Search

Serine protease inhibitors (serpins) are thought to mediate the tick’s evasion of the host’s serine protease-mediated defense pathways such as inflammation and blood clotting. This study describes characterization and target validation of 11 blood meal-responsive serpins that are associated with nymph and adult Ixodes scapularistick feeding as revealed by quantitative (q)RT-PCR and RNAi silencing analyses. Given the high number of targets, we used combinatorial (co) RNAi silencing to disrupt candidate serpins in two groups (G): seven highly identical and four non-identical serpins based on amino acid identities, here after called GI and GII respectively. We show that injection of both GI and GII co-dsRNA into unfed nymph and adult I. scapularisticks triggered suppression of cognate serpin mRNA. We show that disruption of GII, but not GI serpins significantly reduced feeding efficiency of both nymph and adult I. scapularisticks. Knockdown of GII serpin transcripts caused significant respective mortalities of ≤40 and 71% of nymphal and adult ticks that occurred within 24–48 h of attachment. This is significant, as the observed lethality preceded the tick feeding period when transmission of tick borne pathogens is predominant. We suspect that some of the GII serpins (S9, S17, S19 and S32) play roles in the tick detachment process in that upon detachment, mouthparts of GII co-dsRNA injected were covered with a whitish gel-like tissue that could be the tick cement cone. Normally, ticks do not retain tissue on their mouthparts upon detachment. Furthermore, disruption of GII serpins reduced tick blood meal sizes and the adult tick’s ability to convert the blood meal to eggs. We discuss our data with reference to tick feeding physiology and conclude that some of the GII serpins are potential targets for anti-tick vaccine development.

Studies on the transcriptional control of gene expression are crucial to understand changes in organism's physiological or cellular conditions. To obtain reliable data on mRNA amounts and the estimation of gene expression levels, it is crucial to normalize the target gene with one or more internal reference gene(s). However, the use of constitutive genes as reference genes is controversial, as their expression patterns are sometimes more complex than previously thought. In various arthropod vectors, including ticks, several constitutive genes have been identified by studying gene expression in different tissues and life stages. The cattle tick Rhipicephalus microplusis a major vector for several pathogens and is widely distributed in tropical and subtropical regions globally. Tick developmental physiology is an essential aspect of research, particularly embryogenesis, where many important developmental events occur, thus the identification of stable reference genes is essential for the interpretation of reliable gene expression data. This study aimed to identify and select R. microplushousekeeping genes and evaluate their stability during embryogenesis. Reference genes used as internal control in molecular assays were selected based on previous studies. These genes were screened by quantitative PCR (qPCR) and tested for gene expression stability during embryogenesis. Results demonstrated that the relative stability of reference genes varied at different time points during the embryogenesis. The GeNorm tool showed that elongation factor 1α(Elf1a) and ribosomal protein L4(Rpl4) were the most stable genes, while H3 histone family 3A(Hist3A) and ribosomal protein S18(RpS18) were the least stable. The NormFinder tool showed that Rpl4was the most stable gene, while the ranking of Elf1awas intermediate in all tested conditions. The BestKeeper tool showed that Rpl4and cyclophilin A(CycA) were the more and less stable genes, respectively. These data collectively demonstrate that Rpl4, Elf1a, and GAPDHare suitable internal controls for normalizing qPCR during R. microplusembryogenesis. These genes were consistently identified as the most stable in various analysis methods employed in this study. Thus, findings presented in this study offer valuable information for the study of gene expression during embryogenesis in R. microplus.

In this study we characterized Amblyomma americanum(Aam) tick calreticulin (CRT) homolog in tick feeding physiology. In nature, different tick species can be found feeding on the same animal host. This suggests that different tick species found feeding on the same host can modulate the same host anti-tick defense pathways to successfully feed. From this perspective it's plausible that different tick species can utilize universally conserved proteins such as CRT to regulate and facilitate feeding. CRT is a multi-functional protein found in most taxa that is injected into the vertebrate host during tick feeding. Apart from it's current use as a biomarker for human tick bites, role(s) of this protein in tick feeding physiology have not been elucidated. Here we show that annotated functional CRT amino acid motifs are well conserved in tick CRT. However our data show that despite high amino acid identity levels to functionally characterized CRT homologs in other organisms, AamCRT is apparently functionally different. Pichia pastorisexpressed recombinant (r) AamCRT bound C1q, the first component of the classical complement system, but it did not inhibit activation of this pathway. This contrast with reports of other parasite CRT that inhibited activation of the classical complement pathway through sequestration of C1q. Furthermore rAamCRT did not bind factor Xa in contrast to reports of parasite CRT binding factor Xa, an important protease in the blood clotting system. Consistent with this observation, rAamCRT did not affect plasma clotting or platelet aggregation. We discuss our findings in the context of tick feeding physiology.

Foreign body reactions may occur in patients who receive bioabsorbable implants during orthopedic surgery for fractures and ligament repair. The authors describe a 34-year-old man who presented with a palpable tender mass on the lateral aspect of the left knee of 1 month’s duration. He underwent posterior cruciate ligament and posterolateral corner reconstruction 3 years earlier. Physical examination showed a 1×1-cm soft, nontender mass without localized warmth on the lateral epicondyle of the distal femur. Magnetic resonance imaging showed a broken screw fragment surrounded by a cyst-like mass. Under general anesthesia, the surgeon excised the screw fragment and the fibrotic mass, enclosing it in the subcutaneous tissue at the lateral epicondyle, the site at which a poly-L-lactic acid bioabsorbable screw had been inserted to fix the graft for posterolateral corner reconstruction. Histologic evaluation showed a foreign body reaction to the degraded screw particles. To the authors’ knowledge, this report is the first description of a patient presenting with a delayed foreign body reaction to a broken poly-L-lactic acid bioabsorbable screw at the lateral femoral epicondyle after posterolateral corner reconstruction. Because delayed foreign body reactions can occur at any site of poly-L-lactic acid bioabsorbable screw insertion, care should be taken to avoid screw protrusion during ligament reconstruction because it can lead to screw breakage and delayed foreign body reaction. [Foreign body reactions may occur in patients who receive bioabsorbable implants during orthopedic surgery for fractures and ligament repair. The authors describe a 34-year-old man who presented with a palpable tender mass on the lateral aspect of the left knee of 1 month’s duration. He underwent posterior cruciate ligament and posterolateral corner reconstruction 3 years earlier. Physical examination showed a 1×1-cm soft, nontender mass without localized warmth on the lateral epicondyle of the distal femur. Magnetic resonance imaging showed a broken screw fragment surrounded by a cyst-like mass. Under general anesthesia, the surgeon excised the screw fragment and the fibrotic mass, enclosing it in the subcutaneous tissue at the lateral epicondyle, the site at which a poly-L-lactic acid bioabsorbable screw had been inserted to fix the graft for posterolateral corner reconstruction. Histologic evaluation showed a foreign body reaction to the degraded screw particles. To the authors’ knowledge, this report is the first description of a patient presenting with a delayed foreign body reaction to a broken poly-L-lactic acid bioabsorbable screw at the lateral femoral epicondyle after posterolateral corner reconstruction. Because delayed foreign body reactions can occur at any site of poly-L-lactic acid bioabsorbable screw insertion, care should be taken to avoid screw protrusion during ligament reconstruction because it can lead to screw breakage and delayed foreign body reaction. [Orthopedics.2014; 37(12):e1129–e1132.]

This study was conducted to obtain information of the oral dose toxicity of low molecular fucoidan (LMF) in male and female mice. In order to calculate 50% lethal dose (LD50) and approximate lethal dose (LD), test material was once orally administered to male and female ICR mice at dose levels of 2000, 1000, 500, 250, 125 and 0 (vehicle control) mg/kg (body wt.). The mortality and the changes on body weight, clinical signs, gross observation and organ weight and histopathology of principle organs were monitored 14 days after LMF treatment. We could not find any mortalities, clinical signs, body weight changes and gross findings. In addition, significant changes in the organ weight and histopathology of principal organs were not observed except for some sporadic findings. The results obtained in this study suggest that LMF may not be toxic in mice and may be therefore safe for clinical use. The LD50and approximate LD in mice after single oral dose of LMF were considered over 2000 mg/kg in both female and male mice.

Abstract: The catalytic properties of β-cyclodextrin glucanotransferase (β-CGTase) from alkalophilicBacillus sp. BL-12 specific for the intermolecular transglycosylation of stevioside were investigated. The molecular mass of purified β-CGTase by ultra-filtration and β-cyclodextrin polymer affinity chromatography was estimated to be 90 kDa, which is high compared to other known bacterial CGTases. The optimal pH and temperature were 9.0 and 50°C, respectively, and thermal stability at 40°C was elevated 10-fold in the presence of 1% maltodextrin. The kinetic parameters of the new β-CGTase from alkalophilicBacillus sp. BL-12 indicate that it is more suitable for transglycosylation than the cyclization reaction. Maltodextrin was the most suitable glycosyl donor for transglycosylation of stevioside. The transglycosylation of stevioside was carried out using 60 units of CGTase per gram of maltodextrin, 20 g/L stevioside as the glycosyl acceptor, and 50 g/L maltodextrin as the gycosyl donor at 40°C for 6 h, and a conversion yield of stevioside as high as 76% was obtained.

A hydrophobic substrate triolein was hydrolyzed by lipase in a mono-phase reaction system containing cyclodextrin(CD) as emulsifier. The triolein was transformed to an emulsion-like state in the CD containing reaction system in contrast to the oil-droplet like state without CD due to the formation of an inclusion complex between the lipids and CDs. The hydrolysis reaction increased substantially in the CD containing reaction system, and the optimum reaction conditions including the amount of lipase, β-CD concentration, and mixing ratio of triolein and β-CD, were determined. The performance of the enzyme reaction in a mono-phase reaction system was compared with that of a two-phase reaction system which used water immiscible hexane as the organic solvent. The role of a CD in the mono-phase reaction system was elucidated by comparing the degree of the inclusion complex formation with triolein and oleic acid, Kmand Vmaxvalues, and product inhibition by oleic acid in aqueous and CD containing reaction systems. The resulting enhanced reaction seems to be caused by two phenomena; the increased accessibility of lipase to triolein and reduced product inhibition by oleic acid through the formation of an inclusion complex.

Electrochemical performance of porous NiO films was investigated as anodes for lithium ion batteries. Different porous morphologies of NiO films were fabricated by the electrostatic spray deposition (ESD) technique in different spray distances. Prepared porous NiO films were assembled in a half cell using lithium metal as a counter electrode and tested by cyclic voltammetry and galvanostatic charge/discharge. The test results showed that ESD derived porous NiO films exhibited highly reversible capacities and good cycle performance. The superior electrochemical performance might be attributed to their porous structures, which provide large electrode/electrolyte contact area. The porous structured NiO film electrodes could be promising anodes for use in high-energy density lithium ion batteries.