Journal: Scientific Reports

Article Title: A Novel and Conserved Plasmodium Sporozoite Membrane Protein SPELD is Required for Maturation of Exo-erythrocytic Forms

doi: 10.1038/srep40407

Figure Lengend Snippet: ( A ) The amino acid sequence of PbSPELD was aligned with other Plasmodium orthologues P. chabaudi , PCHAS_0712200; P. yoelii , PY17X_0912300; P. cynomolgi , PCYB_094370; P. falciparum , PF3D7_1137800; P. knowlesi , PKNH_0936000 and P. vivax PVX_092505 using the Clustal Omega program. The conserved residues are shaded turquoise and physico-chemically similar residues are shaded grey. All orthologues contain a tyrosine-rich domain (underlined) and a putative transmembrane region (boxed). ( B ) The table shows amino acid identities between the indicated proteins. ( C ) The expression of pbspeld across P. berghei life cycle stages. The gene expression was analysed by quantitative real time PCR that revealed highest gene expression in the salivary gland sporozoites (SG Spz) followed by day 14 midgut sporozoites (MG Spz). The normalized data was expressed as a ratio of absolute copy numbers of pbspeld versus Pb18srRNA (internal control) for each stage of the Plasmodium life cycle. Ring: Ring stages, MBS: mixed blood stages, MG Spz: Midgut sporozoites, SG Spz: salivary gland sporozoites, LS16H: Liver stage 16 h, LS25H: Liver stage 25 h, LS42H: Liver stage 42 h, LS50H: Liver stage 50 h, LS65H: Liver stage 65 h.

Article Snippet: For microarray analysis of speld mutants, an Agilent Custom Plasmodium berghei gene expression microarray slide having 4 × 44k format designed by Genotypic Technology Private Limited was used that comprised of a total number of 43803 features including 5155 numbers of probes, and 1417 Agilent control features.

Techniques: Sequencing, Expressing, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: A Novel and Conserved Plasmodium Sporozoite Membrane Protein SPELD is Required for Maturation of Exo-erythrocytic Forms

doi: 10.1038/srep40407

Figure Lengend Snippet: ( A ) Genomic locus of pbspeld showing ORF, 3′ region and position of the primers. ( B ) Cloning of ORF (without stop codon) and 3′ region in the targeting vector pBC-mCherry-hDHFR. The pbspeld ORF was PCR amplified using primer set FP6/RP6 and cloned into ApaI/XhoI site of the targeting vector. The 3′ region of pbspeld was PCR amplified using primer set FP2/RP2 and cloned in the NotI/AscI site of the targeting vector. ( C ) Agarose gel showing products of diagnostic PCR with primer set FP7/RP7 and primer set FP8/RP8 indicating correct integration respectively in pbspeldmCherry transgenic clones C1 and C2. No product was amplified from genomic DNA of WT parasites using same set of primers. ( D ) pbspeldmCherry transgenic parasites express the reporter in oocyst and salivary gland sporozoite stages. (i) Dissected mosquito midgut showing oocyst expressing mCherry. Scale bar 200 μm. (ii) Higher magnification (100X) of oocyst showing mCherry expression in sporozoites within oocyst. Scale bar 10 μm. (iii) Dissected salivary gland lobe showing sporozoites expressing mCherry. Scale bar 10 μm. ( E ) Circles with white outline indicate sporozoites showing SPELD mCherry localization to the plasma membrane with in dissected salivary gland lobes. Scale bar 5 μm. ( F ) and ( G ) Colocalisation of mCherry with CSP in midgut sporozoites and salivary gland sporozoites respectively. The sporozoites were incubated with 3D11, a monoclonal antibody that recognizes the repeats of P. berghei CSP protein. Scale bar 10 μm. ( H ) pbspeldmCherry transgenic express reporter only at 17 h EEF development. The development of EEFs was monitored at indicated post-invasion time points by IFA using UIS4 antibody specific for the parasitophorous vacuolar membrane (PVM) and DAPI that facilitated visualizing host and parasite nuclei. Scale bar 10 μm.

Article Snippet: For microarray analysis of speld mutants, an Agilent Custom Plasmodium berghei gene expression microarray slide having 4 × 44k format designed by Genotypic Technology Private Limited was used that comprised of a total number of 43803 features including 5155 numbers of probes, and 1417 Agilent control features.

Techniques: Clone Assay, Plasmid Preparation, Amplification, Agarose Gel Electrophoresis, Diagnostic Assay, Transgenic Assay, Expressing, Incubation

Journal: Scientific Reports

Article Title: A Novel and Conserved Plasmodium Sporozoite Membrane Protein SPELD is Required for Maturation of Exo-erythrocytic Forms

doi: 10.1038/srep40407

Figure Lengend Snippet: ( A ) Heat map showing global gene expression changes in P. berghei liver stages at 36 hrs from WT and pbspeld ko. B) Pie diagram indicating the major functional pathways upregulated ( B ) and downregulated ( C ) in pbspeld ko. ( D ) Few of the well characterised liver stage specific genes downregulated in pbspeld ko. The plasmoDB ID of each of these genes with their probable functions are indicated. ( E ) Quantitative real time PCR showing downregulation of transcripts: LISP2, UIS4, FABL and EXP1 in pbspeld ko. (*p < 0.05,**p < 0.005, ***p < 0.0005 as compared to WT).

Article Snippet: For microarray analysis of speld mutants, an Agilent Custom Plasmodium berghei gene expression microarray slide having 4 × 44k format designed by Genotypic Technology Private Limited was used that comprised of a total number of 43803 features including 5155 numbers of probes, and 1417 Agilent control features.

Techniques: Expressing, Functional Assay, Real-time Polymerase Chain Reaction

Journal: BMC Genomics

Article Title: Transcription profile of Trichophyton rubrum conidia grown on keratin reveals the induction of an adhesin-like protein gene with a tandem repeat pattern

doi: 10.1186/s12864-016-2567-8

Figure Lengend Snippet: Real-time RT-PCR of selected genes from the microarray hybridization of T. rubrum genes during growth on keratin and elastin compared to control ( a ). Modulation of the adhesin-like protein gene of T. rubrum co-cultured with keratinocytes compared to fungal conidia ( b )

Article Snippet: The transcriptome profile of T. rubrum after growth on protein substrates was analysed using a microarray custom slide containing 6,091 sequences, which correspond to approximately 70 % of T. rubrum protein coding genes (according to the latest update released by the Broad Institute on 02/12/2014, available at www.broadinstitute.org/annotation/genome/dermatophyte_comparative ).

Techniques: Quantitative RT-PCR, Microarray, Hybridization, Control, Cell Culture