Whole Genomic Analysis of Human G12P[6] and G12P[8] Rotavirus Strains that Have Emerged in Myanmar

G12 rotaviruses are emerging rotavirus strains causing severe diarrhea in infants and young children worldwide. However, the whole genomes of only a few G12 strains have been fully sequenced and analyzed. In this study, we sequenced and characterized the complete genomes of six G12 strains (RVA/Human-tc/MMR/A14/2011/G12P[8], RVA/Human-tc/MMR/A23/2011/G12P[6], RVA/Human-tc/MMR/A25/2011/G12P[8], RVA/Human-tc/MMR/P02/2011/G12P[8], RVA/Human-tc/MMR/P39/2011/G12P[8], and RVA/Human-tc/MMR/P43/2011/G12P[8]) detected in six stool samples from children with acute gastroenteritis in Myanmar. On whole genomic analysis, all six Myanmarese G12 strains were found to have a Wa-like genetic backbone: G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 for strains A14, A25, P02, P39, and P43, and G12-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1 for strain A23. Phylogenetic analysis showed that most genes of the six strains examined in this study were genetically related to globally circulating human G1, G3, G9, and G12 strains. Of note is that the NSP4 gene of strain A23 exhibited the closest relationship with the cognate genes of human-like bovine strains as well as human strains, suggesting the occurrence of reassortment between human and bovine strains. Furthermore, strains A14, A25, P02, P39, and P43 were very closely related to one another in all the 11 gene segments, indicating derivation of the five strains from a common origin. On the other hand, strain A23 consistently formed distinct clusters as to all the 11 gene segments, indicating a distinct origin of strain A23 from that of strains A14, A25, P02, P39, and P43. To our knowledge, this is the first report on whole genome-based characterization of G12 strains that have emerged in Myanmar. Our observations will provide important insights into the evolutionary dynamics of spreading G12 rotaviruses in Asia.     

Differential rate of dentin formation on human deciduous anterior tooth surfaces deduced from tetracycline labeling

Distances between the intersecting points of two fluorescent lines with the outer dentin surface were measured on enlarged microphotographs of ground sections of human deciduous anterior teeth. The results suggested that: extension of odontoblast differentiation is greater on the lingual than on the labial side during the 8 months following birth in incisors, and during the first year in canines; it is greater on the distal than on the mesial side during the 3 months following birth in incisors, and during the first year in canines; in the incisors, dentin formation of newly differentiated odontoblasts is more active on the distal than on the mesial side during the 8 months following birth.     

Some Enzymic Activities in the Germinating Oil Palm (Elaeis guineensis) Seedling

In germinating oil palm (Elaeis guineensis var D × P) seedling, an active lipase was present in the shoot but absent from both the kernel and the haustorium. It has an optimum pH of 6.2 and a smaller peak at pH 8.6. The shoot lipase was active against a number of mono-, di-, and triacylglycerols as well as the endogenous lipids present in the shoot, haustorium, and kernel. Activity against related substrates were in the order: trilaurin > dilaurin > monolaurin but monopalmitin > dipalmitin > tripalmitin. The level of the enzyme in the seedling was highest at a relatively early stage of growth (18-21 days) and also higher in dark-grown seedlings. Glyoxylate bypass enzymes (malate synthetase and isocitrate lyase), glutamate-oxaloacetate transaminase, phosphoenolpyruvate carboxykinase and lauroyl-coenzyme A oxidase were located in the haustorium. The levels of the enzymes paralleled seedling development and were slightly higher in light-grown seedlings. Fatty acyl-coenzyme A synthetase activity was very low and was found in both the shoot and haustorium.     

Extracellular export of sphingosine kinase-1a contributes to the vascular S1P gradient

Sphingosine 1-phosphate (S1P), produced by Sphks (sphingosine kinases), is a multifunctional lipid mediator that regulates immune cell trafficking and vascular development. Mammals maintain a large concentration gradient of S1P between vascular and extravascular compartments. Mechanisms by which S1P is released from cells and concentrated in the plasma are poorly understood. We recently demonstrated [Ancellin, Colmont, Su, Li, Mittereder, Chae, Stefansson, Liau and Hla (2002) J. Biol. Chem. 277, 6667-6675] that Sphk1 activity is constitutively secreted by vascular endothelial cells. In the present study, we show that among the five Sphk isoforms expressed in endothelial cells, the Sphk-1a isoform is selectively secreted in HEK-293 cells (human embryonic kidney cells) and human umbilical-vein endothelial cells. In sharp contrast, Sphk2 is not secreted. The exported Sphk-1a isoform is enzymatically active and produced sufficient S1P to induce S1P receptor internalization. Wild-type mouse plasma contains significant Sphk activity (179 pmol x min(-1) x g(-1)). In contrast, Sphk1-/- mouse plasma has undetectable Sphk activity and approx. 65% reduction in S1P levels. Moreover, human plasma contains enzymatically active Sphk1 (46 pmol x min(-1) x g(-1)). These results suggest that export of Sphk-1a occurs under physiological conditions and may contribute to the establishment of the vascular S1P gradient.     

Validation of G6PD Point-of-Care Tests among Healthy Volunteers in Yangon,Myanmar

Primaquine and other 8-amnoquinoline based anti-malarials can cause haemolysis in subjects with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Correct diagnosis of G6PD status in patients is crucial for safe treatment of both relapsing stages of Plasmodium vivax and transmitting forms of Plasmodium falciparum. Lack of suitable point-of-care tests has hampered a much needed wide use of primaquine for malaria elimination. In this study we have assessed the performances of two qualitative tests, the fluorescent spot test (FST) and the G6PD CareStart test (CST), against the gold standard quantitative spectrophotometric assay in a population of 1000 random adult healthy volunteers living in Yangon, Myanmar. The prevalence of G6PD deficiency in the Bamar, Karen and in the whole sample set was 6.6% (10.1% in males), 9.2% (21.0% in males) and 6.8% (11.1% in males) respectively. The FST and CST showed comparable performances with sensitivity over 95% and specificity over 90%, however for cases with severe G6PD activity the FTS had improved performance. If used with a conservative interpretation of the signal, the CareStart test has the potential to be used in the field and, by allowing a wider use of primaquine, to help malaria elimination.     

Intracellular role for sphingosine kinase 1 in intestinal adenoma cell proliferation

Sphingosine kinase (Sphk) enzymes are important in intracellular sphingolipid metabolism as well as in the biosynthesis of sphingosine 1-phosphate (S1P), an extracellular lipid mediator. Here, we show that Sphk1 is expressed and is required for small intestinal tumor cell proliferation in Apc Min/+ mice. Adenoma size but not incidence was dramatically reduced in Apc Min/+ Sphk(-/-) mice. Concomitantly, epithelial cell proliferation in the polyps was significantly attenuated, suggesting that Sphk1 regulates adenoma progression. Although the S1P receptors (S1P1R, S1P2R, and S1P3R) are expressed, polyp incidence or size was unaltered in Apc Min/+ S1p2r(-/-), Apc Min/+ S1p3r(-/-), and Apc Min/+ S1p1r(+/-) bigenic mice. These data suggest that extracellular S1P signaling via its receptors is not involved in adenoma cell proliferation. Interestingly, tissue sphingosine content was elevated in the adenomas of Apc Min/+ Sphk1(-/-) mice, whereas S1P levels were not significantly altered. Concomitantly, epithelial cell proliferation and the expression of the G1/S cell cycle regulator CDK4 and c-myc were diminished in the polyps of Apc Min/+ Sphk1(-/-) mice. In rat intestinal epithelial (RIE) cells in vitro, Sphk1 overexpression enhanced cell cycle traverse at the G1/S boundary. In addition, RIE cells treated with sphingosine but not C6-ceramide exhibited reduced cell proliferation, reduced retinoblastoma protein phosphorylation, and cyclin-dependent kinase 4 (Cdk4) expression. Our findings suggest that Sphk1 plays a critical role in intestinal tumor cell proliferation and that inhibitors of Sphk1 may be useful in the control of intestinal cancer.     

A Randomised Controlled Trial of Artemether-Lumefantrine Versus Artesunate for Uncomplicated Plasmodium falciparum Treatment in Pregnancy

Background

To date no comparative trials have been done, to our knowledge, of fixed-dose artemisinin combination therapies (ACTs) for the treatment of Plasmodium falciparum malaria in pregnancy. Evidence on the safety and efficacy of ACTs in pregnancy is needed as these drugs are being used increasingly throughout the malaria-affected world. The objective of this study was to compare the efficacy, tolerability, and safety of artemether-lumefantrine, the most widely used fixed ACT, with 7 d artesunate monotherapy in the second and third trimesters of pregnancy.

Methods and Findings

An open-label randomised controlled trial comparing directly observed treatment with artemether-lumefantrine 3 d (AL) or artesunate monotherapy 7 d (AS7) was conducted in Karen women in the border area of northwestern Thailand who had uncomplicated P. falciparum malaria in the second and third trimesters of pregnancy. The primary endpoint was efficacy defined as the P. falciparum PCR-adjusted cure rates assessed at delivery or by day 42 if this occurred later than delivery, as estimated by Kaplan-Meier survival analysis. Infants were assessed at birth and followed until 1 y of life. Blood sampling was performed to characterise the pharmacokinetics of lumefantrine in pregnancy. Both regimens were very well tolerated. The cure rates (95% confidence interval) for the intention to treat (ITT) population were: AS7 89.2% (82.3%–96.1%) and AL 82.0% (74.8%–89.3%), p = 0.054 (ITT); and AS7 89.7% (82.6%–96.8%) and AL 81.2% (73.6%–88.8%), p = 0.031 (per-protocol population). One-third of the PCR-confirmed recrudescent cases occurred after 42 d of follow-up. Birth outcomes and infant (up to age 1 y) outcomes did not differ significantly between the two groups. The pharmacokinetic study indicated that low concentrations of artemether and lumefantrine were the main contributors to the poor efficacy of AL.

Conclusion

The current standard six-dose artemether-lumefantrine regimen was well tolerated and safe in pregnant Karen women with uncomplicated falciparum malaria, but efficacy was inferior to 7 d artesunate monotherapy and was unsatisfactory for general deployment in this geographic area. Reduced efficacy probably results from low drug concentrations in later pregnancy. A longer or more frequent AL dose regimen may be needed to treat pregnant women effectively and should now be evaluated. Parasitological endpoints in clinical trials of any antimalarial drug treatment in pregnancy should be extended to delivery or day 42 if it comes later. Trial Registration: Current Controlled Trials ISRCTN86353884