Stress has been shown to affect brain activity and exert potent and complex modulatory effects on pain. Several behavioral tests have shown that acute stress produces hyperalgesia, depending on the stress conditions. In the present study, we investigated the effects of single restraint stress on the tactile threshold and anxiety sensitivity in mice. Mice were evaluated for the tactile threshold using von Frey filaments and for anxiety sensitivity using the elevated plus maze (EPM) test. Tactile thresholds were lowered by both 2 and 4 hour of restraint stress, but anxiety-like behaviors were observed only after 4 hour of restraint stress in the EPM test. In addition, we found that alfaxalone, which is a positive allosteric modulator of the γ-aminobutyric acid (GABA)A receptor, prevented restraint stress-induced hyperalgesia-like and anxiety-like behaviors. These results indicate that GABAergic function appears to be critical in the regulation of physical stress-induced hyperalgesia and anxiety.

Pancreatic ductal adenocarcinoma has a particularly poor prognosis as it is often detected at an advanced stage and acquires resistance to chemotherapy early during its course. Stress adaptations by mitochondria, such as metabolic plasticity and regulation of apoptosis, promote cancer cell survival; however, the relationship between mitochondrial dynamics and chemoresistance in pancreatic ductal adenocarcinoma remains unclear. We here established human pancreatic cancer cell lines resistant to gemcitabine from MIA PaCa-2 and Panc1 cells. We compared the cells before and after the acquisition of gemcitabine resistance to investigate the mitochondrial dynamics and protein expression that contribute to this resistance. The mitochondrial number increased in gemcitabine-resistant cells after resistance acquisition, accompanied by a decrease in mitochondrial fission 1 protein, which induces peripheral mitosis, leading to mitophagy. An increase in the number of mitochondria promoted oxidative phosphorylation and increased anti-apoptotic protein expression. Additionally, enhanced oxidative phosphorylation decreased the AMP/ATP ratio and suppressed AMPK activity, resulting in the activation of the HSF1-heat shock protein pathway, which is required for environmental stress tolerance. Synergistic effects observed with BCL2 family or HSF1 inhibition in combination with gemcitabine suggested that the upregulated expression of apoptosis-related proteins caused by the mitochondrial increase may contribute to gemcitabine resistance. The combination of gemcitabine with BCL2 or HSF1 inhibitors may represent a new therapeutic strategy for the treatment of acquired gemcitabine resistance in pancreatic ductal adenocarcinoma.

The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology.

Colorectal cancer with a Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) gene mutation is considered to be resistant to anti-EGFR agents. G12D is the most common KRAS mutation in colorectal cancer, followed by G12V and G13D. According to clinical and basic research data, patients with colorectal cancer exhibiting G12D and G12V KRAS mutations are resistant to anti-EGFR agents; however, this is not true of G13D and other minor mutations, which are still not well understood. The current study focused on minor KRAS mutations (G12A, G12C, G12S, Q61H and A146T) and evaluated whether these were resistant to anti-EGFR antibodies using a mix culture assay. The results demonstrated that all KRAS mutations, including minor mutations, were resistant to two anti-EGFR agents: Cetuximab and panitumumab. The combined effect of MEK and BCL-XL inhibition on colorectal cancer cells with KRAS minor mutations were subsequently evaluated. The combined effect of MEK and BCL-XL inhibitors was confirmed in all KRAS minor mutations. The sensitivity of AMG510, a novel KRAS G12C selective inhibitor, was also assessed. The mix culture assay revealed that AMG510 selectively exerted an antitumor effect on colon cancer cells with a G12C KRAS mutation. The combination of MEK and BCL-XL inhibition markedly enhanced the effect of AMG510 in colon cancer cells. The current study suggested that AMG510 may have potential clinical use in combination with MEK and BCL-XL inhibitors in the treatment of patients with colorectal cancer exhibiting the G12C KRAS mutation.

The Kirsten rat sarcoma (KRAS) oncogene was "undruggable" until sotorasib, a KRASG12C selective inhibitor, was developed with promising efficacy. However, inhibition of mutant KRAS in colorectal cancer cells (CRC) is ineffective due to feedback activation of MEK/ERK downstream of KRAS. In this study, we screened for combination therapies of simultaneous inhibition to overcome sotorasib resistance using our previously developed Mix Culture Assay. We evaluated whether there was an additive effect of sotorasib administered alone and in combination with two or three drugs: trametinib, a MEK inhibitor, and cetuximab, an anti-epidermal growth factor receptor (EGFR) antibody. The MAPK pathway was reactivated in KRASG12C-mutated cell lines treated with sotorasib alone. Treatment with KRAS and MEK inhibitors suppressed the reactivation of the MAPK pathway, but upregulated EGFR expression. However, the addition of cetuximab to this combination suppressed EGFR reactivation. This three-drug combination therapy resulted in significant growth inhibition in vitro and in vivo. Our data suggest that reactive feedback may play a key role in the resistance signal in CRC. Simultaneously inhibiting KRAS, MEK, and EGFR is a potentially promising strategy for patients with KRASG12C-mutated CRC.

Type 2 diabetes (T2D) is a major global health issue, and it has also become one of the major diseases in Arab countries. In addition to the exome databases that have already been established, whole exome sequencing data for the Greater Middle East are now available. To elucidate the genetic features of T2D in the Arabian Peninsula, we integrated two exome databases (gnomAD exome and the Greater Middle East Variome Project) with clinical information from the ClinVar. After the integration, we obtained 18 single nucleotide polymorphisms and found two statistically and clinically significant variants in two genes, SLC30A8 rs13266634 and KCNJ11 rs5219. Interestingly, the two genes are linked to the uptake of the metals, Zn and K respectively, which indicating the regional features of the genetic variants. The frequency of the risk allele of rs13266634 among individuals in the Arabian Peninsula was higher than among individuals in other regions. On the other hand, the frequency of the risk allele of rs5219 in the Arabian Peninsula was lower than that in other regions. We identified and characterized T2D-related variants that show unique tendencies in the Arabian Peninsula. Our analyses contribute to and provide guidance for the clinical research of T2D in the Arabian Peninsula.

Viral spike proteins play important roles in the viral entry process, facilitating attachment to cellular receptors and fusion of the viral envelope with the cell membrane. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein binds to the cellular receptor angiotensin converting enzyme-2 (ACE2) via its receptor-binding domain (RBD). The cysteine residue at position 488, consisting of a disulfide bridge with cysteine 480 is located in an important structural loop at ACE2-binding surface of RBD, and is highly conserved among SARS-related coronaviruses. We showed that the substitution of Cys-488 with alanine impaired pseudotyped SARS-CoV-2 infection, syncytium formation, and cell-cell fusion triggered by SARS-CoV-2 spike expression. Consistently, in vitro binding of RBD and ACE2, spike-mediated cell-cell fusion, and pseudotyped viral infection of VeroE6/TMPRSS2 cells were inhibited by the thiol-reactive compounds N-acetylcysteine (NAC) and a reduced form of glutathione (GSH). Furthermore, we demonstrated that the activity of variant spikes from the SARS-CoV-2 alpha and delta strains were also suppressed by NAC and GSH. Taken together, these data indicate that Cys-488 in spike RBD is required for SARS-CoV-2 spike functions and infectivity, and could be a target of anti-SARS-CoV-2 therapeutics.

Peracetic acid (PAA) disinfectants are effective against a wide range of pathogenic microorganisms, including bacteria, fungi, and viruses. Several studies have shown the efficacy of PAA against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); however, its efficacy in SARS-CoV-2 variants and the molecular mechanism of action of PAA against SARS-CoV-2 have not been investigated. SARS-CoV-2 infection depends on the recognition and binding of the cell receptor angiotensin-converting enzyme 2 (ACE2) via the receptor-binding domain (RBD) of the spike protein. Here, we demonstrated that PAA effectively suppressed pseudotyped virus infection in the Wuhan type and variants, including Delta and Omicron. Similarly, PAA reduced the authentic viral load of SARS-CoV-2. Computational analysis suggested that the hydroxyl radicals produced by PAA cleave the disulfide bridges in the RBD. Additionally, the PAA treatment decreased the abundance of the Wuhan- and variant-type spike proteins. Enzyme-linked immunosorbent assay showed direct inhibition of RBD-ACE2 interactions by PAA. In conclusion, the PAA treatment suppressed SARS-CoV-2 infection, which was dependent on the inhibition of the interaction between the spike RBD and ACE2 by inducing spike protein destabilization. Our findings provide evidence of a potent disinfection strategy against SARS-CoV-2.

Transcription is one of the most fundamental nuclear functions and is an enzyme complex-mediated reaction that converts DNA sequences into mRNA. Analyzing DNA sequences of 5'-flanking regions of several human genes that respond to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in HL-60 cells, we have identified that the ets (GGAA) motifs are duplicated, overlapped, or clustered within a 500-bp distance from the most 5'-upstream region of the cDNA. Multiple protein factors including Ets family proteins are known to recognize and bind to the GGAA containing sequences. In addition, it has been reported that the ets motifs play important roles in regulation of various promoters. Here, we propose a molecular mechanism, defined by the presence of duplication and multiplication of the GGAA motifs, that is responsible for the initiation of transcription of several genes and for the recruitment of binding proteins to the transcription start site (TSS) of TATA-less promoters.

Genome Information Broker for Viruses (GIB-V) is a comprehensive virus genome/segment database. We extracted 18 418 complete virus genomes/segments from the International Nucleotide Sequence Database Collaboration (INSDC, http://www.insdc.org/) by DNA Data Bank of Japan (DDBJ), EMBL and GenBank and stored them in our system. The list of registered viruses is arranged hierarchically according to taxonomy. Keyword searches can be performed for genome/segment data or biological features of any virus stored in GIB-V. GIB-V is equipped with a BLAST search function, and search results are displayed graphically or in list form. Moreover, the BLAST results can be used online with the ClustalW feature of the DDBJ. All available virus genome/segment data can be collected by the GIB-V download function. GIB-V can be accessed at no charge at http://gib-v.genes.nig.ac.jp/.

Here we report the new features and improvements in our latest release of the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/), a comprehensive annotation resource for human genes and transcripts. H-InvDB, originally developed as an integrated database of the human transcriptome based on extensive annotation of large sets of full-length cDNA (FLcDNA) clones, now provides annotation for 120 558 human mRNAs extracted from the International Nucleotide Sequence Databases (INSD), in addition to 54 978 human FLcDNAs, in the latest release H-InvDB_4.6. We mapped those human transcripts onto the human genome sequences (NCBI build 36.1) and determined 34 699 human gene clusters, which could define 34 057 (98.1%) protein-coding and 642 (1.9%) non-protein-coding loci; 858 (2.5%) transcribed loci overlapped with predicted pseudogenes. For all these transcripts and genes, we provide comprehensive annotation including gene structures, gene functions, alternative splicing variants, functional non-protein-coding RNAs, functional domains, predicted sub cellular localizations, metabolic pathways, predictions of protein 3D structure, mapping of SNPs and microsatellite repeat motifs, co-localization with orphan diseases, gene expression profiles, orthologous genes, protein-protein interactions (PPI) and annotation for gene families. The current H-InvDB annotation resources consist of two main views: Transcript view and Locus view and eight sub-databases: the DiseaseInfo Viewer, H-ANGEL, the Clustering Viewer, G-integra, the TOPO Viewer, Evola, the PPI view and the Gene family/group.

RAS homolog family member A (RhoA), a member of the Rho family of small GTPases, and Vav1, a guanine nucleotide exchange factor for Rho family GTPases, have been reported to activate pathways related to the actin cytoskeleton and regulation of cell shape, attachment, and motility. The interaction between these molecules in lymphoma is involved in malignant signaling, but its function in epithelial malignancy is unknown. Here, we investigated the malignant signal of mutant RhoA in gastric cancer and demonstrated the potential of RhoA G17E/Vav1 as a therapeutic target for diffuse gastric cancer.