Pump-probe X-ray holographic imaging of laser-induced cavitation bubbles with femtosecond FEL pulses.

Cavitation bubbles can be seeded from a plasma following optical breakdown, by focusing an intense laser in water. The fast dynamics are associated with extreme states of gas and liquid, especially in the nascent state. This offers a unique setting to probe water and water vapor far-from equilibrium. However, current optical techniques cannot quantify these early states due to contrast and resolution limitations. X-ray holography with single X-ray free-electron laser pulses has now enabled a quasi-instantaneous high resolution structural probe with contrast proportional to the electron density of the object. In this work, we demonstrate cone-beam holographic flash imaging of laser-induced cavitation bubbles in water with nanofocused X-ray free-electron laser pulses. We quantify the spatial and temporal pressure distribution of the shockwave surrounding the expanding cavitation bubble at time delays shortly after seeding and compare the results to numerical simulations.

Correlative microscopy approach for biology using X-ray holography, X-ray scanning diffraction and STED microscopy.

We present a correlative microscopy approach for biology based on holographic X-ray imaging, X-ray scanning diffraction, and stimulated emission depletion (STED) microscopy. All modalities are combined into the same synchrotron endstation. In this way, labeled and unlabeled structures in cells are visualized in a complementary manner. We map out the fluorescently labeled actin cytoskeleton in heart tissue cells and superimpose the data with phase maps from X-ray holography. Furthermore, an array of local far-field diffraction patterns is recorded in the regime of small-angle X-ray scattering (scanning SAXS), which can be interpreted in terms of biomolecular shape and spatial correlations of all contributing scattering constituents. We find that principal directions of anisotropic diffraction patterns coincide to a certain degree with the actin fiber directions and that actin stands out in the phase maps from holographic recordings. In situ STED recordings are proposed to formulate models for diffraction data based on co-localization constraints.

Erratum: X-Ray Optics on a Chip: Guiding X Rays in Curved Channels [Phys. Rev. Lett. 115, 203902 (2015)].

This corrects the article DOI: 10.1103/PhysRevLett.115.203902.

Combined in-situ imaging of structural organization and elemental composition of substantia nigra neurons in the elderly.

Human dopaminergic system in general, and substantia nigra (SN) neurons, in particular, are implicated in the pathologies underlying the human brain aging. The interplay between aberrations in the structural organization and elemental composition of SN neuron bodies has recently gained in importance as selected metals: Fe, Cu, Zn, Ca were found to trigger oxidative-stress-mediated aberration in their molecular assembly due to concomitant protein (alpha-synuclein, tau-protein) aggregation, gliosis and finally oxidative stress. In the present study, we demonstrate an integrated approach to the analysis of the structural organization, assembly, and metals' accumulation in two distinct areas of SN: in the neuromelanin neurons and neuropil. By using the highly brilliant source of PETRA III and the Kirkpatrick-Baez nano-focus, large area histological brain slices are scanned at the sub-neuronal resolution, taking advantage of continuous motor movement and reduced acquisition time. Elemental analysis with synchrotron radiation based X-ray Fluorescence (SRXRF) is combined with X-ray Phase Contrast Imaging (XPCI) to correct for inherent aberrations in the samples' density and thickness, often referred to as the mass thickness effect. Based on the raw SRXRF spectra, we observed the accumulation of P, S, Cl, K, Ca, Fe, Cu and Zn predominantly in the SN neurons. However, upon the mass thickness correction, the distributions of Cl became significantly more uniform. Simultaneously with the fluorescence signal, the Small Angle X-ray Scattering (SAXS) is recorded by a pixel detector positioned in the far-field, enabling fast online computation of the darkfield and differential phase contrast (DPC). The data has demonstrated the SN neurons and neuropil produces excellent contrast which is due to their different mass density and scattering strength, indicative of differences in local structure and assembly therein. In all, the results show that combined SRXRF-XPCI-SAXS experiments can robustly serve as a unique tool for understanding the interplay between the chemical composition and structural organization that may drive the biochemical age-related processes occurring in the human dopaminergic system.

Regulation of the clock gene expression in human adipose tissue by weight loss.

BACKGROUND: The circadian clock coordinates numerous metabolic processes to adapt physiological responses to light-dark and feeding regimens and is itself regulated by metabolic cues. The implication of the circadian clock in the regulation of energy balance and body weight is widely studied in rodents but not in humans. Here we investigated (1) whether the expression of clock genes in human adipose tissue is changed by weight loss and (2) whether these alterations are associated with metabolic parameters. SUBJECTS/METHODS: Subcutaneous adipose tissue (SAT) samples were collected before and after 8 weeks of weight loss on an 800 kcal per day hypocaloric diet (plus 200 g per day vegetables) at the same time of the day. Fifty overweight subjects who lost at least 8% weight after 8 weeks were selected for the study. The expression of 10 clock genes and key metabolic and inflammatory genes in adipose tissue was determined by quantitative real-time PCR. RESULTS: The expression of core clock genes PER2 and NR1D1 was increased after the weight loss. Correlations of PERIOD expression with body mass index (BMI) and serum total, high-density lipoprotein and low-density lipoprotein (LDL) cholesterol levels and of NR1D1 expression with total and LDL cholesterol were found that became non-significant after correction for multiple testing. Clock gene expression levels and their weight loss-induced changes tightly correlated with each other and with genes involved in fat metabolism (FASN, CPT1A, LPL, PPARG, PGC1A, ADIPOQ), energy metabolism (SIRT1), autophagy (LC3A, LC3B) and inflammatory response (NFKB1, NFKBIA, NLRP3, EMR1). CONCLUSION: Clock gene expression in human SAT is regulated by body weight changes and associated with BMI, serum cholesterol levels and the expression of metabolic and inflammatory genes. Our data confirm the tight crosstalk between molecular clock and metabolic and inflammatory pathways involved in adapting adipose tissue metabolism to changes of the energy intake in humans.

X-Ray Optics on a Chip: Guiding X Rays in Curved Channels.

We study the propagation of hard x rays in single curved x-ray waveguide channels and observe waveguide effects down to surprisingly small radii of curvature R≃10 mm and a large contour length s≃5 mm, deflecting beams up to 30°. At these high angles, about 2 orders of magnitude above the critical angle of total reflection θ(c), most radiation modes are lost by "leaking" into the cladding, while certain "survivor" modes persist. This may open up a new form of integrated x-ray optics "on a chip," requiring curvatures mostly well below the extreme values studied here, e.g., to split and to delay x-ray pulses.

Collective lipid bilayer dynamics excited by surface acoustic waves.

We use standing surface acoustic waves to induce coherent phonons in model lipid multilayers deposited on a piezoelectric surface. Probing the structure by phase-controlled stroboscopic x-ray pulses we find that the internal lipid bilayer electron density profile oscillates in response to the externally driven motion of the lipid film. The structural response to the well-controlled motion is a strong indication that bilayer structure and membrane fluctuations are intrinsically coupled, even though these structural changes are averaged out in equilibrium and time integrating measurements. Here the effects are revealed by a timing scheme with temporal resolution on the picosecond scale in combination with the sub-nm spatial resolution, enabled by high brilliance synchrotron x-ray reflectivity.

Sub-5 nm hard x-ray point focusing by a combined Kirkpatrick-Baez mirror and multilayer zone plate.

Compound optics such as lens systems can overcome the limitations concerning resolution, efficiency, or aberrations which fabrication constraints would impose on any single optical element. In this work we demonstrate unprecedented sub-5 nm point focusing of hard x-rays, based on the combination of a high gain Kirkpatrick-Baez (KB) mirror system and a high resolution W/Si multilayer zone plate (MZP) for ultra-short focal length f. The pre-focusing allows limiting the MZP radius to below 2 µm, compatible with the required 5 nm structure width and essentially unlimited aspect ratios, provided by enabling fabrication technology based on pulsed laser deposition (PLD) and focused ion beam (FIB).

Is mesenteric panniculitis truely a paraneoplastic phenomenon? A matched pair analysis.

PURPOSE: Mesenteric panniculitis (MP) is an underdiagnosed inflammatory condition of mesenteric adipose tissue. Prior studies suggested an association of MP with malignancy. To reassess this hypothesis, we performed the first matched case-control study comparing prevalence of malignancy and other disease in patients with and without MP. MATERIAL AND METHODS: With a keyword search we identified CT examinations of MP patients between 2010 and 2012. Each MP patient was matched with two control patients for age, gender, abdominal diameter and CT protocol. Manifestation and extent of mesenteric panniculitis was classified independently by two investigators according to established criteria. Concomitant disease, laboratory parameters and follow up CTs were recorded and analyzed for all patients. RESULTS: 77 of 13485 CT patients were diagnosed with MP (prevalence 0.58%). 50.6% of MP patients suffered from malignancy vs. 60.2% in the control group (p=0.157). Over up to 4 years of follow up in 35 of these 77 MP patients no association between development of MP and the course of tumor diseases could be identified. There was also no significant difference in the rate of frequent concomitant diseases such as hypertension, diabetes or previous surgery between the two groups. CONCLUSION: In this first case-control-study we could show that, contrary to previous reports, mesenteric panniculitis is neither paraneoplastic nor is it associated with other diseases.

Elevated hepatic chemerin mRNA expression in human non-alcoholic fatty liver disease.

OBJECTIVE: Adipose tissue-derived factors link non-alcoholic fatty liver disease (NAFLD) with obesity, which has also been reported for circulating chemerin. On the other hand, hepatic chemerin and chemokine-like receptor 1 (CMKLR1) mRNA expression has not yet been studied in an extensively characterized patient collective. DESIGN: This study was cross-sectional and experimental in design. METHODS: Liver tissue samples were harvested from 47 subjects and histologically examined according to the NAFLD activity score (NAS). The concentrations of chemerin and CMKLR1 were measured using semi-quantitative real-time PCR, and the concentration of serum chemerin was measured using ELISA. To evaluate potential effects of chemerin and CMKLR1, cultured primary human hepatocytes (PHHs) were exposed to selected metabolites known to play a role in NAFLD (insulin, glucagon, palmitoic acid, and interleukin-6 (IL6)). RESULTS: Chemerin and CMKLR1 mRNA levels were elevated in the human liver. Their expression was correlated with the NAS (R(2)=0.543; P<0.001 and R(2)=0.355; P=0.014 respectively) and was significantly elevated in patients with definite non-alcoholic steatohepatitis (NASH) (P<0.05 respectively). Linear regression analysis confirmed an independent association of liver fibrosis, steatosis, inflammation, and hepatocyte ballooning with hepatic chemerin mRNA expression (P<0.05 respectively). The expression of hepatic chemerin and CMKLR1 was correlated with the measures of obesity (P<0.05). The incubation of PHHs with IL6 significantly increased the expression of CMKLR1 mRNA (P=0.027), while that of chemerin remained unaffected (P>0.05). None of the other metabolites showed an influence (P>0.05). CONCLUSION: This is the first study to show that chemerin mRNA expression is significantly elevated in the liver of NASH patients and that CMKLR1 expression is upregulated in liver inflammation, whereby IL6 could play a causal role.

Single pulse coherence measurements in the water window at the free-electron laser FLASH.

The spatial coherence of free-electron laser radiation in the water window spectral range was studied, using the third harmonic (λ<(3rd) = 2.66 nm) of DESY's Free-electron LASer in Hamburg (FLASH). Coherent single pulse diffraction patterns of 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) multilamellar lipid stacks have been recorded. The intensity histogram of the speckle pattern around the first lamellar Bragg peak, corresponding to the d = 5 nm periodicity of the stack, reveals an average number of transverse modes of M¯ = 3.0 of the 3rd harmonic. Using the lipid stack as a 'monochromator', pulse-to-pulse fluctuations in the third harmonic λ(3rd) have been determined to be 0.033 nm.

Nonequilibrium collective dynamics in photoexcited lipid multilayers by time resolved diffuse x-ray scattering.

We study the nonequilibrium shape fluctuations in fluorescence labeled phospholipid multibilayers composed of the model lipid DOPC and the well-known lipid dye Texas red, driven out of equilibrium by short laser pulses. The temporal evolution of the lipid bilayer undulations after excitation was recorded by time resolved x-ray diffraction. Already at moderate peak intensities (Pp≤10(5) W/cm2), pulsed laser illumination leads to significant changes of the undulation modes in a well-defined lateral wavelength band. The observed phenomena evolve on nano- to microsecond time scales after optical excitation, and can be described in terms of a modulation instability in the lipid multilamellar stack.

Levothyroxine medication is associated with adiposity independent of TSH.

AIM: In euthyroidism, higher TSH levels are weakly associated with increased BMI. Furthermore, a considerable number of patients complain of weight gain during thyroid hormone replacement after thyroidectomy. We therefore investigated the association between levothyroxine medication and BMI in a large cross-sectional study group. METHODS: We included euthyroid participants from the MeSyBePo study group (TSH between 0.3 and 4.5 µU/ml) that did not take thyreostatic drugs. Linear regression analyses were performed to address the association between levothyroxine medication and obesity. Additionally, pairs matched by sex, age and TSH but discordant in levothyroxine medication were compared. RESULTS: 1663 subjects (569 males) were eligible for inclusion. 151 participants were taking levothyroxine. Adjusted for sex and age both TSH (standardised beta 0.1, p<0.001) and levothyroxine medication (standardised beta 0.05, p=0.03) were significantly associated with BMI. There was no significant interaction between TSH and levothyroxine medication with respect to BMI. Further adjustment for smoking and the restriction to those subjects with normal glucose metabolism (947 participants (314 males, 82 on levothyroxine medication) did not alter the result. In matched pair analysis (133 pairs), BMI was significantly increased in subjects taking levothyroxine compared to controls. CONCLUSION: Independently from TSH, levothyroxine medication was associated with a higher BMI. The mechanisms, however, responsible for this association need to be elucidated.

Partially coherent nano-focused x-ray radiation characterized by Talbot interferometry.

We have studied the spatial coherence properties of a nano-focused x-ray beam by grating (Talbot) interferometry in projection geometry. The beam is focused by a fixed curvature mirror system optimized for high flux density under conditions of partial coherence. The spatial coherence of the divergent exit wave emitted from the mirror focus is measured by Talbot interferometry The results are compared to numerical calculations of coherence propagation. In view of imaging applications, the magnified in-line image of a test pattern formed under conditions of partial coherence is analyzed quantitatively. Finally, additional coherence filtering by use of x-ray waveguides is demonstrated. By insertion of x-ray waveguides, the beam diameter can be reduced from typical values of 200 nm to values below 15 nm. In proportion to the reduction in the focal spot size, the numerical aperture (NA) of the projection imaging system is increased, as well as the coherence length, as quantified by grating interferometry.

T3/rT3-ratio is associated with insulin resistance independent of TSH.

Thyroid dysfunction has been shown to be associated with insulin resistance (IR). This may involve peripheral thyroid hormone metabolism, which is assumed to be reflected by the ratio triiodothyronine/reverse triiodothyronine (T3/rT3-ratio). To explore a potential association between the T3/rT3-ratio and IR we investigated pairs which differed in IR, but were matched by sex, age, body mass index (BMI), and thyroid stimulating hormone (TSH). For this purpose, matched pair analyses were embedded into a cross sectional study group. 22 pairs were matched from either the first or the third tertile of HOMA%S of a cohort of 353 euthyroid subjects with normal glucose metabolism who did not take any medication. The T3/rT3-ratio was compared in the matched pairs. The T3/rT3-ratio was significantly increased in the insulin resistant subjects compared to their insulin sensitive partners (8.78 ± 0.47 vs. 7.33 ± 0.33, p=0.019). Furthermore the T3/rT3-ratio was lower in men compared to women (p for the within-subject effect=0.046) both in the insulin sensitive and the insulin resistant subjects. Here we show that the T3/rT3-ratio, which is supposed to reflect the tissue thyroid hormone metabolism, is significantly increased in insulin resistant subjects. This further supports a link between thyroid function and IR.

Free fatty acids increase androgen precursors in vivo.

CONTEXT: There is considerable evidence that metabolic factors such as insulin resistance may induce hyperandrogenemia in polycystic ovary syndrome. However, other metabolic factors such as free fatty acids (FFAs) may also contribute to androgen excess. OBJECTIVE: The objective was to study effects of FFAs on adrenal production of androgen precursors in vivo. DESIGN AND PARTICIPANTS: We investigated eight healthy young men, because male individuals produce the androgen precursors dehydroepiandrosterone (DHEA), DHEA sulfate, and androstenedione predominantly in the adrenal gland. A randomized controlled crossover trial was performed. INTERVENTION: After a 10-h overnight fast, 20% lipid/heparin or saline/heparin infusion was given at a rate of 1.5 ml/min. Four hours after start of lipid infusion, a euglycemic hyperinsulinemic clamp was performed. MAIN OUTCOME MEASURES: DHEA, androstenedione, 17-OH-progesterone, testosterone, estrone, LH, FSH, ACTH, and cortisol were measured. RESULTS: The adrenal androgen precursors DHEA and androstenedione showed a circadian decline during saline/heparin infusion (P < 0.05 vs. baseline, respectively), whereas no significant changes were observed during lipid/heparin infusion (P = not significant vs. baseline, respectively). Correspondingly, DHEA and androstenedione values were significantly elevated during lipid compared with saline infusion (P < 0.05, respectively), and areas under curve of both androgen precursors were significantly increased with lipid compared with saline infusion. Notably, all changes were detected before induction of insulin resistance. CONCLUSIONS: This study demonstrates that FFAs increase production of androgen precursors in vivo in men. These data tentatively suggest that hyperandrogenemia in polycystic ovary syndrome may be induced, at least in part, by elevated FFAs.

Impact of cereal fibre on glucose-regulating factors.

AIMS/HYPOTHESIS: Insoluble dietary fibre intake is associated, by unknown mechanisms, with a reduced risk of type 2 diabetes. We investigated whether a short-term dietary intervention with purified insoluble fibres influences acute and delayed responses of glucose, insulin, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1. METHODS: Fourteen healthy women with NGT were studied for 300 min on six to eight occasions. Subjects consumed three matched portions of control (C) or fibre-enriched bread (10.4-10.6 g/portion; wheat fibre [WF], oat fibre [OF], and, in a substudy [n=9], resistant starch [RS]) followed by control (C-C, C-WF, C-OF, C-RS) on subsequent days. RESULTS: Fibre enrichment accelerated the early insulin response (fibrextime interaction p=0.026 for WF, p<0.001 for OF, p=0.126 for RS; time of maximal concentration [T(max)], C 57.9+/-5.9, WF 49.3+/-2.5 [p=0.086], OF 46.1+/-2.9 [p=0.026], RS 46.7+/-5.8 min [p=0.029]). It was also associated with an earlier postprandial GIP response after OF (T(max), C 83.6+/-7.2, WF 70.7+/-6.0 [p=0.054], OF 64.3+/-6.9 [p=0.022], RS 60.0+/-5.0 [p>0.15]). Increased fibre intake for 24 h was further associated with a reduced postprandial glucose response on the following day subsequent to ingestion of a control meal (AUC(C-C) 4,140+/-401, AUC(C-WF) 2,850+/-331 [p=0.007], AUC(C-OF) 2,830+/-277 [p=0.011]), with no difference in maximal concentration and T(max) of glucose responses. No differences in insulin responses were observed 24 h after the fibre-enriched diets compared with control (p>0.15). Colonic fermentation was increased only on study days C-OF (p=0.017) and C-RS (p=0.016). CONCLUSIONS/INTERPRETATION: The consumption of highly purified insoluble dietary fibres accelerated the acute GIP and insulin response and was further associated with enhanced postprandial carbohydrate handling the following day upon ingestion of a control meal.

[Significance of biomarkers for metabolic syndrome during weight reduction]. / Bedeutung von Biomarkern des metabolischen Syndroms bei Gewichtsreduktion.

INTRODUCTION: Obesity is a risk factor for type 2 diabetes, hypertension, dyslipidemia and cardiovascular disease. We aimed to analyse the changes of parameters of the metabolic syndrome and to investigate which markers are useful in the prediction of a successful weight loss. Preliminary data of an ongoing study are presented. METHODS: 18 obese individuals (15 female, 3 male, mean age 50.9 years, mean BMI 36.1) finished a 12 month weight loss program. This weight loss program was based on a hypocaloric diet (50 % carbohydrates, 30 % fat, 20 % protein) and at least 60 min physical activity per week. At baseline, 6 months and 12 months physical examination, indirect calorimetry, bioimpedance analysis were performed and blood was taken for routine laboratory. An oral glucose tolerance test and an euglycemic hyperinsulinemic clamp (n = 13) were carried out at baseline and after 6 months. RESULTS: There was a decrease of the BMI (+/- SEM) from 36.1 +/- 1.3 to 33.4 +/- 1.2 after 6 months and 32.8 +/- 1.3 after 12 months. Waist circumference (-8.8 cm), fasting blood glucose (98.0 to 91.2 and 92.5 mg/dl) and HDL cholesterol (47.2 to 64.6 mg/dl after 12 months) improved significantly. Other parameters of the metabolic syndrome (blood pressure, lipids, insulin resistance) and adiponectin improved slightly, but changes failed to be significant. In a linear regression analysis age, insulin resistance (M-value) and adiponectin at baseline were significant and independent predictors of a successful weight loss. CONCLUSION: In conclusion, most parameters of the metabolic syndrome improved after successful weight reduction, although changes of most parameters were modest and did not reach statistical significance.

Ca2+/calmodulin-dependent protein kinase II delta2 regulates gene expression of insulin in INS-1 rat insulinoma cells.

Ca(2+)/calmodulin-dependent protein kinase II is a member of a broad family of ubiquitously expressed Ca(2+) sensing serine/threonine-kinases. Ca(2+)/calmodulin-dependent protein kinase II is highly expressed in insulin secreting cells and is associated with insulin secretory granules and has been proposed to play an important role in exocytosis or in insulin granule transport to release sites. To elucidate its function the antisense sequence of the major beta-cell subtype, Ca(2+)/calmodulin-dependent protein kinase II delta(2), was stably expressed in INS-1 rat insulinoma cells. This caused a loss of Ca(2+)/calmodulin-dependent protein kinase II delta(2) expression at the mRNA and protein level, while the expression of the 95% homologous Ca(2+)/calmodulin-dependent protein kinase II gamma and of beta-cell specific proteins such as the homeodomain factor pancreatic-duodenal homeobox factor-1 (PDX-1, also referred to as islet/duodenum homeobox-1, IDX-1, insulin promoter factor-1, IPF-1 and somatostatin transactivating factor-1, STF-1), the glucagon-like peptide-1 (GLP-1) receptor and K(ATP)-channels K(IR)6.2/SUR-1 (sulfonylurea receptor-1) was not altered. Unexpectedly, the cells showed a large reduction of insulin gene expression, which was due to reduced insulin gene transcription. Electrophoretic mobility shift assays of PDX-1 binding to the insulin promoter A1 and E2/A3A4 elements showed additional bands indicating alterations of PDX-1 complex formation. Stable over expression of Ca(2+)/calmodulin-dependent protein kinase II delta(2), by contrast, was associated with elevated expression of insulin mRNA. Therefore, we conclude that Ca(2+)/calmodulin-dependent protein kinase II delta(2) links fuel-dependent increases in intracellular Ca(2+) concentrations to transcriptional regulation of genes related to the metabolic control of insulin secretion.