Effects of Low-Level Persistent Infection on Maintenance of Immunity by CD4 T Cell Subsets and Th1 Cytokines.

CD4 T cells are required, along with antibodies, for complete protection from blood-stage infection with Plasmodium spp., which cause malaria. Without continuous exposure, as on emigration of people from endemic areas, protection from malaria decays. As in other persistent infections, low-level Plasmodium chabaudi infection protects the host from reinfection at 2 months postinfection, a phenomenon termed premunition. Premunition is correlated with T cell responses, rather than antibody levels. We previously showed that while both effector T cells (Teff) and memory T cells (Tmem) are present after infection, Teff protect better than Tmem. Here, we studied T cell kinetics post-infection by labeling dividing Ifng+ T cells with 5-bromo-2'-deoxyuridine (BrdU) in infected Ifng reporter mice. Large drops in specific T cell numbers and Ifng+ cells upon clearance of parasites suggest a mechanism for decay of protection. Although protection decays, CD4 Tmem persist, including a highly differentiated CD27- effector memory (Tem) subset that maintains some Ifng expression. In addition, pretreatment of chronically infected animals with neutralizing antibody to interferon gamma (IFN-γ) or with clodronate liposomes before reinfection decreases premunition, supporting a role for Th1-type immunity to reinfection. A pulse-chase experiment comparing chronically infected to treated animals showed that recently divided Ifng+ T cells, particularly IFN-γ+ TNF+ IL-2- T cells, are promoted by persistent infection. These data suggest that low-level persistent infection reduces CD4+ Tmem and multifunctional Teff survival, but promotes IFN-γ+ TNF+ IL-2- T cells and Ifng+ terminally differentiated effector T cells, and prolongs immunity.

Protection by and maintenance of CD4 effector memory and effector T cell subsets in persistent malaria infection.

Protection at the peak of Plasmodium chabaudi blood-stage malaria infection is provided by CD4 T cells. We have shown that an increase in Th1 cells also correlates with protection during the persistent phase of malaria; however, it is unclear how these T cells are maintained. Persistent malaria infection promotes protection and generates both effector T cells (Teff), and effector memory T cells (Tem). We have previously defined new CD4 Teff (IL-7Rα-) subsets from Early (TeffEarly, CD62LhiCD27+) to Late (TeffLate, CD62LloCD27-) activation states. Here, we tested these effector and memory T cell subsets for their ability to survive and protect in vivo. We found that both polyclonal and P. chabaudi Merozoite Surface Protein-1 (MSP-1)-specific B5 TCR transgenic Tem survive better than Teff. Surprisingly, as Tem are associated with antigen persistence, Tem survive well even after clearance of infection. As previously shown during T cell contraction, TeffEarly, which can generate Tem, also survive better than other Teff subsets in uninfected recipients. Two other Tem survival mechanisms identified here are that low-level chronic infection promotes Tem both by driving their proliferation, and by programming production of Tem from Tcm. Protective CD4 T cell phenotypes have not been precisely determined in malaria, or other persistent infections. Therefore, we tested purified memory (Tmem) and Teff subsets in protection from peak pathology and parasitemia in immunocompromised recipient mice. Strikingly, among Tmem (IL-7Rαhi) subsets, only TemLate (CD62LloCD27-) reduced peak parasitemia (19%), though the dominant memory subset is TemEarly, which is not protective. In contrast, all Teff subsets reduced peak parasitemia by more than half, and mature Teff can generate Tem, though less. In summary, we have elucidated four mechanisms of Tem maintenance, and identified two long-lived T cell subsets (TemLate, TeffEarly) that may represent correlates of protection or a target for longer-lived vaccine-induced protection against malaria blood-stages.

Early Inhibition of Fatty Acid Synthesis Reduces Generation of Memory Precursor Effector T Cells in Chronic Infection.

Understanding the mechanisms of CD4 memory T cell (Tmem) differentiation in malaria is critical for vaccine development. However, the metabolic regulation of CD4 Tmem differentiation is not clear, particularly in persistent infections. In this study, we investigated the role of fatty acid synthesis (FAS) in Tmem development in Plasmodium chabaudi chronic mouse malaria infection. We show that T cell-specific deletion and early pharmaceutical inhibition of acetyl CoA carboxylase 1, the rate limiting step of FAS, inhibit generation of early memory precursor effector T cells (MPEC). To compare the role of FAS during early differentiation or survival of Tmem in chronic infection, a specific inhibitor of acetyl CoA carboxylase 1, 5-(tetradecyloxy)-2-furoic acid, was administered at different times postinfection. Strikingly, the number of Tmem was only reduced when FAS was inhibited during T cell priming and not during the Tmem survival phase. FAS inhibition during priming increased effector T cell (Teff) proliferation and strongly decreased peak parasitemia, which is consistent with improved Teff function. Conversely, MPEC were decreased, in a T cell-intrinsic manner, upon early FAS inhibition in chronic, but not acute, infection. Early cure of infection also increased mitochondrial volume in Tmem compared with Teff, supporting previous reports in acute infection. We demonstrate that the MPEC-specific effect was due to the higher fatty acid content and synthesis in MPEC compared with terminally differentiated Teff. In conclusion, FAS in CD4 T cells regulates the early divergence of Tmem from Teff in chronic infection.

Dissemination of non-typhoidal Salmonella during Plasmodium chabaudi infection affects anti-malarial immunity.

Malaria-associated bacteremia accounts for up to one-third of deaths from severe malaria, and non-typhoidal Salmonella (NTS) has been reported as a major complication of severe malarial infection. Patients who develop NTS bacteremia during Plasmodium infection show higher mortality rates than individuals with malaria alone. Systemic bacteremia can be caused by a wound or translocation from epithelial or endothelial sites. NTS is an intestinal pathogen, however the contribution of bacterial translocation from the intestinal tract during Plasmodium infection is not well studied. Here, we investigated the integrity of the intestinal barrier function of P. chabaudi-infected mice using large molecules and Salmonella infection. Intestinal histology and the adaptive immune response to malaria were also studied using light microscopy and flow cytometry. P. chabaudi infection compromised intestinal barrier function, which led to increased intestinal cellular infiltration. In addition, we observed increased serum lipopolysaccharide binding protein and leakage of soluble molecules from the intestine into the blood in infected mice. Plasmodium infection also increased intestinal translocation and dissemination of NTS to the liver. The adaptive immune response to P. chabaudi infection was also significantly impacted by NTS translocation. Reduced B and T cell activation were observed in co-infected animals, suggesting interference in the malaria-specific immune responses by bacteremia. These studies demonstrate that P. chabaudi infection induces failure of the barrier function of the intestinal wall and enhanced intestinal bacterial translocation, affecting anti-malarial immunity.

Early effector cells survive the contraction phase in malaria infection and generate both central and effector memory T cells.

CD4 T cells orchestrate immunity against blood-stage malaria. However, a major challenge in designing vaccines to the disease is poor understanding of the requirements for the generation of protective memory T cells (Tmem) from responding effector T cells (Teff) in chronic parasite infection. In this study, we use a transgenic mouse model with T cells specific for the merozoite surface protein (MSP)-1 of Plasmodium chabaudi to show that activated T cells generate three distinct Teff subsets with progressive activation phenotypes. The earliest observed Teff subsets (CD127(-)CD62L(hi)CD27(+)) are less divided than CD62L(lo) Teff and express memory genes. Intermediate (CD62L(lo)CD27(+)) effector subsets include the most multicytokine-producing T cells, whereas fully activated (CD62L(lo)CD27(-)) late effector cells have a terminal Teff phenotype (PD-1(+), Fas(hi), AnnexinV(+)). We show that although IL-2 promotes expansion, it actually slows terminal effector differentiation. Using adoptive transfer, we show that only early Teff survive the contraction phase and generate the terminal late Teff subsets, whereas in uninfected recipients, they become both central and effector Tmem. Furthermore, we show that progression toward full Teff activation is promoted by increased duration of infection, which in the long-term promotes Tem differentiation. Therefore, we have defined markers of progressive activation of CD4 Teff at the peak of malaria infection, including a subset that survives the contraction phase to make Tmem, and show that Ag and cytokine levels during CD4 T cell expansion influence the proportion of activated cells that can survive contraction and generate memory in malaria infection.

The significance of extramural venous invasion in R1 positive rectal cancer.

BACKGROUND AND AIMS: Evidence has shown that a positive resection margin (R1) is a key determinant of subsequent local recurrence and a poor prognostic factor in rectal cancer. The aim of this study was to evaluate the outcomes and prognosticators in patients with R1 resection of rectal cancer. MATERIAL AND METHODS: Retrospective study of all patients operatively managed within our institution between April 2008 and April 2013 for rectal cancer. Baseline demographics and multiple outcome measures recorded. Overall survival (OS), disease-free survival (DFS) and recurrence were the primary outcome measures. RESULTS: Overall, there were 306 primary rectal cancers. Seventy-six percent were grade T3/4 tumours. OS was 30 months. R1 rate was 16 % (48 patients). Thirty-one patients underwent APR and 17 AR. In patients who responded to neoadjuvant chemotherapy (NAC), overall survival was 55 months, with no extramural venous invasion (EMV) seen in this cohort. In non-responders OS was 29 months, with EMV in 48 %. In patients who did not receive NAC, OS was 23 months, with EMV in 74 %. EMV is a strong predictor for poor survival following R1 (p = 0.001). We also found a correlation between number of positive nodes and OS/DFS (p = 0.004). CONCLUSIONS: In this small cohort of patients with R1 positive rectal cancers, response to NAC is the strongest predictor of poor overall and disease-free survival. In patients who respond to NAC, OS and DFS has been shown to be positive, with a reduced rate of EMV.

New Record of Catharanthus yellow mosaic virus and a Betasatellite Associated with Lethal Leaf Yellowing of Kalmegh (Andrographis paniculata) in Northern India.

Andrographis paniculata (Family Acanthaceae), also called Kalmegh, is a medicinal herb in India well-known for its various pharmaceutical properties (1). In August 2012, during a survey in the northern parts of India, several Kalmegh plants in Barabanki District of Uttar Pradesh Province showed typical virus-like symptoms along with prominent lethal leaf yellowing. The infected plants initially showed some chlorotic streaks, which later turned completely yellow, ultimately leading to premature death. Mechanical/sap inoculation failed to transmit the pathogen. Based on the symptomology, a heavy infestation of whiteflies (Bemisia tabaci) in the infected fields, and lack of mechanical transmission, the association of a begomovirus was suspected. The disease incidence was calculated to be about 15 to 20% on the basis of plant population. Twenty samples from naturally infected plants of A. paniculata were collected from various field locations. Total genomic DNA from the symptomatic and non-symptomatic samples was isolated by the modified CTAB method (4). The initial PCR-based detection was performed using begomovirus coat protein gene specific primers (forward 5'-ATGGCGAAGCGACCAG-3' and reverse 5'-TTAATTTGTGACCGAATCAT-3'), which generated an amplicon of 771 bp in most of the (17/20) symptomatic samples. No amplification was obtained in healthy or non-symptomatic plant samples. The full-length genome was amplified via rolling-circle amplification (RCA) according to the manufacturer's instructions using random hexamer primers and φ29 DNA polymerase. A portion of the RCA product (1 µl) was subjected to digestion with different restriction enzymes, out of which BamHI yielded DNA fragments of approximately 2.7 and 1.3 kb, corresponding to DNA-A and ß satellite molecules, respectively. These fragments were eluted from the gel and cloned into the suitable restriction site of pGreen0029 vector. The positive clones were checked by restriction digestion. Twelve out of 20 clones were found to be positive and sequenced. The complete genome sequences of DNA A (2,754 bp) and ß (1,366 bp) satellites were deposited in the GenBank database with the accession numbers KM359406 and KM359407, respectively. The absence of DNA-B molecule was ascertained, as no PCR amplification was detected with DNA-B-specific primers. Sequence analysis showed highest nucleotide identity (90%) with Catharanthus yellow mosaic virus (CYMV) (HE580234) and ≤85% identity with other begomoviruses of the database. Sequence analysis of the associated betasatellite showed 96% identity with Andrographis yellow vein leaf curl betasatellite (KC967282). CYMV was first reported on Catharanthus roseus with no associated betasatellite from Pakistan (2). However, this is the first report of CYMV along with a betasatellite infecting A. paniculata in India. Recently a begomovirus (Eclipta yellow vein virus) infection was reported on A. paniculata in association with Andrographis yellow vein leaf curl betasatellite from India for the first time (3); now the crop has also become a host of CYMV. Thus, this study highlights the spread of CYMV from its preliminary host to a new host plant (A. paniculata), across the South Asian countries. Therefore, it is important to take measures for the management of its transmitting vector so as to curtail the spread of the virus to new economically and commercially important crops. References: (1) S. Akbar. Altern. Med. Rev. 16:1, 2011. (2) M. Ilyas et al. Arch. Virol. 158:505, 2013. (3) A. Khan and A. Samad. Plant Dis. 98:698, 2014. (4) S. P. S. Khanuja et al. Plant Mol. Biol. Rep. 17:1, 1999.

First Report on the Molecular Identification of Phytoplasma (16SrII-D) Associated with Witches' Broom of Kalmegh (Andrographis paniculata) in India.

Andrographis paniculata (family Acanthaceae), also known as "King of Bitters" or Kalmegh, is an important medicinal plant used for the treatment of various diseases. It has antimicrobial, antiviral, anti-inflammatory, hepatoprotective, antidiabetic, antihyperglycemic, and antioxidant properties (1). During June 2014, while performing a routine survey of the commercial trial fields of Kalmegh at Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow, India, typical phytoplasma disease symptoms such as virescence, proliferation, and witches' broom along with little leaf and stunted growth were observed. The disease incidence was estimated to be approximately 7 to 10%. To ascertain the presence of phytoplasma, 16 samples of leaves were collected from nine different field sites, and total genomic DNA was extracted from the symptomatic and symptomless Kalmegh plants by the CTAB method. Direct and nested PCR assays were performed targeting the 16S rDNA using generic phytoplasma primer pairs P1/P6 followed by R16F2n/R16R2 (2). Resulting bands of the expected size (1.5 kb and 1.2 kb, respectively) were amplified from symptomatic plants. No amplification was observed with DNA from asymptomatic plant samples. The purified nested PCR products were cloned into E. coli DH5α, using the pGEM-T Easy vector (Promega, United States) and sequenced with primers M13For/M13Rev using an automatic sequencer (ABI Prism, Perkin Elmer) at CIMAP. The sequence was analyzed by BLASTn and found to share 99% similarity with Echinacea witches'-broom phytoplasma and Sesame phyllody phytoplasma strain (GenBank Accession Nos. JF340080 and KF612966, respectively), which belong to the 16SrII-D group. The sequence was deposited in NCBI as GenBank Accession No. KM359410. A phylogenetic tree using MEGA v5.0 (4) was constructed with 16S rDNA; consensus sequences of phytoplasmas belonging to distinct groups revealed that the present phytoplasma clustered with the 16SrII group. iPhyClassifier software was used to perform sequence comparison and generate a virtual restriction fragment length polymorphism (RFLP) profile (5). On the basis of iPhyClassifier, the 16S rDNA sequence analysis of our isolate showed 99.2% similarity with that of the 'Candidatus Phytoplasma australasiae' reference strain (GenBank Accession No. Y10097), which belongs to 16Sr group II. The virtual RFLP pattern of F2n/R2 fragment was most similar to the 16SrII-D subgroup (similarity coefficient of 0.91) but showed a difference in profile with HpaI, HhaI, and MseI enzymes. Several bacterial/fungal and viral diseases have been reported on A. paniculata (3); however, to our knowledge, this is the first report of witches' broom disease in India and the first record of a 16SrII-D group phytoplasma on Kalmegh. Its presence in Kalmegh is of great significance due to its commercial interest. References: (1) S. Akbar. Altern. Med. Rev. 16:1, 2011. (2) D. E. Gundersen and M. Lee. Phytopathol. Mediterr. 35:144, 1996. (3) A. Khan and A. Samad. Plant Dis. 98:698, 2014. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011. (5) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

New Report of a Sweet Basil Leaf Blight Caused by Cochliobolus lunatus in India.

Sweet basil (Ocimum basilicum), a member of the Lamiaceae, is used as an ornamental as well as a culinary herb. It is a rich source of the phenolic compound methyl chavicol and is used as a traditional medicinal plant in India, where the crop is grown on ~2,500 ha annually (4). The species is native to India, where it has been cultivated for >5,000 years. During the rainy season, August of 2013, a severe leaf blight was observed on 30- to 45-day-old sweet basil plants in experimental fields (approximately 5 ha) at the CSIR-CIMAP and adjoining areas in Lucknow. Initial symptoms comprised small, irregular, necrotic lesions that coalesced into a leaf blight. Infected parts of the leaves turned black during wet and humid conditions. The incidence of symptoms ranged from 20 to 30%. Infected leaf samples were cut into small pieces and surface-sterilized with 1% sodium hypochlorite for 1 min, followed by two rinses in sterilized, distilled water. The leaf pieces were then blotted dry with sterilized filter paper, placed onto potato dextrose agar (PDA), and incubated at 28°C for 3 to 5 days. Blackish-brown fungal colonies developed. Microscopic examination revealed the presence of brown conidiophores that were cylindrical, septate, unbranched, and straight or geniculate near the apex. Conidia were three-septate, mostly curved at the third cell from the base, which was usually larger and darker than the other cells; intermediate cells were brown or dark brown; terminal cells were subhyaline or pale brown and 16 to 23.5 × 8.5 to 11.5 µm (the average size of 100 conidia was 19.9 × 10.18 µm). On the basis of these characteristics, the fungus was identified as Cochliobolus lunatus (anamorph Curvularia lunata (Wakk.) Boedijin) (1,2). The identification was confirmed by sequencing the internal spacer (ITS) region of ribosomal DNA (rDNA). Genomic DNA was extracted from five fungal isolates, using the 5 Prime Archive Pure DNA Cell/Tissue kit, and subjected to a polymerase chain reaction (PCR) assay with the universal primers ITS1 and ITS4 (5). The amplified product was cloned and sequenced. An NCBI-BLASTn search showed greatest homology (98% similarity) with the ITS sequence of C. lunatus (GenBank Accession No. DQ836800). The sequence was deposited in Genbank (KM272001). A pathogenicity test was carried out using 10, 30-day-old sweet basil (cv. CIM Soumya) plants in pots, by spraying a spore suspension (105 spores/ml) onto the leaves of each plant. Five plants treated similarly with sterilized, distilled water served as a control treatment. The plants were kept at 27 ± 2°C and 85 ± 3% RH for 8 to 10 days. Small, irregular, necrotic lesions appeared after 4 days on all inoculated leaves, while leaves of control plants remained asymptomatic. Fungi re-isolated from inoculated leaves resembled C. lunatus on the basis of microscopic and sequence data, fulfilling Koch's postulates. The fungus was not re-isolated from the control plants. C. guatemalensis has been reported to cause a leaf spot on sweet basil in Korea (3). To our knowledge, this is the first report of a sweet basil leaf blight caused by C. lunatus in India. C. lunatus has the potential to reduce the yield of sweet basil. References: (1) L. M. Liu et al. Plant Dis. 98:686, 2014. (2) D. S. Manamgoda et al. Fungal Divers. 56:131, 2012. (3) J. H. Park et al. Plant Dis. 96:580, 2012. (4) H. A. A. Taie et al. Not. Bot. Hort. Agrobot. Cluj. Napoca 38:119, 2010. (5) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

A New Isolate of Eclipta yellow vein virus Along with a Betasatellite Associated with Yellow Vein Leaf Curl Disease of Andrographis paniculata in India.

Geminiviruses are ssDNA viruses that infect a wide range of plant species. Andrographis paniculata (family Acanthaceae), an herb commonly known as Kalmegh, is famous for its medicinal properties such as anti-inflammatory, antibiotic, antimalarial, anticancerous, antidiabetic, antihepatotoxic, antioxidant, and is helpful in curing various diseases (1). Surveys of kalmegh fields carried out in September and October 2010 in Lucknow, India, revealed symptomatic plants that initially showed yellow veins on younger leaves and later upward leaf curling, vein clearing, chlorosis, reduced leaf size, poor inflorescence, and stunted growth leading to drastic reduction in herb yield. The disease incidence was estimated at 25 to 40%. Aphids (Myzus persicae and Aphis crassivora) failed to transmit the disease; however, similar disease symptoms developed on healthy plants after transmission by viruliferous whiteflies (Bemesia tabaci). Healthy whiteflies were used for acquisition feeding on the naturally infected twig of A. paniculata and then transferred to healthy seedlings for an overnight inoculation feeding. After inoculation feeding, whiteflies were killed by insecticide. Four out of six plants were positive after whitefly transmission. Total nucleic acids were extracted from the leaves of symptomatic and symptomless plants by modified CTAB method. PCR amplification of a 771-bp fragment of DNA, with begomovirus CP gene-specific primers (forward 5'-ATGGCGAAGCGACCAG-3' and reverse 5'-TTAATTTGTGACCGAATCAT-3') from symptomatic samples only, supported the presence of a begomovirus. The amplified DNA fragment was revealed in 13 out of 17 symptomatic samples. The full length DNA-A was amplified using two sets of overlapping primer pairs (F1For/F1Rev and F2For/F2Rev), generating the amplicons of ~1,200 bp and ~1,700 bp in size, respectively (3). Nine PCR positive samples were eluted from agarose gel by QIAquick gel extraction kit (Qiagen), cloned into pGEM-T Easy vector (Promega), and 16 clones were sequenced. The complete DNA-A sequence (2,739 nt) was deposited in GenBank (Accession No. KC476655). Sequence analysis showed 96% nucleotide identity with Eclipta yellow vein virus (EYVV, GQ478343) and more distant affinities (≤89%) with other begomoviruses. No DNA-B was detected in any of the samples with the universal primer pair PBL1v2040/PCRc1 (4). However, a betasatellite was identified by PCR amplification of a 1,379-bp fragment using universal primers ß01 and ß02 (2). Sequence analysis of this betasatellite (KC967282) associated with the present disease showed 83% to 89% identity with sequences of other betasatellites, like Ageratum yellow vein betasatellite (AJ542498), available in GenBank. There was no evidence of the presence of alphasatellites. The presence of a begomovirus and an associated betasatellite was also validated using rolling circle amplification with TempliPhi 100 Amplification system (GE Healthcare), which generated two fragments of 2.7 kb and 1.3 kb, respectively, after digestion with enzymes EcoRI, EcoRV, and HincII. EYVV (family Geminiviridae; genus Begomovirus) was reported for the first time from Pakistan in 2006 on Eclipta prostrata (GQ478343.1). To our knowledge, this is the first report of a new isolate of EYVV infecting A. paniculata in India. Kalmegh is cultivated as a mixed crop in some areas and it could potentially be a reservoir of inoculum to other hosts susceptible to begomoviruses. References: (1) S. Akbar. Altern Med Rev. 16:1, 2011. (2) R. W. Briddon et al. Mol Biotechnol. 20:315, 2002. (3) A. Kumar et al. New Dis. Rep. 24:18, 2011. (4) M. R. Rojas et al. Plant Dis. 77:340, 1993.

First Report of Chilli leaf curl India virus Infecting Mentha spicata (Neera) in India.

Mint (Mentha spp.; family Lamiaceae) is an important essential oil-bearing crop cultivated on the Indian subcontinent as a cash crop for the international market and industrial purposes. Since May 2010, typical symptoms such as yellow vein, leaf yellowing, mosaic, crinkling, and cupping were observed, which led to significant yield loss in spearmint (M. spicata var. Neera) at CIMAP experimental fields and farmers' fields of Badaun, Rampur, and Moradabad regions of Uttar Pradesh province, India. Disease incidence was recorded in the range of 40 to 50%. Mentha spp. has been reported to be affected by many viral diseases (3). Due to the absence of fungal/bacterial infection, lack of mechanical transmission of the pathogen, and presence of whiteflies in the fields, the causal pathogen was suspected to be a begomovirus. Total genomic DNA was extracted from the leaves of naturally infected and healthy samples of Mentha by the CTAB protocol. Eighteen symptomatic samples were collected from different location of fields and screened for the presence of begomovirus. DNA from these samples was used as PCR template to amplify a 771-bp fragment using begomovirus coat protein (CP) gene specific primers. Eleven of 18 (61.1%) samples were found positive. PCR products were cloned into the pGEM-T Easy (Promega) and sequenced using the universal M13F/M13R primers showed sequence similarity with Chilli leaf curl India virus. To amplify the full-length DNA-A/B and a possible ß-satellite, a second detection method was used: rolling circle amplification (RCA) using the TempliPhi 100 Amplification System (GE Healthcare). RCA products were digested independently with various restriction enzymes: BamHI, EcoRI, EcoRV, HincII, HindIII, SacI, and KpnI. Digested products were resolved on 1% agarose gel and the bands corresponding to ~2.7 and ~1.3 kb were purified using Nucleospin Gel and PCR Clean-up Kit and cloned into the respective sites of pGreen0029 vector. The sequence of full-length DNA-A (2,749 bp) and ß-satellite component (1,347-bp) were obtained and deposited in NCBI GenBank with accession nos. KF312364 and KF364485, respectively. The sequence analysis showed maximum nucleotide identity (99%) with Chilli leaf curl India virus (FM877858) and distant affinities (≤88%) with other begomoviruses. The sequence analysis of isolated ß-satellite showed 93% identity with Ageratum yellow vein virus satellite (AJ252072.1). No presence of DNA-B was detected using the universal primer PBL1v2040/PCRc1 (2), thus confirming it to be a monopartite begomovirus (1). Viruliferous whiteflies (Bemisia tabaci) proved Koch's postulation by inducing similar symptoms on healthy plants while aphids (Myzus persicae) failed to transmit the virus. To our knowledge, this is the first report of Chilli leaf curl India virus infecting M. spicata var. Neera in India. Mint is widely grown together with other reported hosts of begomoviruses, and thus could pose a serious threat as future expansion of begomovirus to new crops. Hence, the development of resistant varieties coupled with the implementation of adapted integrated pest management strategies would be essential for successful production of mint crops. References: (1) Y. Kumar et al. Plant Pathol. 60:1040, 2011. (2) M. R. Rojas et al. Plant Dis. 77:340, 1993. (3) I. E. Tzanetakis et al. Plant Dis. 94:4, 2010.

New Report of Black Leaf Spot Mold (Pseudocercospora fuligena) on Withania somnifera from India.

Withania somnifera (family solanaceae) commonly known as ashwagandha and Indian ginseng, originated in India is one of the most powerful medicinal plants for more than 3,000 years (1). It is commercially cultivated for its roots, a natural rich source of glycowithanolides, tannins, potassium nitrate, etc., which are an anti-inflammatory, anti-tumor, anti-oxidant, anti-ulcer, and regulator of the nervous system and sleep (2). During the monsoon of July 2011, black spots on the leaves of infected plants were observed in the ashwagandha growing Lucknow, Raibareilly, and adjoining areas of Uttar Pradesh province with 10 to 20% disease incidence. Early stage of disease were characterized by the presence of light chlorotic spots on both sides of old leaves that later turned into dark black spots resulting in early defoliation. About 27 samples were collected from different locations of the fields for isolation of the causal organism and microscopic studies. Infected leaves were cut into small pieces, surface sterilized with 1% sodium hypochlorite for 1 min, rinsed thrice with sterilized distilled water, and placed onto potato dextrose agar (PDA) plates. After 21 days of dark incubation at 25°C, 8- to 10-mm grayish-brown colonies were observed. Microscopic studies at early and mature stages of infection showed production of conidia in conidiophores. Conidiophores were mostly 5 to 9, few dense pale brown, simple unbranched, septate, geniculate and 14 to 55 × 3 to 5.5 µm. Conidia were subhyline, obclavate to cylindrical, some were straight to slightly curved, multiseptate, base long obconic to long obconically truncate, and 12 to 85 × 3.5 to 5 µm. On the basis of cultural and morphological studies, the pathogen was identified as Pseudocercospora fuligena (3). The pathogen identity was further confirmed at molecular level using universal primers ITS1/ITS4 through PCR (4). An amplification of the expected size (~550 bp) was generated, eluted from agarose gel by QIAquick gel extraction kit (Qiagen), cloned into pGEM-T Easy vector (Promega), sequenced, and deposited in GenBank (Accession No. KF881898). NCBI BLASTn showed 99% identity with P. fuligena (GU214675) strain CPC 12296, isolated from Lycopersicon sp. Pathogenicity test was carried out on 10 plants of W. somnifera cv. Poshita through two approaches, one using mycelia from culture and another using spore suspension from naturally infected leaves. In the first approach, fungal mycelia were applied onto the healthy ashwagandha leaves, whereas in the second approach, infected leaves were washed with distilled water and spore suspension of 106 spores/ml was sprayed on healthy plants. Plants sprayed with sterilized distilled water served as controls. Inoculated plants were placed in a growth chamber at 28°C under 90% humidity for 3 days. After, pots were placed in the glasshouse at 27 ± 2°C with 70 to 80% humidity for 21 days. Initial symptoms appeared on the 7th day while typical symptoms appeared on all the inoculated plants after 12 to 17 days. Control plants remained free of infection. Re-isolation of the pathogen on PDA fulfilled Koch's postulates. Black leaf mold caused by P. fuligena has been reported on tomato (5). This is the first report of black leaf mould caused by P. fuligena on W. somnifera from India. P. fuligena has the potential to reduce yield of W. somnifera. References: (1) Anonymous. Alt. Med Rev. 9:211, 2004. (2) B. D. Basu and K. R. Kirtikar. Indian Medicinal Plants: Plates, vol. 1-4. Bishen Singh Mahendra Pal Singh, Dehradun, India, 1991. (3) T. C. Wang et al. Plant Dis. 79:661, 1995. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990. (5) S. Yamada. Ann. Phytopathol. Soc. Jpn. 15:13, 1951.

The Pictorial Fit-Frail Scale Malay Version (PFFS-M): Predictive Validity Testing in Malaysian Primary Care.

The purpose of this study was to evaluate the association between Pictorial Fit Frail Scale-Malay version (PFFS-M) and adverse outcomes, such as falls, new disability, hospitalisation, nursing home placement, and/or mortality, in patients aged 60 and older attending Malaysian public primary care clinics. We assessed the baseline PFFS-M levels of 197 patients contactable by phone at 18 months to determine the presence of adverse outcomes. 26 patients (13.2%) reported at least one adverse outcome, including five (2.5%) who fell, three (1.5%) who became disabled and homebound, 15 (7.6%) who were hospitalized, and three (1.5%) who died. Using binary multivariable logistic regression adjusted for age and gender, we found that patients who were at-risk of frailty and frail at baseline were associated with 5.97(95% CI [1.89-18.91]; P=0.002) and 6.13 (95% CI [1.86-20.24]; P= 0.003) times higher risk of developing adverse outcomes at 18 months, respectively, than patients who were not frail. The PFFS-M was associated with adverse outcomes.

Functional Grading Between Soft-Magnetic Fe-Co/Fe-Ni Alloys and the Effect on Magnetic and Microstructural Properties.

Producing soft magnetic alloys by additive manufacturing has the potential to overcome cracking and brittle fracture issues associated with conventional thermomechanical processing. Fe-Co alloys exhibit high magnetic saturation but low ductility that makes them difficult to process by commercial methods. Ni-Fe alloys have good ductility and high permeability in comparison to Fe-Co, but they suffer from low magnetic saturation. Functional grading between Fe-Co and Ni-Fe alloys through blown powder directed energy deposition can produce soft magnetic materials that combine and enhance properties beyond the strengths of the individual magnetic materials. This work focuses on the microstructure, crystal structure, and magnetic properties of functionally graded Fe49Co49V2/Ni80Fe16Mo4 coupons. The grading between the two materials is found to refine the microstructure, thereby improving the mechanical hardness without the use of a nonmagnetic element. Postbuild thermal treatments are found to recrystallize the microstructure and increase the grain size, leading to improved magnetic properties. Analysis of crystal structures provides an understanding of the solubility limits and phase equilibria between the BCC (Fe-Co) and FCC (Ni-Fe) structures. Success in functional grading of soft magnets may provide a pathway toward improving energy conversion efficiency through strategic combinations of high saturation and high strength materials.

First Report of Tagetes erecta Damping Off Caused by Ceratobasidium sp. from India.

The Mexican marigold (Tagetes erecta) is cultivated commercially in India for medicinal, ceremonial, and decorative purposes. It is native to Mexico and the United States. The natural phytochemical thiophene extracted from T. erecta has been shown to have antibacterial activity. It is also grown to extract lutein, a common yellow/orange food color. During winter of 2011, approximately 15% marigold seedlings exhibited damping off symptoms at CSIR-CIMAP, Lucknow, India, and its adjoining areas. Infected seedlings initially produced water-soaked lesions on the stem at the soil level that later turned pinkish with a brownish halo in the center. The infected seedlings were cut into small pieces, surface disinfected with 1% sodium hypochlorite, rinsed thrice with sterile distilled water, and placed onto potato dextrose agar (PDA) plates. The plates were incubated at 25°C for 3 days. The isolation yielded whitish fungal growth that later turned tan brown. The mycelium was binucleate, septate, sub branching at right angles, with distinct constriction at the origin of branching. Bisbenzamide (Hoechst 33258; Chemical Abstracts no. 23491-45-4) was used as fluorescent dye for the staining of nuclei. Based on cultural as well as morphological characteristic features, the fungus was identified as Ceratobasidium sp. (1,2). The molecular identification was on the basis of internal transcribed spacer (ITS) region sequence. Amplification of the ITS of rDNA using primers ITS1/ITS4 yielded a ~700 bp band and sequenced data were deposited in the NCBI GenBank (KC193238). The ITS region (700 bp) was 100% identical to Ceratobasidium sp. AG-F strain BAGF101 isolated from Musa spp. in Georgia, United States (GenBank Accession No. HQ168370). The pathogenicity of the fungus was tested under glasshouse conditions. The inoculum of the fungus was prepared on sterile maize seeds in Erlenmeyer flasks by inoculating seeds with three disks (1 mm) of 5-day-old culture, and kept at 25 ± 2 °C for 14 days in the dark. The healthy, 5 to 7 day old seedlings were inoculated with five artificially infested maize seeds per pot. The uninoculated seedlings served as control. Both inoculated and uninoculated seedlings were kept at 28 ± 2°C in a humidity (95%) chamber for 3 days and thereafter placed in the glasshouse at 28 ± 2°C for development of disease symptoms. Initial symptoms developed as water-soaked lesions on the infected seedlings in 2 to 3 days, while typical disease symptoms appeared after 4 to 5 days of inoculation. Uninoculated seedlings were free from infection. The fungus was reisolated from the artificially infected seedlings on PDA and its identification as Ceratobasidium sp. was confirmed by morphological and molecular characteristics. Recently, Ceratobasidium sp. was reported as causal organism of root rot on Atractylodes macrocephala and banana (3,4). To the best of our knowledge, marigold damping off disease caused by Ceratobasidium sp. has not been reported so far on T. erecta. Hence, it is the first report from India. During fungal disease management for marigold, association of Ceratobasidium sp. should not be ignored for better crop protection. References: (1) R. T. Moore. Mycotaxon 29:91, 1987. (2) B. Sneh et al. Identification of Rhizoctonia Species. The American Phytopathological Society, St. Paul, MN. 1991. (3) J. Yin et al. Plant Dis. 95:490, 2011. (4) J. M. You et al. Plant Dis.97:139, 2013.

Influence of particle shape on plasma protein adsorption and macrophage uptake.

The purpose of this study was to evaluate the plasma protein adsorption behavior onto different LIPOMER nanoparticles, especially looking for the first time, if the particle shape affects the protein adsorption pattern. The potential in vivo fate is discussed and compared with previous in vivo animal studies. The two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was used for identification of adsorbed plasma proteins. Qualitative similar patterns were obtained from the protein adsorption analysis and four apolipoproteins with considerable quantitative differences were identified. Besides the quantitative differences in the adsorbed apolipoproteins, in vitro uptake in the human macrophage cell line U-937 of histocytic lymphoma organ revealed significantly lower uptake of the irregular glycerol monostearate LIPOMER nanoparticles. Therefore, protein adsorption does not seem to play a role in the splenotropic behavior in the sense, that adsorption of opsonins, especially spleen-specific opsonins are required for the uptake. The splenotropic uptake might be favored because all LIPOMER nanoparticles did not adsorb opsonins at all, mediating competitive uptake by liver macrophages. Differences in the in vivo uptake by the spleen were attributed to differences in particle shape with potential super position effect by the quantitative differences in the adsorbed proteins.

Complete Heparin Reversal by Protamine during Off-Pump Coronary Artery Bypass Surgery (OPCAB): A Necessity or Myth?

In our country majority of the coronary artery bypass surgery (CABG) are done off-pump and was reported having excellent clinical outcome along with cost efficiency by various investigators. Heparin is commonly used as most effective anticoagulant, and protamine sulfate is now generally used to reverse the anticoagulant action of heparin. While under dosing of protamine may result in incomplete heparin reversal and prolonged anticoagulation, protamine overdosing is associated with impaired clot formation exerted by the intrinsic anti-coagulation properties of protamine itself, moreover protamine administration is associated with mild to severe cardiovascular and pulmonary complications. Apart from traditional full neutralization of heparin now-a-days, half dose protamine was also introduced showing good outcome regarding lower activated clotting time (ACT), overall, less surgical bleeding with less transfusion. This comparative study was designed to detect differences between traditional and decreased protamine dosing in Off-Pump Coronary Artery Bypass (OPCAB) surgery. Four hundred (400) patients who underwent Off-Pump Coronary Artery Bypass Surgery (OPCAB) surgery at our institution over a period of 12 months were analyzed and were divided into two groups. Group A- received 0.5mg of protamine per 100 unit of heparin; Group B-received 1.0mg of protamine per 100 unit of heparin. ACT, blood loss, hemoglobin and platelet count units of blood and blood product transfusion requirements, clinical outcome and hospital stay were assessed in each patient. This study showed that 0.5mg of protamine per 100 unit of heparin was always able to reverse the anticoagulant effect of heparin with no significant difference in hemodynamic parameters, amount of blood loss and requirements of blood transfusion in between the groups. A standard protamine dosing formula (protamine-heparin at ratio of 1:1) adequate for on-pump cardiac surgical procedures significantly overestimates protamine requirements for OPCAB. Patients treated with decreased protamine do not appear to have adverse outcomes in terms of post-operative bleeding.

Boosting Live Malaria Vaccine with Cytomegalovirus Vector Can Prolong Immunity through Innate and Adaptive Mechanisms.

Vaccines to persistent parasite infections have been challenging, and current iterations lack long-term protection. Cytomegalovirus (CMV) chronic vaccine vectors drive protection against SIV, tuberculosis and liver-stage malaria correlated with antigen-specific CD8 T cells with a Tem phenotype. This phenotype is likely driven by a combination of antigen-specific and innate adjuvanting effects of the vector, though these mechanisms are less well understood. Sterilizing immunity from live Plasmodium chabaudi vaccination lasts less than 200 days. While P. chabaudi-specific antibody levels remain stable after vaccination, the decay of parasite-specific T cells correlates with loss of challenge protection. Therefore, we enlisted murine CMV as a booster strategy to prolong T cell responses against malaria. To study induced T cell responses, we included P. chabaudi MSP-1 epitope B5 (MCMV-B5). We found that MCMV vector alone significantly protected against a challenge P. chabaudi infection 40-60 days later, and that MCMV-B5 was able to make B5-specific Teff, in addition to previously-reported Tem, that survive to the challenge timepoint. Used as a booster, MCMV-B5 prolonged protection from heterologous infection beyond day 200, and increased B5 TCR Tg T cell numbers, including both a highly-differentiated Tem phenotype and Teff, both previously reported to protect. B5 epitope expression was responsible for maintenance of Th1 and Tfh B5 T cells. In addition, the MCMV vector had adjuvant properties, contributing non-specifically through prolonged stimulation of IFN-γ. In vivo neutralization of IFN-γ, but not IL-12 and IL-18, late in the course of MCMV, led to loss of the adjuvant effect. Mechanistically, sustained IFN-γ from MCMV increased CD8α+ dendritic cell numbers, and led to increased IL-12 production upon Plasmodium challenge. In addition, neutralization of IFN-γ before challenge reduced the polyclonal Teff response to challenge. Our findings suggest that, as protective epitopes are defined, an MCMV vectored booster can prolong protection through the innate effects of IFN-γ.

Identification of levomenthol derivatives as potential dipeptidyl peptidase-4 inhibitors: a comparative study with gliptins.

Dipeptidyl peptidase-4 (DPP4) inhibitors are a potent therapeutic treatment for type 2 diabetes mellitus (T2DM). There is a family of compounds used as DPP4 inhibitors (DPP4Is) called gliptins. They bind tightly to DPP4 to form an inactive protein-ligand complex. However, there remains a need to identify novel DPP4Is that are more efficacious and safer due to the increasing prevalence of T2DM and the undesirable side effects of gliptins. To identify potential DPP4Is, we screened over 1800 novel compounds in a comparative study with gliptins. We performed dual-factor molecular docking to assess the binding affinity of the compounds to DPP4 and found four compounds with a higher binding affinity to DPP4 than currently used gliptins. The newly identified compounds interacted with the dyad glutamate (GLU205 and GLU206) and tyrosine (TYR662 and TYR666) residues in DPP4's active site. We performed molecular dynamics simulations to determine the stability of the protein-ligand complexes formed by the compounds and DPP4. Furthermore, we examined the toxicity and pharmacological profile of the compounds. The compounds are drug-like, easy to synthesize, and relatively less toxic than gliptins. Collectively, our results suggest that the novel compounds are potential DPP4Is and should be considered for further studies to develop novel antidiabetics.Communicated by Ramaswamy H. Sarma.

Pharmacokinetic-pharmacodynamic equivalence of three gliclazide formulations in healthy human male subjects.

AIMS: To find out the pharmacokinetic (PK) and pharmacodynamic (PD) parameters for assessing the bioequivalence of three marketed products. To study the relationship between the pharmacokinetics of gliclazide and pharmacodynamic effect in healthy male volunteers. METHODS: This was an open label, balanced, randomized, 3-treatment, 3-sequence, 3 period, single-dose, cross-over bioavailability study in which 18 healthy adults were randomized to receive gliclazide 80 mg with 7 days wash out between treatments. The drug was administered with 240 ml of 20% glucose solution after a 10 h overnight fasting. Pharmacokinetic parameters like t(max), C(max), AUC(0-t), AUC(0-∞), AUC(0-t) / AUC(0-∞) and t(1/2) and pharmacodynamic parameters like maximum effect (minimum glucose level in the body, C(minglu)), time to minimum glucose level in the body (T(cminglu)) and partial AUC were calculated for all the products. RESULTS: The values for mean ± SD for age, height and weight of the volunteers were 28.00 ± 22.68, 165.78 ± 5.56 and 56.78 ± 13.37 respectively. There were total 4 withdrawn subjects and 1 drop out. Within batch accuracy of the method were in the range of 95.5 - 101.7%, 99.1 - 106.1% and 96.2 - 104.2% for three consecutive batches. The 90% CI for log transformed data of the PK and PD were within the acceptance range of 80.0 - 125.0%. CONCLUSIONS: This population PKPD analysis has characterized the relationship between the exposure to gliclazide and its hypoglycemic effect. The test products A & B compared to reference product R were bioequivalent on the basis of pharmacokinetic and pharmacodynamic parameters. Finally it is recommended that the more costly product R can be safely switched with less costly products i.e. A and B.