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Disposable microfluidic micromixers for effective capture of Cryptosporidium parvum oocysts from water samples
L. Diéguez ORCID: orcid.org/0000-0003-3695-69631,2,
M. Winter1,
S. Molan1,
P. Monis1,3,
B. King3 &
B. Thierry1
Journal of Biological Engineering volume 12, Article number: 4 (2018) Cite this article
Protecting drinking water supplies from pathogens such as Cryptosporidium parvum is a major concern for water utilities worldwide. The sensitivity and specificity of current detection methods are largely determined by the effectiveness of the concentration and separation methods used. The purpose of this study is to develop micromixers able to specifically isolate and concentrate Cryptosporidium, while allowing in situ analysis.
In this study, disposable microfluidic micromixers were fabricated to effectively isolate Cryptosporidium parvum oocysts from water samples, while allowing direct observation and enabling quantification of oocysts captured in the device using high quality immunofluorescence microscopy. In parallel, quantitative analysis of the capture yield was carried out by analyzing the waste from the microfluidics outlet with an Imaging Flow Cytometer. At the optimal flow rate, capture efficiencies up to 96% were achieved in spiked samples.
Scaled microfluidic isolation and detection of Cryptosporidium parvum will provide a faster and more efficient detection method for Cryptosporidium compared to other available laboratory-scale technologies.
Cryptosporidium is a highly resistant protozoan commonly encountered in surface waters. Although Cryptosporidium infections are self-limiting in healthy individuals, the consequences can be far more serious in infants and young children causing moderate to severe diarrhea [1]. Cryptosporidium outbreaks are often linked to treatment failures or treatment deficiencies at water treatment plants, allowing contamination of drinking water. Risk management of source waters requires cost effective, rapid and efficient monitoring of Cryptosporidium [2]. Procedures for Cryptosporidium detection typically includes collection of a large volume of water sample (10 - 1000 L), followed by concentration using various techniques including filtration and centrifugation to obtain a concentrated sample. The standardized procedure EPA 1623 describes a complete isolation/detection protocol based on filtration, elution from the filter and centrifugation to obtain a concentrated sample. The recovered sample then undergoes immunomagnetic separation to isolate oocysts from debris. Isolated oocysts are stained with specific fluorescent antibodies and nuclear stain for detection by immunofluorescence and microscopy. This method has demonstrated a recovery yield of 61% in spiked pure water, 48.8% in spiked filter tap samples and 19.5% in raw sources, with a limit of detection of approximately 10 oocysts/L [3]. The final steps in the EPA 1623 method require skilled technicians and are time- and resource-consuming. While immunofluorescence microscopy is the gold standard for the detection of the oocysts, other antigen based detection methods including ELISA (enzyme-linked immunosorbent assay) and immunochromatographic assays are also commercially available [4].
A number of alternative approaches have been reported to detect waterborne pathogens [5], including those based on Surface Plasmon Resonance [6], nucleic acid detection [7,8,9,10,11], immunocantilevers [12], Surface Enhanced Raman Scattering [13], dielectrophoresis [14] and impedance spectroscopy [15]. But all detection methods rely on efficient concentration of large volume of water sample and isolation of the pathogen. A number of microfabricated filters [16,17,18] have been described and yielded oocysts capture efficiencies as high as 97% in spiked pure water samples. Microfluidic methods have also been actively investigated in recent years for oocysts detection due to their excellent reliability and efficiency. Owing to the inability of microfluidic systems to deal with very large volumes, these approaches have typically focused on treating samples that have already been subjected to the initial concentration step. Microfluidic filters achieved an efficiency of 86% when capturing Cryptosporidium oocysts from spiked concentrated water samples [19]. Inertial microfluidics has also been applied with some success to enrich water and food pathogens, yielding 100% efficiency sorting Cryptosporidium from concentrated water [20] and 68.4% when recovering Giardia from food samples [21]. The Nano-DEP enrichment system provided a 10 times concentration of the raw sample [13]. These microfluidic systems rely on physical filtration or separation of oocysts from a pre-concentrated sample to deliver the sample for the final detection step. The pre-concentrated samples still need to be further processed to be analyzed using for example fluorescence microscopy. In addition, approaches based on physical features lack specificity in comparison to standard immunomagnetic separation. With this in mind, the integration of immunomagnetic separation with microfluidics has been advanced [22]. In addition, good recovery yield using microfabricated microwells functionalized with antibodies has also been reported, although the static nature of this approach limits its application to only a small volume of sample [23]. Recently, McGrath et al. reported on the interesting concept of high throughput Microfluidic Impedance Cytometry (MIC) for rapid enumeration and identification of different types of Cryptosporidium spiked in saline buffer and achieved over 92% accuracy in discriminating Cryptosporidium parvum, Cryptosporidium muris and Giardia lamblia [24].
Microfluidic devices bioconjugated with specific molecular probes have been used with remarkable success in the isolation of circulating tumor cells from the blood of cancer patients [25]. To perform efficient immunocapture of cells in functionalized microfluidic devices, it is necessary to maximize the surface to volume ratio and to create appropriate mixing to optimize the chances of the target cells coming into contact with the immunoconjugated surface. To our knowledge, the application of a biofunctionalised micromixer for the immunospecific capture of Cryptosporidium has not been investigated thus far. Towards simplifying the workflow of Cryptosporidium oocysts detection from a pre-concentrated water sample with increasing accuracy, the aim of this work is to evaluate a streamlined microfluidic technology for its ability to specifically isolate Cryptosporidium oocysts from concentrated water samples while simultaneously allowing for high quality immunofluorescence observation. In this way, oocysts present in pre-concentrated samples (using any of the above mentioned approaches) can be specifically isolated and quantified in a single system. A positive enrichment strategy has been used due to sample/technical specificities including multiple contamination source in water samples and the existence of an antibody that binds the target oocysts with high specificity and affinity. For this purpose, disposable microfluidic devices were fabricated and functionalized with an antibody (Cry 104) specific against antigens expressed on the oocyst surface. The microfluidic device consists of an array of 25 μm thin micromixers designed to enhance the surface interactions between the Cryptosporidium and the channel walls. Once isolated within the micromixer, the oocysts can be readily stained using standard fluorescent tags and observed in situ under fluorescence microscopy, providing a simpler alternative to the current method based on immunomagnetic separation. Under optimal conditions, a recovery yield of up to 96% could be obtained for Cryptosporidium oocysts spiked in water.
The design of this microfluidic device enhances mixing and hence binding of the Cryptosporodium with its many parallel channels which not only increase the area for binding but also provides some redundancy for blockage as would be expected with environmental water.
The micromixers were fabricated in SU8 on a silicon wafer. The design consists of an array of 25 μm high microchannels favoring chaotic mixing. Standard soft lithography was used to produce disposable microfluidic devices, sealed with oxygen plasma. Using a silane-based functionalization strategy, antibodies were immobilized on the channel surface. Known numbers of Cryptosporidium were spiked in saline buffer and introduced through the microfluidic devices at different flow rates. The oocysts captured in the device were washed, fixed, permeabilized and stained prior to microscope examination. The waste solution recovered from the devices was also analyzed with an Imaging Flow Cytometer Image Stream X (ISX, AMNIS, Seattle, WA, USA).
Phosphate buffered saline (PBS), (3-aminopropyl) trimethoxysilane (APTMS), trichloro 1,1,2,2-perfluorooctyl-silane, bovine serum albumin (BSA), fetal bovine serum (FBS), glutaraldehyde, formaldehyde, Triton™ X-100, and 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma Aldrich (USA). γ-irradiated Cryptosporidium parvum were kindly donated from SA Water (Australia). Specific monoclonal antibody Cry104 was obtained from BTF Biomerieux (Australia). A FITC goat anti mouse IgG secondary antibody was purchased from Sigma Aldrich (USA). Polydimethylsiloxane (PDMS) elastomer SYLGARD 184 was obtained from Dow Corning (USA) and SU-8 10 photoresist was purchased from MicroChem (USA). All other chemicals were analytical grade. Silicon wafers with a 3″ diameter were obtained from Micro Materials & Research Cons. Pty Ltd (Australia), and the syringe pumps KDS-212-CE and KDS-210 used in this study were purchased from KD Scientific.
Fabrication of microfluidic devices
Standard photolithography was used to fabricate chaotic mixing silicon masters for PDMS molding, as previously described [26]. Briefly, silicon substrates were cleaned with acetone in a sonic bath for 5 min and then with isopropanol for 5 min. After rinsing with isopropanol, substrates were dried with a nitrogen gun and activated with oxygen plasma for 10 min. The negative photoresist, SU-8 10, was then spin-coated onto the wafer. The SU-8 was then patterned using a film photomask (JD tools) with a UV dose of 225 mJ/cm2 and post-baked at 65 °C for 1 min and at 95 °C for 2 min. SU-8 was then developed during 2 min to form a template and hard-baked with a ramping temperature from 65 °C to 200 °C.
The template was hydrophobized submitting it to a trichloro 1,1,2,2-perfluorooctyl-silane vapor in a desiccator for 1 h at 80 °C, and covered with liquid PDMS (the PDMS prepolymer was mixed with the cross-linker at a 10:1 ratio and degassed). PDMS was then degassed, cured at 80 °C for 1 h and unmolded from the silicon master. Inlet and outlet were punched in the PDMS replica that was then irreversibly sealed against a clean glass slide upon treatment in oxygen plasma at low power for 15 s.
Functionalization of the microfluidic devices
After attaching tubing to the inlet and outlet ports, the micromixers were connected to a syringe pump and filled with ethanol at a flow rate of 100 μl/min. Once the devices were stabilized, 2% APTMS in ethanol was withdrawn into the device for 30 min and rinsed with ethanol for 10 min. The buffer solution was then changed with MilliQ water and stabilized for 10 min prior to withdrawal of 1% glutaraldehyde in water for another 30 min and rinsing in ultrapure water for 10 min. PBS was then withdrawn into the device and equilibrated for 10 min just before introducing 200 μl of 50 μg/ml Cry104 in PBS that was left to react overnight at 4 °C. Unreacted antibodies were rinsed with PBS and the surface blocked with 2% BSA in PBS. All the functionalization steps were done at the same flow rate. Control devices were functionalized following the same protocol, but without the antibody conjugation.
Capture of Cryptosporidium parvum oocysts
To optimize the parameters for the isolation of Cryptosporidium, small volumes (50 μl) of high concentrated spiked samples in PBS (1.5 × 106 and 1.5 × 104 oocysts/ml) were injected into the functionalized micromixer at flow rates of 0.5, 2 or 5 μl/min to allow binding of the oocysts to the specific antibodies and explore the effect of flow rate. Washing of unbound oocysts was conducted at the same flow as for binding of oocysts, i.e. 0.5, 2 or 5 μl/min.
Fluorescence microscopy studies
The microfluidic devices were examined under a Nikon Ti Eclipse inverted fluorescence microscope. The oocysts isolated in the device were fixed with 4% formaldehyde, permeabilised with 0.05% Triton X-100 and stained with 1:10,000 DAPI at 0.5, 2 or 5 μl/min (same as flow rate used for oocyte binding) to allow identification by fluorescence microscopy as a proof of concept. The presence of DAPI fluorescent bodies identified as oocysts captured inside the device was quantified in situ by imaging 50 randomly chosen low magnification fields of view in different sections of the device, using a 10× objective.
Towards demonstrating the relevance of this approach for environmental samples, the presence of Cryptosporidium oocysts was also confirmed using the Cry104 antibody. Briefly, after capture, the device was incubated with the Cry104 antibody (1/50) for 60 min. After washing with PBS, permeabilisation and blocking was performed with 0.05% Triton X-100 and 2% BSA for 10 min. The devices were then incubated with the FITC goat anti mouse IgG secondary antibody for 30 min as per the manufacturer's instructions, before washing and imaging.
Imaging flow cytometry studies
To quantitatively characterise the performance of the device and calculate the isolation yield of the micromixer, the input solution and the solution eluted from the device were recovered and analyzed with an Imaging Flow Cytometer (IFC) Image Stream X (AMNIS, Seattle, WA, USA). Solutions were centrifuged at 10,000 RCF for 15 min at 4 °C. The supernatants were removed and the pellets resuspended to 100 μl for Imaging Flow analysis. Since Cryptosporidium particles were already stained with DAPI during the microfluidic procedure, channel 1 of the flow cytometer was set to record all the DAPI stained events, with the 405 laser. Channel 4 and 6 were set for brightfield and darkfield images, respectively. In order to discriminate the oocysts from autofluorescent debris, a size classification was applied.
To confirm that the DAPI-stained bodies identified as oocysts were indeed Cryptosporidium parvum, the recovered sample was labelled with the specific Cry104 antibody and a goat anti mouse FITC IgG secondary antibody was used. Briefly, after blocking with 10% fetal bovine serum in PBS for 10 min on ice, the sample was incubated with the Cry104 antibody (1/50) for 60 min. After washing with PBS, 0.05% Triton X-100 and 1% FBS, the sample was incubated with the FITC secondary antibody (1/5000) for 30 min. After washing with PBS samples were run through the IFC. The FITC events were recorded in Channel 2 of the IFC with the 488 laser. Controls were performed to discriminate non-specific staining.
The capture efficiency was calculated based on eq. 1.
$$ \boldsymbol{Capture}\ \boldsymbol{Efficiency}\ \left(\%\right)=\frac{\left(\#\boldsymbol{oocytes}\ \boldsymbol{in}\boldsymbol{troduced}\right)-\left(\#\boldsymbol{oocytes}\ \boldsymbol{in}\ \boldsymbol{waste}\right)}{\left(\#\boldsymbol{oocytes}\ \boldsymbol{in}\boldsymbol{troduced}\right)}\times \mathbf{100} $$
Statistical analysis was performed in Matlab (2017b, Mathworks) and Excel. Student's t-tests were used for directly comparing two variables. A 1-way ANOVA (confidence interval 0.05) adjusted using Bonferroni correlation was used for multi-variable analysis.
Capture of Cryptosporidium in the micromixer microfluidic devices
To investigate the effect of the velocity on the capture efficiency, 50 μl of Cryptosporidium oocyst solutions were initially flowed at a high concentration (1.5 × 106oocysts / mL, ~ 75,000 oocysts) into the micromixers under 3 different flow rates. Microfluidic micromixers conjugated with BSA instead of the anti- Cryptosporidium antibody were used as controls. All experiments were performed in duplicate. IFC was used as a tool to quantitatively determine the capture efficiency of Cryptosporidium in the microfluidic micromixers. To this end, the unbound oocysts were recovered from the outlet of the microfluidic devices and prepared for analysis with IFC. Figure 1 shows Cryptosporidium oocysts recorded from different channels in the IFC: brightfield, DAPI, and Cry104 antibody labelled with a goat anti mouse FITC IgG secondary antibody, brightfield and darkfield.
Cryptosporidium images from the different channels in the Imaging Flow Cytometer: Brightfield (BF), Darkfield, nuclear staining in DAPI and the Cryptosporidium specific Cry104 antibody (goat anti mouse FITC IgG secondary antibody), and the combined image. Images taken at 400× magnification and have been adjusted to enhance visual appearance
Once the protocol to identify and count the oocysts with IFC was optimized, the capture efficiency for each flow rate was calculated. At 0.5 μL/min, the average capture efficiency was 92%, a maximum average capture efficiency of 96% was determined at 2 μL/min – only 3375 of the initial 75,000 oocysts were counted in the device's waste (Fig. 2). There was statistical differences between bioconjugated and unconjugated control chip capture efficiencies for both 0.5 and 2 μl/min (students t-test, P < 0.05). There was, however, no difference in the capture efficiency between the 0.5 and 2 μl/min flow rates for the functionalised device. On the other hand, increased flow rates led to drastic decreases in the recovery yield with only 40% of the oocysts being recovered at 5 μl/min (45,225 in the waste). There was no statistical difference in capture between the 5 μl/min experiment and its respective control. This is due to the specific design of the F-shape micromixer, which maximizes mixing at lower flow rates. All control experiments resulted in a low non-specific capture of the oocysts within the non biofunctionalized micromixers (18%, 10% and 9% for 0.5, 2 and 5 μL/min, respectively).
Capture efficiency of Cryptosporidium oocysts in the microfluidic devices at different flow rates calculated with Imaging Flow Cytometry. Light grey bars are the functionalized experimental results and the dark grey bars are the non-functionalized control devices. The average capture efficiency was 92%, 96% and 40% at 0.5, 2 and 5 μl/min respectively. Capture efficiency in the control devices was 18%, 10% and 9% for 0.5, 2 and 5 μL/min, respectively
The use of IFC for characterization of the device performance is highly relevant, since it provides excellent quality of images for the micron-scale Cryptosporidium as well as the required high throughput quantification of the eluted samples. Standard flow cytometry, in contrast with IFC, encounters problems with the presence of auto fluorescent debris and clumps of Cryptosporidium, inhibiting proper identification and therefore, quantification [27]. The quantitative data obtained using IFC was confirmed using fluorescence microscopy (Fig. 3) of the micromixers. In good agreement with IFC, the highest number of Cryptosporidium oocysts retained in the micromixer was at the operating flow-rate of 2 μl/min. Minimal non-specific binding in control (non-functionalized) devices was also observed, confirming the specific nature of the binding. A flow rate of 2 μl/min was thus chosen to enhance throughput of the system without compromising capture efficiency.
Microscopic images at 100× magnification of Cryptosporidium oocysts, stained in blue (DAPI), captured at the entrance of the micromixers, from PBS samples spiked with 1500 000 oocysts per ml
An important feature of the proposed microfluidic micromixer approach is the possibility to carry out high quality imaging directly within the devices themselves. Standard staining of the captured Cryptosporidium oocysts with the specific antibody Cry104 and DAPI, enabled straightforward visualization of the oocysts inside the micromixer as shown in Fig. 4.
Microscopic images of the Cryptosporidium oocysts captured inside the functionalized microfluidic devices at the flow rate of 2 μl/min. Cryptosporidium oocysts are specifically recognized by Cry104 antibody, stained with a secondary FITC antibody and imaged at 100×, scale bar 100 μm (1). Composite image with brightfield (BF), FITC and DAPI channels (1a), DAPI channel (1b), FITC channel (1c) and BF image (1d). Images were also taken at higher magnification, 400×, scale bar 50 μm (2, 3). Composite images with BF and FITC (2a) or BF and DAPI (3a), and individual FITC channel (2b), and DAPI channel (3b)
The oocysts had a strong binding affinity to the antibody-functionalized micromixers as shown by preferential binding towards the inlet side (Fig. 5a). To confirm this observation, the number of oocysts captured at different lengths in the device was systematically counted using the following protocol: the number of oocysts captured was counted at 10 randomly chosen areas at different lengths into the device (0, 4, 8, 12 and 16 mm), 0 and 16 being the inlet and the outlet of the micromixer, respectively. Then the number of oocysts per area was averaged at each length and plotted in Fig. 5b. For each position there was statistically more oocysts captured in the Cry104 functionalised device than in the control (p < 0.05, students t-test). It was observed that the number of oocysts found in the microfluidic device is statistically different across the device decreasing with the penetration length with most binding occurring in the first part of the device (F statistic 0.0012, 1-way ANOVA). Specifically, at 0 mm penetration into the device there are statistically more Cryptosporidium than at 12 and 16 mm. For the control there was no difference in binding between any of the positions (F statistic 0.1039, 1-way ANOVA). This clearly demonstrates that Cryptosporidium oocysts have specific and strong binding affinity to the antibody bioconjugated device.
The surface density of Cryptosporidium oocysts in the microfluidic micromixers decreased from inlet to outlet. a Merged fluorescent microscopy images show the capture of oocysts in regions of the device at different distances from the inlet. (Left to right are distances of 0, 4, 8, and 12 mm from the inlet). b Average number of Cryptosporidium oocysts captured in a micromixer at 2 μl/min at different penetration lengths from inlet to outlet. The black line shows a functionalized experiment device and the red is the non-functionalized control device
To further confirm the validity of our system to isolate and quantify Cryptosporidium presence in water samples, the capture efficiency of Cryptosporidium oocysts was also studied at a lower concentration. For this purpose, 50 μl of two different concentrations of Cryptosporidium, 1.5 × 106 and 1.5 × 104 oocysts/ml (75,000 and 750 oocysts, respectively), were introduced at 2 μl/min, in the micromixers and the mean capture efficiencies were determined with IFC. As shown in Fig. 6, the capture efficiency remains constant independently of the Cryptosporidium concentration. For both, 75,000 and 750, there was a statistical difference between the functionalised device and the control (p < 0.05, students, t-test), with significally more binding in the functionalised device.
Dependence of the capture efficiency of Cryptosporidium oocysts in microfluidic micromixers at 2 μl/min at concentrations 15,000 and 1,500,000 crypto/ml, using imaging flow cytometry. Light grey bars are the functionalized experimental results and the dark grey bars are the non-functionalized control devices
Once the ability of the system to capture and detect Cryptosporidium oocytes has been confirmed, for the system to be an analytical tool for quantification, a counting protocol needs to be defined. For this purpose, a calibration curve needs to be established to relate the number of oocytes counted by fluorescence microscopy to the real concentration of a given tested sample. Similarly to Fig. 5, we propose that the number of oocysts captured at different lengths in the device should be systematically counted at several randomly chosen areas. Then the average number of oocysts per area at different lengths could be plotted and fitted to a classical kinetics exponential decay, as in eq. 2:
$$ \boldsymbol{y}\left(\boldsymbol{x}\right)=\boldsymbol{A}{\boldsymbol{e}}^{-\boldsymbol{kx}} $$
where y(x) is the average number of oocytes in the device at a given length x. Following this protocol at different initial concentrations of Cryptosporidium and normalizing y(x), a calibration curve could be theoretically established for the dynamic range between the constant of decay k and the concentration of the solution. Alternatively, if the number of oocysts is very low, the total area of the microfluidic device can be directly scanned to find the concentration.
The detection of pathogenic oocysts in water is an important health and environmental issue. A microfluidic micromixer device was developed to capture Cryptosporidium and enable direct in situ high quality microscopic observation. Imaging flow cytometry and fluorescence microscopy demonstrated that the oocysts had a strong binding affinity to the antibody-functionalized micromixers. Only minimal non-specific binding was observed in control devices, confirming the specific nature of the binding. A capture efficiency of 96% was determined under optimal conditions. Further studies are warranted beyond this proof of principle work to validate the proposed approach for its analytical potential for quantification in real samples. The main limitation of this microfluidic technology is its limited throughput, which restricts its application to water concentrates. However, it is anticipated that a minor redesign and/or multiplexing of the microfluidic mixers could lead to higher throughputs that would be readily compatible with volumes of water typically obtained from standard concentration methods as described in the EPA guideline. Besides its excellent efficiency, the main advantage of this technology is that its application would have strong potential to simplify the overall workflow, leading to notable time and resource savings. In summary, the microfluidic micromixer approach can capture Cryptosporidium in water concentrates and has the potential to accelerate and simplify the detection of Cryptosporidium and other microorganisms of concern in surface or drinking water.
APTMS:
(3-aminopropyl) trimethoxysilane
BSA:
DAPI:
4′,6-Diamidino-2-phenylindole dihydrochloride
IFC:
Imaging Flow Cytometer
MIC:
Microfluidic Impedance Cytometry
PDMS:
Polydimethylsiloxane
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This work was performed in part at the South Australian node of the Australian National Fabrication Facility, a company established under the National Collaborative Research Infrastructure Strategy to provide nano and micro-fabrication facilities for Australia's researchers.
This work has been supported by the NHMRC Project grant APP1045841 and by the NORTE-45-2015-02 program under grant NORTE-01-0145-FEDER-000029. Thierry is supported by a NHMRC CDA Fellowship.
Future Industries Institute and ARC Centre of Excellence in Convergent Bio and Nano Science and Technology, University of South Australia, Mawson Lakes Campus, Mawson Lakes, South Australia, Australia
L. Diéguez
, M. Winter
, S. Molan
, P. Monis
& B. Thierry
International Iberian Nanotechnology Laboratory, Braga, Portugal
South Australian Water Corporation, Adelaide, SA, Australia
P. Monis
& B. King
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LD designed and fabricated the microfluidic masters and devices, designed the surface functionalization strategy and the experimental protocol for cryptosporidium isolation. She was involved in the planning of all experiments and was the primary responsible of manuscript writing. MW participated in the fluorescent labelling of the cryptospodirium, was in charge of the imaging flow cytometry and participated extensively in manuscript writing. SM fabricated the disposable PDMS microfluidic devices, and carried out all the experiments for the optimisation of oocytes isolation, labelling and quantification. PM and BK contributed to the design of the isolation strategy, provided the irradiated oocytes and developed the antibody used for the isolation. They also participated in the manuscript writing. BT directed the work, had the initial idea of the project and followed closely the experimental procedures. He also supported the work financially and was involved in the manuscript writing. All authors read and approved the final manuscript.
Correspondence to L. Diéguez or B. Thierry.
Diéguez, L., Winter, M., Molan, S. et al. Disposable microfluidic micromixers for effective capture of Cryptosporidium parvum oocysts from water samples. J Biol Eng 12, 4 (2018) doi:10.1186/s13036-018-0095-6
Cryptosporidium parvum oocysts
Disposable microfluidic micromixers
Imaging flow cytometry


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Anyone ever tried this ultra light fishing vest?
Looks like the perfect thing for warmer weather.
Looks cool, beware of Allen products though. They are very hit or miss quality wise. I'd have to see it in person first.


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Theatre Review: The Legend of Mike Smith by Soweto Kinch. The Studio, Birmingham Repertory Theatre
By ljazzn on 18 September 2013 • ( Leave a comment )
Soweto Kinch (musician / performer)
Photo Credit: Graeme Braidwood
The Legend of Mike Smith by Soweto Kinch
(The Studio, Birmingham Repertory Theatre, Mon. 16th Sept 2013. Review by Alison Bentley)
A new studio theatre hosting a new jazz/hip-hop drama written and performed by Birmingham saxophonist Soweto Kinch. Protagonist Mike Smith is on an an episodic quest, featuring jazz improvisation, rap, contemporary dance and video installation. It's a profound and often funny study of human motivation (or lack of it). It's like a Medieval Morality Play where the soliloquies are rapped in alliterative wordplay; there's a gentle recorded hip-hop accompaniment, enhanced live by bassist Nick Jurd and drummer Shane Forbes.
Jurd and Forbes- both excellent- are visible throughout, flanking a gauzy screen that doubles as backdrop for video projections and a curtain for characters to climb through on to the small square stage. Mike Smith (Ricardo da Silva) is a slight figure creating arcane poetical raps; he's in his own world in his room. The disembodied voice of a record company exec (minor characters are recorded voices) phones, asking him to convince the senior board to sign him. He has one night to prepare…the screen lights up with fire and Lord of the Rings-like fantasy figures. Kinch has drawn on Dante's vision of the underworld, but Mike is guided by 'a Gandalf-looking geezer'. A mystical golden microphone appears onscreen, amid (not always audible) booming voices- a Holy Grail and game show object of desire- a moment of comic bathos.
Soweto Kinch (musician / performer), Ricardo Da Silva (performer) and Tyrone Isaac – Stuart (performer)
But Seven Deadly Sins lure Mike from his goal. Sloth takes the form of his alter ego (the taller, more robust-looking Tyrone Isaac-Stuart) who argues him out of getting fit and into getting stoned, with a certain amount of slapstick. As they channel-hop, they see images of a man surrounded by cars, money, bling and the green lights and smoke of Envy. Isaac-Stuart mimes Mike's dissatisfaction brilliantly, turning the stage into a cage, with Kinch's nervy behind-screen sax improv and machine-gun fire rap.
External forces conspire against Mike too- transport delays, racist police. They provoke his Wrath, and he walks through onscreen fire to an M-BASE style jazz funk piece from the trio. There's a comic anti-climax as he furiously breaks his friend's front door. One of the most moving scenes is where Mike dances his frustration, Martha Graham-style, to the sax trio, in a moonlit street.
Mike's waylaid again in the mall as Gluttony and Lust take over. Mike 'vomits' designer logos on the floor before climbing hilariously over various audience members in pursuit of his lust, while Kinch raps about 'X-rated exchanges'. Avarice emerges in the amusement arcade, as Mike rolls on the roulette wheel. We chanted, 'Privatise the gains, socialise the losses', in Kinch's clever analogy between Monopoly and the recession. A black-suited figure in red stocking mask appears: a puppet master pulling Mike's strings. In one of the most powerful scenes, beautiful and chilling, they dance a pas de deux till the strings are cut and Mike slumps. Kinch's sax solo heightens the pathos. As Mike and alter egos walk home through faceless buildings, as if in a video game, the tension builds: they start to run and stumble as Forbes' thrilling drum solo doubles the tempo. Pride finally takes over. 'It's my turn at the top- I'm somebody special' says Mike, as Kinch leaps into the audience delivering comic freestyle rap insults to anyone who catches his notice. Cue more scary hellfire and gargoyle screen images, and a fine Ornette Coleman-like piece for Kinch and Isaac-Stuart on saxes, as Mike asks: 'Which passions contain genuine treasure?'
He reaches the record company but the room is symbolically empty. What will Mike do with the golden mic? He returns to creating his poetical raps, and doing things his way. The three 'Mikes' rap on an upbeat note: 'I recognise my mic's a cutlass to stifle injustice…creativity means you can't chain and limit me…breaking free of the sins caging me in.' The vulnerable, solitary figure of Mike is back alone on stage for the final words: 'It's freedom time'.
It's a spectacular piece of theatre, constantly engaging, drawing together ancient ideas of sin and their modern counterparts in social satire. Jonzi D and the team's well-paced staging is thoroughly entertaining as well as thought-provoking- and the jazz is superb.
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Home > Insights > Tax > Compensation & Benefits > The Impact of Tax Reform on Startup Equity: New Rules Could Make It Easier For Employees to Exercise
The Impact of Tax Reform on Startup Equity: New Rules Could Make It Easier For Employees to Exercise Options
This article originally posted on the Founders Circle blog and is reposted here with Founders Circle's permission.
Countless workers have been hit with surprise tax bills after they exercise options, the result of Internal Revenue Service rules that haven't always been friendly to startups.
The good news is that the new tax law offers relief for some employees who might otherwise have been forced to hold options or let them expire because of a large tax bill. But the new code doesn't remove every obstacle in the way of employee liquidity—and one provision ostensibly designed to do so looks like it will fall well short.
Peter Klinger, a principal in BDO's Compensation and Benefits practice, recently spoke with Founder's Circle about the new law and what it means for startups and their employees. The conversation has been edited for length and clarity.
What's the biggest change for option holders?
The biggest change is on the individual income tax side related to the alternative minimum tax (AMT). The AMT is a parallel set of tax rules that were enacted in 1969 to capture more revenue from very high net worth individuals. Prior to the passage of the tax reform law, the AMT was expanded to a point where a lot of folks that aren't high-net-worth individuals were impacted as well.
When you exercise an incentive stock option (ISO), assuming you don't sell the shares in the same calendar year, there's no impact on your regular income tax. But the spread between the strike price and the fair-market value at the time of exercise is included as income in the formula that determines AMT. So, if you already owe the AMT, then you're going to pay tax on the spread. If you're not subject to the AMT, exercising may put you there.
Following tax reform, the AMT threshold is higher and after-income phaseouts may also be significantly higher, which means fewer people will have to pay taxes when they exercise. That could help them lock in long term capital gains. However, behavior has to follow suit: Employees have to exercise and hold their stock, which in the past, very few have done.
Did the AMT affect a lot of startup employees?
Let's just say there's a complicated history. Back in the dot-com days, startups granted ISOs to employees as much as they could. Many of these employees didn't understand the workings of AMT. I used to do a lot of seminars back in those days, and I'd ask the audience, "How many people have heard of AMT?" And maybe one or two out of 30 or 40 hands would go up.
During the dot-com boom, employees would exercise at a time when stock prices or valuations were very high, but would not sell or were not able to sell. Then the market crashed, and their stock was worth next to nothing. But their tax bill stayed the same because it was set at the time of exercise. The result was a large number of employees with a huge tax bill and no way to pay it.
There's a new section in the tax code that seems to relate to startups called 83i. What is it?
Section 83(i) is intended to allow employees to defer paying tax on stock awards for up to five years, although there are events like an IPO that would cause employees to owe the tax sooner. In theory, an employee could exercise early, and then not have to pay the tax bill until there's a liquidity event, avoiding the problem some face now.
It's well-intentioned, but I don't really see 83(i) as practical because of the way it's set up. First, it's a broad-based plan in which the stock options (or RSUs) must be offered to essentially all full-time1 employees with the "same rights and privileges"—rather unclear language when it comes to implementation. Second, deferral elections may be made by the rank-and-file employees, while the top executives (CEO, CFO, four highest paid officers, and more-than-1 percent owners) cannot defer taxes on their options. But if a company repurchased stock in a prior year not connected with an 83(i) election, as many companies did, then they would be precluded from offering these plans in the current year, in most cases.
83(i) also has a lot of administrative requirements for employers, which might be enough of a burden for them to choose not to bother offering a plan. Every year, employers would need to report for each employee the amount includible in income at the end of the 83(i) deferral period and the aggregate amount of income being deferred under 83(i) elections at the close of each year2. Further, the company must notify the employees at the time the stock option is exercised or a reasonable time before. The company is liable for a penalty of $100 for each required notice they fail to provide. They would also have to attach forms to their corporate tax returns that would include summaries of all the amounts that they reported on W-2s.
Complying with these requirements would be a significant undertaking for a small private company.
Are there other problems with 83i?
Employees usually sell their stock awards as soon as they can. Here, you're asking them to do the opposite, essentially deferring paying the tax, holding the shares, and then paying a (presumably) lower amount of tax later. However, you run the risk of repeating the problem from the dot-com boom, as employees may not be able to sell until the value of the shares has already declined. If the value of the shares drops from the date they exercise, they still owe the tax on the amount that was due at the time of exercise.
And then there's the FICA (Federal Insurance Contribution Act) problem. If I exercise an ISO as part of an 83(i) plan, it immediately becomes a non-qualified stock option, which doesn't change its value, but does change the way it's treated under the tax code. For tax purposes, unlike ISOs, the spread between the strike price and fair-market value for non-qualified stock options count as ordinary income. Section 83(i) allows employees to defer paying income tax, but not FICA tax, which includes Social Security and Medicare. So the employee and the employer will both owe FICA at the time that the election is made.
There's one other problem, too. The tax deferral ends on the date of an IPO. So employees would owe the tax on any options they exercised under 83(i) that day. It's the employer's responsibility to collect and remit the tax payments to the IRS, also that day. But there's no liquidity at that point because the employees can't sell their stock for another six months. So, how is the company going to collect and remit the tax payment?
Facebook faced a similar problem when it went public. Vested RSUs were due to employees at the IPO. That was a taxable event, except those employees couldn't sell the stock right away because they were subject to a six-month lockup. Facebook had to go to the bank and take out billions of dollars in loans to pay the tax on behalf of the employees, who then repaid those loans when the six-month lockup period had ended.
With all these headaches, it's hard to see many companies deciding to offer 83(i) programs.
What's stayed the same in this law?
The $100,000 limit with respect to ISO hasn't changed. You can only get options that qualify as ISOs to the extent that in any calendar year, the strike price multiplied by the number of shares that become first exercisable in that year is $100,000 or less. Once you exceed that number, then those automatically become non-qualified stock options, which, as I mentioned earlier, are taxable as ordinary income upon exercise.
Also, there's a lot of gray area in the tax code around equity compensation that the language of the new law didn't clarify. And it's unclear if or when the IRS will shed light on these details. The agency is facing severe resource constraints, and has publicly said they're prioritizing other issues at this time.
What would you recommend startups do?
The smartest thing startups can do is have a plan for how to treat equity well in advance of any possible liquidity event, and to make sure employees understand the tax impact of exercising options.
Companies should also take the time to determine whether an 83(i) plan is right for them, but as I said earlier, they need to consider the potential administrative burden before moving forward.
The new tax law is complicated, and changes in one area can have a ripple effect throughout the tax liability of both companies and individuals. Mapping out the impact of these changes is critical to informing any tax strategy moving forward.
1§§ 83(i)(2)(C)(iii); 4980E(d)(4).
2§§ 6051(a)(16),(17).
This material has been prepared by BDO USA, LLP for informational purposes only, and is not intended to provide, and should not be relied on for, tax, legal or accounting advice. You should consult your own tax, legal and accounting advisors before engaging in any transaction.
Founders Circle Capital Disclaimer: The information contained herein is provided for informational and discussion purposes only and is not, and may not be relied on in any manner, as a personal recommendation or as legal, regulatory, tax, accounting, valuation, or investment advice. Neither Founders Circle nor any related person (i) is acting as a fiduciary or financial or investment adviser to you or (ii) is providing any investment advice, opinion or other information in respect of whether any proposed sale of securities is prudent.
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Web pages, on-line voting, nine candidates – the University Students' Council presidential elections have come a long way over the last 30 years.
In 1965, the first paid student council president was elected. Peter White was given $100 a week during that summer but was not paid throughout the school year. "My fellow students thought it was an atrocious amount," White said.
Today the USC president is paid about $20,000 for a full-time 14-month job.
White said there was tremendous unrest on university campuses across North America at the time of his tenure. "I felt that some of the things that needed to be done on behalf of the students could be done without demonstrations, without burning flags, but by very forceful negotiation."
Although Vietnam was not an issue on Western's campus it was certainly an issue in people's minds among other things, White said. "John Kennedy was assassinated in 1963 and that was a very symbolic event."
White was a 26-year-old economics major with a full course load when he became president and his council hired the first full-time paid general manager of the council.
"I think we were a pretty successful council in that there was a tremendous amount of change going on. We managed that change really well considering how young we were."
Major issues at Western included a shortage of housing and the desire for a large theatre. There were also traditional issues like tuition fees.
"There was no Radio Western, there was no TV Western and the electoral system was different too." The presidents were chosen through a federal electoral system. White was enrolled at University College so he had to be elected by the voters in UC.
Campaigning consisted of putting up posters. "Occasionally there were some meetings which were generally not very well attended."
Once elected in University College, all elected members from colleges and faculties gathered and elected officers. White was unopposed at that stage.
White remembers a rapidly changing organization. Enrolment was going straight up and the campus was exploding in terms of construction. The medical school, dentistry school, Alumni Hall, Middlesex College and Talbot College were all being built. "John Robarts of London was the premier and so Western was unquestionably favoured in terms of construction fund."
Delaware Hall was also being built and the joke at the time was it was going to be named after the administrator responsible for student residences – Mr. Virtue.
White is now the president of Cariff Corporation, an industrial textile manufacturer near Charlotte, North Carolina.


text:
How J.J. Culver scored a Kobe-inspired 100 points in a NAIA game
J.J. Culver had never even thought about 100 points. Never even thought about posing like Wilt Chamberlain. But he had thought about 50.
That was the school record at Wayland Baptist University heading into Tuesday night. It was a mark that had eluded J.J. over three-plus years at the small NAIA school in Plainview, Texas. It had eluded his younger brother, Minnesota Timberwolves rookie Jarrett Culver, throughout his career as well. So on occasion, the brothers would playfully urge each other: "J, you gotta get that 50-piece!"
On Tuesday night, at halftime of a performance for the ages, J.J. had 51.
By the end of the night, the 6-5 senior guard had scored 100 points in Wayland Baptist's 124-60 victory over Southwestern Adventist. It was the second 100-point game in NAIA history. It was the first since 1954.
2 of JJ's 100. #WaylandBaptist pic.twitter.com/FWefAEqqWg
— WBU Basketball (@WBUBasketball) December 11, 2019
When Jarrett FaceTimed J.J. after the game, the No. 6 pick in the 2019 NBA draft was at a loss for words. The call began with incoherent screams. "I don't even know what to say, bro," Jarrett told the newly-minted Most Famous Culver.
Hours later, as text messages inundated his phone, J.J. spoke with Yahoo Sports, in part to answer one simple question: 100 points … How?!?
How Kobe inspired Culver
The answer begins this past summer, and not quite with Wilt, but with the second-highest single-game scorer in NBA history, Kobe Bryant.
"I saw some of his videos," J.J. said of the former Laker great. "He said when he scored 81, he was conditioned really well. So in the summer, I ran a lot."
100 points for Wayland's own JJ Culver tonight. That's insane. pic.twitter.com/G7Lnmw6pdQ
— Tommy King (@RTKingWT) December 11, 2019
He practiced with Jarrett. Worked out at Texas Tech. Grinded. All of which is why, on Tuesday night, in the second half, with the game out of hand but history within reach, when Wayland Baptist coach Ty Harrelson asked J.J. whether he was conditioned to keep going, his answer came naturally.
"All summer, coach."
Harrelson had emptied his bench, but left Culver, the NAIA's leading scorer, in to chase 100. To drive by and shoot over double-teams and denials and diamond-and-ones and triangle-and-twos. He shot 62 times from the field and made 34. He was 12-of-33 from beyond the arc. He hit 20 of his 27 free throws.
With around a minute to go, he was at 98. He beat his man on a backdoor cut, finished at the rim, and got fouled.
Sitting at 98 points, here's JJ scoring to make it 100! Look at the reaction from the crowd and his brother Trey! pic.twitter.com/oCP3ACGel8
— Pete Christy (@pchristy11) December 11, 2019
He missed the free throw, unintentionally. "I really tried to make it," he assured. But then he admitted: That round "100" does sound a bit better.
Why Culver's coach left him in the game
The box score, from afar, reads like a gimmick. No other Wayland Baptist player took more than three shots. None scored more than eight points.
But Harrelson, the head coach, insists it wasn't. That many of the shots were within the flow of the offense. "Out of those 62," Harrelson told Yahoo Sports, "there were probably only two that I didn't like."
"A hundred points wasn't planned," Harrelson continued. "Getting him the ball is planned. It's always planned. It's been planned for at least the last two years. The amazing thing was that he was making so many of them. And our guys were going, Let's just let him keep going."
But with the game over before halftime, should Harrelson have left Culver in?
He's aware it's a legitimate question. He's aware there'll be criticism. He's already gotten some in his inbox. But his response?
Essentially, that he was in a no-win situation. Because he loves J.J. He raves about the player, and raves about the human. In the second half, as Culver crept toward triple digits, he thought: "I don't want to be the coach that stops that from happening."
"I coach him really hard," Harrelson reasoned. "And there's been times where he probably could've broken school records, and I stopped it. I've either taken him out of the game, or I've told him, 'Go get your assists.' … And today I just said, 'As long as [you] take the right shots…'
"If I woulda had one player say, 'Coach, I don't like this,' or, 'No, we don't want to do this,' we woulda changed," Harrelson said. "But I think they realized they had a chance to be a part of something special."
So they kept feeding Culver. "All the guys on our team like him – that was obvious in the way they kind of manipulated the game," Harrelson said. Around the 80-point mark, Culver said, he started hearing from teammates in huddles. Around 92, he started hearing from fans in the bleachers: J.J., you need eight more!
"So I tried to get it," Culver says.
And he did. In the locker room afterward, he recreated Wilt's famous photo.
JJ Culver scored 100! pic.twitter.com/FzxJURHEnO
And he created memories for everybody – including his older brother, Trey – who bore witness.
"It's something we'll never forget for the rest of our lives," Harrelson said. "We'll be telling our kids and our grandkids about the time J.J. scored 100."
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George teases 'tell-all' after boos rain down from Pacers fans


My reflections are based of course on the fundamental canonical principles of the Curia and on its own history, but also on the personal vision that I have sought to share with you in my addresses of recent years, within the context of the reform currently under way. Speaking of reform, I think of the amusing yet pointed remark of Archbishop Frédéric-François-Xavier de Mérode: "Making reforms in Rome is like cleaning the Sphinx with a toothbrush". His mot points to the patience, tenacity and sensitivity needed to attain that goal. For the Curia is an ancient, complex and venerable institution made up of people of different cultures, languages and mindsets, and bound, intrinsically and from the outset, to the primatial office of the Bishop of Rome in the Church, that is, to the "sacred" office willed by Christ the Lord for the good of the entire Church (ad bonum totius corporis).
Recently, in greeting the Fathers and Heads of the Oriental Catholic Churches, and reflecting on this ministerial, petrine and curial finality of service, I used the expression "diaconal primacy", which immediately calls to mind the image of the Servus servorum Dei, so beloved of Saint Gregory the Great. This definition, in its Christological dimension, is above all the expression of a firm desire to imitate Christ, who took on the form of a servant (cf. Phil 2:7). Benedict XVI, in this regard, has said that on the lips of Gregory this phrase was "no mere pious formula, but a true manifestation of his way of living and acting. Gregory was deeply moved by the humility of God, who in Christ made himself our servant, who washed and continues to wash our dirty feet".
A diaconal primacy "with regard to the Pope", and consequently diaconal as well, is the work which is carried out within the Roman Curia ad intra and outside of it, ad extra. This theme of a ministerial and curial diaconia reminds me of a phrase in the ancient Didascalia Apostolorum, which states that "the deacon must be the ear and the mouth of the Bishop, his heart and his soul". For this agreement between the two is linked to communion, harmony and peace in the Church, inasmuch as "the deacon is the guardian of service in the Church". I do not believe that it is by chance that the ear is the organ of hearing but also of balance; and that the mouth is the organ of both taste and speech.
The relationship that these images suggest is that of communion in filial obedience for the service of God's holy people. There can be doubt, then, that such must be also the relationship that exists between all those who work in the Roman Curia. From the Dicastery heads and superiors to the officials and all others. Communion with Peter reinforces and reinvigorates communion between all the members.
Antennae that "send", inasmuch as they are capable of faithfully transmitting the will of the Pope and the Superiors. For those working in the Holy See, the word "fidelity" is particularly important, "since they spend so much of their energy, their time and their daily ministry in the service of the Successor of Peter. This entails a serious responsibility but also a special gift, which as time goes by should lead to a relationship of closeness to the Pope, a closeness marked by interior trust, a natural idem sentire, which is expressed precisely by the word 'faithfulness'".
I have used the expression "diaconal primacy" and the images of the body, the senses and antennae to make clear that, in order to reach the places where the Spirit speaks to the Churches (history) and to achieve the aim of our work (salus animarum), it is necessary, indeed indispensable, to practice discernment of the signs of the times, communion in service, charity in truth, docility to the Holy Spirit and trusting obedience to Superiors.
Meetings with Heads of State and with various Delegations, together with the Apostolic Journeys, are its means and its goal. For this reason, the Third Section of the Secretariat of State has been established. It is meant to show the concern and closeness of the Pope and of the Superiors of the Secretariat of State for diplomatic personnel and for the men and women religious and lay people serving in the Nunciatures. The Third Section will deal with issues involving persons working in the diplomatic service of the Holy See or preparing for this service, in close cooperation with the Section for General Affairs and the Section for Relations with States.
The Visits ad Limina Apostolorum, in this sense, represent a great opportunity for encounter, dialogue and mutual enrichment. I have preferred, when meeting with Bishops, to have an open and sincere conversation that remains private and goes beyond the formalities of protocol and the customary exchange of speeches and recommendations. Dialogue between the bishops and the various Dicasteries is also important. In the course of the Visits ad Limina that resumed this year, the Bishops told me that they were received well and listened to by all the Dicasteries. This makes me very happy.
Here allow me, at this particular moment of the Church's life, to draw our attention to the forthcoming Fifteenth Ordinary General Assembly of the Synod of Bishops, which has as its theme Young People, the Faith and Vocational Discernment. To call upon the Curia, the bishops and the entire Church to give particular attention to young people does not mean considering them alone. It also means focusing on a critical theme for a combination of relationships and pressing issues, such as intergenerational relationships, the family, pastoral work, social life, and so forth. The Preparatory Document makes this clear in its Introduction: "The Church has decided to examine herself on how she can lead young people to recognize and accept the call to the fullness of life and love, and to ask young people to help her in identifying the most effective ways to announce the Good News today. By listening to young people, the Church will once again hear the Lord speaking in today's world. As in the days of Samuel (cf. 1 Sam 3:1-21) and Jeremiah (cf. Jer 1:4-10), young people know how to discern the signs of our times, indicated by the Spirit. Listening to their aspirations, the Church can glimpse the world which lies ahead and the paths the Church is called to follow".
In this regard, at my last meeting with the Patriarchs and Heads of the Oriental Churches, I spoke of the "diaconal primacy" and likewise stressed the importance of further study and review of the sensitive question of the election of new Bishops and Eparchs. This must correspond, on the one hand, to the autonomy of the Oriental Churches and, at the same time, to their spirit of evangelical responsibility and desire to strengthen constantly their unity with the Catholic Church. "Everything should be done with the thorough application of that authentic synodal praxis which distinguishes the Oriental Churches". The election of each bishop must reflect and strengthen unity and communion between the Successors of Peter and the entire College of Bishops.
The relationship of the Roman Curia to other religions is based on the teaching of the Second Vatican Council and the need for dialogue. "For the only alternative to the civility of encounter is the incivility of conflict". Dialogue is grounded in three fundamental lines of approach: "The duty to respect one's own identity and that of others, the courage to accept differences, and sincerity of intentions. The duty to respect one's own identity and that of others, because true dialogue cannot be built on ambiguity or a willingness to sacrifice some good for the sake of pleasing others. The courage to accept differences, because those who are different, either culturally or religiously, should not be seen or treated as enemies, but rather welcomed as fellow-travellers, in the genuine conviction that the good of each resides in the good of all. Sincerity of intentions, because dialogue, as an authentic expression of our humanity, is not a strategy for achieving specific goals, but rather a path to truth, one that deserves to be undertaken patiently, in order to transform competition into cooperation".
My meetings with religious leaders during the various Apostolic Visits and here in the Vatican, are a concrete proof of this. These are only some aspects, important but not comprehensive, of the work of the Curia ad extra. They are aspects linked to the theme of "diaconal primacy", "institutional senses", and of "faithful antennae that transmit and receive".


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Studying the culture and folklore of Turks, we can not limit ourselves to the borders of today's Turkey.
Turks have a proud cultural history that started out in the steppes of central Asia, and at the height of the Ottoman Empire, spread all the way to The Balkans; not to mention the Asia Minor" and most of Arabia.
All of these regions can be divided into eight dance categories: HORA, ZEYBEK, KASIK, TEKE, HALAY, HORON, BAR and AZERI.
Since it is rather impossible in a short article such as this to articulate on all the songs, sayings, dances, costumes and musical instruments of all these regions, I will give the reader a glimpse into some of these categories. "
HORA; Edirne, Kirklareli, Tekirdag province and Uskup.
Out of the four regions listed under HORA, Uskup is the one which is most intriging due to the fact that it does no longer lies within the borders of Turkey. Known to the Westerners as Skopia, Uskup is a city of 650,000 in Yugoslavia, majority of residents of which are Turks.
Uskup, with. its culture havily influenced by, theTurks since 1389, with all of its music, dances, costumes and local customes display an all-Turkish city in the heart of the Balkans.
Dances of Uskup can be performed as line or couple dances. The basic costuming for the men is: A white, .long_sleeve, button-down shirt worn under ,a navy blue vest covered with lace and embroidery of black gilt thread. For pants, they wear either navy blue or black rather tigh-gitting "potur". Besides the white socks and open-front black shoes, the costume is completed with a white sash and a watch chain.
Womens' costume consists o a long-sleeve, white shirt a vest shaped like the mens' but always in brighter colors, a baggy pair of pants (shalvar), and a red pair of shoes. Accessories of women. are a necklace with a"variety of gold coins attached and ahead scarf, called "yazma".
Zeybeks are Brave mountaineers who live in western Turkey area such as lzmir etc. This type of western folkdance has 9/8 tempo counted in music:3/4 plus 3/8.
The main instruments of this type of dance are.: darbuka (hour-glass-shaped drum), clarinet, kanun ( a flat string instrument, finger-picked on one's lap), and rills. This type of dance is a slow motion dance, portraying the man as a brave, heroic and freemountain eagle.
The Zeybek dancer is instantly recognized by the odd-looking costume he wears, which consists of short, loose trousers leaving the knees bare, heavy boots with spat-like attachments, ,and colorful flannel shirts that have. bat-like sleeves, signifying the eagle.
In western Turkey, this dance is still very popular and is important part of the cultural and social life of the people. The difficult part of this dance is taught and handed down from generation to generation.
TEKE: Burdur, Isparta, Antalya, Muğla, Fethiye provices.
Widely used in this area are sipsi, vulum (bagpipe) and Kabak kemane.
Sipsi: This, in some other regions is called "coban dudugu" (a shephard's whistle). Its length is approximately is six to eight inches, and is made from bamboo of quarter-inch diameter.
Tulum: This Turkish bagpipe is basically a half of a goat skin. The remainder of the goat skin, consists of one-half of the body and the two legs.
Certain other regions of Turkey recognizes tulum as culum zurna, gayda, Kayda. Gayda and Kayda, both of which meaning the same depending on the Turkish, dialect, is derived from the word "kaide;" meaning the rule-in music; the tuning or the tune.
KASIK: Bursa, Bilecik, Eskisehir, Bolu, Kastamonu, Kirsehir, Konya, Silifke provices.
Kasik means "spoon" in Turkey is a widely used rhythm and dance instrument in Turkey and are an integral part of the styling of this region. Men and women click the rhythm of the dance with .a pair of wooden spoons in each hand. This type of dance symbolizes the every-day. life of the people of Anatolia and is danced to the accompaniment of clarinet and davul, a very large double-headed drum, although in other regions played with sticks, played in this region with the snappy movements of fingers.
Some of the most well known dances from this region are named: "Silifke'nin Yogurdu" and "keklik:" "Yogurt of Silifke" and "Partridge," respectively.
In "Silifke'nin Yogurdu", the dancers immitate the acts involved in yogurt making, and in "Keklik", they skip and hop with the footwork and .sway arms wildly on sides, come to a sudden stop and seeds and then suddenly start flying again.
HALAY: Corum, Yozgat, Amasya, Tokat, Sivas, Malatya, Gumushane Elazig, Bingol, Adana, Gazi Antep, Sanli Urfa, Adiyaman, Diyarbakir, Mardin, Siirt, Bitlis, Mus, Erzincan, Kars (partial), Van, Tunceli, Bayburt provices.
Village Socks: This is, one of the most common hand crafts of Anatolia. They are made in all corners of the land and each region can be recognized by the special motives attributed only to them. As a matter of fact these elaborate socks are named according to the type of pattern of the motive on them. They are made out of wool for the winter and of cotton for the summer.
Yazma: This is the head scarf used by all women in all the rural areas. Just like the socks, they are an ancient art from of the Turks. What distinguishes one yazma from the other is not really the material, but rather the "oya" sewn around the edges. Long winter nights are the times for many hours of careful knitting of "oya". "oya" is such a significant hand-art form that many scientific books have been written on them, mostly in an attempt to preserve this art form.
Kese: These are little bags, usually tied down from one's sash, used for variety of purposes. They can be made out of leather or cloth. They were mostly used to carry money, tobacco or men's watches.
Guvercin Oyunu: This Elazig dance is one of the animal imitation dances. The, name translates as the Dance of the Pigeon.
Pigeons, as well as in many other parts of eastern Turkey, are in Elazığ too are known as holy animals. This is due to the commonly known story that when Prophet Mohammed was running away from his enemies, with a few of his men, had hidden in a cave (Hira). A spider instantly formed a web and then a pigeon laid an egg just in 'front of the cave, leading the searchers to think that' no 'One could have recently been in the cave and Mohammed's life was Saved.
Not only in Elazig, but in many other areas of Turkey it is possible to see dances by the same name.
HORON: Ordu, Ciresun, Rize, Artvin, Trabzon provices.
The two main regions under this category are Artvin and Trabzon. Both regions dances are characterized with the sharp footwork that the dancers-both men and women-display imitating the turbulent waters of Black Sea to which they live next. With the slow footwork, shimmies, and sometimes arms being raised with straight elbow, fingers pointing down, the dancers imitates :the fish that the fisherman have just caught.
When a dance group from Artvin, during a festival of the Balkan countries in Istanbul, before Ataturk , demonstrates this dance in 1937. Ataturk became so enthesuastic that he got up and did the dance with the group. And ever since then the dance, which was originally called Artvin Bari has been called Atabari. The types of musical instruments used for Artvin dances are mostly the accordion, tulum (the bagpipe), and the drums. In Trabzon region, the instruments used for Horon dance are zurna, drum and kemence.
Zurna : This indispensable accompaniment of the davul ( big, double-headed drum) traces back to the early days of Turks in Central Asia.
Kemence : This is the instrument of the Black Sea region and is used in no other region. The origins of both the kemence and the kabak kemane is the old, central Asian Turkish instrument, the iklig.
Iklig was brought out of central Asia by Gokturks and was further expanded into the Azerbaijan and Anatolian areas by the Seljuk Turks.
Kemence has only three strings, and its bow is the horse hair. The horse hair is normally very loose, but is tightened by the player during playing, often tightened and released, allowing loudness or softness of the sound.
Horon dances of Black Sea are often performed to cura zurna. However especially indoors, kemaenche is a must. Total length of this instrument is about 20 inches.
Erzurum, Kars, Ağrı, Artvin, Gümüşhane, Bayburt, Erzincan provinces.
Parts of Kars, Gole, Ani, Igdir provinces.
Azerbaijani music is characterized by liberal melodic embellishment and improvisational passages. Both indigenous folk instruments such as the, saz, zurna and Western instruments such as the accordion and clarinet are used.
Ercument Kilic was born in Ankara, Turkey in 1958.
He began his dance and music career at an age of four and was eventually admitted, as culture specialist/ artist, to the Turkish National Ensemble with which he remained until coming to the U.S. in 1977.
Since that time, Ercument has given lectures on Turkish Culture for dance companies and universities in various countries as he traveled extensively numerous times to Canada, Great Britain, West Germany, Netherlands, Turkey, Switzerland, Republic of China, Hong Kong, Singapore, Japan as well as all 50 states in the U.S.


text:
Final Fantasy XII Official Movie 13
By Gamespot Staff on May 25, 2019 at 4:03AM PDT
Final Fantasy XII More Info
Final Fantasy XII stars a princess named Ashe, who teams up with Vaan, an urchin, and several other characters to protect their homeland of Ivalice against invaders.


43.3 million Americans and counting are struggling under the weight of their student debts, according to the New York Federal Reserve. With so many voters dealing with the same issue, potential presidential candidates are all trying to climb onboard the student debt support bandwagon.
But some politicians are proposing plans that sound helpful in theory, while in practice could actually prove entirely ineffective for many Americans with student debt. Earlier this week, Hillary Clinton jumped to share her own plan for easing the total $1.2 trillion debt burden of post-college Americans.
One of her most radical suggestions was implementing a new four-year degree from public schools, completely debt-free. This is a plan that has worked in favor of universities in other developed countries to great educational and financial success. But it's a plan that many Americans may not support.
Her main plan is one that is meant to assist people currently juggling student debt rather than preventing future students the same debt burden. The concept is a straightforward one: lower the interest rates.
It seems like a solid idea. The proposed strategy estimates that it would assist 25 million Americans by an average of about $2,000 each. It aims to make getting out from under student debt easier, faster, more affordable, and also ensure the government wouldn't be the one profiting from the interest rates.
But does it actually work? Jason Delisle, director of the Federal Education Budget Project at the New America Foundation says no. This type of plan will only work for a few Americans, and not the right ones.
Reducing the interest rates on students loans will only benefit higher-earning borrowers. These are the graduates who typically completed more than four years of schooling to earn degrees in high-paying jobs as lawyers and doctors. While they did manage to rack up higher amounts of student debt, they're also the borrowers who are most capable of comfortably paying off that debt.
In 2014, the Brookings Institute found that higher-income/higher-educated families are more likely to hold large figures of student debt than their lower-income/less-educated counterparts. So refinancing student loans in order to take advantage of the reduced interest rates will automatically help borrowers who don't particularly need the help, rather than the borrowers that do need assistance.
"You're showering people with money who don't necessarily need it and are struggling in order to make the problem go away for people who actually need a bit of help," says Delisle.
A percentage cut will be a greater help to those with a six-figure student debt rather than those with just a few thousand in debt, because the borrowers with higher amounts of debt are of significantly higher income.
While it's a good thought, it's not the correct solution for the student debt problem that is preventing Americans from owning homes, getting married, having children, and creating retirement funds. A one-size-fits-all solution won't cut it when there's such a large income discrepancy between those with student debt.
Other plans designed to assist college-debt-ridden Americans are being proposed by politicians of every party, and will continue to be at the forefront of the debate in order to appeal to the issues faced by young voters.


Never one to duck controversy, Kentucky's Republican governor has feuded with teachers and insulted judges. He stunned GOP lawmakers recently by vetoing a pension bill counted on by an array of state-funded agencies, provoking a spat with the state Senate's top leader.
Sen. Elizabeth Warren on Friday became the first 2020 Democratic presidential candidate to make a full-throated call for the House of Representatives to begin impeachment proceedings against President Donald Trump after the release of special counsel Robert Mueller's redacted report.


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Limonit eller järnockra är en malmmineral bestående av en blandning av olika järnoxider och den innehåller som högst 62 viktprocent järn. Limonit kan anses som järn(III)oxidhydroxid FeO(OH) med varierande vattenhalt. Man har i limonit påvisat mineralen goethit, α-FeO(OH), och lepidokrokit, γ-FeO(OH), med samma sammansättning men olika kristallstruktur. Också ferrihydrit, Fe2O3·1,4H2O (äldre namn järn(III)-hydroxid), är vanligt i limonit.

Förekomst
Limonit återfinns i naturen som brun järnsten, järnsandsten, sjömalm, myrmalm och rödjord. Malmen kan anta olika former, den kan se ut som kornig rödsand, som ärtor, penningar eller ta formen av stora tjocka skivor som helt kan täcka botten på sjöar. Mångfalden av namn visar var man kan finna malmen.

Jämte järnockran som finns i den rödmull som bildas vid mineraliseringen av kopparfattig malm, vid bland annat koppargruvor runt Falun, finns också kiselsyra. Den bedöms ha en viss konserverande effekt som verkar vara av mer mekanisk art. Denna effekt eller så kallat rötskydd är inte helt fastställd och inte heller specifik för den kända röda kulören som kallas falu rödfärg. Rödmullens olika färgämnen finns bland annat också som ljusrött, svart, grått, ockra (ljusbrunt), brunt, grönt och går att få även i gult och violett.

Sjömalm
Sjömalm räknas ibland som en hård typ av limonit, ibland som en malm med limonit som huvudsakligt innehåll. Malmen bildas ofta i kalkfattiga områden på botten av näringsfattiga (oligotrofa) sjöar. Beroende på utseendet av sjömalmen så har den fått namn som ärt-, bön-, pärl-, krut-, hagel-, penning- och skraggmalm (kakformad). Sjömalmen kan ofta innehålla en hel del inneslutna sand- och gruskorn som minskar järnhalten och ökar mängden slagg.

Historia
Limonit var viktig som råvara för järnframställning från järnåldern och framåt, i Småland ända in på mitten av 1800-talet. Först då slogs den ut som råvara av gruvorna framför allt i Bergslagen. Detta ledde till en järnbruksdöd i Småland, många järnbruk omvandlades till glasbruk, ett av de mest kända är Orrefors glasbruk.

Se även
 Blästbruk

Referenser

Noter

Gruvdrift
Industrihistoria
Järn

redpajama_set_name: RedPajamaWikipedia


Microsoft founder and philanthropist Bill Gates has announced his newest initiative is to help cure Alzheimer's, a disease which several men in his family have had. He will be investing 50 million dollars with the Dementia Discovery Fund, a venture fund focused entirely on discovering and developing novel therapies for dementia.
Alzheimer's is now the sixth leading cause of death in the United States, and the most common form of dementia.
Launched in 2015, the Dementia Discovery Fund (DDF) has made significant progress building an initial portfolio of 12 investments in drug discovery companies and projects predominantly in the UK and US in areas including microglial biology and inflammation, mitochondrial dynamics, trafficking and membrane biology and synaptic physiology and function.
The DDF team believes there is a significant opportunity to develop dementia drugs targeting biological pathways beyond the prevailing amyloid beta hypothesis and to apply insights from areas such as oncology and immunology to develop novel drugs targeting these other biological pathways that may drive different forms of dementia.
This is the first time that Bill Gates has made a commitment to fighting a noncommunicable disease. Previous health initiatives taken on by his foundation has focused primarily on infectious diseases such as HIV, malaria and polio.
The DDF is uniquely positioned to benefit from the expertise of its world-class Scientific Advisory Board (SAB) which includes heads of Neuroscience and/or R&D from seven major pharma companies (Biogen, Eli Lilly and Company, GSK, Johnson & Johnson, Otsuka (Astex), Pfizer and Takeda) and ARUK, who collectively have a large network and experience in neuroscience drug discovery.
The SAB provides ongoing advice and knowledge, offers insights on different approaches and historical failures, suggests priority areas to explore new approaches to treat dementia and advises on strategies to drugging these new pathways.
With this increased investment the DDF team will grow, including the appointment of a Chief Executive to lead the organization.


A Skin Walker is by definition a person who is able to change their form either into an animal or something supernatural.
Skinwalker is an RPG PC horror game about four friends going out for camping into a dark forest. It's an adventure they are not going to forget until the rest of their lives - if and only if they survive. In short, they go out for camping into the woods. They set up the tent near the family cabin, start the fire and have few drinks. As the night gets colder they decide to go into the tent and get some rest. But strange sounds start to appear constantly walking them up. The game is really really creepy! Great game actually with really bad end - literally!
A Skin Walker is by definition a person who is able to change their form either into an animal or something supernatural. Now, let's see if this game has anything to do with this. It's a horror game, so for now, supernatural and horror go hand in hand, but we'll see.
To start the game, you just need to unpack it, go into 'Skinwalker' directory and double click Game.exe. That means no special installation is needed. The game simply runs from the folder. And it's only 13MB in size.
Controls are explained on start: Z is 'action key', arrow keys to move and hold shift to move faster. That's all.
The story: Four friends are going out for camping in the woods: Darren, Celeste, Michael and me - Joe. You will get to know them all at the start. When they got to the cabin, one of them had to go and get some fire woods. And then a strange sound could be heard. Very uncommon and unusual for a forest sound.
That first night was very foggy and while our friends were sitting by the fire, drinking beer, the strange metallic sound appears again. Boys ignored the sound and went to the tent to get some sleep.
A few hours later, something woke them up ... Joes' the bravest. He gets out of the tent to check out what's going on.
Here's the DOWNLOAD LINK for Skinwalker Horror Game.
i wish there wasnt a game id have to download... WAAHHHHHH!!!!