Author Correction: Heterochromatin links to centromeric protection by recruiting shugoshin.

An Amendment to this Letter has been published and is linked from the HTML version of this paper.

Propagation-based phase-contrast imaging method for full-field X-ray microscopy using advanced Kirkpatrick-Baez mirrors.

We demonstrate a propagation-based phase-contrast imaging method for full-field X-ray microscopy based on advanced Kirkpatrick-Baez (AKB) mirrors to achieve high-contrast observations of weak phase objects and correct field curvature aberrations. Through a demonstration performed at SPring-8, the phase contrast of weak phase objects such as polystyrene spheres and chemically fixed cells was successfully observed with high sensitivity (∼0.03 rad). Furthermore, the field of view of the AKB mirrors was expanded to the full area of the obtained images (25 × 30 µm) by correcting the field curvature aberration using reconstructed complex wavefields.

Soft X-ray ptychography system using a Wolter mirror for achromatic illumination optics.

A soft X-ray ptychography system using a Wolter mirror for the illumination optics has been developed. By taking advantage of the achromaticity of the optics, the system is capable of seamlessly imaging at half-period resolution of 50 nm with a broad photon-energy range from 250 eV to 2 keV while maintaining the focal position. Imaging a mammalian cell at various wavelengths was demonstrated, and high-resolution visualization of organelle was achieved. Stereo imaging was also performed with a long working distance of 20 mm. In combination with in-situ/operando and tomographic measurements, this system will be a powerful tool for observing biological and material targets with complex features.

Formation Mechanism and Toxicological Significance of Biogenic Mercury Selenide Nanoparticles in Human Hepatoma HepG2 Cells.

It is widely recognized that the toxicity of mercury (Hg) is attenuated by the simultaneous administration of selenium (Se) compounds in various organisms. In this study, we revealed the mechanisms underlying the antagonistic effect of sodium selenite (Na2SeO3) on inorganic Hg (Hg2+) toxicity in human hepatoma HepG2 cells. Observations by transmission electron microscopy indicated that HgSe (tiemannite) granules of up to 100 nm in diameter were accumulated in lysosomal-like structures in the cells. The HgSe granules were composed of a number of HgSe nanoparticles, each measuring less than 10 nm in diameter. No accumulation of HgSe nanoparticles in lysosomes was observed in the cells exposed to chemically synthesized HgSe nanoparticles. This suggests that intracellular HgSe nanoparticles were biologically generated from Na2SeO3 and Hg2+ ions transported into the cells and were not derived from HgSe nanoparticles formed in the extracellular fluid. Approximately 85% of biogenic HgSe remained in the cells at 72 h post culturing, indicating that biogenic HgSe was hardly excreted from the cells. Moreover, the cytotoxicity of Hg2+ was ameliorated by the simultaneous exposure to Na2SeO3 even before the formation of insoluble HgSe nanoparticles. Our data confirmed for the first time that HepG2 cells can circumvent the toxicity of Hg2+ through the direct interaction of Hg2+ with a reduced form of Se (selenide) to form HgSe nanoparticles via a Hg-Se soluble complex in the cells. Biogenic HgSe nanoparticles are considered the ultimate metabolite in the Hg detoxification process.

Wound fluid of rats fed protein-free diets delays wound healing through the suppression of the IGF-1/ERK(1/2) signaling pathway.

Adequate nutrition is required to maintain healthy skin integrity, and malnourished patients with poor protein diet often experience delayed wound healing. Understanding the cellular mechanisms of protein malnutrition will justify the importance of optimal protein diets in health and disease defense. Therefore in the present study, we examined the effects of changes in wound fluid composition and its function caused by protein malnutrition on wound healing. Rats were fed a control (CO; 20% protein) diet or a protein-free (PF) diet for 2 weeks; we then created full-thickness wounds on the dorsolateral skin. On day 5 after wounding, frozen sections of the wounds were created to investigate the state of granulation tissues, and wound fluid obtained from the rats was collected to examine variations in cytokine levels and its function. Wound closure was significantly delayed from day 4 until total wound closure in rats fed a PF diet. Thickness of granulation tissue, which is composed of mainly dermal fibroblasts, and Ki67 immunohistochemical staining were significantly decreased in rats fed PF diets. PF diets decreased insulin-like growth factor (IGF)-I, which promotes wound healing, and increased IGF-binding protein-1, which inhibits IGF-I bioavailability, in wound fluid. Wound fluid obtained from rats fed a PF diet suppressed dermal fibroblast proliferation. Furthermore, the wound fluid remarkably decreased the phosphorylation level of IGF-I receptor ß (IGF-IR) and extracellular signal-regulated kinase (ERK)(1/2) in dermal fibroblasts. These results show that wound fluid of rats fed PF diets delays wound healing by inhibiting granulation tissue formation through the suppression of the IGF-1/ERK(1/2) signaling pathway.

Imaging of intracellular fatty acids by scanning X-ray fluorescence microscopy.

Fatty acids are taken up by cells and incorporated into complex lipids such as neutral lipids and glycerophospholipids. Glycerophospholipids are major constituents of cellular membranes. More than 1000 molecular species of glycerophospholipids differ in their polar head groups and fatty acid compositions. They are related to cellular functions and diseases and have been well analyzed by mass spectrometry. However, intracellular imaging of fatty acids and glycerophospholipids has not been successful due to insufficient resolution using conventional methods. Here, we developed a method for labeling fatty acids with bromine (Br) and applied scanning X-ray fluorescence microscopy (SXFM) to obtain intracellular Br mapping data with submicrometer resolution. Mass spectrometry showed that cells took up Br-labeled fatty acids and metabolized them mainly into glycerophospholipids in CHO cells. Most Br signals observed by SXFM were in the perinuclear region. Higher resolution revealed a spot-like distribution of Br in the cytoplasm. The current method enabled successful visualization of intracellular Br-labeled fatty acids. Single-element labeling combined with SXFM technology facilitates the intracellular imaging of fatty acids, which provides a new tool to determine dynamic changes in fatty acids and their derivatives at the single-cell level.-Shimura, M., Shindou, H., Szyrwiel, L., Tokuoka, S. M., Hamano, F., Matsuyama, S., Okamoto, M., Matsunaga, A., Kita, Y., Ishizaka, Y., Yamauchi, K., Kohmura, Y., Lobinski, R., Shimizu, I., Shimizu, T. Imaging of intracellular fatty acids by scanning X-ray fluorescence microscopy.

Transforming growth factor-ß1 induces cholesterol synthesis by increasing HMG-CoA reductase mRNA expression in keratinocytes.

In this study, we investigated the effect of TGF-ß1 on cholesterol synthesis in human keratinocytes. TGF-ß1 increased the level of cholesterol and the mRNA level of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in human keratinocytes. These results show that TGF-ß1 induces cholesterol synthesis by increasing HMG-CoA reductase mRNA expression in human keratinocytes.

Transcription factors interfering with dedifferentiation induce cell type-specific transcriptional profiles.

Transcription factors (TFs) are able to regulate differentiation-related processes, including dedifferentiation and direct conversion, through the regulation of cell type-specific transcriptional profiles. However, the functional interactions between the TFs regulating different transcriptional profiles are not well understood. Here, we show that the TFs capable of inducing cell type-specific transcriptional profiles prevent the dedifferentiation induced by TFs for pluripotency. Of the large number of TFs expressed in a neural-lineage cell line, we identified a subset of TFs that, when overexpressed, strongly interfered with the dedifferentiation triggered by the procedure to generate induced pluripotent stem cells. This interference occurred through a maintenance mechanism of the cell type-specific transcriptional profile. Strikingly, the maintenance activity of the interfering TF set was strong enough to induce the cell line-specific transcriptional profile when overexpressed in a heterologous cell type. In addition, the TFs that interfered with dedifferentiation in hepatic-lineage cells involved TFs with known induction activity for hepatic-lineage cells. Our results suggest that dedifferentiation suppresses a cell type-specific transcriptional profile, which is primarily maintained by a small subset of TFs capable of inducing direct conversion. We anticipate that this functional correlation might be applicable in various cell types and might facilitate the identification of TFs with induction activity in efforts to understand differentiation.

Hydrocellular foam dressing increases the leptin level in wound fluid.

Hydrocellular foam dressing (HCF) absorbs excessive wound fluid, which contains various cytokines and growth factors, and ensures a moist environment to promote wound healing. However, the molecular mechanisms underlying the wound fluid component changes induced by HCF are poorly understood. In the present study, we examined the effect of HCF on wound healing and the associated regulatory mechanisms in relation to variations in cytokine levels in the wound fluid. We created full-thickness wounds on the dorsolateral skin of rats and collected the resulting wound fluid samples. HCF was immersed in a plate containing the wound fluids. HCF was then removed and the excess wound fluid remaining in the plate was examined by cytokine array and enzyme-linked immunosorbent assay. We also used a rat model and human dermal fibroblast cultures to examine the effect of wound fluid component changes during the wound healing process. Upon treatment with HCF, leptin levels were upregulated in the wound fluid. Fibroblast proliferation was enhanced and the effect was suppressed in the presence of leptin antagonist. In our in vivo model, HCF increased wound contraction compared with film dressings and this positive effect of HCF was suppressed by addition of leptin antagonist. Our results suggest that dermal fibroblast proliferation is upregulated by HCF due to increased leptin level at the wound surface, and these effects promote wound healing. We believe that the present study contributes to furthering the understanding of the mechanisms underlying the effects of HCF-induced wound healing.

Hydrocellular foam dressings promote wound healing associated with decrease in inflammation in rat periwound skin and granulation tissue, compared with hydrocolloid dressings.

The effects of modern dressings on inflammation, which represent the earliest phase of wound healing, are poorly understood. We investigated the effects of modern hydrocellular foam dressings (HCFs) on wound healing and on the gene expression levels of the inflammatory markers--interleukin (IL)-1ß, IL-6, and IL-10--in rat periwound skin and granulation tissue by quantitative reverse transcription-polymerase chain reaction. HCF absorbed significantly higher volume of water than hydrocolloid dressing (HCD) and increased the contraction of wounds. Polymorphonuclear neutrophils were massively infiltrated to the wound edge and boarded between granulation and dermis in the HCD group. IL-1ß, IL-6, and IL-10 mRNA levels were significantly decreased in the periwound skin around the wounds and granulation tissue covered with HCF. These findings suggest that HCF may promote wound healing along with decrease in inflammation by reducing gene expression levels of IL-1ß, IL-6, and IL-10.

Heterochromatin links to centromeric protection by recruiting shugoshin.

The centromere of a chromosome is composed mainly of two domains, a kinetochore assembling core centromere and peri-centromeric heterochromatin regions. The crucial role of centromeric heterochromatin is still unknown, because even in simpler unicellular organisms such as the fission yeast Schizosaccharomyces pombe, the heterochromatin protein Swi6 (HP1 homologue) has several functions at centromeres, including silencing gene expression and recombination, enriching cohesin, promoting kinetochore assembly, and, ultimately, preventing erroneous microtubule attachment to the kinetochores. Here we show that the requirement of heterochromatin for mitotic chromosome segregation is largely replaced by forcibly enriching cohesin at centromeres in fission yeast. However, this enrichment of cohesin is not sufficient to replace the meiotic requirement for heterochromatin. We find that the heterochromatin protein Swi6 associates directly with meiosis-specific shugoshin Sgo1, a protector of cohesin at centromeres. A point mutation of Sgo1 (V242E), which abolishes the interaction with Swi6, impairs the centromeric localization and function of Sgo1. The forced centromeric localization of Sgo1 restores proper meiotic chromosome segregation in swi6 cells. We also show that the direct link between HP1 and shugoshin is conserved in human cells. Taken together, our findings suggest that the recruitment of shugoshin is the important primary role for centromeric heterochromatin in ensuring eukaryotic chromosome segregation.

Identification of viral protein R of human immunodeficiency virus-1 (HIV) and interleukin-6 as risk factors for malignancies in HIV-infected individuals: A cohort study.

BACKGROUND: Despite effective antiretroviral therapy, patients with human immunodeficiency virus type-1 (HIV) suffer from a high frequency of malignancies, but related risk factors remain elusive. Here, we focused on blood-circulating viral protein R (Vpr) of HIV, which induces proinflammatory cytokine production and genotoxicity by exogenous functions. METHODS AND FINDINGS: A total 404 blood samples of HIV patients comprising of 126 patients with malignancies (tumor group) and 278 patients without malignancies (non-tumor group), each of 96 samples was first selected by one-to-one propensity score matching. By a detergent-free enzyme-linked immunosorbent assays (detection limit, 3.9 ng/mL), we detected Vpr at a higher frequency in the matched tumor group (56.3%) than in the matched non-tumor group (39.6%) (P = 0.030), although there was no different distribution of Vpr levels (P = 0.372). We also detected anti-Vpr immunoglobulin (IgG), less frequently in the tumor group compared with the tumor group (22.9% for tumor group vs. 44.8% for non-tumor group, P = 0.002), and the proportion of patients positive for Vpr but negative of anti-Vpr IgG was significantly higher in the tumor group than in the non-tumor group (38.6% vs. 15.6%, respectively, P < 0.001). Additionally, Interleukin-6 (IL-6), the levels of which were high in HIV-1 infected patients (P < 0.001) compared to non-HIV-infected individuals, was significantly higher in advanced cases of tumors (P < 0.001), and IL-6 level was correlated with Vpr in the non-tumor group (P = 0.010). Finally, multivariate logistic regression analysis suggested a positive link of Vpr with tumor occurrence in HIV patients (P = 0.002). CONCLUSION: Vpr and IL-6 could be risk factors of HIV-1 associated malignancies, and it would be importance to monitor these molecules for well managing people living with HIV-1.

Ultracompact mirror device for forming 20-nm achromatic soft-X-ray focus toward multimodal and multicolor nanoanalyses.

Nanoscale soft-X-ray microscopy is a powerful analysis tool in biological, chemical, and physical sciences. To enhance its probe sensitivity and leverage multimodal soft-X-ray microscopy, precise achromatic focusing devices, which are challenging to fabricate, are essential. Here, we develop an ultracompact Kirkpatrick-Baez (ucKB) mirror, which is ideal for the high-performance nanofocusing of broadband-energy X-rays. We apply our advanced fabrication techniques and short-focal-length strategy to realize diffraction-limited focusing over the entire soft-X-ray range. We achieve a focus size of 20.4 nm at 2 keV, which represents a significant improvement in achromatic soft-X-ray focusing. The ucKB mirror extends soft-X-ray fluorescence microscopy by producing a bicolor nanoprobe with a 1- or 2-keV photon energy. We propose a subcellular chemical mapping method that allows a comprehensive analysis of specimen morphology and the distribution of light elements and metal elements. ucKB mirrors will improve soft-X-ray nanoanalyses by facilitating photon-hungry, multimodal, and polychromatic methods, even with table-top X-ray sources.

A lipid index for risk of hyperlipidemia caused by anti-retroviral drugs.

HIV-associated lipodystrophy has been reported in people taking anti-retroviral therapy (ART). Lipodystrophy can cause cardiovascular diseases, affecting the quality of life of HIV-infected individuals. In this study, we propose a pharmacological lipid index to estimate the risk of hyperlipidemia caused by anti-retroviral drugs. Lipid droplets were stained in cells treated with anti-retroviral drugs and cyclosporin A. Signal intensities of lipid droplets were plotted against the drug concentrations to obtain an isodose of 10 µM of cyclosporin A, which we call the Pharmacological Lipid Index (PLI). The PLI was then normalized by EC50. PLI/EC50 values were low in early proteinase inhibitors and the nucleoside reverse transcriptase inhibitor, d4T, indicating high risk of hyperlipidemia, which is consistent with previous findings of hyperlipidemia. In contrast, there are few reports of hyperlipidemia for drugs with high PLI/EC50 scores. Data suggests that PLI/EC50 is a useful index for estimating the risk of hyperlipidemia.

Viral protein R of human immunodeficiency virus type-1 induces retrotransposition of long interspersed element-1.

BACKGROUND: Viral protein R (Vpr), a protein of human immunodeficiency virus type-1 (HIV-1) with various biological functions, was shown to be present in the blood of HIV-1-positive patients. However, it remained unclear whether circulating Vpr in patients' blood is biologically active. Here, we examined the activity of blood Vpr using an assay system by which retrotransposition of long interspersed element-1 (L1-RTP) was detected. We also investigated the in vivo effects of recombinant Vpr (rVpr) by administrating it to transgenic mice harboring human L1 as a transgene (hL1-Tg mice). Based on our data, we discuss the involvement of blood Vpr in the clinical symptoms of acquired immunodeficiency syndrome (AIDS). RESULTS: We first discovered that rVpr was active in induction of L1-RTP. Biochemical analyses revealed that rVpr-induced L1-RTP depended on the aryl hydrocarbon receptor, mitogen-activated protein kinases, and CCAAT/enhancer-binding protein ß. By using a sensitive L1-RTP assay system, we showed that 6 of the 15 blood samples from HIV-1 patients examined were positive for induction of L1-RTP. Of note, the L1-RTP-inducing activity was blocked by a monoclonal antibody specific for Vpr. Moreover, L1-RTP was reproducibly induced in various organs, including the kidney, when rVpr was administered to hL1-Tg mice. CONCLUSIONS: Blood Vpr is biologically active, suggesting that its monitoring is worthwhile for clarification of the roles of Vpr in the pathogenesis of AIDS. This is the first report to demonstrate a soluble factor in patients' blood active for L1-RTP activity, and implies the involvement of L1-RTP in the development of human diseases.

Zinc and iron dynamics in human islet amyloid polypeptide-induced diabetes mouse model.

Metal homeostasis is tightly regulated in cells and organisms, and its disturbance is frequently observed in some diseases such as neurodegenerative diseases and metabolic disorders. Previous studies suggest that zinc and iron are necessary for the normal functions of pancreatic ß cells. However, the distribution of elements in normal conditions and the pathophysiological significance of dysregulated elements in the islet in diabetic conditions have remained unclear. In this study, to investigate the dynamics of elements in the pancreatic islets of a diabetic mouse model expressing human islet amyloid polypeptide (hIAPP): hIAPP transgenic (hIAPP-Tg) mice, we performed imaging analysis of elements using synchrotron scanning X-ray fluorescence microscopy and quantitative analysis of elements using inductively coupled plasma mass spectrometry. We found that in the islets, zinc significantly decreased in the early stage of diabetes, while iron gradually decreased concurrently with the increase in blood glucose levels of hIAPP-Tg mice. Notably, when zinc and/or iron were decreased in the islets of hIAPP-Tg mice, dysregulation of glucose-stimulated mitochondrial respiration was observed. Our findings may contribute to clarifying the roles of zinc and iron in islet functions under pathophysiological diabetic conditions.

Differential anti-APOBEC3G activity of HIV-1 Vif proteins derived from different subtypes.

Antiretroviral cytidine deaminase APOBEC3G, which is abundantly expressed in peripheral blood lymphocytes and macrophages, strongly protects these cells against HIV-1 infection. The HIV-1 Vif protein overcomes this antiviral effect by enhancing proteasome-mediated APOBEC3G degradation and is key for maintaining viral infectivity. The 579-bp-long vif gene displays high genetic diversity among HIV-1 subtypes. Therefore, it is intriguing to address whether Vif proteins derived from different subtypes differ in their viral defense activity against APOBEC3G. Expression plasmids encoding Vif proteins derived from subtypes A, B, C, CRF01_AE, and CRF02_AG isolates were created, and their anti-APOBEC3G activities were compared. Viruses produced from cells expressing APOBEC3G and Vif proteins from different subtypes showed relatively different viral infectivities. Notably, subtype C-derived Vif proteins tested had the highest activity against APOBEC3G that was ascribed to its increased binding activity, for which the N-terminal domain of the Vif protein sequences was responsible. These results suggest that the biological differences of Vif proteins belonging to different subtypes might affect viral fitness and quasispecies in vivo.

Occupational exposure of platinum-based anti-cancer drugs: five-year monitoring of hair and environmental samples in a single hospital.

BACKGROUND: Occupational exposure to chemotherapeutic agents in hospitals is a critical issue. Here, we focused on occupational exposure to platinum-based anti-cancer drugs (PDs) by evaluating platinum concentrations in hair and environmental workplace samples to monitor the risk among workers. METHODS: Hospital workers who dealt with or without PDs, patients treated with PDs, and non-medical office workers outside the hospital donated hair samples and completed a questionnaire regarding their history of handling PDs, including any incidents. Hair samples were collected and surface wipe sampling was performed in July 2010 and April 2015, before and after moving to a new building and introducing a revised safety program in August 2010. Samples were analyzed by inductively coupled plasma-mass spectrometry. RESULTS: Platinum concentrations in hair from PDs-handling workers was significantly higher than in non-PDs-handling workers (P = 0.045), although 50 times lower than that from PDs-treated patients. Platinum concentrations in the hospital environment had decreased at the second survey 5 years later but had not changed significantly in the hair samples from hospital workers. CONCLUSION: Platinum concentrations in hair are likely dependent on the frequency of handling PDs. Reduced environmental contamination from PDs did not influence platinum levels in hospital workers' hair. Continuous monitoring by measuring platinum concentrations in the environment and in hair would provide information regarding these issues.

Cu and Zn isotope ratio variations in plasma for survival prediction in hematological malignancy cases.

We have examined potential changes in the isotopic compositions of Fe, Cu and Zn (using multi-collector inductively coupled plasma-mass spectrometry) and the corresponding concentrations (using inductively coupled plasma-atomic emission spectrometry) in plasma from hematological malignancy (HM) patients and assessed their prognostic capability. Together with clinical laboratory test values, data were examined in view of a 5-years survival prediction. Plasma Cu and Zn isotope ratios and their concentrations were significantly different in HM patients compared to matched controls (P < 0.05). Both δ65Cu and δ66Zn values showed significant mortality hazard ratios (HRs) in HM. The group of patients with decreased δ65Cu and increased δ66Zn values showed significantly poorer survival from the early phase (HR 3.9; P = 0.001), forming a unique cohort not identified based on laboratory test values. Well-known prognostic factors for HM, such as the creatinine level, and anemia-related values were highly correlated with the δ66Zn value (P < 0.05). Time-dependent ROC curves based on the δ65Cu or δ66Zn value were similar to that based on the creatinine concentration (a well-known prognostic factor in HM), indicating that δ65Cu or δ66Zn values are useful for prognosis of HM. Variations in stable isotope ratios of essential mineral elements have thus been shown to reflect alterations in their homeostasis due to physiological changes in malignancies with higher sensitivity than concentrations do.

All APOBEC3 family proteins differentially inhibit LINE-1 retrotransposition.

Approximately 17% of the human genome is comprised of long interspersed nuclear element 1 (LINE-1, L1) non-LTR retrotransposons. L1 retrotransposition is known to be the cause of several genetic diseases, such as hemophilia A, Duchene muscular dystrophy, and so on. The L1 retroelements are also able to cause colon cancer, suggesting that L1 transposition could occur not only in germ cells, but also in somatic cells if innate immunity would not function appropriately. The mechanisms of L1 transposition restriction in the normal cells, however, are not fully defined. We here show that antiretroviral innate proteins, human APOBEC3 (hA3) family members, from hA3A to hA3H, differentially reduce the level of L1 retrotransposition that does not correlate either with antiviral activity against Vif-deficient HIV-1 and murine leukemia virus, or with patterns of subcellular localization. Importantly, hA3G protein inhibits L1 retrotransposition, in striking contrast to the recent reports. Inhibitory effect of hA3 family members on L1 transposition might not be due to deaminase activity, but due to novel mechanism(s). Thus, we conclude that all hA3 proteins act to differentially suppress uncontrolled transposition of L1 elements.