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OSA Publishing > Biomedical Optics Express > Volume 1 > Issue 2 > Page 729
Christoph Hitzenberger, Editor-in-Chief
Dual-point dual-wavelength fluorescence monitoring of DNA separation in a lab on a chip
Chaitanya Dongre, Jasper van Weerd, Nicola Bellini, Roberto Osellame, Giulio Cerullo, Rob van Weeghel, Hugo J. W. M. Hoekstra, and Markus Pollnau
Chaitanya Dongre,1 Jasper van Weerd,2 Nicola Bellini,3 Roberto Osellame,3 Giulio Cerullo,3 Rob van Weeghel,2 Hugo J. W. M. Hoekstra,1 and Markus Pollnau1,*
1Integrated Optical Microsystems (IOMS), MESA + Institute for Nanotechnology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands
2Zebra Bioscience BV, Wethouder Beversstraat 185, 7543 BK Enschede, The Netherlands
3Istituto di Fotonica e Nanotecnologie (IFN-CNR), Dipartimento di Fisica, Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milan, Italy
*Corresponding author: M.Pollnau@ewi.utwente.nl
C Dongre
J van Weerd
N Bellini
R Osellame
G Cerullo
R van Weeghel
H Hoekstra
M Pollnau
Vol. 1,
•https://doi.org/10.1364/BOE.1.000729
Chaitanya Dongre, Jasper van Weerd, Nicola Bellini, Roberto Osellame, Giulio Cerullo, Rob van Weeghel, Hugo J. W. M. Hoekstra, and Markus Pollnau, "Dual-point dual-wavelength fluorescence monitoring of DNA separation in a lab on a chip," Biomed. Opt. Express 1, 729-735 (2010)
Combined microfluidic-optical DNA analysis with single-base-pair sizing capability (BOE)
Fluorescence lifetime biosensing with DNA microarrays and a CMOS-SPAD imager (BOE)
Fluorescence monitoring of microchip capillary electrophoresis separation with monolithically integrated waveguides (OL)
Channeled waveguides
Laser induced fluorescence
Glass waveguides (130.2755)
Fluorescence (260.2510)
Biological sensing and sensors (280.1415)
Original Manuscript: July 12, 2010
Revised Manuscript: August 6, 2010
Manuscript Accepted: August 23, 2010
2. Experimental approach to highly accurate dual-wavelength fluorescent DNA analysis
3. Monitoring of single base-pair insertion / deletion through a single DW
4. Intrinsic referencing / calibration of DNA analysis by monitoring through two DWs
We present a simple approach in electrophoretic DNA separation and fluorescent monitoring that allows to identify the insertion or deletion of base-pairs in DNA probe molecules from genetic samples, and to perform intrinsic calibration/referencing for highly accurate DNA analysis. The principle is based on dual-point, dual-wavelength laser-induced fluorescence excitation using one or two excitation windows at the intersection of integrated waveguides and microfluidic channels in an optofluidic chip and a single, color-blind photodetector, resulting in a limit of detection of ~200 pM for single-end-labeled DNA molecules. The approach using a single excitation window is demonstrated experimentally, while the option exploiting two excitation windows is proposed theoretically.
©2010 Optical Society of America
Lab-on-a-chip (LOC) [1] systems have become increasingly popular for DNA analysis. In microchip capillary electrophoresis (MCE) [2] labeled DNA molecules are separated in a microfluidic (MF) channel. Typically, the molecules are monitored by laser-induced fluorescence (LIF) [3] either in a confocal setting using bulky bench-top optics [4] or in an integrated optofluidic approach [5]. To enhance analysis capabilities, multi-wavelength fluorescence sensing has been implemented, mostly in bulk capillaries [6], e.g. by use of a broadband light source such as a Xe lamp and extracting the multiple wavelengths with monochromators and complex optical schemes to achieve an unambiguous separation of different wavelengths. Other reports include the use of multiple photodiodes [7], external wavelength selective gratings [8], color CCD arrays [9], or external spectrum analyzers [10].
In several fundamentally important diagnostic applications only two independent samples, each consisting of a number of differently sized DNA molecules, have to be monitored simultaneously, hence only two excitation beams and detection of fluorescence signals at merely two wavelengths are required. These applications include, firstly, internal calibration of the set-up during a MCE experiment by adding to the sample under investigation a reference sample with several DNA molecules of known base-pair sizes, thereby making the experiment insensitive to environmental influences such as operating temperature, condition of the sieving gel matrix and inner wall coating of the MF channels, or changes of the applied electric field. Secondly, an unknown, potentially malign sample exhibiting single base-pair insertions or deletions can be separated together with its healthy counterpart, thereby providing unprecedented sizing accuracy to the experiment.
In this paper we propose a simple method for spatial and wavelength duplexing, which applies dual-point, dual-wavelength detection from either one or two detection windows (DWs). It does not require any external apparatus to separate the wavelengths, thereby allowing for fluorescence detection with a single, ultrasensitive photomultiplier tube (PMT), which enables an ultra-low limit of detection. After introducing the set-up used for on-chip DNA separation and fluorescence detection in Section 2, we describe two applications, namely detection of single base-pair insertion/deletion in genetic samples through a single DW, which we demonstrate experimentally in Section 3, and internal calibration of DNA separation through two DWs, which we propose theoretically in Section 4.
Optofluidic chips were fabricated in a two-step procedure. Firstly, the MF channel network and MF reservoirs were patterned photolithographically and wet-etched into fused silica glass and then sealed off by bonding another piece of fused silica glass on top. This commercially mass-produced chip (LioniX BV) has dimensions of 55 mm width × 5.5 mm depth × 1 mm height and the MF channels have a cross section of ~110 µm depth and ~50 µm height. In a second step, we applied femtosecond-laser micromachining to post-process optical waveguides (WGs) into the bulk of such LOCs on a flexible, chip-by-chip basis [11] for integrated fluorescence excitation of DNA molecules [12]. An elliptical WG cross section was obtained, with a height of ~50 µm, in order to excite the maximum possible volume of the MF channel, while its width is ~12 µm in order to retain a high spatial resolution along the direction of DNA flow and separation. Single-mode optical fibers carrying the excitation laser light were butt-coupled to the end-facets of WGs in the optofluidic chip with coupling losses < 2 dB. The WG propagation losses were in the range of 0.5–0.9 dB/cm at the wavelength of 543 nm [11].
The lay-out of our optofluidic chip is presented in Fig. 1 . Each of the two DWs consists of a region in which two WGs carrying two different excitation wavelengths intersect the MCE separation channel in plane at two nearby locations. From the MF crossing junction, at which the separation commences, the four WGs are distanced by 2.0 cm and 2.1 cm (WG 1 and WG 2 within DW1, respectively) as well as 3.5 cm and 3.6 cm (WG 3 and WG 4 within DW2, respectively). Depending on the application (see Sections 3 and 4) only a single DW or both DWs can be used. In the latter case, the order of the two excitation wavelengths is swapped between the two DWs to ensure unambiguous analysis.
Fig. 1 Layout of the optofluidic chip indicating the MF reservoirs, MF channels, and the two DWs, each comprising two WGs crossing the MCE separation channel perpendicularly in plane
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Prior to the experimental runs the inner walls of the MF channel network were coated with a polymer to suppress the electro-osmotic flow and minimize adsorption of DNA molecules on the MF channel wall [13]. Subsequently, the channels were filled with a sieving gel matrix consisting of hydroxypropyl-cellulose [14] to maximize the resolution of the DNA CE separation. The reagents were sterilized, filtered, and stored at 269 K until their use. The CE sample loading and separation protocol was based on actuation voltages of up to 1.5 kV, provided by a MF control system (CapiliX BV) and delivered via Pt electrodes integrated into the MF reservoirs. Application of a high voltage forced the negatively charged DNA molecules to migrate into the CE injection channel from sample inlet reservoir 1 to sample outlet reservoir 2. By switching the voltages at all four reservoirs simultaneously a well-confined plug of DNA molecules – with a volume of ~605 picoliters at the crossing junction of the two MF channels – was injected into the CE separation channel toward separation driver reservoir 4. The electric field applied to the CE separation channel was ~180 V/cm and the corresponding speed of the DNA molecules was ~320 μm/s. During their migration along the CE separation channel the DNA molecules contained in the plug volume were separated according to their size.
A cooled PMT (H7421-40, Hamamatsu Photonics K.K.) was built onto the output port of an inverted microscope (DMI5000M, Leica Microsystems GmbH) with the objective lens (10 × , NA = 0.25) aligned to collect light from either of the two DWs. In the experiments presented in Section 3 we used DW2. Through a butt-coupled fiber-array unit, 0.9 mW of power from the 543-nm and 633-nm lines of a green and a red He-Ne laser were coupled into WG3 and WG4, respectively. We estimate ~0.4 mW of power from each laser to be incident on the MF channel. Upon laser excitation of fluorescently labeled, migrating DNA molecules in the CE separation channel through such a WG, a sharp fluorescent segment 12 µm in width is observed along the intersection of WG and MF channel [12]. An appropriate multi-band filter (XF57, Omega Optical, Inc.) ensured that only the desired fluorescence signals reached the PMT. With this setup, separation of a set of DNA molecules in the diagnostically relevant size range (150-1000 bp) and investigation of the migration times of the individual molecules as a function of their a priori known sizes resulted in a mean square deviation from the expected behavior of < 1% – corresponding to a relative sizing accuracy of > 99% [15]. A limit of detection of 2.1 pM was estimated with intercalating dyes as fluorescent labels [15].
Dual-point, dual-wavelength fluorescence monitoring is valuable in diagnostic applications to compare an unknown sample, e.g. from a gene region under investigation for base-pair insertion / deletion, with a reference sample, e.g. from the same gene region corresponding to a healthy person. The base-pair difference between the corresponding analyte DNA molecules would be small (1-2 bp), if any, thus challenging the resolution capabilities of standard MCE systems. Here we propose to determine these differences by exclusively labeling the two samples, flowing them against each other, and monitoring their separation with dual-point, dual-wavelength fluorescence excitation / detection through a single DW. If no insertion / deletion is present, the two species migrate with the same speed (with an error of < 1%), but will nevertheless be optically separated due to the spatial separation between the two excitation WGs in the DW.
While diagnostically relevant genetic probes are usually double-stranded DNA molecules sized >100 bp, clinically produced by a polymerase chain reaction, for this proof of principle we used commercially produced (Molecular Probes, Inc.) single-stranded DNA primer molecules sized 19 and 20 nucleotides (nt), end-labeled with either Alexa Fluor 647 (AF647) or Cyanine 3 (Cy3). These two fluorophores can be selectively excited in the red (633 nm, coupled through WG3) and in the green (543 nm, coupled through WG4), respectively.
Initially, only a single species (19-nt-AF647) was injected in the separation channel, with either the red or green He-Ne laser turned on. In the former case a strong signal peak was observed when the fluorescently labeled DNA plug crossed the excitation WG, while in the latter case the measured signal equaled the PMT baseline signal. This procedure was repeated for the same molecule labeled with the other dye (19-nt-Cy3), leading to the complementary result (i.e., strong signal peak with the green He-Ne laser and no signal with the red He-Ne laser). This confirmed that cross-excitation between the two fluorophores was, for all practical purposes, absent. Based on the signal-to-noise ratio of the electropherograms and the known concentrations of single-end-labeled DNA molecules in the plugs, we estimate for this experiment a limit of detection of ~200 pM. The limit of detection achieved earlier (2.1 pM) is valid for DNA molecules with intercalating dyes that attach at numerous positions on the same molecule, leading to a much larger effective fluorescence intensity.
The two differently labeled, but equally sized DNA molecules were then mixed and the resulting polychromatic mixture was resolved into the individual monochromatic DNA components by dual-point, dual wavelength excitation in the MCE separation channel. Figure 2(a) depicts the corresponding electropherogram. Since cross-excitation is absent, the two peaks appearing after a migration time of ~108-110 s and separated by ~1.8 s correspond to the two species, each excited by the corresponding laser wavelength of 633 nm or 543 nm coupled into the adjacent WGs. This is further confirmed by repeating the MCE separation of the two molecules twice, alternately turning off one of the lasers. The corresponding electropherograms displayed in Fig. 2(b) show a good match with the corresponding peak heights and positions in Fig. 2(a). This result demonstrates the impact of the described dual-point, dual-wavelength technique on the DNA separation resolution. Even equally sized, but differently labeled molecules that naturally flow at the same speed and could electrophoretically never be separated, have nevertheless been optically separated by the inherent spatial separation of the two WGs carrying the corresponding unique excitation wavelengths. By changing the distance between the adjacent WGs, the effective separation can be adjusted.
Fig. 2 Electropherograms depicting the MCE separation of two fluorescently labeled DNA molecules: (a) cumulative signal during simultaneous dual-wavelength excitation of migrating 19-nt-AF647 and 19-nt-Cy3 molecules; (b) individual signals detected during different flow experiments applying single-wavelength excitation with only one of the two lasers switched on, temporally superimposed on each other; (c) and (d) the same for 19-nt-AF647 and 20-nt-Cy3 molecules.
Furthermore, considering the potential application, i.e. the detection of a genetic abnormality, to visualize the case of single base-pair insertion / deletion, we repeated the experiment with a new sample mixture (19-nt-AF647 + 20-nt-Cy3), where the size difference of a single base-pair between the two molecules was chosen to represent the potential application scenario. The corresponding electropherograms are shown in Figs. 2(c) and 2(d), with an evidently larger peak separation of ~6.2 s. The lack of perfect match in the temporal positions of the peaks and their intensities between Fig. 2(a) and 2(b) and Fig. 2(c) and 2(d) is attributed to slight, unintentional differences in the experimental flow parameters. Such effects justify the need for an internal calibration / referencing as described in Section 4.
MCE separation and, consequently, sizing accuracy depend critically on multiple flow parameters, such as fluid and chip temperature, condition of the sieving gel matrix and MF wall coating, and applied electric field. Hence, for achieving single-base-pair accuracy of DNA analysis – for molecules in the diagnostically relevant range of 150-1000 bp – in an optofluidic chip, reliable intrinsic calibration of the CE separation setup is required. In our approach, a set of sample molecules under investigation and a set of well-known reference molecules (for calibration) can be exclusively labeled with two different fluorophores and separated simultaneously in a single MCE experiment. The reference molecules provide an intrinsic 'ruler' allowing one to accurately sort the sample molecules according to their size independently of the fluctuating experimental conditions. Building upon the experimental proof of principle presented in Section 3, we discuss our approach for intrinsic calibration by means of a numerical simulation based on data obtained from earlier experiments [15].
Monitoring through two well separated DWs, each comprising a WG pair carrying two different excitation wavelengths whose spatial order is swapped in DW2 (Fig. 1), provides the following advantages. (i) An unfortunate size difference between two differently labeled DNA species may occur, such that the difference in migration speed exactly compensates for the distance between the two excitation WGs within one DW. As a result, the two fluorescence peaks are simultaneously detected by the color-blind PMT in this DW. However, the peaks do not coincide in the other DW, because the swapping of the excitation wavelength would indeed enhance the effect of different migration speeds instead of creating a degeneracy. (ii) From the double detection, additional information can be deduced about flow speed, plug broadening, etc. (iii) Low-concentrated species that disappear under the background noise owing to plug broadening while migrating to DW2, will still give rise to weak fluorescence peaks at DW1, thus providing extra information that would otherwise be lost.
In principle, such a set-up can be operated with a single PMT, e.g., by collecting the fluorescence from the two DWs by two fibers glued to the top of the chip [11] or two 3D-integrated WGs [16], and transporting the signals to the same PMT, thereby simplifying the whole setup and reducing its size. In this case, to achieve an unambiguous detection, it is a critical prerequisite to choose a sufficiently large distance between the two DWs, such that the first peak in DW2 appears only after the last peak in DW1 has disappeared. Since our experimental setup requires detection by a PMT through a microscope, only one of the two DWs can be monitored at a time. Hence, it is currently not possible in our setup to monitor both DWs simultaneously.
Therefore, to demonstrate the potential of dual-point, dual-wavelength monitoring for internal size calibration, we present a simulation of a MCE experiment based on the logarithmic relationship between DNA size and migration speed, as well as the square-root dependence of plug broadening on the DNA size, as established in an earlier experimental analysis [15]. The Poissonian dependence of the electropherogram peak width on the DNA size is represented in Fig. 3 as the evidently broader peaks in DW2 in comparison with DW1, with the broadening being related to the DNA size by a square-root dependence, while the migration times of the DNA molecules have been simulated based on their estimated migraton speeds, which depend on their sizes. In this simulation, we consider the separation of a (usually unknown) DNA sample under investigation, consisting of green-labeled molecules S1 (250 bp), S2 (300 bp), S3 (450 bp), and S4 (700 bp), flown against a (known) DNA reference consisting of red-labeled molecules R1 (150 bp), R2 (355 bp), and R3 (1000 bp).
Fig. 3 Simulated electropherograms as would be detected from the two DWs with swapped excitation wavelengths during an experiment with internal calibration using a green-labeled DNA sample ("S") consisting of four different molecule sizes (250 bp, 300 bp, 450 bp, and 700 bp) and a red-labeled DNA reference ("R") consisting of three different molecule sizes (150 bp, 355 bp, and 1000 bp). Fluorescence signals S were excited through WG1, R through WG2, R' through WG3 and S' through WG4.
Figure 3 (top, black curve) shows a simulated electropherogram that would be generated if the fluorescence is detected by a color-blind PMT simultaneously from both DWs as a result of laser excitation (with a mutually swapped order of excitation wavelengths) through the four WGs. The distance between the two DWs and/or the minimum and maximum sizes of DNA molecules are chosen such that all DNA molecules migrate first through DW1 and then through DW2, i.e., the luminescence signals occurring from the two DWs are separated in time, as indicated by the dashed line.
The deconvolution of the detected signal into the individual signals obtained from the sample and reference DNA molecules is presented in Fig. 3 (center, red curve, and bottom, green curve). The sizes of the reference molecules are chosen such that the smallest and largest base-pair sizes belong to reference molecules, hence they can be easily identified. Based on the a priori known sizes of the reference molecules, one can make use of the known dependence of the relative migration time on DNA size [15] to identify the third, medium-sized reference molecule in both windows, because its peak principally shifts from DW1 to DW2 in the same manner with respect to the peaks of the unknown molecules as the first and last reference molecule, and calibrate the remaining peaks of the electropherogram for varying environmental conditions, i.e., to accurately determine the sizes of the corresponding sample molecules based on their migration times relative to the migration times of the known reference molecules. The third, medium-sized reference molecule provides an indication of the experimental deviation from the expected migration due to parameter drifts. While the R2 and S3 peaks incidentally overlap in DW1 owing to their unfortunate size difference, they are well resolved as R'2 and S'3 in DW2 as a result of swapping the excitation wavelengths. In such a setting, it is straight forward to identify each peak in both parts of the diagram, DW1 and DW2, thus enabling unambiguous analysis of two sets of exclusively color-labeled DNA molecules of different origin by detection with a single color-blind PMT.
We have analyzed the electrophoretic separation of fluorescently labeled DNA molecules along a microfluidic channel with integrated waveguides in a dual-point, dual-wavelength setting. Using a single detection window, we demonstrate highly accurate detection of single-nucleotide insertion/deletion, as is relevant in genetic diagnostics – detection of anomalies in genetic samples obtained from a patient with respect to their healthy genetic counterparts. Furthermore, based on earlier experimental DNA separation data, we present a simulation of a separation experiment in a setup that uses two detection windows, with the two excitation wavelengths swapped in the second one, for internal calibration/referencing of DNA sizes. This can be a highly effective approach for intrinsically eliminating the undesirable effects of several flow parameters on calibration of a MCE separation system by flowing a well-known reference sample simultaneously with the unknown sample. The results presented in this paper bear the potential of leading to a new generation of compact optofluidic devices for use in point-of-care diagnostics.
We acknowledge financial support by the EU Project HIBISCUS (IST-2005-034562).
1. A. Manz, N. Graber, and H. M. Widmer, "Miniaturized total chemical analysis systems: A novel concept for chemical sensing," Sens. Actuators B Chem. 1, 244–248 (1990).
2. J. P. Landers, "Molecular diagnostics on electrophoretic microchips," Anal. Chem. 75(12), 2919–2927 (2003). [CrossRef] [PubMed]
3. B. Kuswandi, J. Nuriman, J. Huskens, and W. Verboom, "Optical sensing systems for microfluidic devices: a review," Anal. Chim. Acta 601(2), 141–155 (2007). [CrossRef] [PubMed]
4. M. E. Johnson and J. P. Landers, "Fundamentals and practice for ultrasensitive laser-induced fluorescence detection in microanalytical systems," Electrophoresis 25(21-22), 3513–3527 (2004). [CrossRef] [PubMed]
5. D. Psaltis, S. R. Quake, and C. Yang, "Developing optofluidic technology through the fusion of microfluidics and optics," Nature 442(7101), 381–386 (2006). [CrossRef] [PubMed]
6. R. Iten, "Apparatus for emitting and detecting light in a nucleic acid amplification reaction," Eur. Patent 1962084 (2007).
7. L. Zhu, W. J. Stryjewski, and S. A. Soper, "Multiplexed fluorescence detection in microfabricated devices with both time-resolved and spectral-discrimination capabilities using near-infrared fluorescence," Anal. Biochem. 330(2), 206–218 (2004). [CrossRef] [PubMed]
8. S. Götz and U. Karst, "Wavelength-resolved fluorescence detector for microchip capillary electrophoresis separations," Sens. Actuators B Chem. 123(1), 622–627 (2007). [CrossRef]
9. A. E. Karger, J. M. Harris, and R. F. Gesteland, "Multiwavelength fluorescence detection for DNA sequencing using capillary electrophoresis," Nucleic Acids Res. 19(18), 4955–4962 (1991). [CrossRef] [PubMed]
10. H. Sugino, K. Ozaki, Y. Shirasaki, T. Arakawa, S. Shoji, and T. Funatsu, "On-chip microfluidic sorting with fluorescence spectrum detection and multiway separation," Lab Chip 9(9), 1254–1260 (2009). [CrossRef] [PubMed]
11. R. M. Vazquez, R. Osellame, D. Nolli, C. Dongre, H. H. van den Vlekkert, R. Ramponi, M. Pollnau, and G. Cerullo, "Integration of femtosecond laser written optical waveguides in a lab-on-chip," Lab Chip 9(1), 91–96 (2009). [PubMed]
12. C. Dongre, R. Dekker, H. J. W. M. Hoekstra, M. Pollnau, R. Martinez-Vazquez, R. Osellame, G. Cerullo, R. Ramponi, R. van Weeghel, G. A. J. Besselink, and H. H. van den Vlekkert, "Fluorescence monitoring of microchip capillary electrophoresis separation with monolithically integrated waveguides," Opt. Lett. 33(21), 2503–2505 (2008). [CrossRef] [PubMed]
13. R. Martinez Vazquez, R. Osellame, M. Cretich, M. Chiari, C. Dongre, H. J. W. M. Hoekstra, M. Pollnau, H. Vlekkert, R. Ramponi, and G. Cerullo, "Optical sensing in microfluidic lab-on-a-chip by femtosecond-laser-written waveguides," Anal. Bioanal. Chem. 393(4), 1209–1216 (2009). [CrossRef] [PubMed]
14. J. C. Sanders, M. C. Breadmore, Y. C. Kwok, K. M. Horsman, and J. P. Landers, "Hydroxypropyl cellulose as an adsorptive coating sieving matrix for DNA separations: artificial neural network optimization for microchip analysis," Anal. Chem. 75(4), 986–994 (2003). [CrossRef] [PubMed]
15. C. Dongre, J. van Weerd, G. A. J. Besselink, R. van Weeghel, R. M. Vazquez, R. Osellame, G. Cerullo, M. Cretich, M. Chiari, H. J. W. M. Hoekstra, and M. Pollnau, "High-resolution electrophoretic separation and integrated-waveguide excitation of fluorescent DNA molecules in a lab on a chip," Electrophoresis 31(15), 2584–2588 (2010). [CrossRef] [PubMed]
16. A. Crespi, Y. Gu, B. Ngamsom, H. J. W. M. Hoekstra, C. Dongre, M. Pollnau, R. Ramponi, H. H. van den Vlekkert, P. Watts, G. Cerullo, and R. Osellame, "Three-dimensional Mach-Zehnder interferometer in a microfluidic chip for spatially-resolved label-free detection," Lab Chip 10(9), 1167–1173 (2010). [CrossRef] [PubMed]
Article Order
A. Manz, N. Graber, and H. M. Widmer, "Miniaturized total chemical analysis systems: A novel concept for chemical sensing," Sens. Actuators B Chem. 1, 244–248 (1990).
J. P. Landers, "Molecular diagnostics on electrophoretic microchips," Anal. Chem. 75(12), 2919–2927 (2003).
[Crossref] [PubMed]
B. Kuswandi, J. Nuriman, J. Huskens, and W. Verboom, "Optical sensing systems for microfluidic devices: a review," Anal. Chim. Acta 601(2), 141–155 (2007).
M. E. Johnson and J. P. Landers, "Fundamentals and practice for ultrasensitive laser-induced fluorescence detection in microanalytical systems," Electrophoresis 25(21-22), 3513–3527 (2004).
D. Psaltis, S. R. Quake, and C. Yang, "Developing optofluidic technology through the fusion of microfluidics and optics," Nature 442(7101), 381–386 (2006).
R. Iten, "Apparatus for emitting and detecting light in a nucleic acid amplification reaction," Eur. Patent 1962084 (2007).
L. Zhu, W. J. Stryjewski, and S. A. Soper, "Multiplexed fluorescence detection in microfabricated devices with both time-resolved and spectral-discrimination capabilities using near-infrared fluorescence," Anal. Biochem. 330(2), 206–218 (2004).
S. Götz and U. Karst, "Wavelength-resolved fluorescence detector for microchip capillary electrophoresis separations," Sens. Actuators B Chem. 123(1), 622–627 (2007).
[Crossref]
A. E. Karger, J. M. Harris, and R. F. Gesteland, "Multiwavelength fluorescence detection for DNA sequencing using capillary electrophoresis," Nucleic Acids Res. 19(18), 4955–4962 (1991).
H. Sugino, K. Ozaki, Y. Shirasaki, T. Arakawa, S. Shoji, and T. Funatsu, "On-chip microfluidic sorting with fluorescence spectrum detection and multiway separation," Lab Chip 9(9), 1254–1260 (2009).
R. M. Vazquez, R. Osellame, D. Nolli, C. Dongre, H. H. van den Vlekkert, R. Ramponi, M. Pollnau, and G. Cerullo, "Integration of femtosecond laser written optical waveguides in a lab-on-chip," Lab Chip 9(1), 91–96 (2009).
C. Dongre, R. Dekker, H. J. W. M. Hoekstra, M. Pollnau, R. Martinez-Vazquez, R. Osellame, G. Cerullo, R. Ramponi, R. van Weeghel, G. A. J. Besselink, and H. H. van den Vlekkert, "Fluorescence monitoring of microchip capillary electrophoresis separation with monolithically integrated waveguides," Opt. Lett. 33(21), 2503–2505 (2008).
R. Martinez Vazquez, R. Osellame, M. Cretich, M. Chiari, C. Dongre, H. J. W. M. Hoekstra, M. Pollnau, H. Vlekkert, R. Ramponi, and G. Cerullo, "Optical sensing in microfluidic lab-on-a-chip by femtosecond-laser-written waveguides," Anal. Bioanal. Chem. 393(4), 1209–1216 (2009).
J. C. Sanders, M. C. Breadmore, Y. C. Kwok, K. M. Horsman, and J. P. Landers, "Hydroxypropyl cellulose as an adsorptive coating sieving matrix for DNA separations: artificial neural network optimization for microchip analysis," Anal. Chem. 75(4), 986–994 (2003).
C. Dongre, J. van Weerd, G. A. J. Besselink, R. van Weeghel, R. M. Vazquez, R. Osellame, G. Cerullo, M. Cretich, M. Chiari, H. J. W. M. Hoekstra, and M. Pollnau, "High-resolution electrophoretic separation and integrated-waveguide excitation of fluorescent DNA molecules in a lab on a chip," Electrophoresis 31(15), 2584–2588 (2010).
A. Crespi, Y. Gu, B. Ngamsom, H. J. W. M. Hoekstra, C. Dongre, M. Pollnau, R. Ramponi, H. H. van den Vlekkert, P. Watts, G. Cerullo, and R. Osellame, "Three-dimensional Mach-Zehnder interferometer in a microfluidic chip for spatially-resolved label-free detection," Lab Chip 10(9), 1167–1173 (2010).
Arakawa, T.
Besselink, G. A. J.
Breadmore, M. C.
Cerullo, G.
Chiari, M.
Crespi, A.
Cretich, M.
Dekker, R.
Dongre, C.
Funatsu, T.
Gesteland, R. F.
Götz, S.
Graber, N.
Gu, Y.
Harris, J. M.
Hoekstra, H. J. W. M.
Horsman, K. M.
Huskens, J.
Johnson, M. E.
Karger, A. E.
Karst, U.
Kuswandi, B.
Kwok, Y. C.
Landers, J. P.
Manz, A.
Martinez Vazquez, R.
Martinez-Vazquez, R.
Ngamsom, B.
Nolli, D.
Nuriman, J.
Osellame, R.
Ozaki, K.
Pollnau, M.
Psaltis, D.
Quake, S. R.
Ramponi, R.
Sanders, J. C.
Shirasaki, Y.
Shoji, S.
Soper, S. A.
Stryjewski, W. J.
Sugino, H.
van den Vlekkert, H. H.
van Weeghel, R.
van Weerd, J.
Vazquez, R. M.
Verboom, W.
Vlekkert, H.
Watts, P.
Widmer, H. M.
Yang, C.
Zhu, L.
Anal. Bioanal. Chem. (1)
Anal. Biochem. (1)
Anal. Chem. (2)
Anal. Chim. Acta (1)
Lab Chip (3)
Nucleic Acids Res. (1)
Opt. Lett. (1)
Sens. Actuators B Chem. (2)
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Coinmen
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How India's Ministry Of Corporate Affairs Took Steps To Help Businesses Amidst The 2019-20 Coronavirus Pandemic
The Big Bang: When Coronavirus Took Over The World In 2020
Coronavirus, otherwise known as Covid-19 or novel coronavirus, is a disease-turned pandemic which started from China's Wuhan region and spread at a rapid pace in various parts of Asia and Europe. As it stands, the virus has virtually affected almost all parts of the world, with new cases coming in every day. The global population is leaving no stone unturned to try and slow the speed of the pandemic via a variety of measures, ranging from social distancing, experimental medication, and nation-wide lockdowns as scientists and researchers looks for a cure.
Mission Lockdown India And Its Impact
Followed by the PM's address on 19th March 2020 to impose a "Janta Curfew" (Public Curfew) for a day, a big news came in on 24th March 2020. The PM again addressed the nation on the 24th and imposed complete lockdown of the country at least till 14th April 2020, for a period of 21 days. This lockdown ruling was followed by clarifications to ensure no mass hysteria, as well as by guidelines on how things would go on for the next 3 weeks.
Due to the imposition of lockdown in various states and then in the entire country, all factories, offices, shops, establishments etc. have been closed and wherever possible, employees have been asked to work from home. All means of transportation such as railways, flights, metro rail, cabs, auto rickshaws, etc. have been stopped as well. Even public transport buses have been deployed only for the transportation of people involved in essential services.
Due to all these steps taken by the Government, companies have been forced to temporarily cease all operation which has not only resulted in financial loss for their business but also in issues in carrying out statutory compliances such as payment of taxes, conducting board meetings, issue of statutory notices, financial statement closing, etc.
Response By The Ministry Of Corporate Affairs Towards Businesses Operating In India
Considering the above-mentioned issues and impact of Covid-19 and keeping in mind the lockdown of the entire country, the Ministry of Finance and the Ministry of Corporate affairs ("MCA" or "Ministry") came out with various circulars and notifications pertaining to the above issues.
A. Companies To Comply With COVID Guidelines
Firstly, Form CAR (Companies Affirmation of Readiness towards Covid-19) has been asked to be filed voluntarily by all companies/LLPs to check whether all the companies/LLPs are complying with the COVID Guidelines, including work from home policy as a confidence-building measure to assess readiness. No fee / penalty / enforcement-related action is applicable on the said form.
B. Impact On Board Meetings
Further, the Ministry has relaxed the requirement of holding Board Meetings with physical presence of the directors. Prior to the mandatory lockdown, matters pertaining to approval of financial statements, board reports, mergers and restructuring, etc. were required to be taken in a board meeting with a physical presence of the company's directors. However, on account of the lockdown, the Ministry has allowed for the meetings to be conducted with aforesaid agendas through video conferencing or other audio visual means till 30th June, 2020.
This move was much needed, especially for foreign directors who could not travel for such meetings due to travel restrictions; and this provision enables the boards of these companies to continue functioning.
The Ministry has also relieved the mandatory requirement of holding board meetings within the intervals. The previous interval of 120 days between two consecutive board meetings has now been extended to 180 days for the next 2 financial year quarters, i.e. till 30th September 2020.
C. Impact On Residency Criteria
Further, if due to the Coronavirus pandemic, where virtually all major countries are under lockdown, the resident directors of the companies are not able to comply with the requirement of minimum residency in India, i.e., to stay in India for a period of at least 182 days in a financial year, the same shall not be treated as a non-compliance for FY 2019-20.
Also, the Ministry has allowed the spending on activities related to Covid-19 as eligible CSR CSR (Corporate Social Responsibility) expenses as per the applicable provisions of the Act. Funds may be spent for various activities related to Covid-19 relating to promotion of healthcare, including preventive healthcare, sanitation, and disaster management.
Major Relief Measures Related Filing Of Documentation For Companies
Further, I believe the major step which has been taken by the Ministry due to the outbreak of Corona Virus is by announcing that no additional fee shall be charged for late filing during a moratorium period from 01st April 2020 to 30th September 2020 in respect of any document, return, statement etc. required to be filed in the MCA-21 registry, irrespective of the due date.
The motive behind this move is not only to reduce the compliance burden on companies / LLPs at large but also to enable the non-compliant companies / LLPs to make a fresh start without paying an additional fee.
Listed below are the other major reliefs provided by MCA related to regulatory matters:
The Companies (Auditor's Report) Order, 2020 shall be made applicable from F.Y. 2020-21 instead of being applicable from F.Y. 2019-20 notified earlier
Independent directors required to hold at least one meeting without the attendance of non-independent directors and members of management. For FY 2019-20, if independent directors of a company have not been able to hold such a meeting, the same shall not be viewed as a violation
Requirement to create a deposit repayment reserve of 20% of deposits maturing during FY 2020-21 before 30th April 2020 shall be allowed to be complied with till 30th June 2020
Requirement to invest or deposit at least 15% of amount of debentures maturing in specified methods of investments or deposits before 30th April 2020 maybe complied with till 30th June 2020
Newly incorporated companies are required to file a declaration for commencement of business within 180 days of incorporation. An additional period of 180 more days is allowed for this compliance
The above measures and steps taken by the MCA are welcome steps and will help the businesses/companies in times of crisis, while the world attempts to recover from a deadly pandemic. These steps will help the businessmen and their employees to shift their focus from statutory compliances and focus on health of their own self and others, as well.
Amrita Deol
Amrita Deol currently leads the Corporate Secretarial Services at Coinmen.
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3/1/2017 - NRA Video Mocks New York Times "The Truth" Ad
3/1/2017 - Would You Buy It? The Obamas get a Book Deal
3/1/2017 - Why does the Associated Press Fact Check Trump, Trump's Cabinet but didn't Fact Check Obama?
3/1/2017 - Social Media Erupted after Photo of Kellyanne Conway Sitting on a Couch Upset People
3/1/2017 - Liberal Media Upset that President Trump Allegedly Questioned Who was Behind High-Profile Anti-Semitic Acts?
3/1/2017 - Anti-Trump Protest Takes Place in front of New Trump Hotel in Vancouver
3/1/2017 - Jon Stewart, former host of 'The Daily Show', tells Media to 'Stop Your Whining'
3/1/2017 - CNN Published Anti-Trump Piece by Losing Candidate Evan McMullin
2/28/2017 - L.A. Times Now Selling " We Will Not Shut Up" T-Shirts
2/28/2017 - NRA President LaPierre Criticized CNN, Violent Leftist Protesters in CPAC 2017 Speech
2/28/2017 - SCOTUS Justice Ruth Bader Ginsburg Reassured Liberal Audience She Won't Retire Soon
2/28/2017 - NRA Counters New York Times 'Truth' Ad, Highlighted Liberal Media Bias and Missed Stories
2/28/2017 - Go Figure: Liberal Hollywood Critical of Trump at the Oscars
2/28/2017 - Liberal Media Ignores Democratic Party's Problems, Only Two Articles Wonder about DNC's Future
2/27/2017 - Slight Majority of Americans Think Media Coverage of Trump is Too Tough
2/27/2017 - Media Floored that Trump will Skip Annual White House Correspondent's Dinner
2/27/2017 - Liberal Media Covers Jewish Cemetery Vandalisms
2/27/2017 - Liberal Media Goes after Trump's CPAC 2017 Comments on 'Fake News Media'
2/27/2017 - Liberal Media Outlets Not Permitted to Attend Press 'Gaggle' with Sean Spicer
2/27/2017 - Retired WNBA Player said 98% of League is Gay, was Bullied as a Straight Player
2/26/2017 - Trump Knocks NYT "Truth" Ad, Challenges Paper to Report "Accurately and Fairly"
2/26/2017 - Outcry after Police Chief placed 'Punisher', 'Blue Lives Matter' Decals on Patrol Cars
2/26/2017 - Movement in France to make Obama a French President
2/25/2017 - Politico Published Leaked GOP ObamaCare Repeal Plan
2/25/2017 - LA Times Wonders if Mexico can 'Expect Less-Hostile Days' from Trump after Visits
2/24/2017 - Fox Boosts Ad Rates for Tucker Carlson Show by 27%
2/24/2017 - VP Mike Pence's CPAC 2017 Remarks on Repealing Obamacare Highlighted by Liberal Media
2/24/2017 - NRA's Wayne LaPierre to Speak Today at CPAC 2017
2/24/2017 - Much-Hyped NYC Security Cost to Protect Trumps is $10 Million Lower than the First Estimate
2/24/2017 - Liberal Media Blast Trump Adviser Steve Bannon's Comment that the Media is Wrong about Trump
2/24/2017 - How did the Liberal Media Cover VP Mike Pence's Visit to Clean Up Vandalized Jewish Cemetery
2/23/2017 - New York Times Targets Trump Administration with "The Truth" Ad Campaign
2/23/2017 - The Bias Buzz Podcast- Milo Yiannopoulos Controversy, Trump vs. the Media, Left Wing Protests Losing Steam, Immigration Policy and More
2/23/2017 - CPAC 2017 is Underway: Short List of Featured Speakers
2/23/2017 - Washington Post Criticizes Trump's Deference to States on Transgender Bathroom Issue
2/23/2017 - Los Angeles Times Heralds Protesters Disrupting GOP Town Halls
2/23/2017 - DAPL Protesters Set Tents on Fire before Eviction Order
2/23/2017 - Liberal Narrative Debunked: Poll finds 80% of Americans Oppose Sanctuary Cities
2/22/2017 - Former Democratic Rhode Island Gov. Lincoln Chafee Says Media Has Conducted a "Full Onslaught" Against Trump
2/22/2017 - Media Quotes Jewish Leaders, Neglected to Mention Their Opposition to Trump
2/22/2017 - Rich Irony? USA Today Quoted anti-Israel Democrat Keith Ellison's Tweets about Anti-Semitism and Trump
2/22/2017 - USA Today Compared Trump's Campaign Slogan to Anti-Semitism, Claimed Breitbart is an 'Alt-Right' Website
2/22/2017 - Liberal Media Makes It Seem that President Trump Doesn't Denounce Anti-Semitism
2/21/2017 - NRA Taps Dana Loesch as Major National Spokesperson
2/21/2017 - Milo Yiannopoulos Resigns from Breitbart after Video Surfaced
2/21/2017 - 'Not My President's Day' Protests Held on President's Day
2/21/2017 - 'A Day Without Immigrants' Protesters Lost Their Day Jobs for Skipping Out on Work Without Notice
2/21/2017 - CPAC Rescinded Invitation for Milo Yiannopoulos to Speak after Comments Emerge
2/21/2017 - Liberal Media Pounced on Trump's Sweden Remark at Rally
2/21/2017 - Trump Clarified Rally Remarks on Sweden and Crime
2/21/2017 - Border Wall, or Border Fence? CNN Notes Difference of Opinion between Trump, Government Experts
2/20/2017 - President Trump Upset Liberal Media with Tweet on Media as the 'Enemy of the American People'
2/20/2017 - CSPAN Presidents Survey: Lincoln, Washington are Top Two Presidents to Historians
2/20/2017 - Flashback: Democratic Pollster Pat Caddell Called the Media the "Enemy of the American People" in 2012 [Video]
2/19/2017 - Norma McCorvey, Jane Roe of Roe v. Wade, Passes Away
2/19/2017 - While Liberal Media Gripes over Trump on Press Freedom, Socialist Venezuela Kicks CNN Network Off the Air
2/18/2017 - Women's March Strike is Set for March 8
2/18/2017 - Liberal Arts College Museum Removes Artwork Done by Immigrants for Protest Day
2/17/2017 - Trump Blasts "Out of Control" Media at Press Conference
2/17/2017 - CNN's Jake Tapper: Trump's Press Conference was 'Unhinged'
2/17/2017 - Pro-Clinton New York Times Reporters Criticize Trump during 'Live Analysis' of Press Conference
2/17/2017 - Liberal Media Criticized by Trump for 'Fake News' and Stories of 'Chaos'
2/17/2017 - Donald Trump Blasted Liberal Media for 'Unfair' Coverage of his Wife Melania
2/17/2017 - Liberal Media Upset over Case of Mexican National who Committed Voter Fraud
2/17/2017 - Senate Confirmation Hearings for Judge Neil Gorsuch for Supreme Court Set for March
2/17/2017 - New Labor Secretary Nominee is R. Alexander Acosta, a Law School President
2/16/2017 - Under Armour CEO Caves to Social Media Pressure, Walks Back Statement Supporting Trump
2/16/2017 - Bias Buzz Podcast: Leaks Force Flynn's Resignation, Liberal Outrage over Immigration Raids
2/16/2017 - 'Day Without Immigrants" Strike Protests Trump, Immigrant Workers Didn't Go to Work Today
2/16/2017 - CNN Reporter Jim Acosta Alleged "Fix is In" when Trump Called on Conservative Outlets for Questions
2/16/2017 - ABC News Analyst: Trump's Selection of Questions from Conservatives Shuts Down the First Amendment
2/16/2017 - Rebuffed by Trump, CNN Writes 21 Questions They'd Like to Ask Him
2/16/2017 - CBS News Blamed Penguin Depopulation on Climate Change
2/16/2017 - President Trump Won't Fill Out a College Basketball Bracket
2/16/2017 - Political Correctness Propagates Radical Liberalism and Undermines The Truth
2/16/2017 - The Bias Buzz Podcast- Why Did Mike Flynn Resign?, Immigration Enforcement Roils Illegal Aliens, Grammys, Town Halls and the Tea Party
2/15/2017 - Deborah Turness Out as NBC News President
2/15/2017 - Liberal Media Botched Story about Muslim American Olympian Detained at the Airport
2/15/2017 - CNN Misreported GOP Senators' Support of Trump Cabinet Nominee
2/15/2017 - New York Times Reporter Called Melania Trump a 'Hooker', but Michelle Obama didn't face Liberal Media Insults
2/15/2017 - Lost in Flynn's Resignation is the Pro-Iran Deal Obama Supporters' Push to Hide the Iran Deal Details
2/15/2017 - Dakota Access Pipeline Protesters Left Behind 250 Truckloads of Trash
2/14/2017 - New York Times Reprimands Reporter for Calling Melania Trump a "Hooker"
2/14/2017 - Joy Villa Cashes in on Trump Dress, Sets Liberal Media Ablaze
2/14/2017 - CNN Debuts Donald Trump Valentine's Day Card Generator
2/14/2017 - NBC News: Illegal Immigrants 'Risk Perilous Winter Trek' to get to America
2/14/2017 - CNN Highlighted Mexican Protester who said Trump is a Mean Man
2/14/2017 - New Liberal Media Narrative: Beware of Donald Trump's Alleged Mental Health Concerns
2/13/2017 - Unorthodox for Liberal Hollywood: Singer Wears 'Make America Great Again' Dress to Grammy's
2/13/2017 - Fmr. Sen. Jim Webb: Media, Academia Trying to Personally Discredit Donald Trump [Video]
2/13/2017 - Dear Liberal Media, Athletes have Skipped White House Meeting Before
2/13/2017 - Journalism? CNN Focused on Trump-Abe Handshake lasting 19 Seconds
2/13/2017 - The Irony: Women's March Twitter Praised Protesters for Blocking Education Secretary Betsy DeVos from Entering DC School
2/13/2017 - Protesters Stop New Education Secretary Betsy DeVos from Entering DC Public School
2/13/2017 - Virginia High School Students Walk Out of Class to Protest Trump's Executive Order
2/12/2017 - Is the NAACP Deciding Their Policy on Charter Schools?
2/12/2017 - Museum Shuts Down Actor's Anti-Trump Display after it created Safety Issues
2/11/2017 - Ethics Concerns Raised after Kellyanne Conway Suggested Viewers to Buy Ivanka Trump's Merchandise
2/11/2017 - Mocking Obama? Syria's Assad welcomes US Troops into Syria to Fight 'Terrorists'
2/10/2017 - 9th Circuit Court Ruled against Trump's Executive Order
2/10/2017 - LA Times: 1 Million Illegal Immigrants Live in LA, or Nearly 10% of Total Illegal Immigrant Population
2/10/2017 - Study finds 92% of Anti-Fascist Protesters in Germany Live with their Parents
2/10/2017 - Anti-Trump Protesters Block Road, Force EMTs to Do Procedure in Ambulance
2/10/2017 - Obamacare is Still Failing, Liberal Media
2/10/2017 - Dear Media: Australia's Refugees aren't America's Problem
2/10/2017 - The Bias Buzz Podcast- DeVos Confirmed, Immigration Policy, Political Super Bowl Commercials, Elizabeth Warren Rebuked [Video]
2/9/2017 - Dan Rather Launches "News and Guts" Facebook Page to Combat Fake News
2/9/2017 - ESPN's Sage Steele was Right to Call Out Airport Protesters
2/9/2017 - Trump Tells U.S. Military He has Their Back, Will Fight Radical Islam
2/8/2017 - MSNBC's Joe Scarborough Assails Fake News [Video]
2/8/2017 - John Stossel Looks Back at Liberal Media Bias at ABC's 20/20
2/8/2017 - The Berkeley Riot Expresses Dominant Anti-American Campus Culture
2/8/2017 - Liberal Media Headlines Claim Trump White House is in 'Chaos'
2/8/2017 - He's Right: Columnist said Journalists are 'Having a Nervous Breakdown' over Trump
2/8/2017 - National Geographic Twitter Goes PC, Talks Gender Pronouns
2/7/2017 - She's Baack! Hillary Clinton Says "The Future is Female" [Video]
2/7/2017 - Betsy DeVos, Despite Opposition from Democrats, Teachers Unions, Confirmed as New Education Secretary
2/7/2017 - Women's March Organizers Plan General Strike, 'A Day without a Woman'
2/7/2017 - Liberal Media Championed 97 Companies which Oppose Trump's Executive Order
2/7/2017 - The Anti-Trump Fake News Stories that the Liberal Media Loved to Believe
2/7/2017 - Liberal Media Highlighted Russia's Criticism of Fox News for Calling Putin a Killer
2/7/2017 - Washington Post Dives into Kellyanne Conway's 'Bowling Green' Comment
2/6/2017 - Super Bowl Ads Take Shots at Trump Policies
2/6/2017 - Liberal Media Fawns over Anti-Trump Federal Workers
2/6/2017 - CNN Celebrated GOP won't Repeal ObamaCare At Once, but Forgot How Onerous Obamacare is
2/6/2017 - USA Today: GOP, Trump are Hypocrites on Executive Action, but Not Obama
2/6/2017 - CNN: Democrats 'facing a bleak reality: they have virtually no power in Washington'
2/5/2017 - Tom Brokaw Praises Gorsuch, Warns Dems on Filibuster [Video]
2/5/2017 - CNN Claimed We're Living in a Trump 'Photo Op Presidency'
2/5/2017 - Study: Majority of Children in Immigrant Families are US-Born
2/4/2017 - Outrage over Trump White House's Holocaust Remembrance Day Statement
2/4/2017 - Liberal Media Outlet Laments Week of Hell under Trump
2/3/2017 - Fake News: Buzzfeed, Media Caught Publishing a Debunked Story of Iraqi Mom Dying due to Trump Refugee Vetting Order
2/3/2017 - Liberal Media Gets Two Stories Wrong about Trump SCOTUS Nominee Neil Gorsuch
2/3/2017 - Bias Buzz Podcast: Trump's SCOTUS Nominee Neil Gorsuch, Trump and Refugees, Boy Scouts Transgender Policy, March for Life & More
2/3/2017 - Liberal Media Outlet Criticized Trump's Top Advisers, Refers to their 'Dark' History
2/3/2017 - Liberal Media Fell in Love with the Word 'Dark' in Describing Trump, Trump's Inaugural Address
2/3/2017 - Trump's Heartland Voters Unfazed by Liberal Media Panic over Alleged 'Muslim Ban'
2/3/2017 - What We Saw at the March for Life: Liberal Media Ignored Millennial Attendees
2/3/2017 - WATCH: Videos of March for Life 2017
2/3/2017 - PHOTOS: Remembering Donald Trump's Inauguration Day
2/3/2017 - WATCH: On-the-Ground Footage of Trump's Inauguration Day
2/2/2017 - U.C. Berkeley Burns Free Speech
2/2/2017 - Why did the New York Times Omit 'Gosnell' from Best-Seller List?
2/2/2017 - Wow: Violent Protesters Shut Down Milo Yiannopoulos' Cal-Berkeley Event
2/2/2017 - NBC News Analysis Criticized Trump's Foreign Policy, Quoted Six Critics of Trump and Only One Trump Ally
2/2/2017 - NBC News Focused on Trump Nominee DeVos's Comment on Grizzly Bears, but Not Rebuttal of Bernie Sanders
2/2/2017 - CNN "Frozen Out" of Trump's White House
2/2/2017 - Forgot about Obama? Even LA Times Admits Obama's Legacy was Leading 'the Nation in War'
2/2/2017 - Washington Post Points Out Frustrated Democrats Cannot Hold Up Trump's Cabinet Nominees for Much Longer
2/2/2017 - The Bias Buzz Podcast- Gorsuch Nomination, Misguided Immigration Order Backlash, Media's Coverage of March for Life, Boy Scouts Transgender Policy and More
2/2/2017 - Late Night TV is Relevant? NBC News Heralds Jon Stewart Mocking Trump's Executive Orders
2/1/2017 - Fox News' January Ratings Soar on Strength of Inaugural Coverage, MacCallum, Carlson Shows
2/1/2017 - Bias Buzz Podcast: Trump's Refugee Order, Future of American Immigration Laws with Center for Immigration Studies' Mark Krikorian
2/1/2017 - Who is Judge Neil Gorsuch? SCOTUS Blog's Take
2/1/2017 - New York Times Ran 'President Bannon' Op-Ed, but Didn't Do That with Obama's Advisor Valerie Jarrett
2/1/2017 - Washington Post Claimed that Trump doesn't have a Mandate due to Losing Popular Vote
2/1/2017 - Underreported March for Life News: At least 600,000 Attended, 75% were Millennials
1/31/2017 - Donald Trump Announces SCOTUS Nominee: Judge Neil Gorsuch
1/31/2017 - The Bias Buzz Podcast Special Edition- Immigration Under the Trump Presidency with Mark Krikorian
1/31/2017 - Oprah Winfrey Joins "60 Minutes" as Special Contributor
1/31/2017 - Trump Fired Acting Attorney General over Insubordination on Refugee Vetting Executive Order
1/31/2017 - Obama Comments on Trump's Refugee Executive Order, Bucks Trend from George W. Bush
1/31/2017 - Washington Post Soccer Op-Ed Criticized Trump Refugee Executive Order, Can't Back It Up with Data
1/31/2017 - Trump to Announce Supreme Court Nominee at 8 PM Tuesday (Today)
1/31/2017 - What Glass Ceiling? Liberal Media Continues to Ignore Trump Counselor Kellyanne Conway's Success
1/30/2017 - WaPo Media Columnist: Media Needs More Facts, Fewer Pundits, to Regain Public Trust
1/30/2017 - Liberal Media Jumps on Trump's Refugee Vetting Executive Order
1/30/2017 - Separating Liberal Hysteria from Facts about Trump's Refugee Vetting Executive Order
1/30/2017 - Surprise! Liberal Media Referred to 'March for Life' by Name
1/30/2017 - Liberal Media Used 'Anti-Abortion' Phrase to Describe 'March for Life' by 19-2 Margin
1/30/2017 - CBS News Accidentally Makes Case for Building a Border Wall
1/30/2017 - Grasping for Straws? Boston City Hall Lit Up in American Flag Colors to Protest Trump's Immigration Proposals
1/30/2017 - NBC News Insinuated George W. Bush was Weaker than UK's Tony Blair Post-9/11
1/29/2017 - NBC News Fearful of GOP-Backed Bill to Preserve Religious Beliefs in Discrimination Cases
1/29/2017 - Liberal Media Jumps on Pentagon's Tweet about a Refugee-Turned-US Marine
1/28/2017 - CNN Photo Article Highlighted America's Current Border Wall
1/28/2017 - Virginia Legislature Takes Look at Combatting Anti-Semitism at Colleges
1/27/2017 - Morning Joe Congratulates Tucker Carlson's Success at Fox News
1/27/2017 - The Bias Buzz Podcast Special Edition- Gosnell: The Untold Story of America's Most Prolific Serial Killer with Phelim McAleer
1/27/2017 - That's the Best Headline for the March for Life, New York Times? 'Pence will speak at anti-abortion rally'
1/27/2017 - Deafening Silence: Businesses Ignore March for Life But Sponsor LGBT Movement
1/27/2017 - CNN: Donald Trump is doing exactly what he said he would do, but "Power Emboldens Him"
1/27/2017 - Washington Post Confused that Trump is Fulfilling Campaign Promises
1/27/2017 - Schism in the Democratic Party as Anti-Trump Element Resists Working with Trump
1/27/2017 - Liberal Media is Worried about Trump's Unsecured Phone Used for Twitter
1/27/2017 - LA Times Took 'Alternative Facts' Shot at Trump Counselor Kellyanne Conway in Article about Sales Spike of Orwell's '1984'
1/27/2017 - Austin, a 'Sanctuary City', could be Punished for Sanctuary Status
1/27/2017 - Delayed Pipelines under Obama get Renewed Life via Trump Executive Orders
1/26/2017 - Bias Buzz Podcast: Special Edition with Best-Selling Author Phelim McAleer
1/26/2017 - Drain the Swamp? Trump's Worker Hiring Freeze Disappoints Feds
1/26/2017 - Temporary Media Blackout for EPA, Other Departments
1/26/2017 - No, Liberal Media, Obama's White House Pages were Archived and NOT Deleted
1/26/2017 - United Nations Resolution on Israel, if Implemented, could Affect Schools
1/26/2017 - Obama Administration Sneaked $221 Million to the Palestinian Authority
1/26/2017 - The Bias Buzz Podcast- Inauguration Recap, Alternative Facts,Trump's CIA Visit,Women's March on D.C., Executive Orders and More
1/25/2017 - CNN's Tapper: "The Media Is in a Rough Place Right Now" After Blowing the Election
1/25/2017 - Bias Buzz Podcast: What Really Happened at the Women's March on Washington
1/25/2017 - Liberal Media Ignores Annual March for Life
1/25/2017 - Trump's Supreme Court Pick will be Announced Next Week
1/25/2017 - Trump Didn't Fire FBI Director James Comey After All
1/25/2017 - Abortion Activists Concerned about Trump Overturning Roe v. Wade via Supreme Court Nominees
1/25/2017 - Trump's Press Secretary Sean Spicer Gives First Three Questions to Conservative-Leaning Outlets
1/25/2017 - First Foreign Leader to Visit Trump? UK's Theresa May
1/25/2017 - The Bias Buzz Podcast Special Edition- Women's March on Washington, March for Life with Beverly Hallberg
1/25/2017 - Liberal Media Ate Up Story of National Parks Twitter Account Contradicting Trump Administration on Climate Change
1/25/2017 - Comedians Rally Around SNL Writer Suspended for Tweet about Barron Trump on Twitter
1/24/2017 - Time Magazine Posts Note to Readers Over Inaccurate Report on MLK Bust
1/24/2017 - Liberal Media Narratives Crash
1/24/2017 - CNN Does Behind-the-Scenes Video of Anti-Trump Women's March on Washington
1/24/2017 - Taken Out of Context, Madonna said about Her Blowing-Up-The-White-House Comment at Anti-Trump Women's March
1/24/2017 - Liberal Media Praised Anti-Trump Women's March on Washington, Felt Compelled to Point Out They Were Peaceful
1/24/2017 - Who Was Behind the Women's March? George Soros-Backed Groups
1/24/2017 - Liberal Media Slams Kellyanne Conway for 'Alternative Facts' Comment
1/23/2017 - Columbia Journalism Review Issues Open Letter to Trump on Press Relations
1/23/2017 - Fox News Cuts Ties With George Will, Stacey Dash, Adds Trump Supporter Nigel Farage
1/23/2017 - 7 Reasons, According to NBC News, Why the Democrats are Lost Without Obama
1/22/2017 - CNN Remembered Obama's Legacy through Hip-Hop Lyrics
1/22/2017 - Surprisingly, CBS News Didn't Cite Climate Change in Recent Crack in Antarctica Ice Shelf
1/21/2017 - CNN Highlighted So-Called Climate Change Consensus Narrative in Discussing Trump's EPA Pick
1/21/2017 - CNN Runs with Headline of How Trump Nominee Betsy DeVos 'Painted by Dems as Unqualified'
1/20/2017 - We Remember Ronald Reagan's 1981 Inaugural Address
1/20/2017 - Trump's Inauguration Estimated to Cost $200 Million, but Obama's Inauguration Cost $170 Million
1/20/2017 - Clarence Thomas to Swear-In Mike Pence, the First African-American to Administer Oath of Office
1/20/2017 - House Democrats, 60 of Them, Plan to Skip Trump's Inauguration
1/19/2017 - Fox News' Ratings Soar With Tucker Carlson, While Van Susteren Finishes Third at MSNBC
1/19/2017 - In Last Press Conference, Obama says Voter Fraud is 'Fake News'
1/19/2017 - Remember This? Democrats Paid Protesters to Disrupt Trump's Rallies
1/19/2017 - New York Times' Upshot Tries Hand at Obama Legacy Data and Omits Key Stats
1/19/2017 - Women's March on Washington Booted Pro-Life Activists from the March
1/19/2017 - Obama at Final Press Conference: Chelsea Manning Suffered Long Enough
1/19/2017 - The Bias Buzz Podcast- Chelsea Manning, Dems Boycott Inauguration, Obama Blames Fox News, Limbaugh for Failures and More
1/18/2017 - Trump Tweets to Fight Back Against "Dishonest Media"
1/18/2017 - Former DNC Chair Wasserman Schultz Won't Say If Trump is a "Legitimate" President [Video]
1/18/2017 - Protesters Show Up in Droves for Nixon and Bush, but Not For Obama
1/18/2017 - In Interview, Obama Blamed Gerrymandering for Democratic Party's Election Losses
1/18/2017 - Obama, in Dateline NBC Interview, says He Doesn't Personalize Politics
1/18/2017 - Lester Holt's Questions to Obama in Dateline NBC Interview were Softballs
1/18/2017 - Obama in Dateline NBC Interview Admitted Instant 'Hope and Change' was 'Unrealistic'
1/18/2017 - Not the First Time: John Lewis, Democratic Congressman, Did Skip George W. Bush's Inauguration
1/17/2017 - Hypocrisy? Obama Commuted Sentence of Intel Leaker Chelsea Manning
1/17/2017 - Obama Blames Fox News, Rush Limbaugh, for His Failures
1/17/2017 - Over Two-Thirds of Police Officers Surveyed Think Post-Incident Protests are Anti-Police
1/17/2017 - Dakota Access Pipeline Protests Will Re-Emerge after a Winter Break
1/17/2017 - NBC News Praised Obama's Legacy, Forgetting Key Economic Indicator
1/16/2017 - NBC News Highlighted 6 Gay Events 'Worth Traveling for in 2017'
1/16/2017 - NBC News: Obamacare Repeal can Affect Your Workday
1/15/2017 - Dan Rather: Clinton Campaign, Press Were "Tone Deaf" About Trump's Appeal
1/15/2017 - GOP-Democrats Keep Taking Down, Putting Up Painting of Cops as Animals
1/15/2017 - Obama Could Commute Chelsea Manning's 35-Year Sentence
1/14/2017 - ABC News Tours DNC's War Room during Trump's Cabinet Nominee Hearings
1/14/2017 - Liberal Media Confused? Donald Trump Compared Intel Leaks to Nazi Germany
1/13/2017 - Michael Moore: Press Must Stick Together Because Trump Will "Attack the Truth Tellers" [Video]
1/13/2017 - Lots of Speculation from CNN on How 2 Days Showed What'll Happen Under a Trump Presidency
1/13/2017 - Is Cory Booker a Hypocrite on Jeff Sessions? He Once Praised Him
1/13/2017 - Obama's 'Nostalgic Goodbye' Heralded by CNN
1/12/2017 - Bias Buzz Podcast: Obama's Farewell Speech was a Legacy Speech, Meryl Streep, Trump Cabinet and More!
1/12/2017 - The Bias Buzz Podcast- Obama's Legacy, Buzzfeed's Fake News on Trump, Meryl Streep and More
1/12/2017 - Trump: Buzzfeed a 'Failing Pile of Garbage', CNN 'is Terrible' and is 'Fake News'
1/12/2017 - NBC News: Trump wasn't Told about Unverified Report Published by Buzzfeed
1/12/2017 - CNN Issued Statement to Clarify Differences between Their Trump-Russia Report and Buzzfeed's


Am J Surg Pathol. 1990 Dec;14(12):1112-25.
Travis WD1, Colby TV, Lombard C, Carpenter HA.
Based on a clinicopathologic study of 34 patients with biopsy-confirmed diffuse pulmonary hemorrhage (DPH), we present an approach to the differential diagnosis of DPH with attention to histologic features such as capillaritis and the importance of laboratory tests such as anticytoplasmic autoantibodies (ACPA). The following DPH syndromes were encountered: antibasement membrane antibody (ABMA) disease (four cases); idiopathic pulmonary hemorrhage (four cases); Wegener's granulomatosis (WG) (five cases); probable WG (six cases); systemic necrotizing vasculitis otherwise unclassified (three cases); systemic lupus erythematosus (two cases); rheumatoid arthritis (one case); seronegative juvenile rheumatoid arthritis (one case); IgA nephropathy (one case); idiopathic glomerulonephritis (two cases--one with and one without immune complexes); and unclassified pulmonary-renal syndromes (five cases). Capillaritis was found in lung biopsy samples from 30 of the 34 patients (88%) and included patients with every type of DPH syndrome. Serologic testing for ACPA was useful in the diagnosis of WG. Identification of ABMA in the serum, kidney, or lung was the defining feature for the diagnosis of ABMA-mediated disease. Subclassification of the cases could not be done solely on histologic grounds except for cases of WG that showed granulomatous inflammation, foci of necrosis, or vasculitis. Classification of the remaining cases required correlation with (a) clinical and laboratory data; (b) biopsy samples from other sites such as the kidney, nasal sinuses, or skin; and (c) results of immunofluorescence or electron microscopy of kidney or lung biopsy samples.


Andrew Bogut is a popular man in the Golden State Warriors building.
He was a large part of the organisation's transformation from what was once a struggling franchise into an NBA giant, and his old, and potentially new teammate, Stephen Curry, addressed the big man's impending arrival on Wednesday (AEDT).
"It's so exciting, weird, blast from the past," Curry told reporters.
"Obviously, I know they've got a couple more steps before it's official, but he's a guy that we have a lot of experience with.
"He knows our system, he knows how we do things around here. He was instrumental in that turnaround to become a championship calibre team.
Curry said he was "for sure" shocked that Bogut was open to the idea of returning to the NBA.
"I figured once he went to Australia, that would probably be the last we saw of him in NBA circles," Curry said.
The Warriors' core from 2015 — Curry, Draymond Green, Klay Thompson, Andre Iguodala and Shaun Livingston — remain, but Bogut will come off the bench with the addition of All-Star big man DeMarcus Cousins and Durant.
"Who knows how many minutes he'll play or what the situations will be obviously with DeMarcus back," Curry said.


text:
Tag: Duke Blue Devils
November 11, 2020 Danny Thompson
Georgia Tech's Alvarado Voted Second Team All-ACC
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Zion Williamson-The Destination Arbitration
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March Madness- Let The Insanity Begin!
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Five Prospects The Philadelphia Eagles Should Draft
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Zion Williamson: The Man Child, The Myth, The Legend
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September 13, 2018 Georgia Tech
Georgia Tech's ACC Basketball Schedule Announced
THE FLATS – Georgia Tech will open its Atlantic Coast Conference schedule at home for the first time since the 2012-13 season and will have five of its first eight ACC games at home in...
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Teen Towers Series – Volume Two Wendell Carter Jr.
The NBA Draft brings new hope and anticipation for teams, especially the team's locked into a lottery pick. Its been some time since we've had a plethora of big men at the head of...
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By Danny Thompson
/Tags: Clemson Tigers, Duke Blue Devils, Florida State Seminoles, Georgia Tech Basketball, Jose Alvarado, Louisville Cardinals, Miami Hurricanes, Virginia Cavaliers
On Wednesday, the Atlantic Coast Conference's media members voted Georgia Tech Yellow Jackets' senior guard Jose Alvarado to it's preseason All-Conference second team. He was joined by David Johnson (Louisville), M.J. Walker (Florida State), Kihei Clark (Virginia) and Wendell Moore Jr. (Duke). The 6'0″ senior averaged 16.0 points per...
/Tags: Anthony Davis, Duke Blue Devils, NBA, New Orleans Pelicans, Zion Williamson
With the first pick in the 2019 NBA Draft, the New Orleans Pelicans already know exactly who they are selecting. Duke forward Zion Williamson going number one is as much of a sure thing as a Teenage Mutant Ninja Turtle asking for a slice of pizza. Well, him being...
/Tags: Auburn Tigers, Carson Edwards, Chuma Okeke, Duke Blue Devils, Final Four, Gonzaga Bulldogs, Kentucky Wildcats, March Madness, Michigan State Spartans, Purdue Boilermakers, Road To The Final Four, Texas Tech Red Raiders, Virginia Cavaliers
The Elite 8 produced an array of great games this past weekend. As good as the young talent has been this season, the upperclassmen owned the weekend. Experience won out in a big way, all the remaining four teams are littered with pivotal older players. Best Game of...
/Tags: Auburn Tigers, Duke Blue Devils, LSU Tigers, Michigan State Spartans, North Carolina Tar Heels, R.J. Barrett, Virginia Tech Hokies, Zion Williamson
East Region Preview The first game has the LSU Tigers taking on the Michigan State Spartans. This will be a fun game to watch because of the contrasting styles. The Tigers have the kind of offense that gives the Spartans defense problems and Michigan State wants to ugly the...
/Tags: Duke Blue Devils, Gonzaga Bulldogs, Kentucky Wildcats, March Madness, Michigan Wolverines, Murray State, North Carolina, Tennessee Volunteers, Virginia Cavaliers
The NCAA Men's Basketball Tournament is the single greatest postseason event and it's not close. There is no shortage of storylines to write or talk about. They're dedications to those who are no longer here to players overcoming long odds just to be here and of course, the upsets....
By TBelcher
/Tags: Arizona State Sun Devils, Byron Murphy, Duke Blue Devils, Joe Giles-Harris, Miami Hurricanes, N'Keal Harry, Philadelphia Eagles, Rock Ya-Sin, Temple Owls, Tyree St. Lous, Washington Huskies
Defending a championship in any sport is never easy. Especially in a city like Philadelphia, where fans expect players to give it there all even if it means crawling off the field to secure the victory. Injuries, lack of depth and inconsistent play have bitten this Philadelphia Eagles' team this...
/Tags: College Basketball, Duke Blue Devils, Karl Malone, LeBron James, sports, Zion Williamson
Unless you've been living under a rock, in a cave or high in a secluded mountain, you have heard the name Zion Williamson. In this day and age of social media, no other other basketball player has had this much fanfare or coverage since (gasp) LeBron James. It's borderline...
By Georgia Tech
/Tags: Clemson Tigers, College Basketball, Duke Blue Devils, Florida State Seminoles, Georgia Tech Basketball, Georgia Tech Yellow Jackets, Miami Hurricanes, NC State Wolfpack, NCAA Basketball, North Carolina Tar Heels
THE FLATS – Georgia Tech will open its Atlantic Coast Conference schedule at home for the first time since the 2012-13 season and will have five of its first eight ACC games at home in the 2018-19 season. The ACC announced the full conference basketball schedules for all 15 members Thursday morning....
/Tags: 2018 NBA Draft, Duke Blue Devils, Wendell Carter Jr.
The NBA Draft brings new hope and anticipation for teams, especially the team's locked into a lottery pick. Its been some time since we've had a plethora of big men at the head of the field. In the 2018 draft the top ten picks will be filled with players...
/Tags: Duke Blue Devils, Indiana Hoosiers, Jaren Jackson Jr., Marvin Bagley III, Michigan State Spartans, NBA, NBA Draft, Wendell Carter Jr.
The NBA Draft brings new hope and anticipation for teams, especially the team's locked into a lottery pick. Its been some time since we've had a plethora of big men at the head of the field. In the 2018 draft, the top ten picks will be filled with players...


The University of Glasgow has issued a tender for graphic design framework, which will feature at least five consultancies.
The university says the framework will cover both 'complex' and 'simple' graphics and branding projects across a range of touchpoints.
Two consultancies will be appointed to work on the 'complex' projects, which will include brand development work, while three will work on 'simple' projects, which the university says will include print and digital design work.
The framework will run for four years.
The deadline for receipt of tenders or requests to participate is 24 June. For more information visit ted.europa.eu.
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'Definitive' New Wave Of British Heavy Metal history gets February release date
By Paul Brannigan ( Classic Rock ) published 7 January 22
Denim And Leather promises to reveal how "a group of unlikely looking blokes from the provinces wearing spandex trousers changed heavy music forever"
(Image credit: Fin Costello/Redferns)
A new book chronicling the rise and fall of the New Wave Of British Heavy Metal is to be published by Constable in February.
Denim and Leather, written by Guardian/Spectator/The Quietus contributor Michael Hann, promises to tell 'the definitive story about the greatest days of British heavy rock'.
An oral history, the book features input from artists such as Jimmy Page (Led Zeppelin), Ritchie Blackmore (Deep Purple), Rob Halford (Judas Priest), Joe Elliott (Def Leppard), Cronos (Venom), Phil Mogg (UFO), Biff Byford (Saxon), Lars Ulrich (Metallica) and scores of less celebrated names from the grass roots British metal community.
The publishers say: "In Denim and Leather, these stars tell their own stories – their brilliant, funny tales of hubris and disaster, of ambition and success – and chart how, over a handful of years from the late 1970s to the early 1980s, a group of unlikely looking blokes from the provinces wearing spandex trousers changed heavy music forever."
(Image credit: Constable)
"Denim and Leather captures a moment in time and a genre of music that had a brief moment in the sun," says Def Leppard guitarist Phil Collen. "The book is an honest portrayal, directly from the horse's mouth. The interesting thing is that it was people trying to discover their own identities. The book sums it up by the people who experienced it."
Denim and Leather is available to pre-order now.
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