Description: Guanosine triphosphatases (GTPases) function as molecular switches in signal transduction pathways that enable cells to respond to extracellular stimuli. Saccharomyces cerevisiae yeast protein two 1 protein (Ypt1p) is a monomeric small GTPase that is essential for endoplasmic reticulum-to-Golgi trafficking. By size-exclusion chromatography, SDS-PAGE, and native PAGE, followed by immunoblot analysis with an anti-Ypt1p antibody, we found that Ypt1p structurally changed from low-molecular-weight (LMW) forms to high-molecular-weight (HMW) complexes after heat shock. Based on our results, Ypt1p exhibited dual functions both as a GTPase and a molecular chaperone, and furthermore, heat shock induced a functional switch from that of a GTPase to a molecular chaperone driven by the structural change from LMW to HMW forms. Subsequently, we found, by using a galactose-inducible expression system, that conditional overexpression of YPT1 in yeast cells enhanced the thermotolerance of cells by increasing the survival rate at 55°C by ~60%, compared with the control cells expressing YPT1 in the wild-type level. Altogether, our results suggest that Ypt1p is involved in the cellular protection process under heat stress conditions. Also, these findings provide new insight into the in vivo roles of small GTP-binding proteins and have an impact on research and the investigation of human diseases.—Kang, C. H., Lee, S. Y., Park, J. H., Lee, Y., Jung, H. S., Chi, Y. H., Jung, Y. J., Chae, H. B., Shin, M. R., Kim, W. Y., Yun, D.-J., Lee, S. Y. Stress-driven structural and functional switching of Ypt1p from a GTPase to a molecular chaperone mediates thermo tolerance in Saccharomyces cerevisiae .

Description: CD4 + T cells play a central role in orchestrating adaptive immunity. To better understand the roles of CD4 + T cells in the effects of adjuvants, we investigated the efficacy of a T-dependent influenza virus split vaccine with MF59 or alum in CD4 knockout (CD4KO) and wild-type (WT) mice. CD4 + T cells were required for the induction of IgG antibody responses to the split vaccine and the effects of alum adjuvant. In contrast, MF59 was found to be highly effective in raising isotype-switched IgG antibodies to a T-dependent influenza virus split vaccine in CD4KO mice or CD4-depleted WT mice equivalent to those in intact WT mice, thus overcoming the deficiency of CD4 + T cells in helping B cells and inducing immunity against influenza virus. Vaccination with the MF59-adjuvanted influenza virus vaccine was able to induce protective CD8 + T cells and long-lived antibody-secreting cells in CD4KO mice. The effects of MF59 adjuvant in CD4KO mice might be associated with uric acid, inflammatory cytokines, and the recruitment of multiple immune cells at the injection site, but their cellularity and phenotypes were different from those in WT mice. These findings suggest a new paradigm of CD4-independent adjuvant mechanisms, providing the rationales to improve vaccine efficacy in infants, the elderly, immunocompromised patients, as well as healthy adults. IMPORTANCE MF59-adjuvanted influenza vaccines were licensed for human vaccination, but the detailed mechanisms are not fully elucidated. CD4 + T cells are required to induce antibody isotype switching and long-term memory responses. In contrast, we discovered that MF59 was highly effective in inducing isotype-switched IgG antibodies and long-term protective immune responses to a T-dependent influenza vaccine independent of CD4 + T cells. These findings are highly significant for the following reasons: (i) MF59 can overcome a defect of CD4 + T cells in inducing protective immunity to vaccination with a T-dependent influenza virus vaccine; (ii) a CD4-independent pathway can be an alternative mechanism for certain adjuvants such as MF59; and (iii) this study has significant implications for improving vaccine efficacies in young children, the elderly, and immunocompromised populations.

Description: Vaccine adjuvant effects in the CD4-deficient condition largely remain unknown. We investigated the roles of combined monophosphoryl lipid A (MPL) and aluminum hydroxide (Alum) adjuvant (MPL+Alum) in inducing immunity after immunization of CD4 knockout (CD4KO) and wild-type (WT) mice with T-dependent influenza vaccine. MPL+Alum adjuvant mediated IgG isotype-switched Abs, IgG-secreting cell responses, and protection in CD4KO mice, which were comparable to those in WT mice. In contrast, Alum adjuvant effects were dependent on CD4 + T cells. MPL+Alum adjuvant was effective in recruiting monocytes and neutrophils as well as in protecting macrophages from Alum-mediated cell loss at the injection site in CD4KO mice. MPL+Alum appeared to attenuate MPL-induced inflammatory responses in WT mice, likely improving the safety. Additional studies in CD4-depleted WT mice and MHC class II KO mice suggest that MHC class II + APCs contribute to providing alternative B cell help in the CD4-deficient condition in the context of MPL+Alum–adjuvanted vaccination.

Description: Force-based atomic force microscopy (AFM) was used to detect HCV ( hepatitis C virus ) RNA directly and to quantitatively analyse it without the need for reverse transcription or amplification. Capture and detection DNA probes were designed. The former was spotted onto a substrate with a conventional microarrayer, and the latter was immobilized on an AFM probe. To control the spacing between the immobilized DNAs on the surface, dendron self-assembly was employed. Force–distance curves showed that the mean force of the specific unbinding events was 32 ± 5 pN, and the hydrodynamic distance of the captured RNA was 30–60 nm. Adhesion force maps were generated with criteria including the mean force value, probability of obtaining the specific curves and hydrodynamic distance. The maps for the samples whose concentrations ranged from 0.76 fM to 6.0 fM showed that cluster number has a linear relationship with RNA concentration, while the difference between the observed number and the calculated one increased at low concentrations. Because the detection limit is expected to be enhanced by a factor of 10 000 when a spot of 1 micron diameter is employed, it is believed that HCV RNA of a few copy numbers can be detected by the use of AFM.

Description: All cancers harbor molecular alterations in their genomes. The transcriptional consequences of these somatic mutations have not yet been comprehensively explored in lung cancer. Here we present the first large scale RNA sequencing study of lung adenocarcinoma, demonstrating its power to identify somatic point mutations as well as transcriptional variants such as gene fusions, alternative splicing events, and expression outliers. Our results reveal the genetic basis of 200 lung adenocarcinomas in Koreans including deep characterization of 87 surgical specimens by transcriptome sequencing. We identified driver somatic mutations in cancer genes including EGFR , KRAS , NRAS , BRAF , PIK3CA , MET , and CTNNB1 . Candidates for novel driver mutations were also identified in genes newly implicated in lung adenocarcinoma such as LMTK2 , ARID1A , NOTCH2 , and SMARCA4 . We found 45 fusion genes, eight of which were chimeric tyrosine kinases involving ALK , RET , ROS1 , FGFR2 , AXL , and PDGFRA . Among 17 recurrent alternative splicing events, we identified exon 14 skipping in the proto-oncogene MET as highly likely to be a cancer driver. The number of somatic mutations and expression outliers varied markedly between individual cancers and was strongly correlated with smoking history of patients. We identified genomic blocks within which gene expression levels were consistently increased or decreased that could be explained by copy number alterations in samples. We also found an association between lymph node metastasis and somatic mutations in TP53 . These findings broaden our understanding of lung adenocarcinoma and may also lead to new diagnostic and therapeutic approaches.

Description: Epstein-Barr virus (EBV) is a human herpesvirus associated with various tumors. Rather than going through the lytic cycle, EBV maintains latency by limiting the expression of viral genes in tumors. Viral microRNAs (miRNAs) of some herpesviruses have been reported to directly target immediate early genes and suppress lytic induction. In this study, we investigated whether BamHI-A rightward transcript (BART) miRNAs targeted two EBV immediate early genes, BZLF1 and BRLF1 . Bioinformatic analysis predicted that 12 different BART miRNAs would target BRLF1 . Of these, the results of a luciferase reporter assay indicated that only one interacted with the 3' untranslated region (UTR) of BRLF1 : miR-BART20-5p. miR-BART20-5p's effect on gene expression involved two putative seed match sites in the BRLF1 3' UTR, but a mutant version of the miRNA, miR-BART20-5pm, had no effect on expression. As expected from the fact that the entire 3' UTR of BZLF1 resides within the 3' UTR of BRLF1 , miR-BART20-5p interacted with the 3' UTR of BZLF1 as well. BZLF1 and BRLF1 mRNA and protein expression was suppressed in cells of an AGS cell line infected with the recombinant Akata strain of EBV (AGS-EBV) transfected with a miR-BART20-5p mimic. The expression of various EBV early proteins was also suppressed by the miR-BART20-5p mimic. In contrast, BZLF1 and BRLF1 expression in AGS-EBV cells transfected with a miR-BART20-5p inhibitor was enhanced. Furthermore, progeny virus production was suppressed by the miR-BART20-5p mimic and enhanced by the miR-BART20-5p inhibitor in AGS-EBV cells induced for the lytic cycle. Our data suggest that miR-BART20-5p plays a key role in latency maintenance in EBV-associated tumors by directly targeting immediate early genes. IMPORTANCE Herpesviruses maintain latency using various mechanisms and establish lifelong infection in the host. From time to time, herpesviruses are reactivated and express immediate early genes which trigger a lytic cascade, leading to the production of progeny viruses. Recently, some herpesviruses have been shown to use their own microRNAs (miRNAs) to downregulate immediate early genes to inhibit the lytic cycle. This study presents evidence that EBV also downregulates two immediate early genes by miR-BART20-5p to suppress the lytic cycle and progeny virus production. Overall, this is the first study to report the direct regulation of EBV immediate early genes by an EBV miRNA, implying its likely importance in latency maintenance in EBV-associated tumors.

Description: Objective Few studies have reported on injury of the mammillothalamic tract (MTT) in patients with stroke. However, no study in patients with subarachnoid haemorrhage (SAH) has been reported. Using diffusion tensor tractography, we attempted to investigate injury of the MTT in patients with SAH. Methods We recruited 16 patients with SAH and 15 control participants. DTI was obtained at 5.7±1.5 weeks after onset and reconstruction of the MTT was performed using the probabilistic tractography method. The fractional anisotropy (FA) value and tract number of the MTT and the Mini-Mental State Examination (MMSE) score were determined. Values of FA and tract volume showing a decrement of more than two SDs that of normal control were defined as abnormal. Results The FA value and tract volume in the patient group were significantly lower than those in the control group (p〈0.05). In addition, MMSE showed strong (r=0.67, p=0.005) positive correlation with tract volume without correlation with FA. In the individual analysis, 16 MTTs of 32 MTTs in 16 patients showed abnormalities of the MTT in terms of the FA value, the tract volume or the presence of a reconstructed MTT. As a result, 10 (62.5%) of 16 patients showed abnormality of the MTT in at least one hemisphere. Conclusions We found that patients with SAH showed injury of the MTT and this injury showed correlation with cognitive dysfunction.

Description: Background and Purpose— Clear elucidation of the exact pathophysiological mechanisms of motor weakness in patients with subarachnoid hemorrhage has not yet been achieved. We attempted to investigate injury to the corticospinal tract in patients with subarachnoid hemorrhage using diffusion tensor imaging. Methods— Twenty-two patients with subarachnoid hemorrhage and 24 control subjects were recruited for this study. DTI-Studio software was used for reconstruction of the corticospinal tract. We measured fractional anisotropy and apparent diffusion coefficient values at 5 regions of interest along the corticospinal tract pathway including: the corona radiata, the posterior limb of the internal capsule, the upper midbrain, the midpons, and the upper medulla. Results— Fractional anisotropy value for the midbrain region of interest was lower in the patient group compared with the control group without change of apparent diffusion coefficient value ( P 〈0.05). By contrast, fractional anisotropy and apparent diffusion coefficient values of the other 4 regions of interest were not different between the patient and control groups. Conclusions— Injury of the corticospinal tract at the midbrain was observed in patients with subarachnoid hemorrhage. Injury of the corticospinal tract at the midbrain appears to be one of the various pathophysiological mechanisms for motor weakness after subarachnoid hemorrhage.