Efficient expression of γ-glutamyl transpeptidase in Bacillus subtilis via CRISPR/Cas9n and its immobilization.

In this study, we successfully applied the strategy of combining tandem promoters and tandem signal peptides with overexpressing signal peptidase to efficiently express and produce γ-glutamyl peptidase (GGT) enzymes (BsGGT, BaGGT, and BlGGT) from Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus licheniformis in Bacillus subtilis ATCC6051Δ5. In order to avoid the problem of instability caused by duplicated strong promoters, we assembled tandem promoters of different homologous genes from different species. To achieve resistance marker-free enzyme in the food industry, we first removed the replication origin and corresponding resistance marker of Escherichia coli from the expression vector. The plasmid was then transformed into the B. subtilis host, and the Kan resistance gene in the expression plasmid was directly edited and silenced using the CRISPR/Cas9n-AID base editing system. As a result, a recombinant protein expression carrier without resistance markers was constructed, and the enzyme activity of the BlGGT strain during shake flask fermentation can reach 53.65 U/mL. The recombinant BlGGT was immobilized with epoxy resin and maintained 82.8% enzyme activity after repeated use for 10 times and 87.36% enzyme activity after storage at 4 °C for 2 months. The immobilized BlGGT enzyme was used for the continuous synthesis of theanine with a conversion rate of 65.38%. These results indicated that our approach was a promising solution for improving enzyme production efficiency and achieving safe production of enzyme preparations in the food industry. KEY POINTS: • Efficient expression of recombinant proteins by a combination of dual promoter and dual signal peptide. • Construction of small vectors without resistance markers in B. subtilis using CRISPR/Cas9n-AID editing system. • The process of immobilizing BlGGT with epoxy resin was optimized.

Enhancement of Astaxanthin Bioaccessibility by Encapsulation in Liposomes: An In Vitro Study.

Astaxanthin was encapsulated in liposomes by a thin layer dispersion and ultrasound method using soybean phospholipid. The digestion properties of liposomes for encapsulating astaxanthin were investigated in light of particle size, size distribution, zeta potential, and microstructure during in vitro digestion as a function of time. These results exhibited that the average particle size increased gradually with liposomal vesicles retained round shapes and a fairly uniform distribution after passage through the simulated gastric fluid digestion. The result revealed that astaxanthin-loaded liposomes were stable in low pH conditions. It was also found that the mixed micelles formed in a simulated intestinal fluid. The zeta potential of astaxanthin-loaded liposomes had a decrease in negativity after digestion. In comparison with free astaxanthin, there was an appreciable increase in the bioaccessibility of astaxanthin after encapsulation in liposomes. This enhancement can be attributed to more soluble astaxanthin in the mixed micelles for astaxanthin-loaded liposomes. It indicated that the barrier of the liposomal bilayer could inhibit astaxanthin fading and leaking after encapsulation in liposomes. These results provide useful information for designing more stable delivery systems in the gastrointestinal tract and improving the bioaccessibility of lipophilic nutraceuticals.

Multiple-Site SUMOylation of FMDV 3C Protease and Its Negative Role in Viral Replication.

Protein SUMOylation represents an important cellular process that regulates the activities of numerous host proteins as well as of many invasive viral proteins. Foot-and-mouth disease virus (FMDV) is the first animal virus discovered. However, whether SUMOylation takes place during FMDV infection and what role it plays in FMDV pathogenesis have not been investigated. In the present study, we demonstrated that SUMOylation suppressed FMDV replication by small interfering RNA (siRNA) transfection coupled with pharmaceutical inhibition of SUMOylation, which was further confirmed by increased virus replication for SUMOylation-deficient FMDV with mutations in 3C protease, a target of SUMOylation. Moreover, we provided evidence that four lysine residues, Lys-51, -54, -110, and -159, worked together to confer the SUMOylation to the FMDV 3C protease, which may make SUMOylation of FMDV 3C more stable and improve the host's chance of suppressing the replication of FMDV. This is the first report that four lysine residues can be alternatively modified by SUMOylation. Finally, we showed that SUMOylation attenuated the cleavage ability, the inhibitory effect of the interferon signaling pathway, and the protein stability of FMDV 3C, which appeared to correlate with a decrease in FMDV replication. Taken together, the results of our experiments describe a novel cellular regulatory event that significantly restricts FMDV replication through the SUMOylation of 3C protease. IMPORTANCE FMD is a highly contagious and economically important disease in cloven-hoofed animals. SUMOylation, the covalent linkage of a small ubiquitin-like protein to a variety of substrate proteins, has emerged as an important posttranslational modification that plays multiple roles in diverse biological processes. In this study, four lysine residues of FMDV 3C were found to be alternatively modified by SUMOylation. In addition, we demonstrated that SUMOylation attenuated FMDV 3C function through multiple mechanisms, including cleavage ability, the inhibitory effect of the interferon signaling pathway, and protein stability, which, in turn, resulted in a decrease of FMDV replication. Our findings indicate that SUMOylation of FMDV 3C serves as a host cell defense against FMDV replication. Further understanding of the cellular and molecular mechanisms driving this process should offer novel insights to design an effective strategy to control the dissemination of FMDV in animals.

Inhibition of Antiviral Innate Immunity by Foot-and-Mouth Disease Virus Lpro through Interaction with the N-Terminal Domain of Swine RNase L.

Foot-and-mouth disease virus (FMDV) is the pathogen of foot-and-mouth disease (FMD), which is a highly contagious disease in cloven-hoofed animals. To survive in the host, FMDV has evolved multiple strategies to antagonize host innate immune responses. In this study, we showed that the leader protease (Lpro) of FMDV, a papain-like proteinase, promoted viral replication by evading the antiviral interferon response through counteracting the 2',5'-oligoadenylate synthetase (OAS)/RNase L system. Specifically, we observed that the titers of Lpro deletion virus were significantly lower than those of wild-type FMDV (FMDV-WT) in cultured cells. Our mechanistic studies demonstrated that Lpro interfered with the OAS/RNase L pathway by interacting with the N-terminal domain of swine RNase L (sRNase L). Remarkably, Lpro of FMDV exhibited species-specific binding to RNase L in that the interaction was observed only in swine cells, not human, monkey, or canine cells. Lastly, we presented evidence that by interacting with sRNase L, FMDV Lpro inhibited cellular apoptosis. Taken together, these results demonstrate a novel mechanism that Lpro utilizes to escape the OAS/RNase L-mediated antiviral defense pathway. IMPORTANCE FMDV is a picornavirus that causes a significant disease in agricultural animals. FMDV has developed diverse strategies to escape the host interferon response. Here, we show that Lpro of FMDV antagonizes the OAS/RNase L pathway, an important interferon effector pathway, by interacting with the N-terminal domain of sRNase L. Interestingly, such a virus-host interaction is species-specific because the interaction is detected only in swine cells, not in human, monkey, or canine cells. Furthermore, Lpro inhibits apoptosis through interacting with sRNase L. This study demonstrates a novel mechanism by which FMDV has evolved to inhibit host innate immune responses.

Development of an indirect ELISA using a novel linear epitope at the C-terminal region of the VP2 protein to specifically detect antibodies against Senecavirus A.

BACKGROUND: Senecavirus A (SVA) is a pathogen that has recently caused porcine idiopathic vesicular disease (PIVD). The clinical signs are similar to those of foot-and-mouth disease, porcine vesicular disease, and vesicular stomatitis. Therefore, identification of SVA as a cause of PIVD is important to eliminate this emerging pathogen. METHODS: In this study, an indirect ELISA based on the VP2 epitope (VP2-epitp-ELISA) was developed to detect antibodies directed against SVA. RESULTS: A novel linear epitope (271GLRNRFTTGTDEEQ284) was first identified at the C-terminus of the VP2 protein by epitope mapping. The diagnostic performance of VP2-epitp-ELISA was estimated by testing a panel of known background sera from swine. Under the optimum test conditions, when the cutoff value was 37%, the diagnostic sensitivity (Dn) and diagnostic specificity (Dp) of the assay were 91.13% and 91.17%, respectively. The accuracy of VP2-epitp-ELISA was validated and further compared with that of commercial diagnostic kits. The diagnostic results showed that VP2-epitp-ELISA did not cross-react with serum positive for other idiopathic vesicular diseases and had a concordance rate of 90.41% with the Swinecheck® SVA bELISA. CONCLUSIONS: These results indicate that VP2-epitp-ELISA is suitable for specific detection of antibodies against SVA in swine.

Convolution Neural Networks and Targeted Fluorescent Nanoparticles to Detect and ICDAS Score Caries.

Previous work has shown targeted fluorescent starch nanoparticles (TFSNs) can label the subsurface of carious lesions and assist dental professionals in the diagnostic process. In this study, we aimed to evaluate the potential of using artificial intelligence (AI) to detect and score carious lesions using ICDAS in combination with fluorescent imaging following application of TFSNs on teeth with a range of lesion severities, using ICDAS-labeled images as the reference standard. A total of 130 extracted human teeth with ICDAS scores from 0 to 6 were selected by a calibrated cariologist. Then, the same surface was imaged with a stereomicroscope under white light illumination, without visible fluorescence, and blue light illumination with an orange filter following application of the TFSNs. Both sets of images were labeled by another blinded ICDAS-calibrated cariologist to demarcate lesion position and severity. Convolutional neural networks, state-of-the-art models in imaging AI, were trained to determine the presence, location, ICDAS score (severity), and lesion surface porosity (as an indicator of activity) of carious lesions, and tested by 30 k-fold validation for white light, blue light, and the combined image sets. The best models showed high performance for the detection of carious lesions (sensitivity 80.26%, PPV 76.36%), potential for determining the severity via ICDAS scoring (accuracy 72%, SD 5.67%), and the detection of surface porosity as an indicator of the activity of the lesions (accuracy 90%, SD 7.00%). More broadly, the combination of targeted biopolymer nanoparticles with imaging AI is a promising combination of novel technologies that could be applied to many other applications.

Effects and mechanisms of histatins as novel skin wound-healing agents.

Wound healing is a complex and important physiological process that maintains the integrity of skin after various injuries. Abnormal wound healing, especially of chronic wounds, impairs normal physical function. Therefore, the search for effective and safe healing agents is one of the main concerns. Histatins are histidine-rich low molecular weight peptides that are expressed in the saliva of both humans and higher primates. Histatins have two main biological effects, cell stimulation and bacteria killing, with the former playing an important role in wound healing by promoting epithelial cell and fibroblast migration and angiogenesis and enhancing the re-epithelialization of the wounded area. Because of these biological effects, histatins have been shown to be promising agents of improved wound healing. Histatins are categorized into many subtypes, of which histatin 1 and its hydrolysates are the most effective in promoting wound healing. This review addresses the bioactivity of histatins in wound healing, such as their stimulatory effects on epithelial cells and fibroblasts, and elucidates the possible mechanisms by which histatin subtypes induce their biological effects.

Comparison of immune responses in guinea pigs by intranasal delivery with different nanoparticles-loaded FMDV DNA vaccine.

To compare different nanoparticle-based nasal vaccines against foot-and-mouth disease (FMD), chitosan (CS)-coated poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) (CS/PLGA-NPs) and amino-functionalized mesoporous silica nanoparticles (Am/MSNs) loaded with FMDV recombinant plasmid (pP12A3C/IFN-CS/PLGA-NPs and pP12A3C/IFN-Am/MS-NPs) were used to induce mucosal and systemic immune responses in guinea pigs via intranasal delivery. Simultaneously, CpG oligodeoxy nucleotides (ODNs) as a vaccine adjuvant were encapsulated in chitosan-coated poly (lactic-co-glycolic acid) nanoparticles (CpG-CS/PLGA-NPs). The pP12A3C/IFN-CS/PLGA-NPs and CpG-CS/PLGA-NPs generated displayed good morphology, high stability, mean diameters of 500 and 400 nm and encapsulation efficiencies of 83.8% and 88.4%, respectively. Data from the in vitro release assay showed that plasmid and CpG were sustainably released from nanoparticles (up to 66.73% and 64%, respectively, of the total amount loaded). Guinea pigs immunized with pP12A3C/IFN-CS/PLGA-NPs + CpG-CS/PLGA-NPs showed markedly higher mucosal, cellular and humoral immune responses than those administered pP12A3C/IFN-CS/PLGA-NPs or naked plasmid vaccine alone. FMDV-specific secretory immunoglobulin A (sIgA) antibodies in nasal washes were initially detected at 3 days post-vaccination with CS/PLGA-NPs loaded with plasmid. Guinea pigs immunized with pP12A3C/IFN-CS/PLGA-NPs also displayed higher cellular and humoral immune responses than pP12A3C-CS/PLGA-NPs and naked plasmid vaccine alone. FMDV-specific immunoglobulin G (IgG) antibodies in serum were initially detected at 5 days post-vaccination (intramuscularly) with the naked plasmid. Finally, challenge experiments 42 days post-vaccine revealed 100% protection in guinea pigs immunized with pP12A3C/IFN-CS/PLGA-NPs + CpG-CS/PLGA-NPs and pP12A3C/IFN-CS/PLGA-NPs. However, plasmid DNA was burst released from pP12A3C/IFN-Am/MS-NPs. Our attempts to use pP12A3C/IFN-Am/MS-NPs to immunize guinea pigs failed to induce immune responses. In conclusion, CpG and IFN-α adjuvant based FMD vaccines elicit protection in guinea pigs. Moreover, CS-coated PLGA NPs present an efficient and safe mucosal immune delivery system for FMDV DNA vaccine. Data from the current study provide a foundation for understanding and further evaluating protective immune responses in pigs.

The immune response to a recombinant Lactococcus lactis oral vaccine against foot-and-mouth disease virus in mice.

OBJECTIVE: Development of an effective mucosal vaccine to induce specific immune responses against Foot-and-mouth disease virus (FMDV). RESULTS: For this purpose, the FMDV VP1 gene (SPVP1) was optimized and synthesized based on the codon bias of Lactococcus lactis (L. lactis), and then incorporated in the plasmid pNZ8148. L. lactis NZ9000 containing the pNZ8148-SPVP1 recombinant plasmid was used as an oral delivery vehicle to induce anti-FMDV mucosal and systemic immune responses in mice. After confirmation that the SPVP1 protein was expressed successfully in the recombinant L. latic, the mice were orally challenged with NZ9000-pNZ8148, NZ9000-pNZ8148-SPVP1, phosphate-buffered saline as a mock infection group, or with inactivated vaccine as a positive group. Mice immunized with NZ9000-pNZ8148-SPVP1 produced high levels of mucosal secretory IgA (sIgA), antigen-specific serum IgG, IgA, and neutralizing antibodies, and developed stronger cell-mediated immune reactions and significant T spleen lymphocyte proliferation. Furthermore, the recombinant group generated much higher levels of IFN-γ, IL-2, IL-4, IL-5, and IL-10 than the other groups. CONCLUSIONS: Potent immune responses were successfully elicited in mice with FMDV VP1 delivered through L. lactis.

Nanoliposomes as Vehicles for Astaxanthin: Characterization, In Vitro Release Evaluation and Structure.

Astaxanthin was encapsulated in nanoliposomes by a film dispersion-ultrasonic technique using soybean phosphatidyl choline. The astaxanthin-loaded nanoliposomes displayed advantages in the aspects of high encapsulation efficiency and less particle size with a remarkably homodisperse size distribution. Based on X-ray diffraction and differential scanning calorimetry the analysis, it has been demonstrated that there could be interactions of astaxanthin with the lipid bilayer, resulting in the forming of astaxanthin-loaded nanoliposomes. The thermal gravimetric analysis revealed that the thermal stability of astaxanthin after encapsulation in nanoliposomes was remarkably enhanced as compared to astaxanthin alone. Furthermore, encapsulation could greatly enhance the water dispersibility of astaxanthin. This study also confirmed that encapsulation of astaxanthin in nanoliposomes could be an effective way to supply astaxanthin continuously in the body. The effects of astaxanthin incorporation on structural changes of the liposomal membrane were investigated through steady-state fluorescence measurements. This study revealed that the incorporation of astaxanthin into the lipid bilayer decreased membrane fluidity, but increased micropolarity in the membrane within a certain range of astaxanthin concentrations. Additionally, it indicated that the encapsulation of astaxanthin in the lipid bilayer could be applied to modulate the structural properties of membranes.

Estimation of the daily soil/dust (SD) ingestion rate of children from Gansu Province, China via hand-to-mouth contact using tracer elements.

A total of 60 children (31 males and 29 females) between the ages of 3 and 12 years were randomly selected from Lanzhou City in Gansu Province, northwest China. Hand (soil/dust) SD samples from these children were collected using hand wipes. We determined the approximate amounts of hand SD and the concentrations of three tracer soil elements (Ce, Y, and V) in these samples. The approximate amounts of hand SD ranged from 42.28 to 173.76 mg, with a median value of 85.42 mg. In addition, the mean amounts of hand SD estimated using the concentrations of Ce, Y, and V in the samples were 4.63, 3.43, and 3.42 mg, respectively. The amount of hand SD varied greatly among the age groups: primary school children had more hand SD than kindergarten children, males had more hand SD than females, and children from rural areas had more hand SD than those from urban areas. The rates of daily ingestion of hand SD for kindergarten and primary school children were estimated to be 7.73 and 6.61 mg/day, respectively.

[Povidone-iodine versus the benzethonium chloride wipe for penile skin disinfection before semen collection from sperm donors].

Objective: To study the influence of povidone-iodine (PI) versus that of the benzethonium chloride wipe (BCW) on semen collection and semen quality of sperm donors undergoing penile skin disinfection and provide some evidence for the selection of disinfection methods for semen collection. METHODS: We used PI from August to December 2015 and BCWs from January to July 2016 for penile skin disinfection before semen collection, with two samples from each donor, one collected with and the other without penis skin disinfection (the blank control group). After semen collection, we conducted a questionnaire investigation on the influence of the two disinfection methods on semen collection and compared the semen parameters between the two groups of sperm donors. RESULTS: Totally, 185 sperm donors were included in this study, of whom 63 underwent penile skin disinfection with PI and the other 122 with BCWs before semen collection. Statistically significant differences were found between the PI and BCW groups in the adaptability to the disinfectant and rigid disinfection procedures (P <0.05), but not in the other items of the questionnaire (P >0.05). Compared with the sperm donors of the blank control group, those of the PI group showed statistically significant difference in the percentage of progressively motile sperm (PMS) (ï¼»63.02 ± 3.18ï¼½% vs ï¼»61.45 ± 4.78ï¼½%, P<0.05), but not in the abstinence time (ï¼»4.97 ± 1.79ï¼½ vs ï¼»4.7 ± 0.94ï¼½ d, P >0.05), semen volume (ï¼»4.11 ± 1.54ï¼½ vs ï¼»4.15 ± 1.61ï¼½ ml, P >0.05), sperm concentration (ï¼»110 ± 29.6ï¼½ vs ï¼»107.5 ± 31.79ï¼½ ×106/ml, P >0.05), or total sperm count (ï¼»439.10 ± 170.13ï¼½ vs ï¼»434.02 ± 186.91ï¼½ ×106/ejaculate, P >0.05), while those of the BCW group exhibited no remarkable difference in any of the above parameters (P >0.05). Among the samples with abnormal semen quality, significantly fewer were found with abnormal PMS in the BCW than in the PI group (1.64% ï¼»2/122ï¼½ vs 9.68% ï¼»6/62ï¼½, P <0.05). However, there were no significant differences between the PI and BCW groups in the abnormal semen volume, abnormal sperm concentration, or the rate of semen bacterial contamination (P >0.05). CONCLUSIONS: Before semen collection from donors, penile skin disinfection with povidone-iodine may affect both the semen collection process and the quality of donor sperm, while the benzethonium chloride wipe can reduce the influence on the semen collection process and does not affect the semen parameters.

eEF1G interaction with foot-and-mouth disease virus nonstructural protein 2B: Identification by yeast two-hybrid system.

Foot-and-mouth disease virus (FMDV) is a picornavirus that causes an economically significant disease in cattle and swine. Replication of FMDV is dependent on both viral proteins and cellular factors. Nonstructural protein 2B of FMDV plays multiple roles during viral infection and replication. We investigated the roles of 2B in virus-host interactions by constructing a cDNA library obtained from FMDV-infected swine tissues, and used a split-ubiquitin-based yeast two-hybrid system to identify host proteins that interacted with 2B. We found that 2B interacted with amino acids 208-437 in the C-terminal region of the eEF1G subunit of eukaryotic elongation factor 1, which is essential for protein synthesis. The 2B-eEF1G interaction was confirmed by co-immunoprecipitation of 2B and eEF1G in HEK293T cells. Collectively, our results suggest that eEF1G interacts with the 2B protein of FMDV. The identified 2B interaction partner may help to elucidate the mechanisms of FMDV infection and replication.

Chimeric virus-like particles elicit protective immunity against serotype O foot-and-mouth disease virus in guinea pigs.

Foot-and-mouth disease (FMD) is an acute and highly contagious disease caused by foot-and-mouth disease virus (FMDV) that can affect cloven-hoofed animal species, leading to severe economic losses worldwide. Therefore, the development of a safe and effective new vaccine to prevent and control FMD is both urgent and necessary. In this study, we developed a chimeric virus-like particle (VLP) vaccine candidate for serotype O FMDV and evaluated its protective immunity in guinea pigs. Chimeric VLPs were formed by the antigenic structural protein VP1 from serotype O and segments of the viral capsid proteins (VP2, VP3, and VP4) from serotype A. The chimeric VLPs elicited significant humoral and cellular immune responses with a higher level of anti-FMDV antibodies and cytokines than the control group. Furthermore, four of the five guinea pigs vaccinated with the chimeric VLPs were completely protected against challenge with 100 50% guinea pig infectious doses (GPID50) of the virulent FMDV strain O/MAY98. These data suggest that chimeric VLPs are potential candidates for the development of new vaccines against FMDV.

Efficacy of synthetic peptide candidate vaccines against serotype-A foot-and-mouth disease virus in cattle.

Foot-and-mouth disease (FMD) remains a major threat to livestock worldwide, especially in developing countries. To improve the efficacy of vaccination against FMD, various types of vaccines have been developed, including synthetic peptide vaccines. We designed three synthetic peptide vaccines, 59 to 87 aa in size, based on immunogenic epitopes in the VP1, 3A, and 3D proteins of the A/HuBWH/CHA/2009 strain of the foot-and-mouth disease virus (FMDV), corresponding to amino acid positions 129 to 169 of VP1, 21 to 35 of 3A, and 346 to 370 of 3D. The efficacies of the vaccines were evaluated in cattle and guinea pigs challenged with serotype-A FMDV. All of the vaccines elicited the production of virus-neutralizing antibodies. The PB peptide, which contained sequences corresponding to positions 129 to 169 of V P1 and 346 to 370 of 3D, demonstrated the highest levels of immunogenicity and immunoprotection against FMDV. Two doses of 50 µg of the synthetic PB peptide vaccine provided 100% protection against FMDV infection in guinea pigs, and a single dose of 100 µg provided 60% protection in cattle. These findings provide empirical data for facilitating the development of synthetic peptide vaccines against FMD.

Evolutionary analysis of structural protein gene VP1 of foot-and-mouth disease virus serotype Asia 1.

Foot-and-mouth disease virus (FMDV) serotype Asia 1 was mostly endemic in Asia and then was responsible for economically important viral disease of cloven-hoofed animals, but the study on its selection and evolutionary process is comparatively rare. In this study, we characterized 377 isolates from Asia collected up until 2012, including four vaccine strains. Maximum likelihood analysis suggested that the strains circulating in Asia were classified into 8 different groups (groups I-VIII) or were unclassified (viruses collected before 2000). On the basis of divergence time analyses, we infer that the TMRCA of Asia 1 virus existed approximately 86.29 years ago. The result suggested that the virus had a high mutation rate (5.745 × 10(-3) substitutions/site/year) in comparison to the other serotypes of FMDV VP1 gene. Furthermore, the structural protein VP1 was under lower selection pressure and the positive selection occurred at many sites, and four codons (positions 141, 146, 151, and 169) were located in known critical antigenic residues. The remaining sites were not located in known functional regions and were moderately conserved, and the reason for supporting all sites under positive selection remains to be elucidated because the power of these analyses was largely unknown.

Origin of product selectivity in a prenyl transfer reaction from the same intermediate: exploration of multiple FtmPT1-catalyzed prenyl transfer pathways.

FtmPT1 is a fungal indole prenyltransferase that catalyzes the reaction of tryptophan derivatives with dimethylallyl pyrophosphate to form various biologically active compounds. Herein, we describe detailed studies of FtmPT1 catalysis involving dimethylallyl pyrophosphate and Brevianamide F following the native pathway (yielding Tryprostatin B) and an alternate pathway observed in the Gly115Thr mutant of FtmPT1 yielding a novel cyclized product. Importantly, these two products arise from the same intermediate state, meaning that a step other than the cleavage of the dimethylallyl pyrophosphate (DMAPP; C-O) bond is differentiating between the two product reaction channels. From detailed potential of mean force (PMF) and two-dimensional PMF analyses, we conclude that the rate-limiting step is the cleavage of the C-O bond in DMAPP, while the deprotonation/cyclization step determines the final product distribution. Hence, in the case of FtmPT1, the optimization of the necessary catalytic machinery guides the generation of the final product after formation of the intermediate carbocation.

Cellular microRNAs and picornaviral infections.

microRNAs (miRNAs) are a subtype of short, endogenous, and non-coding RNAs, which post-transcriptionally regulate gene expression. The miRNA-mediated gene silencing mechanism is involved in a wide spectrum of biological processes, such as cellular proliferation, differentiation, and immune responses. Picornaviridae is a large family of RNA viruses, which includes a number of causative agents of many human and animal diseases viz., poliovirus, foot-and-mouth disease virus (FMDV), and coxsackievirus B3 (CVB3). Accumulated evidences have demonstrated that replication of picornaviruses can be regulated by miRNAs and picornaviral infections can alter the expression of cellular miRNAs. Herein, we outline the intricate interactions between miRNAs and picornaviral infections.

Angiogenic sprouting into neural tissue requires Gpr124, an orphan G protein-coupled receptor.

The vasculature of the CNS is structurally and functionally distinct from that of other organ systems and is particularly prone to developmental abnormalities and hemorrhage. Although other embryonic tissues undergo primary vascularization, the developing nervous system is unique in that it is secondarily vascularized by sprouting angiogenesis from a surrounding perineural plexus. This sprouting angiogenesis requires the TGF-ß and Wnt pathways because ablation of these pathways results in aberrant sprouting and hemorrhage. We have genetically deleted Gpr124, a member of the large family of long N-terminal group B G protein-coupled receptors, few members of which have identified ligands or well-defined biologic functions in mammals. We show that, in the developing CNS, Gpr124 is specifically expressed in the vasculature and is absolutely required for proper angiogenic sprouting into the developing neural tube. Embryos lacking Gpr124 exhibit vascular defects characterized by delayed vascular penetration, formation of pathological glomeruloid tufts within the CNS, and hemorrhage. In addition, they display defects in palate and lung development, two processes in which TGF-ß and/or Wnt pathways also play important roles. We also show that TGF-ß stimulates Gpr124 expression, and ablation of Gpr124 results in perturbed TGF-ß pathway activation, suggesting roles for Gpr124 in modulating TGF-ß signaling. These results represent a unique function attributed to a long N-terminal group B-type G protein-coupled receptor in a mammalian system.

Salivary Testosterone Levels and Pain Perception Exhibit Sex-Specific Association in Healthy Adults But Not in Patients With Migraine.

This study investigated the sex-specific associations between pain perception and testosterone levels in healthy controls (HCs) and patients with migraine. Male and female HCs and migraine patients were recruited. A series of questionnaires were completed by the participants to evaluate their psychosocial profiles, which included data on mood, stress, and sleep quality. Heat pain thresholds and suprathreshold pain ratings at 45 °C (referred to as the pain perception score [PPS]) were assessed using the Thermode system. Salivary testosterone levels were analyzed using a commercial enzyme-linked immunosorbent assay kit. A total of 88 HCs (men/women: 41/47, age: 29.9 ± 7.7 years) and 75 migraine patients (men/women: 30/45, age: 31.1 ± 7.7 years) completed all assessments. No significant differences were observed in either the psychosocial profiles or heat pain thresholds and PPSs between the sexes in the control and migraine groups. A positive correlation between testosterone levels and PPSs was identified in the male controls (r = .341, P = .029), whereas a negative correlation was identified in the female controls (r = -.407, P = .005). No such correlations were identified in the migraine group. This study confirms that a negative association is present between PPSs and testosterone levels in female controls, which is in line with the findings that testosterone is associated with reduced pain perception. Our study is the first to demonstrate a sex-specific association between PPSs and testosterone levels in HCs. Moreover, this study also revealed that the presence of migraine appears to disrupt this association. PERSPECTIVE: This study revealed that testosterone levels demonstrate opposite associations with pain perception in healthy men and women. However, the presence of migraine appears to disrupt this sex-specific association.