Effective Preparation of Plasmodium vivax Field Isolates for High-Throughput Whole Genome Sequencing

Whole genome sequencing (WGS) of Plasmodium vivax is problematic due to the reliance on clinical isolates which are generally low in parasitaemia and sample volume. Furthermore, clinical isolates contain a significant contaminating background of host DNA which confounds efforts to map short read sequence of the target P. vivax DNA. Here, we discuss a methodology to significantly improve the success of P. vivax WGS on natural (non-adapted) patient isolates. Using 37 patient isolates from Indonesia, Thailand, and travellers, we assessed the application of CF11-based white blood cell filtration alone and in combination with short term ex vivo schizont maturation. Although CF11 filtration reduced human DNA contamination in 8 Indonesian isolates tested, additional short-term culture increased the P. vivax DNA yield from a median of 0.15 to 6.2 ng µl⁻¹ packed red blood cells (pRBCs) (p = 0.001) and reduced the human DNA percentage from a median of 33.9% to 6.22% (p = 0.008). Furthermore, post-CF11 and culture samples from Thailand gave a median P. vivax DNA yield of 2.34 ng µl⁻¹ pRBCs, and 2.65% human DNA. In 22 P. vivax patient isolates prepared with the 2-step method, we demonstrate high depth (median 654X coverage) and breadth (≥89%) of coverage on the Illumina GAII and HiSeq platforms. In contrast to the A+T-rich P. falciparum genome, negligible bias was observed in coverage depth between coding and non-coding regions of the P. vivax genome. This uniform coverage will greatly facilitate the detection of SNPs and copy number variants across the genome, enabling unbiased exploration of the natural diversity in P. vivax populations.     

Giemsa-Stained Wet Mount Based Method for Reticulocyte Quantification: A Viable Alternative in Resource Limited or Malaria Endemic Settings

The quantity of circulating reticulocytes is an important indicator of erythropoietic activity in response to a wide range of haematological pathologies. While most modern laboratories use flow cytometry to quantify reticulocytes, most field laboratories still rely on ‘subvital’ staining. The specialist ‘subvital’ stains, New Methylene Blue (NMB) and Brilliant Crésyl Blue are often difficult to procure, toxic, and show inconsistencies between batches. Here we demonstrate the utility of Giemsa''s stain (commonly used microbiology and parasitology) in a ‘subvital’ manner to provide an accurate method to visualize and count reticulocytes in blood samples from normal and malaria-infected individuals.     

Proteomic identification of alterations in metabolic enzymes and signaling proteins in hypokalemic nephropathy

Hypokalemic nephropathy caused by prolonged K(+) deficiency is associated with metabolic alkalosis, polydipsia, polyuria, growth retardation, hypertension, and progressive tubulointerstitial injury. Its pathophysiology, however, remains unclear. We performed gel-based, differential proteomics analysis of kidneys from BALB/c mice fed with high-normal-K(+) (HNK), low-normal-K(+) (LNK), or K(+)-depleted diet for 8 wk (n = 6 in each group). Plasma K(+) levels were 4.62 +/- 0.35, 4.46 +/- 0.23, and 1.51 +/- 0.21 mmol/L for HNK, LNK, and KD mice, respectively (p < 0.0001; KD vs. others). With comparable amounts of food intake, the KD mice drank significantly more water than the other two groups and had polyuria. Additionally, the KD mice had growth retardation, metabolic alkalosis, markedly enlarged kidneys, renal tubular dilation, intratubular deposition of amorphous and laminated hyaline materials, and tubular atrophy. A total of 33 renal proteins were differentially expressed between the KD mice and others, whereas only eight proteins were differentially expressed between the HNK and LNK groups, as determined by quantitative intensity analysis and ANOVA with Tukey's post hoc multiple comparisons. Using MALDI-MS and/or quadrupole-TOF MS/MS, 30 altered proteins induced by K(+)-depletion were identified as metabolic enzymes (e.g., carbonic anhydrase II, aldose reductase, glutathione S-transferase GT41A, etc.), signaling proteins (14-3-3 epsilon, 14-3-3 zeta, and cofilin 1), and cytoskeletal proteins (gamma-actin and tropomyosin). Some of these altered proteins, particularly metabolic enzymes and signaling proteins, have been demonstrated to be involved in metabolic alkalosis, polyuria, and renal tubular injury. Our findings may lead to a new road map for research on hypokalemic nephropathy and to better understanding of the pathophysiology of this medical disease when the functional and physiological significances of these altered proteins are defined.     

Development and mapping of functional expressed sequence tag-derived simple sequence repeat markers in a rubber tree RRIM600 × PB217 population

Sets of polymorphic expressed sequence tag–simple sequence repeat (EST-SSR) markers from the rubber tree (Hevea brasiliensis) have been published by many researchers, but none has been specifically developed to study latex and wood yield traits. In this study, a total 10,321 rubber tree EST sequences, generated from suppression subtractive hybridization-cDNA libraries of bark and latex of high- and low-yielding clones, were used as sources for SSR searching. A total of 432 EST-SSR loci were identified and it was possible to design primer pairs for a subset of 298 EST-SSRs. The highest proportion of EST-SSRs was represented by dinucleotide repeats (46.6 %), followed by trinucleotide repeats (44.3 %). Based on BLASTX analysis, 234 ESTs (80 %) showed similarity to genes in NCBI databases and could be divided into 120 putative proteins with known function and 114 unknown proteins. To enhance the resolution of an existing linkage map from previous work on a rubber tree RRIM600 × PB217 population, 69 EST-SSR markers from the above set were tested to be integrated into the reference genetic map. The enriched map of 18 linkage groups spanned 2054.2 cM in length, showed an average genetic distance of 4.3 cM between adjacent markers, and included 63 new EST-SSR markers. The enhanced map from this study provides a basis for comparative mapping using PCR-based markers and identification of expressed genes possibly affecting important traits of interest.     

Secreted Products of Macrophages Exposed to Calcium Oxalate Crystals Induce Epithelial Mesenchymal Transition of Renal Tubular Cells via RhoA-Dependent TGF-β1 Pathway

Kidney stone disease is associated with renal fibrosis by the unclear mechanisms. We hypothesized that calcium oxalate (CaOx), a major crystalline component of kidney stones, could induce secretion of fibrotic factors from macrophages leading to “epithelial mesenchymal transition/transdifferentiation” (EMT) of renal tubular cells. Western blot analysis revealed an increased level of vimentin (mesenchymal marker) but decreased levels of E-cadherin and cytokeratin (epithelial markers) in MDCK cells treated with “secreted products from CaOx-exposed macrophages” (CaOx-M-Sup). Immunofluorescence study confirmed the increased level of vimentin and decreased level of cytokeratin, and also revealed the increased level of fibronectin (another mesenchymal marker). The data also showed decreased levels and disorganization of F-actin (cytoskeletal marker) and zonula occludens-1 (ZO-1) (tight junction marker) induced by CaOx-M-Sup. ELISA demonstrated the increased level of transforming growth factor-β1 (TGF-β1), the well-defined EMT inducer, in CaOx-M-Sup. Downstream signaling of TGF-β1 was involved as demonstrated by the decreased level of RhoA. Interestingly, pretreatment with a proteasome inhibitor (MG132) could restore RhoA to its basal level, most likely through ubiquitin-proteasome pathway (UPP). Moreover, MG132 successfully sustained cytoskeletal assembly and tight junction, and could prevent the cells from EMT. Altogether, these data demonstrate for the first time that CaOx-M-Sup could induce EMT in renal tubular cells by TGF-β1 signaling cascade via RhoA and UPP. This may be, at least in part, the underlying mechanism for renal fibrosis in kidney stone disease.     

EGCG decreases binding of calcium oxalate monohydrate crystals onto renal tubular cells via decreased surface expression of alpha-enolase

Crystal retention on tubular cell surface inside renal tubules is considered as the earliest and crucial step for kidney stone formation. Therapeutics targeting this step would cease the development of kidney stone. This study thus aimed to investigate the potential role of epigallocatechin-3-gallate (EGCG), a major antioxidant found in green tea leaves, in the reduction of calcium oxalate monohydrate (COM) crystal binding onto renal tubular cells. Pretreatment of the cells with EGCG for up to 6 h significantly diminished crystal-binding capability in a dose-dependent manner. Indirect immunofluorescence assay without and with cell permeabilization followed by laser-scanning confocal microscopy revealed that EGCG significantly reduced surface expression of alpha-enolase, whereas its intracellular level was increased. Western blot analysis confirmed such contradictory changes in membrane and cytosolic fractions of EGCG-treated cells, whereas the total level in whole cell lysate remained unchanged. Moreover, overexpression of surface alpha-enolase and enhancement of cell–crystal adhesion induced by 10 mM sodium oxalate were completely abolished by EGCG. Taken together, these data indicate that EGCG decreases binding of COM crystals onto renal tubular cells by decreasing the surface expression of alpha-enolase via re-localization or inhibition of alpha-enolase shuttling from the cytoplasm to the plasma membrane. These findings may also explain the effects of EGCG in reducing COM crystal deposition in previous animal models of kidney stone disease. Thus, EGCG may be useful for the prevention of new or recurrent stone formation.     

Macropinocytosis is the Major Mechanism for Endocytosis of Calcium Oxalate Crystals into Renal Tubular Cells

During an initial phase of kidney stone formation, the internalization of calcium oxalate (CaOx) crystals by renal tubular cells has been thought to occur via endocytosis. However, the precise mechanism of CaOx crystal endocytosis remained unclear. In the present study, MDCK renal tubular cells were pretreated with inhibitors specific to individual endocytic pathways, including nystatin (lipid raft/caveolae-mediated), cytochalasin D (actin-dependent or macropinocytosis), and chlorpromazine (CPZ; clathrin-mediated) before exposure to plain (non-labeled), or fluorescence-labeled CaOx monohydrate (COM) crystals. Quantitative analysis by flow cytometry revealed that pretreatment with nystatin and CPZ slightly decreased the crystal internalization, whereas the cytochalasin D pretreatment caused a marked decrease in crystal uptake. Immunofluorescence study and laser-scanning confocal microscopic examination confirmed that the cytochalasin D-pretreated cells had dramatic decrease of the internalized crystals, whereas the total number of crystals interacted with the cells was unchanged (crystals could adhere but were not internalized). These data have demonstrated for the first time that renal tubular cells endocytose COM crystals mainly via macropinocytosis. These novel findings will be useful for further tracking the endocytosed crystals inside the cells during the course of kidney stone formation.     

Significant Biochemical,Biophysical and Metabolic Diversity in Circulating Human Cord Blood Reticulocytes

Background

The transition from enucleated reticulocytes to mature normocytes is marked by substantial remodeling of the erythrocytic cytoplasm and membrane. Despite conspicuous changes, most studies describe the maturing reticulocyte as a homogenous erythropoietic cell type. While reticulocyte staging based on fluorescent RNA stains such as thiazole orange have been useful in a clinical setting; these ‘sub-vital’ stains may confound delicate studies on reticulocyte biology and may preclude their use in heamoparasite invasion studies.

Design and Methods

Here we use highly purified populations of reticulocytes isolated from cord blood, sorted by flow cytometry into four sequential subpopulations based on transferrin receptor (CD71) expression: CD71high, CD71medium, CD71low and CD71negative. Each of these subgroups was phenotyped in terms of their, morphology, membrane antigens, biomechanical properties and metabolomic profile.

Results

Superficially CD71high and CD71medium reticulocytes share a similar gross morphology (large and multilobular) when compared to the smaller, smooth and increasingly concave reticulocytes as seen in the in the CD71low and CD71negativesamples. However, between each of the four sample sets we observe significant decreases in shear modulus, cytoadhesive capacity, erythroid receptor expression (CD44, CD55, CD147, CD235R, and CD242) and metabolite concentrations. Interestingly increasing amounts of boric acid was found in the mature reticulocytes.

Conclusions

Reticulocyte maturation is a dynamic and continuous process, confounding efforts to rigidly classify them. Certainly this study does not offer an alternative classification strategy; instead we used a nondestructive sampling method to examine key phenotypic changes of in reticulocytes. Our study emphasizes a need to focus greater attention on reticulocyte biology.