Hybrid Assembly and Annotation of the Genome of the Indian Punica granatum, a Superfood.

The wonder fruit pomegranate (Punica granatum, family Lythraceae) is one of India's economically important fruit crops that can grow in different agro-climatic conditions ranging from tropical to temperate regions. This study reports high-quality de novo draft hybrid genome assembly of diploid Punica cultivar "Bhagwa" and identifies its genomic features. This cultivar is most common among the farmers due to its high sustainability, glossy red color, soft seed, and nutraceutical properties with high market value. The draft genome assembly is about 361.76 Mb (N50 = 40 Mb), ∼9.0 Mb more than the genome size estimated by flow cytometry. The genome is 90.9% complete, and only 26.68% of the genome is occupied by transposable elements and has a relative abundance of 369.93 SSRs/Mb of the genome. A total of 30,803 proteins and their putative functions were predicted. Comparative whole-genome analysis revealed Eucalyptus grandis as the nearest neighbor. KEGG-KASS annotations indicated an abundance of genes involved in the biosynthesis of flavonoids, phenylpropanoids, and secondary metabolites, which are responsible for various medicinal properties of pomegranate, including anticancer, antihyperglycemic, antioxidant, and anti-inflammatory activities. The genome and gene annotations provide new insights into the pharmacological properties of the secondary metabolites synthesized in pomegranate. They will also serve as a valuable resource in mining biosynthetic pathways for key metabolites, novel genes, and variations associated with disease resistance, which can facilitate the breeding of new varieties with high yield and superior quality.

Designing an Escherichia coli Strain for Phenylalanine Overproduction by Metabolic Engineering.

The phenylalanine pathway flux is controlled by two types of regulators, those that are specific to the pathway, as well as by global regulators. In order to demonstrate the importance of these global regulators, we first removed the pathway-specific regulators using all possible combinations of gene knockouts and knockins. We found that genes like aroG fbr performed best individually as well as in combination with other genes, while other genes like tyrA and tyrR worked only in combination with other modifications. Knocking in the tktA gene under a tyrR promoter and knocking out pykF further increased phenylalanine production demonstrating that the supply of precursor via PEP and E4P is also a rate-limiting step. Finally, we tested the role of global regulators on this deregulated pathway and found that Fis overexpression helps in both enhancing and sustaining the flux through this pathway. This work opens up the possibility of using global regulators in synergy with pathway-specific modifications to enhance product yields.

Evaluation of Apis mellifera syriaca Levant region honeybee conservation using comparative genome hybridization.

Apis mellifera syriaca is the native honeybee subspecies of Jordan and much of the Levant region. It expresses behavioral adaptations to a regional climate with very high temperatures, nectar dearth in summer, attacks of the Oriental wasp and is resistant to Varroa mites. The A. m. syriaca control reference sample (CRS) in this study was originally collected and stored since 2001 from "Wadi Ben Hammad", a remote valley in the southern region of Jordan. Morphometric and mitochondrial DNA markers of these honeybees had shown highest similarity to reference A. m. syriaca samples collected in 1952 by Brother Adam of samples collected from the Middle East. Samples 1-5 were collected from the National Center for Agricultural Research and Extension breeding apiary which was established for the conservation of A. m. syriaca. Our objective was to determine the success of an A. m. syriaca honey bee conservation program using genomic information from an array-based comparative genomic hybridization platform to evaluate genetic similarities to a historic reference collection (CRS). Our results had shown insignificant genomic differences between the current population in the conservation program and the CRS indicated that program is successfully conserving A. m. syriaca. Functional genomic variations were identified which are useful for conservation monitoring and may be useful for breeding programs designed to improve locally adapted strains of A. m. syriaca.

Next generation sequencing of Apis mellifera syriaca identifies genes for Varroa resistance and beneficial bee keeping traits.

Apis mellifera syriaca exhibits a high degree of tolerance to pests and pathogens including varroa mites. This native honey bee subspecies of Jordan expresses behavioral adaptations to high temperature and dry seasons typical of the region. However, persistent honey bee imports of commercial breeder lines are endangering local honey bee population. This study reports the use of next-generation sequencing (NGS) technology to study the A. m. syriaca genome and to identify genetic factors possibly contributing toward mite resistance and other favorable traits. We obtained a total of 46.2 million raw reads by applying the NGS to sequence A. m. syriaca and used extensive bioinformatics approach to identify several candidate genes for Varroa mite resistance, behavioral and immune responses characteristic for these bees. As a part of characterizing the functional regulation of molecular genetic pathway, we have mapped the pathway genes potentially involved using information from Drosophila melanogaster and present possible functional changes implicated in responses to Varroa destructor mite infestation toward this. We performed in-depth functional annotation methods to identify ∼600 candidates that are relevant, genes involved in pathways such as microbial recognition and phagocytosis, peptidoglycan recognition protein family, Gram negative binding protein family, phagocytosis receptors, serpins, Toll signaling pathway, Imd pathway, Tnf, JAK-STAT and MAPK pathway, heamatopioesis and cellular response pathways, antiviral, RNAi pathway, stress factors, etc. were selected. Finally, we have cataloged function-specific polymorphisms between A. mellifera and A. m. syriaca that could give better understanding of varroa mite resistance mechanisms and assist in breeding. We have identified immune related embryonic development (Cactus, Relish, dorsal, Ank2, baz), Varroa hygiene (NorpA2, Zasp, LanA, gasp, impl3) and Varroa resistance (Pug, pcmt, elk, elf3-s10, Dscam2, Dhc64C, gro, futsch) functional variations genes between A. mellifera and A. m. syriaca that could be used to develop an effective molecular tool for bee conservation and breeding programs to improve locally adapted strains such as syriaca and utilize their advantageous traits for the benefit of apiculture industry.

Draft genome sequence of the Algerian bee Apis mellifera intermissa.

Apis mellifera intermissa is the native honeybee subspecies of Algeria. A. m. intermissa occurs in Tunisia, Algeria and Morocco, between the Atlas and the Mediterranean and Atlantic coasts. This bee is very important due to its high ability to adapt to great variations in climatic conditions and due to its preferable cleaning behavior. Here we report the draft genome sequence of this honey bee, its Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession JSUV00000000. The 240-Mb genome is being annotated and analyzed. Comparison with the genome of other Apis mellifera sub-species promises to yield insights into the evolution of adaptations to high temperature and resistance to Varroa parasite infestation.

Management and prognosis of teeth with trauma induced crown fractures and large periapical cyst like lesions following apical surgery with and without retrograde filling.

Crown fractures are common detrimental consequences of dental traumatic injuries. Early management of such cases is mandatory in order to prevent subsequent pathological changes that could further complicate the treatment. Pulp necrosis, chronic and cystic apical periodontitis can be the fate if these teeth are left untreated. Despite these serious complications, root canal treatment followed by apical surgery is considered a valid treatment option when such cases become complicated with large periapical lesions. However, whether a retrograde filling is essential to be placed or not is still a matter of debate. This case report discusses the orthograde endodontic management, the surgical approach and the clinical outcomes of longstanding crown fractured teeth with large cyst-like periapical lesions with and without retrograde filling.

Cytotoxicity evaluation of a new fast set highly viscous conventional glass ionomer cement with L929 fibroblast cell line.

AIM: This study aims to evaluate the cytotoxicity of a new fast set highly viscous conventional glass ionomer cement (GIC) with L929 fibroblasts. MATERIALS AND METHODS: The cement capsule was mixed and introduced into a paraffin wax mould. After setting, the cement was incubated in Dulbecco's Modified Eagle's Medium. Six replicates of the material extract were added to the culture medium in 96-well plates. L929 mouse fibroblast cells were added into the wells and then incubated for 48 h. Dimethylthiazol diphenyltetrazolium bromide test was performed for cytotoxicity evaluation. RESULTS: The results showed that this GIC brand did not yield a half-maximal inhibitory concentration value, IC50, as the cell viability was above 50% at all concentrations. Cell viability over 90% was observed at the concentrations of 3.125 and 1.5625 mg/ml. Maximum concentration of the material showed cell viability of 59.4%. CONCLUSIONS: This new fast set highly viscous conventional GIC showed low cytotoxicity to mouse fibroblast cells, and it can be suggested as a substitute for dental cements exhibiting a long setting time.

Alloimmunity-induced autoimmunity as a potential mechanism in the pathogenesis of chronic rejection of human lung allografts.

BACKGROUND: Bronchiolitis obliterans syndrome (BOS) is a major cause of morbidity and mortality after lung transplantation (LTx). We sought to better understand the relationship between alloimmune responses and autoimmunity and, subsequently, how autoimmunity leads to chronic rejection. METHODS: We analyzed the development of donor-specific antibodies (Abs) in LTx by flow PRA and the development of Abs to K-α1 tubulin (K-α1T) and collagen V (ColV) by ELISA. The frequency of K-α1T- and ColV-specific T cells that secrete IFN-γ, IL-17 and IL-10 in LTx recipients was measured by ELISPOT. RESULTS: In a retrospective analysis of 42 LTx recipients, we demonstrated a strong correlation between development of donor-specific anti-HLA Abs, Abs to self-antigens and BOS (p < 0.05). To test the hypothesis that alloimmunity is related to an immune response to self-antigens, we analyzed 103 LTx patients prospectively for the development of donor-specific Abs (DSA) and Abs to self-antigens. A total of 42.7% of recipients developed DSA and 30.1% developed Abs to K-α1T and ColV. Development of DSA preceded development of Abs to self-antigens. BOS(+) patients had higher frequency of T cells secreting IL-17 (p < 0.01) and IFN-γ (p < 0.05) with decreased IL-10 (p < 0.05) when compared with BOS(-) patients. CONCLUSIONS: Based on these results we propose that alloimmune responses to donor HLA can induce autoimmune responses to airway epithelial self-antigens, characterized by activation of the IL-17 pathway. These immune responses to self-antigens along with alloimmunity contribute to the pathogenesis of BOS. Strategies to prevent development of autoimmunity may be play a key role in preventing the development of chronic rejection.

Significant association of high-risk human papillomavirus (HPV) but not of p53 polymorphisms with oral squamous cell carcinomas in Malaysia.

PURPOSE: The purpose of this study was to evaluate the role of HPV and p53 polymorphisms in oral squamous cell carcinomas (OSCC) affecting Malaysian population. METHODS: We analysed frozen samples from 105 OSCC as well as 105 oral specimens derived from healthy individuals. PCR assays targeting two regions of the virus were used. PCR amplification for the analysis of p53 codon 72 arginine/proline alleles was carried out in a separate reaction. RESULTS: HPV DNA was detected in 51.4% OSCC samples, while 24.8% controls were found to be HPV positive. HPV was found to be significantly associated with OSCC (P < 0.001, OR = 4.3 after adjustment for habits) when compared to controls. High-risk HPV was found to be significantly associated with OSCC cases (P < 0.05). Demographic profiles of age, gender, race and habits were not associated with HPV presence in cases and controls. However, significantly less HPV positivity was seen in poorly differentiated compared to well-differentiated OSCCs. No significant association was found between HPV positivity and p53 polymorphisms in cases and control groups. Additionally, we found no association of codon 72 polymorphism with oral cancer. CONCLUSIONS: This study indicates that high-risk HPV infection is one of the contributing factors for OSCCs. HPV 16 was the predominant type found in Malaysian patients with OSCC. Further, we did not find any association between p53 codon 72 polymorphism and HPV infection or between the p53 polymorphism and the risk of oral cancer.

Respiratory virus-induced dysregulation of T-regulatory cells leads to chronic rejection.

BACKGROUND: Lower respiratory viral infections predispose to bronchiolitis obliterans syndrome (BOS). In addition, there is emerging evidence to support the role of autoimmunity in the pathogenesis of BOS. Because CD4(+)CD25(+)Foxp3(+) regulatory T-cells (Treg) control autoimmunity, we tested the hypothesis that respiratory virus-induced Treg dysfunction leads to BOS. METHODS: Treg frequency was monitored using flow cytometry. Apoptosis, cytokines, and antibodies were analyzed using annexin V assay, LUMINEX, and enzyme-linked immunosorbent assay, respectively. Murine studies were performed using the orthotopic tracheal transplant model. RESULTS: (A) Human studies: Treg troughs (decrease >50% of baseline) were found in 13 (43.3%) of 30 lung transplant recipients. Treg isolated during troughs revealed increased apoptosis (37.8%). Patients with Treg troughs had increased prevalence of antibodies to self-antigens collagen type I (23.1% vs 5.8% pretrough), collagen V (7.7% vs 0%), and k-alpha tubulin (30.7% vs 11.7%, p < 0.01) at 6 months post-trough. Increased number of Treg troughs correlated with more rapid onset of BOS. (B) Murine studies: Infection of tracheal transplant recipients with murine parainfleunza sendai virus led to increased Treg apoptosis (50.5%) in the draining lymph nodes. Vaccination against sendai virus prior to transplant abrogated apoptosis of Treg. In vitro, sendai virus-infected, but not naive, tracheal epithelial cells demonstrated upregulation of FasL (>3.5-fold) and induction of co-cultured Treg apoptosis (5.6-fold increase). CONCLUSIONS: Respiratory viral infections cause Treg apoptosis which leads to the development of de novo autoimmunity that may play a role in the pathogenesis of BOS.

Antibodies to self-antigens predispose to primary lung allograft dysfunction and chronic rejection.

BACKGROUND: Primary graft dysfunction (PGD) is a known risk factor for bronchiolitis obliterans syndrome (BOS) after lung transplantation. Here, we report that preformed antibodies to self-antigens increase PGD risk and promote BOS. METHODS: Adult lung transplant recipients (n = 142) were included in the study. Primary graft dysfunction and BOS were diagnosed based on International Society for Heart and Lung Transplantation guidelines. Antibodies to self-antigens k-alpha-1 tubulin, collagen type V, and collagen I were quantitated using standardized enzyme-linked immunosorbent assays, and cytokines were analyzed using Luminex immunoassays (Biosource International, Camirillo, CA). Human leukocyte antigen (HLA) antibodies were measured using Flow-PRA (One Lambda, Canoga Park, CA). RESULTS: Lung transplant recipients with pretransplant antibodies to self-antigens had increased risk of PGD (odds ratio 3.09, 95% confidence interval: 1.2 to 8.1, p = 0.02) compared with recipients without. Conversely, in patients with PGD, 34.7% were positive for pretransplant antibodies whereas in the PGD negative group, only 14.6% had antibodies (p = 0.03). Antibody positive patients demonstrated high levels of proinflammatory cytokines interleukin (IL)-1ß (2.1-fold increase), IL-2 (3.0), IL-12 (2.5), IL-15 (3.0), and chemokines interferon-inducible protein-10 (3.9) and monocyte chemotactic protein-1 (3.1; p < 0.01 for all). On 5-year follow-up, patients without antibodies showed greater freedom from development of HLA antibodies compared with patients who had antibodies (class I: 67% versus 38%, p = 0.001; class II: 71% versus 41%, p < 0.001). Patients with pretransplant antibodies were found to have an independent relative risk of 2.3 (95% confidence interval: 1.7 to 4.5, p = 0.009) for developing BOS. CONCLUSIONS: Presence of antibodies to self-antigens pretransplant increases the risk of PGD immediately after transplant period and BOS on long-term follow-up. Primary graft dysfunction is associated with an inflammatory cascade that augments the alloimmune (anti-HLA) response that predisposes to BOS.

Synergistic effect of antibodies to human leukocyte antigens and defensins in pathogenesis of bronchiolitis obliterans syndrome after human lung transplantation.

BACKGROUND: This study aims to determine the role of antibodies to donor-mismatched human leukocyte antigen (HLA) developed during the post-transplant period in inducing defensins and their synergistic role in the pathogenesis of chronic rejection, bronchiolitis obliterans syndrome (BOS), after human lung transplantation (LTx). METHODS: Bronchoalveolar lavage (BAL) and serum from 21 BOS+ LTx patients were assayed for ß-defensins human neutrophil peptides (HNP) 1-3 (enzyme-linked immunosorbent assay [ELISA]) and anti-HLA antibodies (Luminex, Luminex Corp, Austin, TX). Human airway epithelial cells (AEC) were treated with anti-HLA antibodies, HNP-1/2, or both, and the levels of ß-defensin were measured by ELISA. Using a mouse model of obliterative airway disease induced by anti-major histocompatibility (MHC) class-I antibodies, we quantitatively and qualitatively determined neutrophil infiltration by myeloperoxidase (MPO) staining and activity by MPO assay, and defensin levels in the BAL. RESULTS: In human LTx patients, higher defensin levels correlated with presence of circulating anti-HLA antibodies (p < 0.05). AEC treated with anti-HLA antibodies or HNP-1/2, produced ß-defensin with synergistic effects in combination (612 ± 06 vs 520 ± 23 pg/ml anti-HLA antibody, or 590 ± 10 pg/ml for HNP treatment; p < 0.05). Neutrophil numbers (6-fold) and activity (5.5-fold) were higher in the lungs of mice treated with anti-MHC antibodies vs control. A 2-fold increase in α-defensin and ß-defensin levels was also present in BAL on Day 5 after anti-MHC administrations. CONCLUSIONS: Anti-HLA antibodies developed during the post-transplant period and α-defensins stimulated ß-defensin production by epithelial cells, leading to increased cellular infiltration and inflammation. Chronic stimulation of epithelium by antibodies to MHC and resulting increased levels of defensins induce growth factor production and epithelial proliferation contributing to the development of chronic rejection after LTx.

Regulation of Golgi structure and secretion by receptor-induced G protein ßγ complex translocation.

We show that receptor induced G protein betagamma subunit translocation from the plasma membrane to the Golgi allows a receptor to initiate fragmentation and regulate secretion. A lung epithelial cell line, A549, was shown to contain an endogenous translocating G protein gamma subunit and exhibit receptor-induced Golgi fragmentation. Receptor-induced Golgi fragmentation was inhibited by a shRNA specific to the endogenous translocating gamma subunit. A kinase defective protein kinase D and a phospholipase C beta inhibitor blocked receptor-induced Golgi fragmentation, suggesting a role for this process in secretion. Consistent with betagamma translocation dependence, fragmentation induced by receptor activation was inhibited by a dominant negative nontranslocating gamma3. Insulin secretion was shown to be induced by muscarinic receptor activation in a pancreatic beta cell line, NIT-1. Induction of insulin secretion was also inhibited by the dominant negative gamma3 subunit consistent with the Golgi fragmentation induced by betagamma complex translocation playing a role in secretion.

Oral mucosal lesions in non oral habit diabetic patients and association of diabetes mellitus with oral precancerous lesions.

AIMS: This study was aimed to determine the prevalence of oral mucosal lesions (OML) in patients with diabetes mellitus (DM) and non-diabetic subjects without any oral habits and to investigate the association of DM with oral precancerous lesions. METHODS: This cross-sectional study involved 420 diabetic and 420 non-diabetic control subjects without any oral habits. Detailed oral examination was performed based on international criteria. RESULTS: A significantly greater proportion of subjects with DM (45%) had one or more OML in comparison to non-diabetics (38.3%). Patients with DM showed a significantly greater prevalence of geographic tongue, denture stomatitis and angular cheilitis than non-diabetics (p<0.05). The results also showed an association between occurrence of one or more OML and metabolic control of diabetic patients (p<0.05). For precancerous lesions, lichen planus was found in two diabetic patients while none of controls had any precancerous lesion (p>0.05). CONCLUSIONS: Prevalence of OML was significantly higher in diabetic patients than non-diabetics and this prevalence was associated with the metabolic control of the patients. However, no association was observed between DM and oral precancerous lesions.

Development of antibodies to human leukocyte antigen precedes development of antibodies to major histocompatibility class I-related chain A and are significantly associated with development of chronic rejection after human lung transplantation.

The development of antibodies (Abs) to major histocompatibility (MHC) class I-related chain A (MICA) and human leukocyte antigen (HLA) and their role in the immunopathogenesis of chronic rejection (bronchiolitis obliterans syndrome [BOS]) after human lung transplantation (LTx) was analyzed. Sera from 80 LTx recipients were analyzed for anti-MICA and anti-HLA Abs using Luminex and flow PRA (panel reactive assay). Development of Abs either to MICA alone or MICA and HLA together significantly correlated (p < 0.01) with development of BOS. Kinetic analysis in the post-LTx period revealed that development of anti-HLA Abs (7.6 +/- 4.7 months) preceded the development of anti-MICA Abs (10.0 +/- 3.5 months). Abs to MICA alleles (*001 and *009) developed approximately 6 months after LTx and peak titers were present at the time of clinical diagnosis of BOS (16.3 +/- 2.7 months). The development of Abs to both MICA and HLA was strongly associated with the development of BOS thereby suggesting a synergistic effect. Furthermore, immune response to mismatched HLA can lead to development of Abs to other MHC related antigens expressed on the airway epithelial cells. Cumulatively, these immune responses contribute to the pathogenesis of chronic rejection following human LTx.

Disruption of the mGsta4 gene increases life span of C57BL mice.

The lipid peroxidation product 4-hydroxynonenal (4-HNE) forms as a consequence of oxidative stress. By electrophilic attack on biological macromolecules, 4-HNE mediates signaling or may cause toxicity. A major route of 4-HNE disposal is via glutathione conjugation, in the mouse catalyzed primarily by glutathione transferase mGSTA4-4. Unexpectedly, mGsta4-null mice, in which 4-HNE detoxification is impaired, have an extended life span. This finding could be explained by the observed activation of the transcription factor Nrf2 in the knockout mice, which in turn leads to an induction of antioxidant and antielectrophilic defenses. Especially, the latter could provide a detoxification mechanism that contributes to enhanced longevity. We propose that disruption of 4-HNE conjugation elicits a hormetic response in which an initially increased supply of 4-HNE is translated into activation of Nrf2, leading to a new steady state in which the rise of 4-HNE concentrations is dampened, but life-extending detoxification mechanisms are concomitantly induced.

Increased erythrocyte C4D is associated with known alloantibody and autoantibody markers of antibody-mediated rejection in human lung transplant recipients.

BACKGROUND: Immune responses to mismatched donor human leukocyte antigens (HLA) are important in the pathogenesis of chronic rejection. This study evaluated whether erythrocyte-bound C4d (E-C4d) is associated with known alloimmune and autoimmune markers of antibody-mediated rejection after human lung transplantation (LTx). METHODS: Flow cytometry was used to analyze 22 LTx recipients and 15 healthy individuals for E-C4d. Development of antibodies to donor-mismatched HLA (donor-specific antibody [DSA]) and antibodies to HLA were determined using the solid-phase method by Luminex. Development of antibodies to self-antigens, K-alpha-1-tubulin (KA1T) and collagen V (Col-V), were measured by enzyme-linked immunosorbent assay. C3d deposition in lung biopsy specimens was determined by immunohistochemical staining. RESULTS: Percent E-C4d (%E-C4d) levels were 19.9% in LTx patients vs 3.7% in healthy individuals (p = 0.02). DSA+ patients had higher E-C4d levels than DSA- patients (34.1% vs 16.7%, p = 0.02). In 5 patients with preformed anti-HLA, E-C4d levels were not significantly different vs 13 patients without detectable anti-HLA (p = 0.1). E-C4d levels were higher in patients who developed antibodies to KA1T (p = 0.02) and Col-V (p = 0.03). Recipients with C3d-positive tissue deposition had higher E-C4d levels than patients with C3d-negative biopsy results (p = 0.01). CONCLUSIONS: Increased %E-C4d levels are found in patients with positive DSA, high antibody titers to KA1T and Col-V, and have C3d+ lung biopsy findings. Therefore, %E-C4d can serve as a potential marker for antibody-mediated rejection after LTx.

Oleanolic Acid, a plant triterpenoid, significantly improves survival and function of islet allograft.

BACKGROUND.: Oleanolic acid (OA) is a ubiquitous triterpenoid, with potent antioxidant and anti-inflammatory properties. Here, we tested whether these combined properties of OA can prevent nonimmunologic primary nonfunctioning and immunologic phenomena ascribed to graft rejection hence prolong islet allograft survival. METHODS.: Islet transplants were performed under kidney capsule of streptozotocin-induced diabetic C57BL/6 mice with BALB/c islets. Recipients were treated with 0.5 mg/day of OA intraperitoneally, and serum samples were collected once in 2 days and used for luminex, ELISA, and donor-specific antibody screening. Transplanted mice were killed at different time intervals to obtain splenocytes and kidney samples for ELISPOT, mixed leukocyte reaction, and immunohistochemical studies. RESULTS.: After transplantation, the decrement of blood glucose was significantly faster in mice receiving OA less than 2+/-1 days compared with untreated (4+/-2 days). OA prolonged survival of transplanted islets up to 23+/-3 days and reversed diabetes even with 250 islets. Treatment group showed increased serum interleukin (IL)-10 (twofold) and decreased inducible protein-10 and IL-4 (threefold) in luminex. Significantly reduced frequency of interferon-gamma (4.5-fold), IL-4 (3.5-fold), IL-2 (2.3-fold), and IL-17 (fourfold) producing T-cell populations were found in ELISPOT. OA-treated grafts had significant reduced and delayed infiltration of CD4+ and CD8+ T cells. OA also delayed donor-specific antibody generation up to 19 days after transplantation. Combined treatment with cyclosporine A, OA further prolonged the islet allograft survival to 34+/-3 days. CONCLUSIONS.: In conclusion, OA is an attractive, dietary nontoxic plant triterpenoid, which suppresses the production of proinflammatory cytokines and delays graft-specific immune responses to prolong islet allograft survival.

Phage displayed scFv: pIII scaffold may fine tune binding specificity.

The fine specificity of antibodies is important for their discriminating powers during diagnostics and in vivo therapy. We have attempted to isolate human scFv antibodies to the oncofetal antigen, the placental isozyme of alkaline phosphatase (PLAP) in which it is important to distinguish between the closely related intestinal alkaline phosphatase (IAP) and bone alkaline phosphatase (BAP) isozymes. As the antibodies are selected in the phage displayed form and might be finally used as different entities, including the soluble scFv form, it may be important to look at the influence of scaffolds in determining specificity. There have been earlier reports of the role of the constant region and other scaffolding proteins in determining specificity. In this paper, we report isolation of one such clone, E6, which showed specificity to PLAP in phage antibody form but lost the specificity when soluble scFv was tested for same, and showed partial cross reactivity to BAP. We suggest that the altered specificity of scFv might be the result of loss of phage pIII scaffold, which is present in phage-displayed antibody and may help the displayed antibody to assume specific conformational structure, which may govern binding characteristics of the same.

Single-tube seminested PCR assay for detecting human papillomavirus in clinical samples.

There is a growing appreciation of the potential value for routine screening for the presence of HPV not only for cervical specimens but also from oral cavity. The purpose of this study was to develop and clinically evaluate a single-tube seminested PCR assay for the detection of HPV. Several parameters such as PCR primers, primer annealing temperature, the number of PCR cycles and concentration of PCR components were optimized. The assay was evaluated using HPV inserts of type 6, 11, 16, 18, 31, 33, 38 and 51. Evaluation of seminested PCR assay was performed with cervical scrapings from 30 patients and buccal swabs from 30 head and neck cancer patients and results were compared with those of two-tube nested PCR. The results were found to be comparable with a total of 60% (36/60) of samples being positive for HPV using the single-tube assay, while 62% (37/60) positivity was found with two-tube PCR assay. We succeeded in developing a single-tube seminested PCR method for HPV DNA detection which is easier than the conventional nested PCR and can be further evaluated as a potential screening tool for detecting HPV in oral and cervical regions.