Detection of abnormal behaviors in prenatal Poly(I:C) exposed mice in a group-rearing environment.

During pregnancy, the maternal environment is critical for normal ontogeny and central nervous system development. Occasionally, prenatal exposure to environmental factors affects tissue architecture and functional development of the brain, which causes developmental disorders, including disorders of the autism spectrum. One of these environmental factors is the exposure to infectious diseases during pregnancy. In this study, we generated mice with infectious disease-induced inflammation by prenatal exposure to 200 µg/kg polyinosinic-polycytidylic acid sodium salt [Poly(I:C)] at embryonic day 12.5 and analyzed their phenotypes on 30-weeks-old. We attempted to detect abnormalities in spontaneous activity and social interaction, which may be indicators of developmental disorder-like behavioral abnormalities, in free-ranging behaviors in multiple rearing environments using multiple animal positioning systems and UMATracker in mice with fetal inflammation. Increased spontaneous activity and abnormal social interactions were observed in mice in the Poly(I:C)-treated group compared with those in the control group. Prenatal exposure to Poly(I:C) increased motor activity and decreased social interaction, and social behavior in prenatally treated mice in a multiple-individual rearing environment. Poly(I:C) exposure during the fetal period resulted in developmental disorder-like behavioral abnormalities, such as increased activity and abnormal social interactions, even after maturation in a multiple-individual rearing environment. This experimental method may provide a new way to analyze the behavior of mouse models of developmental disorders in a multiple-individual rearing environment, in which free-ranging behavior is possible.

Characterization of cytoskeletal and structural effects of INF2 variants causing glomerulopathy and neuropathy.

Focal segmental glomerulosclerosis (FSGS) is a common glomerular injury leading to end-stage renal disease. Monogenic FSGS is primarily ascribed to decreased podocyte integrity. Variants between residues 184 and 245 of INF2, an actin assembly factor, produce the monogenic FSGS phenotype. Meanwhile, variants between residues 57 and 184 cause a dual-faceted disease involving peripheral neurons and podocytes (Charcot-Marie-Tooth CMT/FSGS). To understand the molecular basis for INF2 disorders, we compared structural and cytoskeletal effects of INF2 variants classified into two subgroups: One (G73D, V108D) causes the CMT/FSGS phenotype, and the other (T161N, N202S) produces monogenic FSGS. Molecular dynamics analysis revealed that all INF2 variants show distinct flexibility compared to the wild-type INF2 and could affect stability of an intramolecular interaction between their N- and C-terminal segments. Immunocytochemistry of cells expressing INF2 variants showed fewer actin stress fibers, and disorganization of cytoplasmic microtubule arrays. Notably, CMT/FSGS variants caused more prominent changes in mitochondrial distribution and fragmentation than FSGS variants and these changes correlated with the severity of cytoskeletal disruption. Our results indicate that CMT/FSGS variants are associated with more severe global cellular defects caused by disrupted cytoskeleton-organelle interactions than are FSGS variants. Further study is needed to clarify tissue-specific pathways and/or cellular functions implicated in FSGS and CMT phenotypes.

Electrogenerated base-promoted cyclopropanation using alkyl 2-chloroacetates.

The electrochemical reduction conditions of the reaction of alkyl 2-chloroacetates in Bu4NBr/DMF using a divided cell equipped with Pt electrodes to produce the corresponding cyclopropane derivatives in moderate yields were discovered. The reaction conditions were optimized, the scope and limitations, as well as scale-up reactions were investigated. The presented method for the electrochemical production of cyclopropane derivatives is an environmentally friendly and easy to perform synthetic procedure.

Protein-ligand binding affinity prediction of cyclin-dependent kinase-2 inhibitors by dynamically averaged fragment molecular orbital-based interaction energy.

Fragment molecular orbital (FMO) method is a powerful computational tool for structure-based drug design, in which protein-ligand interactions can be described by the inter-fragment interaction energy (IFIE) and its pair interaction energy decomposition analysis (PIEDA). Here, we introduced a dynamically averaged (DA) FMO-based approach in which molecular dynamics simulations were used to generate multiple protein-ligand complex structures for FMO calculations. To assess this approach, we examined the correlation between the experimental binding free energies and DA-IFIEs of six CDK2 inhibitors whose net charges are zero. The correlation between the experimental binding free energies and snapshot IFIEs for X-ray crystal structures was R2  = 0.75. Using the DA-IFIEs, the correlation significantly improved to 0.99. When an additional CDK2 inhibitor with net charge of -1 was added, the DA FMO-based scheme with the dispersion energies still achieved R2  = 0.99, whereas R2 decreased to 0.32 employing all the energy terms of PIEDA.

Hemiplegic migraine type 2 with new mutation of the ATP1A2 gene in Japanese cases.

We analyzed the clinical symptoms of hemiplegic migraine (HM) and their relevance in four Japanese patients considered to have ATP1A2 mutations as a cause. Sequencing of ATP1A2 was performed using the Sanger method in 43 blood samples from clinically suspected patients with familial HM. Subsequently, algorithm analysis, allele frequency determination, and three-dimensional structure analysis of the recognized variants were performed, and the recognized variants were evaluated. We found four heterozygous missense mutations in ATP1A2 (Case 1: p.R51C; Case 2: p.R65L; Case 3: p.A269P; Case 4: p.D999H), three of which had not been reported to date. These four mutations may also affect the structure of the protein products, as assessed using a three-dimensional structural analysis. In all four cases, the clinical symptoms included visual, sensory, motor, and verbal symptoms and the frequency and duration of headache attacks varied. Additionally, oral administration of a combination of lomerizine hydrochloride and topiramate had a partial effect in three cases. We report four missense mutations in ATP1A2. This report will be useful for the future analysis of mutations and clinical types in Asians, as well as Westerners, with migraine.

Double mutations in the H9N2 avian influenza virus PB2 gene act cooperatively to increase viral host adaptation and replication for human infections.

Avian H9N2 influenza viruses in East Asia are genetically diversified and multiple genotypes (A-W) have been established in poultry. Genotype S strains are currently the most prevalent strains, have caused many human infections and pose a public health threat. In this study, human adaptation mutations in the PB2 polymerase in genotype S strains were identified by database screening. Several PB2 double mutations were identified that acted cooperatively to produce higher genotype S virus polymerase activity and replication in human cells than in avian cells and to increase viral growth and virulence in mice. These mutations were chronologically and phylogenetically clustered in a new group within genotype S viruses. Most of the relevant human virus isolates carry the PB2-A588V mutation together with another PB2 mutation (i.e. K526R, E627V or E627K), indicating a host adaptation advantage for these double mutations. The prevalence of PB2 double mutations in human H9N2 virus isolates has also been found in genetically related human H7N9 and H10N8 viruses. These results suggested that PB2 double mutations in viruses in the field acted cooperatively to increase human adaptation of the currently prevalent H9N2 genotype S strains. This may have contributed to the recent surge of H9N2 infections and may be applicable to the human adaptation of several other avian influenza viruses. Our study provides a better understanding of the human adaptation pathways of genetically related H9N2, H7N9 and H10N8 viruses in nature.

Intermolecular Interaction Analyses on SARS-CoV-2 Spike Protein Receptor Binding Domain and Human Angiotensin-Converting Enzyme 2 Receptor-Blocking Antibody/Peptide Using Fragment Molecular Orbital Calculation.

The spike glycoprotein (S-protein) mediates SARS-CoV-2 entry via intermolecular interaction with human angiotensin-converting enzyme 2. The receptor binding domain (RBD) of the S-protein has been considered critical for this interaction and acts as the target of numerous neutralizing antibodies and antiviral peptides. This study used the fragment molecular orbital method to analyze the interactions between the RBD and antibodies/peptides and extracted crucial residues that can be used as epitopes. The interactions evaluated as interfragment interaction energy values between the RBD and 12 antibodies/peptides showed a fairly good correlation with the experimental activity pIC50 (R2 = 0.540). Nine residues (T415, K417, Y421, F456, A475, F486, N487, N501, and Y505) were confirmed as being crucial. Pair interaction energy decomposition analyses showed that hydrogen bonds, electrostatic interactions, and π-orbital interactions are important. Our results provide essential information for understanding SARS-CoV-2-antibody/peptide binding and may play roles in future antibody/antiviral drug design.

FMODB: The World's First Database of Quantum Mechanical Calculations for Biomacromolecules Based on the Fragment Molecular Orbital Method.

We developed the world's first web-based public database for the storage, management, and sharing of fragment molecular orbital (FMO) calculation data sets describing the complex interactions between biomacromolecules, named FMO Database (https://drugdesign.riken.jp/FMODB/). Each entry in the database contains relevant background information on how the data was compiled as well as the total energy of each molecular system and interfragment interaction energy (IFIE) and pair interaction energy decomposition analysis (PIEDA) values. Currently, the database contains more than 13 600 FMO calculation data sets, and a comprehensive search function implemented at the front-end. The procedure for selecting target proteins, preprocessing the experimental structures, construction of the database, and details of the database front-end were described. Then, we demonstrated a use of the FMODB by comparing IFIE value distributions of hydrogen bond, ion-pair, and XH/π interactions obtained by FMO method to those by molecular mechanics approach. From the comparison, the statistical analysis of the data provided standard reference values for the three types of interactions that will be useful for determining whether each interaction in a given system is relatively strong or weak compared to the interactions contained within the data in the FMODB. In the final part, we demonstrate the use of the database to examine the contribution of halogen atoms to the binding affinity between human cathepsin L and its inhibitors. We found that the electrostatic term derived by PIEDA greatly correlated with the binding affinities of the halogen containing cathepsin L inhibitors, indicating the importance of QM calculation for quantitative analysis of halogen interactions. Thus, the FMO calculation data in FMODB will be useful for conducting statistical analyses to drug discovery, for conducting molecular recognition studies in structural biology, and for other studies involving quantum mechanics-based interactions.

Characterization of a variant CD4 molecule in Japanese Black cattle.

Monoclonal antibodies (mAbs) that recognize cluster of differentiation (CD) molecules on lymphocytes are useful tools for the study of different lymphocyte subsets in flow cytometry (FCM) analysis. CD4 is a glycoprotein found on the surfaces of helper T cells, monocytes, macrophages, and dendritic cells. In this study, we describe Japanese Black (JB) calves in a farm whose peripheral blood mononuclear cells (PBMCs) did not react with a CD4-specific mAb. To identify calves with PBMCs with low mAb reactivity, PBMCs from 21 JB calves (1-12 months of age) bred at the same farm were examined using two different bovine CD4 mAbs (clones #CC8 and #CACT138A). FCM analysis indicated that the calves fell into two groups based on reactivity against the two mAbs, i.e., double-positive (DP) calves, whose PBMCs were recognized by both mAbs clones, and single-positive (SP) calves, whose PBMCs were only recognized by #CACT138A. PBMCs from seven calves were not recognized by #CC8, although they had normal reactivity with another mAb, #CACT138A. Sequencing analysis of the CD4 gene in these calves revealed four nucleotide substitutions (G918 T, A930C, G970A, and G1074A) in the coding region in the SP group when compared to the DP group. Three of the four mutations were associated with amino acid substitution (Q306H, K310 N, and A324 T). The substitution at A324 T was located in the D4 domain of CD4 gene. Homology modeling based on the amino acid sequences revealed that the surface structure of this part of the molecule was significantly different between the SP and the DP groups. Therefore, the epitope recognized by the #CC8 CD4 mAb was altered in calves with this genetic mutation, and this led to the low reactivity of the PBMCs from calves in the SP group aginst the #CC8 mAb. In conclusion, this is the first study to identify CD4 variants in JB cattle. We confirmed that the variants did not affect lymphocyte functions, such as mitogen stimulation or lipopolysaccharide-induced cytokine gene expression.

Knowledge, attitudes, and practices toward COVID-19 among university students in Japan and associated factors: An online cross-sectional survey.

The coronavirus disease (COVID-19) pandemic has greatly altered peoples' daily lives, and it continues spreading as a crucial concern globally. Knowledge, attitudes, and practices (KAP) toward COVID-19 are related to individuals' adherence to government measures. This study evaluated KAP toward COVID-19 among university students in Japan between May 22 and July 16, 2020, via an online questionnaire, and it further investigated the associated determining KAP factors. Among the eligible respondents (n = 362), 52.8% were female, 79.0% were undergraduate students, 32.9% were students whose major university subjects were biology-related, 35.4% were from the capital region, and 83.7% were Japanese. The overall KAP of university students in Japan was high. All respondents (100%) showed they possessed knowledge on avoiding enclosed spaces, crowded areas, and close situations. Most respondents showed a moderate or higher frequency of washing their hands or wearing masks (both at 96.4%). In addition, 68.5% of respondents showed a positive attitude toward early drug administration. In the logistic regressions, gender, major subjects, education level, nationality, residence, and psychological factors (private self-consciousness and extroversion) were associated with knowledge or attitudes toward COVD-19 (p < 0.05). In the logistic and multiple linear regressions, capital regions, high basic knowledge, high information acquisition, correct information explanations contributed positively to preventative action (p < 0.05). Non-capital regions, male gender, non-bio-backgrounds, high public self-consciousness, high advanced knowledge, incorrect information explanations, and high extroversion contributed negatively to self-restraint (p < 0.05). Moreover, self-restraint was decreasing over time. These findings clarify the Japanese university students' KAP and the related factors in the early period of the COVID-19 pandemic, and they may help university managers, experts, and policymakers control the future spread of COVID-19 and other emerging infections.

PA Mutations Inherited during Viral Evolution Act Cooperatively To Increase Replication of Contemporary H5N1 Influenza Virus with an Expanded Host Range.

Adaptive mutations and/or reassortments in avian influenza virus polymerase subunits PA, PB1, and PB2 are one of the major factors enabling the virus to overcome the species barrier to infect humans. The majority of human adaptation polymerase mutations have been identified in PB2; fewer adaptation mutations have been characterized in PA and PB1. Clade 2.2.1 avian influenza viruses (H5N1) are unique to Egypt and generally carry the human adaptation PB2-E627K substitution during their dissemination in nature. In this study, we identified other human adaptation polymerase mutations by analyzing phylogeny-associated PA mutations that H5N1 clade 2.2.1 viruses have accumulated during their evolution in the field. This analysis identified several PA mutations that produced increased replication by contemporary clade 2.2.1.2 viruses in vitro in human cells and in vivo in mice compared to ancestral clade 2.2.1 viruses. The PA mutations acted cooperatively to increase viral polymerase activity and replication in both avian and human cells, with the effect being more prominent in human cells at 33°C than at 37°C. These results indicated that PA mutations have a role in establishing contemporary clade 2.2.1.2 virus infections in poultry and in adaptation to infect mammals. Our study provided data on the mechanism for PA mutations to accumulate during avian influenza virus evolution and extend the viral host range.IMPORTANCE Clade 2.2.1 avian influenza viruses (H5N1) are unique to Egypt and have caused the highest number of human H5N1 influenza cases worldwide, presenting a serious global public health threat. These viruses may have the greatest evolutionary potential for adaptation from avian hosts to human hosts. Using a comprehensive phylogenetic approach, we identified several novel clade 2.2.1 virus polymerase mutations that increased viral replication in vitro in human cells and in vivo in mice. These mutations were in the polymerase PA subunit and acted cooperatively with the E627K mutation in the PB2 polymerase subunit to provide higher replication in contemporary clade 2.2.1.2 viruses than in ancestral clade 2.2.1 viruses. These data indicated that ongoing clade 2.2.1 dissemination in the field has driven PA mutations to modify viral replication to enable host range expansion, with a higher public health risk for humans.

A Comparison between the Cycloadditions of Allenyl- and Vinyl-Cyclopentanes Using Density Functional Theory and GRRM Program.

Cycloaddition catalyzed by transition metals such as rhodium (I) is an important way to synthesize functionalized molecules in medicinal chemistry. When the reagent has a saturated ring containing more than five carbons (or heavy atoms), the reaction can progress when the compound has an allenyl group, but not for a vinyl group. Here, we constructed two computational models for allenylcyclopentane-alkyne and vinylcyclopentane-alkyne, and obtained their reaction pathways using density functional theory (DFT). From the reaction pathways, we confirmed that the former model has a much lower reaction energy than the latter. We also found that the molecular orbitals of the transition state structure at the rate-controlling step contribute significantly to the difference in reactivity between the two models.

Metal-Free Nitrogen-Containing Polyheterocyclic Near-Infrared (NIR) Absorption Dyes: Synthesis, Absorption Properties, and Theoretical Calculation of Substituted 5-Methylisoindolo[2,1-a]quinolines.

We have synthesized and theoretically calculated 5-methylisoindolo[2,1-a]quinoline derivatives as novel near-infrared absorption dyes via a ruthenium-catalyzed one-pot metathesis/oxidation/1,3-dipolar cycloaddition protocol. The reactivity in 1,3-dipolar cycloaddition was governed by the electronic effect of aromatic ring substituents. Substrates with an electron-withdrawing group on the aromatic ring afforded higher yields. The maximal absorption wavelength of 3,5-dimethyl-11-phenylisoindolo[2,1-a]quinoline-7,10-dione and 11-(4-methoxyphenyl)-5-methylisoindolo[2,1-a]quinoline-7,10-dione in MeOH increased to 736 and 737 nm, although that of 3a was 727 nm.

Correction to: Genetic and genomic basis of the mismatch repair system involved in Lynch syndrome.

In the original publication, part d of Figure 2 was mistakenly not included.

Genetic and genomic basis of the mismatch repair system involved in Lynch syndrome.

Lynch syndrome is a cancer-predisposing syndrome inherited in an autosomal-dominant manner, wherein colon cancer and endometrial cancer develop frequently in the family, it results from a loss-of-function mutation in one of four different genes (MLH1, MSH2, MSH6, and PMS2) encoding mismatch repair proteins. Being located immediately upstream of the MSH2 gene, EPCAM abnormalities can affect MSH2 and cause Lynch syndrome. Mismatch repair proteins are involved in repairing of incorrect pairing (point mutations and deletion/insertion of simple repetitive sequences, so-called microsatellites) that can arise during DNA replication. MSH2 forms heterodimers with MSH6 or MSH3 (MutSα, MutSß, respectively) and is involved in mismatch-pair recognition and initiation of repair. MLH1 forms a complex with PMS2, and functions as an endonuclease. If the mismatch repair system is thoroughly working, genome integrity is maintained completely. Lynch syndrome is a state of mismatch repair deficiency due to a monoallelic abnormality of any mismatch repair genes. The phenotype indicating the mismatch repair deficiency can be frequently shown as a microsatellite instability in tumors. Children with germline biallelic mismatch repair gene abnormalities were reported to develop conditions such as gastrointestinal polyposis, colorectal cancer, brain cancer, leukemia, etc., and so on, demonstrating the need to respond with new concepts in genetic counseling. In promoting cancer genome medicine in a new era, such as by utilizing immune checkpoints, it is important to understand the genetic and genomic molecular background, including the status of mismatch repair deficiency.

PB2 mutations arising during H9N2 influenza evolution in the Middle East confer enhanced replication and growth in mammals.

Avian influenza virus H9N2 has been endemic in birds in the Middle East, in particular in Egypt with multiple cases of human infections since 1998. Despite concerns about the pandemic threat posed by H9N2, little is known about the biological properties of H9N2 in this epicentre of infection. Here, we investigated the evolutionary dynamics of H9N2 in the Middle East and identified phylogeny-associated PB2 mutations that acted cooperatively to increase H9N2 replication/transcription in human cells. The accumulation of PB2 mutations also correlated with an increase in H9N2 virus growth in the upper and lower airways of mice and in virulence. These mutations clustered on a solvent-exposed region in the PB2-627 domain in proximity to potential interfaces with host factors. These PB2 mutations have been found at high prevalence during evolution of H9N2 in the field, indicating that they have provided a selective advantage for viral adaptation to infect poultry. Therefore, continuous prevalence of H9N2 virus in the Middle East has generated a far more fit or optimized replication phenotype, leading to an expanded viral host range, including to mammals, which may pose public health risks beyond the current outbreaks.

Three-Dimensional Classification Structure-Activity Relationship Analysis Using Convolutional Neural Network.

Quantitative structure-activity relationship (QSAR) techniques, especially those that possess three-dimensional attributes, such as the comparative molecular field analysis (CoMFA), are frequently used in modern-day drug design and other related research domains. However, the requirement for accurate alignment of compounds in CoMFA increases the difficulties encountered in its use. This has led to the development of several techniques-such as VolSurf, Grid-independent descriptors (GRIND), and Anchor-GRIND-which do not require such an alignment. We propose a technique to construct the prediction model that uses molecular interaction field grid potentials as inputs to convolutional neural network. The proposed model has been found to demonstrate higher accuracy compared to the conventional descriptor-based QSAR models as well as Anchor-GRIND techniques. In addition, the method is target independent, and is capable of providing useful information regarding the importance of individual atoms constituting the compounds contained in the chemical dataset used in the proposed analysis. In view of these advantages, the proposed technique is expected to find wide applications in future drug-design operations.

Multiple polymerase gene mutations for human adaptation occurring in Asian H5N1 influenza virus clinical isolates.

The role of the influenza virus polymerase complex in host range restriction has been well-studied and several host range determinants, such as the polymerase PB2-E627K and PB2-D701N mutations, have been identified. However, there may be additional, currently unknown, human adaptation polymerase mutations. Here, we used a database search of influenza virus H5N1 clade 1.1, clade 2.3.2.1 and clade 2.3.4 strains isolated from 2008-2012 in Southern China, Vietnam and Cambodia to identify polymerase adaptation mutations that had been selected in infected patients. Several of these mutations acted either alone or together to increase viral polymerase activity in human airway cells to levels similar to the PB2-D701N and PB2-E627K single mutations and to increase progeny virus yields in infected mouse lungs to levels similar to the PB2-D701N single mutation. In particular, specific mutations acted synergistically with the PB2-D701N mutation and showed synergistic effects on viral replication both in human airway cells and mice compared with the corresponding single mutations. Thus, H5N1 viruses in infected patients were able to acquire multiple polymerase mutations that acted cooperatively for human adaptation. Our findings give new insight into the human adaptation of AI viruses and help in avian influenza virus risk assessment.

Dengue Virus and Its Inhibitors: A Brief Review.

The global occurrence of viral infectious diseases poses a significant threat to human health. Dengue virus (DENV) infection is one of the most noteworthy of these infections. According to a WHO survey, approximately 400 million people are infected annually; symptoms deteriorate in approximately one percent of cases. Numerous foundational and clinical investigations on viral epidemiology, structure and function analysis, infection source and route, therapeutic targets, vaccines, and therapeutic drugs have been conducted by both academic and industrial researchers. At present, CYD-TDV or Dengvaxia® is the only approved vaccine, but potent inhibitors are currently under development. In this review, an overview of the viral life circle and the history of DENVs is presented, and the most recently reported antiviral candidates and newly discovered promising targets are focused and summarized. We believe that these successes and failures have enabled progress in anti-DENV drug discovery and hope that our review will stimulate further innovation in this area.

Characterization of H5N1 Influenza Virus Quasispecies with Adaptive Hemagglutinin Mutations from Single-Virus Infections of Human Airway Cells.

Transmission of avian influenza (AI) viruses to mammals involves phylogenetic bottlenecks that select small numbers of variants for transmission to new host species. However, little is known about the AI virus quasispecies diversity that produces variants for virus adaptation to humans. Here, we analyzed the hemagglutinin (HA) genetic diversity produced during AI H5N1 single-virus infection of primary human airway cells and characterized the phenotypes of these variants. During single-virus infection, HA variants emerged with increased fitness to infect human cells. These variants generally had decreased HA thermostability, an indicator of decreased transmissibility, that appeared to compensate for their increase in α2,6-linked sialic acid (α2,6 Sia) binding specificity and/or in the membrane fusion pH threshold, each of which is an advantageous mutational change for viral infection of human airway epithelia. An HA variant with increased HA thermostability also emerged but could not outcompete variants with less HA thermostability. These results provided data on HA quasispecies diversity in human airway cells.IMPORTANCE The diversity of the influenza virus quasispecies that emerges from a single infection is the starting point for viral adaptation to new hosts. A few studies have investigated AI virus quasispecies diversity during human adaptation using clinical samples. However, those studies could be appreciably affected by individual variability and multifactorial respiratory factors, which complicate identification of quasispecies diversity produced by selective pressure for increased adaptation to infect human airway cells. Here, we found that detectable HA genetic diversity was produced by H5N1 single-virus infection of human airway cells. Most of the HA variants had increased fitness to infect human airway cells but incurred a fitness cost of less HA stability. To our knowledge, this is the first report to characterize the adaptive changes of AI virus quasispecies produced by infection of human airway cells. These results provide a better perspective on AI virus adaptation to infect humans.