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1.  ProxiMAX randomization: a new technology for non-degenerate saturation mutagenesis of contiguous codons 
Biochemical Society Transactions  2013;41(Pt 5):1189-1194.
Back in 2003, we published ‘MAX’ randomization, a process of non-degenerate saturation mutagenesis using exactly 20 codons (one for each amino acid) or else any required subset of those 20 codons. ‘MAX’ randomization saturates codons located in isolated positions within a protein, as might be required in enzyme engineering, or else on one face of an α-helix, as in zinc-finger engineering. Since that time, we have been asked for an equivalent process that can saturate multiple contiguous codons in a non-degenerate manner. We have now developed ‘ProxiMAX’ randomization, which does just that: generating DNA cassettes for saturation mutagenesis without degeneracy or bias. Offering an alternative to trinucleotide phosphoramidite chemistry, ProxiMAX randomization uses nothing more sophisticated than unmodified oligonucleotides and standard molecular biology reagents. Thus it requires no specialized chemistry, reagents or equipment, and simply relies on a process of saturation cycling comprising ligation, amplification and digestion for each cycle. The process can encode both unbiased representation of selected amino acids or else encode them in predefined ratios. Each saturated position can be defined independently of the others. We demonstrate accurate saturation of up to 11 contiguous codons. As such, ProxiMAX randomization is particularly relevant to antibody engineering.
doi:10.1042/BST20130123
PMCID: PMC3782830  PMID: 24059507
antibody engineering; gene library; MAX randomization; overlap PCR; protein engineering; saturation mutagenesis; CDR3, complementarity-determining region 3
2.  Emerging roles of mistletoes in malignancy management 
3 Biotech  2013;4(1):13-20.
Mistletoes are a group of obligate plant semi-parasites in the order Santalales. These clumps of plants growing on a wide range of host plants have been traditionally regarded as medicinal repositories. However, current scientific discoveries have validated their health potentials like never before. Their extracts containing alkaloids, viscotoxins, lectins, and polysaccharides have been evidenced to possess a myriad biological potentials including cancer inhibition. Mistletoes have emerged as promising alternative therapy against colon, oral, lung, and pancreas cancers. The plant extracts bolster immunity, delay tumour initiation and progression, kill malignant tumours, stabilize DNA, alleviate side effects of chemotherapeutics, improve the lifespan, and coping ability of cancer patients and survivors. A range of proprietary formulations viz. Iscador, Eurixor, Helixor, Lektinol, Isorel, Iscucin, Abnoba-viscum and recombinant lectin ML-1 are already being commercialized. This review presents an informative account on the recent developments in mistletoe-mediated cancer management. The underlying mechanisms, possibilities and limitations in cancer therapeutic development are outlined for kindling both researcher and public interest.
doi:10.1007/s13205-013-0124-6
PMCID: PMC3909578
Mistletoe; Anticancer; Lectin; Polysaccharide; Immune modifier
3.  Potentials of Exopolysaccharides from Lactic Acid Bacteria 
Recent research in the area of importance of microbes has revealed the immense industrial potential of exopolysaccharides and their derivative oligosaccharides from lactic acid bacteria. However, due to lack of adequate technological knowledge, the exopolysaccharides have remained largely under exploited. In the present review, the enormous potentials of different types of exopolysaccharides from lactic acid bacteria are described. This also summarizes the recent advances in the applications of exopolysaccharides, certain problems associated with their commercial production and the remedies.
doi:10.1007/s12088-011-0148-8
PMCID: PMC3298600  PMID: 23449986
Lactic acid bacteria; Exopolysaccharides; Probiotic; Prebiotic; Oligosaccharides
4.  Therapeutic importance of sulfated polysaccharides from seaweeds: updating the recent findings 
3 Biotech  2012;2(3):171-185.
Seaweeds, being prolific sources of bioactive components have garnered unprecedented interest in recent times. The complex polysaccharides from the brown, red and green seaweeds possess broad spectrum therapeutic properties. Especially, the sulfated polysaccharides, viz. fucans, carrageenans and ulvans have exhibited strong antioxidant, antitumor, immunostimulatory, anti-inflammatory, pulmonary fibrosis anticoagulant/antithrombotic, lipid lowering, antiviral, antibacterial, antiprotozoan, hyperplasia prevention, gastrointestinal, regenerative and nano medicine applications. Considering the immense biomedical prospects of sulfated polysaccharides, the profound and emerging functional properties published in recent times will be discussed here with experimental evidences. The limitations of the seaweed-derived sulfated polysaccharides in healthcare will be summarized. Strategies to maximize extraction and bioavailability will be pondered.
doi:10.1007/s13205-012-0061-9
PMCID: PMC3433884
Sulfated polysaccharides; Antioxidant; Antitumor; Anticoagulant; Antiviral
5.  Alloimmunization against RBC or PLT Antigens Is Independent of TRIM21 Expression in a Murine Model 
Molecular immunology  2011;48(6-7):909-913.
Generation of alloantibodies to transfused RBCs can be a serious medical problem for patients who require chronic RBC transfusion therapy. Patients with sickle cell disease have a substantially increased rate of alloimmunization compared to other chronically transfused populations. A recent study has forwarded the hypothesis that a polymorphism in an immunoregulatory gene in close proximity to beta-globin (TRIM21 rs660) plays a role in the increased rates of RBC alloimmunization in sickle cell patients. In particular, it was hypothesized that rs660C/T decreases expression of TRIM21, resulting in loss of a negative feedback pathway in immune responses and increased RBC alloimmunization. To test the effects of TRIM21 expression on alloimmunization, we analyzed antibody responses to alloantigens on RBCs and platelets transfused into wild-type and TRIM21 KO mice. No significant increases were seen in the frequency or magnitude of humoral immunization to alloantigens on transfused RBCs or platelets in adult or juvenile TRIM 21 KO recipients compared to wild-type controls. Moreover, recipient inflammation with poly (I:C) enhanced RBC alloimmunization to similar degrees in both TRIM 21 KO and wild-type control recipients. Together, these data rule out the hypothesis that decreased TRIM 21 expression enhances transfusion induced humoral alloimmunization, in the context of a reductionist murine model.
doi:10.1016/j.molimm.2010.12.017
PMCID: PMC3090620  PMID: 21269695
Transfusion; alloimmunization; TRIM21; red blood cell; platelet
6.  The current trends and future perspectives of prebiotics research: a review 
3 Biotech  2012;2(2):115-125.
Prebiotics are non-digestible food ingredients that stimulate the growth of bifidogenic and lactic acid bacteria in the gastro-intestinal tract. Typically, the prebiotics consist of dietary fibers and oligosaccharides. Prebiotics exert a plethora of health-promoting effects, owing to which multi million food and pharma industries have been established. Prebiotics are being implicated in starter culture formulation, gut health maintenance, colitis prevention, cancer inhibition, immunopotentiaton, cholesterol removal, reduction of cardiovascular disease, prevention of obesity and constipation, bacteriocin production, use in fishery, poultry, pig, cattle feed and pet food. Looking at the ever-increasing demand of prebiotics, in this review, recent trends in prebiotic production from new novel sources, from food industrial wastes, prebiotic supplementation in food, commercially available prebiotic agents, prebiotic production by various techniques and future perspectives has been discussed. The critical insight into this hot research area aims to stimulate further ponderance.
doi:10.1007/s13205-012-0044-x
PMCID: PMC3376865
Prebiotics; Oligosaccharides; Anticancer; Colitis prevention; Cholesterol reduction
7.  Recent developments in mushrooms as anti-cancer therapeutics: a review 
3 Biotech  2011;2(1):1-15.
From time immemorial, mushrooms have been valued by humankind as a culinary wonder and folk medicine in Oriental practice. The last decade has witnessed the overwhelming interest of western research fraternity in pharmaceutical potential of mushrooms. The chief medicinal uses of mushrooms discovered so far are as anti-oxidant, anti-diabetic, hypocholesterolemic, anti-tumor, anti-cancer, immunomodulatory, anti-allergic, nephroprotective, and anti-microbial agents. The mushrooms credited with success against cancer belong to the genus Phellinus, Pleurotus, Agaricus, Ganoderma, Clitocybe, Antrodia, Trametes, Cordyceps, Xerocomus, Calvatia, Schizophyllum, Flammulina, Suillus, Inonotus, Inocybe, Funlia, Lactarius, Albatrellus, Russula, and Fomes. The anti-cancer compounds play crucial role as reactive oxygen species inducer, mitotic kinase inhibitor, anti-mitotic, angiogenesis inhibitor, topoisomerase inhibitor, leading to apoptosis, and eventually checking cancer proliferation. The present review updates the recent findings on the pharmacologically active compounds, their anti-tumor potential, and underlying mechanism of biological action in order to raise awareness for further investigations to develop cancer therapeutics from mushrooms. The mounting evidences from various research groups across the globe, regarding anti-tumor application of mushroom extracts unarguably make it a fast-track research area worth mass attention.
doi:10.1007/s13205-011-0036-2
PMCID: PMC3339609  PMID: 22582152
Polysaccharides; β-Glucan; Anti-tumor agent; Apoptosis; Caspase; Chemistry; Bioinformatics; Agriculture; Stem Cells; Biomaterials; Biotechnology; Cancer Research
8.  Recent developments in mushrooms as anti-cancer therapeutics: a review 
3 Biotech  2011;2(1):1-15.
From time immemorial, mushrooms have been valued by humankind as a culinary wonder and folk medicine in Oriental practice. The last decade has witnessed the overwhelming interest of western research fraternity in pharmaceutical potential of mushrooms. The chief medicinal uses of mushrooms discovered so far are as anti-oxidant, anti-diabetic, hypocholesterolemic, anti-tumor, anti-cancer, immunomodulatory, anti-allergic, nephroprotective, and anti-microbial agents. The mushrooms credited with success against cancer belong to the genus Phellinus, Pleurotus, Agaricus, Ganoderma, Clitocybe, Antrodia, Trametes, Cordyceps, Xerocomus, Calvatia, Schizophyllum, Flammulina, Suillus, Inonotus, Inocybe, Funlia, Lactarius, Albatrellus, Russula, and Fomes. The anti-cancer compounds play crucial role as reactive oxygen species inducer, mitotic kinase inhibitor, anti-mitotic, angiogenesis inhibitor, topoisomerase inhibitor, leading to apoptosis, and eventually checking cancer proliferation. The present review updates the recent findings on the pharmacologically active compounds, their anti-tumor potential, and underlying mechanism of biological action in order to raise awareness for further investigations to develop cancer therapeutics from mushrooms. The mounting evidences from various research groups across the globe, regarding anti-tumor application of mushroom extracts unarguably make it a fast-track research area worth mass attention.
doi:10.1007/s13205-011-0036-2
PMCID: PMC3339609  PMID: 22582152
Polysaccharides; β-Glucan; Anti-tumor agent; Apoptosis; Caspase
9.  Potential Peripheral Biomarkers for the Diagnosis of Alzheimer's Disease 
Advances in the discovery of a peripheral biomarker for the diagnosis of Alzheimer's would provide a way to better detect the onset of this debilitating disease in a manner that is both noninvasive and universally available. This paper examines the current approaches that are being used to discover potential biomarker candidates available in the periphery. The search for a peripheral biomarker that could be utilized diagnostically has resulted in an extensive amount of studies that employ several biological approaches, including the assessment of tissues, genomics, proteomics, epigenetics, and metabolomics. Although a definitive biomarker has yet to be confirmed, advances in the understanding of the mechanisms of the disease and major susceptibility factors have been uncovered and reveal promising possibilities for the future discovery of a useful biomarker.
doi:10.4061/2011/572495
PMCID: PMC3202136  PMID: 22114744
10.  Dextransucrase from the mutant of Pediococcus pentosaceus (PPm) is more stable than the wild type 
3 Biotech  2011;1(4):199-205.
A comparative study on both wild type and mutant of Pediococcus pentosaceus for dextransucrase activity, its stability, dextran synthesizing activity, antibiotic sensitivity and carbohydrate utilization was performed. The wild type P. pentosaceus had specific activity of 0.58 U/mg whereas the mutant showed that of 1.0 U/mg with 72% enhancement. The antibiogram of 27 antibiotics tested against mutant showed significant differences with 9 antibiotics when compared to wild type. In carbohydrate fermentation profile, trehalose, galactose, maltose, lactose and fructose are metabolized by both the strains, but weakly in case of mutant. Stabilization of purified dextransucrase from wild type and mutant with various stabilizers was studied at 30 and 4 °C. Both enzymes were more stable at 4 °C. Among various stabilizers such as dextran (100 kDa, 10 μg/ml), glycerol (0.5%, v/v), PEG 8000 (10 μg/ml) and Tween 80 (0.5%, v/v), Tween 80 provided maximum stabilization at 4 and 30 °C. The mutant showed better stabilization than that of the wild type at both 30 and 4 °C. The loss of activity at 30 °C after 24 h in wild type and mutant in the presence of Tween 80 was only 34 and 32%, respectively, whereas the loss of activity in control of wild type and mutant was 76 and 59%, respectively. After 15 days at 4 °C, the loss of activity in control of wild type and mutant in the presence of Tween 80 was only 15 and 8%, respectively, whereas at 30 °C, the loss of activity in control of wild type and mutant was 49 and 42% respectively. Half-life of the enzyme with Tween 80 was 28.5 and 33.5 h for wild type and mutant, respectively, at 30 °C and 52.1 and 106.6 days for wild type and mutant respectively, at 4 °C.
doi:10.1007/s13205-011-0018-4
PMCID: PMC3339597  PMID: 22558538
Pediococcus; Dextransucrase; Antibiogram; Carbohydrate; Fermentation; Stabilization; Chemistry; Stem Cells; Biotechnology; Biomaterials; Agriculture; Bioinformatics; Cancer Research
11.  Dextransucrase from the mutant of Pediococcus pentosaceus (PPm) is more stable than the wild type 
3 Biotech  2011;1(4):199-205.
A comparative study on both wild type and mutant of Pediococcus pentosaceus for dextransucrase activity, its stability, dextran synthesizing activity, antibiotic sensitivity and carbohydrate utilization was performed. The wild type P. pentosaceus had specific activity of 0.58 U/mg whereas the mutant showed that of 1.0 U/mg with 72% enhancement. The antibiogram of 27 antibiotics tested against mutant showed significant differences with 9 antibiotics when compared to wild type. In carbohydrate fermentation profile, trehalose, galactose, maltose, lactose and fructose are metabolized by both the strains, but weakly in case of mutant. Stabilization of purified dextransucrase from wild type and mutant with various stabilizers was studied at 30 and 4 °C. Both enzymes were more stable at 4 °C. Among various stabilizers such as dextran (100 kDa, 10 μg/ml), glycerol (0.5%, v/v), PEG 8000 (10 μg/ml) and Tween 80 (0.5%, v/v), Tween 80 provided maximum stabilization at 4 and 30 °C. The mutant showed better stabilization than that of the wild type at both 30 and 4 °C. The loss of activity at 30 °C after 24 h in wild type and mutant in the presence of Tween 80 was only 34 and 32%, respectively, whereas the loss of activity in control of wild type and mutant was 76 and 59%, respectively. After 15 days at 4 °C, the loss of activity in control of wild type and mutant in the presence of Tween 80 was only 15 and 8%, respectively, whereas at 30 °C, the loss of activity in control of wild type and mutant was 49 and 42% respectively. Half-life of the enzyme with Tween 80 was 28.5 and 33.5 h for wild type and mutant, respectively, at 30 °C and 52.1 and 106.6 days for wild type and mutant respectively, at 4 °C.
doi:10.1007/s13205-011-0018-4
PMCID: PMC3339597  PMID: 22558538
Pediococcus; Dextransucrase; Antibiogram; Carbohydrate; Fermentation; Stabilization
12.  Evaluation of in-vitro antibiotic susceptibility of different morphological forms of Borrelia burgdorferi 
Background:
Lyme disease is a tick-borne illness caused by the spirochete Borrelia burgdorferi. Although antibiotic therapy is usually effective early in the disease, relapse may occur when administration of antibiotics is discontinued. Studies have suggested that resistance and recurrence of Lyme disease might be due to formation of different morphological forms of B. burgdorferi, namely round bodies (cysts) and biofilm-like colonies. Better understanding of the effect of antibiotics on all morphological forms of B. burgdorferi is therefore crucial to provide effective therapy for Lyme disease.
Methods:
Three morphological forms of B. burgdorferi (spirochetes, round bodies, and biofilm-like colonies) were generated using novel culture methods. Minimum inhibitory concentration and minimum bactericidal concentration of five antimicrobial agents (doxycycline, amoxicillin, tigecycline, metronidazole, and tinidazole) against spirochetal forms of B. burgdorferi were evaluated using the standard published microdilution technique. The susceptibility of spirochetal and round body forms to the antibiotics was then tested using fluorescent microscopy (BacLight™ viability staining) and dark field microscopy (direct cell counting), and these results were compared with the microdilution technique. Qualitative and quantitative effects of the antibiotics against biofilm-like colonies were assessed using fluorescent microscopy and dark field microscopy, respectively.
Results:
Doxycycline reduced spirochetal structures ∼90% but increased the number of round body forms about twofold. Amoxicillin reduced spirochetal forms by ∼85%–90% and round body forms by ∼68%, while treatment with metronidazole led to reduction of spirochetal structures by ∼90% and round body forms by ∼80%. Tigecycline and tinidazole treatment reduced both spirochetal and round body forms by ∼80%–90%. When quantitative effects on biofilm-like colonies were evaluated, the five antibiotics reduced formation of these colonies by only 30%–55%. In terms of qualitative effects, only tinidazole reduced viable organisms by ∼90%. Following treatment with the other antibiotics, viable organisms were detected in 70%–85% of the biofilm-like colonies.
Conclusion:
Antibiotics have varying effects on the different morphological forms of B. burgdorferi. Persistence of viable organisms in round body forms and biofilm-like colonies may explain treatment failure and persistent symptoms following antibiotic therapy of Lyme disease.
doi:10.2147/IDR.S19201
PMCID: PMC3132871  PMID: 21753890
Lyme disease; spirochetes; cysts; round bodies; biofilms
13.  Harmful and beneficial aspects of Parthenium hysterophorus: an update 
3 Biotech  2011;1(1):1-9.
Parthenium hysterophorus is a noxious weed in America, Asia, Africa and Australia. This weed is considered to be a cause of allergic respiratory problems, contact dermatitis, mutagenicity in human and livestock. Crop production is drastically reduced owing to its allelopathy. Also aggressive dominance of this weed threatens biodiversity. Eradication of P. hysterophorus by burning, chemical herbicides, eucalyptus oil and biological control by leaf-feeding beetle, stem-galling moth, stem-boring weevil and fungi have been carried out with variable degrees of success. Recently many innovative uses of this hitherto notorious plant have been discovered. Parthenium hysterophorus confers many health benefits, viz remedy for skin inflammation, rheumatic pain, diarrhoea, urinary tract infections, dysentery, malaria and neuralgia. Its prospect as nano-medicine is being carried out with some preliminary success so far. Removal of heavy metals and dye from the environment, eradication of aquatic weeds, use as substrate for commercial enzyme production, additives in cattle manure for biogas production, as biopesticide, as green manure and compost are to name a few of some other potentials. The active compounds responsible for hazardous properties have been summarized. The aim of this review article is to explore the problem P. hysterophorus poses as a weed, the effective control measures that can be implemented as well as to unravel the latent beneficial prospects of this weed.
doi:10.1007/s13205-011-0007-7
PMCID: PMC3339593  PMID: 22558530
Parthenium hysterophorus; Sesquiterpene lactone; Dermatitis; Biocontrol; Green manure; Bioremediation; Chemistry; Biotechnology; Stem Cells; Biomaterials; Bioinformatics; Agriculture; Cancer Research
14.  Harmful and beneficial aspects of Parthenium hysterophorus: an update 
3 Biotech  2011;1(1):1-9.
Parthenium hysterophorus is a noxious weed in America, Asia, Africa and Australia. This weed is considered to be a cause of allergic respiratory problems, contact dermatitis, mutagenicity in human and livestock. Crop production is drastically reduced owing to its allelopathy. Also aggressive dominance of this weed threatens biodiversity. Eradication of P. hysterophorus by burning, chemical herbicides, eucalyptus oil and biological control by leaf-feeding beetle, stem-galling moth, stem-boring weevil and fungi have been carried out with variable degrees of success. Recently many innovative uses of this hitherto notorious plant have been discovered. Parthenium hysterophorus confers many health benefits, viz remedy for skin inflammation, rheumatic pain, diarrhoea, urinary tract infections, dysentery, malaria and neuralgia. Its prospect as nano-medicine is being carried out with some preliminary success so far. Removal of heavy metals and dye from the environment, eradication of aquatic weeds, use as substrate for commercial enzyme production, additives in cattle manure for biogas production, as biopesticide, as green manure and compost are to name a few of some other potentials. The active compounds responsible for hazardous properties have been summarized. The aim of this review article is to explore the problem P. hysterophorus poses as a weed, the effective control measures that can be implemented as well as to unravel the latent beneficial prospects of this weed.
doi:10.1007/s13205-011-0007-7
PMCID: PMC3339593  PMID: 22558530
Parthenium hysterophorus; Sesquiterpene lactone; Dermatitis; Biocontrol; Green manure; Bioremediation
15.  Evidence for an early role for BMP4 signaling in thymus and parathyroid morphogenesis 
Developmental biology  2010;339(1):141-154.
The thymus and parathyroids are pharyngeal endoderm-derived organs that develop from common organ primordia, which undergo a series of morphological events resulting in separate organs in distinct locations in the embryo. Previous gene expression and functional analyses have suggested a role for BMP4 signaling in early thymus organogenesis. We have used conditional deletion of Bmp4 or Alk3 from the pharyngeal endoderm and/or the surrounding mesenchyme using Foxg1-Cre, Wnt1-Cre or Foxn1-Cre. Deleting Bmp4 from both neural crest cells (NCC) and early endoderm-derived epithelial cells in Foxg1-Cre;Bmp4 conditional mutants resulted in defects in thymus-parathyroid morphogenesis. Defects included reduced condensation of mesenchymal cells around the epithelium, partial absence of the thymic capsule, a delay in thymus and parathyroid separation, and failed or dramatically reduced organ migration. Patterning of the primordia and initial organ differentiation were not affected in any of the mutants. Deleting Bmp4 from NCC-derived mesenchyme or differentiating thymic epithelial cells (TECs) had no effects on thymus-parathyroid development, while loss of Alk3 from either neural crest cells or TECs resulted in only a mild thymic hypoplasia. These results show that the processes of cell specification and morphogenesis during thymus-parathyroid development are independently controlled, and suggest a specific temporal and spatial role for BMP4-mediated epithelial-mesenchymal interactions during early thymus and parathyroid morphogenesis.
doi:10.1016/j.ydbio.2009.12.026
PMCID: PMC2823926  PMID: 20043899
Bmp4; Alk3; Thymus; Parathyroid; Pharyngeal pouch; Endoderm; Mesenchyme; Organogenesis; Morphogenesis; Thymic epithelium; Foxg1-Cre; Wnt1-Cre; Foxn1-Cre
16.  Transfusion of minor histocompatibility antigen–mismatched platelets induces rejection of bone marrow transplants in mice 
The Journal of Clinical Investigation  2009;119(9):2787-2794.
Bone marrow transplantation (BMT) represents a cure for nonmalignant hematological disorders. However, compared with the stringent conditioning regimens used when performing BMT to treat hematological malignancies, the reduced intensity conditioning regimen used in the context of nonmalignant hematological disorders leads to substantially higher rates of BMT rejection, presumably due to an intact immune system. The relevant patient population typically receives transfusion support, often including platelets, and the frequency of BMT rejection correlates with the frequency of transfusion. Here, we demonstrate that immunity to transfused platelets contributes to subsequent BMT rejection in mice, even when the BMT donor and recipient are MHC matched. We used MHC-matched bone marrow because, although immunity to transfused platelets is best characterized in relation to HLA-specific antibodies, such antibodies are unlikely to play a role in clinical BMT rejection that is HLA matched. However, bone marrow is not matched in the clinic for minor histocompatibility antigens, such as those carried by platelets, and we report that transfusion of minor histocompatibility antigen–mismatched platelets induced subsequent BMT rejection. These findings indicate previously unappreciated sequelae of immunity to platelets in the context of transplantation and suggest that strategies to account for minor histocompatibility mismatching may help to reduce the chance of BMT rejection in human patients.
doi:10.1172/JCI39590
PMCID: PMC2735899  PMID: 19726874
17.  Sustained desensitization to bacterial Toll-like receptor ligands after resolutionof respiratory influenza infection 
The World Health Organization estimates that lower respiratory tract infections (excluding tuberculosis) account for ∼35% of all deaths caused by infectious diseases. In many cases, the cause of death may be caused by multiple pathogens, e.g., the life-threatening bacterial pneumonia observed in patients infected with influenza virus. The ability to evolve more efficient immunity on each successive encounter with antigen is the hallmark of the adaptive immune response. However, in the absence of cross-reactive T and B cell epitopes, one lung infection can modify immunity and pathology to the next for extended periods of time. We now report for the first time that this phenomenon is mediated by a sustained desensitization of lung sentinel cells to Toll-like receptor (TLR) ligands; this is an effect that lasts for several months after resolution of influenza or respiratory syncytial virus infection and is associated with reduced chemokine production and NF-κB activation in alveolar macrophages. Although such desensitization may be beneficial in alleviating overall immunopathology, the reduced neutrophil recruitment correlates with heightened bacterial load during secondary respiratory infection. Our data therefore suggests that post-viral desensitization to TLR signals may be one possible contributor to the common secondary bacterial pneumonia associated with pandemic and seasonal influenza infection.
doi:10.1084/jem.20070891
PMCID: PMC2271005  PMID: 18227219
18.  Molecular interactions of ASPP1 and ASPP2 with the p53 protein family and the apoptotic promoters PUMA and Bax 
Nucleic Acids Research  2008;36(16):5139-5151.
The apoptosis stimulating p53 proteins, ASPP1 and ASPP2, are the first two common activators of the p53 protein family that selectively enable the latter to regulate specific apoptotic target genes, which facilitates yes yet unknown mechanisms for discrimination between cell cycle arrest and apoptosis. To better understand the interplay between ASPP- and p53-family of proteins we investigated the molecular interactions between them using biochemical methods and structure-based homology modelling. The data demonstrate that: (i) the binding of ASPP1 and ASPP2 to p53, p63 and p73 is direct; (ii) the C-termini of ASPP1 and ASPP2 interact with the DNA-binding domains of p53 protein family with dissociation constants, Kd, in the lower micro-molar range; (iii) the stoichiometry of binding is 1:1; (iv) the DNA-binding domains of p53 family members are sufficient for these protein–protein interactions; (v) EMSA titrations revealed that while tri-complex formation between ASPPs, p53 family of proteins and PUMA/Bax is mutually exclusive, ASPP2 (but not ASPP1) formed a complex with PUMA (but not Bax) and displaced p53 and p73. The structure-based homology modelling revealed subtle differences between ASPP2 and ASPP1 and together with the experimental data provide novel mechanistic insights.
doi:10.1093/nar/gkn490
PMCID: PMC2532732  PMID: 18676979
19.  Endothelial Progenitor Cells, Cardiovascular Risk Factors, Cytokine Levels and Atherosclerosis – Results from a Large Population-Based Study 
PLoS ONE  2007;2(10):e975.
Background
EPC number and functionality are assumed to reflect the endogenous vascular repair capacity with the EPC pool declining in higher ages and being exhausted by unfavorable life-style and risk factors. This intriguing and clinically highly relevant concept, however, has so far been derived from small case-control studies and patient series.
Methodology and Principle Findings
In the population-based Bruneck Study EPC number and EPC-colony forming units (EPC-CFU) were assessed as part of the fourth follow-up evaluation (2005) in 571 and 542 subjects, respectively. EPC number declined with age (p = 0.013), was significantly lower in women (p = 0.006) and higher in subjects on statin, hormone replacement or ACE inhibitor/angiotensin-receptor blockers, and correlated positively with moderate alcohol consumption. Unexpectedly, a positive relation between EPC number and several vascular risk factors emerged. In a step forward multivariate linear regression analysis EPC number was independently related with SDF1α, MMP-9, triglycerides, alcohol consumption, and Hba1c. EPC-CFU in turn was related to SDF1α and diastolic blood pressure. Moreover, EPC number showed a significant positive association with the Framingham risk score (P = 0.001). Finally, there was an inverse association between EPC number and common carotid artery intima-media thickness (p = 0.02) and the carotid artery atherosclerosis score (p = 0.059).
Conclusions
Our population-based data confirm the decline of EPC number with advancing age and lend first epidemiological support to a role of SDF-1α and MMP9 in EPC differentiation, mobilization and homing, but are conflict with the view that EPC number is unfavorably affected by cardiovascular risk factors. EPC number increases with the cardiovascular risk estimated by the Framingham risk score (FRS), which in the absence of similar changes for EPC-CFU. Finally, we demonstrate a significant inverse association between EPC number and extent of carotid atherosclerosis even though this association was only of moderate strength and not entirely consistent in other vascular territories.
doi:10.1371/journal.pone.0000975
PMCID: PMC1995762  PMID: 17925881
20.  HDAC3 is crucial in shear- and VEGF-induced stem cell differentiation toward endothelial cells 
The Journal of Cell Biology  2006;174(7):1059-1069.
Reendothelialization involves endothelial progenitor cell (EPC) homing, proliferation, and differentiation, which may be influenced by fluid shear stress and local flow pattern. This study aims to elucidate the role of laminar flow on embryonic stem (ES) cell differentiation and the underlying mechanism. We demonstrated that laminar flow enhanced ES cell–derived progenitor cell proliferation and differentiation into endothelial cells (ECs). Laminar flow stabilized and activated histone deacetylase 3 (HDAC3) through the Flk-1–PI3K–Akt pathway, which in turn deacetylated p53, leading to p21 activation. A similar signal pathway was detected in vascular endothelial growth factor–induced EC differentiation. HDAC3 and p21 were detected in blood vessels during embryogenesis. Local transfer of ES cell–derived EPC incorporated into injured femoral artery and reduced neointima formation in a mouse model. These data suggest that shear stress is a key regulator for stem cell differentiation into EC, especially in EPC differentiation, which can be used for vascular repair, and that the Flk-1–PI3K–Akt–HDAC3–p53–p21 pathway is crucial in such a process.
doi:10.1083/jcb.200605113
PMCID: PMC2064396  PMID: 16982804
21.  Identification and Molecular Characterization of EatA, an Autotransporter Protein of Enterotoxigenic Escherichia coli  
Infection and Immunity  2004;72(3):1786-1794.
Enterotoxigenic Escherichia coli (ETEC) strains remain a formidable cause of diarrheal disease. To identify novel surface proteins of ETEC, we performed TnphoA mutagenesis of prototype ETEC strain H10407 and discovered a secreted protein not previously recognized in ETEC. DNA sequencing of the interrupted locus in mutant TnphoA.977 revealed a candidate 4,095-bp open reading frame without significant homology to commensal E. coli K-12 genomic DNA. Translation of this sequence revealed that it encoded a predicted peptide of 147.7 kDa that bears significant homology to members of the autotransporter family of bacterial virulence factors, particularly the serine protease autotransporters of the Enterobacteriaceae proteins. The gene identified in H10407, eatA (ETEC autotransporter A), encodes a potential serine protease motif (GDSGSP) in the secreted amino-terminal domain, and the predicted peptide shows more than 80% homology with SepA, a virulence protein secreted by Shigella flexneri. DNA hybridization and PCR demonstrated that eatA resides on the 92-kDa pCS1 virulence plasmid of H10407 and that it is present in multiple clinical ETEC strains. Immunoblots with antisera directed against a recombinant EatA passenger protein fragment identified a 110-kDa protein in supernatants purified from H10407 but not from the TnphoA.977 mutant or H10407-P, which lacks pCS1. EatA possesses serine protease activity that is abolished by mutations within a serine protease catalytic triad formed by residues H134, D162, and S267. Finally, interruption of the eatA gene retarded fluid accumulation in the rabbit ileal loop model, suggesting that this autotransporter contributes to the virulence of ETEC.
doi:10.1128/IAI.72.3.1786-1794.2004
PMCID: PMC356008  PMID: 14977988
22.  The impact of specific HIV treatment-related adverse events on adherence to antiretroviral therapy: A systematic review and meta-analysis 
AIDS Care  2012;25(4):400-414.
Poor adherence to antiretroviral therapies (ARTs) in human immunodeficiency virus (HIV)-infected patients increases the risk of incomplete viral suppression, development of viral resistance, progression to acquired immune deficiency syndrome and death. This study assesses the impact of specific treatment-related adverse events (AEs) on adherence to ART in the adult HIV patient population. A systematic review of studies involving adult HIV-infected patients aged ≥ 16 years that reported an odds ratio (OR) for factors affecting adherence to ART was conducted through a search of the EMBASE® and Medline® databases. Database searches were complemented with a search of titles in the bibliographies of review papers. Studies conducted in populations limited to a particular demographic characteristic or behavioural risk were excluded. To qualify for inclusion into a meta-analysis, treatment-related AEs had to be defined similarly across studies. Also, multiple ORs from the same study were included where study sub-groups were distinct. Random effects models were used to pool ORs. In total, 19 studies and 18 ART-related AEs were included in meta-analyses. Adherence to ART was significantly lower in patients with non-specific AEs than in patients who did not experience AEs [OR = 0.623; 95% confidence interval (CI): 0.465–0.834]. Patients with specific AEs such as fatigue (OR = 0.631; 95% CI: 0.433–0.918), confusion (OR = 0.349; 95% CI: 0.184–0.661), taste disturbances (OR = 0.485; 95% CI: 0.303–0.775) and nausea (OR = 0.574; 95% CI: 0.427–0.772) were significantly less likely to adhere to ART compared to patients without these AEs. Knowledge of specific treatment-related AEs may allow for targeted management of these events and a careful consideration of well-tolerated treatment regimens to improve ART adherence and clinical outcomes.
doi:10.1080/09540121.2012.712667
PMCID: PMC3613968  PMID: 22908886
antiretroviral therapy; adherence; adverse events; HIV

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