Transcriptional Silencing of Multiple Genes in Trophozoites of Entamoeba histolytica

In a previous work we described the transcriptional silencing of the amoebapore A (AP-A) gene (Ehap-a) of Entamoeba histolytica strain HM-1:IMSS. The silencing occurred following transfection with a plasmid containing a 5′ upstream region (473 bp) of Ehap-a that included a truncated segment (140 bp) of a short interspersed nuclear element (SINE1). Silencing remained in effect even after removal of the plasmid (clone G3). Neither short interfering RNA nor methylated DNA were detected, but the chromatin domain of Ehap-a in the gene-silenced trophozoites was modified. Two other similar genes (Ehap-b and one encoding a Saposin-like protein, SAPLIP 1) also became silenced. In the present work we demonstrate the silencing of a second gene of choice, one that encodes the light subunit of the Gal/GalNAc inhibitable lectin (Ehlgl1) and the other, the cysteine proteinase 5 (EhCP-5). This silencing occurred in G3 trophozoites transfected with a plasmid in which the 473 bp 5′ upstream Ehap-a fragment was directly ligated to the second gene. Transcriptional silencing occurred in both the transgene and the chromosomal gene. SINE1 sequences were essential, as was a direct connection between the Ehap-a upstream region and the beginning of the open reading frame of the second gene. Gene silencing did not occur in strain HM-1:IMSS with any of these plasmid constructs. The trophozoites with two silenced genes were virulence-attenuated as were those of clone G3. In addition, trophozoites not expressing Lgl1 and AP-A proteins had a significantly reduced ability to cap the Gal/GalNAc-lectin to the uroid region when incubated with antibodies against the heavy (170 kDa) subunit of the lectin. Lysates of trophozoites lacking cysteine proteinase 5 and AP-A proteins had 30% less cysteine proteinase activity than those of HM-1:IMSS strain or the G3 clone. Silencing of other genes in G3 amoebae could provide a model to study their various functions. In addition, double gene-silenced, virulence-attenuated trophozoites may be an important tool in vaccine development

Examination of Hardy-Weinberg equilibrium in papers of Kidney International: An underused tool

Background. Population-genetic studies investigating genetic polymorphisms of Mendelian inheritance should always test whether the measured genotype frequencies deviate from the expected one. For this purpose Hardy-Weinberg (HW) criteria are generally used. If genotype distribution of control population misses HW equilibrium, the results should be treated cautiously because the observed genotype distribution in control population does not represent genotype distribution in the overall population. If HW criteria are not fulfilled in the investigated population, this may be further evidence for the correlation between genotype and investigated condition. Methods. Between September, 1998, and September, 2003, we tested papers published in Kidney International if HW criteria were ordinarily and correctly checked in studies investigating genetic polymorphisms. Seventy-five genotype distributions of the selected 39 articles were reanalyzed. Results. HW calculation was reportedly performed in 25 papers (64%). The observed genotype distribution deviated significantly from the expected one in three control, and in 16 patient populations and in three populations of association studies of 15 papers overall; however, this fact was not mentioned in 12 papers. Conclusion. Although the deviation of genotype distribution from the expected one is important information, HW calculations are not performed routinely for each investigated subject groups in these papers investigating genetic polymorphisms

No Association Between MTHFR A1298C and MTRR A66G Polymorphisms, and MS in an Australian Cohort

Multiple sclerosis (MS) is a complex neurological disease that affects the central nervous system (CNS) resulting in debilitating neuropathology. Pathogenesis is primarily defined by CNS inflammation and demyelination of nerve axons. Methionine synthase reductase (MTRR) is an enzyme that catalyzes the remethylation of homocysteine (Hcy) to methionine via cobalamin and folate dependant reactions. Cobalamin acts as an intermediate methyl carrier between methylenetetrahydrofolate reductase (MTHFR) and Hcy. MTRR plays a critical role in maintaining cobalamin in an active form and is consequently an important determinant of total plasma Hcy (pHcy) concentrations. Elevated intracellular pHcy levels have been suggested to play a role in CNS dysfunction, neurodegenerative, and cerebrovascular diseases. Our investigation entailed the genotyping of a cohort of 140 cases and matched controls for MTRR and MTHFR, by restriction length polymorphism (RFLP) techniques. Two polymorphisms: MTRR A66G and MTHFR A1298C were investigated in an Australian age and gender matched case-control study. No significant allelic frequency difference was observed between cases and controls at the α = 0.05 level (MTRR χ^2 = 0.005, P = 0.95, MTHFR χ^2 = 1.15, P = 0.28). Our preliminary findings suggest no association between the MTRR A66G and MTHFR A1298C polymorphisms and MS

Dietary intervention modifies DNA methylation age assessed by the epigenetic clock

Scope: Alterations in DNA methylation patterns are correlated with aging, environmental exposures and disease pathophysiology; the possibility of reverting or preventing these processes through dietary intervention is gaining momentum. In particular, methyl donors that provide S-adenosyl-methionine for one-carbon metabolism and polyphenols such as flavanols that inhibit the activity of DNA methyltransferases (DNMTs) could be key modifiers of epigenetic patterns. Methods and results: We assessed DNA methylation patterns in publicly available Illumina Infinium 450K methylation datasets from intervention studies with either folic acid + vitamin B12 (GSE74548) or monomeric and oligomeric flavanols (MOF) (GSE54690) in 44 and 13 participants, respectively. Global DNA methylation levels increased in unmethylated regions such as CpG islands and shores following folic acid + vitamin B12 supplementation and decreased in highly methylated regions, including shelves and open-seas following intervention with MOF. After supplementation with folic acid + vitamin B12, epigenetic age, estimated by the Horvath ‘epigenetic clock’ model, was reduced in women with the MTHFR 677CC genotype. Conclusions: The effects of supplementation with folic acid + vitamin B12 and MOF on DNA methylation age are dependent upon gender and MTHFR genotype. Additionally, our findings demonstrate the potential for these dietary factors to modulate global DNA methylation profiles