Cytokine and Antibody Responses to Plasmodium falciparum in Na?ve Individuals during a First Malaria Episode: Effect of Age and Malaria Exposure

Age- and exposure-dependent immune responses during a malaria episode may be key to understanding the role of these factors in the acquisition of immunity to malaria. Plasma/serum samples collected from naïve Mozambican children (n = 48), European adults (naïve travelers, n = 22; expatriates with few prior malaria exposures, n = 15) and Mozambican adults with long-life malaria exposure (n = 99) during and after a malaria episode were analyzed for IgG against merozoite proteins by Luminex and against infected erythrocytes by flow cytometry. Cytokines and chemokines were analyzed in plasmas/sera by suspension array technology. No differences were detected between children and adults with a primary infection, with the exception of higher IgG levels against 3D7 MSP-1₄₂ (P = 0.030) and a P. falciparum isolate (P = 0.002), as well as higher IL-12 (P = 0.020) in children compared to other groups. Compared to malaria-exposed adults, children, travelers and expatriates had higher concentrations of IFN-γ (P≤0.0090), IL-2 (P≤0.0379) and IL-8 (P≤0.0233). Children also had higher IL-12 (P = 0.0001), IL-4 (P = 0.003), IL-1β (P = 0.024) and TNF (P = 0.006) levels compared to malaria-exposed adults. Although IL-12 was elevated in children, overall the data do not support a role of age in immune responses to a first malaria episode. A T_H1/pro-inflammatory response was the hallmark of non-immune subjects.     

A human cancer cell line initiates DNA replication normally in the absence of ORC5 and ORC2 proteins

The origin recognition complex (ORC), composed of six subunits, ORC1–6, binds to origins of replication as a ring-shaped heterohexameric ATPase that is believed to be essential to recruit and load MCM2–7, the minichromosome maintenance protein complex, around DNA and initiate DNA replication. We previously reported the creation of viable cancer cell lines that lacked detectable ORC1 or ORC2 protein without a reduction in the number of origins firing. Here, using CRISPR-Cas9–mediated mutations, we report that human HCT116 colon cancer cells also survive when ORC5 protein expression is abolished via a mutation in the initiator ATG of the ORC5 gene. Even if an internal methionine is used to produce an undetectable, N terminally deleted ORC5, the protein would lack 80% of the AAA+ ATPase domain, including the Walker A motif. The ORC5-depleted cells show normal chromatin binding of MCM2–7 and initiate replication from a similar number of origins as WT cells. In addition, we introduced a second mutation in ORC2 in the ORC5 mutant cells, rendering both ORC5 and ORC2 proteins undetectable in the same cells and destabilizing the ORC1, ORC3, and ORC4 proteins. Yet the double mutant cells grow, recruit MCM2–7 normally to chromatin, and initiate DNA replication with normal number of origins. Thus, in these selected cancer cells, either a crippled ORC lacking ORC2 and ORC5 and present at minimal levels on the chromatin can recruit and load enough MCM2–7 to initiate DNA replication, or human cell lines can sometimes recruit MCM2–7 to origins independent of ORC.     

Isolation of L-ethionine resistant cell lines in Vigna sinensis L. after mutagen treatment

Chemical mutagenesis was employed for the isolation of variant cell lines resistant to L-Ethionine (L-Eth) in Vigna sinensis L. We have measured cell survival after treatment of Vigna sinensis cell suspensions with different mutagens: Ethyl methanesulfonate (EMS), N-methyl-N-nitrosoguanidine (NTG) and Acridine Orange (AO). NTG was more toxic than EMS and AO.L-Eth resistant colonies were isolated by plating on selective medium after NTG treatment. The frequencies of appearance of resistant cells of MS-3 media supplemented with 20 g/ml L-Eth were 1.3 × 10^-6 to 1.8 × 10^-5. The highest number of L-Eth resistant calli were recorded in cells treated with 10 g/ml NTG. Few resistant colonies also appeared spontaneously from non-mutagenized cultures with a frequency of 2.9 × 10^-7 to 5.7 × 10^-7. However, a number of isolated colonies were discarded after successive retesting. The number of resistant calli dropped from 204 to 22 during successive retests. The significance of these observations has also been discussed.     

Identifying dynamical persistent biomarker structures for rare events using modern integrative machine learning approach

The evolution of omics and computational competency has accelerated discoveries of the underlying biological processes in an unprecedented way. High throughput methodologies, such as flow cytometry, can reveal deeper insights into cell processes, thereby allowing opportunities for scientific discoveries related to health and diseases. However, working with cytometry data often imposes complex computational challenges due to high-dimensionality, large size, and nonlinearity of the data structure. In addition, cytometry data frequently exhibit diverse patterns across biomarkers and suffer from substantial class imbalances which can further complicate the problem. The existing methods of cytometry data analysis either predict cell population or perform feature selection. Through this study, we propose a “wisdom of the crowd” approach to simultaneously predict rare cell populations and perform feature selection by integrating a pool of modern machine learning (ML) algorithms. Given that our approach integrates superior performing ML models across different normalization techniques based on entropy and rank, our method can detect diverse patterns existing across the model features. Furthermore, the method identifies a dynamic biomarker structure that divides the features into persistently selected, unselected, and fluctuating assemblies indicating the role of each biomarker in rare cell prediction, which can subsequently aid in studies of disease progression.     

Nondestructive evaluation of the photosynthetic properties of micropropagated plantlets by imaging photochemical reflectance index under low light intensity

The photochemical reflectance index (PRI) of micropropagated potato leaves was estimated nondestructively from outside the culture vessel using a PRI imaging system developed by the present group. The PRI was determined under low light intensity conditions after dark treatment and compared with the chlorophyll fluorescence parameter Fv/Fm, which denotes photosystem II maximum quantum yield. Short-term high-light treatment decreased Fv/Fm of the plantlets. Culture conditions such as temperature and sucrose concentration also affected Fv/Fm. A linear relationship between the PRI and Fv/Fm was observed in both cases of high-light treatment and different culture conditions, suggesting the potential of the PRI to be used as a substitute for Fv/Fm. PRI estimated from reflection images under low light intensity conditions may be used for rapid and noninvasive evaluation of photosynthetic properties of micropropagated plantlets in a similar manner to Fv/Fm.     

Cloning and Expression of Phytase appA Gene from Shigella sp. CD2 in Pichia pastoris and Comparison of Properties with Recombinant Enzyme Expressed in E. coli

The phytase gene appA_S was isolated from Shigella sp. CD2 genomic library. The 3.8 kb DNA fragment contained 1299 bp open reading frame encoding 432 amino acid protein (AppA_S) with 22 amino acid signal peptide at N-terminal and three sites of N-glycosylation. AppA_S contained the active site RHGXRXP and HDTN sequence motifs, which are conserved among histidine acid phosphatases. It showed maximum identity with phytase AppA of Escherichia coli and Citrobacter braakii. The appA_S was expressed in Pichia pastoris and E. coli to produce recombinant phytase rAppA_P and rAppA_E, respectively. Purified glycosylated rAppA_P and nonglycosylated rAppA_E had specific activity of 967 and 2982 U mg^-1, respectively. Both had pH optima of 5.5 and temperature optima of 60°C. Compared with rAppA_E, rAppA_P was 13 and 17% less active at pH 3.5 and 7.5 and 11 and 18% less active at temperature 37 and 50°C, respectively; however, it was more active at higher incubation temperatures. Thermotolerance of rAppA_P was 33% greater at 60°C and 24% greater at 70°C, when compared with rAppA_E. Both the recombinant enzymes showed high specificity to phytate and resistance to trypsin. To our knowledge, this is the first report on cloning and expression of phytase from Shigella sp.     

Promoter hypermethylation of p16INK4A, p14ARF, CyclinD2 and Slit2 in serum and tumor DNA from breast cancer patients

Epigenetic mechanisms such as DNA methylation play important role in cancer. Epigenetic alterations involved in the onset and progression of breast cancer may serve as biomarkers for early detection and prediction of disease prognosis. Furthermore, using body fluids such as serum offers a non-invasive method to procure multiple samples for biomarker analyses. The aim of this study is to determine the correlation between methylation status of multiple cancer genes, p16(INK4A), p14(ARF), Cyclin D2 and Slit2 in invasive ductal carcinoma of the breast and paired serum DNA and clinicopathological parameters. Of the 36 breast cancer patients investigated, 31 (86%) tumors and 30 (83%) paired sera showed methylation of at least one of these 4 genes. Methylation frequencies varied from 27% for CyclinD2, 44% for p16(INK4A), 47% for p14(ARF) to 58% for Slit2. There was concordance between DNA methylation in tumor and paired serum DNA of each gene. This study underscores the potential utility of DNA methylation based screening of serum as a surrogate marker for tumor DNA methylation status of these genes in breast cancer. Further, expression profile of p16(INK4A) could be linked to epigenetic events, thus suggesting this pathway as a potential target for therapeutic strategies based on reversal of epigenetic silencing.     

Phylogenetic analysis based evolutionary study of 16S rRNA in known Pseudomonas sp

Molecular evolution analysis of 16S rRNA sequences of native Pseudomonas strains and different fluorescent pseudomonads wereconducted on the basis of Molecular Evolutionary Genetics Analysis version 5.2 (MEGA5.2). Topological evaluations showcommon origin for native strains with other known strains with available sequences at GenBank database. Phylogenetic affiliationof different Pseudomonas sp based on 16S rRNA gene shows that molecular divergence contributes to the genetic diversity ofPseudomonas sp. Result indicate direct dynamic interactions with the rhizospheric pathogenic microbial community. The selectionpressure acting on 16S rRNA gene was related to the nucleotide diversity of Pseudomonas sp in soil rhizosphere community amongdifferent agricultural crops. Besides, nucleotide diversity among the whole population was very low and tajima test statistic value(D) was also slightly positive (Tajima׳s test statistics D value 0.351). This data indicated increasing trends of infection of soil-bornepathogens under gangetic-alluvial regions of West Bengal due to high degree of nucleotide diversity with decreased population ofplant growth promoting rhizobacteria like fluorescent Pseudomonads in soil.     

Molecular Identification of Cryptosporidium viatorum Infection in a Patient Suffering from Unusual Cryptosporidiosis in West Bengal,India

In this study, we have collected and screened a total of 268 stool samples from diarrheal patients admitted to an Infectious disease hospital in Kolkata for the presence of Cryptosporidium spp. The initial diagnosis was carried out by microscopy followed by genus specific polymerase chain reaction assays based on 70 kDa heat shock proteins (HSP70). DNA sequencing of the amplified locus has been employed for determination of genetic diversity of the local isolates. Out of 268 collected samples, 12 (4.48%) were positive for Cryptosporidium spp. Sequences analysis of 70 kDa heat shock proteins locus in 12 Cryptosporidium local isolates revealed that 2.24% and 1.86% of samples were showing 99% to 100% identity with C. parvum and C. hominis. Along with the other 2 major species one recently described globally distributed pathogenic species Cryptosporidium viatorum has been identified. The HSP70 locus sequence of the isolate showed 100% similarity with a previously described isolate of C. viatorum (Accession No. {"type":"entrez-nucleotide","attrs":{"text":"JX978274.1","term_id":"441431305","term_text":"JX978274.1"}}JX978274.1, {"type":"entrez-nucleotide","attrs":{"text":"JX978273.1","term_id":"441431303","term_text":"JX978273.1"}}JX978273.1, and {"type":"entrez-nucleotide","attrs":{"text":"JN846706.1","term_id":"385399407","term_text":"JN846706.1"}}JN846706.1) present in GenBank.