S1 Region Research Articles

Effects of Mavacamten and Blebbistatin on the Small-Angle X-ray Scattering Structure of Human β-cardiac Myosin

Cardiac myosin is a motor protein that converts chemical energy from ATP hydrolysis to mechanical work that keeps the heart beating. Full-length myosin and even the smaller sub-fragment, heavy meromyosin (HMM) are large and flexible molecules and have not been possible to crystallize and therefore has been unavailable for structural studies. These properties make myosin a good candidate for small-angle X-ray scattering (SAXS) studies. Here we use size exclusion chromatography coupled with SAXS to study structural changes of human β-cardiac 2-hep and 25-hep HMM constructs in relaxing conditions treated with two different myosin inhibitors: blebbistatin and mavacamten. Under resting conditions, the 25-hep HMM had a radius of gyration (Rg) of ∼ 118 Å (Guinier fit) to ∼ 137 Å (GNOM fit) and the maximum dimension of the molecule (Dmax) of 526 Å. The Rg of 25-hep HMM decreased by about 5-10% with either blebbistatin or mavacamten treatment. The Dmax of 25-hep HMM decreased ∼ 20 Å by blebbistatin treatment while it decreased ∼ 80 Å by mavacamten treatment. There were only moderate structural changes (< 2%) for the 2-hep HMM with either blebbistatin or mavacamten treatment. Our results indicate that most of the HMM molecules in relaxing conditions maintain an open configuration and that the myosin S2 region is essential for HMM to adopt the folded back closed configuration in presence of mavacamten and blebbistatin. Supported, in part, by NIH P41 GM103622.

Chemical Structure and Mechanical Properties of Wood Cell Walls Treated with Acid and Alkali Solution

The mechanical properties of individual fibers are related to the production and performance of papers and fiberboards. This paper examines the behavior of the microstructure constituents of wood subjected to acid and alkali treatments. The chemical structure and mechanical properties of wood cell walls with different acid or alkali treatments was analyzed. The results show that, compared with acid treatment, the crystal size and crystallinity index of cellulose increased after alkali treatment, resulting in an increase in the cell wall elastic modulus. The mechanical properties of the wood cell wall S2 region were higher than those of the compound middle lamella (CML) region. There was a topochemical effect between the CML and the S2 region in acid and alkali-treated samples, which provided a major threshold that facilitates the production/separation of wood fibers for better strength fiber properties.

Comparison of fiber tract low frequency stimulation to focal and ANT stimulation in an acute rat model of focal cortical seizures

BackgroundCurrent implementations of direct brain stimulation for epilepsy in patients involve high-frequency (HFS) electrical current and targeting of grey matter. Studies have shown that low-frequency (LFS) fiber-tract stimulation may also prove effective. To compare the efficacy of high-frequency grey matter stimulation to the low-frequency fiber tract stimulation technique a well-controlled set of experiments using a single animal model of epilepsy is needed. ObjectiveThe goal of this study was to determine the relative efficacy of different direct brain stimulation techniques for suppressing seizures using an acute rat model of focal cortical seizures. Methods4-AP was injected into the S1 region of cortex in rodents over 3 h. LFPs were recorded from the seizure focus and mirror focus to monitor seizure frequency during the experiments. CC-LFS, HFS-ANT, Focal-HFS, or a transection of the CC was applied. ResultsStimulation of the CC yielded a 65% ±14% (p = 0.0014) reduction of seizures in the focus and a 97% ±15% (p = 0.0026) reduction in the mirror focus (n = 7). By comparison transection of the CC produced a 65% ±18% reduction in the focus and a non-statistically significant reduction of 57% ±18% (p = 0.1381) in the mirror focus (n = 5). All other methods of stimulation failed to have a statistically significant effect on seizure suppression. ConclusionsLFS of the CC is the only method of stimulation to significantly reduce seizure frequency in this model of focal cortical seizures. These results support the hypothesis that LFSof fiber tracts has significant potential for seizure control.

Middle East Respiratory Syndrome Coronavirus in Dromedaries in Ethiopia Is Antigenically Different From the Middle East Isolate EMC.

Middle East respiratory syndrome (MERS) is an emerging respiratory disease caused by the MERS coronavirus (MERS-CoV). MERS has been endemic to Saudi Arabia since 2012. The reservoir of MERS-CoV is the dromedary camel, suggesting that MERS is primarily a zoonotic disease. MERS-CoV is common in dromedaries throughout the Middle East, North Africa, and East Africa as evidenced by neutralizing antibodies against MERS-CoV; however, human cases have remained limited to the Middle East. To better understand the cause of this difference, the virological properties of African camel MERS-CoV were analyzed based on the spike (S) protein in Ethiopia. Nasal swabs were collected from 258 young dromedaries (≤ 2 years old) in the Afar region of Ethiopia, of which 39 were positive for MERS-CoV, as confirmed by genetic tests. All positive tests were exclusive to the Amibara woreda region. Using next-generation sequencing, two full-length genomes of Amibara isolates were successfully decoded; both isolates belonged to the C2 clade based on phylogenetic analysis of full-length and S protein sequences. Recombinant EMC isolates of MERS-CoV, in which the S protein is replaced with those of Amibara isolates, were then generated to test the roles of these proteins in viral properties. Amibara S recombinants replicated more slowly in cultured cells than in EMC S recombinants. In neutralizing assays, Amibara S recombinants were neutralized by lower concentrations of sera from both Ethiopian dromedaries and EMC isolate (wild-type)-immunized mouse sera, relative to the EMC S recombinants, indicating that viruses coated in the Amibara S protein were easier to neutralize than the EMC S protein. Neutralization experiments performed using S1/S2 chimeric recombinants of the EMC and Amibara S proteins showed that the neutralization profile was dependent on the S1 region of the S protein. These results suggest that the slower viral replication and the ease of neutralization seen in the Ethiopian MERS-CoV are due to strain-specific differences in the S protein and may account for the absence of human MERS-CoV cases in Ethiopia.

Construction of a Recombinant Lactococcus lactis Strain Expressing a Variant Porcine Epidemic Diarrhea Virus S1 Gene and Its Immunogenicity Analysis in Mice.

Porcine epidemic diarrhea caused by porcine epidemic diarrhea virus (PEDV) is a highly contagious disease in newborn piglets. The spike (S) protein is the surface glycoprotein of PEDV, which can induce specific neutralization antibodies and is a candidate antigen for vaccination attempts. In our study, the S1 region of PEDV strain CH/JLDH/2016 spike gene was inserted into the Lactococcus lactis expression vector, pNZ8149, resulting in recombinant plasmid pNZ8149-S1, and the immunogenicity of recombinant L. lactis pNZ8149-S1/NZ3900 was evaluated in mice. After immunization, significantly higher levels of anti-PEDV serum IgG antibodies and mucosal sIgA antibodies were detected in mice orally administered with pNZ8149-S1/NZ3900, compared with control groups pNZ8149/NZ3900, NZ3900, and phosphate buffered saline (p < 0.01). Lymphocyte proliferation assay results showed that the recombinant L. lactis pNZ8149-S1/NZ3900 significantly stimulated the proliferation of splenic lymphocytes (p < 0.01). In addition, the recombinant L. lactis vaccine could induce high levels of IL-4 and IFN-γ in immunized mice (p < 0.01). The results of our study suggest that the recombinant L. lactis pNZ8149-S1/NZ3900 can provide a promising vaccine strategy against PEDV infection.

Effects of thalamic infarction on the structural and functional connectivity of the ipsilesional primary somatosensory cortex.

To identify regions causally influenced by thalamic stroke by measuring white matter integrity, cortical volume, and functional connectivity (FC) among patients with thalamic infarction (TI) and to determine the association between structural/functional alteration and somatosensory dysfunction. Thirty-one cases with TI-induced somatosensory dysfunction and 32 healthy controls underwent magnetic resonance imaging scanning. We reconstructed the ipsilesional central thalamic radiation (CTR) and assessed its integrity using fractional anisotropy (FA), assessed S1 ipsilesional changes with cortical volume, and identified brain regions functionally connected to TI locations and regions without TI to examine the potential effects on somatosensory symptoms. Compared with controls, TI patients showed decreased FA (F = 17.626, p < 0.001) in the ipsilesional CTR. TI patients exhibited significantly decreased cortical volume in the ipsilesional top S1. Both affected CTR (r = 0.460, p = 0.012) and S1 volume (r = 0.375, p = 0.049) were positively correlated with somatosensory impairment in TI patients. In controls, the TI region was highly functionally connected to atrophic top S1 and less connected to the adjacent middle S1 region in FC mapping. However, T1 patients demonstrated significantly increased FC between the ipsilesional thalamus and middle S1 area, which was adjacent to the atrophic S1 region. TI induces remote changes in the S1, and this network of abnormality underlies the cause of the sensory deficits. However, our other finding that there is stronger connectivity in pathways adjacent to the damaged ones is likely responsible for at least some of the recovery of function. • TI led to secondary impairment in the CTR and cortical atrophy in the ipsilesional top of S1. • TI patients exhibited significantly higher functional connectivity with the ipsilateral middle S1 which was mainly located within the non-atrophic area of S1. • Our results provide neuroimaging markers for non-invasive treatment and predict somatosensory recovery.

Drug–DNA interaction, a joint DFT-D3/MD study on safranal as an anticancer and DNA nanostructure model

In this research, using a combination of quantum mechanics and molecular dynamic (MD) simulations, the interaction of safranal (2,6,6-trimethylcyclohexa-1,3-dien-1-carboxaldehyde) as an anti-cancer drug and Dickerson B-DNA was studied. MD simulations were executed for 35 ns in water. Binding energy analysis in three definite parts of the B-DNA and comparison between different contributions of the binding energy shows that the van der Waals energy part of the interaction is impressive among the standard molecular mechanic energy terms. On the basis of Gibbs energies, it is confirmed that the most important interactions in the safranal complex are related to the A–T and C–G rich regions, which is in agreement with the experimental data. Quantum theory of atoms in molecules and natural bond orbital analyses were applied. A diminution in the electronic chemical potential of the safranal–DNA complex in comparison with the isolated DNA, 0.026 and 0.022 au for the S1 region and 0.012 and 0.017 au for the S2 region, was obtained in the gas phase and water, respectively, which increases the complex stability. An enhancement in the electrophilicity character, during the complexation process, shows the electron charge flux between the safranal and DNA, especially in water. The strengths of the CH⋯O bonds at the center of safranal–DNA interaction were also evaluated. A mean value of 0.06 au for the electron density of the bond critical point of the H⋯O in the complex confirms the H-bond formation during the complexation.

Single Chain Fragment Variable (scFv) Antibodies Targeting the Spike Protein of Porcine Epidemic Diarrhea Virus Provide Protection against Viral Infection in Piglets.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that causes severe diarrhea and death in neonatal piglets. Passive immunization with neutralizing antibodies against PEDV is an effective prevention measure. In this study, single chain fragment variable (scFv) antibodies against PEDV were screened from the porcine scFv phage display library. After four rounds of biopanning, scFvs that showed higher affinity to the PEDV antigen were selected for further study. The scFv genes were cloned into the expression plasmid for recombinant protein expression. These scFvs were shown to inhibit PEDV infectivity by the plaque reduction neutralization assay. Immunofluorescence assay (IFA) revealed that the epitopes recognized by these scFvs were in the S1 region of the spike protein. The potential of scFvs to provide prevention against PEDV infections in piglets was further investigated. Piglets orally administered scFvs showed no to mild clinical symptoms, significantly less viral shedding, no mortality and no intestinal lesions. The field application also revealed that the survival rate of piglets was significantly increased by oral administration of scFvs. Our data support the potential role of scFvs in the prevention and treatment of PEDV infection.

A newly isolated Chinese virulent genotype GIIb porcine epidemic diarrhea virus strain: Biological characteristics, pathogenicity and immune protective effects as an inactivated vaccine candidate

Since October 2010, severe porcine epidemic diarrhea (PED) outbreaks caused by highly virulent PED virus (PEDV) strains have occurred continuously in the Chinese pig population and caused considerable economic losses. Although PEDV vaccines based on classical PEDV strains have been widely used in China in recent years, the morbidity and mortality in piglets remain high. Therefore, virulent genotype GII PEDV strains that are prevalent in the field should be isolated and used to develop next-generation vaccines. In the present study, a Chinese virulent genotype GIIb PEDV strain, CH/HNPJ/2017, was serially propagated in Vero cells for up to 90 passages. The S genes contained typical insertions and deletions that were also found in other recently isolated highly virulent PEDV strains from China and other countries and had two neighboring unique insertion mutations, which resulted in four amino acid changes in the S1 region of passages P10 and P60. Pig infection studies revealed that the CH/HNPJ/2017 strain was highly virulent in piglets, and the median pig diarrhea dose (PDD50) was 7.68 log10PDD50/3 mL. Furthermore, the cell-adapted CH/HNPJ/2017 strain elicited potent serum IgG and neutralizing antibody responses in immunized pigs when it was used as an inactivated vaccine candidate. In addition, the pigs that received the experimental inactivated vaccines were partially protected (3/5) against subsequent viral challenge. In brief, these data indicate that the CH/HNPJ/2017 strain is a promising candidate for developing a safe and effective PEDV vaccine in the future.

Graphite particle electrodes that enhance the detoxification of municipal solid waste incineration fly ashes in a three-dimensional electrokinetic platform and its mechanisms

This paper investigated the application of graphite particle electrodes to the removal of Zn, Pb, Cu, and Cd from municipal solid waste incineration (MSWI) fly ashes in a three-dimensional (3D) electrokinetic reactor. The influences of the voltage gradient, mass ratio of graphite powers to fly ashes, nitric acid concentrations, proposing times, and liquid-solid (L-M) ratios on the remedial efficiencies of MSWI fly ashes were comprehensively studied in an orthogonal deign and a sequential double-factor setup. Significant analysis showed that changes in the mass ratios and nitric acid concentrations both had a statistically significant effect on the removals of Zn and Pb. Proposing times and L-M ratios both remarkably affected the removals of heavy metals (HMs) in a 3D electrochemical system. The graphite powers had a narrower distribution interval and slightly larger surface areas compared with MSWI fly ashes, which relented pH gradients over the time in the electrochemical experiments and minimized the bubble barricade caused by the hydrolysis. The particle electrode had increased the residue factions of Zn, Pb, Cu, and Cd in S1 region by approximately 216%, 136%, 309%, and 950%, respectively, compared with the raw MSWI fly ashes. The addition of graphite powders to a two-dimensional (2D) electrochemical process strengthened hydrolysis reactions, shortened time for the redistribution of pH balance, decreased the tortuosity of migration path, and increased the desorption concentrations of HMs in the sample area.

Neuropeptide Y affects thalamic reticular nucleus neuronal firing and network synchronization associated with suppression of spike-wave discharges.

Neuropeptide Y (NPY) potently suppresses spike-wave discharges (SWDs) in a genetic rat model of absence epilepsy (GAERS), but the underlying neurophysiologic mechanisms are not clear. We therefore sought to determine the in vivo effects of NPY on neuronal firing in the cortico-thalamo-cortical network activity, known to play a critical role in the generation of SWDs in these rats. NPY was administered intracerebroventricularly (ICV) or in separate experiments locally on the neurons of caudal thalamic reticular nucleus (NRT) by use of juxtacellular iontophoresis in triple-barrel electrodes in male GAERS aged 12-15 weeks, in vivo under neuroleptic anesthesia. Drug infusions and electroencephalography (EEG) monitoring were performed simultaneously with juxtacellular single neuronal recordings. Effect of NPY on electrically induced SWD induction threshold were also measured. NPY administration ICV led to a decrease in the total length of SWDs in EEG recordings. Both ICV administration and iontophoresis of NPY on NRT neurons led to an increase in interictal neuronal firing of NRT neurons. During ictal periods, ICV NPY administration reduced the number of thalamic action potentials per SWDs, as well as reduced waveform correlations between field potentials within the NRT and the cortical EEG. NPY administration ICV did not significantly alter the firing patterns of relay thalamic neurons interictally and cortical neurons during ictal and interictal periods. In addition, SWD induction threshold in the S2 region of the cortex was significantly increased after NPY administration. Our results show alterations in cortico-thalamo-cortical local and network properties following ICV administration of NPY, suggesting mechanisms of SWD suppression in GAERS. Cellular and network alteration of NRT activity, resulting from a direct action of NPY, may be a contributor to this effect.

Identification of Novel Linear Epitopes Located in the Infectious Bronchitis Virus Spike S2 Region.

We identified novel linear epitopes on the infectious bronchitis virus (IBV) spike S2 region. The conformational structure of the IBV spike protein was predicted from a homologous protein, human coronavirus NL63 spike. Although the obtained structure was incomplete, most of the IBV spike protein structure was predicted; the N-terminus of the S1 region could not be predicted due to its variability. In the model, the region located in the proximity of the fusion peptide appeared to be well conserved, and we evaluated the antigenicity of these domains, which are involved in the membrane fusion machinery. Western blotting revealed that IBV TM86 spike residues 686-723 were antigenic. Epitope mapping analysis using synthesized peptides revealed that IBV TM86 spike 669-685 (SNFSTGAFNISLLLTPP), 686-697 (SNPRGRSFIEDL), and 692-703 (SFIEDLLFTSVE) residues were major linear epitopes; two identified epitopes (686-697 and 692-703) were covered by the fusion peptide, and the other epitope (669-685) was adjacent to the fusion peptide. Although the identified epitopes are identically located as the neutralizing epitope in severe acute respiratory syndrome coronavirus, the recombinant protein that includes those epitopes could not elicit neutralizing antibodies against IBV. This is the first report describing IBV spike S2 epitopes located in the proximity of the fusion peptide, and it is suggested that the spike fusion machinery of IBV may differ from that of severe acute respiratory syndrome coronavirus, or, alternatively, IBV may have another mechanism to penetrate the cell membrane.

Applicability analysis of the solar heating system with parabolic trough solar collectors in different regions of China

This study aimed to investigate the applicability of the solar heating system in different geographical regions with different meteorological conditions, of which parabolic trough solar collectors (PTCs) were operated with the absorption heat pumps (AHP) and oil/water heat exchanger (OWHE) at medium and low operating temperature respectively. The heat transfer model for PTCs was constructed with a lumped parameter method and validated by experimental results. The thermal performance of the system was evaluated by the index of primary energy ratio (PER). The results showed that on overcast days with low direct normal irradiance (DNI), the operation of the PTC + AHP/OWHE system was not cost-effective. On cloudy days with high DNI lasting for a short period (e.g. 2 h), the operation of the PTC + OWHE system was better than that of the PTC + AHP system as the latter needed more preheating energy before the system operation. While on sunny days with high DNI lasting for a longer period (e.g. 8 h), the PTC + AHP system was suggested to be operated as it owned higher PER values. Besides, the solar energy distributions in China could be divided into four categories, i.e. rich (S1), relatively rich (S2), available (S3) and absent (S4). In S1 region the PTC + AHP system was suggested to be operated. In S4 region the PTC + AHP/OWHE system was not suggested to be operated. While in S2 and S3 region, whether the operation of the PTC + AHP/OWHE system was suggested depending on the meteorological conditions. With these findings the developing strategy of the solar heating system in different geographical regions can be devised and the operating strategy on a specific day with different weather conditions can be developed, which is helpful to guide the application of solar energy and improve the energy structure in domestic heating.

Conformer-Specific and Diastereomer-Specific Spectroscopy of αβα Synthetic Foldamers: Ac-Ala-βACHC-Ala-NHBn.

The folding propensities of a capped, cyclically constrained, mixed α/β diastereomer pair, ( SRSS) Ac-Ala-βACHC-Ala-NHBn (hereafter RS) and ( SSRS) Ac-Ala-βACHC-Ala-NHBn ( SR), have been studied in a molecular beam using single-conformation spectroscopic techniques. These α/β-tripeptides contain a cyclohexane ring across each Cα -Cβ bond, at which positions their stereochemistries differ. This cyclic constraint requires any stable species to adopt one of two ACHC configurations: equatorial C═O/axial NH or equatorial NH/axial C═O. Resonant two-photon ionization (R2PI) and infrared-ultraviolet hole-burning (IR-UV HB) spectroscopy were used in the S0-S1 region of the UV chromophore, revealing the presence of three unique conformational isomers of RS and two of SR. Resonant ion-dip infrared spectra were recorded in both the NH stretch (3200-3500 cm-1) and the amide I (1600-1800 cm-1) regions. These experimental vibrational frequencies were compared with the scaled calculated normal-mode frequencies from density functional theory at the M05-2X/6-31+G(d) level of theory, leading to structural assignments of the observed conformations. The RS diastereomer is known in crystalline form to preferentially form a C11/C9 mixed helix, in which alternating hydrogen bonds are arranged in near antiparallel orientation. This structure is preserved in one of the main conformers observed in the gas phase but is in competition with both a tightly folded C7eq/C12/C8/C7eq structure, in which all four amide NH groups and four C═O groups are engaged in hydrogen bonding, as well as a cap influenced C7eq/NH···π/C11 structure. The SR diastereomer is destabilized by inducing backbone dihedral angles that lie outside the typical Ramachandran angles. This diastereomer also forms a C11/C9 mixed helix as well as a cap influenced bifurcated C7ax-C11/NH···π/C7eq structure as the global energy minimum. Assigned structures are compared with the reported crystal structure of analogous α/β-tripeptides, and disconnectivity graphs are presented to give an overview of the complicated potential energy surface of this tripeptide diastereomer pair.

Somatotopic Organization and Temporal Characteristics of Cerebrocortical Excitation in Response to Nasal Mucosa Stimulation With and Without an Odor in the Rat: An Optical Imaging Study

Nasal mucosa has roles in warming and humidifying inspired air and is highly sensitive to mechanical stimuli. Moreover, the upper part of the nasal mucosa expresses olfactory receptors processing olfactory information. Although the somatosensory map of the face in the primary (S1) and secondary (S2) somatosensory cortices is clearly documented, the map of the nasal mucosa and the effect of odors on their activities are largely unknown. This study aimed to identify the cortical regions in S1 and their temporal features in response to somatosensory stimulation of the nasal mucosa using an optical imaging technique in urethane-anesthetized rats. An air puff application response to nasal mucosa first occurred in a part of contralateral S1 and subsequently, spread toward the rostrally and ventrally adjacent sites. Upper pharynx stimulation initially activated this rostrally expanded site and the excitatory propagation from the initially activated region toward ventral region likely represented S2. Signal intensity and activated area increased dependent on air pressure. Nasal tip stimulation initially excited S1 region caudally adjacent to that of nasal mucosa. Moreover, the amplitude of S1 excitation was similar between air puff stimulation with and without an odor, amyl acetate. In contrast to contralateral S1, air puff stimulation with the odor showed a faint optical signal increase in the ipsilateral piriform cortex. These results suggest that somatosensory information from the nasal mucosa and skin, and upper pharynx are processed in spatially continuous regions of S1, and interaction between somatosensory and olfactory systems is relatively small in contralateral S1.

De novo mutations in GRIN1 cause extensive bilateral polymicrogyria.

Polymicrogyria is a malformation of cortical development. The aetiology of polymicrogyria remains poorly understood. Using whole-exome sequencing we found de novo heterozygous missense GRIN1 mutations in 2 of 57 parent-offspring trios with polymicrogyria. We found nine further de novo missense GRIN1 mutations in additional cortical malformation patients. Shared features in the patients were extensive bilateral polymicrogyria associated with severe developmental delay, postnatal microcephaly, cortical visual impairment and intractable epilepsy. GRIN1 encodes GluN1, the essential subunit of the N-methyl-d-aspartate receptor. The polymicrogyria-associated GRIN1 mutations tended to cluster in the S2 region (part of the ligand-binding domain of GluN1) or the adjacent M3 helix. These regions are rarely mutated in the normal population or in GRIN1 patients without polymicrogyria. Using two-electrode and whole-cell voltage-clamp analysis, we showed that the polymicrogyria-associated GRIN1 mutations significantly alter the in vitro activity of the receptor. Three of the mutations increased agonist potency while one reduced proton inhibition of the receptor. These results are striking because previous GRIN1 mutations have generally caused loss of function, and because N-methyl-d-aspartate receptor agonists have been used for many years to generate animal models of polymicrogyria. Overall, our results expand the phenotypic spectrum associated with GRIN1 mutations and highlight the important role of N-methyl-d-aspartate receptor signalling in the pathogenesis of polymicrogyria.

Detection and phylogenetic analyses of spike genes in porcine epidemic diarrhea virus strains circulating in China in 2016\u20132017

BackgroundLarge-scale outbreaks of porcine epidemic diarrhea (PED) have re-emerged in China in recent years. However, little is known about the genetic diversity and molecular epidemiology of field strains of PED virus (PEDV) in China in 2016–2017. To address this issue, in this study, 116 diarrhea samples were collected from pig farms in 6 Chinese provinces in 2016–2017 and were detected using PCR for main porcine enteric pathogens, including PEDV, porcine deltacoronavirus (PDCoV), porcine transmissible gastroenteritis virus (TGEV) and porcine kobuvirus (PKV). In addition, the complete S genes from 11 representative PEDV strains were sequenced and analyzed.ResultsPCR detection showed that 52.6% (61/116) of these samples were positive for PEDV. Furthermore, sequencing results for the spike (S) genes from 11 of the epidemic PEDV strains showed 93–94% nucleotide identity and 92–93% amino acid identity with the classical CV777 strain. Compared with the CV777 vaccine strain, these strains had an insertion (A133), a deletion (G155), and a continuous 4-amino-acid insertion (56NNTN59) in the S1 region. Phylogenetic analysis based on the S gene indicated that the 11 assessed PEDV strains were genetically diverse and clustered into the G2 group. These results demonstrate that the epidemic strains of PEDV in China in 2016–2017 are mainly virulent strains that belong to the G2 group and genetically differ from the vaccine strain. Importantly, this is the first report that the samples collected in Hainan Province were positive for PEDV (59.2%, 25/42).ConclusionsTo our knowledge, this article presents the first report of a virulent PEDV strain isolated from Hainan Island, China. The results of this study will contribute to the understanding of the epidemiology and genetic characteristics of PEDV in China.

Suppression of cortical seizures by optic stimulation of the reticular thalamus in PV-mhChR2-YFP BAC transgenic mice

Deep brain stimulation in thalamic regions has been proposed as a treatment for epilepsy. The electrical current excites thalamocortical activity which is controlled by γ-aminobutyric acid (GABA)ergic interneurons in the reticular thalamic nucleus (nRT). Previous studies showed that enhancing GABAergic inhibitory strength in the nRT reduces the duration and power of seizures, indicating that the thalamus plays an important role in modulating cortical seizures. The aim of the present study was to apply optogenetics to study the role of the nRT in modulating cortical seizures. We used PV-ChR2-EYFP transgenic mice from Jackson Laboratories, in which only Channelrhodopsin-2 (ChR2) is expressed in parvalbumin-expressing interneurons. Cortical seizure-like activity was induced by electrical stimulation of the corpus callosum after applying 4-aminopyridine. ChR2 expression was abundant in the nRT and cerebellum in PV-ChR2-EYFP transgenic mice. Light stimulation in the nRT caused burst firing in regions of the thalamus and nRT in vitro. Multi-unit activity increased during high-frequency (100 and 50 Hz) light stimulation in the S1 region and thalamus in vivo. Corpus callosum stimulation-induced seizure-like activity was effectively suppressed by high-frequency (100 Hz) and long-duration (10 s) light stimulation. The suppressive effects were reversed by applying a GABAB receptor antagonist but not a GABAA receptor antagonist in the cortex. The results indicated that light stimulation affected thalamocortical relay neurons by activating ChR2-expression neurons in the nRT. High-frequency and long-duration light stimulation was more effective in suppressing cortical seizure-like activity. GABAB receptors may participate in suppressing seizure-like activity.

The S1 helix critically regulates the finely tuned gating of Kv11.1 channels

Congenital mutations in the cardiac Kv11.1 channel can cause long QT syndrome type 2 (LQTS2), a heart rhythm disorder associated with sudden cardiac death. Mutations act either by reducing protein expression at the membrane and/or by perturbing the intricate gating properties of Kv11.1 channels. A number of clinical LQTS2-associated mutations have been reported in the first transmembrane segment (S1) of Kv11.1 channels, but the role of this region of the channel is largely unexplored. In part, this is due to problems defining the extent of the S1 helix, as a consequence of its low sequence homology with other Kv family members. Here, we used NMR spectroscopy and electrophysiological characterization to show that the S1 of Kv11.1 channels extends seven helical turns, from Pro-405 to Phe-431, and is flanked by unstructured loops. Functional analysis suggests that pre-S1 loop residues His-402 and Tyr-403 play an important role in regulating the kinetics and voltage dependence of channel activation and deactivation. Multiple residues within the S1 helix also play an important role in fine-tuning the voltage dependence of activation, regulating slow deactivation, and modulating C-type inactivation of Kv11.1 channels. Analyses of LQTS2-associated mutations in the pre-S1 loop or S1 helix of Kv11.1 channels demonstrate perturbations to both protein expression and most gating transitions. Thus, S1 region mutations would reduce both the action potential repolarizing current passed by Kv11.1 channels in cardiac myocytes, as well as the current passed in response to premature depolarizations that normally helps protect against the formation of ectopic beats.

Simultaneous detection of severe acute respiratory syndrome, Middle East respiratory syndrome, and related bat coronaviruses by real-time reverse transcription PCR.

Since severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoVs) share similar characteristics with respect to clinical signs, etiology, and transmission, methods for a rapid and accurate differential diagnosis are important. Therefore, the aim of this study was to develop a duplex real-time reverse transcription (RT)-PCR method for the simultaneous detection of these viruses. Primers and probes that target the conserved spike S2 region of human SARS-CoV, MERS-CoV, and their related bat CoVs were designed. The results of real-time RT-PCR showed specific reactions for each virus with adequate detection limits of 50–100 copies/mL and 5–100 copies/mL using pUC57-SARS-pS2 (a template for SARS-CoV) and pGEM-MERS-S2 (a template for MERS-CoV), respectively. In addition, this real-time RT-PCR system was able to detect the target viruses SARS-like bat CoV and MERS-CoV in bat fecal samples and sputum of MERS patients, respectively. Therefore, this newly developed real-time RT-PCR method is expected to detect not only SARS-CoV and MERS-CoV in humans but also several bat CoVs that are closely related to these viruses in bats.