Frequency comb assisted measurement of fundamental transitions of cold H-3(+), H2D+ and D2H+

HI and two of its deuterated variants have been trapped and cooled in a 4K trap machine, and their fundamental vibrational transitions probed with the laser induced reactions method. With the help of a frequency comb system the line centers are determined with high accuracy and precision, typically well below 1 MHz. For the deuterated variants, ground state combination differences allow for comparison with existing rotational THz data, and the accurate prediction thereof. (C) 2015 Elsevier Inc. All rights reserved

Intraoperative radiotherapy with low-energy x-rays after neurosurgical resection of brain metastases: an Augsburg University Medical Center experience

Purpose!#!External-beam radiotherapy (EBRT) is the predominant method for localized brain radiotherapy (LBRT) after resection of brain metastases (BM). Intraoperative radiotherapy (IORT) with 50-kV x‑rays is an alternative way to focally irradiate the resection cavity after BM surgery, with the option of shortening the overall treatment time and limiting normal tissue irradiation.!##!Methods!#!We retrospectively analyzed the outcomes of all patients who underwent neurosurgical resection of BM and 50-kV x‑ray IORT between 2013 and 2020 at Augsburg University Medical Center.!##!Results!#!We identified 40 patients with 44 resected BM treated with 50-kV x‑ray IORT. Median diameter of the resected metastases was 2.8 cm (range 1.5-5.9 cm). Median applied dose was 20 Gy. All patients received standardized follow-up (FU) including 3‑monthly MRI of the brain. Mean FU was 14.4 months, with a median MRI FU for alive patients of 12.2 months. Median overall survival (OS) of all treated patients was 26.4 months (estimated 1‑year OS 61.6%). The observed local control (LC) rate of the resection cavity was 88.6% (estimated 1‑year LC 84.3%). Distant brain control (DC) was 47.5% (estimated 1‑year DC 33.5%). Only 25% of all patients needed WBI in the further course of disease. The observed radionecrosis rate was 2.5%.!##!Conclusion!#!IORT with 50-kV x‑rays is a safe and appealing way to apply LBRT after neurosurgical resection of BM, with low toxicity and excellent LC. Close MRI FU is paramount to detect distant brain failure (DBF) early

Focal cavity radiotherapy after neurosurgical resection of brain metastases: sparing neurotoxicity without compromising locoregional control

PURPOSE: Does focal cavity radiotherapy after resection of brain metastasis “spare” whole-brain radiotherapy, which is associated with toxicity for patients, through the complete course of their disease without compromising long-term local control of the brain? METHODS: We retrospectively analyzed outcomes of patients who underwent adjuvant focal cavity radiotherapy between 2014 and 2021 at our center. RESULTS: A total of 83 patients with 86 resected brain metastases were analyzed. 64% had singular, 36% two to four brain metastases. In cases with multiple metastases, omitted lesions were treated with radiosurgery. Median follow-up was 7.3 months (range 0–71.2 months), 1‑year overall survival rate was 57.8% (95% CI 44.9–68.8%). Radiotherapy was administered with a median biologically effective dose (α/β 10) surrounding the planning target volume of 48 Gy (range 23.4–60 Gy). Estimated 1‑year local control rate was 82.7% (95% CI 67.7–91.2%), estimated 1‑year distant brain control rate was 55.7% (95% CI 40.5–68.4%), estimated 1‑year leptomeningeal disease rate was 16.0% (95% CI 7.3–32.9%). Eleven distant brain recurrences could be salvaged with radiosurgery. In the further course of disease, 14 patients (17%) developed disseminated metastatic disease in the brain. Estimated 1‑year free of whole-brain radiotherapy rate was 72.3% (95% CI 57.1–82.9%). All applied treatments led to an estimated 1‑year neuro-control rate of 79.1% (95% CI 65.0–88.0%), estimated 1‑year radionecrosis rate was 23% (95% CI 12.4–40.5%). CONCLUSION: In our single-center study, focal cavity radiotherapy was associated with high local control. In three out of four patients, whole-brain radiotherapy could be avoided in the complete course of disease, using radiosurgery as salvage approach without compromising neuro-control

AFT complex formation and the BiFC principle.

<p>(A) Spherical nuclear AFT complexes in HEK293 cells cotransfected with APP-Citrine, CFP-Tip60 and HA-Fe65 (arrow). Cells that lack CFP-Tip60 accumulate neither AICD-Citrine nor HA-Fe65 in the nucleus (arrowhead). Nuclei were counterstained with DAPI. Length of bar: 20 µm. (B) Schematic depiction of the BiFC-based AFT complex detection system, where APP and Tip60 are fused to YFP halves. Since Fe65 serves as an adaptor between APP and Tip60, fluorescence complementation only occurs in the presence of all three proteins. (C) Fluorescence maturation in AFT-BiFC. Fluorescence maturation at 30°C was allowed for increasing time periods before FC quantification (n=3 per timepoint, error bars represent SEM). If samples were maintained at 37°C, no maturation occurred (samples labeled 37°C).</p

Subcellular localization of BiFC signals.

<p>HEK293 cells were imaged by confocal microscopy. (A) Cotransfection of bFos-YC and bJun-YN generates a BiFC signal distributed throughout the cell nucleus. (B) Cotransfection of APP-YC, HA-Fe65 and Tip 60-YN results in multiple spherical nuclear BiFC signals from AFT complexes. (C) In some cells, cotransfection also results in formation of extranuclear BiFC signals (arrow), which is consistent with an interaction of Tip60 and AICD/Fe65 also outside the nucleus as previously reported by us [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076094#B8" target="_blank">8</a>]. (D) Cotransfection with APP-YC and APP-YN results in perinuclear fluorescence (E+F). Colocalization of the APP/APP-BiFC signal with calnexin and TGN46 is consistent with the localization of APP/APP-dimers in the ER/Golgi. Nuclei were counterstained with DAPI. Length of bar: 13 µm (A, C, E, F) or 10 µm (B, D).</p

Visualization and quantification of APP intracellular domain-mediated nuclear signaling by bimolecular fluorescence complementation

BACKGROUND: The amyloid precursor protein (APP) intracellular domain (AICD) is released from full-length APP upon sequential cleavage by either α- or β-secretase followed by γ-secretase. Together with the adaptor protein Fe65 and the histone acetyltransferase Tip60, AICD forms nuclear multiprotein complexes (AFT complexes) that function in transcriptional regulation. OBJECTIVE: To develop a medium-throughput machine-based assay for visualization and quantification of AFT complex formation in cultured cells. METHODS: We used cotransfection of bimolecular fluorescence complementation (BiFC) fusion constructs of APP and Tip60 for analysis of subcellular localization by confocal microscopy and quantification by flow cytometry (FC). RESULTS: Our novel BiFC-constructs show a nuclear localization of AFT complexes that is identical to conventional fluorescence-tagged constructs. Production of the BiFC signal is dependent on the adaptor protein Fe65 resulting in fluorescence complementation only after Fe65-mediated nuclear translocation of AICD and interaction with Tip60. We applied the AFT-BiFC system to show that the Swedish APP familial Alzheimer's disease mutation increases AFT complex formation, consistent with the notion that AICD mediated nuclear signaling mainly occurs following APP processing through the amyloidogenic β-secretase pathway. Next, we studied the impact of posttranslational modifications of AICD on AFT complex formation. Mutation of tyrosine 682 in the YENPTY motif of AICD to phenylalanine prevents phosphorylation resulting in increased nuclear AFT-BiFC signals. This is consistent with the negative impact of tyrosine phosphorylation on Fe65 binding to AICD. Finally, we studied the effect of oxidative stress. Our data shows that oxidative stress, at a level that also causes cell death, leads to a reduction in AFT-BiFC signals. CONCLUSION: We established a new method for visualization and FC quantification of the interaction between AICD, Fe65 and Tip60 in the nucleus based on BiFC. It enables flow cytometric analysis of AICD nuclear signaling and is characterized by scalability and low background fluorescence

AFT-BiFC requires the presence of Fe65.

<p>(A) After cotransfection of APP-YC, Tip 60-YN and HA-Fe65 the BiFC signal can be detected in a subset of cells in the confocal microscope. In contrast, cotransfection of APP-YC and Tip 60-YN alone does not lead to fluorescence complementation, which indicates absence of direct interaction between AICD and Tip60. Transfection of APP-YC and Tip 60-YN together with the AICD-binding protein MINT1/X11 that traps AICD in the cytosol also does not generate a BiFC signal. Lower panels show BiFC overlay with DAPI nuclear staining. Length of bar: 60 µm. (B) Representative BiFC-FC scatter plots of individual samples. Percentages refer to gated cells. Fluorescence intensity and forward scatter are depicted in arbitrary units. (C) BiFC-FC quantification of HEK293 cells cotransfected with APP-YC and Tip 60-YN together with or without HA-Fe65 or with MINT1/X11 (n=6 vs. 5 vs. 6, error bars represent SEM, *** p<0.005, U-test). The approximately 1% BiFC-positive cells in the APP-Tip60 and APP-X11-Tip60 conditions are most likely due to background autofluorescence of HEK cells as well as fluorescence complementation mediated by endogenous Fe65.</p

Cholesterol 25-hydroxylase on chromosome 10q is a susceptibility gene for sporadic Alzheimer's disease

Alzheimer's disease (AD) is the most common cause of dementia. It is characterized by beta-amyloid (A beta) plaques, neurofibrillary tangles and the degeneration of specifically vulnerable brain neurons. We observed high expression of the cholesterol 25-hydroxylase (CH25H) gene in specifically vulnerable brain regions of AD patients. CH25H maps to a region within 10q23 that has been previously linked to sporadic AD. Sequencing of the 5' region of CH25H revealed three common haplotypes, CH25Hchi2, CH25Hchi3 and CH25Hchi4; CSF levels of the cholesterol precursor lathosterol were higher in carriers of the CH25Hchi4 haplotype. In 1,282 patients with AD and 1,312 healthy control subjects from five independent populations, a common variation in the vicinity of CH25H was significantly associated with the risk for sporadic AD (p = 0.006). Quantitative neuropathology of brains from elderly non-demented subjects showed brain A beta deposits in carriers of CH25Hchi4 and CH25Hchi3 haplotypes, whereas no A beta deposits were present in CH25Hchi2 carriers. Together, these results are compatible with a role of CH25Hchi4 as a putative susceptibility factor for sporadic AD; they may explain part of the linkage of chromosome 10 markers with sporadic AD, and they suggest the possibility that CH25H polymorphisms are associated with different rates of brain A beta deposition