Kallikrein 6 secreted by oligodendrocytes regulates the progression of experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS), and experimental autoimmune encephalomyelitis (EAE) is a well-established animal model of the disease. Here, we examined the pathophysiological role of Kallikrein 6 (Klk6), a serine protease produced by oligodendrocytes (OLs), in EAE using Klk6 knockout (Klk6-/-) mice. Compared with Klk6+/+ (wild-type) mice, Klk6-/- mice showed milder EAE symptoms, including delayed onset and milder paralysis. Loss of Klk6 suppressed matrix metalloprotease-9 expression and diminished the infiltration of peripheral inflammatory cells into the CNS by decreasing blood-brain barrier (BBB) permeability and reducing expression levels of inflammatory cytokines, chemokines and their receptors. Scanning electron microscopic analysis revealed demyelination characterized by myelin detachment from the axons in the early phase of EAE progression (days 3-7) in Klk6+/+ mice but not in Klk6-/- mice. Interestingly, anti-MOG (myelin oligodendrocyte glycoprotein) autoantibody was also detected in the cerebrospinal fluid (CSF) and spinal cord on day 3 after MOG immunization. Furthermore, treatment of primary cultured OLs with anti-MOG autoantibody induced oligodendroglial morphological changes and increases in myelin basic protein and Klk6 expression. We also developed a novel enzyme-linked immunoabsorbent assay method for detecting activated KLK6 in human CSF. In human autopsy brain samples, expression of active KLK6 was detected in OLs using an antibody that specifically recognizes the protein's activated form. Taken together, our findings demonstrate that Klk6 secreted by OLs plays a critical role in the pathogenesis of EAE/MS and that it might serve as a potential therapeutic target for MS.

[Analysis of the Squamous Cell Carcinoma Antigen (SCCA) Which Has a Significant Difference in Values between CLIA and FEIA--Case Report of Extra Glycosylation in SCCA].

Squamous cell carcinoma antigen (SCCA) is a glycoprotein that belongs to the serine protease inhibitor family. Clinically, it has been utilized as a tumor marker for squamous cell carcinoma. In clinical laboratories, SCCA is measured by several immunoassays. Recently, a number of studies have been reported that there is a significant difference in values between the immunoassays, attributing to SCCA-immunoglobulin complex. We found a case with significant difference in the SCCA value between CLIA and FEIA. In this case, SCCA-Immunoglobulin complex was not confirmed by gel filtration analysis. Interestingly, 5 to 10 kDa slightly-high molecular weight SCCA compared to control was detected by immunoblotting assay. It may be suspected that the aberrant glycosyl modification of SCCA influenced the reactivity to immunoassays.

The role of childhood and adulthood trauma and appraisal of self-discrepancy in overgeneral memory retrieval.

The aim of this study was to examine the contributory role of childhood and adulthood trauma events and the appraisal of self-discrepancy (the magnitude and distress) in overgeneral memory retrieval (OGM) using a non-clinical sample. Participants with a history of childhood trauma (n=29); adulthood trauma only (n=17) or no-trauma (n=26) participated in this study. The childhood trauma group showed a significantly higher level of general negative memory retrieval than the no-trauma group. Conversely, the adulthood trauma group showed a significantly higher level of general threat memory retrieval than the no-trauma group. The perceived similarity and distress relating to the perceived similarity between participants' "actual self" and "feared self" significantly predicted the level of OGM, even after controlling for the impact of a history of adult or child trauma.

Kinetic study of the daytime atmospheric fate of (Z)-3-hexenal.

Rate coefficients for three daytime atmospheric reactions of (Z)-3-hexenal (3HA)-photolysis (J(1)), reaction with OH radicals (k(2)), and reaction with ozone (k(3))-were measured at 760 Torr and 298 K using a 6 m(3) photochemical reaction chamber. The UV absorption cross sections (σ(3HA)(λ)) were obtained in the wavelength range 240-350 nm. The photodissociation rate of 3HA relative to that of NO(2) was measured by a solar simulator at 760 Torr and was determined to be J(1)/J(NO2) = (4.7 ± 0.4) × 10(-3). Using the obtained σ(3HA)(λ) and J(1)/J(NO2), the effective photodissociation quantum yield was calculated to be Φ(3HA) = 0.25 ± 0.06. The rate coefficient for the reaction with OH radicals was measured by the relative rate method with three reference compounds and was determined to be k(2) = (6.9 ± 0.9) × 10(-11) cm(3) molecule(-1) s(-1). The rate coefficient for the reaction with ozone was measured by an absolute method and was determined to be k(3) = (3.5 ± 0.2) × 10(-17) cm(3) molecule(-1) s(-1). Using the obtained rate coefficients, the daytime atmospheric lifetime of 3HA was estimated.

A new type of lectin discovered in a fish, flathead (Platycephalus indicus), suggests an alternative functional role for mammalian plasma kallikrein.

A skin mucus lectin exhibiting a homodimeric structure and an S-S bond between subunits of ~40 kDa was purified from flathead Platycephalus indicus (Scorpaeniformes). This lectin, named FHL (FlatHead Lectin), exhibited mannose-specific activity in a Ca(2+)-dependent manner. Although FHL showed no homology to any previously reported lectins, it did exhibit ~20% identity to previously discovered plasma kallikreins and coagulation factor XIs of mammals and Xenopus laevis. These known proteins are serine proteases and play pivotal roles in the kinin-generating system or the blood coagulation pathway. However, alignment analysis revealed that while FHL lacked a serine protease domain, it was homologous to the heavy-chain domain of plasma kallikreins and coagulation factor XI therefore suggesting that FHL is not an enzyme but rather a novel animal lectin. On the basis of this finding, we investigated the lectin activity of human plasma kallikrein and revealed that it could indeed act as a lectin. Other genes homologous to FHL were also found in the genome databases of some fish species, but not in mammals. In contrast, plasma kallikreins and coagulation factor XI have yet to be identified in fish. The present findings suggest that these mammalian enzymes may have originally emerged as a lectin and may have evolved into molecules with protease activity after separation from common ancestors.

Preliminary investigation of misconceptions and expectations of the effects of traumatic brain injury and symptom reporting.

OBJECTIVE: This study aimed, first, to introduce a new measure for examining misconceptions of the effects of traumatic brain injury (TBI) and, secondly, to conduct a preliminary investigation of the impact of misconceptions and expectations on level of symptom reporting. METHODS: The Head Injury Knowledge Scale (HIKS) was piloted for its utility in this study using a sample (n = 13) of individuals with brain injury. A sample of 99 uninjured participants was randomly allocated into either TBI simulation (n = 59) or control (n = 40) groups using a 3:2 allocation ratio. All participants initially completed the HIKS and then controls completed the Symptom Expectancy Checklist (SEC). The TBI simulation group was presented with a simulation scenario concerning severe TBI and then completed the SEC. Retrospective data on the SEC for individuals with a severe TBI (n = 38) were used for comparison purposes. RESULTS: The HIKS was deemed suitable for use in the present study based on pilot data and internal consistency analysis of the two sub-scales (α > 0.60). The uninjured participants displayed a greater tendency to over-generalize than minimize the effects of TBI (p < 0.001). The TBI simulation group reported a higher level of symptoms on the SEC than controls and those with severe TBI (p < 0.001). However, level of symptom reporting on the SEC in the simulation group was not significantly related to misconceptions of the effects of TBI (p > 0.05). CONCLUSIONS: Based on these preliminary findings, the HIKS may provide a useful measure of the relative tendency to over-generalize or minimize the effects of TBI. However, further research is needed to investigate the reliability and validity of the HIKS prior to clinical use.

Efficient ssODN-Mediated Targeting by Avoiding Cellular Inhibitory RNAs through Precomplexed CRISPR-Cas9/sgRNA Ribonucleoprotein.

Combined with CRISPR-Cas9 technology and single-stranded oligodeoxynucleotides (ssODNs), specific single-nucleotide alterations can be introduced into a targeted genomic locus in induced pluripotent stem cells (iPSCs); however, ssODN knockin frequency is low compared with deletion induction. Although several Cas9 transduction methods have been reported, the biochemical behavior of CRISPR-Cas9 nuclease in mammalian cells is yet to be explored. Here, we investigated intrinsic cellular factors that affect Cas9 cleavage activity in vitro. We found that intracellular RNA, but not DNA or protein fractions, inhibits Cas9 from binding to single guide RNA (sgRNA) and reduces the enzymatic activity. To prevent this, precomplexing Cas9 and sgRNA before delivery into cells can lead to higher genome editing activity compared with Cas9 overexpression approaches. By optimizing electroporation parameters of precomplexed ribonucleoprotein and ssODN, we achieved efficiencies of single-nucleotide correction as high as 70% and loxP insertion up to 40%. Finally, we could replace the HLA-C1 allele with the C2 allele to generate histocompatibility leukocyte antigen custom-edited iPSCs.

Targeted Disruption of HLA Genes via CRISPR-Cas9 Generates iPSCs with Enhanced Immune Compatibility.

Induced pluripotent stem cells (iPSCs) have strong potential in regenerative medicine applications; however, immune rejection caused by HLA mismatching is a concern. B2M gene knockout and HLA-homozygous iPSC stocks can address this issue, but the former approach may induce NK cell activity and fail to present antigens, and it is challenging to recruit rare donors for the latter method. Here, we show two genome-editing strategies for making immunocompatible donor iPSCs. First, we generated HLA pseudo-homozygous iPSCs with allele-specific editing of HLA heterozygous iPSCs. Second, we generated HLA-C-retained iPSCs by disrupting both HLA-A and -B alleles to suppress the NK cell response while maintaining antigen presentation. HLA-C-retained iPSCs could evade T cells and NK cells in vitro and in vivo. We estimated that 12 lines of HLA-C-retained iPSCs combined with HLA-class II knockout are immunologically compatible with >90% of the world's population, greatly facilitating iPSC-based regenerative medicine applications.

A meta-analytic review of overgeneral memory: The role of trauma history, mood, and the presence of posttraumatic stress disorder.

A number of studies suggest that a history of trauma, depression, and posttraumatic stress disorder (PTSD) are associated with autobiographical memory deficits, notably overgeneral memory (OGM). However, whether there are any group differences in the nature and magnitude of OGM has not been evaluated. Thus, a meta-analysis was conducted to quantify group differences in OGM. The effect sizes were pooled from studies examining the effect on OGM from a history of trauma (e.g., childhood sexual abuse), and the presence of PTSD or current depression (e.g., major depressive disorder). Using multiple search engines, 13 trauma studies and 12 depression studies were included in this review. A depression effect was observed on OGM with a large effect size, and was more evident by the lack of specific memories, especially to positive cues. An effect of trauma history on OGM was observed with a medium effect size, and this was most evident by the presence of overgeneral responses to negative cues. The results also suggested an amplified memory deficit in the presence of PTSD. That is, the effect sizes of OGM among individuals with PTSD were very large and relatively equal across different types of OGM. Future studies that directly compare the differences of OGM among 4 samples (i.e., controls, current depression without trauma history, trauma history without depression, and trauma history and depression) would be warranted to verify the current findings.

Characterization of two novel mutations of the antithrombin gene observed in Japanese thrombophilic patients.

We investigated the molecular basis of reduced functional levels of antithrombin (AT) in two individuals suffering from thromboembolic events. In each case direct sequencing of amplified DNA revealed 13,260-13,262 del in one patient and 2511C>A in the other patient, predicting a heterozygous E381del and P16H, respectively. Both patients had no 20210A allele and factor V Leiden mutation. To understand the molecular mechanism responsible for antithrombin deficiency, stable expression experiments were performed using HEK293 cells transfected with the expression vector containing the wild-type or the mutated recombinant cDNA. In these experiments, the media levels of the two mutated antithrombins were the same as that of wild type, but the specific activity of the E381del mutant decreased significantly compared with that of wild type. These results showed that the E381del mutation was responsible for type II deficiency, whereas the other mutation, P16H, did not produce any definite abnormality which could contribute to antithrombin deficiency.

A meta-analysis of cognitive impairment and decline associated with adjuvant chemotherapy in women with breast cancer.

A meta-analysis was performed to quantify the magnitude and nature of the association between adjuvant chemotherapy and performance on a range of cognitive domains among breast cancer patients. A total of 27 studies (14 cross-sectional, 8 both cross-sectional and prospective, and 5 prospective) were included in the analyses, involving 1562 breast cancer patients who had undergone adjuvant chemotherapy and 2799 controls that included breast cancer patients who did not receive adjuvant chemotherapy. A total of 737 effect sizes (Cohen's d) were calculated for cross-sectional and prospective longitudinal studies separately and classified into eight cognitive domains. The mean effect sizes varied across cross-sectional and prospective longitudinal studies (ranging from -1.12 to 0.62 and -0.29 to 1.12, respectively). Each cognitive domain produced small effect sizes for cross-sectional and prospective longitudinal studies (ranging from -0.25 to 0.41). Results from cross-sectional studies indicated a significant association between adjuvant chemotherapy and cognitive impairment that held across studies with varied methodological approaches. For prospective studies, results generally indicated that cognitive functioning improved over time after receiving adjuvant chemotherapy. Greater cognitive impairment was reported in cross-sectional studies comparing chemotherapy groups with healthy control groups. Results suggested that cognitive impairment is present among breast cancer patients irrespective of a history of chemotherapy. Prospective longitudinal research is warranted to examine the degree and persisting nature of cognitive impairment present both before and after chemotherapy, with comparisons made to participants' cognitive function prior to diagnosis. Accurate understanding of the effects of chemotherapy is essential to enable informed decisions regarding treatment and to improve quality of life among breast cancer patients.

Dark-induced mRNA instability involves RNase E/G-type endoribonuclease cleavage at the AU-box and SD sequences in cyanobacteria.

Light-responsive gene expression is crucial to photosynthesizing organisms. Here, we studied functions of cis-elements (AU-box and SD sequences) and a trans-acting factor (ribonuclease, RNase) in light-responsive expression in cyanobacteria. The results indicated that AU-rich nucleotides with an AU-box, UAAAUAAA, just upstream from an SD confer instability on the mRNA under darkness. An RNase E/G homologue, Slr1129, of the cyanobacterium Synechocystis sp. strain PCC 6803 was purified and confirmed capable of endoribonucleolytic cleavage at the AU- (or AG)-rich sequences in vitro. The cleavage depends on the primary target sequence and secondary structure of the mRNA. Complementation tests using Escherichia coli rne/rng mutants showed that Slr1129 fulfilled the functions of both the RNase E and RNase G. An analysis of systematic mutations in the AU-box and SD sequences showed that the cis-elements also affect significantly mRNA stability in light-responsive genes. These results strongly suggested that dark-induced mRNA instability involves RNase E/G-type cleavage at the AU-box and SD sequences in cyanobacteria. The mechanical impact and a possible common mechanism with RNases for light-responsive gene expression are discussed.

Identification of simultaneous mutation of fibrinogen alpha chain and protein C genes in a Japanese kindred.

Afibrinogenaemia usually induces a bleeding tendency during infancy, whereas protein C deficiency increases susceptibility to thrombosis in children or adolescence. Mutations of these genes have been, therefore, established as independent risk factors for coagulation disorders. We describe the homozygous mutation of the fibrinogen alpha chain gene and additional heterozygous mutation of the protein C gene in a male infant who showed prolonged umbilical bleeding after birth. On examination, the plasma fibrinogen was undetectable, and the activity and antigen level of protein C were reduced. The patient showed no fibrinogen Aalpha chain as well as Bbeta and gamma chains by Western blotting. The sequencing analysis showed the homozygous deletion of 1238 bases from intron 3 at position 2008 to intron 4 at position 3245 in the fibrinogen alpha chain gene. Both parents were heterozygous carriers of this mutation. In this patient, an additional mutation was also detected in the protein C gene: the heterozygous deletion of exon 7 at position 6161-6163 or 6164-6166, resulting the deletion of one amino acid (Lys150 or 151). His mother was also a carrier of this mutation. As the simultaneous mutation of the fibrinogen alpha chain and protein C genes has not been previously reported, the influence of the interaction between these two mutations on the clinical manifestations of this patient should be carefully monitored for a long period.