UV spectrophotometric methods for simultaneous determination of ketorolac tromethamine and olopatadine hydrochloride: Application of multiple standard addition for assay of ophthalmic solution.

Ophthalmic preparations that contain ketorolac tromethamine (KET) and olopatadine HCl (OLO) are used to relieve seasonal allergies and allergic conjunctivitis. Simultaneous quantification of KET and OLO was held by validated and simple spectrophotometric methods. KET was determined directly from the fundamental UV absorption spectra (at 323 nm), while OLO was determined after performing either dual wavelength or ratio derivative methods. The first method was based on measuring the absorbance difference (ΔA) between 243 and 291 nm, while the second depended on generating first derivative ratio spectra using 3.0 µg/mL KET as a divisor and measuring OLO responses at 234 nm (minima). Multiple standard addition method was applied to enable the determination of OLO which is considered as the weakly absorbing species as well as the minor component in a challenging dosage form ratio (4:1). The linearity ranges of the developed methods were 3-12 µg/mL and 4-40 µg/mL for KET and OLO, respectively. Simultaneous determination of both drugs was successfully implemented to lab prepared eye drops that contain KET, OLO and benzalkonium chloride as an inactive ingredient. Greenness assessment indicates minimal impact on environment. The developed methods determined the cited drugs with % recovery ± SD of 99.63 ± 0.01 for KET, 100.90 ± 0.02 and 100.31 ± 0.01 for OLO using dual wavelength and first derivative ratio methods, respectively. Using F-test and t-test at confidence level %95 to compare between the results of the presented methods and a reported method show no significant difference which allows precise, accurate, rapid, and simple quantification of quality control samples that contain KET and OLO.

Impact of N6-methyladenosine (m6A) modification on immunity.

N6-methyl-adenosine (m6A) is the most prevalent modification on mRNAs and long noncoding RNAs (lnRNAs) in higher eukaryotes. Modulation of m6A relies on m6A writers, erasers and readers. m6A modification contributes to diverse fundamental biological functions at the molecular, cellular, and physiological levels. The dysregulation of m6A modification has been implicated in various human diseases. Thus, m6A modification has now become a research hotspot for its potential therapeutic applications in the treatment of various cancers and diseases. The immune system is essential to provide defense against infections and cancers. This review summarizes the current knowledge about the roles of m6A in regulating immune cell functions and immune responses. Video abstract.

Ginkgo biloba extract (EGb 761) mitigates methotrexate-induced testicular insult in rats: Targeting oxidative stress, energy deficit and spermatogenesis.

Methotrexate (MTX) is commonly used as a therapeutic agent in the treatment of malignancies and autoimmune disorders. Risk of subsequent infertility following MTX administration has been reported as a significant side effect due to testicular toxicity. The aim of the study was to evaluate the modulatory effects of Ginkgo biloba (standardized extract, EGb 761) on MTX-induced testicular oxidative stress, energy deficits and spermatogenic status in rats. All groups received intraperitoneal injection of MTX (0.5 mg/kg) twice weekly for four weeks except the control group that received the corresponding vehicles. Other groups received oral EGb761, at doses 25, 50 or 100 mg/kg/day, for four weeks, concurrently with MTX. Blood and semen sampling followed by testis dissection were performed 24 h after last EGb 761 treatment. Semen was examined for sperm progressive motility, percent of normal spermatozoa and sperm cell concentration as well as seminal plasma essential and non-essential amino acids. Serum LH, FSH and testosterone were detected as well as testicular MDA, GSH, GSSG, TNF-α, IL-1ß, IL-6, NF-κB and the nuclear, cytoplasmic and mRNA expression levels of Nrf-2 besides the testicular cell energy; AMP, ADP and ATP. Histopathological studies of interstitial fibrosis and the severity of germ cell degeneration were also conducted. MTX induced significant decline in sperm quality along with decreased essential and non-essential amino acids in seminal plasma. MTX reduced serum FSH, LH and testosterone as well as testicular ATP, GSH and Nrf2, while increased levels of testicular ADP, AMP, MDA, GSSG and TNF-α. Results were confirmed by prominent interstitial fibrosis and severe germ cell degeneration in MTX group. Concurrent treatment with EGb 761 alleviated MTX-induced testicular insult evidenced by amelioration of oxidative stress biomarkers, energy functions, seminal sperms abnormalities and spermatogenesis status. The present study suggests a beneficial role of EGb 761 in MTX-induced testicular injury and subsequent distortion of spermatogenesis.

Pioglitazone Attenuates Lipopolysaccharide-Induced Oxidative Stress, Dopaminergic Neuronal Loss and Neurobehavioral Impairment by Activating Nrf2/ARE/HO-1.

The aim of the present study was to examine the neuroprotective potential of pioglitazone via activation of Nrf2/ARE-dependent HO-1 signaling pathway in chronic neuroinflammation and progressive neurodegeneration mouse model induced by lipopolysaccharide (LPS). After assessing spatial memory, anxiety and motor-coordination, TH+ neurons in substantia nigra (SN) were counted. The oxidative stress marker carbonyl protein levels and HO-1 enzyme activity were also evaluated. RT-qPCR was conducted to detect HO-1, Nrf2 and NF-κp65 mRNA expression levels and Nrf2 transcriptional activation of antioxidant response element (ARE) of HO-1 was investigated. Pioglitazone ameliorated LPS-induced dopaminergic neuronal loss, as well as mitigated neurobehavioral impairments. It enhanced Nrf2 mRNA expression, and augmented Nrf2/ARE-dependent HO-1 pathway activation by amplifying HO-1 mRNA expression. Moreover, it induced a significant decrease in NF-κB p65 mRNA expression, while reducing carbonyl protein levels and restoring the HO-1 enzyme activity. Interestingly, LPS induced Nrf2/antioxidant response element (ARE) of HO-1 activation, ultimately resulting in slight enhanced HO-1 mRNA expression. However, LPS elicited decrease in HO-1 enzyme activity. Zinc protoporphyrin-IX (ZnPPIX) administrated with pioglitazone abolished its effects in the LPS mouse model. The study results demonstrate that coordinated activation of Nrf2/ARE-dependent HO-1 pathway defense mechanism by the PPARγ agonist pioglitazone mediated its neuroprotective effects.

Therapy-relevant aberrant expression of MRP3 and BCRP mRNA in TCC-/SCC-bladder cancer tissue of untreated patients.

Multidrug resistance (MDR) is a critical factor, which results in suboptimal outcomes in cancer chemotherapy. One principal mechanism of MDR is the increased expression of ATP-binding cassette (ABC) transporters. Of these, multidrug resistance-associated protein 3 (MRP3) and breast cancer resistance protein (BCRP) confer MDR when overexpressed in cancer cell lines. We measured the mRNA expression of MRP3 and BCRP in primary untreated bladder cancer specimens using reverse transcription-quantitative PCR (RT-qPCR) in comparison to normal bladder tissue. The MRP3 and BCRP expression in the two major histotypes of bladder cancer; transitional cell carcinoma (TCC; urothelial type of bladder cancer) and squamous cell carcinoma (SCC; 'Schistosoma-induced' bladder cancer) were compared. Furthermore, the association between MRP3 and BCRP expression and tumor grade and stage were investigated. MRP3 mRNA expression in bladder cancer specimens was increased ~13-fold on average compared to normal bladder tissue (n=36, P<0.0001). BCRP mRNA expression was decreased in bladder cancer specimens to ~1/5 on average, compared to normal bladder tissue (n=38, P<0.0001). TCC showed significantly increased MRP3 mRNA expression compared to SCC of the bladder (P<0.0001). BCRP mRNA expression was similar in TCC and SCC of the bladder (P=0.1072). The increased MRP3 mRNA expression was not related to bladder tumor grade (P=0.3465) but was, however, significantly higher in superficial than in invasive bladder tumors (P=0.0173). The decreased expression of BCRP was not related to bladder tumor grade (P=0.1808) or stage (P=0.8016). The current data show that bladder cancer is associated with perturbed expression of MRP3 and BCRP. Representing drug resistance factors, determining the expression of these transporters in native tumors may be predictive of the outcome of chemotherapy based-treatment of bladder cancer.

Altered expression of miR-181a and miR-146a does not change the expression of surface NCRs in human NK cells.

MicroRNAs (miRNAs) play an important role in regulating gene expression and immune responses. Of interest, miR-181a and miR-146a are key players in regulating immune responses and are among the most abundant miRNAs expressed in NK cells. Bioinformatically, we predicted miR-181a to regulate the expression of the natural cytotoxicity receptor NCR2 by seeded interaction with the 3'-untranslated region (3'-UTR). Whereas, miR-146a expression was not significantly different (P = 0.7361), miR-181a expression was, on average 10-fold lower in NK cells from breast cancer patients compared to normal subjects; P < 0.0001. Surface expression of NCR2 was detected in NK cells from breast cancer patients (P = 0.0384). While cytokine receptor-induced NK cell activation triggered overexpression of miR-146a when stimulated with IL-2 (P = 0.0039), IL-15 (P = 0.0078), and IL-12/IL-18 (P = 0.0072), expression of miR-181a was not affected. Overexpression or knockdown of miR-181a or miR-146a in primary cultured human NK cells did not affect the level of expression of any of the three NCRs; NCR1, NCR2 or NCR3 or NK cell cytotoxicity. Expression of miR-181a and miR-146a did not correlate to the expression of the NCRs in NK cells from breast cancer patients or cytokine-stimulated NK cells from healthy subjects.

Expression of RFC/SLC19A1 is associated with tumor type in bladder cancer patients.

Urinary bladder cancer (UBC) ranks ninth in worldwide cancer. In Egypt, the pattern of bladder cancer is unique in that both the transitional and squamous cell types prevail. Despite much research on the topic, it is still difficult to predict tumor progression, optimal therapy and clinical outcome. The reduced folate carrier (RFC/SLC19A1) is the major transport system for folates in mammalian cells and tissues. RFC is also the primary means of cellular uptake for antifolate cancer chemotherapeutic drugs, however, membrane transport of antifolates by RFC is considered as limiting to antitumor activity. The purpose of this study was to compare the mRNA expression level of RFC/SLC19A1 in urothelial and non-urothelial variants of bladder carcinomas. Quantification of RFC mRNA in the mucosa of 41 untreated bladder cancer patients was performed using RT-qPCR. RFC mRNA steady-state levels were ∼9-fold higher (N = 39; P<0.0001) in bladder tumor specimens relative to normal bladder mRNA. RFC upregulation was strongly correlated with tumor type (urothelial vs. non-urothelial; p<0.05) where median RFC mRNA expression was significantly (p<0.05) higher in the urothelial (∼14-fold) compared to the non-urothelial (∼4-fold) variant. This may account for the variation in response to antifolate-containing regimens used in the treatment of either type. RFC mRNA levels were not associated with tumor grade (I, II and III) or stage (muscle-invasive vs. non-muscle invasive) implying that RFC cannot be used for prognostic purposes in bladder carcinomas and its increased expression is an early event in human bladder tumors pathogenesis. Further, RFC can be considered as a potential marker for predicting response to antifolate chemotherapy in urothelial carcinomas.