in rats. In contrast, FASTE had a better ability to reduce serum adenosine deaminase (ADA) activity than ASTE. Furthermore, FASTE could restore catalase (CAT) in HUA model rats in vivo, and superoxide dismutase (SOD) activity to a better degree, thereby inhibiting the production of excess malondialdehyde (MDA). Under the intervention of ASTE and FASTE, the levels of serum pro-inflammatory and anti-inflammatory factors in rats tended to be normal. However, FASTE could increase the expression of the uric acid secretion protein OAT1 and decrease the expression of reabsorption proteins URAT1 and GLUT9 in model rats, thereby reducing the serum uric acid (SUA) level in model rats. ASTE and FASTE can increase the abundance of beneficial bacteria, such as Bifidobacterium and Akkermansiaceae. Conclusion: Overall, part of diCQAs and CQAs was decomposed after fermentation. FASTE had a more substantial HUA-relieving effect than ASTE. It is expected to be applied to functional foods and contribute to the research and development of uric acid lowering functional foods.

Inhibitory mechanism of xanthine oxidase activity by caffeoylquinic acids in vitro.

In this study, the inhibitory activities of eight caffeoylquinic acids (CQAs) against xanthine oxidase (XOD) in vitro were investigated, and the interaction mechanisms between each compound and XOD were studied. HPLC and fluorescence spectra showed that the inhibitory activities of dicaffeoylquinic acids (diCQAs) were higher than that of monocaffeoylquinic acids (monoCQAs), due to the main roles of hydrophobic interaction and hydrogen bond between XOD and diCQAs. Both the binding constant and the lowest binding energy data indicated that the affinities of diCQAs to XOD were stronger than that of monoCQAs. Circular dichroism showed that the structure of XOD was compacted with the increased of α-helix content, resulting in decreased enzyme catalytic activity. Molecular docking revealed that CQAs preferentially bind to the flavin adenine dinucleotide region in XOD. These results provided the mechanisms of CQAs on inhibiting XOD and the further utilization of CQAs as XOD inhibitors to prevent hyperuricemia.

Geochemical and Geological Characteristics of the Upper Ordovician-Lower Silurian Shales in the Upper Yangtze Basin, South China: Implication for the Shale Gas Exploration.

Shale gas exploration in the Upper Yangtze Basin has been conducted for over ten years. The successful drillings in the region suggest that well-planned geological survey and reservoir evaluation are indispensable for the success of these drillings. In this study, the shale gas potential of Late Ordovician to Early Silurian mudstones/shales of the Wufeng-Longmaxi formations in the Upper Yangtze Basin was evaluated. First, all of the available geochemical and petrological data from Late Ordovician to Early Silurian mudstones/shales in the region were compiled. Distributional patterns of total organic carbon (TOC) values, thickness, thermal maturity, and burial depth of organic-rich mudstones/shales from the targeted formations were then analyzed. Our results show that TOC values range from 1.20 to 5.12%; R o values range from 1.92 to 2.86%; porosity varies from 0.77 to 9.2%; permeability varies from 0.015 to 1.99 md; the composition of quartz group of mineral component ranges from 18 to 85%, 59% in average; and clay mineral component ranges from 7 to 56%, 31% in average. Finally, based on these comprehensive analysis, three areas with great potential, that is, Luzhou-Xishui, western Chongqing, and Wulong-Shizhu, were selected as targets for further shale gas exploration and exploitation.

Particle trapping and beaming using a 3D nanotip excited with a plasmonic vortex.

Recent advances in nanotechnology have prompted the need for tools to accurately and noninvasively manipulate individual nano-objects. Among the possible strategies, optical forces have been widely used to enable nano-optical tweezers capable of trapping or moving a specimen with unprecedented accuracy. Here, we propose an architecture consisting of a nanotip excited with a plasmonic vortex enabling effective dynamic control of nanoparticles in three dimensions. The structure illuminated by a beam with angular momentum can generate an optical field that can be used to manipulate single dielectric nanoparticles. We demonstrate that it is possible to stably trap or push the particle from specific points, thus enabling a new, to the best of our knowledge, platform for nanoparticle manipulation.

Comparison of HLA-A, -B and -DRB1 Loci Polymorphism between Kidney Transplants of Uremia Patients and Healthy Individuals in Central China.

Chronic kidney disease is becoming a global public health problem, which will usually cause uremia at the end stage of chronic kidney failure. So far, kidney transplant is the most effective and proper therapy for uremia, however, the short supply of matched donor kidney has been a persistent bottleneck for transplantation. HLA matching of HLA-A, -B and -DRB1 loci is very important for the allocation of kidney transplants. In this study, we investigated genotypes of HLA-A, -B and -DRB1 loci based on 1,464 uremia patients and 10,000 unrelated healthy individuals in Henan province of China, and compared the frequency distribution of these HLA alleles and corresponding haplotypes between patient and healthy groups. We detected 23 HLA-A, 49 HLA-B and 17 HLA-DRB1 alleles in total. The predominant alleles of HLA-A, -B and -DRB1 loci in patients are the same as those in healthy group. The seven most frequent alleles account for about 87%, 50%, and 77% at HLA-A, -B and -DRB1 loci, respectively. The haplotypes (combinations of HLA-A, -B, and -DRB1) with significantly different frequency between patients and controls mostly account for less than 1%. Overall, this suggests that HLA matching is not a potential difficulty for kidney transplant of uremia patients. However, three of the top seven frequent HLA-DRB1 alleles have a significantly different distribution in patients and controls, while only one alleles for HLA-B and zero for HLA-A loci. These HLA-DRB1 alleles may be closely associated with uremia. This study sheds new lights on the composition and difference of HLA genotypes in uremia patients and healthy populations in Central China that can serve as a guide to HLA matching for kidney transplants and a resource for HLA typing-related studies.

Physical mapping of chromosome 4J of Thinopyrum bessarabicum using gamma radiation-induced aberrations.

KEY MESSAGE: Gamma radiation induced a series of structural aberrations involving Thinopyrum bessarabicum chromosome 4J. The aberrations allowed for deletion mapping of 101 4J-specific markers and fine mapping of blue-grained gene BaThb. Irradiation can induce translocations and deletions to assist physically locating genes and markers on chromosomes. In this study, a 12-Gy dosage of (60)Co-γ was applied to pollen and eggs of a wheat (Triticum aestivum) landrace Chinese Spring (CS)-Thinopyrum bessarabicum chromosome 4J disomic addition line (DA4J), and the gametes from irradiated plants were fertilized with normal CS eggs or pollen to produce M1 seeds. Based on genomic in situ hybridization analysis of 261 M1 plants, we identified 74 lines carrying structural aberrations involving chromosome 4J with the higher aberration rate in treated pollen (31.2 %) than in the treated eggs (21.3 %). We further identified 43 (53.8 %) lines with structural aberrations on chromosome 4J by analyzing another 80 M1 plants with 74 4J-specific markers, indicating that combining molecular and cytological methods was more efficient for detecting chromosome aberrations. Marker analysis thus was performed prior to cytogenetic identification on M2-M4 seeds to detect chromosome structural aberrations. Sixty-eight M3 lines with structural aberrations on chromosome 4J and six previously obtained chromosome 4J alien lines were then analyzed using 101 chromosome 4J-specific markers. After combining marker results with chromosome aberrations in each line, chromosome 4J was physically divided into 24 segmental blocks with 7 in the short arm and 17 in the long arm. The blue-grained gene BaThb was further mapped into the region corresponding to block 4JL-11. The chromosome aberrations and the physical map developed in this research provide useful stocks and tools for introgression of genes on chromosome 4J into wheat.

Phylogenetic affinity of tree shrews to Glires is attributed to fast evolution rate.

Previous phylogenetic analyses have led to incongruent evolutionary relationships between tree shrews and other suborders of Euarchontoglires. What caused the incongruence remains elusive. In this study, we identified 6845 orthologous genes between seventeen placental mammals. Tree shrews and Primates were monophyletic in the phylogenetic trees derived from the first or/and second codon positions whereas tree shrews and Glires formed a monophyly in the trees derived from the third or all codon positions. The same topology was obtained in the phylogeny inference using the slowly and fast evolving genes, respectively. This incongruence was likely attributed to the fast substitution rate in tree shrews and Glires. Notably, sequence GC content only was not informative to resolve the controversial phylogenetic relationships between tree shrews, Glires, and Primates. Finally, estimation in the confidence of the tree selection strongly supported the phylogenetic affiliation of tree shrews to Primates as a monophyly.

TFPP: an SVM-based tool for recognizing flagellar proteins in Trypanosoma brucei.

Trypanosoma brucei is a unicellular flagellated eukaryotic parasite that causes African trypanosomiasis in human and domestic animals with devastating health and economic consequences. Recent studies have revealed the important roles of the single flagellum of T. brucei in many aspects, especially that the flagellar motility is required for the viability of the bloodstream form T. brucei, suggesting that impairment of the flagellar function may provide a promising cure for African sleeping sickness. Knowing the flagellum proteome is crucial to study the molecular mechanism of the flagellar functions. Here we present a novel computational method for identifying flagellar proteins in T. brucei, called trypanosome flagellar protein predictor (TFPP). TFPP was developed based on a list of selected discriminating features derived from protein sequences, and could predict flagellar proteins with ∼92% specificity at a ∼84% sensitivity rate. Applied to the whole T. brucei proteome, TFPP reveals 811 more flagellar proteins with high confidence, suggesting that the flagellar proteome covers ∼10% of the whole proteome. Comparison of the expression profiles of the whole T. brucei proteome at three typical life cycle stages found that ∼45% of the flagellar proteins were significantly changed in expression levels between the three life cycle stages, indicating life cycle stage-specific regulation of flagellar functions in T. brucei. Overall, our study demonstrated that TFPP is highly effective in identifying flagellar proteins and could provide opportunities to study the trypanosome flagellar proteome systematically. Furthermore, the web server for TFPP can be freely accessed at http:/wukong.tongji.edu.cn/tfpp.

Protein structure alignment based on internal coordinates.

Protein structure alignment provides an effective way to detect and analyze fold mechanism, evolutionary history and biological function of proteins. In this work, we introduced a novel method named SABIC for protein structure alignment based on the internal coordinates (i.e. bond lengths, bond angles and torsion angles) of structure representation. SABIC provides multi-alignments as the output, from which various aspects of structural similarities between proteins can be identified. The experimental results on benchmark datasets show that SABIC performs better than other popular algorithms, such as DALI, CE and SSM. Using a new defined mQ-score of alignment, SABIC performs consistently better in detecting structural classifications of proteins. In addition, we detected the extreme value distribution form statistics of mQ-score, and then the statistical significance P-value of alignment can be obtained simultaneously. The presented SABIC algorithm has been implemented in C(++) and the software is available ( http://www.aporc.org/doc/wiki/SABIC ).

Quasi-steady-state laws in enzyme kinetics.

To understand how enzymes work is essential for understanding life processes. And, in enzyme kinetics, a fundamental assumption is the so-called Quasi-Steady-State Assumption, which has the history of more than 80 years and has been proven very fruitful in analyzing the equations of enzyme kinetics. Many experimental results and numerical results have shown the validity of the assumption. So, an important problem is if it is always true. If it is always true, then it should be a law, not only an assumption. In this paper, we prove mathematically rigorously that it is indeed always true. Hence, it is a law, and we name it the Quasi-Steady-State Law. Actually, more precisely, we have two Quasi-Steady-State Laws. In one of them quasi-steady state means that the concentration of the enzyme-substrate complex remains approximately constant, and in the other it means that the change rate of the concentration of enzyme-substrate complex is extremely tiny.