Import Smuggling in the Philippines: An Economic Analysis

This study attempts to generate an estimate of import smuggling using Philippine data on import trade. Through the development of microeconomic model, it tries to explain the level of smuggling activity in terms of factors impinging on the trader’s decision to engage in smuggling.trade sector, overvaluation, import commodities, smuggling

Import Smuggling in the Philippines: An Economic Analysis

This study attempts to generate an estimate of import smuggling using Philippine data on import trade. Through the development of microeconomic model, it tries to explain the level of smuggling activity in terms of factors impinging on the trader’s decision to engage in smuggling.trade sector, overvaluation, import commodities, smuggling

Critical Steps of Plasmodium falciparum Ookinete Maturation

The egress and fertilization of Plasmodium gametes and development of a motile ookinete are the first crucial steps that mediate the successful transmission of the malaria parasites from humans to the Anopheles vector. However, limited information exists about the cell biology and regulation of this process. Technical impediments in the establishment of in vitro conditions for ookinete maturation in Plasmodium falciparum and other human malaria parasites further constrain a detailed characterization of ookinete maturation. Here, using fluorescence microscopy and immunolabeling, we compared P. falciparum ookinete maturation in Anopheles coluzzii mosquitoes in vivo and in cell culture in vitro. Our results identified two critical steps in ookinete maturation that are regulated by distinct mosquito factors, thereby highlighting the role of the mosquito environment in the transmission efficiency of malaria parasites

The Plasmodium falciparum, Nima-related kinase Pfnek-4: a marker for asexual parasites committed to sexual differentiation

<b>Background</b> Malaria parasites undergo, in the vertebrate host, a developmental switch from asexual replication to sexual differentiation leading to the formation of gametocytes, the only form able to survive in the mosquito vector. Regulation of the onset of the sexual phase remains largely unknown and represents an important gap in the understanding of the parasite's complex biology. <b>Methods:</b> The expression and function of the Nima-related kinase Pfnek-4 during the early sexual development of the human malaria parasite Plasmodium falciparum were investigated, using three types of transgenic Plasmodium falciparum 3D7 lines: (i) episomally expressing a Pfnek-4-GFP fusion protein under the control of its cognate pfnek-4 promoter; (ii) episomally expressing negative or positive selectable markers, yeast cytosine deaminase-uridyl phosphoribosyl transferase, or human dihydrofolate reductase, under the control of the pfnek-4 promoter; and (iii) lacking a functional pfnek-4 gene. Parasite transfectants were analysed by fluorescence microscopy and flow cytometry. In vitro growth rate and gametocyte formation were determined by Giemsa-stained blood smears. <b>Results:</b> The Pfnek-4-GFP protein was found to be expressed in stage II to V gametocytes and, unexpectedly, in a subset of asexual-stage parasites undergoing schizogony. Culture conditions stimulating gametocyte formation resulted in significant increase of this schizont subpopulation. Moreover, sorted asexual parasites expressing the Pfnek-4-GFP protein displayed elevated gametocyte formation when returned to in vitro culture in presence of fresh red blood cells, when compared to GFP- parasites from the same initial population. Negative selection of asexual parasites expressing pfnek-4 showed a marginal reduction in growth rate, whereas positive selection caused a marked reduction in parasitaemia, but was not sufficient to completely abolish proliferation. Pfnek-4- clones are not affected in their asexual growth and produced normal numbers of stage V gametocytes. <b>Conclusions:</b> The results indicate that Pfnek-4 is not strictly gametocyte-specific, and is expressed in a small subset of asexual parasites displaying high rate conversion to sexual development. Pfnek-4 is not required for erythrocytic schizogony and gametocytogenesis. This is the first study to report the use of a molecular marker for the sorting of sexually-committed schizont stage P. falciparum parasites, which opens the way to molecular characterization of this pre-differentiated subpopulation

High prevalence of drug-resistance mutations in Plasmodium falciparum and Plasmodium vivax in southern Ethiopia

BACKGROUND: In Ethiopia, malaria is caused by both Plasmodium falciparum and Plasmodium vivax. Drug resistance of P. falciparum to sulfadoxine-pyrimethamine (SP) and chloroquine (CQ) is frequent and intense in some areas. METHODS: In 100 patients with uncomplicated malaria from Dilla, southern Ethiopia, P. falciparum dhfr and dhps mutations as well as P. vivax dhfr polymorphisms associated with resistance to SP and P. falciparum pfcrt and pfmdr1 mutations conferring CQ resistance were assessed. RESULTS: P. falciparum and P. vivax were observed in 69% and 31% of the patients, respectively. Pfdhfr triple mutations and pfdhfr/pfdhps quintuple mutations occurred in 87% and 86% of P. falciparum isolates, respectively. Pfcrt T76 was seen in all and pfmdr1 Y86 in 81% of P. falciparum. The P. vivax dhfr core mutations N117 and R58 were present in 94% and 74%, respectively. CONCLUSION: These data point to an extraordinarily high frequency of drug-resistance mutations in both P. falciparum and P. vivax in southern Ethiopia, and strongly support that both SP and CQ are inadequate drugs for this region

Pfnek-1, a NIMA-related kinase from the human malaria parasite Plasmodium falciparum: Biochemical properties and possible involvement in MAPK regulation

We have cloned Pfnek-1, a gene encoding a novel protein kinase from the human malaria parasite Plasmodium falciparum. This enzyme displays maximal homology to the never-in-mitosis/Aspergillus (NIMA)/NIMA-like kinase (Nek) family of protein kinases, whose members are involved in eukaryotic cell division processes. Similar to other P. falciparum protein kinases and many enzymes of the NIMA/Nek family, Pfnek-1 possesses a large C-terminal extension in addition to the catalytic domain. Bacterially expressed recombinant Pfnek-1 protein is able to autophosphorylate and phosphorylate a panel of protein substrates with a specificity that is similar to that displayed by other members of the NIMA/Nek family. However, the FXXT motif usually found in NIMA/Nek protein kinases is substituted in Pfnek-1 by a SMAHS motif, which is reminiscent of a MAP/ERK kinase (MEK) activation site. Mutational analysis indicates that only one of the serine residues in this motif is essential for Pfnek-1 kinase activity in vitro. We show (a) that recombinant Pfnek-1 is able to specifically phosphorylate Pfmap-2, an atypical P. falciparum MAPK homologue, in vitro, and (b) that coincubation of Pfnek-1 and Pfmap-2 results in a synergistic increase in exogenous substrate labelling. This suggests that Pfnek-1 may be involved in the modulation of MAPK pathway output in malaria parasites. Finally, we demonstrate that recombinant Pfnek-1 can be used in inhibition assays to monitor the effect of kinase inhibitors, which opens the way to the screening of chemical libraries aimed at identifying potential new antimalarials

Neuronal Sirt3 Protects against Excitotoxic Injury in Mouse Cortical Neuron Culture

BACKGROUND: Sirtuins (Sirt), a family of nicotinamide adenine nucleotide (NAD) dependent deacetylases, are implicated in energy metabolism and life span. Among the known Sirt isoforms (Sirt1-7), Sirt3 was identified as a stress responsive deacetylase recently shown to play a role in protecting cells under stress conditions. Here, we demonstrated the presence of Sirt3 in neurons, and characterized the role of Sirt3 in neuron survival under NMDA-induced excitotoxicity. METHODOLOGY/PRINCIPAL FINDINGS: To induce excitotoxic injury, we exposed primary cultured mouse cortical neurons to NMDA (30 µM). NMDA induced a rapid decrease of cytoplasmic NAD (but not mitochondrial NAD) in neurons through poly (ADP-ribose) polymerase-1 (PARP-1) activation. Mitochondrial Sirt3 was increased following PARP-1 mediated NAD depletion, which was reversed by either inhibition of PARP-1 or exogenous NAD. We found that massive reactive oxygen species (ROS) produced under this NAD depleted condition mediated the increase in mitochondrial Sirt3. By transfecting primary neurons with a Sirt3 overexpressing plasmid or Sirt3 siRNA, we showed that Sirt3 is required for neuroprotection against excitotoxicity. CONCLUSIONS: This study demonstrated for the first time that mitochondrial Sirt3 acts as a prosurvival factor playing an essential role to protect neurons under excitotoxic injury

The Mediating Effect of Job Expectations on Selected Human Resource Management Practices and Career Satisfaction

As companies face changes from globalization, new technologies, and a younger workforce, Human Resource Management (HRM) has become more important in keeping employees satisfied and motivated. This study looked into how HRM practices and job expectations affect the career satisfaction of Generation Z alumni. The researchers used a descriptive quantitative method with 91 participants. The data were analyzed using frequency counts, mean, standard deviation, and Pearson correlation. The results showed that HRM practices have a strong and direct effect on career satisfaction. While job expectations also had a positive connection with satisfaction, they did not serve as a link between HRM practices and satisfaction. This means that HR practices alone can already help increase how satisfied employees feel in their careers. Based on the findings, organizations can focus on improving HR practices such as fair salary, open communication, giving feedback, and providing training or growth opportunities. These can make employees feel valued and more engaged at work. The study also suggests that HR planning should support not just work performance but also overall employee well-being

Differential Adhesive Properties of Sequestered Asexual and Sexual Stages of Plasmodium falciparum on Human Endothelial Cells Are Tissue Independent

The protozoan parasite Plasmodium falciparum, responsible for the most severe form of malaria, is able to sequester from peripheral circulation during infection. The asexual stage parasites sequester by binding to endothelial cell receptors in the microvasculature of various organs. P. falciparum gametocytes, the developmental stages responsible for parasite transmission from humans to Anopheles mosquitoes, also spend the almost ten days necessary for their maturation sequestered away from the peripheral circulation before they are released in blood mainstream. In contrast to those of asexual parasites, the mechanisms and cellular interactions responsible for immature gametocyte sequestration are largely unexplored, and controversial evidence has been produced so far on this matter. Here we present a systematic comparison of cell binding properties of asexual stages and immature and mature gametocytes from the reference P. falciparum clone 3D7 and from a patient parasite isolate on a panel of human endothelial cells from different tissues. This analysis includes assays on human bone marrow derived endothelial cell lines (HBMEC), as this tissue has been proposed as a major site of gametocyte maturation. Our results clearly demonstrate that cell adhesion of asexual stage parasites is consistently more efficient than that, virtually undetectable of immature gametocytes, irrespectively of the endothelial cell lines used and of parasite genotypes. Importantly, immature gametocytes of both lines tested here do not show a higher binding efficiency compared to asexual stages on bone marrow derived endothelial cells, unlike previously reported in the only study on this issue. This indicates that gametocyte-host interactions in this tissue are unlikely to be mediated by the same adhesion processes to specific endothelial receptors as seen with asexual forms

A new P. falciparum gametocyte drug screening assay based on pLDH detection

Plasmodium gametocytes (GCT) have recently been proposed as a crucial target for the development of new antimalarials in order to achieve malaria elimination and eventually eradication. At present, however, a widely accepted and routinely used screening method for potential gametocytocidal drugs does not exist. The aim of our work was to adapt the parasite lactate dehydrogenase (pLDH), already standardised for drug screening on asexual stages, to measure gametocyte drug sensitivity. In clinics the GCT- pLDH, which is present during all the five stages of gametocyte development, can be measured with good sensitivity through OptiMAL, an immunochromatographic diagnostic test. The pLDH assay is fast, simple, not expensive and does not require complex equipment or special waste disposal. It can be applied to field isolates since transgenesis is not needed. Gametocytogenesis of two different strains of P. falciparum, 3D7 and NF54, was induced in vitro using a standardized protocol, asexual parasites were removed by N-acetylglucosamine treatment, and GCT were seeded in 96well plates. A linear correlation between the percentage of gametocytemia, microscopically counted by Giemsa staining, and the optical density, measured spectroscopically by pLDH assay, was demonstrated. A good signal to noise ratio was obtained with the pLDH assay, and the Z\u2019factor was calculated as indicator of the robustness of the method. Our data also indicate that GCT have a pLDH activity higher than asexual parasites. GCT were treated for 48-72h with primaquine, the gold standard against mature gametocytes in vivo, which was used to validate most of the GCT screening methods in literature; dihydroartemisinin, active on young GCT; and methylene blue, an old antimalarial recently characterised also for its anti-GCT activity. Finally, epoxomicin was tested since its strong gametocytocidal effect has been recently reported. Dose-response curves were obtained with all the four drugs. However, some discrepancies were observed between the Giemsa staining and the pLDH detection at high concentrations of the drugs, suggesting that morphological abnormalities, detected microscopically, precede the decay of pLDH activity in drug-treated GCT. In order to better understand these observations, we prolonged the treatment for further 72h. This extra-incubation period allowed us to calculate, from the pLDH assay, the IC50 (as the 50% inhibition compared to control untreated GCT) of the tested compounds, which were comparable to those obtained by Giemsa staining. These results demonstrate the feasibility of pLDH assay to measure GCT content in culture. Although more specific probes for GCT viability need to be standardized for measuring stage-specific drug activity, pLDH can be used as the first, fast and cheap screening method to find potential gametocitocydal drugs