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Yousef M Al-saraireh,1 Fatemah OFO Alshammari,2 Ahmed MM youssef,3 Sameeh Al-Sarayreh,4 Ghadeer H Almuhaisen,5 Nedal Alnawaiseh,6 Jehad M Al-Shuneigat,4 Hamzeh M Alrawashdeh7 1Department of Pharmacology, Faculty of Medicine, Mutah University, Al-Karak, Jordan; 2Department of Medical Laboratory Technology, Faculty of Health Sciences, The Public Authority for Applied Education and Training, Shuwaikh, Kuwait; 3Department of Pharmacology, Faculty of Pharmacy, Mutah University, Al-Karak, Jordan; 4Department of Biochemistry and Molecular Biology, Faculty of Medicine, Mutah University, Al-Karak, Jordan; 5Department of Microbiology and Pathology, Faculty of Medicine, Mutah University, Al- Karak, Jordan; 6Department of Public Health, Faculty of Medicine, Mutah University, Al-Karak, Jordan; 7Department of Ophthalmology, Sharif Eye Centers, Irbid, JordanCorrespondence: Yousef M Al-sarairehDepartment of Pharmacology, Faculty of Medicine, Mutah University, PO. Box: 7, Al-Karak, 61710, JordanTel +962799172658Email Yousef.sar@mutah.edu.joPurpose: Colon cancer is a leading cause of mortality worldwide. It has a relatively poor prognosis; therefore, new therapies are needed. One of the tumour-related enzymes that has gained considerable interest is CYP4Z1. This enzyme has been expressed in many tumours and has been hypothesized as a potential biomarker or target for novel anticancer therapies.Patients and Methods: CYP4Z1 overexpression was immunohistochemically examined in a large panel of colon tissue types including normal, benign, primary and metastatic ones, and the enzyme’s relation to histopathological features and patient survival was evaluated.Results: A high CYP4Z1 expression was observed in benign, primary and metastatic colon tissues compared to a weak or lack of expression in normal tissues. Importantly, there was a significant differential in CYP4Z1 expression where it was stronger in metastatic, primary and benign, respectively (p < 0.05). A significantly high rate of CYP4Z1 expression was found in high histological grades and late stages of the disease, where its expression was more evident in patients with metastasis in the lymph nodes (p < 0.05). Interestingly, CYP4Z1 expression was identified an independent prognostic predictor of poor overall survival of colon cancer patients (p = 0.003).Conclusion: CYP4Z1 was distinctly overexpressed in benign, primary and metastatic colon tissues compared to corresponding normal tissues. This differential in CYP4Z1 expression across different types of colon tissues strongly supports CYP4Z1 as potential biomarker and target for novel anticancer therapy development.Keywords: cancer, colon cancer, cytochrome P450, cytochrome 4Z1, immunohistochemistry

BACKGROUND Drug resistance and failure of antibiotics to treat bacterial infections is the most challenging clinical and public health problem. There is ongoing research for new, safe and effective alternatives to antibiotics for the therapeutic management of bacterial infections. Essential oils are one of the outcomes of such type of research. This study was conducted to evaluate the effect of Artemisia judaica essential oil against the bacterial & the biofilm activity and to study its mode of action. METHODS Minimum biofilm inhibitory concentration (MBIC) susceptibility assays were performed using a biofilm inoculator with a 96-well plate with peg lid. Minimum inhibitory concentration (MIC) was performed in normal microtiter plates using a twofold dilution series. RESULTS The values of the minimum inhibitory concentration (MIC), the minimum bactericidal concentration (MBC), minimum biofilm inhibitory concentration (MBIC) and the minimum biofilm eradication concentration (MBEC) were in the range of 0.25 - 1 mg / mL, 1.0 - 4 mg / mL, 0.50 - 2 mg / mL and 1.0 - 8 mg / mL, respectively. There was increase in the leakage of K+ and cellular components through the membrane of the tested bacteria under the effect of essential oil of Artemisia judaica proving that the cell membrane was the site of action of Artemisia judaica. This was shown by its inhibitory effect on the Staphylococcus epidermidis (ATCC 35984) during initial adherence at sub-inhibitory concentrations. CONCLUSIONS The essential oil of Artemisia judaica had the ability to overcome all tested bacterial resistance and also prevents the formation of biofilm. The site of action of Artemisia judaica essential oil was the cell membrane and cell wall. KEY WORDS Antibacterial, Antibiofilm, Artemisia judaica, Mode of Action, Essential Oil

Increased multidrug resistance prompted researchers to search for a new drug that has the ability to overcome antibiotic resistant pathogens. Essential oils have been used in folk medicine for centuries, therefore, they could be employed as an effective alternative to antibiotics without having secondary side effects. The aim of the present study was to test the antibacterial and antibiofilm activity of the essential oil of Achillea santolina and to ascertain its mode of action. Minimum Biofilm Inhibitory Concentration (MBIC) susceptibility assays were performed using a biofilm inoculator with a 96-well plate with peg led. Minimum Inhibitory Concentration (MIC) was performed in normal microtitre plates using a twofold dilution series. Achillea santolina essential oil (ASEO) was able to overcome the resistance of all tested bacteria. The MIC values were in the range of 250-1000 µg/ml, while the MBC values were in the range of 500-2000 µg/ml. ASEO increased leakage of potassium ions from the cell membrane and increased release of cellular materials – suggesting that the cell membrane is the target and site of action of ASEO. Moreover, ASEO was able to inhibit initial adherence of methicillin-resistant Staphylococcus aureus (MRSA) (ATCC 43300) at sub-inhibitory concentrations through alterations to cell membrane.

Yousef M Al-saraireh,1 Fatemah OFO Alshammari,2 Ahmed MM Youssef,3 Fatima Al-Tarawneh,4 Sameeh Al-Sarayreh,5 Ghadeer H Almuhaisen,6 Anas O Satari,7 Jehad Al-Shuneigat,5 Hamzeh Mohammad Alrawashdeh8 1Department of Pharmacology, Faculty of Medicine, Mutah University, Al-Karak, Jordan; 2Department of Medical Laboratory Technology, Faculty of Health Sciences, The Public Authority for Applied Education and Training, Shuwaikh, Kuwait; 3Department of Pharmacology, Faculty of Pharmacy, Mutah University, Al-Karak, Jordan; 4Department of Allied Medical Sciences, Faculty of Al-Karak, Al-Balqa Applied University, Al-Karak, Jordan; 5Department of Biochemistry and Molecular Biology, Faculty of Medicine, Mutah University, Al-Karak, Jordan; 6Department of Microbiology and Pathology, Faculty of Medicine, Mutah University, Al-Karak, Jordan; 7Faculty of Medicine, Mutah University, Al-Karak, Jordan; 8Department of Ophthalmology, Sharif Eye Centers, Irbid, JordanCorrespondence: Yousef M Al-sarairehDepartment of Pharmacology, Faculty of Medicine, Mutah University, P.O. Box: 7, Al-Karak, 61710, JordanTel +962 799172658Email Yousef.sar@mutah.edu.joPurpose: Triple-negative breast cancer (TNBC) is characterized by high mortality rate, and its clinical management is difficult and complex. Therefore, there is a need for extensive efforts aimed at accelerating the discovery of novel therapies for TNBC. CYP4Z1 has been implicated in the development of breast cancer. The current study aimed at characterizing the expression of CYP4Z1 on TNBC.Materials and Methods: Using immunohistochemistry, CYP4Z1 expression was evaluated on 122 TNBC samples, four samples of breast cancers expressing ER, PR, and HER-2, and four samples of normal breast tissues. The association between the enzyme and various histopathological features and survival of patients were determined.Results: CYP4Z1 was strongly expressed in 83.3% of various histopathological subtypes of TNBC, when compared to negative expression in normal breast tissues. Interestingly, there were marked variations in CYP4Z1 expression with respect to histopathology subtype, histological grade, histological stage and tumor diameter. There was a high incidence of CYP4Z1 expression in patients with advanced grades, late stages and larger tumor sizes. Importantly, CYP4Z1 expression was correlated with the survival of TNBC patients, but it was an independent determinant of the poor prognosis of TNBC (p< 0.05).Conclusion: CYP4ZI may be a potential biomarker or target for evolving new CYP4Z1-targeted treatments.Keywords: triple-negative breast cancer, cytochrome P450, immunohistochemistry

Background: Insufficient production of thyroid hormones results in hypothyroidism, while overproduction results in hyperthyroidism. These are common adult disorders, with hypothyroidism more common...

Bladder cancer is the tenth most common cancer worldwide, where its burden remains a challenge and needs new novel therapies. Several reports indicate expression of CYP4Z1 and CYP1B1 in many tumours. Their expressions are associated with a poor prognosis, and therefore proposed as promising biomarkers or targets for anticancer therapy. By using immunohistochemistry, expression of CYP4Z1 and CYP1B1 was evaluated in a panel of different types of bladder cancer, and the enzymes’ relation to histopathological features were assessed. Results showed an increased expression of CYP4Z1 (54.3%) and CYP1B1 (76.9%) in the majority of bladder cancers compared to weak or lack of expression of both enzymes in normal tissues. Their expressions were significantly associated with tumour grade and stage where the expressions were markedly increased in a high grade and advanced stage of the disease ( p < 0.05). Additionally, CYP1B1 expression was also associated with TNM staging ( p < 0.05) and its expression was increased in patients with lymph node metastasis. The expression profiles of CYP4Z1 and CYP1B1 suggest that both enzymes have the potential to be biomarkers or targets for novel anticancer therapy for bladder cancer. Nevertheless, further studies are needed to better delineate whether these enzymes are druggable targets.

Bladder cancer is the tenth most common cancer worldwide, where its burden remains a challenge and needs new novel therapies. Several reports indicate expression of CYP4Z1 and CYP1B1 in many tumours. Their expressions are associated with a poor prognosis, and therefore proposed as promising biomarkers or targets for anticancer therapy. By using immunohistochemistry, expression of CYP4Z1 and CYP1B1 was evaluated in a panel of different types of bladder cancer, and the enzymes’ relation to histopathological features were assessed. Results showed an increased expression of CYP4Z1 (54.3%) and CYP1B1 (76.9%) in the majority of bladder cancers compared to weak or lack of expression of both enzymes in normal tissues. CYP4Z1expression was significantly associated with tumour grade and stage where the expression was markedly increased in a high grade and advanced stage of the disease (p p

Background Cytochromes P450 (CYPs) constitute an enzyme family involved in the oxidative metabolism of a wide variety of endogenous and exogenous compounds, including anti-cancer drugs and carcinogens. Unlike other human CYPs, CYP4Z1 is highly expressed in human breast carcinoma and is associated with poor prognosis. As a result, CYP4Z1 was hypothesised to be a potential biomarker or drug target for the discovery and development of promising anti-cancer therapies. Materials and methods CYP4Z1 expression was immunohistochemically studied in a set of 100 different human tissues, including normal, benign, malignant and metastatic tissues, which originated from 27 anatomical sites. As a tumour model for CYP4Z1 expression, a panel of different breast cancers was evaluated for CYP4Z1 expression and its relation to histopathological features and prognostic immunohistochemical markers. Results The immunohistochemical results revealed that CYP4Z1 was expressed in only one (4.3%) of the normal tissues from the mammary glands, while the expression of the enzyme was positive in 1 (11%), 12 (19%) and 2 (40%) of the benign, malignant and metastatic tissues, respectively. Interestingly, several tumour entities showed prominent expressions of CYP4Z1, including carcinomas of adrenal cortex, squamous cells of oesophagus, lung and cervix, as well as seminoma, astrocytoma, melanoma and lastly endometrial adenocarcinoma. In breast cancers, CYP4Z1 was expressed in 82% of the cases. Its expression was significantly associated with the pathology of tumour, histological grade and status of lymph node metastasis. Importantly, it was also significantly associated with the expressions of Her2, P53 and Ki-67. Conclusion These findings greatly support future plans for the use of CYP4Z1 as a biomarker or target for anti-cancer drugs. However, large-scale validation studies are needed to better delineate the potential use of CYP4Z1 for therapeutic purposes.

In this study, the regulation of ascorbate peroxidase (APX) specific activity, anthocyanin, carotenoid, hydrogen peroxide, lipid peroxidation, and protein levels in cress leaves in response to different abiotic stresses were investigated. The total APX specific activity was significantly elevated after 9 days of drought treatment, short-term (2 h) exposure to 10, 100 and 370 µE of light, long-term exposure (at least 6 days) to 100 mM NaCl versus the specific APX activity in the controls. Furthermore, a significant change in total APX activity was detected in response to treatment with different temperatures; this change was an early response to 4 °C and 30 °C for a maximum of 4 h, while short-term exposure to 35 °C did not change total APX activity. The results of the present study revealed that plants have a wide range of mechanisms to cope with different stresses that possibly involve morphological changes. The results indicated that Lepidium sativum plants launch common protective pathways only under drought, salinity and high light stresses, while other protective mechanisms/strategies could be responsible for increasing the plants tolerance towards temperature and low light. Future studies will investigate changes in the photosynthetic quantum yield and specific target metabolites, proteins, and nonenzymatic antioxidants.

Background and Aim: Thrombocytopenia is a frequent problem in patients with post- hepatitis C (HCV) liver cirrhosis and also occurs in chronic HCV-infected patients without liver cirrhosis. The aim of this study was to evaluate the role of plasma thrombopoietin (TPO) in the occurrence of thrombocytopenia in both conditions. Method: Platelet count and plasma thrombopoietin level and liver function tests were measured in four groups of patients: twenty chronic patients with post-hepatitis C liver cirrhosis and thrombocytopenia (group I), ten chronic HCV-positive patients with liver cirrhosis without

Background: There is a high variability in chemical composition of essential oil from Artemisia herba-alba grown in different countries and different localities in the same country. This has led to the characterization of many oil-dependent chemotypes assigned to the plant. Only one report was published on the essential oil composition of Artemisia herba-alba grown in Jordan. Aim: The current study aims to determines the essential oil composition of Artemisia herba-alba grown wild in south Jordan and test their activity against clinical isolate antibiotics resistant bacteria. Methodology: The essential oils were isolated by hydrodistillation and analysed by Gas Chromatography-Mass Spectrometry (GC-MS). The screening for essential oil activity was carried out using disc diffusion method on methicillin-resistant Staphylococcus aureus , methicillin-sensitive

Colonization with methicillin-resistant Staphylococcus aureus (MRSA) increases the risk for subsequent infections with an increased mortality and morbidity. Children were suggested to be a major asymptomatic reservoir for community-associated (CA) MRSA with an ability to quickly spread the MRSA within community. Therefore, the availability of epidemiological and antibiotic susceptibility data of CA-MRSA will be useful for the infection control and management policies. This study aimed to assess the nasal carriage, molecular characteristics and antibiotic susceptibility of MRSA in primary school-aged children from Jordan.A total of 210 nasal swabs were collected from children aged 6-11 years. Isolated MRSA and its SCCmec typing, Spa type and PVL (Panton-Valentine Leukociden) toxin were identified following culture, biochemical and PCR. Antibiogram was determined by the disc diffusion method.The prevalence of CA-MRSA was 7.1%. Allergic rhinitis and recent antibiotic exposure were the only significant risk factors for MRSA nasal carriage among children. Resistance to erythromycin, trimethoprim-sulfamethoxazole and tetracycline was 33.4, 20 and 13.4%, respectively. All isolates were susceptible to the remaining non-β-lactam antibiotics used in this study, in particular linezolid and mupirocin. All MRSA isolates were SCCmec type IV and PVL toxin negative and the majority were Spa type t223.This is the first study to assess the MRSA prevalence among children aged 6-11 years in Jordan. The prevalence in community children is within the range compared with other studies in other countries. The antibiogram, SCCmec and Spa types of the isolated MRSA are much similar to what was found previously in Jordan. However, all isolates were PVL toxin negative. The study recommends increasing the public awareness of MRSA and the proper antibiotics dispensing. Future studies to follow-up on the changing epidemiology of the CA-MRSA in Jordan are also recommended.

Alkaptonuria is a very rare genetic disorder, characterized by a lack of homogentisate dioxygenase and causes accumulation of homogentisic acid. Clinical manifestations include dark urine, dark-black pigmentation of connective tissues (ochronosis), and arthritis of large joints and spine. The disease is usually diagnosed in adulthood by assessment of signs and symptoms of ochronosis. Confirmation of suspected diagnosis can be achieved by quantitative measurements of homogentisic acid levels. In this study, homogentisic acid was analyzed in 17 alkaptonuria patients. After liquid-liquid extraction, the analyte was determined by Gas Chromatography/ Mass Spectrometry (GC-MS) method. For quantitation purposes, external calibration was applied first, regression coefficient of ≥ 0.995 indicated the linearity in the concentration range of 1-100 ng/µl. The instrumental detection limit (IDL) and lower limit of quantitation were 3.82 and 12.7 µg/L, respectively. Recovery rate was ≥ 89%. Precision given as relative standard deviation (RSD) ranged from 3 – 10 %. The results showed that the concentration of homogentisic acid ranged from 0.46 to 1.5 g/24 hours.

Faculty of Medicine, Department of Biochemistry and Molecular Biology, Mutah University, Mutah, Jordan. Faculty of Medicine, Department of Pharmacology, Mutah University, Mutah, Jordan. Faculty of Science, Department of Chemistry, Albalqa' Applied university, Albalqa, Al-Salt, Jordan. Faculty of Science, Department of Chemistry, Yarmouk University, Irbid, Jordan. Faculty of Medicine, Department of Microbiology and Immunology, Mutah University, Mutah, Jordan.

Background: A biofilm is group of bacteria embedded in a self-produced matrix made of polysaccharide and protein. Bacterial biofilms cause chronic infections because they show increased tolerance to antibiotics and disinfectant chemicals as well as resisting phagocytosis. Biofilm bacteria show much greater resistance to antibiotics than their free-living counterparts. Essential oils are a cheap and effective alternative to antibiotics that could be used to combat antibiotics resistant microorganisms. The aim of the present study was to determine the effects of essential oil of wild Thymus vulgaris grown in Jordan on biofilm-forming clinical bacterial isolates. Results: All of the isolates included in this study were able to produce a stable biofilm that adhered to polystyrene microwells. Thymus vulgaris essential oil produced inhibitory effects against all isolates with considerable variation in susceptibility. The minimum inhibitory concentration (MIC) and biofilm inhibitory concentration (BIC) values varied between 0.0625 to 2% v/v. Isolate 6 (Pseudomonas aeruginosa) was the most resistant for both planktonic and biofilm growth while isolate 4 Escherichia coli was the most sensitive. Isolate 6 was the only isolate to exhibit a specific biofilm-related response to Thymus vulgaris essential oil i.e. the minimum Thymus vulgaris essential oil concentration needed to inhibit biofilm adherence was much lower than that required to inhibit planktonic growth. For the remaining isolates, BIC for the biofilm did not differ from the MIC for planktonic. Thymus vulgaris essential oil was able to inhibit Pseudomonas aeruginosa adherence to polystyrene. Conclusions The MIC and BIC values of Thymus vulgaris essential oil show that the essential oil has strong antibacterial and antibiofilm activity. Thymus vulgaris was able to inhibit biofilm formation in the most tolerant isolate at sub-inhibitory concentrations.

There is a high variability in chemical composition of essential oil from Thymus vulgaris grown in different countries and different localities in the same country. Only three reports were published on the essential oil composition of Thymus vulgaris grown in Jordan and non of them reported from South Jordan. The current study aims to determine the essential oil composition of Thymus vulgaris grown wild in South Jordan and to test their activity against clinical isolate antibiotics resistant bacteria. The composition of the essential oil from Thymus vulgaris was determined using GC-MS while the screening for essential oil activity was carried out using disc diffusion method. The results of GC-MS produced forty-eight components of the oil representing more than 97% of the oil contents, monoterpenes being most abundant (about 85%) with thymol (37.05%), cis- dihydrocarvone (9.34%), carvacrol (8.45%), hydroxy-3-(3-methyl-2-butenyl)-3-cyclopenten-1-one (8.41%), p- cymene (5.73%), cis-sabinene (4.42%), z-isoeugenol (3.342%), and aromadendrene (3.42%) as major constituents. The essential oil from Thymus vulgaris was found to be active against all the tested bacteria except in Pseudomonas aeruginosa. The high concentrations of thymol and carvacrol may account for its high activity against resistant bacteria.

Troponin I plays an essential role in the regulation of cardiac muscle contraction. Together with troponin C and T, troponin I induces Ca2+ dependent cooperative transitions of thin filaments between a blocked, a closed and an open state. 29 mutations were found in cardiac troponin I in families with hypertrophic cardiomyopathy. Although unknown, the mechanism of molecular dysfunction is likely to involve an aberrant thin filament responsiveness to changes in intracellular level of Ca2+. Our hypothesis is that these mutations modify important parameters in the cooperative-allosteric transitions of thin filaments. Here we aimed at using transient kinetics to assess the effect of hypertrophic cardiomyopathy TnI mutations (Q130R, R145G, and A157V) on the rate and equilibria of thin filament switching between the blocked, closed and open states. We found that TnIQ130R and TnIA157V did not affect the equilibrium constant between the blocked and the closed states (KB). In contrast TnIR145G substantially increased KB in the absence of Ca2+. An increase in KB is likely to lead to incomplete relaxation. We also investigated the effect of these mutations on the cooperative behaviour of thin filaments. TnIQ130R and A157V did not affect the size of the cooperative unit n while TnIR145G decrease n value to less than 7. Calcium binding to thin filaments was monitored by change in the fluorescence of IAANS-TnCC84S. Thin filaments reconstituted with TnI mutations showed a change in calcium affinity and the rate of Ca2+ dissociation. These findings suggest that mutations in different regions of troponin I are likely to have different biochemical effect highlighting the unique molecular mechanism for each of these mutations.

Data regarding the levels of inflammatory mediators in patients suffering of the rare disease Alkaptonuria are limited. C-reactive protein (CRP), Interleukin-1 Beta (IL-1β) and Interleukin-6 (IL-6) are acute-phase markers associated with joint inflammation. The aim of the present study is to compare the serum levels of the pro-inflammatory mediators, CRP, IL-1β and IL-6 in alkaptonuria patients (n = 17) with those measured for age-matched healthy controls (n = 17). Moreover, we attempt to determine the association between cytokine levels with the disease severity score and age using the Spearman correlation and multiple linear regression. The results show that the serum concentrations of the IL-1 β, IL-6 and CRP are higher in AKU patients compared with healthy controls, with a significant difference in IL-6 (p = 0.02). Moreover, a positive correlation is found between the patients' serum IL-6 and patients' age and the AKU Severity Score ( r o = 0.73 and 0.7, respectively; p < 0.05). Thus, the patients' IL-6 serum levels can predict the disease severity score in alkaptonuria patients (p < 0.05). These findings suggest that the IL-6 might play a role in the pathogenesis of inflammation in AKU patients and thus targeting it may be one mode of treatment in future. However, these findings need to be supported by further studies, conducted on a larger sample of patients.

Calponin is an actin- and calmodulin-binding protein believed to regulate the function of actin. Low-resolution studies based on proteolysis established that the recombinant calponin fragment 131-228 contained actin and calmodulin recognition sites but failed to precisely identify the actin-binding determinants. In this study, we used NMR spectroscopy to investigate the structure of this functionally important region of calponin and map its interaction with actin and calmodulin at amino-acid resolution. Our data indicates that the free calponin peptide is largely unstructured in solution, although four short amino-acid stretches corresponding to residues 140-146, 159-165, 189-195, and 199-205 display the propensity to form α-helices. The presence of four sequential transient helices probably provides the conformational malleability needed for the promiscuous nature of this region of calponin. We identified all amino acids involved in actin binding and demonstrated for the first time, to our knowledge, that the N-terminal flanking region of Lys(137)-Tyr(144) is an integral part of the actin-binding site. We have also delineated the second actin-binding site to amino acids Thr(180)-Asp(190). Ca(2+)-calmodulin binding extends beyond the previously identified minimal sequence of 153-163 and includes most amino acids within the stretch 143-165. In addition, we found that calmodulin induces chemical shift perturbations of amino acids 188-190 demonstrating for the first time, to our knowledge, an effect of Ca(2+)-calmodulin on this region. The spatial relationship of the actin and calmodulin contacts as well as the transient α-helical structures within the regulatory region of calponin provides a structural framework for understanding the Ca(2+)-dependent regulation of the actin-calponin interaction by calmodulin.