Modulation of the functional interfaces between retroviral intasomes and the human nucleosome.

Infection by retroviruses as HIV-1 requires the stable integration of their genome into the host cells. This process needs the formation of integrase (IN)-viral DNA complexes, called intasomes, and their interaction with the target DNA wrapped around nucleosomes within cell chromatin. To provide new tools to analyze this association and select drugs, we applied the AlphaLISA technology to the complex formed between the prototype foamy virus (PFV) intasome and nucleosome reconstituted on 601 Widom sequence. This system allowed us to monitor the association between both partners and select small molecules that could modulate the intasome/nucleosome association. Using this approach, drugs acting either on the DNA topology within the nucleosome or on the IN/histone tail interactions have been selected. Within these compounds, doxorubicin and histone binders calixarenes were characterized using biochemical, in silico molecular simulations and cellular approaches. These drugs were shown to inhibit both PFV and HIV-1 integration in vitro. Treatment of HIV-1-infected PBMCs with the selected molecules induces a decrease in viral infectivity and blocks the integration process. Thus, in addition to providing new information about intasome-nucleosome interaction determinants, our work also paves the way for further unedited antiviral strategies that target the final step of intasome/chromatin anchoring. IMPORTANCE In this work, we report the first monitoring of retroviral intasome/nucleosome interaction by AlphaLISA. This is the first description of the AlphaLISA application for large nucleoprotein complexes (>200 kDa) proving that this technology is suitable for molecular characterization and bimolecular inhibitor screening assays using such large complexes. Using this system, we have identified new drugs disrupting or preventing the intasome/nucleosome complex and inhibiting HIV-1 integration both in vitro and in infected cells. This first monitoring of the retroviral/intasome complex should allow the development of multiple applications including the analyses of the influence of cellular partners, the study of additional retroviral intasomes, and the determination of specific interfaces. Our work also provides the technical bases for the screening of larger libraries of drugs targeting specifically these functional nucleoprotein complexes, or additional nucleosome-partner complexes, as well as for their characterization.

Association with sagittal alignment and osteoporosis-related fractures in outpatient women with osteoporosis.

The baseline sagittal vertical axis (SVA) and pelvic tilt (PT) are independent risk factors of osteoporosis-related fractures in women with osteoporosis. We clarified the SVA and PT to predict the incidence of osteoporosis-related fractures. PURPOSE: Sagittal alignment with osteoporosis women deteriorates with advancing age and sagittal alignment may indicate osteoporosis-related fractures in the future. However, whether the sagittal alignment predicts future osteoporosis-related fracture in patients with osteoporosis has not been clarified. We aimed to investigate the association between sagittal alignment and future osteoporosis-related fractures. METHODS: This was a retrospective cohort study. Of the 313 participants (mean follow-up period, 2.9 years), 236 were included in the analysis. At baseline, we measured bone mineral density (BMD) of the lumbar spine and the femoral neck, sagittal vertical axis (SVA), thoracic kyphosis, pelvic incidence minus lumbar lordosis, sacral slope, pelvic tilt (PT), geriatric locomotive function scale (GLFS), two-step value, and stand-up test. The information on medications and the duration of treatment were reviewed from the medical records. Additionally, participants reported their history of falls at baseline. Multiple logistic regression analysis was used to determine the association of future osteoporosis-related fracture, and adjusted Odds ratios (OR) and 95% confidence interval (CI) were calculated with all predictors as covariates. All continuous variables were calculated using standardized OR (sOR). RESULTS: Osteoporosis-related fractures occurred in 33 of 313 participants (10.5%). Multiple logistic regression analysis showed that a history of falls (OR =4.092, 95% CI: 1.029-16.265, p =0.045), SVA (sOR =4.228, 95% CI: 2.118-8.439, p <0.001), and PT (sOR =2.497, 95% CI: 1.087-5.733, p =0.031) were independent risk factors for future osteoporosis-related fractures. CONCLUSIONS: This study revealed the SVA and PT to predict osteoporosis-related fractures. TRIAL REGISTRATION NUMBER AND DATE OF REGISTRATION: UMIN000036516 (April 1, 2019).

Large-scale prospective genome-wide association study of oxaliplatin in stage II/III colon cancer and neuropathy.

BACKGROUND: The severity of oxaliplatin (L-OHP)-induced peripheral sensory neuropathy (PSN) exhibits substantial interpatient variability, and some patients suffer from long-term, persisting PSN. To identify single-nucleotide polymorphisms (SNPs) predicting L-OHP-induced PSN using a genome-wide association study (GWAS) approach. PATIENTS AND METHODS: A large prospective GWAS including 1379 patients with stage II/III colon cancer who received L-OHP-based adjuvant chemotherapy (mFOLFOX6/CAPOX) under the phase II (JOIN/JFMC41) or the phase III (ACHIVE/JFMC47) trial. Firstly, GWAS comparison of worst grade PSN (grade 0/1 versus 2/3) was carried out. Next, to minimize the impact of ambiguity in PSN grading, extreme PSN phenotypes were selected and analyzed by GWAS. SNPs that could predict time to recovery from PSN were also evaluated. In addition, SNPs associated with L-OHP-induced allergic reactions (AR) and time to disease recurrence were explored. RESULTS: No SNPs exceeded the genome-wide significance (P < 5.0 × 10-8) in either GWAS comparison of worst grade PSN, extreme PSN phenotypes, or time to recovery from PSN. An association study focusing on AR or time to disease recurrence also failed to reveal any significant SNPs. CONCLUSION: Our results highlight the challenges of utilizing SNPs for predicting susceptibility to L-OHP-induced PSN in daily clinical practice.

Catalytic Chemoselective O-Phosphorylation of Alcohols.

Phosphorylation of alcohols is a fundamentally important reaction in both life science and physical science. Product phosphate monoesters play key roles in living organisms, natural products, pharmaceuticals, and organic materials. Most of the chemical methods to date for synthesizing phosphate monoesters, however, require multistep sequences or are limited to specific types of substrates possibly due to harsh conditions. An alternative way to enable the simple production of phosphate monoesters from highly functionalized precursor alcohols is, thus, highly desired. We report herein a catalytic phosphorylation of alcohols with high functional group tolerance using tetrabutylammonium hydrogen sulfate (TBAHS) and phosphoenolpyruvic acid monopotassium salt (PEP-K) as the catalyst and phosphoryl donor, respectively. This method enables the direct introduction of a nonprotected phosphate group to the hydroxy group of a diverse menu of alcohol substrates, including functionalized small molecules, carbohydrates, and unprotected peptides. Nuclear magnetic resonance, mass spectrometric, and density functional theory analyses suggest that an unprecedented mixed anhydride species, generated from PEP-K and TBAHS, acts as an active phosphoryl donor in this reaction. This operationally simple and chemoselective catalytic phosphorylation allows for the efficient production of densely functionalized O-phosphorylated compounds, which are useful in diverse fields including biology and medicine.

Association of NOD2 Mutations with Aggressive Periodontitis.

Aggressive periodontitis (AgP) is characterized by rapid alveolar bone destruction and tooth loss early in life, and its etiology remains unclear. To explore the genetic risk factors of AgP, we performed genome-wide single-nucleotide polymorphism genotyping for identity-by-descent mapping and identified 32 distinct candidate loci, followed by whole exome sequencing with 2 pedigrees of AgP consisting of 3 cases and 1 control in 1 family and 2 sibling cases in the other. After variant filtering procedures and validation by targeted Sanger sequencing, we identified 2 missense mutations at 16q12 in NOD2 (p.Ala110Thr and p.Arg311Trp), which encodes nucleotide-binding oligomerization domain protein 2. We further examined 94 genetically unrelated AgP patients by targeted sequencing of NOD2 and found that 2 patients among them also carried the p.Arg311Trp variant. Furthermore, we found 3 additional missense mutations in this gene (p.His370Tyr, p.Arg459Cys, and p.Ala868Thr). These mutations either had not been previously observed or are extremely rare (frequency <0.001) in Asian populations. NOD2 plays a crucial role in innate immunity as an intracellular receptor initiating nuclear factor κB-dependent and mitogen-activated protein kinase-dependent gene transcription. These results demonstrated NOD2 as a novel gene involved in AgP.

Large-scale prospective pharmacogenomics study of oxaliplatin-induced neuropathy in colon cancer patients enrolled in the JFMC41-1001-C2 (JOIN Trial).

BACKGROUND: Peripheral sensory neuropathy (PSN) is a dose-limiting toxicity of oxaliplatin-based chemotherapy. Several genetic markers have been shown to predict oxaliplatin-induced PSN; however, results remain to be validated in a large-scale and prospective pharmacogenomics study. PATIENTS AND METHODS: Among 882 patients enrolled in the JFMC41-1001-C2 (JOIN trial), which was designed to investigate the tolerability of adjuvant-modified FOLFOX6 (mFOLFOX6) in Japanese Patients with stage II or III colon cancers undergoing curative resection, 465 patients were eligible for this pharmacogenomics analysis. Twelve single-nucleotide polymorphisms (SNPs) were selected based on published data. The effect of each genotype on time to PSN onset was evaluated in all patients (n = 465) using the Cox proportional hazard model. For the association analysis between severity of PSN and 12 SNP markers, 84 patients who failed to complete 12 cycles of mFOLFOX6 from grade 0/1 PSN group were excluded because the termination of the protocol treatment had been caused by reasons other than PSN. RESULTS: Comparison of grade 0/1 PSN with grade 2/3 PSN or grade 3 PSN showed no significant associations with any of the 12 SNP markers after adjustment for total dose of oxaliplatin. Time-to-onset analysis also failed to reveal any significant differences. CONCLUSIONS: Our large-scale and prospective pharmacogenomics study of Japanese patients receiving protocol treatment of adjuvant mFOLFOX6 could not verify a role for any of the 12 SNP markers reported as being significantly associated with PSN. Considering the OR observed in this study (range: 0.76-1.89), further evaluation of these 12 SNP markers in the context of L-OHP-induced PSN is unlikely to be clinically informative.

Aurora-A is a determinant of tamoxifen sensitivity through phosphorylation of ERα in breast cancer.

Despite the clinical success of tamoxifen, its resistance remains a major challenge in breast cancer. Here we show that Aurora-A determines tamoxifen sensitivity by regulation of oestrogen receptor (ER)α. Ectopic expression of Aurora-A decreases and depletion of Aurora-A enhances tamoxifen sensitivity in ERα-positive breast cancer. Elevated Aurora-A was significantly associated with the recurrence of ERα-positive tumours. Notably, Aurora-A inhibitor MLN8237, which is currently in clinical trial, synergizes with tamoxifen and overcomes tamoxifen resistance. Furthermore, Aurora-A interacts with and phosphorylates ERα on serine-167 and -305, leading to increase in ERα DNA-binding and transcriptional activity. Elevated levels of Aurora-A are significantly associated with disease-free survival in ERα-positive but not ERα-negative breast cancers. These data suggest that Aurora-A has a pivotal role in tamoxifen resistance and ERα is a bona fide substrate of Aurora-A. Thus, Aurora-A represents a prognostic marker in ERα-positive tumour and a critical therapeutic target in tamoxifen-resistant breast cancer, and Aurora-A inhibitor could be used as either an independent or concurrent agent in tamoxifen-resistant tumour.

Assessment of serum KL-6 as a prognostic marker in pulmonary tuberculosis patients.

Although serum Krebs von den Lungen-6 (KL-6) levels are reported to increase in pulmonary tuberculosis (PTB) patients according to disease activity, the relationship between serum KL-6 levels and prognosis remains unclear. In this study, we prospectively examined serum KL-6 levels in 188 PTB patients and assessed 60-day mortality. Univariate and multivariate logistic regression analysis demonstrated that serum KL-6 levels were not significantly associated with prognosis. For receiver operating characteristic analysis, the area under the curve had low accuracy for predicting mortality. These findings indicate that serum KL-6 levels do not perform adequately for use as a prognostic marker in patients with PTB.

Drug lymphocyte stimulation test is not useful for side effects of anti-tuberculosis drugs despite its timing.

BACKGROUND: Some patients have adverse reactions to anti-tuberculosis drugs. We have reported that drug lymphocyte stimulation testing (DLST), which we performed at Week 1 of adverse reactions, provides little useful information (14.9% sensitivity). However, it remains unclear whether the time of performance of the DLST contributed to these results. METHODS: Patients with adverse reactions to anti-tuberculosis drugs, including rash, hepatitis and fever, underwent DLST in the first week of the adverse reaction and were then randomly assigned to Group A (among whom a second DLST was performed 2 months after the reaction) or Group B (among whom a second DLST was performed >12 months after the reaction). We compared Group A with Group B to determine the optimal timing for the performance of DLST. The causative drug was identified by an oral drug provocation test. RESULTS: Consistent with the previous study, the sensitivity of DLST performed in the first week was low (14.3%). For DLST performed later, the sensitivity in Group A and Group B was respectively 5.0% and 6.7%. CONCLUSIONS: DLST is not useful for determining the causative drug in patients with rash, hepatitis or fever reactions to anti-tuberculosis drugs, regardless of when it is performed.

Temporary blood pressure drop after bevacizumab administration is associated with clinical course of advanced colorectal cancer.

BACKGROUND: A blood pressure drop after bevacizumab administration and its clinical significance have not been previously reported. METHODS: Blood pressure data at 0, 90, and 180 min after a total of 162 bevacizumab administrations in 81 advanced colorectal cancer patients were retrospectively investigated. RESULTS: Twenty-five patients (30%) demonstrated an average temporary drop of 20 mm Hg or more in systolic blood pressure. We classified these 25 patients as group A and the others as group B. Median time-to-treatment failure (TTF) was significantly longer in group A than in group B (291 vs 162 days; P=0.02). Furthermore, the proportion of patients who required intervention with antihypertensive drugs during bevacizumab treatment was significantly higher in group A than in group B (36% vs 4%; P<0.01). CONCLUSION: This study suggests that a temporary blood pressure drop after bevacizumab administration could be a predictive marker for bevacizumab treatment.

[Lung adenocarcinoma with mixed subtypes which had been followed up for 5-years].

A 63-year-old female was admitted to our hospital for investigation of serum elevation of carcinoembryonic antigen (CEA). She underwent high anterior resection for a rectal cancer 5-years ago. Chest computed tomography (CT) obtained 5-years ago showed a nodule in the right S10, measuring 1.3 x 0.8 cm in size. The nodule was assessed as benign. Chest CT on admission showed the enlarged nodule with a pleural indentation, measuring 2.2 x 1.6 cm in size. Definitive diagnosis could not be established. Since it was difficult to exclude the possibility of malignancy, video-assisted partial resection was performed. Histological examination of the nodule revealed primary adenocarcinoma in frozen sections. Lobectomy with lymph node dissection was performed. The ultimate diagnosis was adenocarcinoma with mixed subtypes. The tumor was classified as stage IA with T1bN0M0. We reported this case because it was a rare slow-growing adenocarcinoma that had a 5-years clinical history before operation.

A case of progressive digital ischemia after early withdrawal of gemcitabine and S-1 in a patient with systemic sclerosis.

The safety of chemotherapy for patients with systemic sclerosis is unclear, and there are few published reports documenting the side effects of chemotherapy in patients with this condition. Here, we report the case of a patient with systemic sclerosis who developed severe digital ischemia during combination gemcitabine/S-1 chemotherapy for pancreatic cancer. In spite of aggressive treatment, the digital ischemia progressively worsened and gangrenous changes developed in multiple fingers and toes. In this patient, the systemic sclerosis had been well controlled, with no digital ischemic symptoms for the previous 6 years, so this progressive clinical course in spite of aggressive treatment strongly suggests that the chemotherapy triggered or aggravated the digital necrosis. To the best of our knowledge, this is only the third reported case of a patient with systemic sclerosis developing digital necrosis after gemcitabine-based chemotherapy. The incidence of digital necrosis during chemotherapy in patients with systemic sclerosis is unknown, and the mechanism by which it occurs is unclear, but the three reports published to date, including the present case, suggest that physicians should be very cautious about administering gemcitabine-based chemotherapy to patients with systemic sclerosis. Any resulting digital ischemia might be refractory to treatment and worsen progressively, even if chemotherapy is withdrawn in the early stages of digital ischemia.

RhoA and RhoC are both required for the ROCK II-dependent promotion of centrosome duplication.

CDK2-cyclin E triggers centrosome duplication, and nucleophosmin (NPM/B23) is found to be one of its targets. NPM/B23 phosphorylated by CDK2-cyclin E acquires a high binding affinity to Rho-associated kinase (ROCK II), and physically associates with ROCK II. The NPM/B23-binding results in superactivation of ROCK II, which is a critical event for initiation of centrosome duplication. The activation of ROCK II also requires the binding of Rho small GTPase to the auto-inhibitory region; hence the availability of the active Rho protein is an important aspect of the centrosomally localized ROCK II to properly initiate centrosome duplication. There are three isoforms of Rho (RhoA, B and C), all of which are capable of binding to and priming the activation of ROCK II. Here, we investigated which Rho isoform(s) are involved in the activation of ROCK II in respect to the initiation of centrosome duplication. We found that both RhoA and RhoC, but not RhoB, were required for initiation of centrosome duplication, and overactivation of RhoA, as well as RhoC, but not RhoB, promoted centrosome duplication and centrosome amplification.

A history of smoking is inversely correlated with the incidence of gemcitabine-induced neutropenia.

BACKGROUND: Smoking may affect the efficacy of chemotherapy and the incidence of adverse events. We investigated the correlation between smoking history and gemcitabine-induced neutropenia. PATIENTS AND METHODS: Data on smoking history and incidence of grade 3-4 neutropenia were retrospectively gathered for 103 chemo-naive patients treated with gemcitabine monotherapy (59 patients with pancreatic, 41 with hepatobiliary and three with other cancers). RESULTS: There was a significantly higher incidence of grade 3-4 neutropenia among patients without a history of smoking (55.7%) than among those with a history of smoking (including current and ex-smokers; 23.6%) [odds ratio (OR) 0.244, 95% confidence interval (CI) 0.105-0.569; P < 0.001]. After adjustment for age, gender, platelet and baseline neutrophil counts, history of surgery for primary cancer, creatinine concentration, hemoglobin concentration, aspartate aminotransferase concentration, alanine aminotransferase concentration and total bilirubin concentration, logistic regression analysis identified a history of smoking as an independent inverse predictor of gemcitabine-induced neutropenia (OR 0.188, 95% CI 0.057-0.618; P = 0.006). CONCLUSION: Patients without a history of smoking may be at higher risk of developing gemcitabine-induced neutropenia. The mechanism underlying this phenomenon is unclear at this point.

Baicalein 5,6,7-trimethyl ether activates peroxisomal but not mitochondrial fatty acid beta-oxidation.

Recently, we reported that baicalein 5,6,7-trimethyl ether (BTM), a flavonoid, is capable of activating fatty acid beta-oxidation in X-linked adrenoleukodystrophy (X-ALD) fibroblasts (FEBS Lett. 2005; 579: 409-414). The objective of this study was to clarify whether BTM activates peroxisomal and/or mitochondrial fatty acid beta-oxidation. We first analysed the effect of BTM on fatty acid beta-oxidation in fibroblasts derived from healthy controls as well as patients with X-ALD, mitochondrial carnitine-acylcarnitine translocase (CACT) deficiency, and peroxisome biogenesis disorder, Zellweger syndrome. Lignoceric acid (C(24:0)) beta-oxidation in the fibroblasts was stimulated by treatment with BTM, except for Zellweger fibroblasts. In contrasts, palmitic acid (C(16:0)) beta-oxidation was increased (2.8-fold) only in CACT-deficient fibroblasts. In U87 glioblastoma cells, C(24:0) beta-oxidation was also activated by treatment with BTM but C(16:0) beta-oxidation was not. The C(16:0) beta-oxidation was, however, significantly increased in the presence of 2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA), a carnitine palmitoyltransferase I inhibitor. These results indicate that BTM activates peroxisomal but not mitochondrial fatty acid beta-oxidation. In addition, we found that BTM did not upregulate the expression of ABCD2/ALDR, ABCD3/PMP70, ACOX1 and FATP4 genes but slightly increased ACSVL1 gene expression.

Roles of cyclins A and E in induction of centrosome amplification in p53-compromised cells.

Abnormal amplification of centrosomes, which occurs frequently in cancers, leads to high frequencies of mitotic defect and chromosome segregation error, profoundly affecting the rate of tumor progression. Centrosome amplification results primarily from overduplication of centrosomes, and p53 is involved in the regulation of centrosome duplication partly through controlling the activity of cyclin-dependent kinase (CDK) 2-cyclin E, a kinase complex critical for the initiation of centrosome duplication. Thus, loss or mutational inactivation of p53 leads to an increased frequency of centrosome amplification. Moreover, the status of cyclin E greatly influences the frequency of centrosome amplification in cells lacking functional p53. Here, we dissected the roles of CDK2-associating cyclins, namely cyclins E and A, in centrosome amplification in the p53-negative cells. We found that loss of cyclin E was readily compensated by cyclin A for triggering the initiation of centrosome duplication, and thus the centrosome duplication kinetics was not significantly altered in cyclin E-deficient cells. It has been shown that cells lacking functional p53, when arrested in either early S-phase or late G(2) phase, continue to reduplicate centrosomes, resulting in centrosome amplification. In cells arrested in early S phase, cyclin E, but not cyclin A, is important in centrosome amplification, whereas in the absence of cyclin E, cyclin A is important for centrosome amplification. In late G(2)-arrested cells, cyclin A is important in centrosome amplification irrespective of the cyclin E status. These findings advance our understandings of the mechanisms underlying the numeral abnormality of centrosomes and consequential genomic instability associated with loss of p53 function and aberrant expression of cyclins E and A in cancer cells.

Pharmacologic action of oseltamivir on the nervous system.

Oseltamivir, an antiviral drug used for the treatment of influenza, contains the L-glutamic acid motif in its chemical structure. We focused on this structural characteristic of oseltamivir and examined the pharmacologic effects of the drug on the nervous system in invertebrate and vertebrate animal models. Injection of oseltamivir or L-glutamic acid into silkworm (Bombyx mori) larvae induced muscle relaxation. Oseltamivir and L-glutamic acid inhibited kainate-induced rapid muscle contraction, but neither drug affected insect cytokine paralytic peptide-induced slow muscle contraction. In the mammalian system, mice (Mus musculus) treated intracerebrally with oseltamivir developed convulsive seizures. Hydrolyzed oseltamivir, the active form containing a carboxylic acid, evoked epileptiform firing of hippocampal neurons in rat (Rattus norvegicus) organotypic hippocampal slice cultures. These results are the first to demonstrate that oseltamivir exerts pharmacologic effects on the nervous system in insects and mammals.