Effects of ischemic post‐conditioning on neuronal VEGF regulation and microglial polarization in a rat model of focal cerebral ischemia

Ischemic postconditioning is increasingly being investigated as a therapeutic approach for cerebral ischemia. However, the majority of studies are focused on the acute protection of neurons per se . Whether and how postconditioning affects multiple cells in the recovering neurovascular unit remains to be fully elucidated. Here, we asked whether postconditioning may modulate help‐me signaling between injured neurons and reactive microglia. Rats were subjected to 100 min of focal cerebral ischemia, then randomized into a control versus postconditioning group. After 3 days of reperfusion, infarct volumes were significantly reduced in animals treated with postconditioning, along with better neurologic outcomes. Immunostaining revealed that ischemic postconditioning increased expression of vascular endothelial growth factor (VEGF ) in neurons within peri‐infarct regions. Correspondingly, we confirmed that VEGFR 2 was expressed on Iba1‐positive microglia/macrophages, and confocal microscopy showed that in postconditioned rats, these cells were polarized to a ramified morphology with higher expression of M2‐like markers. Treating rats with a VEGF receptor 2 kinase inhibitor negated these effects of postconditioning on microglia/macrophage polarization. In vitro , postconditoning after oxygen‐glucose deprivation up‐regulated VEGF release in primary neuron cultures, and adding VEGF to microglial cultures partly shifted their M2‐like markers. Altogether, our findings support the idea that after postconditioning, injured neurons may release VEGF as a ‘help‐me’ signal that promotes microglia/macrophage polarization into potentially beneficial phenotypes.

     

Solution structure of the PHD finger from the human KIAA1045 protein

Cross‐brace structural motifs are required as a scaffold to design artificial RING fingers (ARFs) that function as ubiquitin ligase (E3) in ubiquitination and have specific ubiquitin‐conjugating enzyme (E2)‐binding capabilities. The Simple Modular Architecture Research Tool database predicted the amino acid sequence 131–190 (KIAA1045ZF) of the human KIAA1045 protein as an unidentified structural region. Herein, the stoichiometry of zinc ions estimated spectrophotometrically by the metallochromic indicator revealed that the KIAA1045ZF motif binds to two zinc atoms. The structure of the KIAA1045ZF motif bound to the zinc atoms was elucidated at the atomic level by nuclear magnetic resonance. The actual structure of the KIAA1045ZF motif adopts a C₄HC₃‐type PHD fold belonging to the cross‐brace structural family. Therefore, the utilization of the KIAA1045ZF motif as a scaffold may lead to the creation of a novel ARF.     

Structure-activity relationship of bile acids and bile acid analogs in regard to FXR activation

The farnesoid X receptor (FXR) is a bile acid-activated nuclear receptor that plays a major role in bile acid and cholesterol metabolism. To obtain an insight into the structure-activity relationships of FXR ligands, we investigated the functional roles of structural elements in the physiological ligands chenodeoxycholic acid [CDCA; (3alpha,7alpha)], cholic acid [CA; (3alpha,7alpha,12alpha)], deoxycholic acid [DCA; (3alpha,12alpha)], and lithocholic acid (3alpha) in regard to FXR activation in a cell-based FXR response element-driven luciferase assay and an in vitro coactivator association assay. Conversion of the carboxyl group of CDCA or CA to an alcohol did not greatly diminish their ability to activate FXR. In contrast, the 7beta-epimers of the alcohols were inactive, indicating that the bile alcohols retained the ligand properties of the original bile acids and that the 7beta-hydroxyl group diminished their FXR-activating effect. Similarly, hydroxyl epimers of DCA exhibited decreased activity compared with DCA, indicating a negative effect of 3beta- or 12beta-hydroxyl groups. Introduction of an alkyl group at the 7beta- or 3beta-position of CDCA resulted in diminished FXR activation in the following order of alkyl groups: 7-ethyl=7-propyl>3-methyl>7-methyl. These results indicate that bulky substituents, whether hydroxyl groups or alkyl residues, at the beta-position of cholanoids decrease their ability to activate FXR.     

Genetic deletion of vesicular monoamine transporter-2 (VMAT2) reduces dopamine transporter activity in mesencephalic neurons in primary culture

Our aim was to investigate whether a defect in vesicular monoamine transporter-2 (VMAT2) activities would affect dopaminergic cell functions or not. We examined mesencephalon dopaminergic cultures prepared from VMAT2 wild-type, heterozygous or homozygous knockout (KO) 14-day-old mouse fetuses to determine the number of tyrosine hydroxylase (TH)-positive cells and dopamine transporter activity. The number of TH-positive cells remained unchanged in the VMAT2-KO cultures. Of interest, the dopamine transporter activity in the homozygous cells was significantly decreased, but not in the heterozygous cells, suggesting that complete deletion of VMAT2 inhibited dopamine transporter function. Furthermore, dopamine transporter activity was prominently decreased in the synaptosomal fraction of neonatal homozygous VMAT2-KO mice compared with that of wild-type/heterozygous VMAT2-KO ones, indicating that VMAT2 activity might be one of the factors regulating dopamine transporter activities. To test this possibility, we used reserpine, a VMAT2 inhibitor. Reserpine (1muM) decreased dopamine transporter activity (approx. 50%) in wild-type and heterozygous VMAT2-KO cultures but not in homozygous ones, indicating that blockade of VMAT2 activity reduced dopamine transporter activity. To investigate possible mechanisms underlying the decreased dopamine transporter activity in VMAT2-KO mice, we measured dopamine transporter activities after 24-48h exposure of primary cultures of mesencephalic neurons to dopamine receptor antagonists, PKC inhibitor, PI(3)K inhibitor, and l-DOPA. Among these drugs, l-DOPA slightly reduced the dopamine transporter activities of all genotypes, but the other drugs could not. Since the ratios of reduction in dopamine transporter activity of each genotype treated with l-DOPA were similar, substrate inhibition of dopamine transporters was not the main mechanism underlying the reduced dopamine transporter activity due to genetic deletion of VMAT2. Our results demonstrate that genetic deletion of VMAT2 did not induce immediate cell death but did markedly inhibit dopamine transporter activity.     

Alignment-based approach for durable data storage into living organisms

The practical realization of DNA data storage is a major scientific goal. Here we introduce a simple, flexible, and robust data storage and retrieval method based on sequence alignment of the genomic DNA of living organisms. Duplicated data encoded by different oligonucleotide sequences was inserted redundantly into multiple loci of the Bacillus subtilis genome. Multiple alignment of the bit data sequences decoded by B. subtilis genome sequences enabled the retrieval of stable and compact data without the need for template DNA, parity checks, or error-correcting algorithms. Combined with the computational simulation of data retrieval from mutated message DNA, a practical use of this alignment-based method is discussed.     

Determination of the pheromone-producing region that has epoxidation activity in the abdominal tip of the Japanese giant looper, Ascotis selenaria cretacea (Lepidoptera: Geometridae)

The epoxydienyl sex pheromone of Ascotis selenaria cretacea can be detected only within a rod-like abdominal tip (RAT) of the female. To clarify which part of the RAT is the sex pheromone-producing region, the RAT was morphologically divided into three sections, defined positionally from the abdomen as sections A, B, and C. GC-MS measurements clearly showed that the sex pheromone compound levels in section B were four times greater than those of the other sections. Microscopic dissection analysis revealed that section B consists of four tissues: rectum, oviduct, musculature, and intersegmental membrane. GC-MS analysis of the individual tissues revealed that approximately 90% of the sex pheromone in section B is localized in the intersegmental membrane. A cell layer was found in the intersegmental membrane after staining with propidium iodide. Furthermore, incubation of tissues dissected from section B with a deuterated trienyl pheromone precursor revealed that the labeled epoxy pheromonal component was detected exclusively in the intersegmental membrane. We have determined that the sex pheromone-producing region of A. s. cretacea is on the terminal side of the intersegmental membrane located between the 8th and 9th abdominal segments.     

Near Infrared Photoimmunotherapy Targeting EGFR Positive Triple Negative Breast Cancer: Optimizing the Conjugate-Light Regimen

Aim

Triple-negative breast cancer (TNBC) is considered one of the most aggressive subtypes of breast cancer. Near infrared photoimmunotherapy (NIR-PIT) is a cancer treatment that employs an antibody-photosensitizer conjugate (APC) followed by exposure of NIR light for activating selective cytotoxicity on targeted cancer cells and may have application to TNBC. In order to minimize the dose of APC while maximizing the therapeutic effects, dosing of the APC and NIR light need to be optimized. In this study, we investigate in vitro and in vivo efficacy of cetuximab (cet)-IR700 NIR-PIT on two breast cancer models MDAMB231 (TNBC, EGFR moderate) and MDAMB468 (TNBC, EGFR high) cell lines, and demonstrate a method to optimize the dosing APC and NIR light.

Method

After validating in vitro cell-specific cytotoxicity, NIR-PIT therapeutic effects were investigated in mouse models using cell lines derived from TNBC tumors. Tumor-bearing mice were separated into 4 groups for the following treatments: (1) no treatment (control); (2) 300 μg of cet-IR700 i.v., (APC i.v. only); (3) NIR light exposure only, NIR light was administered at 50 J/cm² on day 1 and 100 J/cm² on day 2 (NIR light only); (4) 300 μg of cet-IR700 i.v., NIR light was administered at 50 J/cm² on day 1 after injection and 100 J/cm² of light on day 2 after injection (one shot NIR-PIT). To compare different treatment regimens with a fixed dose of APC, we added the following treatments (5) 100 μg of cet-IR700 i.v., NIR light administered at 50 J/cm² on day 1 and 50 μg of cet-IR700 i.v. immediately after NIR-PIT, then NIR light was administered at 100 J/cm² on day 2, which were performed two times every week (“two split” NIR-PIT) and (6) 100 μg of cet-IR700 i.v., NIR light was administered at 50 J/cm² on day 1 and 100 J/cm² on day 2, which were performed three times per week (“three split” NIR-PIT).

Result

Both specific binding and NIR-PIT effects were greater with MDAMB468 than MDAMB231 cells in vitro. Tumor accumulation of cet-IR700 in MDAMB468 tumors was significantly higher (p < 0.05) than in MDAMB231 tumors in vivo. Tumor growth and survival of MDAMB231 tumor bearing mice was significantly lower in the NIR-PIT treatment group (p < 0.05). In MDAMB468 bearing mice, tumor growth and survival was significantly improved in the NIR-PIT treatment groups in all treatment regimens (one shot NIR-PIT; p < 0.05, “two split” NIR-PIT; p < 0.01, “three split” NIR-PIT; p < 0.001) compared with control groups.

Conclusion

NIR-PIT for TNBC was effective regardless of expression of EGFR, however, greater cell killing was shown with higher EGFR expression tumor in vitro. In all treatment regimens, NIR-PIT suppressed tumor growth, resulting in significantly prolonged survival that further improved by splitting the APC dose and using repeated light exposures.     

PCR-Based Simple Subgrouping Is Validated for Classification of Gliomas and Defines Negative Prognostic Copy Number Aberrations in IDH Mutant Gliomas

Genetic subgrouping of gliomas has been emphasized recently, particularly after the finding of isocitrate dehydrogenase 1 (IDH1) mutations. In a previous study, we investigated whole-chromosome copy number aberrations (CNAs) of gliomas and have described genetic subgrouping based on CNAs and IDH1 mutations. Subsequently, we classified gliomas using simple polymerase chain reaction (PCR)-based methods to improve the availability of genetic subgrouping. We selected IDH1/2 and TP53 as markers and analyzed 237 adult supratentorial gliomas using Sanger sequencing. Using these markers, we classified gliomas into three subgroups that were strongly associated with patient prognoses. These included IDH mutant gliomas without TP53 mutations, IDH mutant gliomas with TP53 mutations, and IDH wild-type gliomas. IDH mutant gliomas without TP53 mutations, which mostly corresponded to gliomas carrying 1p19q co-deletions, showed lower recurrence rates than the other 2 groups. In the other high-recurrence groups, the median progression-free survival (PFS) and overall survival (OS) of patients with IDH mutant gliomas with TP53 mutations were significantly longer than those of patients with IDH wild-type gliomas. Notably, most IDH mutant gliomas with TP53 mutations had at least one of the CNAs +7q, +8q, −9p, and −11p. Moreover, IDH mutant gliomas with at least one of these CNAs had a significantly worse prognosis than did other IDH mutant gliomas. PCR-based mutation analyses of IDH and TP53 were sufficient for simple genetic diagnosis of glioma that were strongly associated with prognosis of patients and enabled us to detect negative CNAs in IDH mutant gliomas.