Axela Inc. DNA Research

April 2010
Axela Inc. announces bilateral agreement with diagnostic provider Salomao and Zoppi and Cria Opportunities Inc.

Axela Inc, the Toronto-based producer of multiplex biomarker tools for proteins, DNA and RNA in research and diagnostics, Salomao and Zoppi (S&Z), one of the largest diagnostics providers in Sao Paulo Brazil, and innovations management firm Cria Opportunities Inc (CRIA), have signed a memorandum of understanding to commercialize Axela’s technology in Brazil and South America.

June 2009

Xceed Meets HTX/CIHR Grant Milestone: Decreasing Sample-Prep Time From Days to Less Than Eight Hours
Xceed Molecular announced that it has met the goals of a $127,000 HTX/CIHR grant, reducing sample-preparation time from 2.5 days to less than eight hours.

October 2008

James L. Wittliff, Ph.D., FACB, to Present “Predicting Breast Cancer Outcome with Gene Expression Signatures on the Ziplex® System at the Association for Molecular Pathology Conference, October 30
Xceed Molecular announced that renowned breast cancer researcher and Xceed collaborator, Dr. James L. Wittliff, a professor in the Department of Biochemistry and Molecular Biology at the University of Louisville, will be a featured speaker at the Association for Molecular Pathology (AMP) Annual Meeting.

September 2008

Mt. Sinai Services Toronto Becomes An Xceed Authorized Gene-Expression Service Provider Using Ziplex® System
Xceed Molecular announced that the Mt. Sinai Services unit of the Mt. Sinai Hospital in Toronto, Canada, has become an authorized provider of gene-expression services using Xceed’s
Ziplex System.

Queen’s University Chooses Xceed’s Ziplex® System for Translational Research
Xceed Molecular announced that Queen’s University in Kingston, Ontario, Canada, is the latest high-profile institution to implement Xceed’s Ziplex System as a critical tool to advance its translational research.

August 2008

Xceed Ziplex® System for Automated Gene-Expression Analysis Wins 2008 R&D; 100 Award
Xceed Molecular announced that R&D; Magazine has selected the company’s Ziplex System as a recipient of the magazine’s prestigious R&D; 100 Award.

July 2008

Xceed Molecular to Launch New Inflammation Xpress Chip™ for the Ziplex® Automated Gene-Expression Analysis System at Clinical Lab Expo 2008, Booth 2155
Xceed Molecular will launch its new Inflammation Xpress Chip, a pre-configured array for use with the company’s Ziplex automated gene-expression analysis system, at the 2008 AACC/ASCLS Clinical Lab Expo (booth 2155).

June 2008

Xceed Molecular Licenses Signature from Moffitt Cancer Center and Adds Moffitt to Strategic Collaborator Program
Xceed Molecular announced that the Mt. Sinai Services unit of the Mt. Sinai Hospital in Toronto, Canada, has become an authorized provider of gene-expression services using Xceed’s
Ziplex System.

Xceed Molecular Grants Gen-Probe License to Xceed’s Flow-Thru Chip® Technology for Multiplex Molecular Diagnostics
Xceed Molecular has granted Gen-Probe Incorporated (Nasdaq: GPRO) and its affiliates a non-exclusive license to use Xceed’s novel Flow-Thru Chip technology.

May 2008

Xceed Molecular Features Ziplex® Automated Gene-Expression System at the Biomarker World Congress, Booth 16
Xceed’s Dr. David Englert and Dr. Zhe Zhang of Children’s Hospital of Philadelphia will present data at a luncheon workshop on Wednesday, May 21st

April 2008

Xceed Molecular Signs University of Florida to Strategic Collaborator Program; Focus on Developing New Molecular Test to Diagnose Early-Stage Bladder Cancer
Xceed Molecular announced that the University of Florida has joined Xceed’s Strategic Collaborator program. Xceed will work with primary researchers, Charles Joel Rosser, MD (Department of Urology) and Steve Goodison, MD (Department of Surgery), to perform initial verification and validation of an expression signature that has shown promise as a way to differentiate bladder cancer from other conditions using voided urine samples.

November 2007

Xceed Molecular Launches Strategic Collaborator Program; Signs Center for Molecular Medicine as its Inaugural Partner
Xceed Molecular announced that the company has launched its Strategic Collaborator program and signed its first member, the Center for Molecular Medicine (CMM), of Grand Rapids, Michigan. Specific terms were not disclosed.

Xceed Molecular Launches Ziplex® Automated Gene-Expression System at the Association for Molecular Pathology, Booth 84/85
Xceed Molecular announced that the company will launch its Ziplex Automated Gene Expression System for research use only at the Association for Molecular Pathology (AMP) Annual Meeting and Exhibit, which opens today at the Hyatt Regency Century Plaza Hotel in Los Angeles.

October 2007

Xceed Molecular Names Fran Tuttle to its Board of Trustees
Xceed Molecular Corporation announced today that Fran Tuttle has been appointed to Xceed’s board of directors.

Xceed Molecular to Debut RUO Ziplex Array
One year after rechristening itself Xceed Molecular to emphasize its orientation towards the clinical diagnostics market, the company formerly known as Metrigenix is preparing to launch a low-throughput and relatively affordable microarray platform called Ziplex next month.

February 2007

Xceed Molecular names David Deems President, and Michael L. Cohen, Chairman of the Board.
Xceed Molecular announced today that David Deems has been appointed company president with full executive responsibility for strategic, commercial, and operational development,
effective immediately.

October 2006

MetriGenix Changes Name to Xceed Molecular, Focuses on Translating Novel Biomarker-based Tests from Research into Routine Clinical Use
MetriGenix Corp. announced today that it has changed its name to Xceed Molecular.

December 2005

Appointment of Susan Bromley
Susan Bromley Named Vice President Diagnostic Product Development of MetriGenix, Inc.

June 2005

MetriGenix Completes Acquisition of GeneXP Biosciences
MetriGenix, Inc. announced that it has completed the acquisition of Woburn, Massachusetts-based GeneXP Biosciences, Inc.

home© 2009 Xceed Molecular For Research Use Only

Introduction to multiplex immunohistochemistry

For the development of new immunotherapies, it is essential to research the tumor microenvironment. Immunohistochemistry is a versatile technique for studying the expression, distribution and activation of proteins in situ. For the detection of antigens, specific antibodies are used on thin cross-sections made of shock-frozen or formaldehyde-fixed, paraffin-embedded tissue. The visualization of the antigen is made possible by enzymatic reactions which lead to the precipitation of dye at the site of the antibody-antigen binding, or by fluorescent reporters. Fluorescent reporters can either be conjugated directly to the primary antibody (direct immunofluorescence) or to a secondary antibody that recognizes the species-specific primary antibody (indirect immunofluorescence). The last variant is the more frequently used method, since it still delivers signals with very small amounts of antigen.

In the past, this technique was used individually for each immune marker. Recently, molecular histopathology has evolved from single marker immunohistochemistry to multiplex marker detection. Multiplex immunohistochemistry, also called multiple immunolabeling or multiplex immunostaining, can maximize the amount of data obtained from a single sample. This is particularly important in cases where the amount of sample is limited, such as a tumor biopsy. In contrast to next generation sequencing or mass spectrometry, the spatial arrangement of proteins as well as protein interactions and co-localization can be examined in multiplex immunohistochemistry.

Chromogenic detection is in principle compatible with multiplex immunohistochemistry, but indirect labeling techniques with tyramide-based fluorescence have several advantages. The signal is amplified by the deposition of fluorophore-conjugated tyramide at the antigen binding site. Tyramid Signal Amplification (TSA) enables the detection of very rare targets in a sample and improves the fluorescence signal. It is also important that TSA also allows the use of unlabeled primary antibodies with the flexibility to use multiple antibodies from the same species.

Another not insignificant advantage of tyramide-based fluorescence detection is the resistance of the tyramide-antigen binding, which takes place through the covalent binding of tyrosine residues on or near the antigen. This resistance allows the removal of antibodies using heat, while maintaining the fluorescent signal of the antigen. With this method, different antibodies from the same host species can be used in succession without the otherwise expected cross-reactions. If additional, non-overlapping excitation and emission wavelengths are selected, it is easy to visualize different target proteins at the same time. Multispectral recordings allow the graphic isolation of fluorophores with partially overlapping spectra and the reduction of tissue autofluorescence.

The latest advances in multiplex immunohistochemistry and multispectral imaging enable the precise and simultaneous analysis of multiple tissue markers. For example, the analysis of proliferation and autophagy markers in the intestinal tissue can help with an accurate assessment of the epithelium turnover. In clinical application, the elucidation of the tumor microenvironment and the selection of targeted therapies can be improved by localizing and examining immuncheckpoint proteins. The applications of multiplex immunohistochemistry are versatile and include clinical, translational and basic research.

Detection of human CD3 (turquoise), CD8 (green), CD68 (orange), CK (red), Ki67 (yellow) and PD-L1 (white) in FFPE HNSCC with IHC-IF. Rabbit anti-CD3e recombinant monoclonal [BL-298-5D12], rabbit anti-CD8a recombinant monoclonal [BLR044F], mouse anti-CD68 monoclonal [KP-1], mouse anti-cytokeratin monoclonal [AE1 / AE3], rabbit anti-Ki67 monoclonal [BLR021E] and rabbit anti-PD-L1 recombinant monoclonal [BLR020E]. Secondary antibodies: HRP-conjugated goat anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG. Substrate: Opal ™ 480, 520, 570, 620, 690 and 780. Counterstain: DAPI (blue).

Detection of human CD3 (yellow), CD8 (red), and CD20 (green) in FFPE tonsil by IHC-IF.

Detection of human CD3 (yellow), CD8 (red), and CD20 (green) in FFPE Mundel tissue with IHC-IF. Rabbit anti-CD3e recombinant monoclonal [BL-298-5D12], rabbit anti-CD8a recombinant monoclonal [BLR044F], mouse anti-CD20 monoclonal [L26]. Secondary antibodies: HRP-conjugated goat antirabbit IgG and HRP-conjugated goat anti-mouse IgG. Substrate: Opal ™ 520, 620, and 690. Counterstain: DAPI (blue).

Detection of human CD3 (yellow), CD20 (green), and CD68 (red) in FFPE lung carcinoma by IHC-IF.

Detection of human CD3 (yellow), CD20 (green), and CD68 (red) in FFPE lung cancer tissue with IHC-IF. Rabbit anti-CD3e recombinant monoclonal [BL-298-5D12], mouse anti-CD20 monoclonal [L26], and mouse anti-CD68 monoclonal [KP-1]. Secondary antibodies: HRP-conjugated goat antirabbit IgG and HRP-conjugated goat anti-mouse IgG. Substrate: Opal ™ 520, 620, and 690. Counterstain: DAPI (blue).

Required reagents
Component product
PathPlex ™ antibody panels
PathPlex ™ Panel 1 (CD3E, CD8 alpha, PD-L1)
PathPlex ™ Panel 2 (CD3E, CD8 alpha, CD20)
PathPlex ™ Panel 3 (CD3E, CD68, CD20)
PathPlex ™ Panel 4 (CD3E, Cytokeratin, CD8 alpha, CD68, PD-L1, FOXP3)
PathPlex ™ Panel 5 (CD3E, Cytokeratin, CD8 alpha, CD68, Ki-67, PD-L1)
PathPlex ™ Panel 6 (CD3E, Granzyme B, CD8 alpha, cytokeratin, Ki-67, SOX10)
PathPlex ™ Panel 7 (CD8 alpha, cytokeratin, CD45RO, CD4, FOXP3)
PathPlex ™ Panel 8 (CD3E, Cytokeratin, CD8 alpha, CD4, LAG3, FOXP3)
HRP-conjugated secondary antibodies (depending on primary antibodies)
Anti-Mouse IgG-heavy and light chain, HRP conjugated
Anti-Rabbit IgG-heavy and light highly cross-adsorbed, HRP conjugated
Opal dyes
Opal Polaris 480 (Akoya Biosciences)
 Opal 520 (Akoya Biosciences)
 Opal 540 (Akoya Biosciences)
 Opal 570 (Akoya Biosciences)
 Opal 620 (Akoya Biosciences)
 Opal 650 (Akoya Biosciences)
 Opal 690 (Akoya Biosciences)
 Opal Polaris 780 (Akoya Biosciences)
1x Plus Amplification Diluent
FP1498 (Akoya Biosciences)
DAPI
DAPI. dihydrochloride (AdipoGen Life Sciences)

Advantages of fluorescence multiplex immunohistochemistry using TSA
Fluorescence multiplex immunohistochemistry (mIHC) with tyramide signal amplification (TSA) has several advantages over one-color or traditional mIHC. The advantages below show what makes the mIHC with TSA a powerful technology for the visualization of several interesting target structures:

Visualization of multiple target structures within a single tissue section
This is crucial for experiments where the amount of sample is limited, e.g. for tumor biopsies or other clinical samples. mIHC enables the acquisition of the maximum amount of data from a single sample.

Investigation of the spatial arrangement of the target structures
The visualization of multiple targets within a single tissue section enables the spatial arrangement of the structures to be examined. This leads to a better understanding of protein interaction or co-localization within the conserved tissue architecture. This type of investigation is not possible with other techniques such as polymerase chain reaction, mass spectrometry or next-generation sequencing.

Further dynamic and linear measuring ranges
Compared to chromogenic detection, fluorophore detection offers a wider dynamic and linear measurement range, making it easier to visualize both high and low frequency targets on the same slide. The use of TSA also enables the signal amplification of targets with low frequency by amplifying the antigen-associated fluorescence signal.

Simplified panel design
The permanent existence of the covalent tyramide-tyrosine bond facilitates the heat-mediated removal of primary / secondary antibody pairs without interrupting the fluorescence signal. This means that any primary antibody validated for IHC, regardless of the host species, can be used for any target structure, as long as a specific secondary antibody is used.

Use of DAPI as counterstaining
Counterstaining the DNA with DAPI is preferable to hematoxylin because hematoxylin can be masked by other targets during chromogenic staining.

Spectral segregation
Spectral segregation ensures that the signals of each individual target structure can be distinguished from those of the other target structures. It also enables the subtraction of the signal caused by the autofluorescence of the tissue.

Rationalized quantification
The objective determination of the expression level of several targets can be standardized through the use of a suitable imaging platform and software. Since the tissue architecture is retained in the mIHC, the visualization of tissue reference points can also contribute to precise quantification.

Taken together, these features of fluorescent mIHC with TSA represent a robust approach to tissue analysis. Such analysis enables a variety of applications, such as the characterization of molecular signaling pathways, protein-protein interactions, the elucidation of the complex microenvironment of the tumor or the development of individually tailored therapeutic interventions.

Changes to Recommended Western Blotting Protocols

Cell Signaling Technology (CST) antibodies are validated utilizing our really useful western blotting protocol, developed and optimized by CST scientists. All knowledge proven on product webpages and datasheets was generated utilizing these protocols. We strongly suggest following our protocols to obtain optimum outcomes with all of our antibodies. Below are just a few examples of the results minor adjustments to the usual protocol could have in your outcomes.

Lysate Preparation

Sonication versus no sonication
Phospho-Histone H3 (Ser10) Antibody #9701

CKR/PAEC cell extracts

Primary Antibody Dilution Buffer

Milk versus BSA
Phospho-Akt (Ser473) Antibody #9271

C2C12 cells + insulin

Primary Antibody Incubation

Overnight at 4°C versus 2 hours at room temperature

Phospho-Akt (Ser473) Antibody #9271

HeLa cell extracts

PKCδ Antibody #2058

HeLa cell extracts

Phospho-GSK-3β (Ser9) Antibody #9336

HeLa cell extracts

Wash and Dilution Buffer

TBS-T versus PBS-T

Phospho-c-Jun (Ser63) II Antibody #9261
c-Jun (60A8) Rabbit mAb #9165
Ribosomal Protein S3 Antibody #2579
Calnexin (C5C9) Rabbit mAb #2679
Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb #2855

TBS-T (as really useful in CST protocol)
PBS-T (altered protocol)

Transfer and Antibody Incubations

Cell Signaling Technology recommends moist switch (as really useful by producer) adopted by one hour blocking and in a single day main antibody incubation at 4˚C.

iBlot® is a dry blotting system that completes switch in 7 minutes. SNAPi.d.™ is a vacuum operated incubation system, which reduces antibody incubation instances to lower than 30 minutes.

Phospho-p38 MAPK (Thr180/Tyr182) (3D7) Rabbit mAb #9215

Wet switch CST
 really useful incubations
iBlot® (fast transfer-Invitrogen) SNAPi.d.™ (fast incubations-Millipore) * decided optimum antibody focus for SNAPi.d.™

iBlot® is a trademark of Invitrogen Corporation.
SNAPi.d.™ is a trademark of Millipore Corporation.

Lab-on-a-Chip-Based PCR-RFLP Assay for the Detection of Malayan Box Turtle (Cuora amboinensis) in the Food Chain and Traditional Chinese Medicines.

The Malayan field turtle (Cuora amboinensis) (MBT) is a weak and protected turtle species, however it’s a profitable merchandise in the unlawful wildlife commerce as a result of of its nice attraction as an unique meals merchandise and in conventional medication.

Although a number of polymerase chain response (PCR) assays to determine MBT by numerous routes have been documented, their applicability for forensic authentication stays inconclusive as a consequence of the lengthy size of the amplicon targets, that are simply damaged down by pure decomposition, environmental stresses or physiochemical remedies throughout meals processing.

To tackle this analysis hole, we developed, for the first time, a species-specific PCR-restriction fragment size polymorphism (RFLP) assay with a really brief goal size (120 bp) to detect MBT in the meals chain; this authentication ensured higher safety and reliability via molecular fingerprints. The PCR-amplified product was digested with Bfa1 endonuclease, and distinctive restriction fingerprints (72, 43 and 5 bp) for MBT have been discovered upon separation in a microfluidic chip-based automated electrophoresis system, which reinforces the decision of brief oligos.

The possibilities of any false destructive identifications have been eradicated via the use of a common endogenous management for eukaryotes, and the restrict of detection was 0.0001 ng DNA or 0.01% of the meat below admixed states.

Finally, the optimized PCR-RFLP assay was validated for the screening of uncooked and processed industrial meatballs, burgers and frankfurters, that are very talked-about in most international locations.

The optimized PCR-RFLP assay was additional used to display MBT supplies in 153 conventional Chinese medicines of 17 completely different manufacturers and 62 of them have been discovered MBT constructive; whereby the elements weren’t declared in product labels. Overall, the novel assay demonstrated ample benefit for use in any forensic and/or archaeological authentication of MBT, even below a state of decomposition.

Lab-on-a-Chip-Based PCR-RFLP Assay for the Detection of Malayan Box Turtle (Cuora amboinensis) in the Food Chain and Traditional Chinese Medicines.
Lab-on-a-Chip-Based PCR-RFLP Assay for the Detection of Malayan Box Turtle (Cuora amboinensis) in the Food Chain and Traditional Chinese Medicines.

A novel lab-on-chip platform with built-in strong part PCR and Supercritical Angle Fluorescence (SAF) microlens array for extremely delicate and multiplexed pathogen detection.

Solid-phase PCR (SP-PCR) has develop into more and more fashionable for molecular analysis and there have been just a few makes an attempt to include SP-PCR into lab-on-a-chip (LOC) units.

However, their applicability for on-line analysis is hindered by the lack of delicate and moveable on-chip optical detection expertise. In this paper, we addressed this problem by combining the SP-PCR with tremendous essential angle fluorescence (SAF) microlens array embedded in a microchip.

We fabricated miniaturized SAF microlens array as half of a microfluidic chamber in thermoplastic materials and carried out multiplexed SP-PCR instantly on high of the SAF microlens array. Attribute to the excessive fluorescence assortment effectivity of the SAF microlens array, the SP-PCR assay on the LOC platform demonstrated a excessive sensitivity of 1.6 copies/µL, corresponding to off-chip detection utilizing typical laser scanner.

The mixture of SP-PCR and SAF microlens array permits for on-chip extremely delicate and multiplexed pathogen detection with low-cost and compact optical elements. The LOC platform could be extensively used as a high-throughput biosensor to investigate meals, medical and environmental samples.

Application of lab-on-a-chip multiplex cassette PCR for the detection of enterohemorrhagic Escherichia coli.

Fast molecular detection strategies profit from ready-to-run lab-on-a-chip molecular assays with minimal preparation time.

Detection effectivity of such strategies can enhance if a number of targets are detected concurrently per given response. Detection of meals pathogens, i.e. Escherichia coli (E. coli), is mostly carried out in two levels with the detection of a number of targets in every stage.With simultaneous testing, screening for pathogens is quick and environment friendly.

In this research, we present the software of multiplex PCR carried out on a ready-made cassette to detect 10 targets every for eight samples recognized to harbor E. coli. In cassette PCR, the aluminum cassette (38.6 mm × 31.four mm) comprises 10 trenches having a complete of 50 capillaries with microliter volumes of desiccated acrylamide gels holding all reagents required for the PCR together with inner constructive and unfavorable controls. The gel comprises LCGreen dye to detect double stranded DNA.

Fluorescence monitoring permits the detection of the amplified merchandise by soften curve evaluation. In this software, every of the 5 capillaries in a given trench comprises two of the primer units for the detection of 10 targets in pathogenic E. coli, particularly, O157, Eae, Stx1, Stx2 and 6 O-antigen genes. Primer specificity was confirmed. Each trench assessments one pattern. Eight minimally processed enriched beef carcass swab samples had been analyzed for parallel detection of 10 targets inside 1 h and 15 min. Samples had been delivered to the capillaries by capillary forces thereby hydrating the gels.

Multiplex cassette PCR outcomes had been confirmed with standard multiplex PCRs carried out in a industrial real-time PCR system.Cassette PCR know-how is ideally suited to multi-target detection of pathogens in meals merchandise.

The cassette performs a number of PCR reactions in parallel, with multiplex detection of targets inside every response unit. Cassette PCR/ soften curve evaluation outcomes for the simultaneous detection of 10 targets of pathogenic E.coli in beef carcass swab samples had been confirmed with a traditional real-time PCR/ soften curve evaluation in addition to with agarose gel electrophoresis.

Although designed for the detection of E. coli, this multiplex cassette PCR method could be utilized to every other assay the place the quick detection of a number of targets is required.

Application of lab-on-a-chip multiplex cassette PCR for the detection of enterohemorrhagic Escherichia coli.
Application of lab-on-a-chip multiplex cassette PCR for the detection of enterohemorrhagic Escherichia coli.

Migrating a lab-developed MERS-CoV real-time PCR to three “Sample to Result” methods: experiences on optimization and validation.

The objective of the research was to adapt our Middle East respiratory syndrome coronavirus (MERS-CoV) lab-developed check (LDT) to three “Sample to Result” (S2R) methods: BD MAX (BD), ELITe InGenius (ELITechGroup), and ARIES (Luminex).

The BD MAX and InGenius system allowed use of lab-developed primers and TaqMan probes, whereas ARIES required conversion to MultiCode primers for melting curve evaluation.

Each system required ≤1 day of coaching and assay optimization. No discordant outcomes had been famous after evaluation of 32 External Quality Control (EQC) samples. On a 10-fold dilution sequence of a MERS-CoV-positive EQC pattern, InGenius obtained the highest detection price. Laboratory technicians rated the ARIES as the user-friendliest. It additionally required the least hands-on time.

BD MAX had the lowest turnaround time and highest throughput. While every system had distinguishing system properties with related (dis)benefits, the three S2R methods had been comparable in phrases of assay improvement and validation.

Within-Subject Performance on a Real-Life, Complex Task and Traditional Lab Experiments: Measures of Word Learning, Raven Matrices, Tapping, and CPR.

In this knowledge report, we describe a three-session experiment spanning six months. Several well-controlled laboratory duties (Word Learning, Raven Matrices, and Tapping) and Cardiopulmonary Resuscitation (CPR), a complicated however well-defined real-world process, have been administered.

Data are reported from 50 members for the primary session, 40 for the second, and 34 for the third. CPR is a helpful area for learning real-world efficiency contained in the laboratory as a result of clear efficiency requirements may be utilized to quantifying learners’ proficiency protecting each the primary steps that have to be taken previous to the initiation of CPR (declarative data) in addition to the compressions and ventilations themselves (procedural talent).

This analysis resulted in a wealthy dataset with a vary of completely different measures for all members. The uncooked knowledge specifically will allow different researchers to discover potential analyses and modeling past the scope of our personal. The particulars of the information assortment protocol and obtainable knowledge are documented right here to facilitate this course of.

Within-Subject Performance on a Real-Life, Complex Task and Traditional Lab Experiments: Measures of Word Learning, Raven Matrices, Tapping, and CPR.
Within-Subject Performance on a Real-Life, Complex Task and Traditional Lab Experiments: Measures of Word Learning, Raven Matrices, Tapping, and CPR.

A Lab-on-a-Chip Device Integrated DNA Extraction and Solid Phase PCR Array for the Genotyping of High-Risk HPV in Clinical Samples.

Point-of-care (POC) molecular diagnostics play a essential position within the prevention and therapy of infectious ailments. It is critical to develop transportable, easy-to-use, cheap and fast molecular diagnostic instruments.

In this examine, we proposed a lab-on-a-chip gadget that built-in DNA extraction, solid-phase PCR and genotyping detection. The ingenious design of the pneumatic microvalves enabled the fluid mixing and reagent storage to be organically mixed, considerably lowering the scale of the chip. The stable oligonucleotide array integrated into the chip allowed the spatial separation of the primers and minimized undesirable interactions in multiplex amplification.

As a proof-of-concept for POC molecular diagnostics on the gadget, 5 genotypes of high-risk human papillomavirus (HPV) (HPV16/HPV18/HPV31/HPV33/HPV58) have been examined. Positive high quality management samples and HPV affected person cervical swab specimens have been analyzed on the built-in microdevice.

The platform was succesful of detection roughly 50 copies of HPV virus per response throughout a single step, together with DNA extraction, solid-phase PCR and genotype detection, in 1 h from samples being added to the chip. This easy and cheap microdevice offered nice utility for the screening and monitoring of HPV genotypes.

The sample-to-result platform will pave the best way for wider utility of POC molecular testing within the fields of medical diagnostics, meals security, and environmental monitoring.

Antiviral Articles

Process Chemistry in Antiviral Research.

Journal: Topics in current chemistry (Cham)Juli/19/2017
This article reviews antiviral therapies that have been approved for human use during the last decade, with a focus on the process chemistry that enabled access to these important drugs. In particular, process chemistry highlights from the practical syntheses of the HCV drugs sofosbuvir (Gilead), grazoprevir (Merck), and elbasvir (Merck), the HIV therapy darunavir (Tibotec) and the influenza treatment peramivir (BioCryst) are presented.
Yong-Li Zhong; Nobuyoshi Yasuda; Hongming Li; Mark McLaughlin; David Tschaen

Prevalence of etravirine (ETR)-RAMs at NNRTI failure and predictors of resistance to ETR in a large Italian resistance database (ARCA).

Journal: Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious DiseasesJuli/13/2014
The prevalence of drug resistance associated with the failure of non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimens and the predictors of resistance to Etravirine (ETR) were assessed in 2854 subjects: 39 < 18 (paediatric) and 2815 ≥ 18 (adult) years old. These subjects failed to respond to their current NNRTI treatment, were three-class experienced and had been exposed to NNRTI for ≥3 months. A total of 1827 adult (64.9%) and 32 paediatric subjects (82.1%) harboured the virus with at least one ETR mutation. V179I, Y181C and G190A were the most frequent mutations in both groups. A significantly increased risk of ETR resistance with all three algorithms (Monogram (MGR) >3, Tibotec (TBT) >2 and enhanced MGR (ENH) ≥4) emerged in the paediatric population. Multivariate analysis revealed an increased risk of developing TBT >2 for NNRTI exposure, ENH ≥4 for NNRTI and EFV exposure in paediatric subjects; NVP exposure and higher (≥3.5 log10) HIV-RNA values for all three algorithms in adult subjects, whereas CD4 ≥ 200/μL appeared to be protective. The risk of being ETR resistant was more than doubled for paediatric vs. adult subjects, probably due to a more extensive use of NNRTI and an incomplete virological control.
S Rusconi; F Adorni; B Bruzzone; A Di Biagio; G Meini; A Callegaro+10 authors


Similar predictions of etravirine sensitivity regardless of genotypic testing method used: comparison of available scoring systems.

Journal: Antiviral therapyJuni/9/2013
BACKGROUND
The aims of this study were to compare various genotypic scoring systems commonly used to predict virological outcome to etravirine, and examine their concordance with etravirine phenotypic susceptibility.
METHODS
Six etravirine genotypic scoring systems were assessed: Tibotec 2010 (based on 20 mutations; TBT 20), Monogram, Stanford HIVdb, ANRS, Rega (based on 37, 30, 27 and 49 mutations, respectively) and virco(®)TYPE HIV-1 (predicted fold change based on genotype). Samples from treatment-experienced patients who participated in the DUET trials and with both genotypic and phenotypic data (n=403) were assessed using each scoring system. Results were retrospectively correlated with virological response in DUET. κ coefficients were calculated to estimate the degree of correlation between the different scoring systems.
RESULTS
Correlation between the five scoring systems and the TBT 20 system was approximately 90%. Virological response by etravirine susceptibility was comparable regardless of which scoring system was utilized, with 70-74% of DUET patients determined as susceptible to etravirine by the different scoring systems achieving plasma viral load <50 HIV-1 RNA copies/ml. In samples classed as phenotypically susceptible to etravirine (fold change in 50% effective concentration ≤3), correlations with genotypic score were consistently high across scoring systems (≥70%).
CONCLUSIONS
In general, the etravirine genotypic scoring systems produced similar results, and genotype-phenotype concordance was high. As such, phenotypic interpretations, and in their absence all genotypic scoring systems investigated, may be used to reliably predict the activity of etravirine.
Johan Vingerhoets; Steven Nijs; Lotke Tambuyzer; Annemie Hoogstoel; David Anderson; Gaston Picchio

TMC435 for the treatment of chronic hepatitis C.

Journal: Expert opinion on investigational drugsNovember/19/2012
BACKGROUND
Chronic hepatitis C (CHC) virus infection affects more than 170 million people globally. The aim of treatment of CHC is to effect sustained elimination of the virus (a sustained virological response, SVR). Prior to the development of direct-acting antiviral (DAA) agents, the standard of care (SOC) for CHC comprised combined treatment with pegylated interferon (PegIFN) and ribavirin (RBV).
METHODS
TMC435 (Tibotec pharmaceuticals) is a macrocyclic non-covalent NS3/NS4A protease inhibitor (DAA) that is currently in Phase III clinical development. TMC435 is being developed in treatment regimens both with and without PegIFN and RBV. In Phase IIb clinical trials, the addition of TMC435 to current SOC significantly increased the SVR rate in both treatment-naive and experienced patients with CHC. It differs, however, from the other first-generation protease inhibitors in that it is administered once daily, has a different tolerability and resistance profile and has activity against CHC genotypes 1 – 6 with the exception of genotype 3. Furthermore, the addition of TMC435 to PegIFN/RBV appears to be able to significantly shorten treatment duration in the majority of patients. This article will review the pharmacology, pharmacodynamics, safety and efficacy of TMC435 by evaluating the preclinical and clinical studies to date.
CONCLUSIONS
TMC435 is a ‘second-wave’ protease inhibitor that has the potential to play a pivotal role in the next phase of CHC treatment. The forthcoming results of Phase III trials involving TMC435 are awaited with interest.
Sudeep Tanwar; Paul M Trembling; Geoffrey M Dusheiko


Short communication prevalence of susceptibility to etravirine by genotype and phenotype in samples received for routine HIV type 1 resistance testing in the United States.

Journal: AIDS research and human retrovirusesFebruar/22/2012
Abstract The prevalence of susceptibility to etravirine was investigated among clinical samples submitted for routine clinical testing in the United States using two separate weighted genotypic scoring systems. The presence of etravirine mutations and susceptibility to etravirine by phenotype of clinical samples from HIV-1-infected patients, submitted to Monogram Biosciences for routine resistance testing between June 2008 and June 2009, were analyzed. Susceptibility by genotype was determined using the Monogram and Tibotec etravirine-weighted genotypic scoring systems, with scores of ≤3 and ≤2, respectively, indicating full susceptibility. Susceptibility by phenotype was determined using the PhenoSense HIV assay, with lower and higher clinical cut-offs of 2.9 and 10, respectively. The frequency of individual etravirine mutations and the impact of the K103N mutation on susceptibility to etravirine by genotype were also determined. Among the 5482 samples with ≥1 defined nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations associated with resistance, 67% were classed as susceptible to etravirine by genotype by both scoring systems. Susceptibility to etravirine by phenotype was higher (76%). The proportion of first-generation NNRTI-resistant samples with (n=3598) and without (n=1884) K103N with susceptibility to etravirine by genotype was 77% and 49%, respectively. Among samples susceptible to first-generation NNRTIs (n=9458), >99% of samples were susceptible to etravirine by phenotype (FC <2.9); the remaining samples had FC ≥2.9-10. In summary, among samples submitted for routine clinical testing in the United States, a high proportion of samples with first-generation NNRTI resistance was susceptible to etravirine by genotype and phenotype. A higher proportion of NNRTI-resistant samples with K103N than without was susceptible to etravirine.
Gaston Picchio; Johan Vingerhoets; Lotke Tambuyzer; Eoin Coakley; Mojgan Haddad; James Witek


Response-guided telaprevir combination treatment for hepatitis C virus infection.
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Journal: The New England journal of medicineSeptember/26/2011
BACKGROUND
Patients with chronic infection with hepatitis C virus (HCV) genotype 1 often need 48 weeks of peginterferon-ribavirin treatment for a sustained virologic response. We designed a noninferiority trial (noninferiority margin, -10.5%) to compare rates of sustained virologic response among patients receiving two treatment durations.
METHODS
We enrolled patients with chronic infection with HCV genotype 1 who had not previously received treatment. All patients received telaprevir at a dose of 750 mg every 8 hours, peginterferon alfa-2a at a dose of 180 μg per week, and ribavirin at a dose of 1000 to 1200 mg per day, for 12 weeks (T12PR12), followed by peginterferon-ribavirin. Patients who had an extended rapid virologic response (undetectable HCV RNA levels at weeks 4 and 12) were randomly assigned after week 20 to receive the dual therapy for 4 more weeks (T12PR24) or 28 more weeks (T12PR48). Patients without an extended rapid virologic response were assigned to T12PR48.
RESULTS
Of the 540 patients, a total of 352 (65%) had an extended rapid virologic response. The overall rate of sustained virologic response was 72%. Among the 322 patients with an extended rapid virologic response who were randomly assigned to a study group, 149 (92%) in the T12PR24 group and 140 (88%) in the T12PR48 group had a sustained virologic response (absolute difference, 4 percentage points; 95% confidence interval, -2 to 11), establishing noninferiority. Adverse events included rash (in 37% of patients, severe in 5%) and anemia (in 39%, severe in 6%). Discontinuation of all the study drugs was based on adverse events in 18% of patients overall, as well as in 1% of patients (all of whom were randomly assigned) in the T12PR24 group and 12% of the patients randomly assigned to the T12PR48 group (P<0.001).
CONCLUSIONS
In this study, among patients with chronic HCV infection who had not received treatment previously, a regimen of peginterferon-ribavirin for 24 weeks, with telaprevir for the first 12 weeks, was noninferior to the same regimen for 48 weeks in patients with undetectable HCV RNA at weeks 4 and 12, with an extended rapid virologic response achieved in nearly two thirds of patients. (Funded by Vertex Pharmaceuticals and Tibotec; ILLUMINATE ClinicalTrials.gov number, NCT00758043.).
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3809077/bin/nihms371976f1.jpg
Kenneth E Sherman; Steven L Flamm; Nezam H Afdhal; David R Nelson; Mark S Sulkowski; Gregory T Everson+11 authors

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Rilpivirine versus efavirenz with two background nucleoside or nucleotide reverse transcriptase inhibitors in treatment-naive adults infected with HIV-1 (THRIVE): a phase 3, randomised, non-inferiority trial.

Journal: Lancet (London, England)Juli/28/2011
BACKGROUND
The non-nucleoside reverse transcriptase inhibitor (NNRTI), rilpivirine (TMC278; Tibotec Pharmaceuticals, County Cork, Ireland), had equivalent sustained efficacy to efavirenz in a phase 2b trial in treatment-naive patients infected with HIV-1, but fewer adverse events. We aimed to assess non-inferiority of rilpivirine to efavirenz in a phase 3 trial with common background nucleoside or nucleotide reverse transcriptase inhibitors (N[t]RTIs).
METHODS
We undertook a 96-week, phase 3, randomised, double-blind, double-dummy, non-inferiority trial in 98 hospitals or medical centres in 21 countries. We enrolled adults (≥18 years) not previously given antiretroviral therapy and with a screening plasma viral load of 5000 copies per mL or more and viral sensitivity to background N(t)RTIs. We randomly allocated patients (1:1) using a computer-generated interactive web-response system to receive oral rilpivirine 25 mg once daily or efavirenz 600 mg once daily; all patients received an investigator-selected regimen of background N(t)RTIs (tenofovir-disoproxil-fumarate plus emtricitabine, zidovudine plus lamivudine, or abacavir plus lamivudine). The primary outcome was non-inferiority (12% margin on logistic regression analysis) at 48 weeks in terms of confirmed response (viral load <50 copies per mL, defined by the intent-to-treat time to loss of virologic response [TLOVR] algorithm) in all patients who received at least one dose of study drug. This study is registered with ClinicalTrials.gov, number NCT00543725.
RESULTS
From May 22, 2008, we screened 947 patients and enrolled 340 to each group. 86% of patients (291 of 340) who received at least one dose of rilpivirine responded, compared with 82% of patients (276 of 338) who received at least one dose of efavirenz (difference 3.5% [95% CI -1.7 to 8.8]; p(non-inferiority)<0.0001). Increases in CD4 cell counts were much the same between groups. 7% of patients (24 of 340) receiving rilpivirine had a virological failure compared with 5% of patients (18 of 338) receiving efavirenz. 4% of patients (15) in the rilpivirine group and 7% (25) in the efavirenz group discontinued treatment due to adverse events. Grade 2-4 treatment-related adverse events were less common with rilpivirine (16% [54 patients]) than they were with efavirenz (31% [104]; p<0.0001), as were rash and dizziness (p<0.0001 for both) and increases in lipid levels were significantly lower with rilpivirine than they were with efavirenz (p<0.0001).
CONCLUSIONS
Despite a slightly increased incidence of virological failures, a favourable safety profile and non-inferior efficacy compared with efavirenz means that rilpivirine could be a new treatment option for treatment-naive patients infected with HIV-1.
BACKGROUND
Tibotec.
Calvin J Cohen; Jaime Andrade-Villanueva; Bonaventura Clotet; Jan Fourie; Margaret A Johnson; Kiat Ruxrungtham