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Bone marrow mesenchymal stromal cells (BMSCs) can differentiate into adipocytes and osteoblasts, and are important regulators of the haematopoietic system. Ageing associates with an increased ratio of bone marrow adipocytes to osteoblasts and immune dysregulation. Here, we carried out an integrative multiomics analysis of ATAC-Seq, RNA-Seq and proteomics data from primary human BMSCs in a healthy cohort age between 20 - 60. We identified age-sensitive elements uniquely affecting each molecular level where transcription is mostly spared, and characterised the underlying biological pathways, revealing the interplay of age-related gene expression mechanism changes spanning multiple gene regulatory layers. Through data integration with enhancer-mediated gene regulatory network analysis, we discovered that enhancers and transcription factors influence cell differentiation potential in the ageing BMSCs. By combining our results with genome-wide association study data, we found that age-specific changes could contribute to common traits related to BMSC-derived tissues such as bone and adipose tissue, and to immune-related traits on a systemic level such as asthma. We demonstrate here that a multiomics approach is crucial for unravelling complex information, providing new insights on how ageing contributes to bone marrow- and immune-related disorders.

Self-assembled monolayers of several alkanethiol molecules, with varying chain length and terminal groups, were investigated using x-ray photoelectron spectroscopy (XPS). The chain lengths varied from 10 to 15 methylene units, and the terminal group was either hydrophilic carboxylic acid and hydroxyl or hydrophobic methyl. The alkanethiol molecules were deposited on GaAs surfaces from liquid solutions. The impact of atmospheric exposure was examined by investigating one set of samples stored under atmospheric conditions and a second set that was stored in a nitrogen atmosphere prior to analysis. Carbon, oxygen, gallium, and arsenic core level XPS spectra were obtained on all surfaces. The intensity of the gallium and arsenic core levels indicates a considerable difference in the Ga∕As ratio dependent on the terminal group of the alkanethiol. Additionally, the carbon and oxygen spectra indicate varying chemical bonding on the surface with the alkanethiol’s having a carboxylic acid terminal group showing a ...

Transient processes of laser ablation of a highly laser-absorbing liquid, toluene, were investigated by directly measuring (by using a fast-response pressure gauge) the transient pressure caused by toluene ablation under KrF laser irradiation . The results were compared with time-resolved images . The peak pressure P due to a shock wave decreased slowly with increasing distance d for d=100–1000 μm:P∝d-0.33. By extrapolating P to d=8.9 μm, the optical penetration depth of toluene at λ=248 nm, the estimated initial pressure due to toluene ablation was 65 MPa at 1.0 J cm-2 pulse-1. The estimated initial pressure increased linearly with the fluence. These results help clarify the mechanism of laser-induced backside wet etching.

Microstructures with well-defined 1 μm-scale grating and grid patterns were fabricated on the surfaces of silica glass using a laser-induced backside wet etching (LIBWE) method by nanosecond-pulsed KrF excimer laser irradiation through a mask projection system. In the former case, gratings as narrow as 0.75 μm-sized were successfully etched on the glass surface via fine adjustments to the projection system. To investigate the etching mechanisms, formation and propagation of the shockwave and micro-bubbles after laser irradiation, at the interface of the silica glass and the toluene solution, were monitored using time-resolved shadowgraph microscopy, from the front- and side-view directions.

Laser-induced backside wet etching (LIBWE) of silica glass plates was performed by the excitation of a pure toluene solution with an ns-pulsed KrF excimer laser at 248 nm. Well-defined grid micro-pattern was fabricated without debris and micro-crack around the etched area. The etch rate, which increased linearly with laser fluence, was 30% higher than that in the case of acetone solution of pyrene dye at a concentration of 0.4 mol dm −3 . To understand the etching mechanism, the formation and propagation of shock wave and bubble were monitored by time-resolved optical microscopy at the interface between the silica glass and the toluene solution during etching process. Transient high pressure as well as high temperature generated by UV laser irradiation plays a key role in the etching process.

Laser-induced backside wet etching of fused-silica plates using an aqueous solution of naphthalene-1,3,6-trisulfonic acid trisodium salt (Np(SO3Na)3) is reported. A KrF excimer laser was employed as a light source. The etch rate varied greatly with the concentration of the solution and the laser fluence. For lower concentration solutions, the etch rate increased linearly with laser fluence. For highly concentrated solutions, however, the etch rate increased abruptly at higher fluence. Well-defined line-and-space and grid micropatterns were fabricated using a low etch rate. The etched surface was as flat as the surface of the virgin plates and the etched pattern was free of debris and microcracks. The formation and propagation of shockwaves and bubbles in the solution during the etch process were monitored. High pressure, as well as the high temperature generated by the photothermal process, plays a key role in the etching process.

The laser-induced back-side wet etching of fused silica with aqueous solutions of pyranine (8-hydroxy-1,3,6-pyrenetrisulfonic acid trisodium salt) is reported. KrF and XeF excimer lasers were employed as light sources. Well-defined line-and-space and grid micropatterns, free of debris and microcracks, were obtained. Compared with other organic solutions, the aqueous pyranine etching medium etches more slowly but produces a higher quality etched surface. With the KrF laser, the etch rate ranged from 0.02 to 0.12 nm pulse-1, depending on the dye concentration and the fluence of the laser. The etch rate decreased dramatically when the XeF laser was employed, which was partially attributed to the lower absorption efficiency of the aqueous pyranine solution at the XeF laser wavelength.

A novel asymmetrically substituted amphiphilic phthalocyanine, 1,4-di(2-hydroxyethoxyl)-8,9,16,17,23,24-hexapentyloxylphthalocyaninozinc ( asy - PcZn ), was synthesized. It can be deposited as well-ordered LB films. Significant structural changes of the films were observed by UV-vis absorption spectra and low-angle X-ray diffraction after the films were annealed at 150 °C. What is more interesting is that the initial conductance of the films in air decreases and the response signal to NH 3 is weaker with longer response time after annealing compared with that of the unpretreated films.

Background: Whereas proteomics analyses at a cellular level have been performed in murine HPC, most proteomics studies on human material have thus far focused on examining human tissues with a mixture of cell types and not at a specific cellular level. Whole proteomics analyses might reveal genuine molecular alterations und mechanisms of aging on human hematopoietic cells. Aim: To identify the age-related molecular changes in human hematopoietic progenitor cells (HPC) as well as in the cellular elements of the human bone marrow niche, we have applied mass spectrometry-based platform for proteomics analysis of this network of bone marrow (BM) cells at a cellular level. The overarching goal is to acquire a systems understanding of the molecular mechanisms involved in aging of human HPC as well as other cellular constituents of the marrow niche, and to combine the cell biological, proteomic, and genetic studies for a better understanding of age-related diseases such as MDS. Methods: In this study, we have simultaneously characterized at the proteomics level HPC as well as the cellular microenvironment in BM from each individual human subject. Cells were harvested from the BM of 69 healthy human subjects [Age Group (AG) 20-29 years: n=19; AG 30-39: n=16; AG 40-49: n=9; AG 50+: N=15]. BM cells were separated for nucleated cells using Ficoll, followed by sorting with specific marker constellations, i.e. HPC (CD34+), lymphoid cells (CD34-/CD45+/SSClow), myeloid precursors (CD34-/CD45med/SSChigh), monocyte-macrophages (CD34-/CD45+/CD14+), erythroid precursors (CD34-/CD45-), and mesenchymal stromal cells (MSC) based on plastic adherence upon culture. Results: In 60 of these 69 healthy subjects we were able to harvest an adequate amount of material from 5 to 6 the aforementioned subsets from the respective individual, yielding altogether 342 samples for proteomics analysis. After digestion (trypsinization), isobaric labelling of peptides (TMT) of appropriate quality was successfully performed in 270 of these 342 samples derived from 69 healthy human subjects. These 270 samples underwent tandem mass-spectrometry analyses. The number of proteins identified were >12,000, covering 65% of the estimated human proteome (HPC: >7,500; Lymphoid cells: >8,500; Myeloid Precursors: >7,500; Mono/Macro: >8,500; Erythroid Precursors: >6,500; and in MSC: >9,000 proteins). We have then focused on the molecular mechanisms, specifically proteomic alterations involved in aging of these 6 cell types. Gene Set Enrichment Analysis (GSEA) using previously defined gene sets specific for common lymphoid progenitors (CLP), bi-potent granulocyte/macrophage progenitors (GMP), and megakaryocyte progenitors (MKP) revealed significant down-regulation of CLP related proteins and significant up-regulation of MKP related proteins in HPC upon aging. The same down-regulation of CLP proteins and up-regulation of MKP proteins were also found in the BM lymphoid cells. Further significant alterations of note are a decrease in cell cycle related proteins upon aging in HPC, lymphoid cells, as well as in MSC, and an increase in GMP related proteins upon aging in lymphoid cells, erythroid precursors and in MSC. Extensive pathway and network analyses are underway. Conclusions: We have for the first time accomplished a multiplex and comprehensive analysis of a community of cells in human BM, comprising HPC and 5 other cell types that constitute the niche and have identified >12,000 proteins in this cellular network. Proteomics analyses have demonstrated a significant down-regulation of CLP related proteins in human HPC and in marrow lymphoid cells, as well as an up-regulation of MKP in all the cell types tested upon aging. Of note is also a decrease in cell cycle related proteins with age. This study has thus provided evidence that lineage skewing towards megakaryocytic and decrease in lymphoid development upon aging occur in human HPC as well as in other human BM cells. Disclosures No relevant conflicts of interest to declare.

Two kinds of amphiphilic phthalocyanines(AmPc,AmPcZn) have been deposited as smooth and well ordered LB films. Absorption spectra show that AmPc films are consistent with a stacked cofacial columnar structure while the AmPcZn films consist of aggregates with weakly interacting molecules. These two films exhibit different behavior toward NH3 gas-sensing. AmPcZn films gave strong and fast response on exposure to NH3 whereas AmPc films showed very weak response. It is proposed that coordination of NH3 by AmPcZn results in lowering the energy gap and hence the activation energy of AmpcZn that account for the larger increase in conductance of AmpcZn films compared with AmPc films.

A series of asymmetrically substituted amphiphilic phthalocyanines have been synthesized. They have been deposited as Langmuir–Blodgett films onto glass and/or glass substrates bearing aluminum interdigitated electrode patterns. The films show fast response to NO 2 with almost complete recovery at room temperature. It is indicated that layer ordering and molecular packing within the LB films are important factors determined the gas-sensing properties.

To clarify the initial stage of laser-induced backside wet etching, we directly measured transient pressure upon laser ablation of toluene under KrF excimer laser irradiation by using a fast-response pressure gauge. The propagation time of the pressure peak to the gauge agreed well with the time for the shock wave to reach the gauge on time-resolved images. The peak pressure P decreased slowly with increasing distance d: P=30.4 MPa for d=100 μm to P=11.1 MPa for d=1000 μm at a fluence F=1.0 J cm-2 pulse-1. The initial pressure, estimated to be of the order of 10–200 MPa, impinges on a transparent plate and contributes to the etching.

Sapphire plates were etched with the approach of laser-induced backside wet etching (LIBWE) using neat toluene or saturated pyrene/acetone solutions as the etching medium. Using this approach, flexible patterns on the scale of 4-µm can be fabricated on the surface of the plates. The etching process was observed in situ by a CCD camera, which revealed the deposition of black particles and the formation thereafter of a laser-absorbing film in the etched area. By Raman spectroscopic analysis, the film was characterized to be amorphous carbon resulting from the decomposition of toluene or pyrene upon laser irradiation, which is an essential factor contributing to the etching process.

The effect of nitrogen dioxide on the conductance of LB films of three asymmetrically substituted amphiphilic phthalocyanines is investigated. They showed unique properties of fast and reversible responses to NO 2 at room temperature. UV –Vis-polarized absorption spectra and low angle X-ray diffraction studies show that compound 2 deposited as highly ordered LB films, and is superior to others for gas-sensor applications.

1,4-Di(2-hydroxyethoxy)-9,10,16,17,23,24-hexapentyloxy phthalocyanine (asy-Pc) has been deposited as well-ordered Langmuir–Blodgett (LB) films on a glass substrate and glass-bearing interdigital electrodes. The molecular orientation of the asy-Pc LB films has been characterized by polarized UV-vis absorption spectra and in-plane dichroism has been observed. The LB films show good room temperature response to NO 2 gas and the recovery is almost complete. It has been found that the magnitude of the response increases with increasing gas concentration and is also dependent upon the LB dipping direction relative to the arms of the electrodes, consistent with conduction being favored along the axes of the stacks of asy-Pc molecules.

The novel amphiphile 9-octadecyladenine (AdC18), which has an adenine headgroup and a long hydrocarbon chain, was synthesized. Its monolayer behavior and its interactions with three guest molecules were investigated at the air—water interface by means of surface pressure—area isotherms. The structures of multilayer Langmuir—Blodgett (LB) films of AdC18 were characterized by Fourier transform IR spectroscopy. When the monolayer was transferred from a uridine aqueous solution to a solid substrate, the IR spectra varied significantly from the spectrum of the film transferred from the pure water subphase. Shifts of band positions and particularly the appearance of a broadened band demonstrated that guest molecules were transferred to the solid substrate along with the AdC18 monolayer and that hydrogen bonds were formed between complementary nucleobases within the LB films.

For a semiconductor based biosensor, functionalization of the surface and the stability of the semiconductor-biomolecule interface are the primary issues to be addressed by researchers. We have investigated a variety of strategies to passivate (001) GaAs surface with a long chain hexadecanethiol (C 16 H 33 SH: T16). GaAs substrates were cleaned and etched either with ArF excimer laser irradiation in an atmospheric environment or with conventional wet etchants. The effect of surface passivation and stability of the interface were evaluated using photoluminescence (PL) measurements. Significant cleaning of the (001) GaAs surface has been achieved with an ArF laser, as evidenced by the up to 4-fold increase of the PL signal. This compares to the 12-fold enhancement of the PL signal from samples that were alternately etched in solutions of NH 3 /H 2 O and HCl/ethanol. A combination of a diluted base and an acid possibly provides the cleanest surface and therefore the highest surface functionalization efficacy and long term stability upon thiolation.

We have investigated the deposition of biotinylated nano-beads on the surface of GaAs. The deposition procedure involved either direct coating of (001) GaAs with nano-beads, or binding the nano-beads with avidin immobilized on the surface of (001) GaAs through the interface of biotin and the NH 2 terminal group of 11-amino-1-undecanethiol (HS(CH 2 ) 11 NH 2 ). The efficiency of binding was tested by washing the samples in a solution of a commercial detergent and by subjecting them to a deionized water ultrasonic bath. The results indicate that nano-beads deposited directly on the surface of (001) GaAs withstand the detergent washing test but they are easily removed by ultrasonic washing. In contrast, the nano-beads attached to (001) GaAs through the avidin-biotin-thiol interface survive the ultrasonic washing tests.

Passivation of (001) GaAs surface was investigated with self-assembled monolayers (SAMs) of a variety of thiols having various methylene chain length and terminal groups. The effect of passivation was monitored by measuring the intensity of the GaAs-related photoluminescence (PL) signal excited with lasers operating either at 683 or 248 nm wavelengths. Generally, for each case of the thiol treated surface the PL signal was more intense than that from non-treated samples. Additionally, it was found that the thiol terminal groups play an important role in determining the methylene chain orientation in the SAMs and consequently the efficiency of the passivation. The methyl terminated methylene chain formed a layer of a closely packed and relatively thick film, which resulted in a significantly increased PL signal. In contrast, carboxylic acid group (-CO2H) terminated methylene chains formed thin and less compacted films, leading to only a slightly increased PL signal and less efficient passivation of the GaAs surface.

A novel approach in the fabrication of microarrays of dye and protein on fused silica plates using the laser-induced backside wet etching (LIBWE) technique is described. The surface of fused silica plates was initially precoated using trimethoxysilane self-assembled monolayers (SAMs) and then etched using the LIBWE method to obtain the desired microstructures on the plate surface. Using this technique, the SAMs on the nonirradiated areas were able to survive the LIBWE process and were used as templates for the subsequent deposition of dye molecules or proteins via chemical bonding or physical adsorption. In the case of fused silica plates precoated with fluorinated SAMs, the LIBWE method is used to remove the SAMs to expose the etched silica surfaces, on which a thin layer of pyranine molecules can be site-selectively deposited using an aqueous solution of pyranine. In another application, an ethanol solution of rhodamine 6G was preferentially deposited onto the nonirradiated areas. In yet another application, bovine serum albumin was preferentially deposited onto the laser-irradiated areas; in this case, the fused silica plates were precoated with poly(ethylene oxide) SAMs. Interestingly, when an aqueous suspension of polystyrene (PS) microbeads was cast onto the fused silica precoated with the fluorinated SAMs, hexagonally close-packed PS microbeads were deposited into the etched cavities. Depositions of the dye, protein, and microbeads were confirmed by visualization using a fluorescence microscope and scanning electron microscope.

Silica glass is an important material in optics and optoelectronic devices because of its outstanding properties, such as transparence in a wide wavelength range, strong damage resistance for laser irradiation, and high chemical stability. In order to develop simpler processes of micro-fabricating silica glass using a pulsed laser, we have investigated a one-step method to microfabricate a silica glass plate using laser-induced backside wet etching (LIBWE) upon irradiation with ns-pulsed UV lasers. Two laser irradiation systems were employed for the fabrication; (1) mask projection system with a KrF excimer laser and (2) Galvanometer-based point scanning system with a single mode laser beam from a diode-pumped solid state laser at 266 nm. We have succeeded in a fine micro-fabrication of silica glass surface. In addition, a novel approach in the fabrication of microarrays of dye and protein on fused silica plates using LIBWE technique was achieved. The surace of fused silica plates was initially pre-coated using trimethoxysilane self-assembled monolayers (SAMs), then etched using the LIBWE method to obtain the desired microstructures on the plate surface. Using this technique, the SAMs on the non-irradiated areas were able to survive the LIBWE process, and were used as templates for the subsequent deposition of dye molecules, proteins, or polystyrene microbeads via chemical bonding or physical adsorption.

Surface micro-structuring of fused silica glass plates was performed by single-shot irradiation with a single-mode laser beam from a diode-pumped solid state UV laser at 355 nm. Well-defined micropattern without debris and microcrack formations around the etched area was fabricated by laser ablation with a focused laser-beam in the ambient air. The time-resolved optical emission spectra of plume were measured to elucidate the ablation behavior of silica glass induced by nanosecond-pulsed laser irradiation at 355 nm where absorption of silica glass is negligibly small. This method is suitable for rapid prototyping of surface microstructuing without a clean room environment.

Laser-induced backside wet etching of silica glass and sapphire plates was performed by the excitation of a liquid with a ns-pulsed UV excimer laser. Well-defined micropatterns were fabricated without debris and microcrack around the etched area. To understand the etching mechanism, the formation and propagation of shockwave and bubble were monitored by time-resolved optical microscopy at the interface between the silica glass and the liquid during etching process. Transient high-pressure as well as high-temperature generated by UV laser irradiation plays a key role in the etching process.

A novel approach for the fabrication of micropatterns where dye molecules can be site-selectively desposited is described. The micropatterns were fabricated on the surface of fused silica plates using the technique of laser-induced backside wet etching (LIBWE). Prior to the LIBWE process, the surface properties of the fused silica plates were modified with self-assembling trimethoxysilanes bearing functional moieties. On the non-irradiated areas, the self-assembled monolayers (SAMs) survived the LIBWE process, and the remaining SAM acted as a template for the subsequent dye deposition either with chemical bonding or physical adsorption. Site-selective dye deposition was visualized with fluorescence microscopic observation. These results are applicable for micro-fluidic reactors and chemical sensors.

Silica glass is an important material in optics and optoelectronics because of its outstanding properties, such as transparence in a wide wavelength range, strong damage resistance for laser irradiation, and high chemical stability. In order to develop simpler processes of micro-fabricating silica glass using a pulsed laser, we have investigated a one-step method to microfabricate a silica glass plate using laser-induced backside wet etching (LIBWE) upon irradiation with a ns-pulsed excimer laser. Our idea of LIBWE is based on the deposition of laser energy on the surface of silica glass using ablation of a dye solution. When the dye solution was ablated upon the laser irradiation, the etching of a surface layer was performed on the silica glass. We have succeeded in the micro-fabrication of such transparent materials as silica glass, quartz, calcium fluoride, sapphire and fluorocarbon resin. The advantages of our LIBWE method are as follows, (i) a low laser fluence and constant etch rate, (ii) microfabrication without debris and cracks formation, (iii) large area irradiation with an excimer laser beam through a mask projection, (iv) simple pre-/post-treatment on target substrates. This is a one-step process simpler method at ambient pressure, which would be used for mass production.

Laser-induced backside wet etching of silica glass plates was performed by the excitation of a pure toluene solution with a ns-pulsed KrF excimer laser at 248 nm. Well-defined grid micropattern was fabricated without debris and microcrack around the etched area. To understand the etching mechanism, the formation and propagation of shockwave and bubble were monitored by time-resolved optical microscopy at the interface between the silica glass and the toluene solution after laser irradiation. Transient high-pressure as well as high-temperature generated by UV laser irradiation plays a key role in the etching process.

Laser-induced backside wet etching of fused silica plates using aqueous solutions of naphthalene-1,3,6-trisulfonic acid trisodium salt (Np) and pyranine (py) was performed upon KrF excimer laser irradiation at 248 nm. The two etching media show different etching behavior with changing laser fluence and medium concentrations. Well-defined line-and-space and grid micropatterns at 1 μm scale were fabricated using an aqueous solution of Np and the etched pattern was free of debris and microcracks.

Laser-induced backside wet etching (LIBWE) of silica glass plates was performed to fabricate an imprinting template for hot embossing in polymer substrates such as polystyrene and silicone resin. Well-defined inverse surface-micropatterns of gratings and grid arrays on the substrates were produced by the hot embossing using a surface-structured silica glass as the template. These results indicate that the LIBWE method allows us to generate robust glass molding tools that exhibit the inverse shapes of the intended microstructures.

We report on aging and detergent washing effects in GaAs wafers passivated with hexadecanethiol (HDT) [HS(CH2)15CH3]. The evolution of the photoluminescence (PL) signal from GaAs was measured at room temperature as a function of time, up to 1000h, for thiolated (001) GaAs that was solvent cleaned and etched using standard procedures, and for thiolated (110) GaAs that was obtained by cleaving in air. For 2h of thiolation time, the decay of the PL signal from (001) GaAs and (110) GaAs could be fitted, respectively, with double and single exponential functions. This indicates that at least two different types of non-adiative recombination centers (NRRC) form on the (001) surface. The overall density of surface states formed on (110) GaAs is significantly lower than those on (001) GaAs. A single exponential decay of the PL signal has been observed for 18h thiolated samples. The strong increase of the PL signal after detergent washing of (001) GaAs suggests that this treatment is effective in removal of some o...