Optimized expression of classical swine fever virus E2 protein via combined strategy in Pichia pastoris.

Precaution of classical swine fever (CSF) is an important mission for the worldwide swine industry. Glycoprotein E2 is the leading antigen candidate for subunit vaccine of classical swine fever virus (CSFV). In this study, two Spy-tagged E2 genes were synthesized in vitro and subcloned into pMCO-AOX vector for intracellular expression in Pichia pastoris after methanol induction. Western blot analysis and semi-quantitative analysis showed that the yield of recombinant E2 protein was improved 17.87 folds by using co-translocational signal peptide cSIG. After the construction of the tandem multiple copy expression vectors, further increase of E2 production was observed by repetitive transforming expression vectors into P. pastoris genome. Finally, the yeast transformants harboring 8 or 16 copies of cSIG-E2-Spy increased the E2 expression level by 27.01-fold or 30.72-fold, respectively. These results demonstrate that utilizing co-translocational signal peptide together with multi-copy integration strategy can increase the production of recombinant E2 protein efficiently.

Free Energy Profile and Kinetics of Coupled Folding and Binding of the Intrinsically Disordered Protein p53 with MDM2.

Intrinsically disordered proteins (IDPs) exert their functions by binding to partner proteins via a complex process that includes coupled folding and binding. Because inhibiting the binding of the IDP p53 to its partner MDM2 has become a promising strategy for the design of anticancer drugs, we carried out metadynamics simulations to study the coupled folding and binding process linking the IDP p53 to MDM2 in atomic detail. Using bias-exchange metadynamics (BE-MetaD) and infrequent metadynamics (InMetaD), we estimated the binding free energy, the unbinding rate, and the binding rate. By analyzing the stable intermediates, we uncovered the role non-native interactions played in the p53-MDM2 binding/unbinding process. We used a three-state model to describe the whole binding/unbinding process and to obtain the corresponding rate constants. Our work shows that the binding of p53 favors an induced-fit mechanism which proceeds in a stepwise fashion. Our results can be helpful for gaining an in-depth understanding of the coupled folding and binding process needed for the design of MDM2 inhibitors.

Activity of Antimicrobial Peptide Aggregates Decreases with Increased Cell Membrane Embedding Free Energy Cost.

Antimicrobial peptides (AMPs) are a promising alternative to antibiotics for mitigating bacterial infections, in light of increasing bacterial resistance to antibiotics. However, predicting, understanding, and controlling the antibacterial activity of AMPs remain a significant challenge. While peptide intramolecular interactions are known to modulate AMP antimicrobial activity, peptide intermolecular interactions remain elusive in their impact on peptide bioactivity. Herein, we test the relationship between AMP intermolecular interactions and antibacterial efficacy by controlling AMP intermolecular hydrophobic and hydrogen bonding interactions. Molecular dynamics simulations and Gibbs free energy calculations in concert with experimental assays show that increasing intermolecular interactions via interpeptide aggregation increases the energy cost for the peptide to embed into the bacterial cell membrane, which in turn decreases the AMP antibacterial activity. Our findings provide a route for predicting and controlling the antibacterial activity of AMPs against Gram-negative bacteria via reductions of intermolecular AMP interactions.

In Vivo Detoxification of Lipopolysaccharide by Antimicrobial Peptides.

Abundant lipopolysaccharide (LPS) can result in sepsis and septic shock, indicating a serious Gram-negative bacterial contamination. We have developed a novel strategy based on dendritic antimicrobial peptides that can detoxify LPS. The dendritic antimicrobial peptides bind to LPS at the surface of Gram-negative bacteria, killing the bacteria but removing the LPS from the cell wall of dead Gram-negative bacteria, hence detoxifying pathogenic bacteria in its host cells and effectively improving survival of animals infected with Pseudomonas aeruginosa. Our findings provide a way to detoxify bacterial contamination.

Peptide-Based Probes with an Artificial Anion-Binding Motif for Direct Fluorescence "Switch-On" Detection of Nucleic Acid in Cells.

This work reports two new peptide-based fluorescence probes (1 and 2) for the detection of ds-DNA at physiological pH. Probes 1 and 2 contain a fluorophore, either amino-naphthalimide or diethyl-aminocoumarin, respectively, and two identical peptide arms each equipped with a guanidiniocarbonylpyrrole (GCP) anion-binding motif. These probes show "switch-on" fluorescence response upon binding to ds-DNA, whereby they can differentiate between various types of polynucleotides. For instance, they exhibit more pronounced fluorescence response for AT-rich polynucleotides than GC-rich polynucleotides, and both give only negligible response to ds-RNA. The fluorimetric response of 1 is proportional to the AT-basepair content in DNA, whereas the fluorescence of 2 is sensitive to the secondary structure of the polynucleotide. Fluorescence experiments, thermal melting experiments and circular dichroism studies suggest that 1 interacts with ds-DNA in a combined intercalation and minor groove binding, whereas 2 interacts mainly with the outer surface of DNA/RNA. As 1 and 2 have a very low cytotoxicity, 1 can be applied for the imaging of nuclear DNA in cells.

Recent advances in peptide nucleic acid for cancer bionanotechnology.

Peptide nucleic acid (PNA) is an oligomer, in which the phosphate backbone has been replaced by a pseudopeptide backbone that is meant to mimic DNA. Peptide nucleic acids are of the utmost importance in the biomedical field because of their ability to hybridize with neutral nucleic acids and their special chemical and biological properties. In recent years, PNAs have emerged in nanobiotechnology for cancer diagnosis and therapy due to their high affinity and sequence selectivity toward corresponding DNA and RNA. In this review, we summarize the recent progresses that have been made in cancer detection and therapy with PNA biotechnology. In addition, we emphasize nanoparticle PNA-based strategies for the efficient delivery of drugs in anticancer therapies.

Peptide self-assembly triggered by metal ions.

Through their unique and specific interactions with various metal ions, naturally occurring proteins control structures and functions of many biological processes and functions in organisms. Inspired by natural metallopeptides, chemists have developed artificial peptides which coordinate with metal ions through their functional groups either for introducing a special reactivity or for constructing nanostructures. However, the design of new coordination peptides requires a deep understanding of the structures, assembly properties, and dynamic behaviours of such peptides. This review briefly discusses strategies of peptide self-assembly induced by metal coordination to different natural and non-natural binding sites in the peptide. The structures and functions of the obtained aggregates are described as well. We also highlight some examples of a metal-induced peptide self-assembly with relevance to biotechnology applications.

Hepatic Stellate Cell-Targeting Micelle Nanomedicine for Early Diagnosis and Treatment of Liver Fibrosis.

Diagnosing and treating liver fibrosis is a challenging yet crucial endeavor due to its complex pathogenesis and risk of deteriorating into cirrhosis, liver failure, and even hepatic cancer. Herein, a silica cross-linked micelles (SCLMs) based nano-system is developed for both diagnosing and treating liver fibrosis. The SCLMs are first modified with peptide CTCE9908 (CT-SCLMs) and can actively target CXCR4, which is overexpressed in activated hepatic stellate cells (HSCs). To enable diagnosis, an ONOO--responded near-infrared fluorescent probe NOF2 is loaded into the CT-SCLMs. This nano-system can target the aHSCs and diagnose the liver fibrosis particularly in CCl4-induced liver damage, by monitoring the reactive nitrogen species. Furthermore, a step is taken toward treatment by co-encapsulating two anti-fibrosis drugs, silibinin and sorafenib, within the CT-SCLMs. This combined approach results in a significant alleviation of liver injury. Symptoms associated with liver fibrosis, such as deposition of collagen, expression of hydroxyproline, and raised serological indicators show notable improvement. In summary, the CXCR4-targeted nano-system can serve as a promising theragnostic system of early warning and diagnosis for liver fibrosis, offering hope against progression of this serious liver condition.

A dual-modality photoswitchable supramolecular polymer.

Covalent or noncovalent linked polymers with stimuli-responsive properties have been well researched as a kind of advanced functional materials. However, little effort has been devoted to establishing a bridge for switching between covalent polymers and noncovalent polymers. Actually, such unitive system is promising because it can combine their chemical advantages of two types of polymers in a single and tunable platform. Herein, by taking advantage of reversible photodimerization of coumarins and host-guest assemblies with γ-cyclodextrin (γ-CD), we demonstrate a simple and effective way to construct a dual-modality supramolecular polymer, which can be switched between a noncovalent polymer and its corresponding covalent polymer in response to light stimuli. Moreover, this unique switchable polymer can also be employed to construct a dual-stimuli responsive supramolecular hydrogel with the surfactant cetyl trimethylammonium bromide (CTAB). This methodology establishes a bridge between the two "polymer mansions" and is promising to open a new class of photoswitchable materials.

Changes in brain structure and related functional connectivity during menstruation in women with primary dysmenorrhea.

Background: Neuroimaging studies have identified altered brain structures and functions in women with primary dysmenorrhea (PDM). However, previous studies focused on either structural or functional changes in specific brain regions rather than combining structural and functional analysis. Therefore, this prospective cross-sectional study aimed to investigate the changes in whole brain structure, and functional variation along with structural abnormalities in women with PDM during menstruation. Methods: In all, 31 patients with PDM (PTs) and 31 healthy controls (HCs) were recruited. Voxel-based morphometry (VBM) and surface-based morphometry (SBM) analyses were applied to investigate structural changes based on high-resolution T1-weighted magnetic resonance images. Functional connectivity (FC) analysis was performed to evaluate functional variations related to the brain regions that showed structural group differences. Pearson correlation analysis was performed to assess the relationship between neuroimaging changes and clinical measures. Results: Compared to HCs, PTs had reduced gray matter volume (GMV) in the right superior temporal gyrus (STG) and reduced thickness in the bilateral orbitofrontal cortex (OFC), left postcentral gyrus (PoCG), and left superior occipital gyrus (SOG). Among these areas, the STG and PoCG are responsible for altered resting-state FC patterns in PTs. Results showed decreased FC between the STG and the left cerebellar posterior lobe (poCb), the right dorsolateral prefrontal cortex (DLPFC), and the left precentral gyrus (PrCG). Results also showed decreased FC between the PoCG and the right precuneus and the right DLPFC. We also found greater FCs between the PoCG and the bilateral poCb, the left middle temporal gyrus (MTG), and the left angular gyrus. In addition, the FCs between the STG and poCb, and DLPFC in PTs were positively correlated with history and Cox menstrual symptom scale (CMSS) scores, respectively, while the FCs between STG and PrCG were negatively correlated with the onset age of PDM. Conclusions: Our research found structural abnormalities and related FC changes in several brain regions that were mainly involved in the emotional and sensory aspects of menstrual pain in PDM. These findings could help us understand the occurrence of PDM from a neuroimaging perspective.

A molecular peptide beacon for the ratiometric sensing of nucleic acids.

A pyrene-functionalized cationic oligopeptide 1 efficiently binds to double-stranded DNA, as shown by different spectrophotochemical studies. Upon binding, the conformation of 1 changes from a folded to an extended form, which leads to a distinct change in the fluorescence properties. Thus, 1 functions as a molecular peptide beacon, and as it is easily taken up by cells, 1 can also be used for imaging of nucleic acids within cells.

Abnormal interhemispheric functional connectivity in patients with primary dysmenorrhea: a resting-state functional MRI study.

Background: Neuroimaging studies have confirmed that functional connectivity (FC) disruption of pain-related brain networks may contribute to the cerebral pathophysiology of primary dysmenorrhea (PDM). However, it remains unclear whether FC of symmetrical regions of bilateral hemispheres associated with PDM is abnormal. This functional MRI study aimed to explore the changes of voxel-mirrored homotopic connectivity (VMHC) and seed-based FC in patients with PDM. Methods: A cohort comprising patients with PDM (n=35) and healthy controls (HCs) (n=41) underwent resting-state functional MRI scans during their menstrual phase. Interhemispheric FC was compared between the two groups using VMHC analysis. Brain areas with significant group differences in VMHC were selected as seed regions for FC analysis. Correlation analysis was also conducted to examine the relationship between abnormal connectivity of brain regions and clinical measures of pain and anxiety. Results: Compared with healthy individuals, patients with PDM showed significantly enhanced VMHC in the bilateral orbital part of the superior frontal gyrus and the bilateral middle frontal gyrus. Subsequent seed-based FC analysis showed enhanced connectivity between the aforementioned areas and pain-related brain structures. Hyperconnectivity between the left middle frontal gyrus and the right cingulate gyrus in patients was negatively correlated with an increase in the visual analogue score (VAS) for pain (r=-0.341, P<0.05). Conclusions: Our findings indicate that ongoing dysmenorrhea is accompanied by abnormal interhemispheric functional coordination and enhanced connectivity in pain-related regions, attention networks, and the reward system. These findings may provide a novel perspective on the central mechanism of pain caused by PDM.

Peptide functionalized polydiacetylene liposomes act as a fluorescent turn-on sensor for bacterial lipopolysaccharide.

Mixed polydiacetylene (PDA) liposomes functionalized on their surface with a fluorescent pentalysine peptide derivative and histidine in a ratio of 1:9 can identify bacterial lipopolysaccharide (LPS). Upon photopolymerization of the self-assembled liposomes the initial fluorescence of the peptide-diacetylene amphiphiles is quenched. Interaction with LPS in aqueous solution or on the surface of E. coli DH5α restores the fluorescence. This increase in fluorescence is selective for LPS relative to other negatively charged analytes including nucleotides and ctDNA. This simple turn-on fluorescent sensor allows detecting LPS even at low micromolar concentrations.

Activation of Cellular Antioxidant Defense System by Naturally Occurring Dibenzopyrone Derivatives Confers Neuroprotection against Oxidative Insults.

Seven dibenzopyrone phenolic derivatives, i.e., alternariol (1), alternariol 5-O-methyl ether (2), altenusin B (3), dehydroaltenusin (4), altenuene (5), altenusin (6), and alterlactone (7), were isolated from endophytic fungi Alternaria alternata extract, and these compounds' structures were elucidated based on various spectroscopic data. Compound 3, a diphenic acid derivative, was determined as a new compound. In this study, compounds 3, 4, 6, and 7 displayed remarkable neuroprotective effects against oxidative injuries by acting as potent activators of nuclear factor-erythroid derived 2-like 2 (Nrf2) in PC12 cells. A mechanistic study indicated that these compounds induced the nuclear accumulation of Nrf2, promoted the expression of Nrf2-governed cytoprotective genes, and increased the cellular antioxidant capacity. More importantly, genetic silence of Nrf2 expression deprived the observed cytoprotection, highlighting the important role of Nrf2 in the protection of these compounds.

A crosslinked colloidal network of peptide/nucleic base amphiphiles for targeted cancer cell encapsulation.

The use of peptide amphiphiles (PAs) is becoming increasingly popular, not only because of their unique self-assembly properties but also due to the versatility of designs, allowing biological responsiveness, biocompatibility, and easy synthesis, which could potentially contribute to new drug design and disease treatment concepts. Oligonucleotides, another major functional bio-macromolecule class, have been introduced recently as new functional building blocks into PAs, further enriching the tools available for the fabrication of bio-functional PAs. Taking advantage of this, in the present work, two nucleic base-linked (adenine, A and thymine, T) RGD-rich peptide amphiphiles (NPAs) containing the fluorophores naphthalimide and rhodamine (Nph-A and Rh-T) were designed and synthesized. The two NPAs exhibit distinctive assembly behaviours with spherical (Rh-T) and fibrous (Nph-A) morphologies, and mixing Nph-A with Rh-T leads to a densely crosslinked colloidal network (Nph-A/Rh-T) via mutually promoted supramolecular polymerization via nucleation-growth assembly. Because of the RGD-rich sequences in the crosslinked network, further research on in situ targeted cancer cell (MDA-MB-231) encapsulation via RGD-integrin recognition was performed, and the modulation of cell behaviours (e.g., cell viability and migration) was demonstrated using both confocal laser scanning microscopy (CLSM) imaging and a scratch wound healing assay.

Sonication-triggered instantaneous gel-to-gel transformation.

Two new peptide-based isomers containing cholesterol and naphthalic groups have been designed and synthesized. We found that the position of L-alanine in the linker could tune the gelation properties and morphologies. The molecule with the L-alanine residue positioned in the middle of the linker (1b) shows better gelation behavior than that with L-alanine directly linked to the naphthalimido moiety (1a). As a result, a highly thermostable organogel of 1b with a unique core-shell structure was obtained at high temperature and pressure in acetonitrile. Moreover, the gels of 1a and 1b could undergo an instantaneous gel-to-gel transition triggered by sonication. Ultrasound could break the core-shell microsphere of 1b and the micelle structure of 1a into entangled fibers. By studying the mechanism of the sonication-triggered gel-to-gel transition process of these compounds, it can be concluded that ultrasound has a variety of effects on the morphology, such as cutting, knitting, unfolding, homogenizing, and even cross-linking. Typically, ultrasound can cleave and homogenize pi-stacking and hydrophobic interactions among the gel molecules and then reshape the morphologies to form a new gel. This mechanism of morphology transformation triggered by sonication might be attractive in the field of material storage and controlled release.

Aberrant resting-state cerebral blood flow and its connectivity in primary dysmenorrhea on arterial spin labeling MRI.

PURPOSE: This study aimed to clarify the resting-state cerebral blood flow alteration patterns induced by primary dysmenorrhea, investigate the relationships between cerebral blood flow alterations and clinical parameters of patients with primary dysmenorrhea, and explore whether brain regions with abnormal cerebral blood flow also feature functional connectivity changes. METHODS: Arterial spin labeling imaging and clinical parameters were acquired in 42 patients with primary dysmenorrhea and 41 healthy controls during their menstrual phases. Differences in cerebral blood flow were compared between the two groups, and the clusters with significant group differences were selected as the regions of interest for further statistical analyses. RESULTS: Compared to healthy controls, patients with primary dysmenorrhea exhibited increased cerebral blood flow in the bilateral precuneus, left posterior cingulate cortex, and right rolandic operculum. Among patients with primary dysmenorrhea, we identified a negative correlation between the cerebral blood flow in the right rolandic operculum and the visual analogue score for anxiety, and greater correlation between the functional connectivity in the precuneus/posterior cingulate cortex and the right middle cingulate cortex, and between the right rolandic operculum and the left inferior parietal lobule and the bilateral postcentral gyrus. DISCUSSION: Cerebral blood flow abnormalities associated with primary dysmenorrhea were mainly concentrated in the areas comprising the default mode network in primary dysmenorrhea patients, which could be involved in the central mechanism of primary dysmenorrhea. Cerebral blood flow alteration in the rolandic operculum may underlie an anxiety-induced compulsive tendency in patients with primary dysmenorrhea. Investigating the enhanced connectivity among various pain-related brain regions could improve understanding of the onset and development of primary dysmenorrhea.

Tunable gel formation by both sonication and thermal processing in a cholesterol-based self-assembly system.

A family of asymmetric cholesterol-based fluorescent organogelators containing naphthalimide, connected by two acylamines and different alkyl-chain spacers, have been designed and prepared. These compounds can gelate a variety of organic solvents with both ultrasound stimuli and general sol-gel processes. The self-assembly and gelling properties of the compounds depend on the length of the alkyl chains and can be controlled by ultrasound stimuli and renewed by a thermodynamic process. The morphologies and surface wetabilities of the xerogels prepared from these gelators are strongly affected by environmental stimuli. The mechanism of the process was investigated by confocal laser scanning microscopy, transmission or scanning electron microscopy, wide-angle X-ray scattering analysis, and rheological experiments. The studies reveal that the cooperation and relative competition of multiple intermolecular interactions, influenced by the sonication or thermal stimulus, are the main contributors for the aggregation or nucleation processes; this results in the macrodifferences in morphology and surface properties. These results provide a deeper understanding of the intermediate transition state of the gel during use of an ultrasound stimulus.

Inclusion of tetracycline hydrochloride within supramolecular gels and its controlled release to bovine serum albumin.

Three amphiphilic 3,4,5-trihydroxybenzoic derivatives with alkyl chains of different lengths were designed and synthesized. A small amount of these compounds can trap a large quantity of the water-soluble drug tetracycline hydrochloride (TH) within a stable gel in aqueous ethanol. Release experiments were carried out with solutions of bovine serum albumin (BSA) and various concentrations of L-isoleucine, L-phenylalanine, and L-tryptophan. The results indicate that the release rate of TH for a BSA solution (10 mg/mL) was faster than that with the other solutions because of the strong interaction between TH and BSA. Furthermore, to gain an insight into the release dynamics, we studied the release ratios as a function of the square root of time (t1/2). During the initial 1.75 h, diffusion is the dominant release process in water, whereas intermolecular interaction controls TH release in the BSA solution.

Purification and characterization of anthranilate synthase component I (TrpE) from Mycobacterium tuberculosis H37Rv.

The emergence of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis is the main reason why tuberculosis (TB) continues to be a major health problem worldwide. It is urgent to discover novel anti-mycobacterial agents based on new drug targets for the treatment of TB, especially MDR-TB. Tryptophan biosynthetic pathway, which is essential for the survival of M. tuberculosis and meanwhile absent in mammals, provides potential anti-TB drug targets. One of the promising drug targets in this pathway is anthranilate synthase component I (TrpE), whose role is to catalyze the conversion of chorismate to anthranilate using ammonia as amino source. In order to get a deep understanding of TrpE, a study on purification and characteristic identification of TrpE is required. In this work, the putative trpE gene of M. tuberculosis H37Rv was expressed as a fusion protein with a 6x His-tag on the N-terminal (His-TrpE) in Escherichia coli. The recombinant TrpE protein was successfully purified and then its enzymatic characteristics were analyzed. The native TrpE without His-tag was obtained by removal of the N-terminal fusion partner of His-TrpE using enterokinase. It was found that N-terminal fusion partner had little influence on TrpE catalytic activity. In addition, the key residues related to enzyme catalytic activity and that involved in l-tryptophan inhibition were predicted in the structure of M. tuberculosis H37Rv TrpE. These results would be beneficial to the designing of novel anti-TB drugs with high potency and selectivity.