Utilizing experimental design and desirability function in optimizing RP-HPLC method for simultaneous determination of some skeletal muscle relaxants and analgesics.

An experimental design and response surface methodologies using Plackett-Burman and Box-Behnken designs were applied for selecting and optimizing the most appropriate parameters which significantly affect the separation and quantitative estimation of five skeletal muscle relaxants and four analgesic drugs (baclofen, methocarbamol, dantrolene sodium, orphenadrine citrate, cyclobenzaprine hydrochloride, ketoprofen, etoricoxib, ibuprofen, and mefenamic acid) with a relatively short duration of analysis in a single run. For the separation of the nine drugs, an INERTSIL ODS-V3-5 µm C18 column (250 × 4.6 mm I.D.) was used with the optimum mobile phase conditions (45.15 mM ammonium acetate buffer pH 5.56 adjusted with acetic acid, acetonitrile, and methanol in a ratio of 30.5:29.5:40, v/v/v with a flow rate of 1.5 mL/min) and UV-detection at 220 nm. The optimized method was successfully subjected to the validation steps as described in ICH guidelines for linearity, precision, accuracy, robustness, and sensitivity. The optimized and validated method was effectively applied to determine the content of the studied drugs in their pharmaceutical preparations and to expand its applicability to the counterfeit estimation of etoricoxib in different brands of tablet dosage forms.

Evaluation of anterolateral thigh flap dimensions with virtual flap models.

Free flaps are commonly used for head and neck reconstruction. However, flap dimensions are still evaluated by visual and tactile assessment. The aim of this study was to enable preoperative planning of flap dimensions for soft tissue reconstruction based on clinical parameters. Computed tomography records from 230 patients dated from 2009 to 2019 were analysed retrospectively. A virtual, three-dimensional anterolateral thigh flap model was standardized, aligned to segmented leg models in two positions, and flap thicknesses and volumes were determined. Associations of flap thickness and volume with clinical parameters were evaluated, and an approximative calculation method was derived. The laterally positioned anterolateral thigh flap showed an average (interquartile range) thickness of 15.6 mm (8.7 mm) and volume of 1.5 cm3 (0.9 cm3) per cm2. The medially positioned anterolateral thigh flap showed an average (interquartile range) thickness of 16.3 mm (8.7 mm) and volume of 1.6 cm3 (0.9 cm3) per cm2. For both flap positions, leg circumference was the strongest predictor of flap thickness (ß = 0.545, P < 0.001 and ß = 0.529, P < 0.001) and flap volume (ß = 0.523, P < 0.001 and ß = 0.480, P < 0.001). Flap dimensions can be calculated based on leg circumference, and this preoperative planning of flap dimensions can help the surgeon to select the appropriate flap.

Application of Box-Behnken experimental design and response surface methodology for selecting the optimum RP-HPLC conditions for the simultaneous determination of methocarbamol, indomethacin and betamethasone in their pharmaceutical dosage form.

An isocratic RP-HPLC method has been developed for the separation and determination of methocarbamol (MTL), indomethacin (IND), and betamethasone (BET) in combined dosage form using an Inertsil ODS-3v C18 (250 × 4.6 mm, 5 µm) column with UV- detection at 235 nm. Experimental design using Box-Behnken design (BBD) was applied to study the response surface during method optimization and to achieve a good separation with a minimum number of experimental runs. The three independent parameters were pH of buffer, % of acetonitrile and flow rate of the mobile phase while the peak resolution of IND from MTL and the peak resolution of BET from IND (R2) were taken as responses to obtain mathematical models. The composite desirability was employed to optimize a set of responses overall (peak resolutions). The predicted optimum assay conditions include a mobile phase composition of acetonitrile and phosphate buffer (pH 5.95) in a ratio of 79:21, v/v, pumped at a flow rate of 1.4 mL min-1. With this ideal condition, the optimized method was able to achieve baseline separation of the three drugs with good resolution and a total run time of less than 7 min. The linearity of MTL, IND, and BET was determined in the concentration ranges of 5-600 µg mL- 1, 5-300 µg mL- 1, and 5-300 µg mL- 1 and the regression coefficients were 0.9994, 0.9998, and 0.9998, respectively. The average percent recoveries for the accuracy were determined to be 100.41 ± 0.60%, 100.86 ± 0.86%, and 100.99 ± 0.65% for MTL, IND, and BET, respectively. The R.S.D.% of the intra-day precision was found to be less than 1%, while the R.S.D.% of the inter-day precision was found to be less than 2%. The RP-HPLC method was fully validated with regard to linearity, accuracy, precision, specificity, and robustness as per ICH recommendations. The proposed method has various applications in quality control and routine analysis of the investigated drugs in their pharmaceutical dosage forms and laboratory-prepared mixtures with the goal of reducing laboratory waste, analysis time, and effort.

The impact of tumor-infiltrating lymphocytes on tumor features and pathological characteristics in breast cancer patients: the role of cytotoxic T lymphocytes and regulatory T cells.

OBJECTIVE: Though tumor-infiltrating lymphocytes (TILs) have a predictive impact in cancer patients, their association with presentation and prognosis in breast cancer is less consistent. This study aimed to assess the level of infiltrating cytotoxic T lymphocytes (CTLs) and regulatory T lymphocytes (Tregs) and their association with the clinicopathological features of breast cancer. PATIENTS AND METHODS: Tissue samples from female patients (n=153) diagnosed with primary invasive breast cancer were stained with CD8 (a CTL marker) and Foxp3 (a Treg marker) using immunohistochemistry. RESULTS: CTLs were distributed between tumor bed and stroma whereas Treg cells were mainly located in the stroma. The level of intratumoral CTLs correlated positively with Tregs in both tumor and stroma (rho=0.312, p<0.001 and rho=0.176, p=0.031; respectively). Stromal CTLs correlated positively with stromal Tregs (rho=0.319, p=0.005). Tumor size correlated inversely with the number of Treg cells in the tumor bed (rho= - 0.179, p=0.028). Tregs were associated with lymphovascular invasion status in the tumor bed (p=0.042). The ratio of intratumoral CTLs to Tregs was associated with estrogen receptor positivity and luminal subtype (p=0.029 and p=0.045, respectively). The median number of CTLs was significantly lower in patients using aspirin or antihypertensive medications compared to nonusers (p=0.024 and p=0.03, respectively). CONCLUSIONS: TILs were distributed differently in tumor tissues of breast cancer patients. CTLs infiltrates were found in both tumor bed and stroma while Tregs were dominant in the stroma. TILs were also distinctly associated with tumor features. The impact of TILs on prognosis and treatment outcomes in Jordanian breast cancer patients needs further investigation.

Evaluation of the bactericidal effect of cold atmospheric pressure plasma on contaminated human bone: an in vitro study.

The use of cold atmospheric pressure plasma (CAPP) as a bacterial decontaminant for chronic wounds has shown good results. The purpose of this in vitro study was to evaluate the bactericidal effects of CAPP on the cancellous area of the bone. Sterile glass slides and processed sterile human bone allografts 1, 2, 3, and 4mm thick were used for initial contamination and further CAPP treatment. Each block was contaminated with Staphylococcus aureus suspension on one side. Each slide was turned 180° and treated on the reverse side. The bacterial count in colony-forming units (CFU) was then measured and compared with that of a control group, and the bactericidal effects of CAPP in relation to bone density evaluated. A significant reduction in count was measured between treated and untreated groups (groups A-D: p<0.01 and group E: p=0.04). A strong positive linear relation was found between bone density and the S aureus count (r=0.844, p=0.156). Treatment with CAPP had a bactericidal effect on bone structures with a penetration depth of up to 4mm. It might be used for all diseases involving infected bone, and so extends the existing range of treatments.

Correlation between extent of sinus surgery, radiographic disease, and postoperative outcomes.

BACKGROUND: The extent of endoscopic sinus surgery (ESS) required for optimal outcomes in chronic rhinosinusitis (CRS) is undefined. We evaluated whether concordance between the extent of surgery and degree of radiographic disease influences postoperative outcomes. METHODS: 247 CRS patients who underwent ESS were retrospectively assigned a concordance score reflecting the similarity between the extent of surgery and degree of radiographic disease. 0 points were assigned when sinusotomy was performed on a diseased sinus, or no sinusotomy was performed on a nondiseased sinus; plus 1 for sinusotomy on a nondiseased sinus; and -1 for a diseased sinus left unopened. The total possible score ranged from minus 10 to plus 10. Patients were divided into 5 subgroups according to variance from complete concordance. SNOT-22 scores and revision rates were compared at 6 and 24 months. RESULTS: All five subgroups had similar preoperative SNOT-22 scores and improved at 6 months postoperatively. At 6 months postoperatively, the most conservatively operated and most extensively operated subgroups each achieved equivalent improvements in SNOT-22 as the completely concordant subgroup. At 24 months, the most extensively operated subgroup had a 12.5-point smaller improvement in SNOT-22 scores compared to the completely concordant subgroup. Multivariate analysis showed no association between concordance score and revision rate. CONCLUSIONS: Symptom improvement and revision rates after ESS do not appear to correlate with the degree of concordance between extent of surgery and radiographic disease. More extensive surgery than indicated by CT confers neither greater symptomatic improvement nor long-term detriment.

Evaluation of the lingual fracture patterns after bilateral sagittal split osteotomy according to Hunsuck/Epker modified by an additional inferior border osteotomy using a burr or ultrasonic device.

This study was conducted to compare fracture patterns and operation times after sagittal split osteotomy (SSO) by Hunsuck/Epker approach, performed using a burr or ultrasonic device, with and without osteotomy modification. A total of 80 SSOs were performed in fresh human cadavers using a burr or ultrasonic device to investigate the influence of surgical instruments as well as an additional bone cut on the inferior border of the mandible in terms of lingual fracture patterns. The times required for osteotomy and sagittal split were measured, and postoperative cone beam computed tomography images of all splits were analyzed. Without an additional inferior osteotomy, preferred splits according to Hunsuck/Epker were achieved in 35% of cases (7/20) with the burr and 45% (9/20) with the ultrasonic instrument. The inferior modification resulted in a greater number of unwanted fracture patterns in both groups. There was no relationship between the split technique and the fracture pattern (P=0.7854). Statistically significant differences in osteotomy time were observed between burr osteotomy and modified burr osteotomy (P=0.006), as well as modified ultrasonic osteotomy (P<0.001), but not between burr and ultrasonic surgery both without the inferior cut (P=0.36). The bone cut on the inferior border did not improve split control, but rather increased the risk of unwanted fractures and extended the operation time.

Medial approach for minimally-invasive harvesting of a deep circumflex iliac artery flap for reconstruction of the jaw using virtual surgical planning and CAD/CAM technology.

Donor site morbidity is the most common limitation of the deep circumflex iliac artery (DCIA) flap, so the purpose of this paper is to describe a new, minimally-invasive, approach to its harvest using virtual surgical planning and CAD/CAM technology to reduce functional and aesthetic morbidity at the donor site. Virtual surgical planning was based on preoperative computed tomographic data. A newly-designed surgical guide made using CAD/CAM technology was used to transfer the virtual surgical plan to the site of operation. This enabled us to raise a bicortical flap from the pelvis with preservation of the anterior superior iliac crest from the medial side with minimal muscular stripping. The guide, designed at slightly less than 90° to the lateral cortex, allowed the cut segment of bone to be raised medially. The new virtual surgical planning guide allowed a medial approach with reduced stripping of muscle and lower morbidity. No complications were encountered during the operation or the healing phase. Patients treated in this way had a shorter recovery period, with minimal complaints about walking or loss of profile of the hip. We conclude that virtual surgical planning can aid a minimally-invasive approach with predictable results. This allows a medial approach to the harvest of DCIA with preservation of important anatomical structures, and a reduction in donor site morbidity.

Candidate egg case silk genes for the spider Argiope argentata from differential gene expression analyses.

Orb-web weaving spiders produce a variety of task-specific silks from specialized silk glands. The genetics underlying the synthesis of specific silk types are largely unknown, and transcriptome analysis could be a powerful approach for identifying candidate genes. However, de novo assembly and expression profiling of silk glands with RNA-sequencing (RNAseq) are problematic because the few known gene transcripts for silk proteins are extremely long and highly repetitive. To identify candidate genes for tubuliform (egg case) silk synthesis by the orb-weaver Argiope argentata (Araneidae), we estimated transcript abundance using two sequencing methods: RNAseq reads from throughout the length of mRNA molecules, and 3' digital gene expression reads from the 3' region of mRNA molecules. Both analyses identified similar sets of genes as differentially expressed when comparing tubuliform and nonsilk gland tissue. However, incompletely assembled silk gene transcripts were identified as differentially expressed because of RNAseq read alignments to highly repetitive regions, confounding interpretation of RNAseq results. Homologues of egg case silk protein (ECP) genes were upregulated in tubuliform glands. This discovery is the first description of ECP homologues in an araneid. We also propose additional candidate genes involved in synthesis of tubuliform or other silk types.

Clinical outcomes and cost-effectiveness of an externally applied electroceutical device in managing venous leg ulcers in clinical practice in the UK.

OBJECTIVE: To estimate the cost-effectiveness of treating patients with a venous leg ulcer (VLU) with an externally applied electroceutical (EAE) device, plus dressings and compression bandaging or continuing with their previous care plan, from the perspective of the National Health Service (NHS) in the UK. METHOD: This was a prospective, single-arm, non-blinded, clinical and economic evaluation of EAE therapy performed in 2013/14. Patients' VLUs were treated with six active units of EAE therapy (each unit for two days) plus dressings and compression bandaging over a period of 12 days. Afterwards, patients were managed with a combination of dressings and bandages. Each patient acted as their own control so that clinical outcomes, resource use and costs associated with the wound over 12 months before the start of EAE therapy were retrospectively compared with the first 12 months after the start of treatment. The relative cost-effectiveness of EAE therapy was estimated at 2013/14 prices. RESULTS: Within 12 months of starting EAE therapy 77% of all wounds healed and the other 23% improved. This difference in effectiveness between the 12-months period before and after EAE therapy was estimated to yield a 12% improvement in health gain of 0.09 QALYs (p<0.01), a 34% reduction in the requirement for nurse visits (from a mean 50.7 to 33.3 visits per patient) and a 26% reduction in the number of dressings. This resulted in an 11% reduction in the NHS cost of VLU management over 12 months after the start of treatment when compared with the previous 12 months (from £1,981 to £1,754 per patient). Hence, use of EAE therapy was found to be a dominant treatment (i.e. improved outcome for less cost). CONCLUSION: Within the study's limitations, use of the EAE device potentially affords the NHS a cost-effective treatment for managing VLUs when compared with patients remaining on their previous care plan.

Clinical outcomes and cost-effectiveness of three alternative compression systems used in the management of venous leg ulcers.

OBJECTIVE: To assess clinical outcomes and cost-effectiveness of using a two-layer cohesive compression bandage (TLCCB; Coban 2) compared with a two-layer compression system (TLCS; Ktwo) and a four-layer compression system (FLCS; Profore) in treating venous leg ulcers (VLUs) in clinical practice in the UK, from the perspective of the National Health Service (NHS). METHOD: This was a retrospective analysis of the case records of VLU patients, randomly extracted from The Health Improvement Network (THIN) database (a nationally representative database of clinical practice among patients registered with general practitioners in the UK), who were treated with either TLCCB (n=250), TLCS (n=250) or FLCS (n=175). Clinical outcomes and health-care resource use (and costs) over six months after starting treatment with each compression system were estimated. Differences in outcomes and resource use between treatments were adjusted for differences in baseline covariates. RESULTS: Patients' mean age was 75 years old and 57% were female. The mean time with a VLU was 6-7 months and the mean initial wound size was 77-85 cm2. The overall VLU healing rate, irrespective of bandage type, was 44% over the six months' study period. In the TLCCB group, 51% of wounds had healed by six months compared with 40% (p=0.03) and 28% (p=0.001) in the TLCS and FLCS groups, respectively. The mean time to healing was 2.5 months. Patients in the TLCCB group experienced better health-related quality of life (HRQoL) over six months (0.374 quality-adjusted life years (QALYs) per patient), compared with the TLCS (0.368 QALYs per patient) and FLCS (0.353 QALYs per patient). The mean six-monthly NHS management cost was £2,413, £2,707 and £2,648 per patient in the TLCCB, TLCS and FLCS groups, respectively. CONCLUSION: Despite the systems studied reporting similar compression levels when tested in controlled studies, real-world evidence demonstrates that initiating treatment with TLCCB, compared with the other two compression systems, affords a more cost-effective use of NHS-funded resources in clinical practice, since it resulted in an increased healing rate, better HRQoL and a reduction in NHS management cost. The evidence also highlighted the lack of continuity between clinicians managing a wound, the inconsistent nature of the administered treatments and the lack of specialist involvement, all of which may impact on healing. DECLARATION OF INTEREST: This study was supported by an unrestricted research grant from 3M Health Care, UK. 3M Health Care had no influence on the study design, the collection, analysis, and interpretation of data, or on the writing of, and decision to submit for publication, the manuscript.

Combined γ-tocotrienol and Met inhibitor treatment suppresses mammary cancer cell proliferation, epithelial-to-mesenchymal transition and migration.

OBJECTIVES: Dysregulation of Met signalling is associated with malignant transformation. Combined treatment has been shown to reduce Met activation and mammary tumour cell proliferation. Experiments here, were conducted to determine mechanisms involved in mediating anti-cancer effects of combined γ-tocotrienol and SU11274 (Met inhibitor) treatment in various mammary cancer cell lines. MATERIALS AND METHODS: Treatment effects on mouse (+SA) and human (MCF-7, and MDA-MB-231) mammary cancer cell lines, and normal mouse (CL-S1) and human (MCF10A) mammary epithelial cell lines were compared. Cell proliferation and survival were determined by MTT assay and Ki-67 staining; protein expression was determined by western blot analysis. Immunofluorescence staining was also used to characterize expression and localization of multiple epithelial and mesenchymal markers. Cell migration was determined using a wound-healing assay. RESULTS: Combined treatment with γ-tocotrienol and SU11274 resulted in synergistic inhibition of +SA, MCF-7, and MDA-MB-231, but not CL-S1 or MCF10A cell growth that was associated with reduction in Akt STAT1/5 and NFκB activation and corresponding blockade in epithelial-to-mesenchymal transition, as indicated by increased expression of E-cadherin, ß-catenin, and cytokeratins 8/18 (epithelial markers) and corresponding reduction in vimentin (mesenchymal marker) and reduction in cancer cell motility. CONCLUSIONS: Suggest that combined γ-tocotrienol and Met inhibitor treatment may provide benefit in treatment of breast cancers characterized by aberrant Met activity.

γ-Tocotrienol inhibits HGF-dependent mitogenesis and Met activation in highly malignant mammary tumour cells.

OBJECTIVES: Aberrant Met signalling is associated with aggressive cancer cell phenotypes. γ-tocotrienol displays potent anti-cancer activity that is associated with suppression of HER/ErbB receptor signalling. Experiments were conducted to investigate the effects of γ-tocotrienol treatment on HGF-dependent +SA mammary tumour cell proliferation, upon Met activation. MATERIALS AND METHODS: The +SA cells were maintained in serum-free defined media containing 10 ng/ml HGF as the mitogen. Cell viability was determined using the MTT assay, western blot analysis was used to measure protein expression, and Met expression and activation were determined using immunofluorescent staining. RESULTS AND CONCLUSIONS: Treatment with γ-tocotrienol or Met inhibitor, SU11274, significantly inhibited HGF-dependent +SA cell replication in a dose-responsive manner. Treatment with 4 µmγ-tocotrienol reduced both total Met levels and HGF-induced Met autophosphorylation. In contrast, similar treatment with 5.5 µm SU11274 inhibited HGF-induced Met autophosphorylation, but had no effect on total Met levels. Combined treatment with subeffective doses of γ-tocotrienol (2 µm) and SU11274 (3 µm) resulted in significant inhibition of +SA cell expansion compared to treatment with individual agents alone. These findings show, for the first time, the inhibitory effects of γ-tocotrienol on Met expression and activation, and strongly suggest that γ-tocotrienol treatment may provide significant health benefits in prevention and/or treatment of breast cancer, in women with deregulated HGF/Met signalling.

Investigation of phenolic leaf extract of Heimia myrtifolia (Lythraceae): Pharmacological properties (stimulation of mineralization of SaOS-2 osteosarcoma cells) and identification of polyphenols.

Evaluation of the activity of an aqueous alcoholic extract obtained from the leaves of Heimia myrtifolia (Lythraceae) by determining its stimulating effect on two human osteoblastic cell lines HOS58 and SaOS-2 indicated its potential for use in the prevention and treatment of osteoporosis. In addition, the extract was found to significantly increase the mineralization of cultivated human bone cell SaOS-2, in which a strong dose-dependent increase was observed. A phytochemical investigation of the extract also confirmed that H. myrtifolia is capable of synthesizing and accumulating appreciable amounts of several phenolics, thus leading to the isolation and characterization of sixteen of these constituents. Identified among these isolates were a new natural product, 1,6-di-O-dehydrotrigalloyl-ß-D-(4)C(1)-glucopyranose, and a rare natural product (this marks its second report), 5,7,4'-trihydroxy-3-methoxyflavanone (dihydrokaempferol-3-O-methyl ether). Structures of these isolates were fully elucidated on the basis of conventional methods of analysis and confirmed by ESI/MS and (1)H and (13)C-NMR analysis.

Aphyllin, the first isoferulic acid glycoside and other phenolics from Tamarix aphylla flowers.

The first glycosylated isoferulic acid, isoferulic acid 3-O-beta-glucopyranoside, together with the new phenolics, tamarixetin 3,3'-di-sodium sulphate and dehydrodigallic acid dimetyl ester have been characterized from a flower extract of Tamarix aphylla. The structures were established on the basis of spectral data. The extract exhibited a distinct radical scavenging effect and to improve the viability of human keratinocytes (HaCaT cells). Also, the known isoferulic acid and ferulic acid which have been determined to be the major components of the investigated extract by HPLC/ESI mass spectrometric screening have been separated, characterized and evaluated as active antioxidants and as cell activity stimulating agents as well.

Bone mineralization enhancing activity of a methoxyellagic acid glucoside from a Feijoa sellowiana leaf extract.

The capability of an aqueous methanol extract obtained from the leaves of Feijoa sellowiana Berg. on possible prevention and treatment of osteoporosis has been examined by evaluating its stimulating effect on the two human osteoblastic cell lines HOS58 and SaOS-2. The extract was found to increase significantly the mineralization of cultivated human bone cell, whereby a clear increment (15.3 +/- 2.7%) in von Kossa positive area was determined when administering 25 microg/ml leaf extract. A phytochemical investigation of the extract has demonstrated the high phenolic content and led to the isolation and identification of twenty three of them, among which the new 3-methoxyellagic acid 4-O-beta-glucopyranoside was fully identified. All structures were elucidated on the basis of conventional analytical methods and confirmed by FTMS, 1D- and 2D-NMR data. The new compound was found to cause a significant increase of mineralized area at 20 microg/mL, while at lower concentrations the effect was not significant. However, an increase of the number of mineralized spots (nodules) at all tested concentrations of the compound was observed.