Validation of Plasmodium vivax centromere and promoter activities using Plasmodium yoelii

Plasmodium vivax is the leading cause of malaria outside Africa and represents a significant health and economic burden on affected countries. A major obstacle for P. vivax eradication is the dormant hypnozoite liver stage that causes relapse infections and the limited antimalarial drugs that clear this stage. Advances in studying the hypnozoite and other unique biological aspects of this parasite are hampered by the lack of a continuous in vitro laboratory culture system and poor availability of molecular tools for genetic manipulation. In this study, we aim to develop molecular tools that can be used for genetic manipulation of P. vivax. A putative P. vivax centromere sequence (PvCEN) was cloned and episomal centromere based plasmids expressing a GFP marker were constructed. Centromere activity was evaluated using a rodent malaria parasite Plasmodium yoelii. A plasmid carrying PvCEN was stably maintained in asexual-stage parasites in the absence of drug pressure, and approximately 45% of the parasites retained the plasmid four weeks later. The same retention rate was observed in parasites possessing a native P. yoelii centromere (PyCEN)-based control plasmid. The segregation efficiency of the plasmid per nuclear division was > 99% in PvCEN parasites, compared to ?90% in a control parasite harboring a plasmid without a centromere. In addition, we observed a clear GFP signal in both oocysts and salivary gland sporozoites isolated from mosquitoes. In blood-stage parasites after liver stage development, GFP positivity in PvCEN parasites was comparable to control PyCEN parasites. Thus, PvCEN plasmids were maintained throughout the parasite life cycle. We also validated several P. vivax promoter activities and showed that hsp70 promoter (?1 kb) was active throughout the parasite life cycle. This is the first data for the functional characterization of a P. vivax centromere that can be used in future P. vivax biological research

Clinical impact of albuminuria in diabetic nephropathy

金沢大学医薬保健研究域医学系Patients suffering from diabetic nephropathy, resulting in end-stage renal failure, are increasing in number. The pathophysiology of diabetic nephropathy remains to be fully investigated. In the clinical setting, the presence of albuminuria/overt proteinuria and a low glomerular filtration rate may predict poor renal prognosis, but the prognosis of the normoalbuminuric renally insufficient diabetic patient remains controversial. In addition to the measurement of urinary albumin excretion, biomarker studies to detect diabetic nephropathy more specifically at the early stage have been performed worldwide. There is a growing body of evidence for remission and/or regression of diabetic nephropathy, which may be an indicator for cardiovascular and renal risk reduction. Deeper insights into the pathological characteristics as well as the clinical impact of albuminuria on renal and cardiovascular outcome are required. © 2011 Japanese Society of Nephrology

Extreme hyperglycemia and diabetic ketoacidosis occurring in a patient on chronic dialysis

Department of Urology, North medical center, Kyoto prefectural university of medicine, Kyoto, Japan.Department of Nephrology and Urology, Nishijin Hospital, Kyoto, Japan.Department of Internal medicine, Nishijin Hospital, Kyoto, Japan.Diabetic ketoacidosis (DKA) is a complication that is rarely reported in patients on chronic dialysis. Herein, we describe the case of a patient on chronic hemodialysis who presented to us with acute onset diabetic ketoacidosis. A 50-year-old man with insulin-dependent diabetes mellitus, who was on hemodialysis for 2 years, presented to us with altered consciousness. Laboratory data revealed the following results: blood sugar, 110.1 mmol/L (1984 mg/dL); serum sodium, 107 mmol/L; β -hydroxybutyric acid, 1991 μ M; pH, 7.048. A diagnosis of diabetic ketoacidosis was made, and insulin therapy and hemodialysis were initiated, following which his parameters including blood glucose, and serum potassium and sodium improved. High osmotic dehydration was not observed in our patient owing to his renal dysfunction. The patient’s consciousness normalized following the correction of hyperglycemia and DKA. This case report highlights the importance of early diagnosis of DKA, and prompt initiation of insulin therapy and hemodialysis in patients on chronic dialysis. Therefore, in patients with end stage renal disease, the blood glucose correction should be followed by the restoration of sodium and osmolality, guided by corrected sodium concentration and effective osmolality, and by the appropriate adjustment of insulin and dialysis

Medical Treatment of Echinococcus multilocularis and New Horizons for Drug Discovery: Characterization of Mitochondrial Complex II as a Potential Drug Target

As an efficient drug for alveolar echinococcosis (AE) is still not available, new chemotherapy targets are necessary. The mitochondrial respiratory chain may be a good drug candidate because parasite respiratory chains are quite different from those of mammalian hosts. For example, Ascaris suum possesses an NADH‐fumarate reductase system (fumarate respiration) that is highly adapted to anaerobic environments such as the small intestine. It is composed of mitochondrial complex I (NADH‐ubiquinone reductase), complex II (succinate‐ubiquinone reductase), and rhodoquinone. We previously demonstrated that fumarate respiration is also essential in E. multilocularis. Quinazoline, a complex I inhibitor, inhibited growth of E. multilocularis larvae in vitro. These results indicate that fumarate respiration could be a target for E. multilocularis therapy. In the current chapter, we focused on complex II, which is another component of this system, because quinazoline exhibited strong toxicity to mammalian mitochondria. We evaluated the molecular and biochemical characterization of E. multilocularis complex II as a potential drug target. In addition, we found that ascofuranone, a trypanosome cyanide‐insensitive alternative oxidase inhibitor, inhibited E. multilocularis complex II at the nanomolar order. Our findings demonstrate the potential development of targeted therapy against Echinococcus complex II

The Impacts of Albuminuria and Low eGFR on the Risk of Cardiovascular Death, All-Cause Mortality, and Renal Events in Diabetic Patients: Meta-Analysis

Background:Precise effects of albuminuria and low estimated glomerular filtration rate (eGFR) on cardiovascular mortality, all-cause mortality, and renal events in diabetic patients are uncertain.Materials and Methods:A systematic review was conducted of the literature through MEDLINE, EMBASE, and CINHAL from 1950 to December 2010. Cohort studies of diabetic patients providing adjusted relative risk (RR) of albuminuria and eGFR for risks of cardiovascular mortality, all-cause mortality, and renal events were selected. Two reviewers screened abstracts and full papers of each study using standardized protocol.Results:We identified 31 studies fulfilling the criteria from 6546 abstracts. With regard to the risk of cardiovascular mortality, microalbuminuria (RR 1.76, 95%CI 1.38-2.25) and macroalbuminuria (RR 2.96 95%CI 2.44-3.60) were significant risk factors compared to normoalbuminuria. The same trends were seen in microalbuminuria (RR 1.60, 95%CI 1.42-1.81), and macroalbuminuria (RR 2.64, 95%CI 2.13-3.27) for the risk of all-cause mortality, and also in microalbuminuria (RR 3.21, 95%CI 2.05-5.02) and macroalbuminuria (RR 11.63, 95%CI 5.68-23.83) for the risk of renal events. The magnitudes of relative risks associated with low eGFR along with albuminuria were almost equal to multiplying each risk rate of low eGFR and albuminuria. No significant factors were found by investigating potential sources of heterogeneity using subgroup analysis.Conclusions:High albuminuria and low eGFR are relevant risk factors in diabetic patients. Albuminuria and low eGFR may be independent of each other. To evaluate the effects of low eGFR, intervention, or race, appropriately designed studies are needed. © 2013 Toyama et al

Adverse effect of cake collapse on the functional integrity of freeze-dried bull spermatozoa

Under optimal freeze-drying conditions, solutions exhibit a cake-like porous structure. However, if the solution temperature is higher than the glass transition temperature of the maximally freeze-concentrated phase (Tg′) during drying phase, the glassy matrix undergoes viscous flow, resulting in cake collapse. The purpose of the present study was to investigate the effect of cake collapse on the integrity of freeze-dried bull spermatozoa. In a preliminary experiment, factors affecting the Tg′ of conventional EGTA buffer (consisting of Tris–HCl, EGTA and NaCl) were investigated in order to establish the main experimental protocol because EGTA buffer Tg′ was too low (−45.0 °C) to suppress collapse. Modification of the EGTA buffer composition by complete removal of NaCl and addition of trehalose (mEGTA buffer) resulted in an increase of Tg′ up to −27.7 °C. In the main experiment, blastocyst yields after ooplasmic injection of freeze-dried sperm preserved in collapsed cakes (drying temperature: 0 or −15 °C) were significantly lower than those of sperm preserved in non-collapsed cake (drying temperature: −30 °C). In conclusion, freeze-dried cake collapse may be undesirable for maintaining sperm functions to support embryonic development, and can be inhibited by controlling both Tg′ of freeze-drying buffer and temperature during the drying phase.ArticleCRYOBIOLOGY. 68(3):354-360 (2014)journal articl

Relationship between serum uric acid levels and chronic kidney disease in a Japanese cohort with normal or mildly reduced kidney function

Background: Some observational studies have shown the relationships between hyperuricemia and chronic kidney disease (CKD); however, the threshold of serum uric acid (SUA) for deterioration of kidney function and the association between SUA and kidney injury by baseline kidney function remains unclear. This study aimed to clarify the relationships between SUA and reduced kidney function. Methods: We analyzed a historical cohort of male Japanese individuals who underwent medical checkup between 1998 and 2007. Participants with baseline data and who were followed up for at least one year were included and stratified according to baseline kidney function. Kidney function was classified as normal [estimated glomerular filtration rate (eGFR) ≥ 90 ml/min/1.73 m2] or mildly reduced (eGFR 60-89 ml/min/1.73 m2). The outcome measured was kidney impairment defined as a decrease in eGFR to < 60 ml/min/1.73 m2. Associations between SUA and risk for outcome and eGFR slopes were assessed. Results: A total of 41632 subjects with mean age 45.4 years were included. During a mean follow-up of four years, 3186 (7.6%) subjects developed kidney dysfunction. Subjects with SUA ≥ 6.0 mg/dL had a significantly increased risk for kidney impairment compared with subjects with SUA of 4-4.9 mg/dL. SUA threshold levels were different according to baseline kidney function; SUA ≤ 7.0 and ≤ 6.0 mg/dL for normal and mildly reduced kidney function, respectively. Approximately the same trends were observed for eGFR slopes. Conclusion: In the general population, hyperuricemia appears to be a risk factor for kidney impairment in males. For participants with mild kidney dysfunction, even a slight elevation of SUA can be a risk factor. Copyright: © 2015 Toyama et al.This article has a supplementary figure. Please see the last page of the text