Proteome profile of Leishmania donovani Centrin1-/- parasite-infected human macrophage cell line and its implications in determining possible mechanisms of protective immunity.

Our developed cell division-specific 'centrin' gene deleted Leishmania donovani (LdCen1-/-) the causative parasite of the fatal visceral-leishmaniasis (VL), exhibits a selective growth arrest at the intracellular stage and is anticipated as a live attenuated vaccine candidate against VL. LdCen1-/- immunization in animals has shown increased IFN-γ secreting CD4+ and CD8+ T cells along with protection conferred by a protective proinflammatory immune response. A label-free proteomics approach has been employed to understand the physiology of infection and predict disease interceptors during Leishmania-host interactions. Proteomic modulation after infection of human macrophage cell lines suggested elevated annexin A6, implying involvement in various biological processes such as membrane repair, transport, actin dynamics, cell proliferation, survival, differentiation, and inflammation, thereby potentiating its immunological protective capacity. Additionally, S100A8 and S100A9 proteins, known for maintaining homeostatic balance in regulating the inflammatory response, have been upregulated after infection. The inhibitory clade of serpins, known to inhibit cysteine proteases (CPs), was upregulated in host cells after 48 h of infection. This is reflected in the diminished expression of CPs in the parasites during infection. Such proteome analysis confirms LdCen1-/- efficacy as a vaccine candidate and predicts potential markers in future vaccine development strategies against infectious diseases.

QbD-Assisted Development and Optimization of Doxycycline Hyclate- and Hydroxyapatite-Loaded Nanoparticles for Periodontal Delivery.

The current research aims to develop a carrier system for the delivery of a matrix metalloproteinase (MMP) inhibitor along with a bioceramic agent to the periodontal pocket. It is proposed that the present system, if given along with a systemic antibiotic, would be a fruitful approach for periodontitis amelioration. To fulfill the aforementioned objective, a doxycycline hyclate- and hydroxyapatite-adsorbed composite was prepared by a physical adsorption method and successfully loaded inside sodium alginate-chitosan nanoparticles and optimized based on particle size and drug content. Optimized formulation was then subjected to different evaluation parameters like encapsulation efficiency, hydroxyapatite content, ζ potential, surface morphology, in vitro drug release, cell line studies, and stability studies. For the optimized formulation, particle size, polydispersity index (PDI), entrapment efficiency, ζ potential, and drug content were found to be 336.50 nm, 0.23, 41.77%, -13.85 mV, and 14.00%, respectively. The surface morphology of the placebo and adsorbed composite-loaded nanoparticles as observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed the spherical shape and rough surface of the particles. In gingival crevicular fluid (GCF) 7.6, a sustained drug release profile was obtained up to 36 h. In vitro % viability studies performed on murine fibroblast cells (NIH3T3) and human periodontal ligament (hPDL) cell lines confirmed the proliferative nature of the formulation. Also, when subjected to stability studies for 4 weeks, particle size, PDI, and drug content did not vary considerably, thereby ensuring the stable nature of nanoparticles. Henceforth, sodium alginate-chitosan nanoparticles appeared to be a good carrier system for doxycycline hyclate and hydroxyapatite for periodontal therapy. If given along with a system antibiotic, the system will serve as a fruitful tool for infection-mediated periodontal regeneration and healing.

Facile extraction of berberine from different plants, separation, and identification by thin-layer chromatography, high-performance liquid chromatography, and biological evaluation against Leishmaniosis.

The extraction of berberine was carried out from Berberis vulgaris, Berberis aquifolium, and Hydrastis canadensis plants using ethanol and water (70:30, v/v). The extracted berberine was characterized by ultraviolet-visible and Fourier-transform infrared spectroscopy. The purity of berberine was ascertained by thin-layer chromatography using n-propanol-formic acid-water (95:1:4) and (90:1:9) solvents. hRf values were in the range of 44-49 with compact spots (diameter 0.2-0.4 cm). HPLC was carried out using ammonium acetate buffer and acetonitrile in gradient mode with Zodiac (4.6 × 150 mm, 3 µm) column. The flow rate was 1.0 mL/min and detection was at 220 nm. The values of separation and resolution factors of the standard and the extracted berberine were in the range of 1.13-1.16 and 1.40-1.71, respectively. A comparison has shown that both thin-layer chromatography and high-performance liquid chromatography (HPLC) methods found applications in different situations and requirements. The extracted berberine samples were used to treat Leishmaniosis and the results showed better activity of berberine in comparison to the standard drug Amphotericin B. Briefly, the reported research is a novel and may be used to extract berberine from plants, separation and identification of berberine by thin layer chromatography and HPLC and to treat Leishmaniosis.

High resolution melting based method for rapid discriminatory diagnosis of co-infecting Leptomonas seymouri in Leishmania donovani-induced leishmaniasis.

Leishmania donovani, a protozoan parasite of family Trypanosomatidae, causes fatal visceral leishmaniasis (VL) in the Indian subcontinent and Africa and cutaneous leishmaniasis (CL) in Sri Lanka. Another member of Trypanosomatidae, Leptomonas seymouri, resembling Leishmania was discovered recently to co-exist with L. donovani in the clinical samples from India and Sri Lanka and therefore, interfere with its investigations. We earlier described a method for selective elimination of such co-existing L. seymouri from clinical samples of VL exploiting the differential growth of the parasites at 37 °C in vitro. Here, we explored ways for a rapid discriminatory diagnosis using high resolution melting (HRM) curves to detect co-occurring L. seymouri with L. donovani in clinical samples. Initial attempt with kDNA-minicircle (mitochondrial DNA) based HRM did not display different Tm values between L. donovani and L. seymouri. Surprisingly, all of their minicircle sequences co-existed in similar clades in the dendrogram analysis, although the kDNA sequences are known for its species and strain specific variations among the Trypanosomatids. However, an HRM analysis that targets the HSP70 gene successfully recognized the presence of L. seymouri in the clinical isolates. This discovery will facilitate rapid diagnosis of L. seymouri and further investigations in to this elusive organism, including the clinico-pathological implications of its co-existence with L. donovani in patients.

Association between Altered Expression and Genetic Variations of Transforming Growth Factor ß-Smad Pathway with Chronic Myeloid Leukemia.

Background: Chronic myeloid leukemia (CML) is a hematological disorder caused by fusion of BCR and ABL genes. BCR-ABL dependent and independent pathways play equally important role in CML. TGFß-Smad pathway, an important BCR -ABL independent pathway, has scarce data in CML. Present study investigate the association between TGFß-Smad pathway and CML. Materials and Methods: Sixty-four CML patients and age matched healthy controls (n=63) were enrolled in this study. Patients were segregated into responder and resistant groups depending on their response to Imatinib mesylate (IM). TGFß1 serum levels were evaluated by ELISA and transcript levels of TGFß1 receptors, SMAD4 and SMAD7 were evaluated by Real-Time PCR. Sequencing of exons and exon-intron boundaries of study genes was performed using Next Generation Sequencing (NGS) in 20 CML patients. Statistical analysis was performed using SPSS version 16.0. Results:TGFß1 serum levels were significantly elevated (p = 0.02) and TGFßR2 and SMAD4 were significantly down-regulated (p = 0.012 and p = 0.043 respectively) in the patients. c.69A>G in TGFß1, c.1024+24G>A in TGFßR1 and g.46474746C>T in SMAD7 were the most important genetic variants observed with their presence in 10/20, 8/20 and 7/20 patients respectively. In addition, TGFßR1 transcript levels were reduced in CML patients with c.69A>G mutation. None of the genes differed significantly in terms of expression or genetic variants between responder and resistant patient groups. Conclusion: Our findings demonstrate the role of differential expression and genetic variants of TGFß-Smad pathway in CML. Decreased TGFßR2 and SMAD4 levels observed in the present study may be responsible for reduced tumor suppressive effects of this pathway in CML.

A novel signal sequence negative multimeric glycosomal protein required for cell cycle progression of Leishmania donovani parasites.

Expression of the intracellular form amastigote specific genes in the Leishmania donovani parasite plays a major role in parasite replication in the macrophage. In the current work, we have characterized a novel hypothetical gene, Ld30b that is specifically transcribed in the intracellular stage of the parasite. The recombinant Ld30b protein exists as a pentamer in solution as identified by native-PAGE and size exclusion gel chromatography. Structural analysis using circular dichroism and molecular modeling indicate that Ld30b belongs to family of cAMP-dependent protein kinase type I-alpha regulatory subunit. Co-localization immunofluorescence microscopy and western blot analyses (using anti-Ld30b antibody and anti-hypoxanthine-guanine phosphoribosyl transferase, a glycosome marker) on the isolated parasite glycosome organelle fractions show that Ld30b is localized in glycosome, though lacked a glycosome targeting PTS1/2 signal in the protein sequence. Episomal expression of Ld30b in the parasite caused the arrest of promastigotes and amastigotes growth in vitro. Cell cycle analysis using flow cytometry indicates that these parasites are arrested in 'sub G0/G1' phase of the cell cycle. Single allele knockout of Ld30b in the parasite similarly attenuated its growth by accumulation of cells in the S phase of cell cycle, thus confirming the probable importance of appropriate level of protein in the cells. Studying such intracellular stage expressing genes might unravel novel regulatory pathways for the development of drugs or vaccine candidates against leishmaniasis.