Silencing of xylose isomerase and cellulose synthase by siRNA inhibits encystation in Acanthamoeba castellanii

A key challenge in the successful treatment of Acanthamoeba infections is its ability to transform into a dormant cyst form that is resistant to physiological conditions and pharmacological therapies, resulting in recurrent infections. The carbohydrate linkage analysis of cyst walls of Acanthamoeba castellanii showed variously linked sugar residues, including xylofuranose/xylopyranose, glucopyranose, mannopyranose, and galactopyranose. Here, it is shown that exogenous xylose significantly reduced A. castellanii differentiation in encystation assays (P?<?0.05 using paired t test, one-tailed distribution). Using small interfering RNA (siRNA) probes against xylose isomerase and cellulose synthase, as well as specific inhibitors, the findings revealed that xylose isomerase and cellulose synthase activities are crucial in the differentiation of A. castellanii. Inhibition of both enzymes using siRNA against xylose isomerase and cellulose synthase but not scrambled siRNA attenuated A. castellanii metamorphosis, as demonstrated by the arrest of encystation of A. castellanii. Neither inhibitor nor siRNA probes had any effect on the viability and extracellular proteolytic activities of A. castellanii.     

Helicobacter pylori-Associated Upper Gastrointestinal Disease in Saudi Arabia: A Pathologic Evaluation of 298 Endoscopic Biopsies from 201 Consecutive Patients

In a prospective study, histopathological examination 298 upper gastrointestinal (UGI) biopsies, obtained from 201 consecutive patients, was made. Patients were referred with mild to severe dyspeptic symptoms. The aim of the study was to compare the rate of identification of Helicobacter pylori (H. pylori) in the histologically normal gastric mucosa with that in histologically confirmed gastritis or peptic ulcer disease. The gastroduodenal mucosa was histologically normal in 35 patients (17.4%); among those patients, H. pylori was identified in only three (9%). Chronic gastritis was histologically confirmed in 162 patients (80.6%). H. pylori was identified in 123 (76%) of those patients. The difference was statistically significant (p less than 0.00001). Furthermore, when cases with a histological diagnosis of superficial chronic active gastritis (SCAG) are considered separately, the identification rate of H. pylori increases to 88% (121 of 137). When this rate is compared with that of 8% (two of 25), found in superficial chronic quiescent gastritis (SCQG), the difference is highly significant (p less than 0.00001). Of 38 endoscopically diagnosed peptic ulcers, H. pylori was identified in the gastric mucosa of 34 (89%). The organisms were always seen in the antral gastric mucosa, but never in duodenal mucosa. Identification of H. pylori correlates significantly with the histologic activity of chronic gastritis, in both peptic ulcer disease and non-ulcer dyspepsia.     

Spontaneous idiopathic subcapsular biloma

Spontaneous biloma is an uncommon entity. We report a case of subcapsular biloma in an elderly patient with a nonobstructed biliary channel, without prior history of surgery, instrumentation, or trauma. Computed tomography (CT) and magnetic resonance imaging are described. We believe that this is the first reported case of spontaneous subcapsular biloma of idiopathic origin.     

Photochemotherapeutic Strategy against Acanthamoeba Infections

Acanthamoeba is a protist pathogen that can cause serious human infections, including blinding keratitis and a granulomatous amoebic encephalitis that almost always results in death. The current treatment for these infections includes a mixture of drugs, and even then, a recurrence can occur. Photochemotherapy has shown promise in the treatment of Acanthamoeba infections; however, the selective targeting of pathogenic Acanthamoeba has remained a major concern. The mannose-binding protein is an important adhesin expressed on the surface membranes of pathogenic Acanthamoeba organisms. To specifically target Acanthamoeba, the overall aim of this study was to synthesize a photosensitizing compound (porphyrin) conjugated with mannose and test its efficacy in vitro. The synthesis of mannose-conjugated porphyrin was achieved by mixing benzaldehyde and pyrrole, yielding tetraphenylporphyrin. Tetraphenylporphyrin was then converted into mono-nitrophenylporphyrin by selectively nitrating the para position of the phenyl rings, as confirmed by nuclear magnetic resonance (NMR) spectroscopy. The mono-nitrophenylporphyrin was reduced to mono-aminophenylporphyrin in the presence of tin dichloride and confirmed by a peak at m/z 629. Finally, mono-aminoporphyrin was conjugated with mannose, resulting in the formation of an imine bond. Mannose-conjugated porphyrin was confirmed through spectroscopic analysis and showed that it absorbed light of wavelengths ranging from 425 to 475 nm. To determine the antiacanthamoebic effects of the derived product, amoebae were incubated with mannose-conjugated porphyrin for 1 h and washed 3 times to remove extracellular compound. Next, the amoebae were exposed to light of the appropriate wavelength for 1 h. The results revealed that mannose-conjugated porphyrin produced potent trophicidal effects and blocked excystation. In contrast, Acanthamoeba castellanii incubated with mannose alone and porphyrin alone did not exhibit an antiamoebic effect. Consistently, pretreatment with mannose-conjugated porphyrin reduced the A. castellanii-mediated host cell cytotoxicity from 97% to 4.9%. In contrast, treatment with porphyrin, mannose, or solvent alone had no protective effects on the host cells. These data suggest that mannose-conjugated porphyrin has application for the targeted photodynamic therapy of Acanthamoeba infections and may serve as a model in the development of therapeutic interventions against other eukaryotic infections.     

Synthesis,acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities,and molecular docking studies of a novel compound based on combination of flurbiprofen and isoniazide

Synthesis of a compound with balanced bioactivities against a specific target is always a challenging task. In this study, a novel compound (1) has been synthesized by combination of flurbiprofen and isoniazide and shows ∼2.5 times enhanced acetylcholinesterase (AChE) inhibition activity and ∼1.7 times improved butyrylcholinesterase (BuChE) inhibition activity compared to flurbiprofen and a standard drug (i.e. physostigmine). A comparative AutoDock study has been performed, based on the optimized structure, by the DFT/B3LYP method, which confirmed that compound (1) is more active against AChE and BuChE, with calculated binding energies of −12.9 kcal mol⁻¹ and −9.8 kcal mol⁻¹ respectively as compared to flurbiprofen and an eserine (physostigmine) standard for which the binding energy was calculated to be −10.1 kcal mol⁻¹ and −8.9 kcal mol⁻¹, respectively. A mixed mode of inhibition of AChE and BuChE with compound 1 was confirmed by Lineweaver–Burk plots. AChE and BuChE inhibition activity alongside docking results suggests that compound (1) could be used for treatment of Alzheimer''s disease. Moreover, compound (1) also exhibit better α-chymotrypsin activity compared to flurbiprofen. Furthermore, in vitro and in vivo analysis confirmed that compound (1) exhibit more activity and less toxicity than the parent compounds.

A novel compound (1) shows ∼2.5 and ∼1.7 times enhanced AChE inhibition activity and BuChE inhibition activity respectively compared to flurbiprofen and standard drug (i.e. physostigmine). It has also been confirmed by comparative AutoDock studies.     

Discovery of 4-(2-(dimethylamino)ethoxy)benzohydrazide derivatives as prospective microtubule affinity regulating kinase 4 inhibitors

Microtubule affinity regulating kinase 4 (MARK4) is a Ser/Thr kinase, considered as a potential drug target for cancer, diabetes and neurodegenerative diseases. Due to its significant role in the development and progression of cancer, different in-house libraries of synthesized small molecules were screened to identify potential MARK4 inhibitors. A small library of hydrazone compounds showed a considerable binding affinity to MARK4. The selected compounds were further scrutinized using an enzyme inhibition assay and finally two hydrazone derivatives (H4 and H19) were selected that show excellent inhibition (nM range). These compounds have a strong binding affinity for MARK4 and moderate binding with human serum albumin. Anticancer studies were performed on MCF-7 and A549 cells, suggesting H4 and H19 selectively inhibit the growth of cancer cells. The IC₅₀ value of compound H4 and H19 was found to be 27.39 μM and 34.37 μM for MCF-7 cells, while for A549 cells it was 45.24 μM and 61.50 μM, respectively. These compounds inhibited the colonogenic potential of cancer cells and induced apoptosis. Overall findings reflect that hydrazones/hydrazone derivatives could be exploited as potential lead molecules for developing effective anticancer therapies via targeting MARK4.

Inhibition studies of MARK4 with selected hydrazone derivatives.     

Advanced Age,Obesity and Continuous Femoral Nerve Blockade are Independent Risk Factors for Inpatient Falls After Primary Total Knee Arthroplasty

We asked whether femoral nerve blockade (FNB) was an independent risk factor for inpatient post-operative falls after total knee arthroplasty (TKA). Data on 2197 primary TKAs were collected from our institution between 2003 and 2010. Patient demographics, type and duration of blocks were considered predictors of falls in a logistic regression model. Among 60 (2.7%) falls, the odds ratio was 1.04 (1.0–1.07; p = 0.008) for each 1 year of increased age above the mean (66 years), 2.4 (1.3–4.5; p = 0.005) for BMI > 30 kg/m² and 4.4 (1.04–18.2; p = 0.04) for continuous FNB. Single-shot FNB did not increase risk. No fall resulted in operative morbidity. The use of continuous FNB should be cautioned, especially in patients with other risk factors such as obesity and advanced age.     

Pedicled vascularized fibular graft with Ilizarov external fixator for reconstructing a large bone defect of the tibia after tumor resection

Background

Large bony defects in the middle or distal third of the tibia resulting from surgical resection of malignant bone tumors present a difficult reconstructive challenge. Various methods of reconstruction are available, such as allografts, vascularized fibular graft (either free or pedicled), or endoprothesis replacement for distal defects.

Materials and methods

Twelve patients—eight males and four females with mean age of 18 years at operation (range 14–25 years)—with malignant bone tumors of the tibial shaft were selected as candidates for wide resection of the tumor and reconstruction of the bony defect by ipsilateral vascularized fibular graft based on the peroneal vessels. Preoperative staging studies, including plain radiography, local MRI, isotopic bone scan, and chest CT, were done for every patient before biopsy. Ilizarov external fixation was then applied in all cases. The average length of the bony gap bridged was 14.5 cm (13–16.5 cm) and the mean length of the harvested graft was 16.3 cm (15–18 cm). The average operation time was 7.5 h (5.5–9.5 h).

Results

The mean follow-up period was 38 months (range 32–52 months). Bony union at the proximal and distal ends of the fibula occurred in nine patients (75 %) and at a mean time of 5.5 months (range 4.5–8 months). Graft hypertrophy occurred in all patients. The mean percentage of hypertrophy was 95 % (range 80–160 %). The mean MSTS functional score was 84 % (range 80–92 %). A leg length discrepancy of 2 cm was reported in two patients and was managed using a shoe lift.

Conclusion

Reconstruction of bony defects of the middle or distal tibia after bone tumor resection using pedicled vascularized fibula is a useful limb salvage procedure. The procedure can be performed relatively quickly and inexpensively and has a low rate of late complications. It leads to a good outcome regarding the union, hypertrophy, and function.     

Altered E-cadherin/beta-catenin expression in oral squamous carcinoma with and without nodal metastasis

OBJECTIVES: The aim of this study was to investigate the potential use of E-cadherin, a tumour-suppressor gene product involved in establishing cell-cell adhesion and one of its associated proteins, beta-catenin, as markers of nodal metastasis in oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: Thirty invasive OSCCs in patients with (n = 19) and without (n = 11) nodal metastases, as confirmed on histopathologic examination of the resected regional lymph nodes (n = 30), were examined for E-cadherin and beta-catenin expression by immunohistochemistry. RESULTS: There was a highly significant association (P < 0.0001) between E-cadherin and beta-catenin expression and tumour differentiation by conventional Broders' grading of the whole tumour. Irrespective of the nodal status and invasive tumour front (ITF) grading score, however, loss of expression was recorded at the ITF in 28 (93%) of 30 tumours and 22 (73%) of 30 tumours stained for E-cadherin and beta-catenin respectively. CONCLUSION: The results of this study suggest an association between loss of expression of E-cadherin and beta-catenin and a lower degree of differentiation; however, their use as markers of nodal metastasis in OSCC appears unreliable.