Crystal structure of tick tyrosylprotein sulfotransferase reveals the activation mechanism of the tick anticoagulant protein madanin.

Ticks pose a substantial public health risk as they transmit various pathogens. This concern is related to the adept blood-sucking strategy of ticks, underscored by the action of the anticoagulant, madanin, which is known to exhibit an approximately 1000-fold increase in anticoagulant activity following sulfation of its two tyrosine residues, Tyr51 and Tyr54. Despite this knowledge, the molecular mechanism underlying sulfation by tick tyrosylprotein sulfotransferase (TPST) remains unclear. In this study, we successfully prepared tick TPST as a soluble recombinant enzyme. We clarified the method by which this enzyme proficiently sulfates tyrosine residues in madanin. Biochemical analysis using a substrate peptide based on madanin and tick TPST, along with the analysis of the crystal structure of the complex and docking simulations, revealed a sequential sulfation process. Initial sulfation at the Tyr51 site augments binding, thereby facilitating efficient sulfation at Tyr54. Beyond direct biochemical implications, these findings considerably improve our understanding of tick blood-sucking strategies. Furthermore, combined with the utility of modified tick TPST, our findings may lead to the development of novel anticoagulants, promising avenues for thrombotic disease intervention and advancements in the field of public health.

Crystal structure of Arabidopsis thaliana sulfotransferase SOT16 involved in glucosinolate biosynthesis.

Glucosinolates (GSLs), a class of secondary metabolites found in Brassicaceae plants, play important roles in plant defense and contribute distinct flavors and aromas when used as food ingredients. Following tissue damage, GSLs undergo enzymatic hydrolysis to release bioactive volatile compounds. Understanding GSL biosynthesis and enzyme involvement is crucial for improving crop quality and advancing agriculture. Plant sulfotransferases (SOTs) play a key role in the final step of GSL biosynthesis by transferring sulfate groups to the precursor molecules. In the present study, we investigated the enzymatic reaction mechanism and broad substrate specificity of Arabidopsis thaliana sulfotransferase AtSOT16, which is involved in GSL biosynthesis, using crystal structure analysis. Our analysis revealed the specific catalytic residues involved in the sulfate transfer reaction and supported the hypothesis of a concerted acid-base catalytic mechanism. Furthermore, the docking models showed a strong correlation between the substrates with high predicted binding affinities and those experimentally reported to exhibit high activity. These findings provide valuable insights into the enzymatic reaction mechanisms and substrate specificity of GSL biosynthesis. The information obtained in this study may contribute to the development of novel strategies for manipulating GSL synthesis pathways in Brassica plants and has potential agricultural applications.

Root-knot nematode chemotaxis is positively regulated by l-galactose sidechains of mucilage carbohydrate rhamnogalacturonan-I.

Root-knot nematodes (RKNs) are plant parasites and major agricultural pests. RKNs are thought to locate hosts through chemotaxis by sensing host-secreted chemoattractants; however, the structures and properties of these attractants are not well understood. Here, we describe a previously unknown RKN attractant from flaxseed mucilage that enhances infection of Arabidopsis and tomato, which resembles the pectic polysaccharide rhamnogalacturonan-I (RG-I). Fucose and galactose sidechains of the purified attractant were found to be required for attractant activity. Furthermore, the disaccharide α-l-galactosyl-1,3-l-rhamnose, which forms the linkage between the RG-I backbone and galactose sidechains of the purified attractant, was sufficient to attract RKN. These results show that the α-l-galactosyl-1,3-l-rhamnose linkage in the purified attractant from flaxseed mucilage is essential for RKN attraction. The present work also suggests that nematodes can detect environmental chemicals with high specificity, such as the presence of chiral centers and hydroxyl groups.

Low Seroprevalence of Severe Fever with Thrombocytopenia Syndrome Virus Antibodies in Individuals Living in an Endemic Area in Japan.

Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne infection with a high mortality rate. It is caused by the SFTS virus (SFTSV) and is endemic in some areas in western Japan, including the Kagoshima prefecture. In the present study, healthy individuals living in this prefecture were examined to assess for anti-SFTSV seroprevalence. An initial study was performed using the serum samples collected from a total of 646 individuals living in Kagoshima. At the same time, a questionnaire was used to collect information (such as occupation and a history of tick bite). Enzyme-linked immunosorbent assay and indirect immunofluorescence assay were used for the screening. Finally, the seroprevalence of anti-SFTSV antibodies was confirmed using a neutralization assay. Only 2 (0.3%) out of 646 study participants were positive for anti-SFTSV antibodies. No significant difference was observed between individuals who are at a high or low risk of tick bite in terms of seropositivity. Next, a total of 1,000 serum samples collected from general blood donors by the Japanese Red Cross Kyushu Block Blood Center were tested. None of these samples tested positive for anti-SFTSV antibodies. These results suggest a low seroprevalence of anti-SFTSV antibodies in healthy individuals living in an endemic area in Japan.

Unique transcriptional profile of native persisters in Escherichia coli.

Non-dividing persisters, bacteria that can survive in the presence of antibiotics by pausing their metabolic activity, are among the many causes of the refractory nature of bacterial infections. Here we constructed a recombinant Escherichia coli strain that enables to distinguish non-dividing from dividing cell based on Z-ring during cell division. Then, non-dividing cells and dividing cells were successfully separated using a fluorescence activated cell sorter. The sorted non-dividing cells showed significantly higher tolerance toward ofloxacin than dividing cells, which indicates that persisters were concentrated with the methodology. Transcriptional analysis revealed that genes involved in guanosine tetraphosphate synthesis are upregulated in persisters, which represses transcription and DNA replication and leads to ofloxacin tolerance. Lactate dehydrogenase and several ATP-binding cassette transporters were upregulated in persisters to adapt to anaerobic metabolism. In addition, nitrite and dimethyl sulfoxide (DMSO) may be used as reducible substrates for alternative energy generation pathways. Our methodology revealed a unique transcriptional profile of E. coli persisters.

Structure-activity relationship studies and discovery of a potent transient receptor potential vanilloid (TRPV1) antagonist 4-[3-chloro-5-[(1S)-1,2-dihydroxyethyl]-2-pyridyl]-N-[5-(trifluoromethyl)-2-pyridyl]-3,6-dihydro-2H-pyridine-1-carboxamide (V116517) as a clinical candidate for pain management.

A series of novel tetrahydropyridinecarboxamide TRPV1 antagonists were prepared and evaluated in an effort to optimize properties of previously described lead compounds from piperazinecarboxamide series. The compounds were evaluated for their ability to block capsaicin and acid-induced calcium influx in CHO cells expressing human TRPV1. The most potent of these TRPV1 antagonists were further characterized in pharmacokinetic, efficacy, and body temperature studies. On the basis of its pharmacokinetic, in vivo efficacy, safety, and toxicological properties, compound 37 was selected for further evaluation in human clinical trials.

Imaging neural circuit dynamics with a voltage-sensitive fluorescent protein.

Population signals from neuronal ensembles in cortex during behavior are commonly measured with EEG, local field potential (LFP), and voltage-sensitive dyes. A genetically encoded voltage indicator would be useful for detection of such signals in specific cell types. Here we describe how this goal can be achieved with Butterfly, a voltage-sensitive fluorescent protein (VSFP) with a subthreshold detection range and enhancements designed for voltage imaging from single neurons to brain in vivo. VSFP-Butterfly showed reliable membrane targeting, maximum response gain around standard neuronal resting membrane potential, fast kinetics for single-cell synaptic responses, and a high signal-to-noise ratio. Butterfly reports excitatory postsynaptic potentials (EPSPs) in cortical neurons, whisker-evoked responses in barrel cortex, 25-Hz gamma oscillations in hippocampal slices, and 2- to 12-Hz slow waves during brain state modulation in vivo. Our findings demonstrate that cell class-specific voltage imaging is practical with VSFP-Butterfly, and expand the genetic toolbox for the detection of neuronal population dynamics.

Distinct relations among plasma concentrations required for different pharmacological effects in oxycodone, morphine, and fentanyl.

Several clinical reports showed that adverse effect profiles are not the same in morphine, oxycodone, and fentanyl. The authors investigated whether the relationship between plasma concentrations for antinociceptive effect and for various pharmacological effects differed among oxycodone, morphine, and fentanyl under controlled experimental setting using animal models. Oxycodone induced constipation and an antinociceptive effect in a similar concentration-dependent manner, whereas morphine required approximately 9-fold higher plasma concentration for antinociceptive effect compared with that for constipation when 50% effective plasma concentration (EC(50)) levels were compared. The EC(50) values for inhibition of behavioral activity were 2.1-, 2.7-, and 1.3-fold higher than those for antinociceptive effect in oxycodone, morphine, and fentanyl, respectively. Respiratory inhibition was observed even at higher plasma concentrations in all three opioids, and the differences in the EC(50) values compared with those for antinociceptive effects were 234.5-fold (oxycodone), 233.1-fold (morphine), or 104.2-fold (fentanyl). These results showed that oxycodone, morphine, and fentanyl exhibited unique patterns of plasma concentrations required for different pharmacological effects. The different adverse effect profiles observed in a clinical setting appear to be resulted from, at least in part, distinct intrinsic pharmacological profiles among these µ-opioid receptor agonists.

Optogenetic monitoring of membrane potentials.

Over the last decade, researchers in our laboratory have engineered and developed several series of genetically encoded voltage-sensitive fluorescent proteins (VSFPs) by molecular fusion of a voltage-sensing domain operand with different fluorescent reporter proteins. These genetically encoded VSFPs have been shown to provide a reliable optical report of membrane potential from targeted neurons and muscle cells in culture or in living animals. However, these various reporters also exhibit discrepancies in both their voltage-sensing and targeting properties that are essentially related to the intrinsic characteristics of the fluorescent reporter proteins. It is therefore important carefully to select the sensor that is most appropriate for the particular question being investigated experimentally. Here we examine the current state of this subfield of optogenetics, address current limitations and challenges, and discuss what is likely to be feasible in the near future.

Second and third generation voltage-sensitive fluorescent proteins for monitoring membrane potential.

Over the last decade, optical neuroimaging methods have been enriched by engineered biosensors derived from fluorescent protein (FP) reporters fused to protein detectors that convert physiological signals into changes of intrinsic FP fluorescence. These FP-based indicators are genetically encoded, and hence targetable to specific cell populations within networks of heterologous cell types. Among this class of biosensors, the development of optical probes for membrane potential is both highly desirable and challenging. A suitable FP voltage sensor would indeed be a valuable tool for monitoring the activity of thousands of individual neurons simultaneously in a non-invasive manner. Previous prototypic genetically-encoded FP voltage indicators achieved a proof of principle but also highlighted several difficulties such as poor cell surface targeting and slow kinetics. Recently, we developed a new series of FRET-based Voltage-Sensitive Fluorescent Proteins (VSFPs), referred to as VSFP2s, with efficient targeting to the plasma membrane and high responsiveness to membrane potential signaling in excitable cells. In addition to these FRET-based voltage sensors, we also generated a third series of probes consisting of single FPs with response kinetics suitable for the optical imaging of fast neuronal signals. These newly available genetically-encoded reporters for membrane potential will be instrumental for future experimental approaches directed toward the understanding of neuronal network dynamics and information processing in the brain. Here, we review the development and current status of these novel fluorescent probes.

Spectrally-resolved response properties of the three most advanced FRET based fluorescent protein voltage probes.

Genetically-encoded optical probes for membrane potential hold the promise of monitoring electrical signaling of electrically active cells such as specific neuronal populations in intact brain tissue. The most advanced class of these probes was generated by molecular fusion of the voltage sensing domain (VSD) of Ci-VSP with a fluorescent protein (FP) pair. We quantitatively compared the three most advanced versions of these probes (two previously reported and one new variant), each involving a spectrally distinct tandem of FPs. Despite these different FP tandems and dissimilarities within the amino acid sequence linking the VSD to the FPs, the amplitude and kinetics of voltage dependent fluorescence changes were surprisingly similar. However, each of these fluorescent probes has specific merits when considering different potential applications.

2-Arylimino-5,6-dihydro-4H-1,3-thiazines as a new class of cannabinoid receptor agonists. Part 3: Synthesis and activity of isosteric analogs.

Structure-activity relationships and efforts to optimize the pharmacokinetic profile of isosteric analogs of 2-arylimino-5,6-dihydro-4H-1,3-thiazines as cannabinoid receptor agonists are described. Among those examined, compound 25 showed potent affinity for cannabinoid receptor 1 (CB1) and receptor 2 (CB2). This compound displayed oral bioavailability and analgesic activity.

2-Arylimino-5,6-dihydro-4H-1,3-thiazines as a new class of cannabinoid receptor agonists. Part 2: orally bioavailable compounds.

Structure-activity relationships and efforts to optimize the pharmacokinetic profile of a class of 2-arylimino-5,6-dihydro-4H-1,3-thiazines as cannabinoid receptor agonists are described. Among the compounds examined, compound 14 showed potent affinity and high selectivity for CB2, and compound 23 showed potent affinities against CB1 and CB2. These compounds displayed oral bioavailability.

Molecular cloning of rat Spetex2 family genes mapped on chromosome 15p16, encoding a 23-kilodalton protein associated with the plasma membranes of haploid spermatids.

We used differential display in combination with cDNA cloning to isolate a novel rat gene, designated as Spetex2, that has an open reading frame of 582 nucleotides, encoding a protein of 194 amino acids. Spetex2 mRNA was highly expressed in testis and spleen, and its expression in rat testis was developmentally up-regulated. In situ hybridization revealed that Spetex2 mRNA was predominantly expressed in haploid spermatids at steps 1-13 within the seminiferous epithelium. A BLAST search against rat genome databases at the National Center for Biotechnology Information revealed that the Spetex2 gene is composed of four exons and is mapped to at least 18 loci in a cluster on rat chromosome 15p16, indicating that the genes occur as a repeated tandem array over a long stretch of genomic DNA. By immunocytochemical analysis with confocal laser-scanning microscopy, SPETEX2 protein was detected as a dot-like distribution on the cell periphery of haploid spermatids (steps 1-13) but was not observed in other spermatogenic cells. On the basis of these data, we hypothesize that SPETEX2 might be correlated with cell differentiation of spermaytids in rat testis.

Complete absence of Cockayne syndrome group B gene product gives rise to UV-sensitive syndrome but not Cockayne syndrome.

UV-sensitive syndrome (UVsS) is a rare autosomal recessive disorder characterized by photosensitivity and mild freckling but without neurological abnormalities or skin tumors. UVsS cells show UV hypersensitivity and defective transcription-coupled DNA repair of UV damage. It was suggested that UVsS does not belong to any complementation groups of known photosensitive disorders such as xeroderma pigmentosum and Cockayne syndrome (CS). To identify the gene responsible for UVsS, we performed a microcell-mediated chromosome transfer based on the functional complementation of UV hypersensitivity. We found that one of the UVsS cell lines, UVs1KO, acquired UV resistance when human chromosome 10 was transferred. Because the gene responsible for CS group B (CSB), which involves neurological abnormalities and photosensitivity as well as a defect in transcription-coupled DNA repair of UV damage, is located on chromosome 10, we sequenced the CSB gene from UVs1KO and detected a homozygous null mutation. Our results indicate that previous complementation analysis of UVs1KO was erroneous. This finding was surprising because a null mutation of the CSB gene would be expected to result in CS features such as severe developmental and neurological abnormalities. On the other hand, no mutation in the CSB cDNA and a normal amount of CSB protein was detected in Kps3, a UVsS cell line obtained from an unrelated patient, indicating genetic heterogeneity in UVsS. Possible explanations for the discrepancy in the genotype-phenotype relationship in UVs1KO are presented.