Modulation of apoptosis and Inflammasome activation in chondrocytes: co-regulatory role of Chlorogenic acid.

BACKGROUND: The B-cell lymphoma 2 (Bcl-2) protein regulates programmed cell death throughout the disease conditions by upholding apoptotic pathways. However, the mechanism by which it's expressed in chondrocytes still needs to be studied in chondrocyte-related disorders. Additionally, exploring the potential therapeutic role of Chlorogenic acid (CGA) in confluence with Bcl-2 modulation is of significant interest. METHODS: In vivo and in vitro studies were performed according to our previous methodologies. The chondrocytes were cultured in specific growth media under standard conditions after expression verification of different microRNAs through high-throughput sequencing and verification of Bcl-2 involvement in tibial growth plates. The effect of Bcl-2 expression was investigated by transfecting chondrocytes with miR-460a, siRNA, and their negative controls alone or in combination with CGA. The RNA was extracted and subjected to a reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Western blot analysis and immunofluorescence assays were performed to visualize the intracellular localization of Bcl-2 and associated proteins related to apoptotic and inflammasome pathways. Moreover, apoptosis through flow cytometry was also performed to understand the modulation of concerning pathways. RESULTS: The suppression of Bcl-2 induced higher apoptosis and mitochondrial dysfunction, leading to IL-1ß maturation and affecting the inflammasome during chondrocyte proliferation. Conversely, overexpression attenuated the activation, as evidenced by reduced caspase activity and IL-1ß maturation. In parallel, CGA successfully reduced siRNA-induced apoptosis by decreasing Cytochrome C (Cyto C) release from the mitochondria to the cytoplasm, which in turn decreased Caspase-3 and Caspase-7 cleavage with Bcl-2-associated X protein (Bax). Furthermore, siBcl-2 transfection and CGA therapy increased chondrocyte proliferation and survival. The CGA also showed a promising approach to maintaining chondrocyte viability by inhibiting siRNA-induced apoptosis. CONCLUSIONS: Targeting Bcl-2-mediated regulation might be a possible treatment for chondrocyte-related conditions. Moreover, these results add knowledge of the complicated processes underlying chondrocyte function and the pathophysiology of related diseases, highlighting the significance of target specific therapies. Video Abstract.

A comparison of the activity, lysosomal stability, and efficacy of legumain-cleavable and cathepsin cleavable ADC linkers.

1. Over the past two decades antibody-drug conjugates (ADCs) have emerged as a highly effective drug delivery technology. ADCs utilize a monoclonal antibody, a chemical linker, and a therapeutic payload to selectively deliver highly potent pharmaceutical agents to specific cell types.2. Challenges such as premature linker cleavage and clearance due to linker hydrophobicity have adversely impacted the stability and safety of ADCs. While there are various solutions to these challenges, our team has focused on replacement of hydrophobic ValCit linkers (cleaved by CatB) with Asn-containing linkers that are cleaved by lysosomal legumain.3. Legumain is abundantly present in lysosomes and is known to play a role in tumor microenvironment dynamics. Herein, we directly compare the lysosomal cleavage, cytotoxicity, plasma stability, and efficacy of a traditional cathepsin cleavable ADC to a matched Asn-containing legumain-cleavable ADC.4. We demonstrate that Asn-containing linker sequences are specifically cleaved by lysosomal legumain and that Asn-linked MMAE ADCs are broadly active against a variety of tumors, even those with low legumain expression. Finally, we show that AsnAsn-linked ADCs exhibit comparable or improved efficacy to traditional ValCit-linked ADCs. Our study paves the way for replacement of the traditional ValCit linker technology with more hydrophilic Asn-containing peptide linker sequences.

Prevalence and determinants of symptomatic COVID-19 infection among children and adolescents in Qatar: a cross-sectional analysis of 11 445 individuals.

There is a paucity of evidence about the prevalence and risk factors for symptomatic infection among children. This study aimed to describe the prevalence of symptomatic coronavirus disease 2019 (COVID-19) and its risk factors in children and adolescents aged 0-18 years in Qatar. We conducted a cross-sectional study of all children aged 0-18 years diagnosed with COVID-19 using polymerase chain reaction in Qatar during the period 1st March to 31st July 2020. A generalised linear model with a binomial family and identity link was used to assess the association between selected factors and the prevalence of symptomatic infection. A total of 11 445 children with a median age of 8 years (interquartile range (IQR) 3-13 years) were included in this study. The prevalence of symptomatic COVID-19 was 36.6% (95% confidence interval (CI) 35.7-37.5), and it was similar between children aged <5 years (37.8%), 5-9 years (34.3%) and 10 + years (37.3%). The most frequently reported symptoms among the symptomatic group were fever (73.5%), cough (34.8%), headache (23.2%) and sore throat (23.2%). Fever (82.8%) was more common in symptomatic children aged <5 years, while cough (38.7%) was more prevalent in those aged 10 years or older, compared to other age groups. Variables associated with an increased risk of symptomatic infection were; contact with confirmed cases (RD 0.21; 95% CI 0.20-0.23; P = 0.001), having visited a health care facility (RD 0.54; 95% CI 0.45-0.62; P = 0.001), and children aged under 5 years (RD 0.05; 95% CI 0.02-0.07; P = 0.001) or aged 10 years or older (RD 0.04; 95% CI 0.02-0.06; P = 0.001). A third of the children with COVID-19 were symptomatic with a higher proportion of fever in very young children and a higher proportion of cough in those between 10 and 18 years of age.

Perinatal and infant outcome of fetuses with prenatally diagnosed hyperechogenic kidneys.

OBJECTIVE: Hyperechogenic kidneys are a relatively rare antenatal finding, which can generate significant parental anxiety due to uncertain prognosis. We report on the perinatal and infant outcomes of a large cohort of fetuses with antenatally diagnosed hyperechogenic kidneys. METHODS: This was a retrospective analysis of all cases diagnosed prenatally with hyperechogenic kidneys between 2002 and 2017 in a large tertiary fetal medicine unit. Hyperechogenicity was defined as kidney parenchyma with greater echogenicity than that of the liver. Pregnancy, pathological and postnatal outcomes were collected from hospital and general practitioner records up to 1 year of age. Abnormal renal outcome was defined as elevated creatinine beyond 6 months of age, hypertension requiring medication or major kidney surgery, such as nephrectomy. Severe abnormal renal outcome was defined as the need for dialysis or kidney transplant at any stage. RESULTS: Three-hundred and sixteen fetuses with hyperechogenic kidneys were identified at a mean gestational age of 21 (range, 13-37) weeks. The majority of cases (97%) had bilateral hyperechogenic kidneys. In the 265 cases with available follow-up data, other associated renal tract abnormalities were identified prenatally in 36%, concomitant extrarenal structural abnormalities in 39% and abnormal karyotype in 15% of cases. Of the 316 included cases, 139 did not survive, including 105 terminations of pregnancy, five intrauterine deaths and 29 early neonatal deaths. Only 4.3% (6/139) of these fetuses had isolated hyperechogenic kidneys while 28.1% (39/139) had associated multiple renal tract abnormalities alongside hyperechogenic kidneys and over two-thirds (67.6%; 94/139) had concomitant extrarenal abnormalities. Of the 177 cases that survived beyond 1 month of age, outcome data were available in 126. Of these, based on the antenatal findings, 60 (47.6%) cases had isolated hyperechogenic kidneys, 56 (44.4%) had associated renal structural abnormalities and 10 (7.9%) had additional extrarenal abnormalities. Considering renal outcome alone, kidney function was abnormal in 13 (21.7%), 10 (17.9%) and 0 (0%) infants in these three groups, respectively, although concurrent pathology clearly affected global outcome in the more complex cases. Neonatal mortality of 1.6% was observed in the isolated renal hyperechogenicity group. The presence of oligohydramnios or abnormal renal volume was not associated significantly with abnormal renal function (odds ratio (OR), 2.32 (99% CI, 0.54-10.02) and OR, 0.74 (99% CI, 0.21-2.59), respectively) in this group. CONCLUSIONS: Hyperechogenic kidneys are often complicated by associated renal tract and extrarenal abnormalities, aberrant karyotype and genetic disease, and these factors have a greater effect on overall outcome than does kidney echogenicity. The renal outcome of fetuses with isolated hyperechogenic kidneys is good generally, with over 70% of cases having normal renal function postpartum. Importantly, for prognostic counseling, all of the fetuses in this non-selected series with isolated hyperechogenic kidneys and normal amniotic fluid levels had normal renal outcome in infancy. © 2020 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Sustained release and enhanced oral bioavailability of rivaroxaban by PLGA nanoparticles with no food effect.

The purpose of the currents study was to enhance bioavailability of rivaroxaban (RXB) and reduce the food effect. RXB loaded PLGA nanoparticles (RXB-PLGA-NPs) were prepared by emulsion solvent evaporation method and optimized using central composite design (CDD). The optimized RXB-PLGA-NPs (F8) with composition, PLGA (125 mg), PVA (0.5%w/w) and RXB (20 mg) was found optimum with particle size (496 ± 8.5 nm), PDI (0.607), ZP (- 18.41 ± 3.14 mV), %EE (87.9 ± 8.6) and %DL (9.5 ± 1.6). The optimized NPs (F8) was further evaluated in vitro for DSC, FTIR, SEM and in vitro release studies. A comparative pharmacokinetic studies with commercial tablet (XARELTO®) were conducted on fasted and fed state rats. Compared to commercial tablet (XARELTO®), the RXB-PLGA-NPs (F8) exhibited a significant enhancement of bioavailability in both fasted and fed state. In addition, the bioavailability of RXB from NPs (F8) was found unaffected in the presence of food.

Transmembrane Polar Relay Drives the Allosteric Regulation for ABCG5/G8 Sterol Transporter.

The heterodimeric ATP-binding cassette (ABC) sterol transporter, ABCG5/G8, is responsible for the biliary and transintestinal secretion of cholesterol and dietary plant sterols. Missense mutations of ABCG5/G8 can cause sitosterolemia, a loss-of-function disorder characterized by plant sterol accumulation and premature atherosclerosis. A new molecular framework was recently established by a crystal structure of human ABCG5/G8 and reveals a network of polar and charged amino acids in the core of the transmembrane domains, namely, a polar relay. In this study, we utilize genetic variants to dissect the mechanistic role of this transmembrane polar relay in controlling ABCG5/G8 function. We demonstrated a sterol-coupled ATPase activity of ABCG5/G8 by cholesteryl hemisuccinate (CHS), a relatively water-soluble cholesterol memetic, and characterized CHS-coupled ATPase activity of three loss-of-function missense variants, R543S, E146Q, and A540F, which are respectively within, in contact with, and distant from the polar relay. The results established an in vitro phenotype of the loss-of-function and missense mutations of ABCG5/G8, showing significantly impaired ATPase activity and loss of energy sufficient to weaken the signal transmission from the transmembrane domains. Our data provide a biochemical evidence underlying the importance of the polar relay and its network in regulating the catalytic activity of ABCG5/G8 sterol transporter.

Structural snapshot of the cholesterol-transport ATP-binding cassette proteins 1.

The ATP-binding cassette (ABC) proteins play critical roles in maintaining lipid and sterol homeostasis in higher eukaryotes. In humans, several subfamily-A and -G members function as cholesterol transporters across the cellular membranes. Deficiencies of these ABC proteins can cause dyslipidemia that is associated with health conditions such as atherosclerosis, diabetes, fatty liver disease, and neurodegeneration. The physiological roles of ABC cholesterol transporters have been implicated in mediating cholesterol efflux for reverse cholesterol transport and in maintaining membrane integrity for cell survival. The precise role of these ABC transporters in cells remains elusive, and little is known about the sterol-transport mechanism. The membrane constituents of ABC transporters have been postulated to play a key role in determining the transport substrates and the translocation mechanisms via the transmembrane domains. Recent breakthroughs in determining high-resolution structures of the human sterol transporter ABCG5/G8 and its functional homologs have shed light on new structural features of ABC transporters, providing a more relevant framework for mechanistic analysis of cholesterol-transport ABC proteins. This minireview outlines what is known about ABCG cholesterol transporters, addresses key structural features in the putative sterol translocation pathway on the transmembrane domains, and concludes by proposing a mechanistic model of ABC cholesterol transporters.

ABCG5/G8: a structural view to pathophysiology of the hepatobiliary cholesterol secretion.

The ABCG5/G8 heterodimer is the primary neutral sterol transporter in hepatobiliary and transintestinal cholesterol excretion. Inactivating mutations on either the ABCG5 or ABCG8 subunit cause Sitosterolemia, a rare genetic disorder. In 2016, a crystal structure of human ABCG5/G8 in an apo state showed the first structural information on ATP-binding cassette (ABC) sterol transporters and revealed several structural features that were observed for the first time. Over the past decade, several missense variants of ABCG5/G8 have been associated with non-Sitosterolemia lipid phenotypes. In this review, we summarize recent pathophysiological and structural findings of ABCG5/G8, interpret the structure-function relationship in disease-causing variants and describe the available evidence that allows us to build a mechanistic view of ABCG5/G8-mediated sterol transport.

Augmentation of DTH reaction of mycobacterial antigenic cocktail using synthetic mycobacterial 19-kDa lipoprotein as a TLR-stimulant.

The current study proposed that previously characterized individual antigenic proteins could represent potential replacement for conventional purified protein derivative (PPD) in tuberculosis skin testing when used in cocktails triggered by suitable TLR-stimulants that would provide the missing pro-inflammatory stimulus. Three different cocktails of previously selected antigens, including C1 (ESAT-6/CPF-10/MPB-83); C2 (ESAT-6/MPB-64/MPB-83); and C3 (CPF-10/MPB-64/MPB-83), were evaluated in vitro using lymphocytic proliferation and IFN-γ production assays, as well as mRNA and protein expression levels of TNF-α, IL-12p40, and IL-2 as pro-inflammatory molecules. C1 showed the highest significant induction of pro-inflammatory molecules as compared to other cocktails, yet still significantly lower than that induced by conventional PPD. Interestingly, inclusion of the synthetic Mycobacterium tuberculosis 19-kDa lipoprotein (Pam3Cys-SSNKSTTGSGETTTA) as a TLR-stimulant resulted in obvious augmentation of C1-induced pro-inflammatory molecules to levels comparable to that of PPD. In addition, skin testing using sensitized guinea pig model revealed comparable significant reaction to that of conventional PPD. ESAT-6/CPF-10/MPB-83 cocktail is suggested as a potential alternative skin-testing reagent when used in combination with the M. tuberculosis 19-kDa lipoprotein as a TLR-stimulant.

Structure and function of membrane proteins encapsulated in a polymer-bound lipid bilayer.

New technologies for the purification of stable membrane proteins have emerged in recent years, in particular methods that allow the preparation of membrane proteins with their native lipid environment. Here, we look at the progress achieved with the use of styrene-maleic acid copolymers (SMA) which are able to insert into biological membranes forming nanoparticles containing membrane proteins and lipids. This technology can be applied to membrane proteins from any host source, and, uniquely, allows purification without the protein ever being removed from a lipid bilayer. Not only do these SMA lipid particles (SMALPs) stabilise membrane proteins, allowing structural and functional studies, but they also offer opportunities to understand the local lipid environment of the host membrane. With any new or different method, questions inevitably arise about the integrity of the protein purified: does it retain its activity; its native structure; and ability to perform its function? How do membrane proteins within SMALPS perform in existing assays and lend themselves to analysis by established methods? We outline here recent work on the structure and function of membrane proteins that have been encapsulated like this in a polymer-bound lipid bilayer, and the potential for the future with this approach. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.

A Targeted Mass Spectrometry Assay for Detection of HIV Gag Protein Following Induction of Latent Viral Reservoirs.

During early infection, HIV-1 establishes a reservoir of latently infected cells that persist during antiretroviral therapy. These reservoirs are considered the primary obstacle to eradicating HIV-1 from patients, and multiple strategies are being investigated to eliminate latently infected cells. Measuring the reservoir size using an affordable and scalable assay is critical as these approaches move into clinical trials: the current "gold-standard" viral outgrowth assay is costly, labor-intensive, and requires large numbers of cells. Here, we assessed whether selective reaction monitoring-mass spectrometry (SRM-MS) is sufficiently sensitive to detect latent HIV reservoirs following reactivation of virus. The Gag structural proteins were the most abundant viral proteins in purified virus and infected cells, and tractable peptides for monitoring Gag levels were identified. We then optimized a Gag immunoprecipitation procedure that permitted sampling of more than 107 CD4+ T cells, a requirement for detecting exceedingly rare latently infected cells. Gag peptides were detectable in both cell lysates and supernatants in CD4+ T cells infected in vitro at frequencies as low as ∼1 in 106 cells and in cells from HIV-infected patients on suppressive antiretroviral therapy with undetectable viral loads. To our knowledge, this represents the first detection of reactivated latent HIV reservoirs from patients without signal amplification. Together, these results indicate that SRM-MS is a viable method for measuring latent HIV-1 reservoirs in patient samples with distinct advantages over current assays.

CD4+ memory stem cells are infected by HIV-1 in a manner regulated in part by SAMHD1 expression.

UNLABELLED: CD4(+) and CD8(+) memory T cells with stem cell-like properties (T(SCM) cells) have been identified in mice, humans, and nonhuman primates and are being investigated for antitumor and antiviral vaccines and immunotherapies. Whether CD4(+) T(SCM) cells are infected by human immunodeficiency virus (HIV) was investigated by using a combination HIV reporter virus system in vitro and by direct staining for HIV p24 antigen ex vivo. A proportion of T(SCM) cells were found to express the HIV coreceptors CCR5 and CXCR4 and were infected by HIV both in vitro and in vivo. Analysis of viral outcome following fusion using the combination reporter virus system revealed that T(SCM) cells can become productively or latently infected, although the vast majority of T(SCM) cells are abortively infected. Knockdown of the HIV restriction factor SAMHD1 using Vpx-containing simian immunodeficiency virus (SIV) virion-like particles enhanced the productive infection of T(SCM) cells, indicating that SAMHD1 contributes to abortive infection in these cells. These results demonstrate that CD4(+) T(SCM) cells are targets for HIV infection, that they become productively or latently infected at low levels, and that SAMHD1 expression promotes abortive infection of this important memory cell subset. IMPORTANCE: Here we demonstrate the susceptibility of CD4(+) memory stem cells (T(SCM) cells) to infection by HIV in vitro and in vivo, provide an in-depth analysis of coreceptor expression, demonstrate the infection of naïve and memory CD4(+) T cell subsets with both CCR5- and CXCR4-tropic HIV, and also perform outcome analysis to calculate the percentage of cells that are productively, latently, or abortively infected. Through these outcome studies, we determined that the vast majority of T(SCM) cells are abortively infected by HIV, and we demonstrate that knockdown of SAMHD1 significantly increases the frequency of infection of this CD4(+) T cell subset, indicating that SAMHD1 is an active restriction factor in T(SCM) cells.

The histone deacetylase inhibitor vorinostat (SAHA) increases the susceptibility of uninfected CD4+ T cells to HIV by increasing the kinetics and efficiency of postentry viral events.

UNLABELLED: Latently infected cells remain a primary barrier to eradication of HIV-1. Over the past decade, a better understanding of the molecular mechanisms by which latency is established and maintained has led to the discovery of a number of compounds that selectively reactivate latent proviruses without inducing polyclonal T cell activation. Recently, the histone deacetylase (HDAC) inhibitor vorinostat has been demonstrated to induce HIV transcription from latently infected cells when administered to patients. While vorinostat will be given in the context of antiretroviral therapy (ART), infection of new cells by induced virus remains a clinical concern. Here, we demonstrate that vorinostat significantly increases the susceptibility of CD4(+) T cells to infection by HIV in a dose- and time-dependent manner that is independent of receptor and coreceptor usage. Vorinostat does not enhance viral fusion with cells but rather enhances the kinetics and efficiency of postentry viral events, including reverse transcription, nuclear import, and integration, and enhances viral production in a spreading-infection assay. Selective inhibition of the cytoplasmic class IIb HDAC6 with tubacin recapitulated the effect of vorinostat. These findings reveal a previously unknown cytoplasmic effect of HDAC inhibitors promoting productive infection of CD4(+) T cells that is distinct from their well-characterized effects on nuclear histone acetylation and long-terminal-repeat (LTR) transcription. Our results indicate that careful monitoring of patients and ART intensification are warranted during vorinostat treatment and indicate that HDAC inhibitors that selectively target nuclear class I HDACs could reactivate latent HIV without increasing the susceptibility of uninfected cells to HIV. IMPORTANCE: HDAC inhibitors, particularly vorinostat, are currently being investigated clinically as part of a "shock-and-kill" strategy to purge latent reservoirs of HIV. We demonstrate here that vorinostat increases the susceptibility of uninfected CD4(+) T cells to infection with HIV, raising clinical concerns that vorinostat may reseed the viral reservoirs it is meant to purge, particularly under conditions of suboptimal drug exposure. We demonstrate that vorinostat acts following viral fusion and enhances the kinetics and efficiency of reverse transcription, nuclear import, and integration. The effect of vorinostat was recapitulated using the cytoplasmic histone deacetylase 6 (HDAC6) inhibitor tubacin, revealing a novel and previously unknown cytoplasmic mechanism of HDAC inhibitors on HIV replication that is distinct from their well-characterized effects of long-terminal-repeat (LTR)-driven gene expression. Moreover, our results suggest that treatment of patients with class I-specific HDAC inhibitors could induce latent viruses without increasing the susceptibility of uninfected cells to HIV.

Central memory CD4+ T cells are preferential targets of double infection by HIV-1.

BACKGROUND: Template switching between two distinct HIV-1 RNA genomes during reverse transcription gives rise to recombinant viruses that greatly expand the genetic diversity of HIV-1 and have adverse implications for drug resistance, immune escape, and vaccine design. Virions with two distinct genomes are produced exclusively from cells infected with two or more viruses, or 'doubly infected' cells. Previous studies have revealed higher than expected frequencies of doubly infected cells compared to frequencies based on chance alone, suggesting non-random enhancement of double infection. METHODS: We investigated double infection of unstimulated primary CD4+ T cells using reporter viruses carrying genes for different fluorescent proteins, EGFP and mCherry, combined with sophisticated modeling techniques based on Poisson distribution. Additionally, through the use of multiparameter flow cytometry we examined the susceptibility of naïve and memory subsets of CD4+ T cells to double infection by HIV. RESULTS: Using our double infection system, we confirm non-random enhancement of multiple infection events. Double infection of CD4+ T cells was not found to be a consequence of suboptimal provirus expression rescued by Tat in trans-as has been reported in cell lines-but rather due to a heterogeneous cell population in which only a fraction of primary peripheral blood CD4+ T cells are susceptible to HIV infection regardless of viral titer. Intriguingly, double infection of CD4+ T cells occurred preferentially in memory CD4+ T cells-particularly the central memory (TCM) subset-but was not a consequence of SAMHD1-mediated restriction of HIV infection in naïve cells. CONCLUSIONS: These findings reveal that double infection in primary CD4+ T cells is primarily a consequences of cellular heterogeneity and not rescue of suboptimal provirus expression by Tat in trans. Additionally, we report a previously unappreciated phenomenon of enhanced double infection within primary TCM cells and suggest that these long-lived cells may serve as an archive that drive ongoing viral recombination events in vivo.

Some common fixed point theorems in complex valued b-metric spaces.

Azam et al. (2011), introduce the notion of complex valued metric spaces and obtained common fixed point result for mappings in the context of complex valued metric spaces. Rao et al. (2013) introduce the notion of complex valued b-metric spaces. In this paper, we generalize the results of Azam et al. (2011), and Bhatt et al. (2011), by improving the conditions of contraction to establish the existence and uniqueness of common fixed point for two self-mappings on complex valued b-metric spaces. Some examples are given to illustrate the main results.

Ultrasonic-assisted extraction to enhance the recovery of bioactive phenolic compounds from Commiphora gileadensis leaves.

The "ultrasonic-assisted extraction (UAE)" method was utilized in this work to assess how different process parameters affected the yield and recovery of phenolic compounds from the leaf of Commiphora gileadensis, which is one of the medicinal plants with a variety of biological functions. Its leaf is used for a various of therapeutic applications, such as the treatment of bacterial infections, inflammation, and wound healing. The "One-Factor-At-a-Time (OFAT)" approach was employed to examine the impacts of various UAE process parameters on the process of extraction, which include time of extraction, sample/solvent ratio, ultrasonic frequency, and solvent (ethanol) concentration. The extracts were then investigated for the presence of several phytochemicals using analytical techniques such as "Gas Chromatography-Mass Spectroscopy (GC-MS)" and "Fourier Transform Infrared Spectroscopy (FTIR)" studies. The findings showed that the maximum extraction yield, the total phenolic content (TPC), and the total flavonoids content (TFC) of the ethanolic extract of the leaves of C. gileadensis using the UAE method were at 31.80 ± 0.41 %, 96.55 ± 2.81 mg GAE/g d.w. and 31.66 ± 2.01 mg QE/g d.w. accordingly under a procedure duration of 15 min, ultrasonic frequency of 20 kHz, solvent/sample ratio of 1:20 g/mL, and solvent concentration of 40 % v/v. The leaves extract of C. gileadensis included 25 phenolic compounds that were previously unreported, and GC-MS analysis confirmed their presence. Hence, it follows that the UAE technique can successfully extract the phytochemicals from C. gileadensis for a variety of therapeutic uses.

The hydrolysis kinetics of monobasic and dibasic aminoalkyl esters of ketorolac.

Six aminoethyl and aminobutyl esters of ketorolac containing 1-methylpiperazine (MPE and MPB), N-acetylpiperazine (APE and APB) or morpholine (ME and MB), were synthesized and their hydrolysis kinetics were studied. The hydrolysis was studied at pH 1 to 9 (for MPE, APE and ME) and pH 1 to 8 (for MPB, APB and MB) in aqueous phosphate buffer (0.16 M) with ionic strength (0.5 M) at 37°C. Calculation of k(obs), construction of the pH-rate profiles and determination of the rate equations were performed using KaleidaGraph® 4.1. The hydrolysis displays pseudo-first order kinetics and the pH-rate profiles shows that the aminobutyl esters, MPE, APB and MB, are the most stable. The hydrolysis of the ethyl esters MPE, APE and ME, depending on the pH, is either fast and catalyzed by the hydroxide anion or slow and uncatalyzed for the diprotonated, monoprotonated and nonprotonated forms. The hydrolysis of the butyl esters showed a similar profile, albeit it was also catalyzed by hydronium cation. In addition, the hydroxide anion is 105 more effective in catalyzing the hydrolysis than the hydronium cation. The hydrolysis pattern of the aminoethyl esters is affected by the number and pKa of its basic nitrogen atoms. The monobasic APE and ME, show a similar hydrolysis pattern that is different than the dibasic MPE. The length of the side chain and the pKa of the basic nitrogen atoms in the aminoethyl moiety affect the mechanism of hydrolysis as the extent of protonation at a given pH is directly related to the pKa.

Effectiveness of coating acrylic resin dentures on preventing Candida adhesion.

PURPOSE: The aim of this study was to prevent the adhesion of C. albicans on acrylic resin dentures by modifying their surfaces. MATERIALS AND METHODS: Ninety acrylic resin plates were divided into three groups. Group I: conventionally processed acrylic resin plates. Group II: plates painted with 2-Octyl Cyanoacrylate adhesive. Group III: plates painted with Adper Single Bond Adhesive. All specimens were immersed separately in containers filled with artificial saliva that contained C. albicans and then incubated for 11 days at 37°C. Three methods of evaluation were used to count the adhered Candida: direct culture, slide count, and serial dilutions. RESULTS: C. albicans in 1/10, 1/10², and 1/10³ dilutions showed overgrowth in group I, while overgrowth was noted only with 1/10 dilution in group III. For group III, mean colony numbers of 123, 22, 3.4, and 0 were found for the 1/10², 1/10³, 1/104, and 1/105 dilutions, respectively. Regarding the slide counts, group I showed a mean fungal count of 166 compared to 40 for group III with 1/10 dilution, 21 compared to 9 with 1/10³ dilution, 8.6 compared to 0.7 with 1/10³ dilution, and 1.2 compared to 0 with 1/104 dilution. No plates in group II showed any candidal colonies regardless of the method of evaluation (0%). These differences were statistically significant (p < 0.0001). CONCLUSION: Coating the acrylic resin dentures with Adper Single Bond Adhesive was effective in reducing C. albicans adhesion to dentures, while coating with 2-Octyl Cyanoacrylate adhesive completely inhibited such adhesion.

EFFECT OF PHARMACIST COUNSELING ON PATIENT MEDICATION COMPLIANCE AND HELICOBACTER PYLORI ERADICATION AMONG JORDANIAN OUTPATIENTS.

BACKGROUND: To examine the impact of pharmacist counseling and follow-up on patient's medication compliance and Helicobacter Pylori (H. pylori) eradication and evaluate the efficiency of an eradication regimen consisting of Clarithromycin 500 mg, Amoxicillin 1 g, and Lansoprazole 30 mg, twice daily for 14 days. METHODS: Two hundred patients undergoing endoscopy and positive rapid urease tests were included in the present study. Patients were randomly divided into two groups: an intervention group (n=100) and a control group (n=100). The intervention patients obtained their medications from the hospital pharmacist and received sufficient counseling and follow-up. On the other hand, the control patients received their medications from another hospital pharmacist and went through the routine hospital procedure without good counseling and follow-up. RESULTS: The intervention resulted in a statistically significant improvement in outpatient compliance with medication (45.0% vs 27.5%; P<0.05) and eradication of H. pylori (28.5% vs 42.5%; P<0.05) among those patients. CONCLUSION: This study reflects the importance of pharmacist counseling and patient compliance to medication, as the patients who received pharmacist counseling exhibited perfect compliance to medication, which led to the successful eradication of H. pylori.