text:
- Technical Note
- Open Access
Rapid screening of innate immune gene expression in zebrafish using reverse transcription - multiplex ligation-dependent probe amplification
© Meijer et al; licensee BioMed Central Ltd. 2011
- Received: 15 March 2011
- Accepted: 15 June 2011
- Published: 15 June 2011
With the zebrafish increasingly being used in immunology and infectious disease research, there is a need for efficient molecular tools to evaluate immune gene expression in this model species. RT-MLPA (reverse transcription - multiplex ligation-dependent probe amplification) provides a sensitive and reproducible method, in which fluorescently labelled amplification products of unique lengths are produced for a defined set of target transcripts. The method employs oligonucleotide probes that anneal to adjacent sites on a target sequence and are then joined by a heat-stable ligase. Subsequently, multiplex PCR with universal primers gives rise to amplicons that can be analyzed with standard sequencing equipment and relative quantification software. Allowing the simultaneous quantification of around 40 selected markers in a one-tube assay, RT-MLPA is highly useful for high-throughput screening applications.
We employed a dual-colour RT-MLPA probe design for chemical synthesis of probe pairs for 34 genes involved in Toll-like receptor signalling, transcriptional activation of the immune response, cytokine and chemokine production, and antimicrobial defence. In addition, six probe pairs were included for reference genes unaffected by infections in zebrafish. First, we established assay conditions for adult zebrafish infected with different strains of Mycobacterium marinum causing acute and chronic disease. Addition of competitor oligonucleotides was required to achieve peak heights in a similar range for genes with different expression levels. For subsequent analysis of embryonic samples it was necessary to adjust the amounts of competitor oligonucleotides, as the expression levels of several genes differed to a large extent between adult and embryonic tissues. Assay conditions established for one-day-old Salmonella typhimurium-infected embryos could be transferred without further adjustment to five-day-old M. marinum-infected larvae. RT-MLPA results were compared with results of previous transcriptome analyses and with real-time PCR data, demonstrating a good correlation between all expression analysis methods.
The RT-MLPA assay developed in this study provides a rapid, cheap, and robust analysis tool for simultaneous quantification of a set of 34 innate immune response genes. With adjustment of conditions, the assay is suitable for infection studies in both adult and embryonic zebrafish. Application of RT-MLPA will facilitate high-throughput screening of immune responses in the zebrafish model.
- Zebrafish Embryo
- Probe Pair
- Adult Zebrafish
- Immune Response Gene
- Mycobacterium Marinum
The use of zebrafish models in immunological and infectious disease research has rapidly expanded over the recent years [1, 2]. Important advantages of zebrafish include its amenability to large-scale forward and reverse genetic screening and small molecule library screening [3, 4]. The recent development of a robotic system for bacterial infection of zebrafish embryos will facilitate antimicrobial drug screening in this model at high-throughput level [5]. The availability of robust molecular tools for the rapid evaluation of immune responses is a highly desired addition to zebrafish-based screening approaches.
Multiplex Ligation-dependent Probe Amplification (MLPA) is a high resolution method to establish the copy number of around 40 nucleic acid sequences in a one-tube reaction [6, 7]. The technique was initially developed to determine copy number variation in genomic DNA, but has also been adapted for quantification of mRNA transcript levels [8]. Since its introduction in 2002, MLPA has rapidly become accepted in genetic diagnostic laboratories, where it is used for the detection of disease-associated polymorphisms, deletions, duplications and rearrangements in genomic DNA [7]. Reasons for the rapid adoption of MLPA in diagnostics are the relative simplicity, low costs (ca. 10 euro per reaction), high-throughput capacity, sensitivity, accuracy and robustness of the technique. The use of reverse-transcriptase-MLPA (RT-MLPA) for mRNA expression profiling is less common but has proven useful for example in human immunology and cancer research [9, 10]. In contrast to approximately 250 MLPA kits available for DNA analysis, only three RT-MLPA kits (including a human inflammation probe set, a human apoptosis probe set, and a mouse inflammation probe set) are commercially available (MRC Holland, http://www.mlpa.com). While the application of existing RT-MLPA tests is fast and simple, the development of new tests for detection of other sets of mRNAs is a significant investment.
Probes used in MLPA usually range between 80 to 400 nucleotides in length. Accurate chemical synthesis of MLPA probes is possible up to a length of approximately 180 nucleotides, while M13 cloning (using SALSA vectors, MRC Holland, http://www.mlpa.com) is used to produce probes up to 400 nucleotides in length [6, 7, 11]. To bypass the laborious M13 cloning procedures, a dual-colour approach has been applied [12]. In this approach, two universal primer pairs are used that give similar amplification efficiencies but are labelled with different fluorophores, thereby increasing the maximum possible number of synthetic probe pairs in a single MLPA assay.
Following probe design, a second time-consuming step in development of an RT-MLPA assay is optimization of the assay conditions by addition of competitor oligonucleotides (probes without universal primer sequences). Competitor oligonucleotides need to be used to titrate down the signals of genes with higher expression levels than others, such that all amplicons will produce peak heights in a similar range [8]. Titration conditions may need to be adjusted separately for different sample types, for example for different tissues or for samples from experimental manipulations that lead to large differences in gene expression. Therefore, RT-MLPA is most useful for high-throughput applications that involve repetitive analysis of a targeted transcript set in similar biological samples.
Here we describe the development of a first RT-MLPA assay for application in zebrafish models. The probe set targets 34 genes with functions in the innate immune system and contains 6 probe pairs for reference genes that are not affected under infection conditions. We show that a dual-colour approach previously developed for MLPA [12] can also be adopted for RT-MLPA, allowing us to combine 41 synthetic probe pairs in a single assay. We tested the method using samples from Mycobacterium marinum and Salmonella typhimurium infection experiments and established assay conditions for analysis of both adult and embryonic tissues. RT-MLPA results correlated well with results from previous transcriptomics studies and with results from real-time PCR analysis.
MLPA probe and RT-primer design
MLPA probes and RT-primers were designed for target mRNAs of 34 immune response genes and three reference genes (Additional file 1). For each target a pair of probes was designed, binding adjacently to each other on the target sequence, with the resulting ligation site located maximally 7 nucleotides from an exon-exon boundary. In addition, a control probe pair, designed on an intron sequence of one of the targets (cxcr3.2), was included in the set to check for the interference of genomic DNA in the MLPA run. The probe oligos were designed following the guidelines described in the manual "Designing synthetic MLPA probes" (http://www.mlpa.com, MRC Holland, Amsterdam, The Netherlands). All probe pairs contain the same universal binding site for the reverse PCR primer on the right probe oligo (RPO). The left probe oligo (LPO) for approximately half of the immune response genes harbours a sequence corresponding with forward PCR primer 1 labelled with FAM, while the remaining LPOs harbour a different sequence corresponding with forward PCR primer 2 labelled with Texas Red (TR). Two different probe pairs were designed for each of the three reference genes, one for amplification with FAM-labelled primer 1 and one for amplification with TR-labelled primer 2, thus resulting in a total of six RT-MLPA reference sequences. The probe pairs were designed such that the fragments resulting from PCR amplification are spaced by a minimum of three nucleotides in a range from 91-178 base pairs. Additionally, competitor oligonucleotides were created, consisting of the left probe oligo hybridization sequence (LHS), but lacking the universal primer sequence. A synthetic control DNA template was generated for each target and used to test the probe pairs for efficiency in setting up the RT-MLPA assay and as a positive control during runs. RT-Primers, reverse complement to the strand corresponding to the probe sequence, were designed for all target mRNAs, and were positioned immediately adjacent to the actual MLPA probe with no more than 15 nucleotides between the last nucleotide of the primer and the first nucleotide of the probe sequence, and with a maximum overlap of 7 nucleotides. The RT primers all have a Tm in the range of 55-65 °C and a GC content between 35 and 60%.
RT-MLPA assay
RT-MLPA reactions were performed using SALSA® MLPA® Reagents kit EK1-RT (MRC-Holland, Amsterdam, The Netherlands) according to the manufacturer's instructions for RT-MLPA reactions (version 10, 18-09-2008). All reaction steps were performed in a thermocycler with heated lid (105°C) using 0.2 ml thin-walled PCR tubes. RPOs were treated with T4 polynucleotide kinase (New England Biolabs) before use to establish 5' phosphorylation. A mix of RT-primers and dNTPs was prepared containing 1 pmol/μl of the RT primer for each target transcript and 2.5 mM of dNTPs. The RT primer/dNTP mix (1 μl) was added to the RNA sample (100 ng total RNA in 2.5 μl) and SALSA RT buffer (1 μl) at 4°C. This mixture was subsequently heated for 1 min to 80°C and incubated for 5 min at 45°C. Subsequently, the temperature was cooled down to 37°C, 30 units of M-MLV Reverse Transcriptase in SALSA dilution buffer (1.5 μl) were added, and the RT reaction was performed for 15 min at 37°C. The RT enzyme was then inactivated by heating to 98°C for 2 min and the mixture was cooled down to 25°C. A probe mix containing all probe oligos was prepared containing 2 fmol/μl of each oligo and, if necessary, competitor oligos were added to the required ratio (Additional file 2). The probe mix (1.5 μl) and MLPA buffer (1.5 μl) were added to the RT reaction, and after heating for 1 min at 95°C, the mixture was incubated for 16 h at 60°C to let the LPO and RPO probes hybridize to their respective targets. Subsequently, the mixture was cooled down to 54°C, 32 μl of Ligase-65 mix was added, and ligation of the LPO and RPO probes was performed by incubating for 15 min at 54°C. The ligase enzyme was inactivated by heating to 98°C for 2 min and the mixture was cooled down to 4°C. Next, 10 μl of the ligation reaction was mixed with 4 μl of SALSA PCR buffer and 26 μl water. When the tubes were in the thermal cycler at 60°C, 10 μl of polymerase mix was added containing 2 μl of PCR primer mix (10 pmol/μl of each forward primer and 20 pmol/μl of the reverse primer), 2 μl SALSA Enzyme Dilution Buffer, 5.5 μl water, and 0.5 μl SALSA Polymerase. The PCR reaction was carried out with slight modifications to the manufacturer's protocol to provide higher selectivity of the PCR primers. To this end the amplification temperature was increased to 65°C, and the number of cycles was increased to 38, resulting in the following cycling conditions: 30 s at 95°C; 30 s at 65°C; 1 min at 72°C. PCR cycling was ended with 20 min incubation at 72°C, after which the reaction was cooled down to 4°C. After PCR, the resulting DNA fragments were treated for 10 min at 37°C with 5 μl of Polymerase I Large Klenow fragment (Promega Corp., Madison, WI, USA) diluted in a 2.5 mM dNTP mix to correct for strand overhang. Next, the fragments were column-purified with the ZR DNA Sequencing Clean-up Kit™ (Zymo Research Corporation, Orange, CA, USA), according to the manufacturer's instructions, to prevent for dye-blobs in capillary fragment analysis. After PCR purification, 0.7 μl of sample was mixed with 0.3 μl of GS500-LIZ size standard and fragment analysis was performed on a ABI 3100 sequencer in GeneScan mode. Data were analyzed using GeneMarker® software, version 1.7 (SoftGenetics, LLC. State College, PA, USA). Raw data were imported with the Analysis Type set on 'Fragment (Animal) Analysis' and the following default settings for the Data Process Options: Raw Data Analysis: auto range, smooth, peak saturation, baseline subtraction, pull-up correction, spike removal; Size call: Local Southern; Allele Call: auto range 60-600 with peak detection threshold settings at: intensity > 100, percentage >5, local region > 25%, max call intensity 30000; Stutter peak filter %: left 90, right 40; Plus-A filter. Data were scaled using the GS500-LIZ size standard and peaks of target transcripts were assigned using a template with the fragment sizes of the RT-MLPA amplification products. A table containing the heights of each assigned peak scored in arbitrary units was exported and further analysed in Microsoft Excel. For normalization of the data, the peak heights of the target transcripts were divided by the average peak heights of the reference genes included in the same dye group. Based on parallel runs of the synthetic DNA control template described above there was no need to correct for possible signal sloping. Fold change values were calculated by dividing the normalized peak values of a treatment sample by the normalized peak values of the control sample.
Real-time PCR
From 1 μg of total RNA cDNA was synthesized using the I-Script cDNA synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA). For real-time PCR assays SYBR Green Universal PCR Mastermix 2x (Applied Biosystems, Foster City, CA, USA) was used. PCR primers were designed with Primer Express Software version 1.7 (Applied Biosystems, Foster City, CA, USA). For 33 of the 40 RT-MLPA target transcripts, including all six reference sequences, real-time PCR primer sets were developed, covering the hybridization sequence of each RT-MLPA target (Additional file 1). Each real-time PCR reaction contained 12,5 μl 2X PCR Mastermix, forward and reverse primer in a final concentration of 300nM, 1 μl of cDNA template and the volume adjusted to 25 μl with ddH2O. The reactions were run on an ABI PRISM™ 7500 Sequence Detection System (Applied Biosystems). The solution was subjected to a protocol of subsequently 95 °C for 10 minutes, followed by 40 cycles of 15 s at 95 °C and 1 min. at 60 °C. All reactions were performed in duplicate. Relative quantification was performed according to Vandesompele et al. [13] using three reference genes (coding for tyrosine 3-monooxygenase activation protein (NM_201484), NADH dehydrogenase (AC024175), and 16S ribosomal RNA (CK739347) that were selected from the GeNorm Zebrafish reference kit (PrimerDesign Ltd, Southampton, UK) for normalization.
RNA samples
Total RNA samples from zebrafish adults infected with M. marinum E11 and Mma20 strains and PBS-injected control fish were identical to those used in a previous microarray study [14]. Three biological replicates of infected fish and controls were used for RT-MLPA analysis, similar as in the previous microarray study. Zebrafish embryos at 28 hours post fertilization (hpf) were injected into the caudal vein with approximately 200 colony forming units (CFU) of S. typhimurium infection or injected with PBS as a control. Total RNA was isolated from pools of 15-20 embryos per treatment group at 8 hours post infection (hpi) and subjected to microarray analysis as previously described [15]. Samples were analyzed from three independent S. typhimurium infection experiments. Zebrafish embryos at 48 hpf were injected into the yolk sac with approximately 2000 CFU of M. marinum E11 bacteria in PVP carrier solution or with carrier solution alone as a control [5]. Total RNA was isolated from pools of 15-20 embryos at 3 days post infection (dpi).
Development of an RT-MLPA probe set for the zebrafish innate immune response
Target genes of the zebrafish immune response RT-MLPA assay
Gene name1
GenBank Accession
Entrez Gene ID
UniGene ID2
Immune response genes
chemokine ccl-c24a (si:dkey-150o13.1, ccl20)
transcribed locus, weakly similar to NP_001108573.1 chemokine CCL-Cub
chemokine ccl-c5a (si:ch211-89f7.4)
chitinase, acidic.6
cxc chemokine 46
chemokine cxcl-c13d
chemokine cxcl-c1c (si:ch73-6k14.1)
chemokine cxcl-c5c
chemokine (C-X-C motif), receptor 3.2
chemokine (C-X-C motif), receptor 4b
FK506 binding protein 5
v-fos FBJ murine osteosarcoma viral oncogene homolog
interferon phi 1
interleukin 10
interleukin 1, beta
interleukin 8
v-jun sarcoma virus 17 oncogene homolog (c-Jun)
jun B proto-oncogene
matrix metalloproteinase 9
myeloid-specific peroxidase
myeloid differentiation primary response gene (88)
nuclear factor of kappa light polypeptide gene enhancer in B-cells 2, p49/p100
v-rel reticuloendotheliosis viral oncogene homolog
v-rel reticuloendotheliosis viral oncogene homolog A
sterile alpha and TIR motif containing 1
spleen focus forming virus (SFFV) proviral integration oncogene spi1 (pu.1)
toll-like receptor adaptor molecule 1 (ticam1, trif)
toll-interleukin 1 receptor (TIR) domain containing adaptor protein (tirap, mal)
toll-like receptor 1
toll-like receptor 4b, duplicate b
toll-like receptor 5a
toll-like receptor 5b
tumor necrosis factor a (TNF superfamily, member 2)
TNF receptor-associated factor 6
Reference genes
beta-actin 1
peptidylprolyl isomerase A, like (cyclophilin)
acidic ribosomal protein (ARP)
RT-MLPA analysis of Mycobacterium-infected zebrafish adults
RT-MLPA analysis of Salmonella- and Mycobacterium-infected zebrafish embryos
Next we performed RT-MLPA analyses on samples from infection studies in embryos. Initial tests showed that the assay conditions developed for samples from M. marinum-infected adult fish were not applicable to samples from embryos, because several genes showed strongly different basal or induced expression levels between adult and embryonic tissues. Therefore, titration levels of competitor oligonucleotides had to be adjusted for analysis of embryo samples. The titration was performed on samples from S. typhimurium-infected embryos at 8 hpi, a stage at which many of the genes included in the RT-MLPA probe set were known to be differentially expressed [15, 16, 19]. After establishment of appropriate assay conditions, S. typhimurium-induction of a broad set of immune response genes was detected, as well as repression of the cxcr3.2 and mpx genes (Figure 2B). A clear induction of several immune response genes was also observed in subsequent RT-MLPA analysis of a sample from five-day-old larvae that had been infected three days earlier by injection of M. marinum E11 bacteria into the yolk sac (Additional file 4). Therefore, the titration conditions set-up for one-day-old S. typhimurium-infected embryos were also suitable for analysis of M. marinum infection at a later developmental stage. However, it should be noted that the analysis of other developmental stages or other infection regimes, would still require a further assessment of the normalising function of the reference genes under those conditions.
Comparison of RT-MLPA and transcriptomics data
The same RNA samples as those used for RT-MLPA analysis (Figure 2) were also subjected to microarray analysis and the data were used for comparison with the RT-MLPA results (Additional file 3).
Infection of adult fish with the M. marinum E11 strain was previously shown to lead to chronic infection with no visible disease symptoms at 6 dpi [14]. Of all 34 immune genes included the RT-MLPA set, only one (cxcr4b) showed a significant induction (2.1-fold) in the previous microarray analysis [14]. The induction of this gene was also above the 2-fold change threshold in the RT-MLPA analysis (2.2-fold), which additionally detected minor inductions (2.0-2.5-fold) of 6 other genes.
Infection with the M. marinum Mma20 strain was previously shown to cause acute disease symptoms at 6 dpi accompanied with high induction levels of many immune response genes [14]. In total 22 genes from the RT-MLPA gene set showed a significant change of 2-fold or higher in the microarray analysis. Similarly, 23 genes were above the 2-fold change threshold in the RT-MLPA analysis, of which 18 overlapped with those detected by microarray. All inductions above 3-fold in the microarray analysis (17 genes) were also detected by RT-MPLA, and, vice versa, the majority of inductions above 3-fold in RT-MLPA were also significant in the microarray analysis (15 out of 18 genes). Only the inductions of sarm1 (3.1-fold) and myd88 (4.2-fold) in RT-MLPA were not significant in the microarray results, and the high induction of il8 in RT-MLPA (12.6-fold) could not be verified due to its absence on the microarray platform. Eight genes were induced above 5-fold in both microarray or RT-MLPA, with mmp9 showing the highest induction in both techniques (58-fold in microarray; 72-fold in RT-MLPA). Other genes showing above 5-fold induction with only one of the two methods (7 genes excluding il8, which is absent on the microarray) also showed clear induction in the other method (2.8-3.9-fold). The largest quantitative difference between the results of the two methods was observed for il1b, which was induced 26.6-fold in the microarray analysis and 2.8-fold in RT-MLPA.
Microarray and RT-MLPA data of S. typhimurium-infected embryos were also in good agreement, not only showing similar gene induction profiles but also consistent down-regulation (approx. 2-fold) of cxcr3.2 and mpx expression (Figure 2B, Additional file 3). In total 16 genes in the microarray analysis and 18 in RT-MLPA showed fold-change differences above 2-fold, of which 13 were overlapping between the two methods. With the exception of ccl20 (4-fold induction in microarray), all other genes (9) showing above 3-fold change in the microarray analysis were also picked up by RT-MLPA. Vice versa, with the exception of tlr5b (5-fold induction in RT-MLPA), all other genes (8) with above 3-fold change in RT-MLPA were also significant in the microarray analysis. Although the induction of tlr5b was below the 2-fold change threshold, it showed 1.7-fold induction in the microarray analysis supported by 9 probes with a significant P-value (<0.0001).
In conclusion, both for M. marinum infection of adult fish and for S. typhimurium infection of embryos the results of microarray and RT-MLPA were largely consistent in that differential expression of the majority of genes tested was confirmed by both methods. As both methods are to be considered semi-quantitative and rely on entirely different technology and data processing, it is not surprising that differences in the absolute induction levels were observed. A possible explanation for some discrepancies is that the microarray and RT-MLPA are measuring different transcripts due to alternative splicing.
Validation of RT-MLPA data by real-time PCR
In this work, we have developed an RT-MLPA analysis panel for relative quantification of innate immune gene expression in zebrafish. The method allows the rapid semi-quantitative expression screening of 34 genes involved in TLR signalling, transcriptional activation of the immune response, cytokine and chemokine production, and antimicrobial defence. Expression levels of these genes are determined relative to the expression of three reference genes in a single tube assay. The validity of the assay was demonstrated by comparison with transcriptomic data sets and real-time PCR analysis. Differential expression levels of the immune response genes detected with RT-MLPA covered a range between 2-fold and over 70-fold, similar to the results of microarray and real-time PCR analysis.
In development of the RT-MLPA assay we chose to employ a dual-colour labelling system previously used in DNA diagnostic MLPA applications [12]. In this system, two probe sets, each in the size range of approximately 90 to 180 oligonucleotides, are amplified using FAM-labelling and TR-labelling, respectively. In this size range it is possible to compose the RT-MLPA assay exclusively from synthetic probes, thus avoiding the laborious M13 cloning procedures used previously to extend the size range of probes for (RT-)MLPA analysis [6–8]. For future development it may be possible to further increase the number of testable mRNA targets by 50% with the use of a third label, which has also been reported in DNA diagnostic MLPA [20]. This would also facilitate further increasing the number of different reference genes in the assay.
The RT-MLPA assay was successfully applied to the analysis of RNA samples from infection experiments in adult and embryonic zebrafish. However, analysis of adult and embryonic material required different assay conditions. In RT-MLPA analysis it is necessary to compensate for large differences in expression levels of genes by the addition of competitor oligonucleotides to the probe mix [8]. We found that the basal and infection-induced expression levels of several genes differed to such an extent between adult and embryonic tissues that titration of the probe mix with competitor oligonucleotides had to be adjusted for these different developmental stages. Fortunately, assay conditions established for one-day-old S.typhimurium-infected embryos could be transferred without further adjustment to five-day-old M. marinum-infected larvae. Thus, by employing two types of titration conditions, we have been able to establish an RT-MLPA assay for infection studies in adult zebrafish and a second RT-MLPA assay that is applicable to infection studies in both embryos and larvae and with different pathogens.
Due to the fact that RT-MLPA is a relative quantification method, the composition of the probe mix cannot be easily altered without adjustment of the assay conditions. The replacement of one probe by another, or the addition of a probe for a new target transcript, will affect the peak heights of other transcripts. Therefore, especially if the new probe results in a large peak, this may require re-titrating the probe mix, as was also necessary when the sample-type was changed from adult to embryonic zebrafish. Since re-titration of the assay is a time-consuming step, RT-MLPA analysis is most suitable for recurrent analysis of the same set of target mRNAs under comparable experimental conditions. For such medium or high throughput screening applications, the innate immune response RT-MLPA assay developed in this study provides a rapid, cheap, and robust analysis tool.
Acknowledgements and Funding
We thank Monika Hidding (BaseClear B.V.) for technical assistance, Anna Zakrzewska (Leiden University), Oliver W. Stockhammer (Leiden University), Zakia Kanwal (Leiden University), and Ralph Carvalho (ZF-Screens B.V., Leiden, The Netherlands) for RNA samples, and the staff of MRC Holland B.V. (Amsterdam, The Netherlands) for valuable advice on synthetic RT-MLPA probe design. This work was supported by the European Commission Sixth Framework Project ZF-TOOLS (LSHG-CT-2006-037220).
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Sarah Kirk
Ph.D., 1998, University of Kansas
Dr. Kirk is a clinical faculty member and administrator. She directs all practicum activity, supervises, and facilitates clinical training opportunities. She also teaches graduate courses and provides professional development mentorship to students.
Research Interests
As director of the KU Psychological Clinic I am most interested in promoting clinical training in our graduate program. Specifically, I am focused on increasing our students' assessment, therapy, and supervision experiences, broadening their practicum opportunities, and improving their use of technology as applied to clinical interventions. Additionally, I would like to further promote evidence based treatments in our clinic.
Quality improvement and assurance is a principal goal for me, as well as developing measurements to promote the highest quality of care for our clients and training.
My research interests are in evaluating graduate training processes via outcome and competency based assessment. I am also interested in clinical outcomes, specifically functional outcomes for clients served in our clinic. I do not act as a primary research advisor for any incoming graduate students so any requests for research mentorship should go to other clinical faculty.
Clinical Interests
I have a specialization in health psychology within clinical psychology. I have often worked with persons of all age ranges with chronic health conditions, pain, and somatization conditions. I have a background in pediatric psychology and enjoy working with children who have health concerns that intersect with psychological symptoms.
I have a strong background working with persons of all ages with Autistic Spectrum Disorders. I also have worked a great deal with anxiety disorders and especially obsessive compulsive disorders and specific phobias using exposure and response prevention approaches.
I also do assessment of all types and especially enjoy cognitive, ADHD, and Learning Disability assessment.
Teaching Interests
I teach clinical psychology Assessment (cognitive and diagnostic interviewing). Supervision and Consultation courses (theory, research and field practicum) and History and Ethics. I am also an active supervisor for practicum students and all of our clinic groups.

subset: commoncrawl


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The 12th Annual EcoFest: Saturday, April 18th, 12-5 in Druid Hill Park:
EcoFest is a celebration of our earth and our community! It is a day filled with educational and fun activities for the whole family. Take a bike ride around the reservoir, do a craft project, or shop with local artists, crafters, and other vendors. Enjoy food and entertainment while learning how to lead a more eco-friendly and healthier lifestyle. Get a free tree to plant in your yard and find out what area organizations are doing to make Baltimore better and more sustainable and how you can get involved! Click here for the full, downloadable 2015 Baltimore Green Week Program Guide for all events April 18th-25
Live on the ECOFEST Park Stage
Belly Dance Performance from Mariza Dance Troupe – 12:00
Penny Black – 12:30
The Epic Steve Weber Band – 1:10
Jahiti – 2:20
J. Pope and Funk Friday – 3:00
Deaf Scene – 4:00
Other ECOFEST Activities
Children in Nature Area – Crafts & Games 12 – 5 pm
Whole Foods Green Product Exchange 12 – ? pm
Graffiti Exhibit – 12 – 5 pm
Rides Around the Reservoir 1 – 5 pm
Trees Around the World Tour – 1 pm
Yoga with Jill Archer – 1:35 pm
Gardening with Succulents Workshop – 3pm
Children's Yoga – 3pm
Sign up and volunteer to help make this day happen! There are lots of fun ways to get involved from leading a scavenger hunt, helping with art and craft projects, or helping to plant and maintain trees. Click here to register. THANKS TO AN OVERWHELMING RESPONSE FROM OUR COMMUNITY, ALL OUR ECOFEST VOLUNTEER POSITIONS HAVE BEEN FILLED!
Interested in becoming a vendor or exhibitor? The application deadline has been extended to April 1st. Click here to download the vendor application.
Please note, a 3% convenience fee (sales tax) will be added to all online transactions. For the transaction to be considered complete, all organizations must send in a copy of the transaction receipt with the completed vendor application found in the vendor application (link found above.) Agreements should be mailed to: PO Box 66088, Baltimore, MD 21239
Thank you to our 2014 Baltimore Green Week Sponsors:
Whole Foods Market Baltimore • Baltimore City Department of Recreation and Parks • Waste Neutral Group • Comcast • Honest Tea • 89.7 WTMD


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Lorde is a girl that you will most likely be hearing more about in the near future. Who is she? She is a very talented sixteen-year-old singer from New Zealand. She was signed by Universal Music at the age of 12 in New Zealand. She has since been developing as an artist and her music comes from a very real place and point-of-view. She is the very opposite of what many top-40 singers sing about. She even said she thinks that todays music isn't relatable to people. The average people of the world don't usually get to partake in the fame and excess that many artists such as Kanye West and Jay-Z sing and rap about.
Last night by Twitter was buzzing with the music video from Lorde's "Royals" song. Lorde doesn't really hold back on her beliefs of todays top-40 songs as she sings about how we're never gonna be living that life or be royal like. What is so funny about this, is this song and her future release are probably going to push her into being able to have that life that she sings against. Hopefully Lorde is able to stay grounded and keep it real as her career and fame grows.
You can take a listen to "Royals" below. I like the original music video better, but I decided to post the US version because its on Vevo. Vevo is now influencing Billboard, so the more views she gets on this video will influence her airplay here in North America. The song right now is at #49 on the Billboard Hot 100, #2 on the Billboard US Alternative chart, and #5 on the Billboard Rock Chart.
Tell me in the comment section what you think about "Royals" and Lorde.


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New Year's Fireworks at Riva
This event occurred in the past.
Map | Find Parking
Dine with the best view of the Navy Pier fireworks. Dining room will be open from 5 p.m. to 12 a.m. and the lower level will feature a dance party from 9 p.m. to 1 a.m. The dance party is free to dinner guests or $15 at the door. Must be 21 and older. Reservations suggested.


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This Coconut Panna Cotta with Papaya Gelee recipe makes an easy dessert but will require at least 4 hours in the refrigerator.
Makes 6-8 servings.
•1 14 oz can coconut cream
•1/4 cup water
•2 packages (1/4 ounce each) unflavored gelatin
•1 15 oz can Cream of Coconut (Coco Lopez)
•1 red papaya
•1 cup water, divided
•1-3 tablespoons agave nectar, simple syrup, or sugar (to taste)
•sugar palm fruit (kaong), for garnish (optional)
Coconut Panna Cotta:
In a saucepan, combine 1 cup of coconut cream with 1/4 cup of water. Sprinkle 1 package gelatin over the coconut cream and let it sit for 5 minutes. Warm the saucepan over low heat and stir, until the gelatin dissolves completely.
Add the remaining coconut cream and cream of coconut, and cook over medium heat until steam arises, stirring periodically. Turn off the heat, cover the pan, and allow to sit for about 20 minutes.
Pour the mixture into your ramekins or serving cups and chill in the refrigerator for at least 4 hours.
Papaya Gelee:
Slice the papaya in half and remove the seeds. Dice about 1 cup of papaya and blend thoroughly in a blender. Pass the pulp through a sieve. Set aside.
Sprinkle 1 package gelatin over 1/2 cup cold water, let stand 1 minute. Take other 1/2 cup of water and bring it to a boil, add to gelatin mixture and stir thoroughly, until the gelatin dissolves, about 5 minutes. Stir in the papaya puree. Sweeten to taste, depending on the ripeness and flavor of the papaya with sugar or simple syrup.
When the coconut panna cotta layer is firm, add a layer of papaya gelee to each ramekin/serving cup. Return to refrigerator and chill for another 4 hours.
Garnish before serving with fresh papaya and sugar palm fruit (kaong)*, if desired. Serve within 24 hours.


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- Commentary
- Open Access
Resolving RAD51C function in late stages of homologous recombination
- Shyam K Sharan1Email author and
- Sergey G Kuznetsov1
© Sharan and Kuznetsov; licensee BioMed Central Ltd. 2007
- Received: 16 May 2007
- Accepted: 04 June 2007
- Published: 04 June 2007
DNA double strand breaks are efficiently repaired by homologous recombination. One of the last steps of this process is resolution of Holliday junctions that are formed at the sites of genetic exchange between homologous DNA. Although various resolvases with Holliday junctions processing activity have been identified in bacteriophages, bacteria and archaebacteria, eukaryotic resolvases have been elusive. Recent biochemical evidence has revealed that RAD51C and XRCC3, members of the RAD51-like protein family, are involved in Holliday junction resolution in mammalian cells. However, purified recombinant RAD51C and XRCC3 proteins have not shown any Holliday junction resolution activity. In addition, these proteins did not reveal the presence of a nuclease domain, which raises doubts about their ability to function as a resolvase. Furthermore, oocytes from infertile Rad51C mutant mice exhibit precocious separation of sister chromatids at metaphase II, a phenotype that reflects a defect in sister chromatid cohesion, not a lack of Holliday junction resolution. Here we discuss a model to explain how a Holliday junction resolution defect can lead to sister chromatid separation in mouse oocytes. We also describe other recent in vitro and in vivo evidence supporting a late role for RAD51C in homologous recombination in mammalian cells, which is likely to be resolution of the Holliday junction.
- Homologous Recombination
- Sister Chromatid
- Meiotic Recombination
- Sister Chromatid Cohesion
- Dicentric Chromosome
Eukaryotic resolvases
Although many resolvases have been identified in prokaryotes, few have been found in eukaryotes. The yeast CCE1 is encoded by a nuclear gene but functions as a resolvase in the mitochondria [9, 17]. Identification of the Mus81-Eme1 endonuclease in Saccharomyces pombe raised hopes that the eukaryotic resolvase has been identified [18]. It has a substrate preference for nicked HJs and displacement loops (D-loops) [19]. Genetic studies showed that S. pombe mus81 mutants are infertile due to failed meiotic recombination, which could be rescued by the expression of bacterial RusA resolvase [18]. These studies demonstrated that Mus81 is indeed a eukaryotic resolvase. The homologs of fission yeast Mus81-Eme1 have been identified in other organisms, including Saccharomyces cereviciae (Mus81-Mms4), Arabidopsis (AtMUS81/At4g30870) and humans (Mus81-Eme1 or Mms4) [20–26]. However, unlike the S. pombe mutants, S. cereviciae mus81 mutants are partially fertile, and MUS81-deficient Arabidopsis plants and mice are fully fertile, with no defect in meiotic recombination [21–23, 27, 28]. These observations suggest the presence of other resolvases in these organisms. Also, it was shown that the HJ resolution activity in mammalian cells could be separated from MUS81, suggesting that MUS81 was not the mammalian HJ resolvase [29].
RAD51 paralogs
The idea that RAD51 paralogs may be involved in resolution of HJs came from the observation that the protein fraction with HJ resolution activity contained RAD51C and XRCC3, members of the RAD51-like protein family [30]. This observation was very exciting considering that members of this family were already known to play a role during the early stages of homologous recombination. In higher eukaryotes, including plants, chicken and mammals, there are six members of the RAD51-like protein family that show 20–30% sequence similarity to RAD51 [31, 32]. These are RAD51B/RAD51L1, RAD51C/RAD51L2, RAD51D/RAD51L3, XRCC2, XRCC3 and DMC1. DMC1 shares about 50% sequence identity with RAD51and is its structural and functional homolog that functions specifically in meiotic recombination [33]. The other five paralogs show 20–30% sequence similarity to RAD51 and have been shown to be part of at least two distinct protein complexes, namely, the BCDX2 and CX3 complexes [34]. The BCDX2 complex comprises RAD51B, RAD51C, RAD51D and XRCC2 proteins; the CX3 complex consists of RAD51C and XRCC3. All these RAD51 paralogs have been reported to be required for normal proliferation and play a role in RAD51-mediated homologous recombination [31].
Studies in chicken B-lymphocyte DT40 cells have shown that only RAD51 is essential for cell viability, while loss of other RAD51 paralogs does not affect cell survival [35]. Interestingly, although mutants lacking any of the RAD51 paralogs show sensitivity to DNA-damaging agents, their phenotypes are not identical, suggesting that their function is similar but not redundant [36]. This functional non-redundancy is corroborated by the loss-of-function studies in mice and Arabidopsis [37–40]. Depletion of RAD51C by siRNA in human cells also suggests that RAD51C plays a role in homologous recombinational repair [41]. These findings are consistent with the idea that these paralogs play a role in early stages of homologous recombination. Recent work from Stephen West's laboratory has suggested that RAD51C and XRCC3 may also play a role in the late stages of homologous recombination [30, 42]. Here we discuss various findings that support the dual role of RAD51C.
The role of RAD51C and XRCC3 in HJ resolution
In a very significant study, Liu et al. demonstrated that RAD51C is required for the resolution of HJs in mammalian cells [30]. After an intricate series of HeLa cell nuclear extract fractionations, fractions with HJ resolution and branch migration activity were identified. These fractions lacked some of the possible candidates like Mus81, Flap endonuclease 1 (FEN1), RecQ DNA helicase (BLM) and Werner syndrome helicase (WRN). On the other hand, all the fractions with HJ resolution activity contained RAD51C. Immunodepletion using a RAD51C antibody resulted in the loss of the HJ processing function. This activity could be restored by adding purified RAD51 paralog protein complexes containing RAD51C, but not those lacking it. These studies directly implicated RAD51C in the resolution process. Furthermore, the Chinese hamster ovary cell line irs3 defective in RAD51C was shown to lack the HJ processing function. Recently, mouse embryonic fibroblasts generated from Rad51c knockout embryos revealed marked reduction in HJ activity [43]. Interestingly, a hamster cell line with mutant XRCC3, irs1SF, also showed a defect in HJ resolution activity, while mutation in another RAD51 paralog, XRCC2, had no effect on HJ resolution [30]. Although Liu et al. provided strong evidence to show the involvement of both RAD51C as well as XRCC3 in the resolution of HJ, a direct cleavage of the HJ structure by purified recombinant proteins remains to be demonstrated. It is possible that these proteins undergo posttranslational modifications, which may be essential for the HJ processing function. This theory is supported by the observation that the mobility of the RAD51C present in the fractions of HeLa cell extract that possess HJ resolution function is different from the mobility of those present in unfractionated whole cell extracts. Identifying the nature of this modification may elucidate the structural or conformational change in RAD51C that may be essential to its HJ resolution activity.
More recently, in a follow-up study, Liu et al. provide new biochemical evidence to link RAD51C and XRCC3 to the HJ processing function [42]. Using affinity chromatography, they showed a direct link between RAD51C and HJ activity. In a nickel column loaded with recombinant His-tagged RAD51C protein, HJ activity present in HeLa cell extracts was shown to bind to the column, which could subsequently be eluted. XRCC3 present in the extract was also shown to bind to the column and elute with HJ activity. Using gel filtration, the HJ resolvase activity was found to elute with an average molecular mass of 80–90 kDa, similar to the sum of the molecular masses of RAD51C (~42 kDa) and XRCC3 (~38 kDa). This finding suggests that the RAD51C-XRCC3 heterodimer should have the HJ activity. Because efforts to demonstrate this activity using purified recombinant proteins have failed, it has been speculated that the resolvosome complex may contain an additional component, such as a small nuclease, which may be essential for the HJ resolution function of the RAD51C-XRCC3 dimer. This suggestion may also explain the absence of any apparent nuclease domain in RAD51C or XRCC3. Future studies aimed at determining whether the HJ resolvosome complex involves other component(s) in addition to RAD51C and XRCC3 are important.
Localization of RAD51C on meiotic chromosomes
In addition to the biochemical studies, Liu et al. have presented interesting immunofluorescence data showing the localization of RAD51C on meiotic chromosomes, which further supports its role in late stages of homologous recombination [42]. Since various stages of meiotic prophase I coincide well with different stages of double-strand-break (DSB) repair by homologous recombination, it is possible to associate the function of a protein based on its localization on meiotic chromosomes at any given prophase I stage. Interestingly, RAD51C was first detected at the pachytene stage, when it was observed as one or two distinct foci associated with each synapsed bivalent. This pattern of expression is similar to the foci formed by the mismatch repair protein MLH1 [44]. These foci are believed to represent the sites of crossovers, formed as a result of recombination between homologous chromosomes. In addition, spermatocytes from Mlh1-deficient mice, which have a severely reduced number of crossover sites, also showed a marked reduction in RAD51C foci. This observation strengthens the notion that RAD51C foci formation during pachytene depends on the generation of crossovers, which is one of the last steps of homologous recombination. Surprisingly, co-localization of MLH1 and RAD51C foci on the bivalents was not observed. Also, XRCC3 foci were not detected on these bivalents. These observations raise concerns and are currently difficult to explain. The lack of co-localization of RAD51C and MLH1 may be due to temporal differences in their localization at crossover sites. It is interesting that, although no XRCC3 foci were observed on autosomal bivalents, XRCC3 foci, along with RAD51C and MLH1 foci, were present in the pseudoautosomal region of the sex chromosomes, a region on X and Y chromosomes that undergoes an obligatory crossover event. These foci in the pseudoautosomal region were not detected in Mlh1-deficient spermatocytes, supporting their dependence on a crossover event.
It was surprising that Liu et al. did not detect RAD51C foci during the leptotene and zygotene stages of prophase I, where it is expected to play an important role based on its known function in the early stages of homologous recombination. However, failure to observe RAD51C foci does not rule out its functional importance at this stage. Different antibodies and more sensitive imaging methods may help resolve this discrepancy. In the meantime, it may be interesting to examine the consequence of the loss of these proteins in an in vivo model system. In Drosophila, the loss of RAD51C-like protein encoded by spn-D gene results in a meiosis-specific defect and may play a role similar to DMC1 [45]. Recent studies in Arabidopsis revealed that RAD51C is essential for repair of Spo11-induced DSBs during prophase I of meiosis [40, 46]. Homologous chromosomes in Rad51c-deficient plant meiocytes fail to synapse and become severely fragmented. These results support a role for RAD51C early in meiotic recombination but do not shed light on its role later in the process.
RAD51C function in meiosis
Because RAD51C is predicted to have a dual function, an early and a late role in homologous recombination, it may be difficult to demonstrate the latter function in vivo, as a defect in the former may result in the arrest of meiocytes. Because RAD51C-deficient mice die during embryogenesis and no suitable meiosis-specific Cre transgenic mouse line is available yet, it is a challenge to generate a suitable mouse model to study the meiotic functions of RAD51C. We recently reported the generation of a hypomorphic allele of Rad51c that results in a reduction of the protein level due to aberrant splicing [43]. This aberrant splicing is caused by the presence of the neomycin resistance gene in one of the introns. Mice that are homozygous for the hypomorphic allele and have about 60% reduction in RAD51C protein level are viable and fertile. However, mice with only one copy of the hypomorphic allele (while the other allele is a null) are viable, but 35% of males and 12% of females are infertile. The infertile males and females provide an ideal in vivo model system to study the role of RAD51C in meiotic recombination. In addition, the meiotic phenotype associated with the loss of RAD51C function is sexually dimorphic, showing an early meiotic defect in males and a late defect in females, which provides a unique opportunity to study the dual function of RAD51C in mouse meiosis.
Infertile males revealed an early role for RAD51C in meiosis, marked by the spermatocyte arrest at leptotene and early zygotene stages, reduction of RAD51 foci at leptotene, and persistence of DNA breaks and unsynapsed chromosomes at pachytene. This finding is consistent with the known function of RAD51C in DSB repair but does not provide any evidence to implicate RAD51C in the late steps of the recombination.
How does an HJ resolution defect result in sister chromatid separation?
The broken chromosomes and aneuploidy observed in Rad51c mutant oocytes are consistent with RAD51C playing a role in HJ resolution. One can expect chromosomes to break if the bivalents do not separate in the absence of HJ resolution. Similarly, it is possible that bivalents can go to the same pole if they remain attached. However, in the Rad51c mutant oocytes, these phenotypes were not frequently observed. The most predominant phenotype displayed by the majority of chromosomes was PSSC, a typical characteristic of a defect in sister chromatid cohesion [49]. So, how can an HJ resolution defect result in this phenotype? Coincidently, a Chinese hamster ovary cell line lacking RAD51C was also reported to exhibit a PSSC defect [50]. Together, these findings raised the question of whether RAD51C may indeed be involved in sister chromatid cohesion. Investigation into a possible role for RAD51C as a cohesin by testing its physical interaction with known cohesins, examining the effect of the loss of RAD51C on the localization of other cohesins on meiotic chromosomes, and electron-microscopic analysis of synapsed chromosomes in mutant spermatocytes yielded no supporting data (Kuznetsov and Sharan, unpublished observation).
Although these possibilities remain to be tested, a genetic evidence reported earlier was used to support this hypothesis [43]. Koehler et al. have shown that, in mouse oocytes, segregation of dicentric chromosomes very frequently (>90%) results in PSSC [58]. They proposed that the physical strain exerted on the homologous centromeres of the dicentric chromatid by the poleward microtubules can result in the PSSC cohesion. Kuznetsov et al. have suggested that unresolved chromosomes and dicentric chromosomes are likely to experience a similar mechanical stress at the centromere during anaphase I and therefore have a similar fate [43]. The processing of dicentric chromosomes is sexually dimorphic and so far has been reported only in mouse and human oocytes. In other organisms, such as maize and flies, such dicentric chromosomes are known to undergo a "bridge-fusion-breakage" cycle [59, 60]. Why dicentric chromosomes have a different fate in mice and human oocytes is currently not understood. Interestingly, some of the spermatocytes from Rad51c mutant infertile males that progressed to metaphase II exhibited chromosomes with broken centromeres, but none showed any sister chromatid cohesion defect, which is consistent with the sexually dimorphic behavior of dicentric chromosomes.
In general, resolution of recombination intermediates in meiosis appears to be tightly linked to sister chromatid cohesion. The condensin-dependent removal of cohesin from the chromosome arms is required for efficient homolog separation in meiosis [61]. At the same time, the condensin – polo-like kinase axis is dispensable for cohesin removal in mitosis [62]. It is not clear how RAD51C might be involved in this particular process and whether it could explain the PSSC phenotype of the RAD51C-deficient mouse oocytes. However, it is intriguing that cohesins and RAD51C are now associated with the resolution of recombination intermediates after previously being independently implicated in the homologous recombination process [63].
Three years since the initial report showing a role for RAD51C in HJ resolution in mammalian cells, are we any closer to resolving the biological function of RAD51C? In spite of all the biochemical experiments and examination of loss-of-function mutations in Drosophila, Arabidopsis, and mice, the late function in HJ resolution remains to be unequivocally demonstrated. The model to explain the phenotype of Rad51c mutant oocytes at metaphase II is intriguing but needs to be validated. It will be fascinating to directly observe the oocytes undergoing in vitro maturation by time lapse imaging to visualize the bivalents being pulled to opposite poles but remaining attached at the site of the crossover by chiasmata-like structures during anaphase I. Also, it will be interesting to examine the fate of shugoshin and cohesins on the centromeres that have undergone precocious separation. An alternative approach may be to bypass the early meiotic arrest during male meiosis by using a conditional Rad51c allele and generating appropriate meiosis-specific Cre transgenic lines. This approach may provide a more convincing phenotype and help explain the late role of RAD51C in homologous recombination. Similar studies on XRCC3 in meiotic recombination may also provide valuable clues.
The authors thank Drs. Stephen West and Yilun Liu for critical review of the manuscript and Allen Kane of the Publication Department for the illustrations.
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Nexus 5 with KitKat ArrivesThe Nexus 5 is here with Android KitKat is here and packing a fair weight on the feature front. With the Nexus 5 Google brings us another great phone, checkout the comparison with the previous iteration. On the back of Android 4.3, Android KitKat was a surprise announcement as well...Read More »
Samsung Galaxy GearSamsung may be attempting to take steal some of Apples September Thunder with a confirmed unveiling of the Samsung Galaxy Gear smart watch on September 4th. The rumours continue to fly on features and specs. Feature wise we are looking at peripheral smart watch device as with Google Glass it will...Read More »
Arrival of the Moto XWe've had months of delays but finally the Moto X has arrived. Google purchased the failing Motorola Mobility last year which has struggled to deliver a smartphone to compete with those from Apple and Samsung. It's been a hard year of restructuring to achieve a new flagship product. There have...Read More »
PROTAG: The Credit Card Sized Location TrackerThe PROTAG from Innova Technology is a BlueTooth based location tracker which can be paired to a mobile to allow you to track belongings when on the move. It is roughly the size of a credit card (3mm thick) and is thin enough to fit inside a wallet. PROTAG acts as...Read More »
GamePop Mini from BlueStacksIn May BlueStacks made a surprise announcement they would be releasing GamePop, a subscription based games mini console based on Android to compete with Ouya and a future Google offering. BlueStacks have just added to that announcing GamePop Mini. Over it's bigger brother at $129 the Mini will be free to...Read More »
OUYA Hands OnI'll start by saying I'm not a huge gaming fan. My gaming exploits have been limited to mobile in recent years and I've never owned a major console from Sony or Microsoft. I have tried these consoles and have a good understanding of recent state of the art. What brought...Read More »
The Missing Sense of SmellOur smart devices today are becoming increasingly immersive. Sure we can see and hear things. Google Glass and the likes promise a new tech immersion, but what about smell? What if you could smell you friends, a new recipe, a game or a movie. The ChatPerf from Scentee is a prototype...Read More »
Focus: Overclock Your Brain!Foc.us continues the recent trend of sticking electronics on your head along with Melon and Google Glass. The company recently announced they will be shipping their tDCS gaming headset towards the end of July 2013. You may have read reports in the media over the past few months of studies...Read More »
Can't Concentrate - Try Wearing A Melon on Your HeadBiofeedback is gaining a lot of momentum recently with the Jawbone Up and Fitbit flex activity monitors now reaching market. The next frontier is the mind and a couple of companies are preparing mainstream devices for release this year. The Zeo Sleep manager has been around for a couple of...Read More »
Nvidia Shield Pre-Orders Now AvailablePre Orders have just been announced for Nvidia's Tegra 4 Android powered handheld gaming system Project Shield. Beginning May 20th, 2013 you can pre-order SHIELD direct from Nvidia for gamers following SHIELD via Notify Me list. It will be available for $350 to those in the United States and Canada...Read More »
What We're Doing With Google GlassIt's been a couple of weeks since developers started receiving their Google Glass Explorer Editions, so what have they been doing with it? And what do we think? We've compiled a series of videos below. Sadly none of the developers have been brave enough for some Sky Diving. Since we...Read More »
Older Entries »


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This hour long strong, moving performance is a sketch for a LIFT festival show that will take place in Tottenham in 2020.
I took part collaborating and making songs and voices through the show… and… In only 5 days – our group of amazing women artists from Tottenham who have just met each other for the first time – prepared a show about the dreams and the realities of marginalised women and their struggle.
It was an intense few days with so much artistic creativity and talent, falling in love with all those amazing women fellow artists I have met there, their ideas, and everything they have thought and said. Can't wait for 2020.


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|Emba59-students -- For students of EMBA cohort only
|About Emba59-students
EMBA Cohort 59 wants a mailing list with the students only so that we can stay in touch after graduation and to send messages to classmates only, without the entire teaching staff having to be included.
To see the collection of prior postings to the list, visit the Emba59-students Archives. (The current archive is only available to the list members.)
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version 2.1.24


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Sorry guys it took me exactly 1 week or more to go back to my computer, I had a job. For a week, cleaning stuff. 500 dollars in a week isn't half bad. Was not bad and had some trippy moments with the neighborhood trippy guy that always hang around with me whenever I clean the washrooms.
I'm going to quote one of unforgettable words from him.
|googled "tripping" this looked trippy enough.
"Whoa dude, is this a dream? This is the first time I had been on a woman's washroom. Slap me silly please, because this might be a dream for a 33 year old guy like me."
"Have you ever feel like a big head looking around people and seeing things like normal people don't?"
"Nothing is cash, Whoever said it's cash. It's not cash to me. Cash is something that has a value and for me, my life is cash."
I never had a chance to ask for permission of his picture so I never took one. But yes he is trippy as hell and gives off a "it's all good" vibe. he made me less bored whenever he hangs around
as I clean those washrooms. He is somewhat a
co-owner of some store I have not time to check lol.
So yeah I'm back and glad to. See ya guys and as always Thanks for reading.


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A Father and Son.. "The Bridge"
The first time I saw this movie was at Harvest Bible in Naperville 4 years ago this Good Friday.. It truly gave me a new understanding of Jesus' death on the cross and the relationship between God, us, and His Son.. Whew… Tears well up as I write.. Enjoy.. I pray this touches your heart like it did mine.. John 3:16.
The alarm goes off at 8:00AM, still groggy from getting to bed at 2:30AM, I hit the snooze… 8:30AM…I jumped up and thought I am gonna be late!! Quickly getting in the shower I got ready for Church… Even though I had little sleep I felt something compelling me to go.. I was running late and couldn't even grab my normal cup of coffee.. A weird feeling, but it was as if my feet were walking for me..
Once I arrived it was clear to me as I saw the cups and bread, then listening to the message delivered by
and John Hinger
where my focus needed to be. The message began with them both playing off each others lack of sleep and funny New Year's resolutions, but turned into what "I now understand" is the best way to start the year.. Communion.. To reflect on our hearts… To examine them… Looking past all the surface resolutions like dieting, organization, working harder, and to really look deep into our hearts.. Something I had clearly overlooked..
WOW!! Many people talk about having a balanced life with mind, body, spirit, but here it is apparent that those may be out of order.. As I think about this morning it was a combination of my mind and spirit working at first. The body followed the conscious decision and the heart-felt emotion.. I have even come to the conclusion that when we make the conscious decision to do something our spirit may be affected positively or negatively depending on that action.. I can't help but think back to years past when I would wake up depressed from a hangover or lacking the motivation to do anything because I made the bad decision the night before, yet today it was different.. The decision I made to go to church was blessed by a great message of priorities.. I am refreshed and feel as if my whole perspective of 2012 is aligned in the proper order by the message these two men gave. Thanks Dan and John!
My goal for this year is to be as consistent as possible with putting my mind, spirit, and body in order with becoming best I can be for my Savior Jesus Christ, my Wife, and the people I encounter on a daily basis. We all have a purpose, and it is not the external things that attract others, but how we internally clean our closets that really shine the light.. May your year be filled with JOY, PEACE, and REST!!


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Colours by Phill Martin Bundle Editions
Phill Martin has produced some amazing Colour Collections which are used across many of the fantastic product ranges from Creative Expressions. Here, you'll be able to choose your favourite colour and have this available in several different mediums including Sparkle Texture Paste, Ink Spray Mister, Gilding Wax, Pearl PVA Glue and more. This is the perfect way to have complimenting set of various products to use on all your project designs.
Please contact us if you can't find what you're looking for.


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St Luke (Biographical details)
St Luke (saint/martyr; Male; fl.c. 1stC)
Also known as
St Luke; St Luke the Evangelist; Evangelist; Apostle; Luke
Christian saint and Evangelist, and author of a gospel and 'Acts'; also physician; accompanied St Paul (q.v.) on his missions to Greece and Rome. Patron saint of painters. Symbol: the ox.


text: The restoration of local buildings at Castel del Giudice is nearly compete. Twenty-five of the villages historic houses are currently available, with a further 8 to be completed in the near future. In total the hotel will have 100 beds. The interior of our guest rooms reflects their traditional origins - classic simplicity combined with modern comforts. The hotel houses vary in size according to the number of guests that can be accommodated. Every house offers free Wi-Fi, a mini-fridge bar, a safe and a cooking area. Some houses have a working fireplace.


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Please send all parish information changes to Lorena Gonzalez at .
Masses offered in: English, Spanish
111 N Wood St
Ferris, TX 75125 (Map)
Phone: 972-544-2161
Fax: 972-325-2168
Deacon Administrator:
Deacon Jose Muñiz
Website: Corpus Christi Catholic Church
8:30 a.m. (English)
10:00 a.m. (Spanish)
12:00 p.m. (Spanish)
Thu: 6:30 p.m. (Communion Service only)
Holy Days:
Please visit parish website, or call parish office.
First Sat of the month: 6:00 p.m. - 7:00 p.m.
Every other Thursday 6:00-8:00 p.m.
Thurs: 12:00 p.m., followed by Communion Service 6:00 p.m.


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Quris improves patient care by ensuring healthcare workers have the most up-to-date, relevant and validated clinical resources (guidelines, algorithms, protocols, handbooks, calculators, alerts and much more) in their hands, at the time they need them most.
The information and resources provided through Quris are always the most up to date and are automatically monitored for review with alerts and notifications for review date expiry. Updates to your resources are notified and made instantly available to all relevant users.
Quris ensures your hospital and departmental resources are easily accessible from any mobile device, from any desktop computer and from your Electronic Health Record (EHR) systems. The same information, up to date and available everywhere.
Templates ensures all your resources (e.g. guidelines, handbooks, pathways, protocols, etc.) follow your organizational governance and editorial requirements thereby increasing quality, compliance as well as reducing unnecessary variation.
Use the Quris Clinical Content Management System (CCMS) to create/edit and/or upload/link to existing resources. The CCMS allows you to organise your resources in a way that makes sense to clinicians. Our standardized templates (e.g. for guidelines, etc.) quicken the editorial process and can be customized, if required, for individual departmental needs. Resources can be shared and re-used across departments to provide a single access point to related information.
Once uploaded, the CCMS allows you to apply an editorial and review process that is as sophisticated or as simple as you require. Quickly upload and publish right away, or define one or more review stages that resources must pass through before final approval. Whatever your process a final click of a button publishes your resources to all subscribed mobile devices and the web (if required) immediately for users to access.
Of course that's not the end of the story. Powerful analytics ensure that you can report upon and track use of your resources. For example: what guidelines are most used; when are they used; by what teams; which sections, etc. Users can also send feedback on specific resources thus improving 'buy in' as the entire team is able to actively contribute to the development of clinical knowledge.
Data/Wi-Fi connections are not needed to use the app. Use it anywhere, anytime.
Templates ease the process of creating, editing and managing your guidelines, handbooks, etc.
Quick and flexible cloud-based deployment makes it easy to get up and running.
Quris can be deployed into an organization with litte or no demand on IT department resources.
Security, data privacy and diaster recovery features ensure your resources are safe and protected.
Understand what resources your staff are using, when, how and where. Quris analytics tracks both online and offline activity.
Licensing is based upon organization size and customizations required, keeping costs low.
Your information and resources can be integrated into any clinical system through the Quris HL7 API.
Quris can deliver your clinical resources to mobile devices as well as desktops - same content, multiple access points.


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image: Murasaki from lizadalby.com
Murasaki was released by Shiseido in 1980. It has top notes of gardenia, galbanum, peach, hyacinth and bergamot; middle notes of lily, orris root, jasmine, lily-of-the-valley and rose; and base notes of leather, sandalwood, amber, musk, oakmoss and vetiver. Or is it a tender green floral with notes of roses, chrysanthemums and lilies on a soft, dry powdery base enhanced by galbanum? I think both descriptions fit it fairly. Named perhaps for Murasaki Shikibu, the 11th-century Japanese female poet who became most famous for her novel- the huge 6 volume- 'The Tale of Genji'.
image: scentdirectMurasaki also means the color purple, as in wisteria or violet blossoms and the color purple is featured in all of the perfume's presentations. The older style bottle is of dark colored glass- it appears reddish but the glass color is actually a dark amethyst purple. I think the newer bottle style pictured below with a purple & clear crystal stopper is prettiest, however the juice in the older dark purple style (which is the version I'm reviewing) is best smelling
image: email@example.com a Japanese haiku this scent has a beautiful construction. It is gentle yet with real presence. The fragrance resembles Anais-Anais but Anais-Anais smells overly sweet, plasticy and crude next to Murasaki; the lily of the valley note is especially clunky by comparison. Murasaki is deceptively complex as well; it displays only a fleeting sweetness, balanced by lasting greeness, along with a bit of spice held nicely in check by dry vetiver and musk, and it is all hidden under an impeccable floral. Murasaki lingers on the skin leaving an impression of the lightest dusting of a fine French chypre powder.
image: cg.scs.carleton.caMurasaki was designed for the Asian audience, who are known for preferring a lighter style in perfumes... The richness of the ingredients have been perfected by the lightness of the formula. The green aspect of galbanum is set off a profusion of jasmine, gardenia, rose, lily and hyacinth; the creaminess of sandalwood and musk is balanced by chypre touches of oakmoss and leather. All these sumptuous ingredients are rooted by the incense-like quality of orris root and vetiver. Complex yet transparent, each note is essential but none is allowed to dominate another. Murasaki even inspired me to compose a haiku for it:
"The violet blooms
while trees sleep under silent skies...
while trees sleep under silent skies...
Purple shines through"
"月百姿 石山月" Lady Murasaki at Ishiyama'The Vintage Perfume Vault, where the scent of yesterday's vogue lives.
Tsuki Hyakushi
by Tsukioka Yoshitoshi 月岡芳年 (1839-1892)
Tsuki Hyakushi
by Tsukioka Yoshitoshi 月岡芳年 (1839-1892)


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Waiting on Wednesday: The Raven King by Maggie Stiefvater
7 comments :
Awesome Pick!! I love this series and I'm anxiously awaiting the release as well. I hope you enjoy it!!ReplyDelete
My WoW: http://jacquesbooknook.blogspot.com/2016/01/waiting-on-wednesday-americas-first.html
Ahh, books that have a super vague synopsis scare me. We obviously know the issues arising in TRK, BUT STILL! Who knows what the hell Stiefvater has planned!ReplyDelete
Great pick! I definitely want to hurry up and check out this series.ReplyDelete
Krystianna @ Downright Dystopian
I need to pick up this series! i've heard so many good things!ReplyDelete
My WoW
I just finished Blue Lily, Lily Blue! AND I CANNOT WAIT TO FINALLY BE ABLE TO ANTICIPATE AND PARTICIPATE IN THE FINALE OF A SERIES WITH EVERYONE ELSE ^_^ReplyDelete
Gah! I hope it doesn't end tragically too but I'm so worried that it will D: This is one of my favorite series of life too so I NEED Raven King like air ♥ Eeeep, I can't wait to gush all about it with everyone once it comes out - if we all survive LOL! Awesome pick Lillie^^ReplyDelete
I think I'm one of few people who hasn't read The Raven Boys. I will at some point. Hope you get and enjoy this one!ReplyDelete


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Key eligibility checklist for a prospective Plan Vivo project
1. Start date
Projects will typically use the 2013 Plan Vivo Standard from the outset. However, it is also possible for a project that is already operational to become an approved Plan Vivo Project, provided it can meet the requirements of 2013 Plan Vivo Standard. No retroactive crediting is possible for activities already implemented.
2. Project participants
- Must be small-scale farmers, land-users or forest dwellers with recognised land tenure or user rights (see below)
- Must be organised, or in the process of being organised, into cooperatives, associations, community-based organisations or other organisational forms able to contribute to the social and economic development of their members and communities and democratically controlled by the members
- Must be able to use existing farmland, forest, woodland or other land type for project activities without undermining livelihood needs
- Producers should not be structurally dependent on permanent hired labour, and should manage their land mainly with their own and their family's labour force
3. Project coordinators
- Must be an established legal entity that takes responsibility for the project and meeting the requirements of the Plan Vivo Standard for its duration
- Must have a strong in-country presence and the respect and experience required to work effectively with local communities and partners
- Must be focused and have the organisational capability and an ability to mobilise the necessary resources to develop the project
- Must have the capability to negotiate and deal with government, local organisations & institutions, and buyers of ecosystem services
- Must have the ability to mobilise and train participants, implement and monitor project activities, carry out technical functions
- Must recognise that the decision of producers to participate in project activities is entirely voluntary
- Must recognise that producers own the carbon/ES benefits of the project activities they choose to undertake
- Must ensure that the benefit-sharing arrangement is fair and equitable and that payments are made in a transparent and traceable manner
- Should not draw on more than 40 percent of sales income for ongoing coordination, administration and monitoring costs, save in exceptional circumstances where justification is provided to the Plan Vivo Foundation and a waiver formally agreed
4. Land status
Land that is not owned by or subject to user rights of smallholders or communities may be included in the project area if:
- It represents less than a third of the project area at all times
- It was not acquired from smallholders/communities in order to develop the project
- It bestows clear benefits to the project on a landscape level
- It is managed under an executed agreement between the owners/managers and the project participants
5. Land tenure/ user rights
- Land tenure or user rights must be secure and stable so that there can be clear ownership, traceability and accountability for ecosystem service benefits such as carbon reduction or sequestration, and the ability to commit to project interventions for the duration of PES Agreements
6. Project activities
- Must enable communities to plan and take control of their resources in a sustainable way that promotes rural livelihoods and other environmental and social co-benefits
- Must be able to generate ecosystem service benefits through one or more of the following project intervention types under the Plan Vivo System:
- Ecosystem restoration (e.g. assisted natural generation)
- Ecosystem rehabilitation (e.g. inter-planting naturalised tree species)
- Prevention of ecosystem conversion or degradation (e.g. REDD+)
- Improved land use management (e.g. minimum till agriculture)
- Must be additional, not liable to cause leakage, and provide foundations for permanence, as described in the Plan Vivo Standard
- Must involve the planting and/or promote the restoration or protection of native or naturalised plant and tree species. The use of naturalised (i.e. non-invasive) species is acceptable only where such species are:
- Preferable to any alternative native species owing to compelling livelihood benefits;
- Specifically selected by communities for this purpose;
- Not going to result in any negative effects on biodiversity or the provision of key ecosystem services in the project and surrounding areas
- Must encourage the development of local capacity and minimise dependency on external support
7. Expansion ambitions
- Must be based on an commitment to initiating activities on a pilot basis, gaining experience, and identifying improvements ('learning by doing')
- Must be based on practical capabilities 'on the ground', not on high-level targets imposed from above


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|Welcome to my site!
This site contains information on how to troubleshoot problems in Windows, registry fixes, customization ideas, utilities and scripts to resolve problems in Windows XP. Resources available in this site should help you learn and fix the problems by yourselves.
Update: This site continues at The Winhelponline Blog - Visit there for regular updates. You can subscribe to RSS feed too!
Ramesh Srinivasan
Frequently used Tips & fixes for Windows XP
There are many other articles and problem solvers
The above is not the exhaustive list. For more Tips and Troubleshooting documents for Windows XP, start from this page. Or check the Recent documents page out here. Internet Explorer FAQ is here