Inhibition of choline kinase as an antiamoebic approach in Entamoeba histolytica infection

@#Entamoeba histolytica is the parasite responsible for amoebiasis, which can result in amoebic colitis or amoebic liver abscess. Metronidazole has been the conventional treatment for intestinal amoebiasis, but concerns regarding resistance have emerged due to the identification of resistance pathways in E. histolytica. This study investigates a novel anti-amoebic approach targeting the CDP-choline pathway. Inhibition studies were conducted using potential choline kinase (CK) inhibitors to inhibit the EhCK enzyme, and RNA interference was employed to knock down the EhCK gene. Km and Vmax of purified EhCK and hCKa2 proteins were determined by pyruvate kinase-lactate dehydrogenase (PK-LDH) coupled assay. The IC50 values for EhCK and hCKa2 were determined with several commercial CK inhibitors. Selected inhibitors were incubated with E. histolytica trophozoites for 48 hours to determine the EC50 for each inhibitor. Silencing of gene encoding EhCK was carried out using duplex siRNA and the gene expression level was measured by real-time qPCR. Based on the IC50 values, three of the inhibitors, namely CK37, flavopiridol and H-89 were more potent against EhCK than hCKa2. Trophozoites growth inhibition showed that only HDTAB, H-89 and control drug metronidazole could penetrate and induce cell death after 48-hour incubation. siRNA concentration of 10 µg/mL was used for the transfection of positive control GAPDH, EhCK, and non-targeting GFP siRNAs. RNAi experiment concluded with positive control GAPDH downregulated by 99% while the level of EhCK mRNA was downregulated by 47%. In this study, potential inhibitors of EhCK and siRNA have been identified, paving the way for further refinement and testing to enhance their potency against EhCK while sparing hCK. The utilization of these specific inhibitors and siRNA targeting EhCK represents a novel approach to impede the growth of E. histolytica by disrupting its phospholipid synthesis pathway.

Inhibition of choline kinase as an antiamoebic approach in Entamoeba histolytica infection.

Entamoeba histolytica is the parasite responsible for amoebiasis, which can result in amoebic colitis or amoebic liver abscess. Metronidazole has been the conventional treatment for intestinal amoebiasis, but concerns regarding resistance have emerged due to the identification of resistance pathways in E. histolytica. This study investigates a novel anti-amoebic approach targeting the CDP-choline pathway. Inhibition studies were conducted using potential choline kinase (CK) inhibitors to inhibit the EhCK enzyme, and RNA interference was employed to knock down the EhCK gene. Km and Vmax of purified EhCK and hCKa2 proteins were determined by pyruvate kinase-lactate dehydrogenase (PK-LDH) coupled assay. The IC50 values for EhCK and hCKa2 were determined with several commercial CK inhibitors. Selected inhibitors were incubated with E. histolytica trophozoites for 48 hours to determine the EC50 for each inhibitor. Silencing of gene encoding EhCK was carried out using duplex siRNA and the gene expression level was measured by real-time qPCR. Based on the IC50 values, three of the inhibitors, namely CK37, flavopiridol and H-89 were more potent against EhCK than hCKa2. Trophozoites growth inhibition showed that only HDTAB, H-89 and control drug metronidazole could penetrate and induce cell death after 48-hour incubation. siRNA concentration of 10 µg/mL was used for the transfection of positive control GAPDH, EhCK, and non-targeting GFP siRNAs. RNAi experiment concluded with positive control GAPDH downregulated by 99% while the level of EhCK mRNA was downregulated by 47%. In this study, potential inhibitors of EhCK and siRNA have been identified, paving the way for further refinement and testing to enhance their potency against EhCK while sparing hCK. The utilization of these specific inhibitors and siRNA targeting EhCK represents a novel approach to impede the growth of E. histolytica by disrupting its phospholipid synthesis pathway.

Numerical analysis of flow distribution behavior in a proton exchange membrane fuel cell.

The flow distribution of a proton exchange membrane fuel cell within a manifold plays an important role on its performance. This study presents a numerical analysis of the flow distribution behavior within different manifold configurations. A two-dimensional model with 75 cells was employed to study the flow behavior. The variation in the stoichiometry and number of cells was also studied. Three different flow configurations were considered with different numbers of flow inlets and outlets. The flow characteristics, such as the pressure and velocity variations in the manifold and cells, were measured to determine the effects of the different flow configurations. The results indicated that the double inlet/outlet configuration had the best flow distribution when using 75 cells. Moreover, increasing the stoichiometry resulted in a better flow distribution to the cells in a stack.

A hypothetical oxidative stress regulatory role of alpha giardins in the protozoan parasite Giardia intestinalis.

Giardiasis is an intestinal infection caused by the protozoan parasite Giardia intestinalis, affecting hundreds of millions of people worldwide. This microaerophilic protozoan is capable of surviving in the host intestine in the presence of both oxygen and reactive oxygen species (ROS), despite the lack of conventional ROS-scavenging enzymes. The understanding of how G. intestinalis tolerates free radicals could help to identify essential biological processes that protect themselves against oxidative stress within the human gut. This review outlines the antioxidant mechanisms that are utilized by G. intestinalis, with an emphasis on the potential novel role of alpha giardins. The comprehensive understanding of the processes involved in oxidative stress management may provide new insights into improved treatments for giardiasis, and other medically important protozoan parasitic diseases.

Prevalence and Distribution of Anti-Amoebic IgG Antibody among Orang Asli (Aborigines) in Peninsular Malaysia.

Entamoeba species are commonly detected in stool samples of Orang Asli due to their substandard living conditions and poor hygiene. Among the Entamoeba spp., Entamoeba histolytica is the only known primary pathogenic species. This study determined the prevalence and distribution of anti-amoebic IgG antibody among Orang Asli in Peninsular Malaysia. The results would reflect the prevalence of amoebiasis in the population. This study analysed a total of 375 serum samples from archives of two Orang Asli projects conducted between 2011 and 2014. They were from six different states in Malaysia, namely Johor, Kedah, Kelantan, Pahang, Perak, and Selangor. Anti-amoebic IgG antibody was detected using an enzymelinked immunosorbent assay (ELISA) with crude soluble antigen produced from axenically grown E. histolytica trophozoites. From the analysis, the overall seropositivity was approximately 71% (266/375), while the seropositivity rates for each of the three Orang Asli tribes i.e. Senoi, Negrito and Proto-Malay, were 66% (137/208), 92% (103/112), and 43% (17/ 41) respectively. Orang Asli from Kedah [95% (52/55)] showed the highest seropositivity, followed by Kelantan [79% (54/68)], Perak [73% (78/107)], Pahang [60% (57/95)], Selangor [56% (14/25)], and Johor [48% (10/21)]. Orang Asli from rural [76% (192/254)] and peripheral urban [65% (69/106)] areas showed significantly higher seropositivity (p=0.002) than those from urban areas [36% (4/11)]. The high prevalences of anti-amoebic IgG antibody in these Orang Asli populations comprised both active and past infections. This study provides current insights of amoebiasis in selected Orang Asli settlements in Peninsular Malaysia. The high seropositivity of anti-amoebic IgG antibody suggests that the settlements are endemic for amoebiasis and there is a high risk of acquiring E. histolytica infection among the dwellers.

Prevalence and Distribution of Anti-Amoebic IgG Antibody among Orang Asli (Aborigines) in Peninsular Malaysia

Entamoeba species are commonly detected in stool samples of Orang Asli due to their substandard living conditions and poor hygiene. Among the Entamoeba spp., Entamoeba histolytica is the only known primary pathogenic species. This study determined the prevalence and distribution of anti-amoebic IgG antibody among Orang Asli in Peninsular Malaysia. The results would reflect the prevalence of amoebiasis in the population. This study analysed a total of 375 serum samples from archives of two Orang Asli projects conducted between 2011 and 2014. They were from six different states in Malaysia, namely Johor, Kedah, Kelantan, Pahang, Perak, and Selangor. Anti-amoebic IgG antibody was detected using an enzymelinked immunosorbent assay (ELISA) with crude soluble antigen produced from axenically grown E. histolytica trophozoites. From the analysis, the overall seropositivity was approximately 71% (266/375), while the seropositivity rates for each of the three Orang Asli tribes i.e. Senoi, Negrito and Proto-Malay, were 66% (137/208), 92% (103/112), and 43% (17/ 41) respectively. Orang Asli from Kedah [95% (52/55)] showed the highest seropositivity, followed by Kelantan [79% (54/68)], Perak [73% (78/107)], Pahang [60% (57/95)], Selangor [56% (14/25)], and Johor [48% (10/21)]. Orang Asli from rural [76% (192/254)] and peripheral urban [65% (69/106)] areas showed significantly higher seropositivity (p=0.002) than those from urban areas [36% (4/11)]. The high prevalences of anti-amoebic IgG antibody in these Orang Asli populations comprised both active and past infections. This study provides current insights of amoebiasis in selected Orang Asli settlements in Peninsular Malaysia. The high seropositivity of anti-amoebic IgG antibody suggests that the settlements are endemic for amoebiasis and there is a high risk of acquiring E. histolytica infection among the dwellers.

Re-revision of failed revision Total Hip Arthroplasty acetabular components.

While revision of total hip arthroplasty (THA) is being performed with increasing frequency, outcomes of repeated revisions have been rarely reported in the literature. The purpose of this study was to report mid-term outcomes of re-revision of failed revision THA acetabular components. We performed at least two revisions of the failed acetabular component in 57 patients (57 hips) between August 1996 and April 2008. Of these, 15 patients with infection were excluded and one died before 4-year evaluation. The final study cohort consisted of 41 patients (41 hips) with a mean age of 55.5 years (range, 37 to 82). Preoperative acetabular bone defects was classified as Paprosky Type IIA in 4 hips, Type IIB in 6, Type IIC in 9, Type IIIA in 16, and Type IIIB in 6. The mean duration of follow-up was 7.2 years (range, 4 to 15). Mean Harris hip score improved 45 points preoperatively to 83 points postoperatively. Six hips (14.6%) required additional revision procedure: 3 for aseptic loosening, 2 for deep infection, and 1 for recurrent instability. Complications included 2 dislocations and 1 peroneal nerve palsy. Kaplan-Meier survivorship with an end point of reoperation for any reason was 88.5% (95% CI, 78.0% to 100%) at 7.2 years. For aseptic loosening of the acetabular component, the survival was 91.8% (95% CI, 80.8% to 100%) at 7.2 years. Rerevision with contemporary uncemented cup or antiprotrusio cage for failed revision THA acetabular components showed encouraging mid-term outcomes for this technically challenging condition.

Efficacies of two in-house indirect ELISAs for diagnosis of amoebic liver abscess.

Entamoeba histolytica causes amoebic diarrhoea, colitis and liver abscess (ALA). Diagnosis of ALA is difficult, as most patients do not have simultaneous intestinal amoebic infection. At Hospital Universiti Sains Malaysia (HUSM), diagnosis of ALA relies on a combination of clinical findings, ultrasound examination of the liver and serodiagnosis using a commercial kit. In this study, two in-house indirect ELISAs were developed and evaluated. One of the in-house assays utilises E. histolytica crude soluble antigen (CSA) to detect serum IgG specific to the parasite whereas the other uses E. histolytica ether extract antigen (EEA). Preparation of CSA requires a sonicator to lyse the amoeba whereas EEA was prepared by chemically solubilizing the trophozoites. Based on the cut-off value of mean optical density + 3SD, CSA-ELISA showed 100% (24/24) sensitivity and 93.33% (210/225) specificity; while EEA-ELISA showed 91.67% (22/24) sensitivity and 95.11% (214/225) specificity. In conclusion, both the in-house indirect ELISAs were found to be efficacious for diagnosis of ALA; and the EEA is easier to prepare than the commonly used CSA.

In vivo study of novel nanofibrous intra-luminal guidance channels to promote nerve regeneration.

A novel nanofibrous construct for promoting peripheral nerve repair was fabricated and tested in a rat sciatic nerve defect model. The conduit is made out of bilayered nanofibrous membranes with the nanofibers longitudinally aligned in the lumen and randomly oriented on the outer surface. The intra-luminal guidance channel is made out of aligned nanofibrous yarns. In addition, biomolecules such as laminin and nerve growth factor were incorporated in the nanofibrous nerve construct to determine their efficacy in in vivo nerve regeneration. Muscle reinnervation, withdrawal reflex latency, histological, axon density and electrophysiology tests were carried out to compare the efficacy of nanofibrous constructs with an autograft. Our study showed mixed results when comparing the artificial constructs with an autograft. In some cases, the nanofibrous conduit with aligned nanofibrous yarn as an intra-luminal guidance channel performs better than the autograft in muscle reinnervation and withdrawal reflex latency tests. However, the axon density count is highest in the autograft at mid-graft. Functional recovery was improved with the use of the nerve construct which suggested that this nerve implant has the potential for clinical usage in reconstructing peripheral nerve defects.

Identification of Entamoeba histolytica trophozoites in fresh stool sample: comparison of three staining techniques and study on the viability period of the trophozoites.

Entamoeba histolytica causes about 50 million infections worldwide with a death rate of over 100,000 annually. In endemic developing countries where resources are limited, microscopic examinations based on Wheatley trichrome staining is commonly used for diagnosis of intestinal amoebiasis. Other than being a time-consuming method, it must be performed promptly after stool collection as trophozoites disintegrate rapidly in faeces. The aim of this study was to compare the efficacies of Eosin-Y, Wheatley trichrome and Iodine stains in delineating the diagnostic features of the parasite, and subsequently to determine the suitable microscopy observation period for detection of erythrophagocytic and non-erythrophagocytic trophozoites spiked in semi-solid stool sample. Wheatley trichrome staining technique was performed using the standard method while the other two techniques were performed on the slides by mixing the respective staining solution with the spiked stool sample. One million of axenically cultured non-erythrophagocytic E. histolytica and erythrophagocytic E. histolytica were separately spiked into 2 g of fresh semisolid faeces. Percentage viability of the trophozoites in the spiked stool sample was determined at 30 minute intervals for eight hours using the 0.4% Trypan blue exclusion method. The results showed that Eosin-Y and Wheatley trichrome stained the karyosome and chromatin granules better as compared to Iodine stain. The percentage viability of non-erythrophagocytic trophozoites decreased faster than the erythrophagocytic form in the first 5 hours and both dropped to ~10% in the 6th hour spiked sample. In conclusion, Eosin-Y staining technique was found to be the easiest to perform, most rapid and as accurate as the commonly used Wheatley trichrome technique; Eosin-Y stained slide sealed with DPX could also be kept as a permanent record. A period not exceeding 6 hours after stool collection was found to be the most suitable in order to obtain good microscopy results of viable trophozoites.

Seroprevalence of anti-amoebic antibody among blood donors by indirect hemeagglutination assay.

The screening for anti-amoebic antibody among a group of donors was to obtain negative control serum samples for an on-going antigen development assay in diagnosis of amoebic liver abscess. Out of 200 samples, 125 (62.5%) were negative, whereas 44 (21.5%) had IHA titer of less than 1:128 and 31 (16.0%) of the samples had significant IHA titers of 1:128 or more, in which 2 serum samples gave titers of 1:4096.

Non-invasive prediction of skin flap shrinkage: a new concept based on animal experimental evidence.

A non-invasive, in vivo method has been developed to predict the skin flap shrinkage (retraction) following a harvest. It involves the use of a novel custom-designed extensometer to measure the force-displacement behaviour of skin and subsequent data analysis to estimate the shrinkage. In validation experiments performed on pigs, this method has been shown to produce results with an average absolute error of 6.0% between the actual and predicted shrinkages. This may be close to what an experienced surgeon would estimate subjectively, thus indicating the potential usefulness of this method to predict flap shrinkage of patient's donor sites.

New extensometer to measure in vivo uniaxial mechanical properties of human skin.

Biomechanical properties of skin are important for clinical decision making as well as clinical intervention. Measuring these properties in vivo is critical for estimating dimensional behaviour of skin flap or graft after harvest. However, existing methodologies and devices often suffer from lack of standardisation and unwanted peripheral force contribution due to the deformation of surrounding tissues during measurement. This naturally leads to measurement inaccuracies and lack of reproducibility. In order to improve the measurement accuracy, a new portable extensometer, which measures the non-invasive in vivo biomechanical properties of skin, has been designed and constructed. This design incorporates three pads that attach to the skin, including a C-shaped pad to shield the force sensor from peripheral forces. Such design produces data that are significantly closer to in vitro measurements. The results have been verified by finite element analysis, and experiments on rubber sheets and pig skins. This device can be used to obtain biomechanical properties of skin that will aid doctors in measuring skin elasticity and surgical planning, especially in skin flap surgery.

Comparison of forearm and conventional Bier's blocks for manipulation and reduction of distal radius fractures.

Forearm-based Bier's block has been advocated as a useful anaesthesic technique in hand surgery. However, there is limited data comparing forearm blocks with the conventional Bier's block. We conducted a randomised controlled trial (n=30) comparing the two techniques of anaesthesia for manipulation and reduction of closed distal radius fractures in an emergency room setting. Pain scores measured using the Visual Analogue Scale during the procedure were used as the primary outcome assessment. There was no significant difference in pain scores between the forearm and conventional Bier's block (mean VAS 18.4 SD 22.10 versus 33.7 SD 29.6). No major complications were observed in either group. The forearm-based Bier block is an effective alternative to the conventional block.

Microsurgical skills training: a new concept for simulation of vessel-wall suturing.

Initiation of training in basic microsurgical suturing technique usually involves placing sutures on incisions made on latex sheets, and then progressing to a live anastomosis in a rat model. A straight incision resembles a skin incision and does not mechanically simulate vessel wall suturing, and various modifications and innovative prosthetic models continue to be developed to bring the simulation closer to a live anastomosis. We developed two models which closely simulate vessel wall suturing and require the application of similar skills by the trainee. In the first model (the "I" model), the straight 1-cm incision is converted to an I-shaped incision which increases the instability of the edge to be sutured, depending on the length of the horizontal incision, putting greater demand on dexterity in suture placement similar to a vessel. The length (1 cm) of the edge does not constrain the spacing of the sutures and can be used for the beginner. The second model (the "double triangle" model) creates a narrow and unstable edge for placing sutures, and puts greater demand on bimanual coordination and instrument control for training in accuracy of suture placement as well as spacing between sutures. Both these models are cost-effective and simple to construct.

Strain rate effect on the failure properties of tendons.

The purpose of this study is to evaluate the effect of strain rate on the failure properties of tendons. Seventy-five chicken flexor digitorum profundus (FDP) tendons were tensile tested at 15 strain rates, ranging from 0.05 to 150 per cent/s. Results showed that strain rate had little effect on the shape of the stress-strain curve. No significant change was observed in the toe region, while the slope of the linear region increased with the increase in strain rate. Generally, the ultimate tensile strength (UTS) and elastic modulus increased significantly as strain rate increased (p < 0.01), while no significant change in strain at UTS (p > 0.01) was observed. It was found the change in failure properties of tendons was not significant, with small change in strain rate.

Post-mastectomy radiotherapy after immediate autologous breast reconstruction in primary treatment of breast cancers.

AIM: To assess the clinical outcome of breast cancer patients with immediate autologous breast reconstruction and post-mastectomy radiotherapy (PMRT) as primary treatment. MATERIALS AND METHODS: Twenty-five women with breast cancer treated with immediate autologous breast reconstruction and post-mastectomy radiotherapy as primary treatment between 1995 and 2001 in Pamela Youde Nethersole Eastern Hospital of Hong Kong were retrospectively studied. Radiation doses of 50 Gy (in 2 Gy daily fraction) were given to the reconstructed breasts, except one who was given 45 Gy (in 1.8 Gy daily fraction). Nine women (36%) were treated without bolus, whereas the other 16 women (64%) were treated with 0.5 cm thick bolus on alternate days. The main outcome measures include local control, treatment complications and cosmetic outcome. RESULTS: Median follow-up was 3.7 years (range: 1.0-6.6 years). Two women (8%), who were treated without bolus, developed chest wall recurrences. The overall 5-year, actuarial, local failure-free rate and disease-specific survival rate were 89.8% and 77.9%, respectively. Apart from mild acute skin reactions, no significant acute radiotherapy side-effects were observed. No flap necrosis or flap loss was seen. The cosmesis of the reconstructed breasts were rated as good to excellent in 85% of the surviving patients. There was no observed adverse effect on cosmesis by adding bolus on alternate days. CONCLUSION: PMRT after immediate autologous tissue-flap breast reconstruction is well tolerated and is not associated with increased incidence of complications. Adding 0.5 cm bolus on alternate days might improve local control without causing adverse cosmetic effect. The concern of adverse effects of radiotherapy should not exclude the choice of immediate breast reconstruction in suitable patients.