The human MT1 and MT2 melatonin receptors are G-proteincoupled receptors (GPCRs) that help to regulate circadian rhythm and sleep patterns. Drug development efforts have targeted both receptors for ...the treatment of insomnia, circadian rhythm and mood disorders, and cancer, and MT2 has also been implicated in type 2 diabetes. Here we report X-ray free electron laser (XFEL) structures of the human MT2 receptor in complex with the agonists 2-phenylmelatonin (2-PMT) and ramelteon at resolutions of 2.8 Å and 3.3 Å, respectively, along with two structures of function-related mutants: H2085.46A (superscripts represent the Ballesteros-Weinstein residue numbering nomenclature) and N862.50D, obtained in complex with 2-PMT. Comparison of the structures of MT2 with a published structure of MT1 reveals that, despite conservation of the orthosteric ligand-binding site residues, there are notable conformational variations as well as differences in 3Hmelatonin dissociation kinetics that provide insights into the selectivity between melatonin receptor subtypes. A membrane-buried lateral ligand entry channel is observed in both MT1 and MT2, but in addition the MT2 structures reveal a narrow opening towards the solvent in the extracellular part of the receptor. We provide functional and kinetic data that support a prominent role for intramembrane ligand entry in both receptors, and suggest that there might also be an extracellular entry path in MT2. Our findings contribute to a molecular understanding of melatonin receptor subtype selectivity and ligand access modes, which are essential for the design of highly selective melatonin tool compounds and therapeutic agents.
EGFR mutations and ALK translocation are considered mutually exclusive. We found that a portion of NSCLCs have EGFR and ALK co-alteration. When using sensitive assays for EGFR, the rate of ...co-alteration increased up to 15.4% of ALK-positive cases. These patients were sensitive to ALK inhibitors, but not to gefitinib. The clinical relevance of finding co-alteration with sensitive methods was discussed.
Epidermal growth factor receptor (EGFR) mutation and anaplastic lymphoma kinase (ALK) translocation are considered mutually exclusive in nonsmall-cell lung cancer (NSCLC). However, sporadic cases having concomitant EGFR and ALK alterations have been reported. The present study aimed to assess the prevalence of NSCLCs with concomitant EGFR and ALK alterations using mutation detection methods with different sensitivity and to propose an effective diagnostic and therapeutic strategy.
A total of 1458 cases of lung cancer were screened for EGFR and ALK alterations by direct sequencing and flourescence in situ hybridization (FISH), respectively. For the 91 patients identified as having an ALK translocation, peptide nucleic acid (PNA)-clamping real-time PCR, targeted next-generation sequencing (NGS), and mutant-enriched NGS assays were carried out to detect EGFR mutation.
EGFR mutations and ALK translocations were observed in 42.4% (612/1445) and 6.3% (91/1445) of NSCLCs by direct sequencing and FISH, respectively. Concomitant EGFR and ALK alterations were detected in four cases, which accounted for 4.4% (4/91) of ALK-translocated NSCLCs. Additional analyses for EGFR using PNA real-time PCR and ultra-deep sequencing by NGS, mutant-enriched NGS increased the detection rate of concomitant EGFR and ALK alterations to 8.8% (8/91), 12.1% (11/91), and 15.4% (14/91) of ALK-translocated NSCLCs, respectively. Of the 14 patients, 3 who were treated with gefitinib showed poor response to gefitinib with stable disease in one and progressive disease in two patients. However, eight patients who received ALK inhibitor (crizotinib or ceritinib) showed good response, with response rate of 87.5% (7/8 with partial response) and durable progression-free survival.
A portion of NSCLC patients have concomitant EGFR and ALK alterations and the frequency of co-alteration detection increases when sensitive detection methods for EGFR mutation are applied. ALK inhibitors appear to be effective for patients with co-alterations.
Summary Background The role of adjuvant chemotherapy for patients with rectal cancer is controversial, especially when used after preoperative chemoradiotherapy. Fluoropyrimidine-based adjuvant ...chemotherapy, including fluorouracil and leucovorin, has been widely used; however, the addition of oxaliplatin to fluorouracil and leucovorin (FOLFOX), a standard adjuvant regimen for colon cancer, has not been tested in rectal cancer. We aimed to compare the efficacy and safety of adjuvant fluorouracil and leucovorin with that of FOLFOX in patients with locally advanced rectal cancer after preoperative chemoradiotherapy. Methods In this open-label, multicentre, phase 2, randomised trial, patients with postoperative pathological stage II (ypT3–4N0) or III (ypTany N1–2) rectal cancer after preoperative fluoropyrimidine-based chemoradiotherapy and total mesorectal excision were recruited and randomly assigned (1:1) via a web-based software platform to receive adjuvant chemotherapy with either four cycles of fluorouracil and leucovorin (fluorouracil 380 mg/m2 and leucovorin 20 mg/m2 on days 1–5, every 4 weeks) or eight cycles of FOLFOX (oxaliplatin 85 mg/m2 , leucovorin 200 mg/m2 , and fluorouracil bolus 400 mg/m2 on day 1, and fluorouracil infusion 2400 mg/m2 for 46 h, every 2 weeks). Stratification factors were pathological stage (II vs III) and centre. Neither patients nor investigators were masked to group assignment. The primary endpoint was 3-year disease-free survival, analysed by intention to treat. This study is fully enrolled, is in long-term follow-up, and is registered with ClinicalTrials.gov , number NCT00807911. Findings Between Nov 19, 2008, and June 12, 2012, 321 patients were randomly assigned to fluorouracil and leucovorin (n=161) and FOLFOX (n=160). 141 (95%) of 149 patients in the fluorouracil plus leucovorin group and 141 (97%) of 146 in the FOLFOX group completed all planned cycles of adjuvant treatment. Median follow-up was 38·2 months (IQR 26·4–50·6). 3-year disease-free survival was 71·6% (95% CI 64·6–78·6) in the FOLFOX group and 62·9% (55·4–70·4) in the fluorouracil plus leucovorin group (hazard ratio 0·657, 95% CI 0·434–0·994; p=0·047). Any grade neutropenia, thrombocytopenia, fatigue, nausea, and sensory neuropathy were significantly more common in the FOLFOX group than in the fluorouracil plus leucovorin group; however, we noted no significant difference in the frequency of these events at grade 3 or 4. The most common grade 3 or worse adverse events were neutropenia (38 26% of 149 patients in the fluorouracil plus leucovorin group vs 52 36% of 146 patients in the FOLFOX group), leucopenia (eight 5% vs 12 8%), febrile neutropenia (four 3% vs one <1%), diarrhoea (four 3% vs two 1%), and nausea (one <1% vs two 1%). Interpretation Adjuvant FOLFOX improves disease-free survival compared with fluorouracil plus leucovorin in patients with locally advanced rectal cancer after preoperative chemoradiotherapy and total mesorectal excision, and warrants further investigation. Funding Korea Healthcare Technology R&D Project (South Korean Ministry of Health and Welfare).
The usefulness of arterial spin-labeling for the evaluation of the effect of the antiangiogenic therapy has not been elucidated. Our aim was to evaluate the antiangiogenic effect of bevacizumab in a ...rat glioblastoma model based on arterial spin-labeling perfusion MR imaging.
DSC and arterial spin-labeling perfusion MR imaging were performed by using a 9.4T MR imaging scanner in nude rats with glioblastoma. Rats were randomly assigned to the following 3 groups: control, 3-day treatment, and 10-day treatment after bevacizumab injection. One-way analysis of variance with a post hoc test was used to compare perfusion parameters (eg, normalized CBV and normalized CBF from DSC MR imaging and normalized CBF based on arterial spin-labeling) with microvessel area on histology. The Pearson correlations between perfusion parameters and microvessel area were also determined.
All of the normalized CBV from DSC, normalized CBF from DSC, normalized CBF from arterial spin-labeling, and microvessel area values showed significant decrease after treatment (P < .001, P < .001, P = .005, and P < .001, respectively). In addition, normalized CBV and normalized CBF from DSC and normalized CBF from arterial spin-labeling strongly correlated with microvessel area (correlation coefficient, r = 0.911, 0.869, and 0.860, respectively; P < .001 for all).
Normalized CBF based on arterial spin-labeling and normalized CBV and normalized CBF based on DSC have the potential for evaluating the effect of antiangiogenic therapy on glioblastomas treated with bevacizumab, with a strong correlation with microvessel area.
The emergency department (ED) is where hand hygiene problems are significant as the procedures in the ED are often high risk and invasive. To date, there have been no comprehensive reviews on hand ...hygiene in EDs. The aim of this study was to investigate hand hygiene compliance (HHC) rate, factors affecting the HHC rate, and intervention strategies to improve HHC in EDs. Electronic databases were used to search for research published from 1948 to January 2018. The databases included ovidMEDLINE, ovidEMBASE, the Cochrane Library, CINAHL, Koreamed, and Kmbase. All study designs were included. Two reviewers independently extracted the data and assessed the bias risk using reliable and validated tools. A narrative synthesis was performed. Twenty-four studies, including 12 cross-sectional surveys and 12 interventional studies, were included. Of the 12 interventional studies reviewed, only 33% (N = 4) reported HHC rates of more than 50%. Factors that influenced HHC included types of healthcare worker, hand hygiene indication, ED crowding, positive attitudes towards HHC, patient location, auditing hand hygiene, and type of shift. Almost all of the studies (83.3%) applied multimodal or dual interventions to improve HHC. A range of strategies, including education, monitoring and providing feedback, campaigns, and cues, effectively improved HHC. The review findings indicate that there is a room for improvement in HHC in EDs. Future randomized controlled trials are necessary to determine which intervention modalities are most effective and sustainable for HHC improvement.
The development of biomimetic bone matrices is one of the major goals in the bone‐regeneration and tissue‐engineering fields. Nanocomposites consisting of a natural polymer and hydroxyapatite (HA) ...nanocrystals, which mimic the human bone matrix, are thus regarded as promising bone regenerative materials. Herein, we developed a biomimetic nanocomposite with a novel nanofibrous structure by employing an electrospinning (ES) method. The HA precipitate/gelatin matrix nanocomposites are lyophilized and dissolved in an organic solvent, and then electrospun under controlled conditions. With this process, we can successfully generate a continuous fiber with a diameter of the order of hundreds of nanometers. The internal structure of the nanofiber features a typical nanocomposite, i.e., HA nanocrystals well distributed within a gelatin matrix. These nanocomposite fibers improve the bone‐derived cellular activity significantly when compared to the pure gelatin equivalent. This method of generating a nanofiber of the biomimetic nanocomposite was effective in producing a biomedical membrane with a composition gradient, which is potentially applicable in the field of guided tissue regeneration (GTR).
Nanofibers of gelatin–hydroxyapatite can be generated from a biomimetic nanocomposite solution by electrospinning. This methodology of generating a nanofiber of the biomedical nanocomposite is effective in producing a functional membrane with a composition gradient (gelatin/gelatin–20%HA layered nanofiber matrix, see Figure), which is potentially applicable in the field of guided tissue regeneration.
Summary Objective Hypoxia-inducible factor (HIF)-2α and the zinc-ZIP8-MTF1 axis in chondrocytes serve as catabolic regulators of osteoarthritic cartilage destruction by regulating the expression of ...catabolic factor genes. We explored possible crosstalk between these signaling pathways and its biological significance in osteoarthritis (OA). Methods Microarray analysis, various mRNA and protein assays were conducted using primary cultured mouse articular chondrocytes and experimental OA cartilage to reveal molecular mechanisms underlying the crosstalk between HIF-2α and the zinc-ZIP8-MTF1 axis. Experimental OA in mice was induced by intra-articular (IA) injection of adenovirus expressing HIF-2α (Ad- Epas1 ), ZIP8 (Ad- Zip8 ), or MTF1 (Ad- Mtf1 ) in wild-type mice or mice with cartilage-specific conditional knockout of HIF-2α ( Epas1fl/fl ; Col2a1-Cre ), ZIP8 ( Zip8fl/fl ; Col2a1-Cre ), or MTF1 ( Mtf1fl/fl ; Col2a1-Cre ). Results HIF-2α activated the zinc-ZIP8-MTF1 axis in chondrocytes by upregulating the Zn2+ transporter ZIP8, thereby increasing Zn2+ influx and activating the downstream transcription factor MTF1. The zinc-ZIP8-MTF1 axis, in turn, acted as a novel transcriptional regulator of HIF-2α. HIF-2α-induced activation of the zinc-ZIP8-MTF1 axis amplified HIF-2α regulation of OA cartilage destruction by synergistically promoting expression of matrix-degrading enzymes. Thus, HIF-2α-induced activation of the zinc-ZIP8-MTF1 axis, together with zinc-ZIP8-MTF1 regulation of HIF-2α, acted collectively to synergistically promote expression of matrix-degrading enzymes and OA cartilage destruction. Conclusion Our findings identify a reciprocal activation mechanism involving HIF-2α and the zinc-ZIP8-MTF1 axis during OA pathogenesis that amplifies catabolic signaling and cartilage destruction.
BACKGROUND
Oocyte activation is a crucial step that comprises the release of the oocyte from meiotic arrest, pronuclear formation and subsequent embryo development. Oocytes are activated by ...repetitive increases in the intracellular concentration of free Ca2+, Ca2+i oscillations, which are triggered during fertilization by the introduction of the sperm-specific phospholipase C zeta 1 (PLCZ1). Recent studies have shown that sperm from patients lacking expression of PLCZ1 or expressing mutant forms of PLCZ1 fail to induce Ca2+i oscillations or oocyte activation. We first purified recombinant human PLCZ1 (hPLCZ1) protein and evaluated its Ca2+i oscillation activity in mouse and human oocytes with the view to investigate its application in the clinic for assisted oocytes activation in lieu of chemical agents.
METHODS
Recombinant hPLCZ1 was synthesized using the Escherichia coli system, and subjected to immunoblot analysis with anti-PLCZ1 and anti-His tag antibodies. Ca2+i oscillations by microinjection of recombinant hPLCZ1 into mouse or human oocytes were examined by Ca2+i monitoring with Fluo 4. Ploidy of the oocytes with recombinant hPLCZ1 injection was confirmed with fluorescence in situ hybridization.
RESULTS
A band of 68 kDa on recombinant protein was detected with both antibodies. Injection of recombinant hPLCZ1 induced Ca2+i oscillations in a dose-dependent manner in both mouse and human oocytes. These oscillations, which closely resembled those initiated by the sperm upon fertilization, triggered activation and cleavage in oocytes of both species, although further development of the mice embryos was low. U73122, a PLC inhibitor, blocked the ability of hPLCZ1 to initiate oscillations. Microinjection of recombinant hPLCZ1 into ICSI-failed human oocytes rescued fertilization failure in five of eight attempts.
CONCLUSIONS
Repeated Ca2+i oscillations and oocyte activation were induced in mouse and human oocytes by microinjection of recombinant hPLCZ1 synthesized in E. Coli. Injection of recombinant protein could thus provide a biological solution for inducing artificial activation of oocytes.
Summary
GABAergic interneurons regulate the degree of glutamatergic excitation and output of projection neurons. In this study, we investigated the distribution of calbindinD‐28k (CB) and parvalbumin ...(PV) in the somatosensory area of the pigeon pallium using immunohistochemical method. Our results show that anatomical structures of the somatosensory area of the pigeon pallium consisted of several subdivisions including the hyperpallium, intercalated hyperpallium, mesopallium, nidopallium and basorostralis. Neuronal density was significantly higher in the intercalated hyperpallium and basorostralis than that in the other subdivisions. The density of the CB immunoreactive neurons was generally similar in all the subdivisions; however, the density of PV immunoreactive neurons was particularly prominent in the basorostralis compared with that in the other subdivisions. In addition, the mean proportion of PV immunoreactive neurons to total neurons was higher than that in the CB immunoreactive neurons in all the subdivisions. In brief, our present study shows that PV immunoreactive neurons in the somatosensory area of the pigeon pallium were significantly abundant compared with CB immunoreactive neurons. This finding needs more studies regarding CB‐ and PV‐related functions in the somatosensory area of the avian pallium.
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