Generation of a tyrosine hydroxylase-2A-Cre knockin non-human primate model by homology-directed-repair-biased CRISPR genome editing.

Non-human primates (NHPs) are the closest animal model to humans; thus, gene engineering technology in these species holds great promise for the elucidation of higher brain functions and human disease models. Knockin (KI) gene targeting is a versatile approach to modify gene(s) of interest; however, it generally suffers from the low efficiency of homology-directed repair (HDR) in mammalian cells, especially in non-expressed gene loci. In the current study, we generated a tyrosine hydroxylase (TH)-2A-Cre KI model of the common marmoset monkey (marmoset; Callithrix jacchus) using an HDR-biased CRISPR-Cas9 genome editing approach using Cas9-DN1S and RAD51. This model should enable labeling and modification of a specific neuronal lineage using the Cre-loxP system. Collectively, the current study paves the way for versatile gene engineering in NHPs, which may be a significant step toward further biomedical and preclinical applications.

A novel murine model of autoimmune dysautonomia by α3 nicotinic acetylcholine receptor immunization.

We aimed to establish a novel murine model of autoimmune autonomic ganglionopathy (AAG), which represents autoimmune dysautonomia, associated with MHC class II to understand its pathomechanism and the pathogenicity of nicotinic acetylcholine receptor (nAChR) antibodies. The amino acid sequence of the mouse nAChRα3 protein was analyzed using an epitope prediction tool to predict the possible MHC class II binding mouse nAChRα3 peptides. We focused on two nAChRα3 peptides in the extracellular region, and experimental AAG (EAAG) was induced by immunization of C57BL/6 mice with these two different peptides. EAAG mice were examined both physiologically and histologically. Mice with EAAG generated nAChRα3 antibodies and exhibited autonomic dysfunction, including reduced heart rate, excessive fluctuations in systolic blood pressure, and intestinal transit slowing. Additionally, we observed skin lesions, such as alopecia and skin ulcers, in immunized mice. Neuronal cell density in the sympathetic cervical ganglia in immunized mice was significantly lower than that in control mice at the light microscopic level. We interpreted that active immunization of mice with nAChRα3 peptides causes autonomic dysfunction similar to human AAG induced by an antibody-mediated mechanism. We suggested a mechanism by which different HLA class II molecules might preferentially affect the nAChR-specific immune response, thus controlling diversification of the autoantibody response. Our novel murine model mimics AAG in humans and provides a useful tool to investigate its pathomechanism.

Observed adducts on positive mode direct analysis in real time mass spectrometry - Proton/ammonium adduct selectivities of 600-sample in-house chemical library.

In this study, direct analysis in real time adduct selectivities of a 558 in-house high-resolution mass spectrometry sample library was evaluated. The protonated molecular ion ([M + H]+) was detected in 462 samples. The ammonium adduct ion ([M + NH4]+) was also detected in 262 samples. [M + H]+ and [M + NH4]+ molecular ions were observed simultaneously in 166 samples. These adduct selectivities were related to the elemental compositions of the sample compounds. [M + NH4]+ selectivity correlated with the number of oxygen atom(s), whereas [M + H]+ selectivity correlated with the number of nitrogen atom(s) in the elemental compositions. For compounds including a nitrogen atom and an oxygen atom [M + H]+ was detected; [M + NH4]+ was detected for compounds including an oxygen atom only. Density functional theory calculations were performed for selected library samples and model compounds. Energy differences were observed between compounds detected as [M + H]+ and [M + NH4]+, and between compounds including a nitrogen atom and an oxygen atom in their elemental compositions. The results suggested that the presence of oxygen atoms stabilizes [M + NH4]+, but not every oxygen atom has enough energy for detection of [M + NH4]+. It was concluded that the nitrogen atom(s) and oxygen atom(s) in the elemental compositions play important roles in the adduct formation in direct analysis in real time mass spectrometry.

Nudged elastic band method and density functional theory calculation for finding a local minimum energy pathway of p-benzoquinone and phenol fragmentation in mass spectrometry.

Analysis of the fragmentation pathways of molecules in mass spectrometry gives a fundamental insight into gas-phase ion chemistry. However, the conventional intrinsic reaction coordinates method requires knowledge of the transition states of ion structures in the fragmentation pathways. Herein, we use the nudged elastic band method, using only the initial and final state ion structures in the fragmentation pathways, and report the advantages and limitations of the method. We found a minimum energy path of p-benzoquinone ion fragmentation with two saddle points and one intermediate structure. The primary energy barrier, which corresponded to the cleavage of the C-C bond adjacent to the CO group, was calculated to be 1.50 eV. An additional energy barrier, which corresponded to the cleavage of the CO group, was calculated to be 0.68 eV. We also found an energy barrier of 3.00 eV, which was the rate determining step of the keto-enol tautomerization in CO elimination from the molecular ion of phenol. The nudged elastic band method allowed the determination of a minimum energy path using only the initial and final state ion structures in the fragmentation pathways, and it provided faster than the conventional intrinsic reaction coordinates method. In addition, this method was found to be effective in the analysis of the charge structures of the molecules during the fragmentation in mass spectrometry.

Possible Contribution of Zerumbone-Induced Proteo-Stress to Its Anti-Inflammatory Functions via the Activation of Heat Shock Factor 1.

Zerumbone is a sesquiterpene present in Zinger zerumbet. Many studies have demonstrated its marked anti-inflammatory and anti-carcinogenesis activities. Recently, we showed that zerumbone binds to numerous proteins with scant selectivity and induces the expression of heat shock proteins (HSPs) in hepatocytes. To dampen proteo-toxic stress, organisms have a stress-responsive molecular machinery, known as heat shock response. Heat shock factor 1 (HSF1) plays a key role in this protein quality control system by promoting activation of HSPs. In this study, we investigated whether zerumbone-induced HSF1 activation contributes to its anti-inflammatory functions in stimulated macrophages. Our findings showed that zerumbone increased cellular protein aggregates and promoted nuclear translocation of HSF1 for HSP expression. Interestingly, HSF1 down-regulation attenuated the suppressive effects of zerumbone on mRNA and protein expressions of pro-inflammatory genes, including inducible nitric oxide synthase and interlukin-1ß. These results suggest that proteo-stress induced by zerumbone activates HSF1 for exhibiting its anti-inflammatory functions.

FDG-PET/CT as a post-treatment restaging tool in urothelial carcinoma: Comparison with contrast-enhanced CT.

PURPOSE: To assess the clinical usefulness of FDG-PET/CT in the diagnosis of recurrent and metastatic urothelial carcinoma in comparison with contrast-enhanced CT. MATERIALS AND METHODS: Eighty-three patients who had undergone treatment for histopathologically proven urothelial carcinoma underwent whole-body FDG-PET/CT and contrast-enhanced CT for suspected recurrence within a time interval of two weeks. Patient-based analysis and lesion sites besides the urinary tract, as interpreted by two experienced readers, were compared between the two modalities using McNemar test. Lesion status was determined on the basis of histopathology, radiological imaging and clinical follow-up for longer than 6 months. RESULT: Patient-based analysis showed that the sensitivity, specificity, and accuracy of FDG-PET/CT were 97.4%, 93.3% and 95.2%, respectively, whereas those of contrast-enhanced CT were 86.8%, 93.3% and 90.4%, respectively. The sensitivity and accuracy of FDG-PET/CT were higher than contrast-enhanced CT without significant difference (p=0.13). The sensitivity of FDG-PET/CT for diagnosis of bone metastasis was significantly higher than that of contrast-enhanced CT (93.8% vs. 25%, p=0.0026). CONCLUSION: FDG-PET/CT is a more accurate modality than CT for assessment of recurrence outside the urinary tract in patients with urothelial carcinoma, especially for bone lesion. Cystoscopy, urine cytology, and FDG-PET/CT are complementary procedures and may have a definite management role.

Diagnostic and prognostic value of (18)F-FDG PET/CT for axillary lymph node staging in patients with breast cancer.

PURPOSE: To investigate the diagnostic and prognostic value of (18)F-FDG-PET/CT for axillary lymph node (LN) staging in breast cancer patients, employing histologic evaluation as the reference. METHODS: Among 196 patients with biopsy-proven breast cancer who had undergone (18)F-FDG-PET/CT before mastectomy or breast-conserving surgery with sentinel LN biopsy and/or axillary LN dissection, 200 axillae were retrospectively analyzed by visual assessment and quantitatively using SUVmax. LN SUVmax as well as other clinicopathological features were assessed for their prognostic value using the log-rank test and Cox method. RESULTS: Metastasis was diagnosed histopathologically in 56 (28 %) axillae. The sensitivity, specificity, and accuracy of visual PET/CT for diagnosing node metastasis were 55.4, 95.8, and 84.5 %, respectively. When the optimal discriminative SUVmax cutoff was 1.5, these figures were 51.8, 97.2, and 84.5 %, respectively. Fourteen of 55 patients (25.5 %) with LN metastases suffered a recurrence during follow-up (median 39 months). Patients with a high nodal SUVmax (≥1.7) had a significantly lower progression-free survival rate than those with a low SUVmax (p = 0.0499). Axillary nodal and primary tumor SUVmax as well as estrogen receptor status were significantly associated with recurrence. CONCLUSION: Axillary nodal SUVmax may be a prognostic indicator of disease recurrence in patients with axillary LN metastases.

Near-Infrared Imaging Using a High-Speed Monitoring Near Infrared Hyperspectral Camera (Compovision).

This review paper reports near-infrared (NIR) imaging studies using a newly-developed NIR camera, Compovision. Compovision can measure a significantly wide area of 150 mmX 250 mm at high speed of between 2 and 5 s. It enables a wide spectral region measurement in the 1,000-2,350 nm range at 6 nm intervals. We investigated the potential of Compovision in the applications to industrial problems such as the evaluation of pharmaceutical tablets and polymers. Our studies have demonstrated that NIR imaging based on Compovision can solve several issues such as long acquisition times and relatively low sensitivity of detection. NIR imaging with Compovision is strongly expected to be applied not only to pharmaceutical tablet monitoring and polymer characterization but also to various applications such as those to food products, biomedical substances and organic and inorganic materials.

Association between ¹8F-FDG uptake and molecular subtype of breast cancer.

PURPOSE: To determine whether (18)F-FDG uptake in breast cancer correlates with immunohistochemically defined subtype and is able to predict molecular subtypes. METHODS: This retrospective study involved 306 patients with 308 mass-type invasive breast cancers (mean size 2.65 cm, range 1.0-15.0 cm) who underwent (18)F-FDG PET/CT before therapy. The correlations between primary tumour (18)F-FDG uptake on PET/CT, expressed as SUVmax, and clinicopathological findings and molecular subtype, i.e. luminal A, luminal B (HER2-negative), luminal B (HER2-positive), HER2-positive and triple-negative, were analysed. The predictors of these subtypes were investigated. RESULTS: The mean SUVmax of the 308 tumours was 5.33 ± 3.63 (range 1.15-19.01). Among the subtypes of the 308 tumours, 87 (28.2 %) were luminal A, 111 (36.0 %) were luminal B (HER2-negative), 31 (10.1 %) were luminal B (HER2-positive), 26 (8.4 %) were HER2-positive and 53 (17.2 %) were triple-negative, and the corresponding mean SUVmax were 3.41 ± 2.07 (range 1.18-14.30), 5.17 ± 3.52 (range 1.35-19.01), 6.57 ± 3.84 (range 1.42-15.58), 7.55 ± 3.63 (range 2.30-13.60) and 6.97 ± 4.17 (range 1.15-16.06), respectively. A cut-off value of 3.60 yielded 70.1 % sensitivity and 66.1 % specificity with an area under the receiver operating characteristics curve (AUC) of 0.734 for predicting that a tumour was of the luminal A subtype. A cut-off value of 6.75 yielded 65.4 % sensitivity and 75.2 % specificity with an AUC of 0.704 for predicting a HER2-positive subtype. CONCLUSION: SUVmax, a metabolic semiquantitative parameter, shows a significant correlation with the molecular subtype of breast cancer, and is useful for predicting the luminal A or HER2-positive subtype.

High-speed monitoring of the crystallinity change in poly(lactic acid) during photodegradation by using a newly developed wide area NIR imaging system (Compovision).

We aimed to achieve wide area rapid monitoring of the crystallinity change in poly(lactic acid) (PLA) during photodegradation caused by ultraviolet (UV) light by using a newly developed near-infrared (NIR) camera (Compovison). Several kinds of PLA samples with different crystallinities and their blends with poly[(3)-(R)-hydroxybutyrate] were prepared. Their two-dimensional NIR spectra in the 1,000-2,350-nm region were measured by Compovision at a 5-min interval during photolysis. An intensity decrease of the band in the 1,900-1,925-nm region due to the second overtone of the C = O stretching vibration of PLA was observed during photolysis. This suggests that an anhydride carbonyl is produced during photolysis. The NIR image of the crystallinity change monitored by the band at 1,917 nm in the standard normal variate spectra clearly shows the inhomogeneity of crystal evolution. A logarithmic increase was observed for all identified areas in the PLA film; however, the time to reach the maximum crystallinity was slightly different according to the initial crystallinity of the sample. It is likely that the initial crystallinity of the sample influences the degradation speed more than the degradation amount. These imaging results have provided fundamental chemical insights into the photolytic process for PLA, and at the same time they have demonstrated that the two-dimensional spectral data obtained by Compovision are useful for process monitoring of polymers.