Homologous recombination (HR) has not been demonstrated in the parasitic protists Entamoeba histolytica or Entamoeba invadens, as no convenient method is available to measure it. However, HR must exist to ensure genome integrity, and possible genetic exchange, especially during stage conversion from trophozoite to cyst. Here we show the up regulation of mitotic and meiotic HR genes in Entamoeba during serum starvation, and encystation. To directly demonstrate HR we use a simple PCR-based method involving inverted repeats, which gives a reliable read out, as the recombination junctions can be determined by sequencing the amplicons. Using this read out, we demonstrate enhanced HR under growth stress in E. histolytica, and during encystation in E. invadens. We also demonstrate recombination between chromosomal inverted repeats. This is the first experimental demonstration of HR in Entamoeba and will help future investigations into this process, and to explore the possibility of meiosis in Entamoeba.

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BackgroundThe increase in antimicrobial-resistant infections has led to significant morbidity, mortality, and healthcare costs. The impact of antimicrobial resistance is greatest on low-income countries, which face the double burden of fewer antibiotic choices and higher rates of infectious diseases. Currently, Guyana has no national policy on rational prescribing. This study aims to characterize antibiotic prescribing patterns in children discharged from the emergency department at Georgetown Public Hospital Corporation (GPHC), as per the World Health Organization (WHO) prescribing indicators.MethodsA retrospective chart review of pediatric patients (aged 1 month–13 years) seen in the GPHC emergency department between January and December 2012 was conducted. Outpatient prescriptions for eligible patients were reviewed. Patient demographics, diagnosis, and drugs prescribed were recorded. The following WHO Prescribing Indicators were calculated: i) average number of drugs prescribed per patient encounter, ii) percentage of encounters with an antibiotic prescribed, iii) percentage of antibiotics prescribed by generic name, and iv) percentage of antibiotics prescribed from essential drugs list or formulary.ResultsEight hundred eleven patient encounters were included in the study. The mean patient age was 5.55 years (s = 3.98 years). 59.6 % (n = 483) patients were male. An average of 2.5 drugs were prescribed per encounter (WHO standard is 2.0). One or more antibiotic was prescribed during 36.9 % (n = 299) of all encounters (WHO standard is 30 %). 90.83 % of antibiotics were prescribed from the essential drugs formulary list and 30 % of the prescriptions included the drug’s generic name. The average duration of antibiotic therapy was 5.73 days (s = 3.53 days). Of the 360 antibiotics prescribed, 74.7 % (n = 269) were broad-spectrum. B-lactam penicillins were prescribed most frequently (51.4 %), with amoxicillin being the most popular choice (33.9 %). The most common diagnoses were injuries (25.8 %), asthma (20 %), respiratory infections (19.5 %), and gastrointestinal infections (12.1 %).ConclusionsPer WHO prescribing indicators, the pediatric emergency department at GPHC has higher than standard rates of antibiotic use and polypharmacy. The department excels in adhering to the essential drug formulary. Our findings provide support for investigating drug utilization in other Guyanese settings, and to work towards developing a national rational prescribing strategy.

This study was designed to understand the mechanism and functional implication of the two heterozygous mutations (H391Y and K422R) of human pyruvate kinase M2 isozyme (PKM 2 ) observed earlier in a Bloom syndrome background. The co-expression of homotetrameric wild type and mutant PKM 2 in the cellular milieu resulting in the interaction between the two at the monomer level was substantiated further by in vitro experiments. The cross-monomer interaction significantly altered the oligomeric state of PKM 2 by favoring dimerization and heterotetramerization. In silico study provided an added support in showing that hetero-oligomerization was energetically favorable. The hetero-oligomeric populations of PKM 2 showed altered activity and affinity, and their expression resulted in an increased growth rate of Escherichia coli as well as mammalian cells, along with an increased rate of polyploidy. These features are known to be essential to tumor progression. This study provides insight in understanding the modulated role of large oligomeric multifunctional proteins such as PKM 2 by affecting cellular behavior, which is an essential observation to understand tumor sustenance and progression and to design therapeutic intervention in future.A variety of genetic diseases and experimental situations within the heterozygous state depict an allelic relationship where the mutant recessive allele overrides the function of its normal (wild type) dominant allele. This condition, referred to as dominant negative, is usually observed in the case of oligomeric or multidomain proteins with a possibility of cross-monomer interaction. Such mutant proteins by acting as competitive inhibitors of the normal protein function could generate polymorphic forms in a single cell. A phenomenon observed in collagen, where dominant negative mutations cause the production of abnormal oligomers (1, 2), and in transcription factors like helix-loop-helix and leucine zippers, where mutant monomers sequestering the function of wild type in a dimer bound to DNA, leads to an altered gene expression (3-5). Dominant negative mutations are also reported to affect some multifunctional molecules like p53 with differential impact on cell physiology (6 -10).Pyruvate kinase (EC 2.7.1.40) catalyzes irreversibly the transphosphorylation from P-enolpyruvate to ADP-generating pyruvate and ATP in glycolysis (11,12). Depending upon the differential metabolic requirements of the tissues, the enzyme is expressed in four different isoforms, L, R, M 1 , and M 2 in vertebrates (13). PKM 2 2 is a ubiquitous, prototype enzyme, present in all tissues during embryonic stage, and is gradually replaced by other isozymic forms in specific tissues, during development. It is necessary for cellular division irrespective of the type of tissue and reappears during cellular division and tumor formation (14 -17). PKM 2 is known to regulate its activity by switching between an active tetramer and inactive dimer form in a fructose 1,6-bisphosphate-dependent manner to shift the cellular met...

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