Publications by authors named "Asim Siddiqui"

54 Publications

Single-cell RNA-seq reveals ectopic and aberrant lung-resident cell populations in idiopathic pulmonary fibrosis.

Sci Adv 2020 Jul 8;6(28):eaba1983. Epub 2020 Jul 8.

Section of Pulmonary, Critical Care and Sleep Medicine, Yale School of Medicine, New Haven, CT, USA.

We provide a single-cell atlas of idiopathic pulmonary fibrosis (IPF), a fatal interstitial lung disease, by profiling 312,928 cells from 32 IPF, 28 smoker and nonsmoker controls, and 18 chronic obstructive pulmonary disease (COPD) lungs. Among epithelial cells enriched in IPF, we identify a previously unidentified population of aberrant basaloid cells that coexpress basal epithelial, mesenchymal, senescence, and developmental markers and are located at the edge of myofibroblast foci in the IPF lung. Among vascular endothelial cells, we identify an ectopically expanded cell population transcriptomically identical to bronchial restricted vascular endothelial cells in IPF. We confirm the presence of both populations by immunohistochemistry and independent datasets. Among stromal cells, we identify IPF myofibroblasts and invasive fibroblasts with partially overlapping cells in control and COPD lungs. Last, we confirm previous findings of profibrotic macrophage populations in the IPF lung. Our comprehensive catalog reveals the complexity and diversity of aberrant cellular populations in IPF.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciadv.aba1983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7439502PMC
July 2020

Rapid, deep and precise profiling of the plasma proteome with multi-nanoparticle protein corona.

Nat Commun 2020 07 22;11(1):3662. Epub 2020 Jul 22.

Seer, Inc., Redwood City, CA, 94065, USA.

Large-scale, unbiased proteomics studies are constrained by the complexity of the plasma proteome. Here we report a highly parallel protein quantitation platform integrating nanoparticle (NP) protein coronas with liquid chromatography-mass spectrometry for efficient proteomic profiling. A protein corona is a protein layer adsorbed onto NPs upon contact with biofluids. Varying the physicochemical properties of engineered NPs translates to distinct protein corona patterns enabling differential and reproducible interrogation of biological samples, including deep sampling of the plasma proteome. Spike experiments confirm a linear signal response. The median coefficient of variation was 22%. We screened 43 NPs and selected a panel of 5, which detect more than 2,000 proteins from 141 plasma samples using a 96-well automated workflow in a pilot non-small cell lung cancer classification study. Our streamlined workflow combines depth of coverage and throughput with precise quantification based on unique interactions between proteins and NPs engineered for deep and scalable quantitative proteomic studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-17033-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376165PMC
July 2020

Rab7 of Plasmodium falciparum is involved in its retromer complex assembly near the digestive vacuole.

Biochim Biophys Acta Gen Subj 2020 10 5;1864(10):129656. Epub 2020 Jun 5.

Division of Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, West Bengal, India. Electronic address:

Background: Intracellular protein trafficking is crucial for survival of cell and proper functioning of the organelles; however, these pathways are not well studied in the malaria parasite. Its unique cellular architecture and organellar composition raise an interesting question to investigate.

Methods: The interaction of Plasmodium falciparum Rab7 (PfRab7) with vacuolar protein sorting-associated protein 26 (PfVPS26) of retromer complex was shown by coimmunoprecipitation (co-IP). Confocal microscopy was used to show the localization of the complex in the parasite with respect to different organelles. Further chemical tools were employed to explore the role of digestive vacuole (DV) in retromer trafficking in parasite and GTPase activity of PfRab7 was examined.

Results: PfRab7 was found to be interacting with retromer complex that assembled mostly near DV and the Golgi in trophozoites. Chemical disruption of DV by chloroquine (CQ) led to its disassembly that was further validated by using compound 5f, a heme polymerization inhibitor in the DV. PfRab7 exhibited Mg dependent weak GTPase activity that was inhibited by a specific Rab7 GTPase inhibitor, CID 1067700, which prevented the assembly of retromer complex in P. falciparum and inhibited its growth suggesting the role of GTPase activity of PfRab7 in retromer assembly.

Conclusion: Retromer complex was found to be interacting with PfRab7 and the functional integrity of the DV was found to be important for retromer assembly in P. falciparum.

General Significance: This study explores the retromer trafficking in P. falciparum and describes amechanism to validate DV targeting antiplasmodial molecules.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbagen.2020.129656DOI Listing
October 2020

Indomethacin impairs mitochondrial dynamics by activating the PKCζ-p38-DRP1 pathway and inducing apoptosis in gastric cancer and normal mucosal cells.

J Biol Chem 2019 05 2;294(20):8238-8258. Epub 2019 Apr 2.

Division of Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, Kolkata, West Bengal 700032. Electronic address:

The subcellular mechanism by which nonsteroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in gastric cancer and normal mucosal cells is elusive because of the diverse cyclooxygenase-independent effects of these drugs. Using human gastric carcinoma cells (AGSs) and a rat gastric injury model, here we report that the NSAID indomethacin activates the protein kinase Cζ (PKCζ)-p38 MAPK (p38)-dynamin-related protein 1 (DRP1) pathway and thereby disrupts the physiological balance of mitochondrial dynamics by promoting mitochondrial hyper-fission and dysfunction leading to apoptosis. Notably, DRP1 knockdown or SB203580-induced p38 inhibition reduced indomethacin-induced damage to AGSs. Indomethacin impaired mitochondrial dynamics by promoting fissogenic activation and mitochondrial recruitment of DRP1 and down-regulating fusogenic optic atrophy 1 (OPA1) and mitofusins in rat gastric mucosa. Consistent with OPA1 maintaining cristae architecture, its down-regulation resulted in EM-detectable cristae deformity. Deregulated mitochondrial dynamics resulting in defective mitochondria were evident from enhanced Parkin expression and mitochondrial proteome ubiquitination. Indomethacin ultimately induced mitochondrial metabolic and bioenergetic crises in the rat stomach, indicated by compromised fatty acid oxidation, reduced complex I- associated electron transport chain activity, and ATP depletion. Interestingly, Mdivi-1, a fission-preventing mito-protective drug, reversed indomethacin-induced DRP1 phosphorylation on Ser-616, mitochondrial proteome ubiquitination, and mitochondrial metabolic crisis. Mdivi-1 also prevented indomethacin-induced mitochondrial macromolecular damage, caspase activation, mucosal inflammation, and gastric mucosal injury. Our results identify mitochondrial hyper-fission as a critical and common subcellular event triggered by indomethacin that promotes apoptosis in both gastric cancer and normal mucosal cells, thereby contributing to mucosal injury.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA118.004415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6527165PMC
May 2019

Hydrazonophenol, a Food Vacuole-Targeted and Ferriprotoporphyrin IX-Interacting Chemotype Prevents Drug-Resistant Malaria.

ACS Infect Dis 2019 01 7;5(1):63-73. Epub 2018 Dec 7.

Division of Infectious Diseases and Immunology , CSIR - Indian Institute of Chemical Biology , 4 Raja S. C. Mullick Road , Jadavpur, Kolkata 700032 , West Bengal , India.

The rapid emergence of resistance against frontline antimalarial drugs essentially warrants the identification of new-generation antimalarials. Here, we describe the synthesis of ( E)-2-isopropyl-5-methyl-4-((2-(pyridin-4-yl)hydrazono)methyl)phenol (18), which binds ferriprotoporphyrin-IX (Fe-PPIX) ( K = 33 nM) and offers antimalarial activity against chloroquine-resistant and sensitive strains of Plasmodium falciparum in vitro. Structure-function analysis reveals that compound 18 binds Fe-PPIX through the -C═N-NH- moiety and 2-pyridyl substitution at the hydrazine counterpart plays a critical role in antimalarial efficacy. Live cell confocal imaging using a fluorophore-tagged compound confirms its accumulation inside the acidic food vacuole (FV) of P. falciparum. Furthermore, this compound concentration-dependently elevates the pH in FV, implicating a plausible interference with Fe-PPIX crystallization (hemozoin formation) by a dual function: increasing the pH and binding free Fe-PPIX. Different off-target bioassays reduce the possibility of the promiscuous nature of compound 18. Compound 18 also exhibits potent in vivo antimalarial activity against chloroquine-resistant P. yoelii and P. berghei ANKA (causing cerebral malaria) in mice with negligible toxicity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsinfecdis.8b00178DOI Listing
January 2019

Macrophage migration inhibitory factor regulates mitochondrial dynamics and cell growth of human cancer cell lines through CD74-NF-κB signaling.

J Biol Chem 2018 12 26;293(51):19740-19760. Epub 2018 Oct 26.

From the Division of Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, Jadavpur, Kolkata 700032, West Bengal, India

The indispensable role of macrophage migration inhibitory factor (MIF) in cancer cell proliferation is unambiguous, although which specific roles the cytokine plays to block apoptosis by preserving cell growth is still obscure. Using different cancer cell lines (AGS, HepG2, HCT116, and HeLa), here we report that the silencing of MIF severely deregulated mitochondrial structural dynamics by shifting the balance toward excess fission, besides inducing apoptosis with increasing sub-G cells. Furthermore, enhanced mitochondrial Bax translocation along with cytochrome release, down-regulation of Bcl-xL, and Bcl-2 as well as up-regulation of Bad, Bax, and p53 indicated the activation of a mitochondrial pathway of apoptosis upon MIF silencing. The data also indicate a concerted down-regulation of Opa1 and Mfn1 along with a significant elevation of Drp1, cumulatively causing mitochondrial fragmentation upon MIF silencing. Up-regulation of Drp1 was found to be further coupled with fissogenic serine 616 phosphorylation and serine 637 dephosphorylation, thus ensuring enhanced mitochondrial translocation. Interestingly, MIF silencing was found to be associated with decreased NF-κB activation. In fact, NF-κB knockdown in turn increased mitochondrial fission and cell death. In addition, the silencing of CD74, the cognate receptor of MIF, remarkably increased mitochondrial fragmentation in addition to preventing cell proliferation, inducing mitochondrial depolarization, and increasing apoptotic cell death. This indicates the active operation of a MIF-regulated CD74-NF-κB signaling axis for maintaining mitochondrial stability and cell growth. Thus, we propose that MIF, through CD74, constitutively activates NF-κB to control mitochondrial dynamics and stability for promoting carcinogenesis via averting apoptosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA118.003935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314129PMC
December 2018

Intravascular Papillary Endothelial Hyperplasia (Masson's Tumor) Involving the Knee Synovium.

J Orthop Case Rep 2018 Mar-Apr;8(2):23-25

Department of Trauma and Orthopaedic, Calderdale and Huddersfield NHS Trust, UK.

Introduction: Intravascular papillary endothelial hyperplasia or Masson's tumor is a rare benign lesion of vascular origin characterized by abnormal proliferation of endothelial cells.

Case Report: We present a case of this lesion involving the knee joint of a Caucasian woman who was referred to our institution for the evaluation of recurrent right knee pain. Her symptoms have started insidiously 5 years earlier following minor trauma. Recurrent symptomatic knee effusion was the cardinal symptom. Following the surgical excision of the lesion, recurrence occurred 2 years later. The tumor was successfully treated with embolization of its vascular supply.

Conclusion: Masson's tumor involving the synovium is an uncommon condition. The intermittent symptomatology and normal appearance of biopsies taken arthroscopically during asymptomatic periods can make the diagnosis challenging. Although resection is proposed to prevent recurrence, embolization of the vascular supply of the tumor can be a viable alternative.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.13107/jocr.2250-0685.1032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6114222PMC
September 2018

Detection of retromer assembly in Plasmodium falciparum by immunosensing coupled to Surface Plasmon Resonance.

Biochim Biophys Acta Proteins Proteom 2018 May - Jun;1866(5-6):722-730. Epub 2018 Apr 11.

Division of Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, West Bengal, India. Electronic address:

Retromer complex plays a crucial role in intracellular protein trafficking and is conserved throughout the eukaryotes including malaria parasite, Plasmodium falciparum, where it is partially conserved. The assembly of retromer complex in RBC stages of malarial parasite is extremely difficult to explore because of its complicated physiology, small size, and intra-erythrocytic location. Nonetheless, understanding of retromer assembly may pave new ways for the development of novel antimalarials targeting parasite-specific protein trafficking pathways. Here, we investigated the assembly of retromer complex in P. falciparum, by an immunosensing method through highly sensitive Surface Plasmon Resonance (SPR) technique. After taking leads from the bioinformatics search and literature, different interacting proteins were identified and specific antibodies were raised against them. The sensor chip was prepared by covalently linking antibody specific to one component and the whole cell lysate was passed through it in order to trap the interacting complex. Antibodies raised against other interacting components were used to detect them in the trapped complex on the SPR chip. We were able to detect three different components in the retromer complex trapped by the immobilized antibody specific against a different component on a sensor chip. The assay was reproduced and validated in a different two-component CD74-MIF system in mammalian cells. We, thus, illustrate the assembly of retromer complex in P. falciparum through a bio-sensing approach that combines SPR with immunosensing requiring a very small amount of sample from the native source.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbapap.2018.04.005DOI Listing
July 2018

Acute mental stress induces mitochondrial bioenergetic crisis and hyper-fission along with aberrant mitophagy in the gut mucosa in rodent model of stress-related mucosal disease.

Free Radic Biol Med 2017 12 7;113:424-438. Epub 2017 Oct 7.

Division of Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, West Bengal, India. Electronic address:

Psychological stress, depression and anxiety lead to multiple organ dysfunctions wherein stress-related mucosal disease (SRMD) is common to people experiencing stress and also occur as a side effect in patients admitted to intensive care units; however the underlying molecular aetiology is still obscure. We report that in rat-SRMD model, cold restraint-stress severely damaged gut mitochondrial functions to generate superoxide anion (O), depleted ATP and shifted mitochondrial fission-fusion dynamics towards enhanced fission to induce mucosal injury. Activation of mitophagy to clear damaged and fragmented mitochondria was evident from mitochondrial translocation of Parkin and PINK1 along with enhanced mitochondrial proteome ubiquitination, depletion of mitochondrial DNA copy number and TOM 20. However, excess and sustained accumulation of O-generating defective mitochondria overpowered the mitophagic machinery, ultimately triggering Bax-dependent apoptosis and NF-κB-intervened pro-inflammatory mucosal injury. We further observed that stress-induced enhanced serum corticosterone stimulated mitochondrial recruitment of glucocorticoid receptor (GR), which contributed to gut mitochondrial dysfunctions as documented from reduced ETC complex 1 activity, mitochondrial O accumulation, depolarization and hyper-fission. GR-antagonism by RU486 or specific scavenging of mitochondrial O by a mitochondrially targeted antioxidant mitoTEMPO ameliorated stress-induced mucosal damage. Gut mitopathology and mucosal injury were also averted when the perception of mental stress was blocked by pre-treatment with a sedative or antipsychotic. Altogether, we suggest the role of mitochondrial GR-O-fission cohort in brain-mitochondria cross-talk during acute mental stress and advocate the utilization of this pathway as a potential target to prevent mitochondrial unrest and gastropathy bypassing central nervous system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.freeradbiomed.2017.10.009DOI Listing
December 2017

Selective scavenging of intra-mitochondrial superoxide corrects diclofenac-induced mitochondrial dysfunction and gastric injury: A novel gastroprotective mechanism independent of gastric acid suppression.

Biochem Pharmacol 2016 Dec 28;121:33-51. Epub 2016 Sep 28.

Division of Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, West Bengal, India. Electronic address:

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to treat multiple inflammatory diseases and pain but severe gastric mucosal damage is the worst outcome of NSAID-therapy. Here we report that mitoTEMPO, a mitochondrially targeted superoxide (O) scavenger protected as well as healed gastric injury induced by diclofenac (DCF), the most commonly used NSAID. Common existing therapy against gastric injury involves suppression of gastric acid secretion by proton pump inhibitors and histamine H receptor antagonists; however, dyspepsia, vitamin B12 deficiency and gastric microfloral dysbalance are the major drawbacks of acid suppression. Interestingly, mitoTEMPO did not inhibit gastric acid secretion but offered gastroprotection by preventing DCF-induced generation of O due to mitochondrial respiratory chain failure and by preventing mitochondrial oxidative stress (MOS)-mediated mitopathology. MitoTEMPO even restored DCF-stimulated reduced fatty acid oxidation, mitochondrial depolarization and bioenergetic crisis in gastric mucosa. MitoTEMPO also prevented the activation of mitochondrial pathway of apoptosis and MOS-mediated proinflammatory signaling through NF-κB by DCF. Furthermore, mitoTEMPO when administered in rats with preformed gastric lesions expedited the healing of gastric injury and the healed stomach exhibited its normal physiology as evident from gastric acid and pepsin secretions under basal or stimulated conditions. Thus, in contrast to the existing antiulcer drugs, mitochondrially targeted O scavengers like mitoTEMPO may represent a novel class of gastroprotective molecules that does not affect gastric acid secretion and may be used in combination with DCF, keeping its anti-inflammatory action intact, while reducing its gastrodamaging effects.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bcp.2016.09.027DOI Listing
December 2016

Antimalarial Activity of Small-Molecule Benzothiazole Hydrazones.

Antimicrob Agents Chemother 2016 07 20;60(7):4217-28. Epub 2016 Jun 20.

Division of Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, Jadavpur, Kolkata, West Bengal, India

We synthesized a new series of conjugated hydrazones that were found to be active against malaria parasite in vitro, as well as in vivo in a murine model. These hydrazones concentration-dependently chelated free iron and offered antimalarial activity. Upon screening of the synthesized hydrazones, compound 5f was found to be the most active iron chelator, as well as antiplasmodial. Compound 5f also interacted with free heme (KD [equilibrium dissociation constant] = 1.17 ± 0.8 μM), an iron-containing tetrapyrrole released after hemoglobin digestion by the parasite, and inhibited heme polymerization by parasite lysate. Structure-activity relationship studies indicated that a nitrogen- and sulfur-substituted five-membered aromatic ring present within the benzothiazole hydrazones might be responsible for their antimalarial activity. The dose-dependent antimalarial and heme polymerization inhibitory activities of the lead compound 5f were further validated by following [(3)H]hypoxanthine incorporation and hemozoin formation in parasite, respectively. It is worth mentioning that compound 5f exhibited antiplasmodial activity in vitro against a chloroquine/pyrimethamine-resistant strain of Plasmodium falciparum (K1). We also evaluated in vivo antimalarial activity of compound 5f in a murine model where a lethal multiple-drug-resistant strain of Plasmodium yoelii was used to infect Swiss albino mice. Compound 5f significantly suppressed the growth of parasite, and the infected mice experienced longer life spans upon treatment with this compound. During in vitro and in vivo toxicity assays, compound 5f showed minimal alteration in biochemical and hematological parameters compared to control. In conclusion, we identified a new class of hydrazone with therapeutic potential against malaria.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.01575-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4914622PMC
July 2016

Expression, purification and characterization of Plasmodium falciparum vacuolar protein sorting 29.

Protein Expr Purif 2016 Apr 12;120:7-15. Epub 2015 Dec 12.

Department of Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, West Bengal, India. Electronic address:

Translocation of various proteins to the subcellular organelles is an essential mechanism to regulate the metabolic pathways and often vacuolar protein sorting (VPS) proteins are involved in this transportation. Plasmodium falciparum VPS29 (PfVPS29) is predicted to be a functional component in the assembly of the retromer complex; however, so far detailed characterization of PfVPS29 in its native form is not yet done. We report the successful expression and purification of tag-free recombinant PfVPS29 with a yield of 5.6 mg from 1 L of Escherichia coli culture. PfVPS29 was purified by combined anion-exchange and size exclusion chromatography. The protein showed a single band in SDS-PAGE and it exhibited molecular mass of 21.7 kDa as measured by MALDI-TOF mass spectrometry. Secondary structure was elucidated by circular dichroism spectroscopy. It was found to be a monomeric protein in solution as evident from dynamic light scattering studies, chemical cross-linking experiments and size exclusion chromatography. Subsequently, polyclonal anti-PfVPS29 antibody was generated and used for evaluating protein expression by western blot and following subcellular localization in P. falciparum by confocal immunofluoroscence microscopy. PfVPS29 was found to be located in cytoplasm and expressed from early trophozoite to schizont stages with maximum expression in trophozoite stage. This study provides purification, biophysical characterization and subcellular localization of PfVPS29 in different asexual stages of P. falciparum.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.pep.2015.12.004DOI Listing
April 2016

Quantifying the problem of kneeling after a two incision bone tendon bone arthroscopic anterior cruciate ligament reconstruction.

Muscles Ligaments Tendons J 2015 Jul-Sep;5(3):181-6. Epub 2015 Oct 20.

Department of Orthopaedics and Trauma Surgery, Calderdale and Huddersfield Foundation NHS Trust, Huddersfield, UK.

Introduction: the aims of this study was to investigate the post-operative incidence of anterior knee pain and quantify the problem of kneeling in patients who have underwent anterior cruciate ligament (ACL) reconstruction with a bone tendon bone (BTB) graft.

Methods: prospective study of 71 male patients who participated in competitive sports and underwent BTB ACL reconstruction using a two incision approach between August 2008 and May 2011. The patella defect was packed with bone graft, and the peritenon was preserved and repaired. A questionnaire was used to evaluate pain and kneeling capability. All patients had pre and post operative Lysholm/Tegner scores, KT1000 evaluation and hop tests to assess knee stability and function.

Results: 71 patients were operated and had a follow up of 42 months, mean age 29.8. 22 patients had anterior knee pain on kneeling, paraesthesia of anterior knee was found in 23 patients. 65 patients were still able to kneel and 6 found they were unable. 36 were able to kneel for unrestricted periods, 9 for 5-15 minutes, 15 kneel for 1-5 minutes and 5 for >1 minute. Anterior knee pain was compared to kneeling time (P=0.001). Paraesthesia and kneeling time, (P=0.001). Anterior knee pain when compared with Lysholm score (P=0.540), hop test (P=0.277), and Lachman's (P=0.254).

Conclusions: two incision BTB grafting of the patella and repair of the paritenon minimises the length of scar at the front of the knee. This reduces any palpable defects which could be causation factor for pain whilst kneeling. We have quantified kneeling and pain, thus aiding patients and surgeons in making the right decision for graft choice for ACL reconstruction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.11138/mltj/2015.5.3.181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4617218PMC
November 2015

Ellagic Acid, a Dietary Polyphenol, Inhibits Tautomerase Activity of Human Macrophage Migration Inhibitory Factor and Its Pro-inflammatory Responses in Human Peripheral Blood Mononuclear Cells.

J Agric Food Chem 2015 May 15;63(20):4988-98. Epub 2015 May 15.

†Division of Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Jadavpur, Kolkata 700032, West Bengal, India.

Ellagic acid (EA), a phenolic lactone, inhibited tautomerase activity of human macrophage migration inhibitory factor (MIF) noncompetitively (Ki = 1.97 ± 0.7 μM). The binding of EA to MIF was determined by following the quenching of tryptophan fluorescence. We synthesized several EA derivatives, and their structure-activity relationship studies indicated that the planar conjugated lactone moiety of EA was essential for MIF inhibition. MIF induces nuclear translocation of NF-κB and chemotaxis of peripheral blood mononuclear cells (PBMCs) to promote inflammation. We were interested in evaluating the effect of EA on nuclear translocation of NF-κB and chemotactic activity in human PBMCs in the presence of MIF. The results showed that EA inhibited MIF-induced NF-κB nuclear translocation in PBMCs, as evident from confocal immunofluorescence microscopic data. EA also inhibited MIF-mediated chemotaxis of PBMCs. Thus, we report MIF-inhibitory activity of EA and inhibition of MIF-mediated proinflammatory responses in PBMCs by EA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jafc.5b00921DOI Listing
May 2015

Expanding the scope of noninvasive prenatal testing: detection of fetal microdeletion syndromes.

Am J Obstet Gynecol 2015 Mar 2;212(3):332.e1-9. Epub 2014 Dec 2.

Division of Human Genetics, Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT. Electronic address:

Objective: The purpose of this study was to estimate the performance of a single-nucleotide polymorphism (SNP)-based noninvasive prenatal test for 5 microdeletion syndromes.

Study Design: Four hundred sixty-nine samples (358 plasma samples from pregnant women, 111 artificial plasma mixtures) were amplified with the use of a massively multiplexed polymerase chain reaction, sequenced, and analyzed with the use of the Next-generation Aneuploidy Test Using SNPs algorithm for the presence or absence of deletions of 22q11.2, 1p36, distal 5p, and the Prader-Willi/Angelman region.

Results: Detection rates were 97.8% for a 22q11.2 deletion (45/46) and 100% for Prader-Willi (15/15), Angelman (21/21), 1p36 deletion (1/1), and cri-du-chat syndromes (24/24). False-positive rates were 0.76% for 22q11.2 deletion syndrome (3/397) and 0.24% for cri-du-chat syndrome (1/419). No false positives occurred for Prader-Willi (0/428), Angelman (0/442), or 1p36 deletion syndromes (0/422).

Conclusion: SNP-based noninvasive prenatal microdeletion screening is highly accurate. Because clinically relevant microdeletions and duplications occur in >1% of pregnancies, regardless of maternal age, noninvasive screening for the general pregnant population should be considered.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajog.2014.11.041DOI Listing
March 2015

Humoral immune responses to a recombinant Plasmodium vivax tryptophan-rich antigen among Plasmodium vivax-infected patients and its localization in the parasite.

Appl Biochem Biotechnol 2015 Feb 3;175(4):2166-77. Epub 2014 Dec 3.

Community Health Sciences Department, College of Applied Medical Sciences, King Saud University, P.O. Box 10219, Riyadh, 11433, Kingdom of Saudi Arabia,

Our recent studies have focused on the identification and characterization of the tryptophan-rich proteins of the Plasmodium vivax parasite where their role in the elicitation of humoral and cellular responses and erythrocyte-binding activity was investigated. Here, we report the humoral responses of a 32.4-kDa P. vivax tryptophan-rich antigen (PvTRAg32.4) among the sera of P. vivax-infected patients. PvTRAg32.4 also contains an unusually high percentage of tryptophan residues (10.7 %) that are positionally conserved with its orthologues in Plasmodium yoelii (PypAg1 and PypAg2) and Plasmodium falciparum (PfTryThrA and PfMATRA). Thirty-four of the 40 (85.0 %) P. vivax isolates showed seropositivity to recombinant PvTRAg32.4 by ELISA. The mean ± SD values of optical density (OD) for P. vivax subjects and naïve individuals were 1.02 ± 0.36 and 0.26 ± 0.11, respectively. In the Western blot analysis, majority of the subjects studied (n = 44) showed reactivity to the recombinant, purified PvTRAg32.4. This antigen does not show binding to the erythrocytes, but the immunofluorescence data reveals that it is expressed in the erythrocytic stages of the parasite. Sequence analysis of the clinical isolates from various parts of the country shows that PvTRAg32.4 is highly conserved. Functional in-depth characterization of more such type of novel proteins in the parasite is warranted for the development of successful malaria intervention methods.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12010-014-1428-7DOI Listing
February 2015

Single-nucleotide polymorphism-based noninvasive prenatal screening in a high-risk and low-risk cohort.

Obstet Gynecol 2014 Aug;124(2 Pt 1):210-218

Northwestern Reproductive Genetics, Chicago, Illinois; Columbia University, New York, New York; and Natera Inc, San Carlos, California.

Objective: To estimate performance of a single-nucleotide polymorphism-based noninvasive prenatal screen for fetal aneuploidy in high-risk and low-risk populations on single venopuncture.

Methods: One thousand sixty-four maternal blood samples from 7 weeks of gestation and beyond were included; 1,051 were within specifications and 518 (49.3%) were low risk. Cell-free DNA was amplified, sequenced, and analyzed using the Next-generation Aneuploidy Test Using SNPs algorithm. Samples were called as trisomies 21, 18, 13, or monosomy X, or euploid, and male or female.

Results: Nine hundred sixty-six samples (91.9%) successfully generated a cell-free DNA result. Among these, sensitivity was 100% for trisomy 21 (58/58, confidence interval [CI] 93.8-100%), trisomy 13 (12/12, CI 73.5-100%), and fetal sex (358/358 female, CI 99.0-100%; 418/418 male, CI 99.1-100%), 96.0% for trisomy 18 (24/25, CI 79.7-99.9%), and 90% for monosomy X (9/10, CI 55.5-99.8%). Specificity for trisomies 21 and 13 was 100% (905/905, CI 99.6-100%; and 953/953, CI 99.6-100%, respectively) and for trisomy 18 and monosomy X was 99.9% (938/939, CI 99.4-100%; and 953/954, CI 99.4-100%, respectively). However, 16% (20/125) of aneuploid samples did not return a result; 50% (10/20) had a fetal fraction below the 1.5th percentile of euploid pregnancies. Aneuploidy rate was significantly higher in these samples (P<.001, odds ratio 9.2, CI 4.4-19.0). Sensitivity and specificity did not differ in low-risk and high-risk populations.

Conclusions: This noninvasive prenatal screen performed with high sensitivity and specificity in high-risk and low-risk cohorts. Aneuploid samples were significantly more likely to not return a result; the number of aneuploidy samples was especially increased among samples with low fetal fraction. This underscores the importance of redraws or, in rare cases, invasive procedures based on low fetal fraction.

Level Of Evidence: II.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/AOG.0000000000000363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4144440PMC
August 2014

Effect of pentosans addition on pasting properties of flours of eight hard white spring wheat cultivars.

J Food Sci Technol 2014 Jun 3;51(6):1066-75. Epub 2012 Feb 3.

Department of Food Science & Technology, University of Karachi, Karachi, Pakistan 75270.

The effects of water extractable pentosans (WEP) and water unextractable pentosans (WUP) on pasting properties in flours of eight different hard white spring wheat (HWSW) cultivars was studied. WEP and WUP isolated from a hard wheat flour were added to each of the cultivars at 1% and 2% level. The results indicated that WEP exhibited a pronounced effect on pasting properties as compared to WUP and variety. Univariate analysis of variance (ANOVA) was used to evaluate sources of variation. The variety significantly (P < 0.001) influenced all the pasting parameters. WUP caused significant (P < 0.001) variation in paste viscosities (except breakdown). WEP influenced more pronouncedly the hot paste, cold paste, breakdown and setback viscosities with F values-221.802, 214.286, 98.073 and 120.159, respectively. Variety-by-WEP interaction exhibited significant (P < 0.01) influence on pasting time, peak, hot paste and cold paste viscosities. Whereas, variety-by-WUP interaction only significantly (P < 0.001) influenced the pasting- time and -temperature. Duncan's test was used to analyze the significant difference (P < 0.05) within the variety. The results revealed that WUP did not induce significant (P < 0.05) influence on all the pasting parameters, whereas, WEP influenced significantly (P < 0.05) the paste viscosities of some of the varieties. It was also found that the addition of WEP remarkably reduced the setback, hot paste, cold paste viscosities and increased the breakdown viscosity in all cultivar flours. The effect of WEP was greater at higher level of supplementation on paste viscosities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s13197-012-0629-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4033747PMC
June 2014

Association of heme oxygenase 1 with the restoration of liver function after damage in murine malaria by Plasmodium yoelii.

Infect Immun 2014 Aug 12;82(8):3113-26. Epub 2014 May 12.

Department of Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, Jadavpur, Kolkata, West Bengal, India

The liver efficiently restores function after damage induced during malarial infection once the parasites are cleared from the blood. However, the molecular events leading to the restoration of liver function after malaria are still obscure. To study this, we developed a suitable model wherein mice infected with Plasmodium yoelii (45% parasitemia) were treated with the antimalarial α/β-arteether to clear parasites from the blood and, subsequently, restoration of liver function was monitored. Liver function tests clearly indicated that complete recovery of liver function occurred after 25 days of parasite clearance. Analyses of proinflammatory gene expression and neutrophil infiltration further indicated that hepatic inflammation, which was induced immediately after parasite clearance from the blood, was gradually reduced. Moreover, the inflammation in the liver after parasite clearance was found to be correlated positively with oxidative stress and hepatocyte apoptosis. We investigated the role of heme oxygenase 1 (HO-1) in the restoration of liver function after malaria because HO-1 normally renders protection against inflammation, oxidative stress, and apoptosis under various pathological conditions. The expression and activity of HO-1 were found to be increased significantly after parasite clearance. We even found that chemical silencing of HO-1 by use of zinc protoporphyrin enhanced inflammation, oxidative stress, hepatocyte apoptosis, and liver injury. In contrast, stimulation of HO-1 by cobalt protoporphyrin alleviated liver inflammation and reduced oxidative stress, hepatocyte apoptosis, and associated tissue injury. Therefore, we propose that selective induction of HO-1 in the liver would be beneficial for the restoration of liver function after parasite clearance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/IAI.01598-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4136231PMC
August 2014

Genome and transcriptome sequencing in prospective metastatic triple-negative breast cancer uncovers therapeutic vulnerabilities.

Mol Cancer Ther 2013 Jan 19;12(1):104-16. Epub 2012 Nov 19.

Translational Genomics Research Institute, Phoenix, AZ 85004, USA.

Triple-negative breast cancer (TNBC) is characterized by the absence of expression of estrogen receptor, progesterone receptor, and HER-2. Thirty percent of patients recur after first-line treatment, and metastatic TNBC (mTNBC) has a poor prognosis with median survival of one year. Here, we present initial analyses of whole genome and transcriptome sequencing data from 14 prospective mTNBC. We have cataloged the collection of somatic genomic alterations in these advanced tumors, particularly those that may inform targeted therapies. Genes mutated in multiple tumors included TP53, LRP1B, HERC1, CDH5, RB1, and NF1. Notable genes involved in focal structural events were CTNNA1, PTEN, FBXW7, BRCA2, WT1, FGFR1, KRAS, HRAS, ARAF, BRAF, and PGCP. Homozygous deletion of CTNNA1 was detected in 2 of 6 African Americans. RNA sequencing revealed consistent overexpression of the FOXM1 gene when tumor gene expression was compared with nonmalignant breast samples. Using an outlier analysis of gene expression comparing one cancer with all the others, we detected expression patterns unique to each patient's tumor. Integrative DNA/RNA analysis provided evidence for deregulation of mutated genes, including the monoallelic expression of TP53 mutations. Finally, molecular alterations in several cancers supported targeted therapeutic intervention on clinical trials with known inhibitors, particularly for alterations in the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways. In conclusion, whole genome and transcriptome profiling of mTNBC have provided insights into somatic events occurring in this difficult to treat cancer. These genomic data have guided patients to investigational treatment trials and provide hypotheses for future trials in this irremediable cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1535-7163.MCT-12-0781DOI Listing
January 2013

Fine specificity of Plasmodium vivax Duffy binding protein binding engagement of the Duffy antigen on human erythrocytes.

Infect Immun 2012 Aug 21;80(8):2920-8. Epub 2012 May 21.

Case Western Reserve University, Center for Global Health and Diseases, Cleveland, Ohio, USA.

Plasmodium vivax invasion of human erythrocytes requires interaction of the P. vivax Duffy binding protein (PvDBP) with its host receptor, the Duffy antigen (Fy) on the erythrocyte surface. Consequently, PvDBP is a leading vaccine candidate. The binding domain of PvDBP lies in a cysteine-rich portion of the molecule called region II (PvDBPII). PvDBPII contains three distinct subdomains based upon intramolecular disulfide bonding patterns. Subdomain 2 (SD2) is highly polymorphic and is thought to contain many key residues for binding to Fy, while SD1 and SD3 are comparatively conserved and their role in Fy binding is not well understood. To examine the relative contributions of the different subdomains to binding to Fy and their abilities to elicit strain-transcending binding-inhibitory antibodies, we evaluated recombinant proteins from SD1+2, SD2, SD3, and SD3+, which includes 24 residues of SD2. All of the recombinant subdomains, except for SD2, bound variably to human erythrocytes, with constructs containing SD3 showing the best binding. Antisera raised in laboratory animals against SD3, SD3+, and SD2+3 inhibited the binding of full-length PvDBPII, which is strain transcending, whereas antisera generated to SD1+2 and SD2 failed to generate blocking antibodies. All of the murine monoclonal antibodies generated to full-length PvDBPII that had significant binding-inhibitory activity recognized only SD3. Thus, SD3 binds Fy and elicits blocking antibodies, indicating that it contains residues critical to Fy binding that could be the basis of a strain-transcending candidate vaccine against P. vivax.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/IAI.00206-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3434565PMC
August 2012

RNA-Seq mapping and detection of gene fusions with a suffix array algorithm.

PLoS Comput Biol 2012 5;8(4):e1002464. Epub 2012 Apr 5.

Life Technologies, Foster City, California, United States of America.

High-throughput RNA sequencing enables quantification of transcripts (both known and novel), exon/exon junctions and fusions of exons from different genes. Discovery of gene fusions-particularly those expressed with low abundance- is a challenge with short- and medium-length sequencing reads. To address this challenge, we implemented an RNA-Seq mapping pipeline within the LifeScope software. We introduced new features including filter and junction mapping, annotation-aided pairing rescue and accurate mapping quality values. We combined this pipeline with a Suffix Array Spliced Read (SASR) aligner to detect chimeric transcripts. Performing paired-end RNA-Seq of the breast cancer cell line MCF-7 using the SOLiD system, we called 40 gene fusions among over 120,000 splicing junctions. We validated 36 of these 40 fusions with TaqMan assays, of which 25 were expressed in MCF-7 but not the Human Brain Reference. An intra-chromosomal gene fusion involving the estrogen receptor alpha gene ESR1, and another involving the RPS6KB1 (Ribosomal protein S6 kinase beta-1) were recurrently expressed in a number of breast tumor cell lines and a clinical tumor sample.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pcbi.1002464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3320572PMC
August 2012

Fy(a)/Fy(b) antigen polymorphism in human erythrocyte Duffy antigen affects susceptibility to Plasmodium vivax malaria.

Proc Natl Acad Sci U S A 2011 Dec 28;108(50):20113-8. Epub 2011 Nov 28.

Center for Global Health and Diseases, Case Western Reserve University, Cleveland, OH 44106, USA.

Plasmodium vivax (Pv) is a major cause of human malaria and is increasing in public health importance compared with falciparum malaria. Pv is unique among human malarias in that invasion of erythrocytes is almost solely dependent on the red cell's surface receptor, known as the Duffy blood-group antigen (Fy). Fy is an important minor blood-group antigen that has two immunologically distinct alleles, referred to as Fy(a) or Fy(b), resulting from a single-point mutation. This mutation occurs within the binding domain of the parasite's red cell invasion ligand. Whether this polymorphism affects susceptibility to clinical vivax malaria is unknown. Here we show that Fy(a), compared with Fy(b), significantly diminishes binding of Pv Duffy binding protein (PvDBP) at the erythrocyte surface, and is associated with a reduced risk of clinical Pv in humans. Erythrocytes expressing Fy(a) had 41-50% lower binding compared with Fy(b) cells and showed an increased ability of naturally occurring or artificially induced antibodies to block binding of PvDBP to their surface. Individuals with the Fy(a+b-) phenotype demonstrated a 30-80% reduced risk of clinical vivax, but not falciparum malaria in a prospective cohort study in the Brazilian Amazon. The Fy(a+b-) phenotype, predominant in Southeast Asian and many American populations, would confer a selective advantage against vivax malaria. Our results also suggest that efficacy of a PvDBP-based vaccine may differ among populations with different Fy phenotypes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1109621108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3250126PMC
December 2011

Diagnosis and treatment of greenstick and torus fractures of the distal radius in children: a prospective randomised single blind study.

J Child Orthop 2010 Aug 2;4(4):321-6. Epub 2010 Jul 2.

Objective: The management and the diagnostic modalities used in cases of undisplaced greenstick and torus fractures of the distal radius in children vary between different treatment centres. The aim of this study was twofold: firstly, to analyse the sensitivity of X-rays versus ultrasound to diagnose these fractures; secondly, to compare three available treatment options (plaster cast, Futuro splints, and double Tubigrip) in terms of pain, analgesia requirements, grip strength, deformity, stiffness and interference with a child's activities of daily living.

Methods: We prospectively included 79 patients suffering from undisplaced greenstick and torus fractures of the distal radius. Patients were randomized (single blindly) to the studied treatment groups.

Results: In terms of diagnosis, the ultrasound was found to be more sensitive than X-rays for diagnosing these fractures. Our results also showed that Tubigrip was superior in terms of interference with a child's ADLs, stiffness and grip strength. However, there was no difference in the levels of pain, analgesia required, and deformity.

Conclusion: These results support the idea that ultrasound is an effective and sensitive tool for detecting undisplaced greenstick and torus fractures of the distal radius in children. Treating these fractures with functional nonrigid devices (Tubigrip) results in improved function without increased discomfort or deformity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11832-010-0269-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2908342PMC
August 2010

Tumor transcriptome sequencing reveals allelic expression imbalances associated with copy number alterations.

PLoS One 2010 Feb 19;5(2):e9317. Epub 2010 Feb 19.

Life Technologies Inc., Foster City, California, United States of America.

Due to growing throughput and shrinking cost, massively parallel sequencing is rapidly becoming an attractive alternative to microarrays for the genome-wide study of gene expression and copy number alterations in primary tumors. The sequencing of transcripts (RNA-Seq) should offer several advantages over microarray-based methods, including the ability to detect somatic mutations and accurately measure allele-specific expression. To investigate these advantages we have applied a novel, strand-specific RNA-Seq method to tumors and matched normal tissue from three patients with oral squamous cell carcinomas. Additionally, to better understand the genomic determinants of the gene expression changes observed, we have sequenced the tumor and normal genomes of one of these patients. We demonstrate here that our RNA-Seq method accurately measures allelic imbalance and that measurement on the genome-wide scale yields novel insights into cancer etiology. As expected, the set of genes differentially expressed in the tumors is enriched for cell adhesion and differentiation functions, but, unexpectedly, the set of allelically imbalanced genes is also enriched for these same cancer-related functions. By comparing the transcriptomic perturbations observed in one patient to his underlying normal and tumor genomes, we find that allelic imbalance in the tumor is associated with copy number mutations and that copy number mutations are, in turn, strongly associated with changes in transcript abundance. These results support a model in which allele-specific deletions and duplications drive allele-specific changes in gene expression in the developing tumor.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0009317PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2824832PMC
February 2010

mRNA-sequencing whole transcriptome analysis of a single cell on the SOLiD system.

J Biomol Tech 2009 Dec;20(5):266-71

Applied Biosystems, a division of Life Technologies Corporation, Genetic Analysis Business Unit, Foster City, California 94404, USA.

We have developed a sequencing-based gene expression profiling assay at single-cell resolution by combining a modified single-cell whole transcriptome amplification method with the next generation sequencing technique, the SOLiD system. Using this assay, we have shown that blastomeres in a four-cell stage embryo have similar gene expression, which is compatible with the fact that they have similar developmental potential. We proved that compared with cDNA microarray technique, our single-cell cDNA SOLiD sequencing assay can detect expression of thousands of more genes. Moreover, for the genes detected by microarray and SOLiD sequencing, our assay detected new transcript variants for a large proportion of them, which confirms unambiguously at single-cell resolution that the transcriptome complexity is higher than expected traditionally. Finally, by using our assay to Dicer knockout (KO) and Ago2 KO oocytes, we showed that a significant amount of transposons were up-regulated abnormally in Dicer/Ago2 KO mature oocytes compared with wild-type controls.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2777344PMC
December 2009

mRNA-Seq whole-transcriptome analysis of a single cell.

Nat Methods 2009 May 6;6(5):377-82. Epub 2009 Apr 6.

Wellcome Trust-Cancer Research UK Gurdon Institute of Cancer and Developmental Biology, University of Cambridge, Cambridge, UK.

Next-generation sequencing technology is a powerful tool for transcriptome analysis. However, under certain conditions, only a small amount of material is available, which requires more sensitive techniques that can preferably be used at the single-cell level. Here we describe a single-cell digital gene expression profiling assay. Using our mRNA-Seq assay with only a single mouse blastomere, we detected the expression of 75% (5,270) more genes than microarray techniques and identified 1,753 previously unknown splice junctions called by at least 5 reads. Moreover, 8-19% of the genes with multiple known transcript isoforms expressed at least two isoforms in the same blastomere or oocyte, which unambiguously demonstrated the complexity of the transcript variants at whole-genome scale in individual cells. Finally, for Dicer1(-/-) and Ago2(-/-) (Eif2c2(-/-)) oocytes, we found that 1,696 and 1,553 genes, respectively, were abnormally upregulated compared to wild-type controls, with 619 genes in common.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nmeth.1315DOI Listing
May 2009

Expression, purification, and characterization of the immunological response to a 40-kilodalton Plasmodium vivax tryptophan-rich antigen.

Infect Immun 2008 Jun 24;76(6):2576-86. Epub 2008 Mar 24.

Department of Biotechnology, All India Institute of Medical Sciences, New Delhi 110029, India.

We describe here an approximately 40-kDa Plasmodium vivax tryptophan-rich antigen (PvTRAg40) which contains 321 amino acids and 11.4% tryptophan residues. This protein shows 65% homology (35% identity) with the previously described PvTRAg, besides sharing 23 of 27 positionally conserved tryptophan residues and similar genomic organization. The nucleotide sequence of the entire tryptophan-rich domain of PvTRAg40 was identical among 35 P. vivax clinical isolates. The protein is expressed by ring, trophozoite, and schizont stages of the parasite. The cDNA covering exon 2 of PvTRAg40 was cloned and expressed in the pPROEXHTa vector, and recombinant protein was purified. A high humoral immune response (90.7% seropositivity; n = 43) against this recombinant protein was detected in humans during the course of natural P. vivax infection. Eighty percent of the total of 20 P. vivax-exposed individuals exhibited lymphoproliferative responses against this antigen. The T cells of these individuals produced larger amounts of interleukin-12 (IL-12), IL-4, and IL-10 than gamma interferon and tumor necrosis factor alpha cytokines in response to the recombinant protein. Production of Th2-biased cytokines, conserved T- and B-cell epitopes, and an enhanced humoral immune response indicate that PvTRAg40 could possibly induce antibody-mediated immune protection against infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/IAI.00677-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2423051PMC
June 2008

Expression and purification of a Plasmodium vivax antigen - PvTARAg55 tryptophan- and alanine-rich antigen and its immunological responses in human subjects.

Vaccine 2007 Dec 9;26(1):96-107. Epub 2007 Nov 9.

Department of Biotechnology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India.

Despite the immense global efforts, the malaria vaccine is not yet available and requires the identification of newer target molecules. Since tryptophan-rich proteins of P. yoelii have been proposed as vaccine candidates, we describe here the expression, purification and immunological characterization of a 55kDa Plasmodium vivax tryptophan- and alanine-rich antigen (PvTARAg55). This protein consists of 480 aa residues with a calculated molecular mass of 55.0kDa. It shows 42% aa sequence identity (64% homology) with PyPAg1 of P. yoelii and shares positional conservation of tryptophan residues. Sequence analysis of PvTARAg55 from different P. vivax isolates revealed that typtophan-rich domain which contains most of the B-cell epitopes was highly conserved in the parasite population while the alanine-rich domain showed polymorphism. Exon-2 covering major part (420 aa) of the protein including both the domains was PCR amplified, cloned, expressed in Escherichia coli, and the recombinant protein purified to its homogeneity. Majority of P. vivax-infected individuals (82.5%, n=40) produced antibodies against this antigen. Proliferative responses to the recombinant PvTARAg55 were observed in 60% (n=20) of individuals who had recently been exposed to the P. vivax infection. Measurement of Th1- (IFN-gamma, TNF-alpha, and IL-12) and Th2-type (IL-4 and IL-10) cytokine production in response to this recombinant antigen revealed a mixed type T-cell response with a Th2 response being more pronounced. These results demonstrate that PvTARAg55 elicits high humoral and cellular immune responses thus establishes its immunogenecity in humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.vaccine.2007.10.042DOI Listing
December 2007

A BAC clone fingerprinting approach to the detection of human genome rearrangements.

Genome Biol 2007 ;8(10):R224

BC Cancer Agency Genome Sciences Centre, West 7th Avenue, Vancouver, British Columbia, Canada V5Z 4S6.

We present a method, called fingerprint profiling (FPP), that uses restriction digest fingerprints of bacterial artificial chromosome clones to detect and classify rearrangements in the human genome. The approach uses alignment of experimental fingerprint patterns to in silico digests of the sequence assembly and is capable of detecting micro-deletions (1-5 kb) and balanced rearrangements. Our method has compelling potential for use as a whole-genome method for the identification and characterization of human genome rearrangements.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/gb-2007-8-10-r224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2246298PMC
May 2008