Datta Meghe Institute of Medical Sciences University

Role of adenosine deaminase in pleural fluid in tubercular pleural effusion Suchita Dilip Modi, Anil Kalyandas Agrawal, Arvind S Bhake, Vikas R Agrawal Journal of Datta Meghe Institute of Medical Sciences University 2018 13(4):163-167 Background: Each year approximately 1 million patients develop the pleural effusion. Tuberculosis (TB), the single most frequent infectious cause of death worldwide, also is one of the leading causes of pleural effusion. Despite the advent of novel improvements in the diagnosis and treatment of pulmonary TB, a definite diagnosis of TB in a significant number of patients is problematic. Measurement of the level of adenosine deaminase (ADA) enzyme in body fluids is a helpful diagnostic tool. The level of ADA elevates as the lymphocyte (T-cell) activity increases. The present study was carried out to evaluate the ADA activity in pleural fluid for early diagnosis and management of tubercular pleural effusion (TPE). Objectives: The main objective is to determine the tubercular pleural fluid ADA level and its sensitivity and specificity. Materials and Methods: The prospective analytical study was conducted at Acharya Vinobha Bhave Rural Hospital, Sawangi (Meghe), in 105 patients of suspected case of TPE. A structured proforma was used for documenting the history, symptoms, side of effusion, hemogram and erythrocyte sedimentation rate (ESR), pleural fluid findings which included pH, glucose, proteins, lactate dehydrogenase (LDH), total leukocyte count (TLC) and differential leukocyte counts and serum ADA level was also calculated. Results: ADA sensitivity, specificity, positive predictive value, and negative predictive value (NPV) are 89.47%, 48.28%, 81.93%, and 63.65%, respectively, for TB. Pleural fluid ADA activity has been shown to be a valuable biochemical marker that has a high sensitivity for TB diagnosis. Conclusion: Pleural fluid ADA is markedly raised in tubercular effusions. ADA estimation is a simple, reliable, cost-efficient, and diagnostically useful investigation.

Knowledge of Anganwadi worker with respect to early childhood development Shashank Gotarkar, Abhishek Ingole Journal of Datta Meghe Institute of Medical Sciences University 2018 13(4):168-170 Background: Being world's largest outreach program targeting infants & children below six years of age, expectants & nursing mothers, ICDS has generated interest worldwide amongst academician, planners, policy makers, administrators & those responsible for implementation. Consequently, many research studies have been conducted to evaluate & assess impact of program. The need for revitalization of ICDS has already been recommended toward better maternal and child health especially in rural areas. Inspite of repeated measures to enhance effectivity of ICDS program, impact is not observed as desired by policy makers or administrators. And hence knowledge of AWWs regarding ICDS program can be assessed & evaluated so that some of attributable reasons may be pointed out. Hence present study was undertaken to find out knowledge of AWW. Aim & Objectives: The AIM is to find out knowledge of Anganwadi Workers under ICDS with regards to ECD. Materials and Methods: This was a community based Cross-Sectional study, carried out at Seloo block of Wardha district, having total under five population approx. 7634. Study participants included 167 AWWs were included in the study. The study period was one year. Data were double entered and analysed in Microsoft excel (2007). The quantitative data was presented as proportion or mean with SD. Chi square test of significance was used wherever appropriate. Results: Shows that maximum no. of AWW (37.72%) were non matriculate. AWW with SSC and HSC qualification were 22.76% & 29.34% respectively. 64% AWW were Trained workers, 19% AWW received revised training while 17% were untrained workers. Conclusions: AWWs are actually the main resource person of the program and whose knowledge and skills do have a direct impact on the implementation of the programme. As the Anganwadi Workers play an important role due to their close and continuous contact with the people of community, especially the children and women, so there is an utmost need to assess their knowledge and the level of awareness regarding services provided by them in Anganwadi Centres.

Psychological pain as predictor of impulse control among BAMS new entrants: A correlation study Rupali Dhananjay Sarode, Vaishali Dinesh Tendolkar Journal of Datta Meghe Institute of Medical Sciences University 2018 13(4):171-174 Background: Emotional discomfort in health professional students many times results in mental health-related problem in the future. First-year students face emotionally stormy transitional phase which leads to more psychological pain. Impulse control (patience) is considered as an absolute virtue of health professionals. Health professional students are expected to possess good impulse control for they come across varied natured people with diverse health problems in different stages of severity. Objectives:(1) The primary objective of this study is to identify the relationship between psychological pain and impulse control among new entrants studying in BAMS course. Research Design: This was correlation study. Population: First-year students both male and female from 1st year BAMS course. Sample Size: 100, (50 males and 50 females). Setting: Ayurveda Science Colleges in Nagpur and Wardha districts. Materials and Methods: Orbach Mikulincer Mental Pain Scale and Impulse control scale by Shrivastava and Naydoo. Results: Psychological pain and impulse control among male new entrants show a strong negative nonsignificant correlation (r = −0.475, P > 0.05), and for females, the correlation is statistically significant weak-positive (r = 0.181, P < 0.05). Together male and female new entrants have a negative statistically significant correlation (r = −0.363, P < 0.05). Conclusion: The findings suggest that higher the psychological pain, lower is the impulse control for both male and female new entrants of Ayurveda Science discipline. Male students show the greater magnitude of negative relation as compared to that of female students.

Retrospective analysis of intracranial and intraspinal space-occupying lesions at a tertiary care center: A 5-Year study Meena B Patil, Manjiri N Karandikar Journal of Datta Meghe Institute of Medical Sciences University 2018 13(4):175-182 Background: Incidence of central nervous system (CNS) tumors is 2% of all malignancies. Low-grade tumors progress to high-grade tumors. CNS is a common site for metastasis from other organs. Aims: The aim is to study the incidence of various lesions in light of the World Health Organization (WHO) classification of tumors of the CNS, 2016, and to study relevant statistics. Objectives: The objective of this study is to provide an overview of intracranial and intraspinal space-occupying lesions at a single tertiary care referral center. Materials and Methods: A total of 124 cases were studied. Intraoperative diagnosis was desired in 70 cases. The final diagnoses in all the cases were made by routine histopathology. Results: Neoplastic lesions comprised 83.88%, including metastatic tumors, while 16.12% were nonneoplastic. Classification and grading of CNS tumors was done according to the WHO, 2016. Astrocytic tumors constituted the largest category. Incidence was more in females than males and in the fifth and sixth decades of life. Conclusion: This study highlights histological diversity of CNS tumors in adults and children. The most common destructive but nonneoplastic lesions such as infections/abscesses, infarcts, and cysts can be diagnosed, by crush cytology and frozen section for the definitive management.

Oral health-related quality of life of institutionalized elderly in Satara District, India KM Shivakumar, Snehal Patil, Vidya Kadashetti, Vaishali Raje Journal of Datta Meghe Institute of Medical Sciences University 2018 13(4):183-189 Context: Almost in every country, the proportion of people aged over 60 years is growing rapidly than any other age group as a result of both longer life expectancy and declining fertility rates. Aims: The aim of this study is to assess the oral health status, oral health-related quality of life (OHRQoL), and the dental care utilization of institutionalized and homebound elderly in Satara district, India. Materials and Methods: A descriptive cross-sectional study was conducted among the 150 institutionalized elderly participants of Satara district. Type 3 clinical examination was done to detect dental caries, missing tooth, periodontal diseases, and any other oral findings followed by interview with elderly patients. A questionnaire was administered to assess OHRQoL according to the Geriatric Oral Health Assessment Index (GOHAI), dental visiting pattern, and sociodemographic and self-perceived oral health status of elderly. The descriptive and analytic statistics were used to analyze the data; Chi-square test or Fisher's exact test was performed to determine the prevalence of poor OHRQoL by independent variables. Results: Poor OHRQoL according to GOHAI was observed among 64.7% of the men and 67.1% of the women, and there was no statistically significant difference observed. In community periodontal index, 42.6% of men and 36.6% of women had all sextants excluded, and of those remaining, 95.6% of men and 96.3% of women needed one or other periodontal treatment. Regarding decayed, missing, and filled teeth index, mean was 22.5 in men and 24.2 in women. Conclusion: Institutionalized elderly with 65 years and older showed higher prevalence of poor oral health status and unmet needs for dental care that were associated with poor OHRQoL. Therefore, it is necessary to improve health and social importance of oral health care for elderly.

Significance of differentiation of round cell count in semen in infertile men: A cost-effective study Seema Balasubramaniam, Vishrabdha Rahul Pawar Journal of Datta Meghe Institute of Medical Sciences University 2018 13(4):190-194 Background: The significance of the presence of immature germ cells (IGCs) and leukocytes in semen have not very frequently been highlighted in the diagnostic criteria of male infertility. Round cell count differentiation aids in better clinical correlation. Aim and Objectives: To differentiate the round cells into IGCs and leukocytes in the semen and to find their correlation with the total sperm count and sperm motility. Materials and Methods: Semen samples from patients at Bharati hospital, Sangli for evaluation of infertility from July 2014 to January 2015 were analyzed as per the WHO criteria. After the consent and collection of the semen sample, routine semen analysis was done. Smears of semen were prepared and stained by Leishman stain. The round cells were counted and differentiated into IGCs and leukocytes. Results: We studied 103 cases. Semen parameters such as total sperm count, motility were noted. The smears were studied for differential counts of round cell count into IGC count and leukocyte. The cases were divided into four groups based on total sperm count. Maximum number of cases was found in group III. Our study shows that round cell count is 5%–10% of the total sperm count. Negative correlation was noted between the total sperm count and IGCs. The correlation of total sperm count and leukocytes was negative and significant. Conclusion: The present study shows that differentiation of round cells in the semen can be effectively differentiated into spermatogenic and nonspermatogenic cells with the use of simple and cost-effective stain like Leishman stain. The correlation of sperm count with the IGCs and leukocytes and sperm motility is of immense use to find out the possible cause of infertility.

Association between C-reactive protein and interleukin-6 levels in subclinical hypothyroid patients Satya Devi Uppu, Madhur Gupta Journal of Datta Meghe Institute of Medical Sciences University 2018 13(4):195-198 Background: The relationship between subclinical hypothyroidism (SCH) and cardiovascular disease has been one of the most popular topics recently. Our study aims to investigate the presence of the well-known preceding clinical situations of atherosclerosis such as endothelial dysfunction and inflammation in SCH. Materials and Methods: The study population comprised 50 patients with recently diagnosed SCH and 50 healthy controls. Thyroid-stimulating hormone (TSH), free triiodothyronine (FT3), and free thyroxine (FT4) were estimated by enzyme-linked fluorescent immunoassay for the diagnosis of SCH. Total cholesterol (TC), triglycerides, and high-density lipoprotein cholesterol (HDLC) were estimated by kit method in Siemens machine. Low-density lipoprotein cholesterol (LDLC) and very LDLC were calculated using Friedewald formula. Inflammatory marker C-reactive protein was estimated by kit method in Siemens and IL-6 by enzyme-linked immunosorbent assay. Results: In this study, the level of TSH ± standard deviation (8.56 ± 1.26 vs. 0.63 ± 0.16) was significantly higher (<0.001) in SCH patients, whereas FT3and FT4levels were within the normal range. TC, triglycerides, and LDLC were significantly higher in patients group, while the HDLC was significantly lower in SCH patients compared to euthyroid group. TSH level was positively correlated with inflammatory markers in SCH. Conclusion: This study suggests that SCH patients have increased inflammatory markers along with dyslipidemia due to that the future risk of further development of cardiovascular disorder can occur. Level of inflammatory makers increases in patients as disease progresses if left untreated.

System Analysis of Biomedical Waste Management Across Health Care Clinics of Udupi Taluk Greeshma Tony, Naveen Kumar, Brayal Dsouza, Rajesh Kamath, Sagarika Kamath Journal of Datta Meghe Institute of Medical Sciences University 2018 13(4):199-201 Background: Improper Biomedical Waste (BMW) management by healthcare facilities can pose severe hazardous effects on the larger population, the environment, and its resources. Due to resurgence in number of clinics in urban areas, there is need to understand the practices and compliance to BMW management. It can be understood by studying and observing their practices and processes of BMW management and understanding the knowledge, attitude, and practice among the staff in the clinics. Aim: The objective of the study was to analyze the BMW management process across the healthcare clinics in Udupi taluk. Methods: A cross-sectional, observational study was conducted using self-administered questionnaires and checklists across 15 clinics in Udupi taluk. The sample size is 130 out of which Doctors, Nurses, housekeeping staff and descriptive Statistics were reported. Results: Of the 15 clinics only eight of them had basic requirements of BMW management; lack of trained workforce, non-compliance to segregation of waste, and non-compliance to collection and storage of waste are seen among majority of the clinics. Other findings during our study are 73.3% had inadequate transportation facility, while only 13.3% had adequate storage space. Doctors and nurses had 100% knowledge and positive attitude, environmental health workforce (housekeeping staff) had poor knowledge and attitude.

The Efficacy of Tamsulosin as Medical Expulsion Therapy in Ureteric Calculus of <8 mm Size Nagesha Shivanna, Abhishek Kumar Singh, Prateek Jugalkishore Laddha Journal of Datta Meghe Institute of Medical Sciences University 2018 13(4):202-205 Introduction: Many conservative medical therapies have been researched and studied for ureteric stones. Alpha 1- Adrenergic blocker Tamsulosin reduces muscle spasm in the ureteric wall, decreases peristalsis below and raises pressure above the stone thus facilitating stone passage. Material and Methods: Patients diagnosed with ureteric stones from OPD, Casualty/Emergency Room and wards were included in the study. The patients were randomized into the study and the control arms. This was a prospective, randomized, controlled, open label clinical trial conducted on patients presenting to tertiary care center in north India with ureteric colic. Study group received Tamsulosin along with the analgesics, whereas the control group received only symptomatic treatment and hydration. Results: There were 29 (52.7&#37;) subjects in the study group and 25 (45.4&#37;) subjects in the control group had a subjective score of over 9 on arrival. The minimum pain score at presentation was 4 in the study group whereas it was 5 in the control group. Majority of the patients received Inj. Morphine after the initial Inj. Diclofenac sodium, 33 (60&#37;) in the study group and 36 (65.45&#37;) from the control group. There were 12 and 11 in the study and the control group who received an additional morphine infusion pump for pain control. The mean number of colic episodes during treatment was 1.75 (SD&#61;1.534) in study group which was marginally less when compared to that of the control group 1.93 (SD&#61;1.138) (P&#61;0.288).In the study group 41 patients had expelled the stone whereas in the control group 30 of them had expelled at the end of study period, the difference was statistically significant (P&#61;0.03). Conclusion: There was decreased pain score, number of colic episodes on treatment and duration of expulsion in the study group as compared to the control group (P &#62;0.05). The difference in the number of patients who expelled the calculus among both the groups was statistically significant (P&#61;0.03) noted in this study.

Bilateral axillary accessory breast with ductal ectasia: Ultrasonography and elastographic appearance Samida A Samad, Suresh V Phatak Journal of Datta Meghe Institute of Medical Sciences University 2018 13(4):206-208 Ectopic breast tissue can present in any part of the body except normal breast region. Ectasia of the breast is a condition where the lactiferous ducts are dilated and blocked by secretions. Both accessory breast tissue and duct ectasia are common entities presenting separately, but the coexistence is rare with a normal breast finding. Here, we report a case of a 28-year-old lactating female presented with firm bilateral painful axillary lump of 6-month duration. This case is reported for its rarity and to give an overview of simultaneous coexistence of bilateral axillary accessory breast with duct ectasia, in a lactating normal breast.

Pharmacognosy Research

Secondary metabolites from lichen Usnea longissima and its pharmacological relevance Moumita Dandapat, Santanu Paul Pharmacognosy Research 2019 11(2):103-109 The objective of the study was to isolate chemical compounds from lichen Usnea longissima (UL) and to use lichen metabolites as antibiotics, antifungal, anti-HIV, anticancer, antiprotozoan, and antibacterial agents. Preparation of the lichen extracts: Fine powder of UL lichen was extracted using acetone, methanol, and water in a Soxhlet extractor. In a rotary evaporator, the extracts were filtered and then concentrated under reduced pressure. The dry extracts were stored at 18°C until they were used in the tests. The extracts were used for various assays. Sixteen compounds were isolated from lichen UL using various chromatographic techniques, including silica gel, Sephadex LH-20, operational data store, and semipreparative high-performance liquid chromatography. By spectroscopic data analyses, their structures were identified as (1) useanol, (2) lecanorin, (3) 3-hydroxy-5-methylphenyl 2-hydroxy-4-methoxy-6-methylbenzoate, (4) lecanorin E, (5) 3'-methylevernic acid, (6) evernic acid, (7) barbatinic acid, (8) 3,7-dihydroxy-1,9-dimethyldibenzofuran, (9) orcinol, (10) O-methyl orcinol, (11) methyl orsellinate, (12) methyl everninate, (13) 2,5-dimethyl-1,3-benzenediol, (14) 2-hydroxy-4-methoxy-3,6-dimethyl benzoic acid, (15) ethyl everninate, and (16) ethyl 2,4-dihydroxy-6-methylbenzoate. UL lichen is a great source of secondary metabolites, which can be used as antimicrobial, antiproliferative, and anticancer agents. This lichen can be used as a natural antioxidant or antitumor agent in clinical practice; commercially, it can be used as an anticancer drug.

Biological screening of tri-jannarose as a recipe from Thai traditional medicine Ladachart Taepongsorat, Ampa Konsue Pharmacognosy Research 2019 11(2):110-114 Context: Tri-Jannarose (TJ) is a Thai traditional medicine recipe, the ingredients of which are betel palm seed (Areca catechu L.), Siamese neem tree root (Azadirachta indica A. Juss.), and heart-leaved moonseed vines (Tinospora cordifolia [Thunb.] Miers). The equal mixture of three plants indicated to treatment of antipyretic, diuretic, expectorate, nourishment and appetizing. Aims: Phytochemical screening, antioxidant, and α-glucosidase inhibitory activities of TJ using different extractions were evaluated. Materials and Methods: The three plants of recipe were extracted using different solvents such as aqueous extract (ATJ), 50% ethanolic extract (HETJ), and 95% ethanolic extract (ETJ). The phytochemical screening was determined by total phenolic compounds and total flavonoid contents. The anti-oxidation were tested using by 2,2-diphenyl-1-picrylhydrazy (DPPH) radical scavenging and 2,2 -azinobis-(3-ethylbenzothiazoline-6-sulphonate) (ABTS+) assay. The α-glucosidase inhibitory activity was determined for glucose transferase mechanism. Results: Phytochemical screening found that this recipe had both phenolic and flavonoid substances. The ETJ (IC50 = 0.0463 ± 0.002) was exerted on antioxidation higher than HETJ and ATJ (IC50 = 0.0511 ± 0.000 and 0.1485 ± 0.005 mg/mL). Surprisingly, ABTS+ assay, ETJ (IC50 = 0.015 ± 0.000 mg/mL), and HETJ (IC50 = 0.022 ± 0.000 mg/mL) showed high effect on free radical scavenging activity than the standard controls, ascorbic acid (IC50 = 0.025 ± 0.001 mg/mL), and Trolox (IC50 = 0.032 ± 0.001 mg/mL). The α-glucosidase inhibitory activity found that all of the extract including ATJ (IC50= 0.0127±0.02 mg/mL), ETJ (IC50= 0.0154±0.01 mg/mL) and ETJ (IC50= 0.0202±0.01 mg/mL) were more potent to inhibit α-glucosidase emzyme than acarbose (IC50=0.745±0.026 mg/mL) as a positive control. Conclusion: The pharmaceutical preliminary scarring was confirmed to treatment on Thai traditional medicine. The recipe composed with phenolic compounds and flavonoids contents which chemical substance were more potent anti-oxidation, and bittersweet flavor was stronger to α-glucosidase inhibitory activity.

Antidiabetic activity of hydroalcoholic extract of Myrtus communis (myrtle) fruits in streptozotocin-induced and dexamethasone-induced diabetic rats Mohammad Sharif Talebianpoor, Mohammad Shafee Talebianpoor, Mahboubeh Mansourian, Tahereh Vafaiee-Nejad Pharmacognosy Research 2019 11(2):115-120 Background: Diabetes mellitus is characterized by an increase in blood glucose levels, resulting in insufficiency of insulin secretion, insulin resistance, or both. Objective: The effects of hydroalcoholic extract of the Myrtus communis (myrtle) fruits on Wistar rats were investigated in two types of diabetes mellitus. Materials and Methods: Intraperitoneal administration of streptozotocin (60 mg/kg) was used to induce Type I diabetes. Type II diabetes was induced by subcutaneous injection of 1 mg/kg/day dexamethasone for 10 days. Two groups of the diabetic animals received the hydroalcoholic extract of the fruit by gavage (250 mg/kg and 500 mg/kg for 45 and 10 days) and the diabetic control groups receiving distilled water. Results: The hydroalcoholic extract of M. communis fruits reduced the serum levels of the glucose, triglyceride, urine volume, urine protein, and malondialdehyde at the end of the 45 day. In Type II diabetic rats, there was a significant effect on plasma glucose levels. On this day, blood glucose-lowering effect was significantly observed after insulin administration as C2>D2M500>D2M250>D2 (P ≤ 0.05). The plasma level of insulin was completely reversed. There were no differences in the other biochemical parameters. Conclusions: The hydroalcoholic extract of M. communis fruits has a significant effect on the improvement of diabetes mellitus complications especially Type II diabetic animals, which begins with insulin resistance.

Identification of bioactive diterpenoid lactones using nuclear magnetic resonance from Andrographis paniculata Raghava N Sriramaneni, Amirin Sadikun, Naveen Kumar Hawala Shivashekaregowda, Annegowda Venkatappa, Mohamad Zaini Asmawi Pharmacognosy Research 2019 11(2):121-126 Background: Andrographis paniculata (AP) (Burm. F) Nees, belongs to the family Acanthaceae, it is also known as a highly bitter plant. In South East Asia this plant is commonly used for cold, cough and respiratory tract infections. Andrographolide (ANG), 14-deoxy-11, 12-didehydroandrographolide (DDA) are the major bioactive diterpenoid lactone compounds of the plant shown vasorelaxant effect, anticancer, anti-inflammatory, and to treat variety of diseases which is well-documented. Materials and Methods: The high-performance liquid chromatography (HPLC) and 1H-NMR analysis of AP chloroform extract (APCE) revealed the presence of ANG, 14-deoxyandrographolide (DA), and DDA. The main advantage of 1H-NMR is simple, rapid and successfully applied to quantify the active diterpenoids and an alternative to HPLC method to check the presence of various bioactive compounds and its presence in the active raw materials of AP. Results: The results revealed that most active bioactive compounds such as ANG, DA, and DDA are present in the APCE portion of the AP. The chemical shifts of various groups confirmed through 1H-NMR that all the three ANG, DA, and DDA has potent vasorelaxant effect and majority of these compounds are present in the APCE portion and not in the methanol or aqueous fractions. The 1H-NMR study confirmed that diterpenoids such as ANG, DA, and DDA from APCE has vasorelaxant effect on the animal study.

In vitro screening for antioxidant and antimicrobial properties of three lebanese medicinal plants crude extracts Josiane Alhage, Hoda Elbitar Pharmacognosy Research 2019 11(2):127-133 Background: Despite its small geographic area, Lebanon is characterized by a natural wealth in medicinal plants. Lebanese flora has about 2600 species, where >100 are endemic and are used in traditional medicine for their therapeutic effects. Objective: To evaluate the antioxidant and antimicrobial activities of dichloromethane, methanol, and aqueous crude extracts of three Lebanese medicinal plants: Geranium robertianum, Asphodelus microcarpus, and Alcea setosa. Materials and Methods: The antioxidant activity of different crude extracts was determined using the free radical, 1,1-diphenyl-2-picrylhydrazyl. Antimicrobial activity was evaluated in a plate-hole diffusion assay against two Gram-negative (Pseudomonas aeruginosa and Escherichia coli) bacterial strains, one Gram-positive (Staphylococcus aureus) bacterial strain, and a fungal strain (Candida albicans). Results: Methanol and aqueous extracts of G. robertianum possessed high scavenging activity (Radical scavenging activity (RSA) >85%) at 0.3 mg/mL followed by the extracts of A. microcarpus which showed a moderate activity. G. robertianum extracts exhibited good inhibition diameters against S. aureus growth. Conclusion: Obtained results give an overall view on the bioactivities of three Lebanese medicinal plants crude extracts.

Phytochemical and antimicrobial evaluation of Lauridia tetragona (L.F) R.H. Archer: A medicinal plant used for the management of dysentery in the Eastern Cape Province of South Africa Olubunmi Abosede Wintola, Anthony Jide Afolayan Pharmacognosy Research 2019 11(2):134-139 Objective: The present investigation evaluated the in vitro phytochemical and antimicrobial potential of the methanol and acetone leaf extracts of Lauridia tetragona (L.F), a plant consumed as a herb in South Africa. Materials and Methods: Spectrophotometry assays using Folin–Ciocalteu, aluminum chloride, and vanillin–hydrochloric acid were used for the determination of phenols, flavonoids, and proanthocyanidin contents of the extracts, respectively. The agar dilution technique was used for the antimicrobial evaluation. The inhibitory activity of the extracts was tested on five Gram-negative and five Gram-positive bacteria and four fungi. Results: The methanol extract had a higher phenol (54.87 ± 4.01 mg gallic acid equivalent [GAE]/g) and proanthocyanidin (78.55 ± 0.83 mg catechin equivalent [CE]/g) content than the acetone extracts with 45.27 ± 3.93 mg GAE/g and 63.54 ± 1.67 mg CE/g, respectively. The acetone extract, however, had higher flavonoid content (462.45 ± 1.93 mg quercetin equivalent [QE]/g) than the methanol extract (412.20 ± 3.85 mg QE/g). The minimum inhibitory concentration (MIC) of the antibacterial assay ranged from 2.5 to 5 mg/mL. All the bacteria except Staphylococcus aureus were susceptible to the acetone extract. Five bacteria, three Gram positive and two Gram negative, were resistant while the remaining five were susceptible to the methanol extract. Conversely, all the fungi tested were susceptible to both extracts with a MIC which ranged from 0.63 to 10 mg/mL. Conclusion: The results obtained revealed that Gram-positive and Gram-negative bacteria and fungi showed some degree of susceptibility to the plant extracts. This gives an indication of broad-spectrum activity exhibited by the crude extracts of L. tetragona and supports its ethnomedicinal usage.

Biochemical screening and determination of bioactive components of commercially cultured pacific white shrimp Penaeus vannamei Jayalakshmi Muniyappan, Vanitha Varadharajan, Pushpabharathi Namadevan Pharmacognosy Research 2019 11(2):140-146 Background: Marine waste is an extraordinarily renewable aid for the restoration of several valued metabolites with potential biological applications. Objectives: The investigation is planned to detect the biochemical components present in the prawn shell waste by qualitative, quantitative methods, to assess the antioxidant potential, and also to find the bioactive compounds existing in the prawn shell waste by gas chromatography-mass spectrometry (GC-MS) analysis. Materials and Methods: Penaeus vannamei shell wastes are collected, cleaned, dried, and powdered well. The bioactive compounds present in crude ethyl acetate extract of P. vannamei shell was determined by qualitative, quantitative, and GC-MS analysis. The free radical scavenging activity of the extract was studied by different in vitro antioxidant assays. Results: The bio-compounds such as carbohydrates, saponins, flavonoids, tannins, and quinones show the positive result by qualitative analysis. The higher tannin content 49.2 ± 0.084 mg/g was observed in the ethyl acetate extract of P. vannamei shell and the flavonoid was found to be 5.994 ± 0.044 mg/g. The GC-MS analysis of the P. vannamei shell shows the various numbers of bio-compounds. Some of the identified compounds are Timonacic which has the powerful antioxidant property, Octadecane, 3ethyl5(2ethylbutyl) is a good antifungal agent, Acetamide possesses antioxidant and anti-inflammatory property. The results of the in vitro assays revealed that P. vannamei shell extract possess significant antioxidant activity. Conclusion: The present study suggests that the effective utilization of prawn shell waste enhances biomedical research field for the development of the natural drug for many chronic diseases with no side effects and at the same time can reduce environmental pollution.

Characterization of the gain and loss of resistance to antibiotics versus tolerance to honey as an antimutagenic and antimicrobial medium in extended-time serial transfer experiments Salma G Abdel-Azim, Ahmad G Abdel-Azim, Brian P Piasecki, Gamal A Abdel-Azim Pharmacognosy Research 2019 11(2):147-154 Background: Honey contains several substances with antimicrobial properties that appear more recalcitrant to generating bacterial-evolved resistance than traditional antibiotics. Objectives: This study seeks to characterize the evolution of bacteria grown for successive generations in honey as an antimutagenic medium versus ampicillin. Materials and Methods: A naive strain of Staphylococcus aureus was serially cultured for 17 days in Lysogeny broth (LB) containing sublethal concentrations of medical-grade Manuka honey, polyfloral honey, and ampicillin. Glucose as an osmotic control and pure LB were included for comparison. A portion of each culture was removed every 24 h to (a) determine the amount of growth that occurred during the previous 24 h and (b) use as a genetic stock in serially transferred tubes containing the same inhibitory compound as used previously. Results: As indicated by an increase in growth over sequential 24-h period, bacteria rapidly gained resistance to ampicillin in a step-wise pattern. However, bacteria grown in Manuka and polyfloral honey never exceeded their initial growth levels. Bacteria grown in relatively high concentrations of honey for a single 24-h period consistently lost viability after one transfer, a phenomenon that has not been reported in the literature before and which indicates the inability of bacteria to adapt to honey as an antimutagenic medium. While bacteria grown in honey did not evolve the ability to grow at higher concentrations, a single isolate grown in Manuka honey gained tolerance to Manuka by successfully surviving and proliferating beyond second transfers. Bacteria that developed antibiotic resistance were found to remain sensitive to honey. Moreover, bacteria lost their resistance to ampicillin upon a single exposure to Manuka honey.

Evaluation of acute toxicity of plants' mixture used in traditional treatment of kidney diseases in Morocco Mohamed Chebaibi, Dalila Bousta, Laila Chbani, Imane Iken, Sanae Achour Pharmacognosy Research 2019 11(2):155-161 Background: The use of plants' mixture in the traditional treatment of kidney diseases in Morocco is widespread. Objective: To evaluate the toxic effects of plants' mixture used in the traditional treatment of kidney diseases in Morocco. Materials and Methods: The phytochemical screening was performed. For acute toxicity, single doses of low dose (300 mg/kg), medium dose (500 mg/kg), high dose (2000 mg/kg), lethal dose (6000 mg/kg), and traditional dose (10 ml/kg) body weight of aqueous extracts of plants' mixture were administered orally in Wistar rats. Animals were monitored daily for at least 15 days after an oral administration of aqueous extract of the mixture to detect any changes in body weight, behavior, autonomic profiles, or mortality. Calculation of relative organ weight (ROW), hematological, biochemical analysis, and histopathology evaluation were carried out. Results: The acute oral toxicity study showed diarrhea, somnolence, and agitation of different groups of rats, while no mortality and no statistically significant decrease in body weight was observed. Statistically, the kidneys, liver, and spleen showed significant decrease in the ROW of the treated groups of rats when compared to the control group. In biochemical analysis, there was a significant increase in aspartate aminotransferase, creatinine, urea, and uric acid. Hematological parameters showed a significant decrease in leukocytes, eosinophil, basophil, lymphocytes, monocytes, and hematocrit. Histopathological evaluation which revealed major histology changes of liver sections of rats treated with low and medium doses had lymphocyte and plasma cell inflammatory infiltrates; whereas, the liver sections of rats treated with high, lethal, and traditional doses exhibited lymphocyte, plasma cell, and eosinophilic inflammatory infiltrates. Conclusion: The study finds that the plants' mixture marketed by herbalists for the treatment of kidney diseases is toxic to body organs such as the liver and the hematopoietic system.

Bioactive secondary metabolites from the locally isolated terrestrial fungus, Penicillium sp. SAM16-EGY Mosad A Ghareeb, Manal M Hamed, Amal M Saad, Mohamed S Abdel-Aziz, Ahmed A Hamed, Laila A Refahy Pharmacognosy Research 2019 11(2):162-170 Background: Penicillium is a diverse genus occurring worldwide; its species are of major importance in the natural environment as decomposer of organic materials as well as food and drug production. Objective: Chromatographic isolation and identification of its bioactive secondary metabolites and their evaluation as antimicrobial agents. Materials and Methods: Disc agar plate method has been recognized to assess the antimicrobial activities. The antioxidant activity was determined using phosphomolybdenum method. The fungus strain SAM16-EGY was isolated from soil and was molecularly identified as Penicillium sp. SAM16-EGY using 18S ribosomal ribonucleic acid technique (acc. no., KP125952). Results: Seven compounds namely 3-O-docosyl-4-benzoyloxy methyl-3-oxobicyclo (4.1.0) heptane-1,5,6,7 tetrol (3-O-docosyl-3-debenzol rotepoxide) (1), (4bE, 6Z, 8E, 9aS, 10S)-1,4-dihydroxy-9a, 10-dihydro-10,12-epoxy-5-methylbenzo[a] azulen-12-one (2), 7α,9β,15β-triacetoxy-3-β-hydroxy jatropha-5E, 11E-diene (3), sesquiterpene I diol dihexoside malonate ester (4), piperogalone (5), (5R, 8Z, 11Z)-5 β-(6'-O-malonyl-β-glucopyranozyloxy-6-hydroxy tetradeca-8, 11-dienoic acid (6), and n-trcosanyl-n-octaced-9-enoate (7) were isolated and identified from this fungus. Their structures were determined on the basis of proton nuclear magnetic resonance and carbon-13 nuclear magnetic resonance spectroscopy. Compounds 1, 2, 4, and 5 exhibited antimicrobial activities against Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans only, whereas compound 3 exerted higher antimicrobial activity against S. aureus (9 mm), P. aeruginosa (9 mm), C. albicans (11 mm), and Aspergillus niger (13 mm) as compared to the other compounds. In the phosphomolybdenum assay, compound 5 showed high total antioxidant capacity value of 608.59 mg ascorbic acid equivalent/g compound, followed by compound 2 (443.66 mg) and compound 1 (332.16 mg). Conclusion: The isolated compounds showed promising antimicrobial and antioxidant activities.

Molecular Imaging and Biology

Correction to: Imaging of Tumor Spheroids, Dual-Isotope SPECT, and Autoradiographic Analysis to Assess the Tumor Uptake and Distribution of Different Nanobodies This article was corrected/updated to include the complete graphic legend of Fig. 3.

Evaluation of Glycolytic Response to Multiple Classes of Anti-glioblastoma Drugs by Noninvasive Measurement of Pyruvate Kinase M2 Using [ 18 F]DASA-23 Abstract Purpose Pyruvate kinase M2 (PKM2) catalyzes the final step in glycolysis, the key process of tumor metabolism. PKM2 is found in high levels in glioblastoma (GBM) cells with marginal expression within healthy brain tissue, rendering it a key biomarker of GBM metabolic re-programming. Our group has reported the development of a novel radiotracer, 1-((2-fluoro- 6-[18F]fluorophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine ([18F]DASA- 23), to non-invasively detect PKM2 levels with positron emission tomography (PET). Procedure U87 human GBM cells were treated with the IC50 concentration of various agents used in the treatment of GBM, including alkylating agents (temozolomide, carmustine, lomustine, procarbazine), inhibitor of topoisomerase I (irinotecan), vascular endothelial and epidermal growth factor receptor inhibitors (cediranib and erlotinib, respectively) anti-metabolite (5-fluorouracil), microtubule inhibitor (vincristine), and metabolic agents (dichloroacetate and IDH1 inhibitor ivosidenib). Following drug exposure for three or 6 days (n = 6 replicates per condition), the radiotracer uptake of [18F]DASA-23 and 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) was assessed. Changes in PKM2 protein levels were determined via Western blot and correlated to radiotracer uptake. Results Significant interactions were found between the treatment agent (n = 12 conditions total comprised 11 drugs and vehicle) and the duration of treatment (3- or 6-day exposure to each drug) on the cellular uptake of [18F]DASA-23 (p = 0.0001). The greatest change in the cellular uptake of [18F]DASA-23 was found after exposure to alkylating agents (p < 0. 0001) followed by irinotecan (p = 0. 0012), erlotinib (p = 0. 02), and 5-fluorouracil (p = 0. 005). Correlation of PKM2 protein levels and [18F]DASA-23 cellular uptake revealed a moderate correlation (r = 0.44, p = 0.15). Conclusions These proof of principle studies emphasize the superiority of [18F]DASA-23 to [18F]FDG in detecting the glycolytic response of GBM to multiple classes of anti-neoplastic drugs in cell culture. A clinical trial evaluating the diagnostic utility of [18F]DASA-23 PET in GBM patients (NCT03539731) is ongoing.

Comparison of [ 18 F]Fluoroethyltyrosine PET and Sodium MRI in Cerebral Gliomas: a Pilot Study Abstract Purpose Positron emission tomography (PET) using O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET) improves the diagnostics of cerebral gliomas compared with conventional magnetic resonance imaging (MRI). Sodium MRI is an evolving method to assess tumor metabolism. In this pilot study, we explored the relationship of [18F]FET-PET and sodium MRI in patients with cerebral gliomas in relation to the mutational status of the enzyme isocitrate dehydrogenase (IDH). Procedures Ten patients with untreated cerebral gliomas and one patient with a recurrent glioblastoma (GBM) were investigated by dynamic [18F]FET-PET and sodium MRI using an enhanced simultaneous single-quantum- and triple-quantum-filtered imaging of 23Na (SISTINA) sequence to estimate total (NaT), weighted non-restricted (NaNR, mainly extracellular), and restricted (NaR, mainly intracellular) sodium in tumors and normal brain tissue. [18F]FET uptake and sodium parameters in tumors with a different IDH mutational status were compared. After biopsy or resection, histology and the IDH mutational status were determined neuropathologically. Results NaT (p = 0.05), tumor-to-brain ratios (TBR) of NaT (p = 0.02), NaNR (p = 0.003), and the ratio of NaT/NaR (p < 0.001) were significantly higher in IDH-mutated than in IDH-wild-type gliomas (n = 5 patients each) while NaR was significantly lower in IDH-mutated gliomas (p = 0.01). [18F]FET parameters (TBR, time-to-peak) were not predictive of IDH status in this small cohort of patients. There was no obvious relationship between sodium distribution and [18F]FET uptake. The patient with a recurrent GBM exhibited an additional radiation injury with strong abnormalities in sodium MRI. Conclusions Sodium MRI appears to be more strongly related to the IDH mutational status than are [18F]FET-PET parameters. A further evaluation of the combination of the two methods in a larger group of high- and low-grade gliomas seems promising.

Quantitative and Qualitative Improvement of Low-Count [ 68 Ga]Citrate and [ 90 Y]Microspheres PET Image Reconstructions Using Block Sequential Regularized Expectation Maximization Algorithm Abstract Purpose There are several important positron emission tomography (PET) imaging scenarios that require imaging with very low photon statistics, for which both quantitative accuracy and visual quality should not be neglected. For example, PET imaging with the low photon statistics is closely related to active efforts to significantly reduce radiation exposure from radiopharmaceuticals. We investigated two examples of low-count PET imaging: (a) imaging [90Y]microsphere radioembolization that suffers the very small positron emission fraction of Y-90's decay processes, and (b) cancer imaging with [68Ga]citrate with uptake time of 3–4 half-lives, necessary for visualizing tumors. In particular, we investigated a type of penalized likelihood reconstruction algorithm, block sequential regularized expectation maximization (BSREM), for improving both image quality and quantitative accuracy of these low-count PET imaging cases. Procedures The NEMA/IEC Body phantom filled with aqueous solution of Y-90 or Ga-68 was scanned to mimic the low-count scenarios of corresponding patient data acquisitions on a time-of-flight (TOF) PET/magnetic resonance imaging system. Contrast recovery, background variation, and signal-to-noise ratio were evaluated in different sets of count densities using both conventional TOF ordered subset expectation (TOF-OSEM) and TOF-BSREM algorithms. The regularization parameter, beta, in BSREM that controls the tradeoff between image noise and resolution was evaluated to find a value for improved confidence in image interpretation. Visual quality assessment of the images obtained from patients administered with [68Ga]citrate (n = 6) was performed. We also made preliminary visual image quality assessment for one patient with [90Y]microspheres. In Y-90 imaging, the effect of 511-keV energy window selection for minimizing the number of random events was also evaluated. Results Quantitatively, phantom images reconstructed with TOF-BSREM showed improved contrast recovery, background variation, and signal-to-noise ratio values over images reconstructed with TOF-OSEM. Both phantom and patient studies of delayed imaging of [68Ga]citrate show that TOF-BSREM with beta = 500 gives the best tradeoff between image noise and image resolution based on visual assessment by the readers. The NEMA-IQ phantom study with [90Y]microspheres shows that the narrow energy window (460–562 keV) recovers activity concentrations in small spheres better than the regular energy window (425–650 keV) with the beta value of 2000 using the TOF-BSREM algorithm. For the images obtained from patients with [68Ga]citrate using TOF-BSREM with beta = 500, the visual analogue scale (VAS) was improved by 17 % and the Likert score was increased by 1 point on average, both in comparison to corresponding scores for images reconstructed using TOF-OSEM. Conclusion Our investigation shows that the TOF-BSREM algorithm improves the image quality and quantitative accuracy in low-count PET imaging scenarios. However, the beta value in this algorithm needed to be adjusted for each radiopharmaceutical and counting statistics at the time of scans.

Retrospective Brain Motion Correction in Glutamate Chemical Exchange Saturation Transfer (GluCEST) MRI Abstract Purpose To evaluate the feasibility of motion correction in glutamate chemical exchange saturation transfer (GluCEST) imaging, using a rat model of epileptic seizure. Procedures Epileptic seizure was induced in six male Wistar rats by intraperitoneal injection of kainic acid (KA). CEST data were obtained using a 7.0 T Bruker MRI scanner before and 3 h after KA injection. Retrospective motion correction was performed in CEST images using a gradient-based motion correction (GradMC) algorithm. GluCEST signals in the hippocampal regions were quantitatively evaluated with and without motion correction. Results Calculated GluCEST signals differed significantly between the pre-KA injection group, regardless of motion-correction implementation, and the post-KA injection group with motion correction (3.662 ± 1.393 % / 3.726 ± 1.982 % for pre-KA injection group with/without motion correction vs. 6.996 ± 1.684 % for post-KA injection group with motion correction; all P < 0.05). Conclusions Our results clearly show that GradMC can be used in CEST imaging for efficient correction of seizure-like motion. The GradMC can be further implemented in various CEST imaging techniques to increase the accuracy of analysis.

Optical Redox Imaging of Fixed Unstained Muscle Slides Reveals Useful Biological Information Abstract Purpose Optical redox imaging (ORI) technique images cellular autofluorescence of nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp containing FAD, i.e., flavin adenine dinucleotide). ORI has found wide applications in the study of cellular energetics and metabolism and may potentially assist in disease diagnosis and prognosis. Fixed tissues have been reported to exhibit autofluorescence with similar spectral characteristics to those of NADH and Fp. However, few studies report on quantitative ORI of formalin-fixed paraffin-embedded (FFPE) unstained tissue slides for disease biomarkers. We investigate whether ORI of FFPE unstained skeletal muscle slides may provide relevant quantitative biological information. Procedures Living mouse muscle fibers and frozen and FFPE mouse muscle slides were subjected to ORI. Living mouse muscle fibers were imaged ex vivo before and after paraformaldehyde fixation. FFPE muscle slides of three mouse groups (young, mid-age, and muscle-specific overexpression of nicotinamide phosphoribosyltransferase (Nampt) transgenic mid-age) were imaged and compared to detect age-related redox differences. Results We observed that living muscle fiber and frozen and FFPE slides all had strong autofluorescence signals in the NADH and Fp channels. Paraformaldehyde fixation resulted in a significant increase in the redox ratio Fp/(NADH + Fp) of muscle fibers. Quantitative image analysis on FFPE unstained slides showed that mid-age gastrocnemius muscles had stronger NADH and Fp signals than young muscles. Gastrocnemius muscles from mid-age Nampt mice had lower NADH compared to age-matched controls, but had higher Fp than young controls. Soleus muscles had the same trend of change and appeared to be more oxidative than gastrocnemius muscles. Differential NADH and Fp signals were found between gastrocnemius and soleus muscles within both mid-aged control and Nampt groups. Conclusion Aging effect on redox status quantified by ORI of FFPE unstained muscle slides was reported for the first time. Quantitative information from ORI of FFPE unstained slides may be useful for biomedical applications.

Mapping pH at Cancer Cell Surfaces Abstract Purpose To develop a tool to measure the pH at the surfaces of individual cells. Procedures The SNARF pH-sensitive dye was conjugated to a pHLIP® peptide (pH-Low Insertion Peptide) that binds cellular membranes in tumor spheroids. A beam splitter allows simultaneous recording of two images (580 and 640 nm) by a CCD camera. The ratio of the two images is converted into a pH map resolving single spheroid cells. An average pH for each cell is calculated and a pH histogram is derived. Results Surface pH depends on cellular glycolytic activity, which was varied by adding glucose or deoxy-glucose. Glucose was found to decrease the surface pH relative to the pH of the bulk solution. The surface pH of metastatic cancer cells was lower than that of non-metastatic cells indicating a higher glycolytic activity. Conclusions Our method allows cell surface pH measurement and its correlation with cellular glycolytic activity.

Magnetic Resonance Imaging of Hard Tissues and Hard Tissue Engineered Bio-substitutes Abstract Magnetic resonance imaging (MRI) is a non-invasive diagnostic imaging tool based on the detection of protons into the tissues. This imaging technique is remarkable because of high spatial resolution, strong soft tissue contrast and specificity, and good depth penetration. However, MR imaging of hard tissues, such as bone and teeth, remains challenging due to low proton content in such tissues as well as to very short transverse relaxation times (T2). To overcome these issues, new MRI techniques, such as sweep imaging with Fourier transformation (SWIFT), ultrashort echo time (UTE) imaging, and zero echo time (ZTE) imaging, have been developed for hard tissues imaging with promising results reported. Within this article, MRI techniques developed for the detection of hard tissues, such as bone and dental tissues, have been reviewed. The main goal was thus to give a comprehensive overview on the corresponding (pre-) clinical applications and on the potential future directions with such techniques applied. In addition, a section dedicated to MR imaging of novel biomaterials developed for hard tissue applications was given as well.

Prognostic Value of O-(2-[ 18 F]Fluoroethyl)-L-Tyrosine PET/CT in Newly Diagnosed WHO 2016 Grade II and III Glioma Abstract Purpose The use of [18F]fluoroethyl)-l-tyrosine ([18F]FET) positron emission tomography/computed tomography (PET/CT) has proven valuable in brain tumor management. This study aimed to investigate the prognostic value of radiotracer uptake in newly diagnosed grade II or III gliomas according to the current 2016 World Health Organization (WHO) classification. Procedures A total of 35 treatment-naive patients (mean age, 48 ± 17 years) with histologically proven WHO grade II or III gliomas as defined by the current 2016 WHO classification were included. Static PET/CT imaging was performed 20 min after intravenous [18F]FET injection. Images were assessed visually and semi-quantitatively using regions of interest for both tumor (SUVmax, SUVmean) and background (BKGmean) to calculate tumor-to-background (TBR) ratios. The association among histological results, molecular markers (including isocitrate dehydrogenase enzyme and methylguanine-DNA methyltransferase status), clinical features (age), and PET findings was tested and compared with outcome (progression-free [PFS] and overall survival [OS]). Results Fourteen patients presented with grade II (diffuse astrocytoma n = 10, oligodendroglioma n = 4) and 21 patients with grade III glioma (anaplastic astrocytoma n = 15, anaplastic oligodendroglioma n = 6). Twenty-seven out of the 35 patients were PET-positive (grade II n = 8/14, grade III n = 19/21), with grade III tumors exhibiting significantly higher amino acid uptake (TBRmean and TBRmax; p = 0.03 and p = 0.02, respectively). PET-negative lesions demonstrated significantly prolonged PFS (p = 0.003) as compared to PET-positive gliomas. PET-positive disease had a complementary value in prognostication in addition to patient age, glioma grade, and molecular markers. Conclusions Amino acid uptake as assessed by [18F]FET-PET/CT imaging is useful as non-invasive read-out for tumor biology and prognosis in newly diagnosed, treatment-naive gliomas according to the 2016 WHO classification.

Impact of rs12917 MGMT Polymorphism on [ 18 F]FDG-PET Response in Pediatric Hodgkin Lymphoma (PHL) Abstract Purpose The enzyme O6-methylguanine-DNA methyltransferase (MGMT) is an important component of the DNA repair machinery. MGMT removes O6-methylguanine from the DNA by transferring the methyl group to a cysteine residue in its active site. Recently, we detected the single nucleotide polymorphism (SNP) rs12917 (C/T) in the MGMT sequence adjacent to the active site in Hodgkin lymphoma (HL) cell line KM-H2. We now investigated whether this SNP is also present in other HL cell lines and patient samples. Furthermore, we asked whether this SNP might have an impact on metabolic response in 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography ([18F]FDG-PET), and on overall treatment outcome based on follow-up intervals of at least 34 months. Procedures We determined the frequency of this MGMT polymorphism in 5 HL cell lines and in 29 pediatric HL (PHL) patients. The patient cohort included 17 female and 12 male patients aged between 4 and 18 years. After characterization of the sequence, we tested a possible association between rs12917 and age, gender, Ann Arbor stage, treatment group, metabolic response following two courses of OEPA (vincristine, etoposide, prednisone, and doxorubicin) chemotherapy, radiotherapy indication, and relapse status. Results We detected the minor T allele in four of five HL cell lines. 11/29 patients carried the minor T allele whereas 18/29 patients showed homozygosity for the major C allele. Interestingly, we observed significantly better metabolic response in PHL patients carrying the rs12917 C allele resulting in a lower frequency of radiotherapy indication. Conclusion MGMT polymorphism rs12917 seems to affect chemotherapy response in PHL. The prognostic value of this polymorphism should be investigated in a larger patient cohort.

Pathology & Oncology Research

Comparison of CDH1 Gene Hypermethylation Status in Blood and Serum among Gastric Cancer Patients Abstract Hypermethylation is epigenetic alteration, well known for gene silencing. CHD1 gene is known as invasion and tumor suppressor gene, decreased expression due to hypermethylation could promote tumor cell invasion and metastasis. Present study designed to investigate the CDH1 gene promoter hypermethylation status by methylation specific polymerase chain reaction in 100 newly diagnosed gastric cancer patients. 53% of hypermethylation was observed in DNA extracted from blood in Gastric cancer patients while 66% was observed in serum based DNA. Significant differences in CDH1gene promoter hypermethylation was observed in serum based DNA extracted from Gastric cancer patients. Patients in early stage (I & II) vs advanced stage (III & IV), distant organ metastases vs no metastases had 60% vs 7% and 42% 24% of CDH1 promoter hypermethylation in serum DNA (p = 0.006, 0.001) respectively. Patients who were with lymph node invasion, loss of appetite, loss of weight had 55%, 47%, 61% CDH1 gene promoter hypermethylation compare to who were not with lymph node invasion, loss of appetite, loss of weight had 11%, 19%, 5% of hypermethylation and these differences was found to be significant. Strong association was observed with overall median survival of patients (p < 0.0001). Patients who had CDH1 gene promoter hypermethylation in serum based DNA showed poor overall median survival (14.3 months) and unmethylated patients had better overall median survival (33.2 months). CDH1 hypermethylation status was found to be associated with advancement of disease, distant organ metastases and lymph node invasion in Gastric cancer patients.

Prognostic Significance of Serum PSA Level and Telomerase, VEGF and GLUT-1 Protein Expression for the Biochemical Recurrence in Prostate Cancer Patients after Radical Prostatectomy Abstract The aim of the study was to evaluate prognosis for biochemical recurrence (BR) by analysing the pathological and biological characteristics of prostate cancer (PCa) after radical prostatectomy (RP). There were 130 men with clinically localized PCa in whom pretreatment serum PSA level and Ki-67, prostate specific membrane antigen (PSMA), glucose transporter-1 (GLUT-1), vascular endothelial growth factor (VEGF), microvessel density (MVD) and human telomerase reverse transcriptase (hTERT) proteins expression, based on number of immunohistochemically positive cells (labelling index), were retrospectively studied. In order to assess the prognostic significance of analysed variables in univariate and multivariate Cox analysis, patients were dichotomized based on cut-off points chosen by receiver operating characteristic (ROC) curves. There were 83 males (63.8%) at pT stage 1–2 and 47 (36.1%) at pT stage 3–4, respectively, with median (range) age of 62.8 years (49–77), and median follow-up of 78.5 months (12–148). In 42 (32.3%) men BR was found. In univariate analysis, tumour biological features: PSA ≤ 8 ng/mL (p = 0.006), Ki-67LI ≤ 12.7% (p = 0.015), VEGFLI>11.0% (p = 0.030), and hTERTLI>6.7% (p = 0.016), but not clinicopathological parameters, appeared to be positive prognosticators for BRFS. In the Cox analysis, Ki-67 lost its significance, and clinicopathological parameters appeared to be nonsignificant. The independent negative prognostic factors for BRFS were: PSA > 8.0 ng/mL, (Hazard ratio = 2.75, p = 0.003), GLUT-1 > 19.1% (HR = 2.1, p = 0.032), VEGF≤11.0% (HR = 1, p = 0.024) and hTERT≤6.7% (HR = 1, p = 0.017). High PSA level, and GLUT-1 expression and lower VEGF and nuclear hTERT expression may indicate the great role of hypoxia in BR induction in PCa.

Identification of Pathogenic Genes and Transcription Factors in Osteosarcoma Abstract Osteosarcoma (OS) is an aggressive malignant tumor of the bones. Our study intended to identify and analyze potential pathogenic genes and upstream regulators for OS. We performed an integrated analysis to identify candidate pathogenic genes of OS by using three Gene Expression Omnibus (GEO) databases (GSE66673, GSE49003 and GSE37552). GO and KEGG enrichment analysis were utilized to predict the functional annotation and potential pathways of differentially expressed genes (DEGs). The OS-specific transcriptional regulatory network was established to study the crucial transcriptional factors (TFs) which target the DEGs in OS. From the three GEO datasets, we identified 759 DEGs between metastasis OS samples and non-metastasis OS samples. After GO and KEGG analysis, 'cell adhesion' (FDR = 1.27E-08), 'protein binding' (FDR = 1.13E-22), 'cytoplasm' (FDR = 5.63E-32) and 'osteoclast differentiation' (FDR = 0.000992221) were significantly enriched pathways for DEGs. HSP90AA1 exhibited a highest degree (degree = 32) and was enriched in 'pathways in cancer' and 'signal transduction'. BMP6, regulated by Pax-6, was enriched in the 'TGF-beta signaling pathway'. We indicated that BMP6 may be downregulated by Pax-6 in the non-metastasis OS samples. The up-regulated HSP90AA1 and down-regulated BMP6 and 'pathways in cancer' and 'signal transduction' were deduced to be involved in the pathogenesis of OS. The identified biomarkers and biological process in OS may provide foundation for further study.

KIBRA Team Up with Partners to Promote Breast Cancer Metastasis Abstract Among women, breast cancer is the most frequently diagnosed cancer. Most of the breast cancers represent metastasis to distant organs at the time of diagnosis and accounts for the majority of deaths. Metastasis is characterized by many genetic aberrations including mutations, overexpression of oncogenes etc. KIBRA (KIdney/BRAin protein), a scaffolding protein is recently described as an important player in the process of invasion and metastasis. The Kidney/BRAin protein through its different domains interacts with various proteins to couple cytoskeleton arrangement, cell polarity and migration. N terminal and C terminal of the protein contains the WW, Internal C2 & putative class III PDZ domain that interacts with DDR1, DLC1 & PKCζ. These protein-protein interactions equip the breast cancer cells to invade and metastasize. Here, we discuss a comprehensive knowledge about the KIBRA protein, its domains and the interacting partners involved in metastasis of breast cancer.

lncRNA SNHG16 Exerts Oncogenic Functions in Promoting Proliferation of Glioma Through Suppressing p21 Abstract Glioma is a malignant brain tumor that accounts for 30% of all brain tumors and 80% of malignant brain tumors. This poor clinical outcome makes the study of molecular mechanisms in glioma as an urgent subject. However, the certain mechanism remains unclear. Long non-coding RNAs (lncRNAs) plays a key role in glioma development and progression. In the present study, we aimed to explore the potential mechanisms of lncRNA SNHG16 in glioma. The levels of lncRNA SNHG16 were qualified in both glioma tissues and cell lines using qRT-PCR assay. The ability of cell proliferation was tested via CCK-8 and colony formation assays. Transfections were performed to knockdown SNHG16 and its target gene p21. The cell cycles and cell apoptosis were evaluated using flow cytometry, and the expression of SNHG16, p21 and apoptosis biomarkers were qualified with qRT-PCR and western blot assays. The expression of SNHG16 were up-regulated in both glioma tissues and cell lines. Knockdown of SNHG16 was associated with poor proliferation, decreased monoclonal formation rates, but increased apoptosis rates, which also caused the high expression of p21. Moreover, p21 could mediate cell proliferation and monoclonal formation, promote cell apoptosis in glioma, which was negatively correlated with lncRNA SNHG16. The molecule mechanism experiments revealed that SNHG16 could not only inhibit the expression of p21 but also suppressed the level of caspase 3 and 9, while promoted cyclinD1 and cyclinB1 expression. lncRNA SNHG16 could promote the cell proliferation and inhibit the apoptosis of glioma through suppressing p21, indicating that lncRNA SNHG16 might be quite vital for the diagnosis and progression of glioma and could even be a novel therapeutic target for glioma.

Differential Analysis of lncRNA, miRNA and mRNA Expression Profiles and the Prognostic Value of lncRNA in Esophageal Cancer Abstract Integrative central axis of lncRNA-miRNA-mRNA plays pivotal roles in tumor development and progression. However, the regulatory role of lncRNA-miRNA-mRNA in esophageal cancer remains elusive. TCGA database was utilized to investigate the differential expression of lncRNA, miRNA and mRNA in esophageal cancer (ESCA) and normal esophageal tissues, and GEO database was used to further validate the expression profile of key genes. Differential lncRNAs in TCGA database were submitted to Starbase, and lncRNAs related to overall survival were analyzed using Kaplan-Meier and log-rank test. We found 145 lncRNAs, 112 miRNAs and 2000 protein coding mRNAs were differentially expressed in ESCA samples, which were tightly involved in chromosome segregation, extracellular matrix assembly by GO assay, and KEGG assay revealed the correlation of differentially expressed genes with cell cycle, apoptosis and cGMP-PKG signaling pathway. Furthermore, there were 291 nodes in ceRNA network, which consisted of 40 lncRNAs, 28 miRNAs and 233 mRNAs, and formed 677 relations. Furthermore, 6 of 10 lncRNAs in TCGA database were consistent with GEO database, and expressions of 10 mRNAs in TCGA database all exhibited the same tendency with GEO database. Notably, we found 8 lncRNAs (WDFY3-AS2, CASC8, UGDH-AS1, RAP2C-AS1, AC007128.1, AC016205.1, AC092803.2 and AC079949.2) were correlated with overall survival of the patients with ESCA. The key differentially expressed genes participate in the development and progression of ESCA, and thus the elucidation of functions of lncRNA-miRNA-mRNA will provide new novel therapeutic target for the patients with ESCA.

C-X-C Chemokine Receptor Type 7 (CXCR-7) Expression in Invasive Ductal Carcinoma of Breast in Association with Clinicopathological Features Abstract C-X-C chemokine receptor type 7 (CXCR-7) is an atypical receptor for chemokines whose role in different stages of carcinogenesis has been evaluated in breast cancer cell lines and animal models. Moreover, it has been demonstrated to be a target of regulation by the tumor suppressor microRNA (miR)-100. In the present study, we assessed CXCR-7 expression in 60 breast cancer patients in association with clinicopathological and demographic data of patients. We also extracted the results of our previous work on miR-100 expression in the same cohort of patients to assess the correlation between miR-100 and CXCR-7 expression levels. Transcript levels of CXCR-7 were significantly higher in tumoral tissues compared with adjacent non-cancerous tissues (ANCTs) (Tumoral vs. ANCTs: 3.64 ± 1.8 vs. 0.73 ± 1.3, P = 0.000). A significant negative correlation was detected between CXCR-7 protein and miR-100 transcript levels (r = −0.526, P < 0.05). High CXCR-7 mRNA levels were significantly associated with tumor size (P = 0.01). Besides, high protein levels were more prevalent in higher TNM stages (P = 0.000). Moreover, high CXCR-7 protein levels were significantly associated with ER (P = 0.005) and PR (P = 0.02) status. The present work provides further evidence for the role of CXCR-7 in breast cancer and proposes the elimination of inhibitory effects of miR-100 on CXCR-7 expression as a mechanism for its up-regulation in breast cancer tissues.

MicroRNA-182-5p Modulates Oral Squamous Cell Carcinoma Migration and Invasion Via Targeting MTSS1 Gene Abstract Oral squamous cell carcinoma (OSCC) characterized with invasive growth, local metastasis and later stage diagnosis was a common malignancy in head and neck region. The aim of this study was to explore the relationship between miR-182-5p and OSCC, which will contribute to find potential biomarker for OSCC metastasis. MiR-182-5p expression level was detected by the quantitative real-time PCR (qRT-PCR). Cell migration and invasion ability were examined by scratch and transwell assay. Loss-of function together with luciferase reporter assay were used to verify the miR-182-5p modulated OSCC cells migration and metastasis was mediated by MTSS1. The expression of MTSS1 protein was examined by western blotting. MiR-182-5p up-regulated in OSCC, was involved in the migration and invasion of OSCC and the increased miR-182-5p expression was correlated with lower OSCC differentiation grade, higher T and N stage. Bioinformatics analysis predicted MTSS1 gene was a potential target of miR-182-5p. Following co-transfection, qRT-PCR, luciferase activities assay and western blotting confirmed that MTSS1 gene was a direct target of miR-182-5p and silence of MTSS1 could reverse the effects of miR-182-5p on OSCC migration and invasion. MiR-182-5p was up-regulated in OSCC and the ability of miR-182-5p to promote MTSS1 repression may precipitate in the OSCC through bypassing cell migration and invasion control.

Dynamic FDG-PET/CT in the Initial Staging of Primary Breast Cancer: Clinicopathological Correlations Abstract Our aim was to evaluate correlation between clinicopathological features (clinical T and clinical N stages; histological type; nuclear grade; hormone-receptor and HER2 status, proliferation activity and tumor subtypes) of breast cancer and kinetic parameters measured by staging dynamic FDG-PET/CT examinations. Following ethical approval and patients' informed consent we included 34 patients with 35 primary breast cancers in our prospective study. We performed dynamic PET imaging, and assessed plasma activity noninvasively. To delineate primary tumors we applied a frame-by-frame semi-automatic software-based correction of motion artefacts. FDG two-compartment kinetic modelling was applied to assess K1, k2, k3 rate coefficients and to calculate Ki (tracer flux constant) and MRFDG (FDG metabolic rate). We found that k3, Ki and MRFDG were significantly higher in higher grade (p = 0.0246, 0.0089 and 0.0076, respectively), progesterone-receptor negative (p = 0.0344, 0.0217 and 0.0132) and highly-proliferating (p = 0.0414, 0.0193 and 0.0271) tumors as well as in triple-negative and hormone-receptor negative/HER2-positive subtypes (p = 0.0310, 0.0280 and 0.0186). Ki and MRFDG were significantly higher in estrogen-receptor negative tumors (p = 0.0300 and 0.0247, respectively). Ki was significantly higher in node-positive than in node-negative disease (p = 0.0315). None of the assessed FDG-kinetic parameters showed significant correlation with stromal TIL. In conclusion, we confirmed a significant relationship between kinetic parameters measured by dynamic PET and the routinely assessed clinicopathological factors of breast cancer: high-grade, hormone-receptor negative tumors with high proliferation rate are characterized by higher cellular FDG-uptake and FDG-phosphorylation rate. Furthermore, we found that kinetic parameters based on the dynamic examinations are probably not influenced by stromal TIL infiltration.

The Investigation of Lipoxygenases as Therapeutic Targets in Malignant Pleural Mesothelioma Abstract Advanced malignant pleural mesothelioma (MPM) has an extremely poor prognosis with limited chemotherapy options, therefore the identification of new therapeutic targets would aid in disease management. Arachidonic acid is metabolised by cyclooxygenase and lipoxygenase enzymes. The lipoxygenase isoenzymes 5-LOX and 12-LOX have been implicated in carcinogenesis. We aimed to examine 5-LOX and 12-LOX protein expression in a large retrospective series of mesothelioma samples. Further to this, the in vitro cytotoxic effects of lipoxygenase pathway inhibitors were investigated in mesothelioma cells. Archival samples from 83 patients with MPM were examined by immunohistochemistry for expression of the 5-LOX and 12-LOX proteins. The MTS assay was used to assess cell viability following 72 h treatment with the lipoxygenase pathway inhibitors baicalein, licofelone, MK-886 and zileuton in the MPM cell lines NCI-H2052, NCI-H2452 and MSTO-211H. Positive 12-LOX protein expression was recorded in 69/83 (83%) and positive 5-LOX expression was observed in 56/77 (73%) of MPM tissue samples. Co-expression of 5-LOX with 12-LOX was seen in 46/78 (58%) of MPM samples. Positive expression of 5-LOX, 12-LOX and COX-2 proteins was identified in the NCI-H2052, NCI-H2452 and MSTO-211H MPM cell lines. Baicalein (12-LOX and 15-LOX inhibitor) was effective in 3/3 MPM cell lines at low concentrations with an IC50 range of 9.6 μM to 20.7 μM. We have demonstrated that the 5-LOX and 12-LOX proteins are expressed in a significant proportion of MPM samples (73% and 83% respectively) and may represent novel therapeutic targets in this disease. We have demonstrated that the inhibition of the LOX pathway using baicalein may be effective as a novel treatment for MPM, however further human pharmacokinetic studies are required in order to establish whether the concentration used in vitro is clinically achievable.

Cellular Oncology

Deep learning and manual assessment show that the absolute mitotic count does not contain prognostic information in triple negative breast cancer Abstract Purpose The prognostic value of mitotic count for invasive breast cancer is firmly established. As yet, however, limited studies have been aimed at assessing mitotic counts as a prognostic factor for triple negative breast cancers (TNBC). Here, we assessed the prognostic value of absolute mitotic counts for TNBC, using both deep learning and manual procedures. Methods A retrospective TNBC cohort (n = 298) was used. The absolute manual mitotic count was assessed by averaging counts from three independent observers. Deep learning was performed using a convolutional neural network on digitized H&E slides. Multivariable Cox regression models for relapse-free survival and overall survival served as baseline models. These were expanded with dichotomized mitotic counts, attempting every possible cut-off value, and evaluated by means of the c-statistic. Results We found that per 2 mm2 averaged manual mitotic counts ranged from 1 to 187 (mean 37.6, SD 23.4), whereas automatic counts ranged from 1 to 269 (mean 57.6; SD 42.2). None of the cut-off values improved the models' baseline c-statistic, for both manual and automatic assessments. Conclusions Based on our results we conclude that the level of proliferation, as reflected by mitotic count, does not serve as a prognostic factor for TNBC. Therefore, TNBC patient management based on mitotic count should be discouraged.

Curcumin: a potent agent to reverse epithelial-to-mesenchymal transition Abstract Background Epithelial-to-mesenchymal transition (EMT) is involved in tumor progression, invasion, migration and metastasis. EMT is a process by which polarized epithelial cells acquire motile mesothelial phenotypic features. This process is initiated by disassembly of cell-cell contacts through the loss of epithelial markers and replacement of these markers by mesenchymal markers. Reconstruction of the cytoskeleton and degradation of the tumor basement membrane ensures the spread of invasive malignant tumor cells to distant locations. Accumulating evidence indicates that curcumin, as a well-known phytochemical, can inhibit EMT/metastasis through various mechanisms and pathways in human tumors. Conclusions In this review, we summarize the mechanisms by which curcumin may affect EMT in cells under pathological conditions to understand its potential as a novel anti-tumor agent. Curcumin can exert chemo-preventive effects by inhibition and reversal of the EMT process through both TGF-β-dependent (e.g. in hepatoma and retinal pigment epithelial cancer) and -independent (e.g. in oral cancer, colorectal cancer, pancreatic cancer, hepatocellular carcinoma, breast cancer, melanoma, prostate cancer, bladder cancer, thyroid cancer and lung cancer) pathways. Curcumin can also mitigate chemoresistance through EMT suppression and promotion of the antiproliferative effects of conventional chemotherapeutics. Therefore, curcumin has the potential to be used as a novel adjunctive agent to prevent tumor metastasis, which may at least partly be attributed to its hampering of the EMT process.

The tumor suppressor FOXO3a mediates the response to EGFR inhibition in glioblastoma cells Abstract Purpose Although EGFR activation is a hallmark of glioblastoma (GBM), anti-EGFR therapy has so far not yielded the desired effects. Targeting PI3K/Akt has been proposed as a strategy to increase the cellular sensitivity to EGFR inhibitors. Here we evaluated the contribution of FOXO3a, a key Akt target, in the response of GBM cells to EGFR inhibition. Methods FOXO3a activation was assessed by immunofluorescence and gene reporter assays, and by evaluating target gene expression using Western blotting and qRT-PCR. Cellular effects were evaluated using cell viability and apoptosis assays, i.e., Annexin V/PI staining and caspase 3/7 activity measurements. Drug synergism was evaluated by performing isobolographic analyses. Gene silencing experiments were performed using stable shRNA transfections. Results We found that EGFR inhibition in GBM cells led to FOXO3a activation and to transcriptional modulation of its key targets, including repression of the oncogene FOXM1. In addition, we found that specific FOXO3a activation recapitulated the molecular effects of EGFR inhibition, and that the FOXO3a activator trifluoperazine, a FDA-approved antipsychotic agent, reduced GBM cell growth. Subsequent isobolographic analyses of combination experiments indicated that trifluoperazine and erlotinib cooperated synergistically and that their concomitant treatment induced a robust activation of FOXO3a, leading to apoptosis in GBM cells. Using gene silencing, we found that FOXO3a is essential for the response of GBM cells to EGFR inhibition. Conclusions Our data indicate that FOXO3a activation is a crucial event in the response of GBM cells to EGFR inhibition, suggesting that FOXO3a may serve as an actionable therapeutic target that can be modulated using FDA-approved drugs.

Notch pathway in small-cell lung cancer: from preclinical evidence to therapeutic challenges Abstract Background Small-cell lung cancer (SCLC) is an aggressive disease with still limited therapeutic options. Despite being both a chemo- and radiation-sensitive malignancy, SCLC recurrence occurs in most cases and negatively impacts patients' prognosis. Over the last few years, a deeper understanding of SCLC molecular aberrations has led to the identification of Notch pathway deregulation as a crucial event in SCLC tumorigenesis, disease progression and chemoresistance. In particular, the delta-like protein 3 (DLL3), a Notch inhibitory ligand whose expression is directly related to the key neuroendocrine transcription factor ASCL1, was found to be expressed in ~85% of SCLCs, while it exhibits minimal to absent surface expression in normal lungs. DLL3 thus represents an appealing novel biomarker as well as a potential target in SCLC. Conclusions The first DLL3-targeted antibody-drug conjugate rovalpituzumab tesirine (Rova-T, SC16LD6.5) has shown promising results in terms of efficacy and safety for the management of extensive SCLC, supporting further studies on this novel therapeutic approach that combines specific SCLC targeting with the cell-killing ability of a pyrrolobenzodiazepine dimer. In the present review, we discuss currently available evidence on the biological role of Notch signaling in SCLC from early preclinical findings to current and future clinical implications.

IFITM3 knockdown reduces the expression of CCND1 and CDK4 and suppresses the growth of oral squamous cell carcinoma cells Abstract Purpose Oral squamous cell carcinoma (OSCC) is a challenging disease to treat. Up to 50% of OSCC patients with advanced disease develop recurrences. Elucidation of key molecular mechanisms underlying OSCC development may provide opportunities to target specific genes and, thus, to improve patient survival. In this study, we examined the expression and functional role of interferon transmembrane protein 3 (IFITM3) in OSCC development. Methods The expression of IFITM3 in OSCC and normal oral mucosal tissues was assessed by qRT-PCR and immunohistochemistry. The role of IFITM3 in driving OSCC cell proliferation and survival was examined using siRNA-mediated gene knockdown, and the role of IFITM3 in driving cell cycle regulators was examined using Western blotting. Results We found that IFITM3 is overexpressed in more than 79% of primary OSCCs. We also found that IFITM3 knockdown led to impaired OSCC cell growth through inhibition of cell proliferation, induction of cell cycle arrest, senescence and apoptosis. In addition, we found that IFITM3 knockdown led to reduced expressions of CCND1 and CDK4 and reduced RB phosphorylation, leading to inhibition of OSCC cell growth. This information may be instrumental for the design of novel targeted therapeutic strategies. Conclusions From our data we conclude that IFITM3 is overexpressed in OSCC and may regulate the CCND1-CDK4/6-pRB axis to mediate OSCC cell growth.

Secretome profiling of heterotypic spheroids suggests a role of fibroblasts in HIF-1 pathway modulation and colorectal cancer photodynamic resistance Abstract Purpose Previous analyses of the tumor microenvironment (TME) have resulted in a concept that tumor progression may depend on interactions between cancer cells and its surrounding stroma. An important aspect of these interactions is the ability of cancer cells to modulate stroma behavior, and vice versa, through the action of a variety of soluble mediators. Here, we aimed to identify soluble factors present in the TME of colorectal cancer cells that may affect relevant pathways through secretome profiling. Methods To partially recapitulate the TME and its architecture, we co-cultured colorectal cancer cells (SW480, TC) with stromal fibroblasts (MRC-5, F) as 3D-spheroids. Subsequent characterization of both homotypic (TC) and heterotypic (TC + F) spheroid secretomes was performed using label-free liquid chromatography-mass spectrometry (LC-MS). Results Through bioinformatic analysis using the NCI-Pathway Interaction Database (NCI-PID) we found that the HIF-1 signaling pathway was most highly enriched among the proteins whose secretion was enhanced in the heterotypic spheroids. Previously, we found that HIF-1 may be associated with resistance of colorectal cancer cells to photodynamic therapy (PDT), an antitumor therapy that combines photosensitizing agents, O2 and light to create a harmful photochemical reaction. Here, we found that the presence of fibroblasts considerably diminished the sensitivity of colorectal cancer cells to photodynamic activity. Although the biological significance of the HIF-1 pathway of secretomes was decreased after photosensitization, this decrease was partially reversed in heterotypic 3D-spheroids. HIF-1 pathway modulation by both PDT and stromal fibroblasts was confirmed through expression assessment of the HIF-target VEGF, as well as through HIF transcriptional activity assessment. Conclusion Collectively, our results delineate a potential mechanism by which stromal fibroblasts may enhance colorectal cancer cell survival and photodynamic treatment resistance via HIF-1 pathway modulation.

Nuclear localization of PD-L1: artifact or reality? Abstract Background The levels of expression and membrane localization of programmed cell death ligand 1 (PD-L1), an immune checkpoint type I transmembrane glycoprotein, are related to the clinical response of anti-PD-L1/PD-1 therapy. Although the biologically relevant localization of PD-L1 is on the plasma membrane of cancer cells, it has also been reported to be in the cytoplasm and sometimes in the nucleus. Furthermore, it has been claimed that chemotherapeutics can modify PD-L1 expression and/or its nuclear localization. Results Data from our group suggest that the nuclear localization of PD-L1, and other plasma membrane proteins as well, could be an artifact resulting from inadequate experimental conditions during immunocytochemical studies. Mild detergent and rigorous fixation conditions should be used in order to preserve the membrane localization and to prevent an erroneous translocation of PD-L1 and other non-interconnected membrane proteins, such as CD24, into other cellular compartments including the nucleus, of untreated and chemotherapeutically treated breast cancer cells. Conclusion We propose that well-specified and rigorously followed protocols should be applied to immunocytochemical diagnostic techniques, especially to those related to individualized diagnosis and treatment.

Interfering with bromodomain epigenome readers as therapeutic option in mucoepidermoid carcinoma Abstract Purpose Emerging evidence indicates that bromodomains comprise a conserved class of epigenome readers involved in cancer development and inflammation. Bromodomains are associated with epigenetic modifications of gene transcription through interactions with lysine residues of histone tails. Particularly, the bromodomain and extra-terminal domain (BET) family member BRD4 has been found to be involved in the control over oncogenes, including c-MYC, and in the maintenance of downstream inflammatory processes. The objective of this study was to evaluate the effect of pharmacologically displacing BRD4 in mucoepidermoid carcinoma (MEC) cells. Methods We assessed the presence of BRD4 levels in a panel of human MEC tissue samples in conjunction with histological grading and clinical information. In vitro studies were carried out using human MEC-derived cell lines. The BET inhibitor iBET762 was administered to MEC cells to assess the impact of disrupted BRD4 signaling on colony forming capacities and cell cycle status. The activation of cellular senescence induced by iBET762 was determined by immunohistochemical staining for p16ink4. Flow cytometry was used to identify populations of cancer stem cells in MEC-derived cell lines. Results We found that primary human MECs and MEC-derived cell lines are endowed with high BRD4 expression levels compared to those in normal salivary glands. We also found that, by displacing BRD4 from chromatin using the BET inhibitor iBET762, MEC cells lose their colony forming capacities and undergo G1 cell cycle arrest and senescence. Finally, we found that targeted displacement of BRD4 from chromatin results in depletion of cancer stem cells from the overall MEC cell populations. Conclusions Our findings indicate that bromodomain-mediated gene regulation constitutes an epigenetic mechanism that is deregulated in MEC cells and that the use of BET inhibitors may serve as a feasible therapeutic strategy to manage MECs.

MicroRNA-mediated redox regulation modulates therapy resistance in cancer cells: clinical perspectives Abstract Background Chemotherapy and radiation therapy are the most common types of cancer therapy. The development of chemo/radio-resistance remains, however, a major obstacle. Altered redox balances are among of the main factors mediating therapy resistance. Therefore, redox regulatory strategies are urgently needed to overcome this problem. Recently, microRNAs have been found to act as major redox regulatory factors affecting chemo/radio-resistance. MicroRNAs play critical roles in regulating therapeutic resistance through the regulation of antioxidant enzymes, redox-sensitive signaling pathways, cancer stem cells, DNA repair mechanisms and autophagy. Conclusions Here, we summarize current knowledge on microRNA-mediated redox regulatory mechanisms underlying chemo/radio-resistance. This knowledge may form a basis for a better clinical management of cancer patients.

SNHG6 is upregulated in primary breast cancers and promotes cell cycle progression in breast cancer-derived cell lines Abstract Background Long non-coding RNAs (lncRNAs) are known as RNAs that do not encode proteins and that are more than 200 nucleotides in size. Previously, it has been found that LncRNAs play crucial roles in normal cellular processes, including proliferation and apoptosis. A growing body of evidence suggests that lncRNAs may also play regulatory roles in the initiation, progression and metastasis of various malignancies, including breast cancer. SNHG6 is a lncRNA that has previously been found to contribute to the initiation and progression of hepatocellular and gastric carcinomas. In this study, the clinical significance of SNHG6 expression in breast cancer was investigated. Methods SNHG6 expression in primary breast cancer tissues was assessed using RT-qPCR. The functional role of SNHG6 was investigated using RNAi-mediated silencing and exogenous overexpression in breast cancer-derived cells. MTT, colony formation, cell cycle, apoptosis and senescence assays were used to determine the impact of SNHG6 expression on breast cancer-derived cells. The effect of SNHG6 on the migration and epithelial-to-mesenchymal transition (EMT) of breast cancer-derived cells was determined using scratch wound healing and immunofluorescence assays, respectively. Results We found that the expression of SNHG6 was significantly upregulated in primary high-grade and progesterone receptor (PR)-positive breast tumours. Additional siRNA-based experiments revealed that SNHG6 silencing led to G1 cell cycle arrest in SK-BR-3 and MDA-MB-231 breast cancer-derived cells. Moreover, we found that SNHG6 silencing led to suppressed breast cancer cell proliferation by inducing apoptosis and senescence. Our data also indicate that SNHG6 may contribute to the migration and EMT of breast cancer cells. Conclusions Our results indicate that lncRNA SNHG6 is involved in breast cancer development and may be considered as a potential biomarker for the diagnosis, prognosis and treatment of breast cancer.