ISSN: 0377-9777 / e-ISSN: 1308-2523
Turkish Bulletin of Hygiene and Experimental Biology - Turk Hij Den Biyol Derg: 78 (2)
Volume: 78  Issue: 2 - 2021
FULL JOURNAL
1. TBHEB 2021-2 Vol 78 Full Printed Journal
Utku ERCÖMERT
doi: 10.5505/TurkHijyen.2021.58672  Pages 118 - 233
Abstract |Full Text PDF

RESEARCH ARTICLE
2. The investigation of Brucella seropositivity in blood donors
Kamber SÜMER, Hüseyin GÜDÜCÜOĞLU, Sümeyye AKYÜZ, Mehmet PARLAK, Yasemin BAYRAM
doi: 10.5505/TurkHijyen.2020.46343  Pages 119 - 124
INTRODUCTION: Although brucellosis is controlled in developed countries, it is a public health problem that is seen as endemic in our country. Although most people living in endemic areas do not have clinical signs of infection, the serological indicators of brucellosis may be positive. The illness can be transmitted to healthy people from asymptomatic patients through blood transfusion. In this study, it is aimed to investigate Brucella seropositivity in healthy blood donors in our region.
METHODS: The study included 4018 healthy blood donors who donated to the Blood Center of Van Yüzüncü Yıl University Medical Faculty between May 2016 and June 2017. Rose Bengal Plate Test (RBPT) was applied to all blood serums and samples with agglutination were confirmed by Standard Tube Agglutination (STA) test. Serum samples with low titer positive (1/40 and 1/80 titer) who were considered ≥1/160 titer positive as a result of the STA test were studied with Coombs test. ≥1/160 titer positive in Coombs test.
RESULTS: In the study, 107 (2.7 %) of the total 4018 serum samples were positive for RBPT screening. As a result of the STA test applied to positive blood samples, 13 were suspect positive and 13 (0.3 %) were positive. Of the 13 serum samples that gave suspected positivity with the STA test, 3 of them were positive with Coombs test. Therefore, in our study, seropositivity rate among all samples was 2.7 % (n=107) with RBPT, 0.3 % (n=13) with STA test and 0.4 % (n=16) by Coombs test+STA test.
DISCUSSION AND CONCLUSION: It should be considered that patients with positive Brucella serology due to active disease but without clinical findings, carry the risk of Brucellosis associated with blood transfusion in endemic regions including our country.

3. Alzheimer hastalığında Kallikrein-6, 7 ve potasyum kanal proteinlerinin olası rolü
Erkut Baha BULDUK, Filiz YILDIRIM, Zuhal YILDIRIM
doi: 10.5505/TurkHijyen.2021.50374  Pages 125 - 132
INTRODUCTION: Background: Although, the formation mechanism of Alzheimer’s disease (AD) is not known with certainty, two major proteins, beta amyloid of senile plaques and tau protein of neurofibrillary tangels are responsible for AD. One of the major factors in the development of the disease is the formation of in soluble amyloid deposits, and the other one is the increased tau phosphorylation. Kallikreins are a sub-family of serine proteases that play a role in the etiology of AD which is characterized by neuronal damage and loss of function.
Kallikrein (KLK)-6 and KLK-7 are known to be age related protease and found at a high levels in the central nervous system (CNS). It was previously shown to be involved in proteolysis of extracellular proteins implicated in neurodegenerative diseases such as AD.
In this study we aimed to investigate the possible role of KLK-6 and KLK-7 in the pathogenesis of AD and the relationship between potassium channels proteins.

METHODS: Materials and Methods: The 35 Alzheimer’s patients age over 65 years, followed by Polatlı Duatepe State Hospital and 35 healthy individual (control group) admitted to the neurology clinic for routine screening with cognitive status considered normal, were included in this study. After a 12 hour hunger, KLK-6 and KLK-7 with inwards corrective potassium channels (KCNJ3) and two-porous potassium channels (KCNK9) levels were measured by the Enzyme-Linked Immuno Sorbent Assay (ELISA) ) in the serum of the blood samples which taken from the antecubital vein by centrifuged for 10 minutes at 2500xg. The differences between the two groups were tested by T- test. A value of p<0.05 was considered statistically significant.
RESULTS: Results: All the groups were matched for age and gender but not statistically significant difference was observed (p>0.05). According to our findings, serum KLK-6 and KLK-7 levels of Alzheimer group were significantly increased (p<0.05). A significant difference was not detected when the levels of serum KCNJ3 and KCNK9 levels of the Alzheimer’s group compared with the control group (p>0.05).
DISCUSSION AND CONCLUSION: Conclusions: It is thought that the failure in preventing the abnormal protein folding and accumulation leads to AD in brain. According to the findings of the present work a positive correlation was detected between the levels of KLK-6 and KLK-7 and AD’s pathology.

4. Investigation of COVID-19 related web search behaviors in Turkey: A digital epidemiology study using google trends
Keziban AVCI
doi: 10.5505/TurkHijyen.2021.48991  Pages 133 - 146
INTRODUCTION: The use of internet data are being increasingly prefering in health area for predicting and analysing human behavior. In addition online search data are being widely used to predicting and monitor diseases occurrence and outbreaks. The most popular tool in health area for use of internet data is Google Trends, an open online tool that provides both real-time and archived information. The purpose of this study to explore the public awareness of COVID-2019 pandemic in Turkish using quantitative analysis of its digital footprints on the internet from January 1, 2020 to May 1, 2020. In addition, this study aims to investigate the relationship between online search behaviors about COVID-19 and the spreading speed of virus among different region and the effect of applied some precautionary strategies on search behavior.
METHODS: The following search terms were used to investigate awareness of COVID-19; “Corona+ Korona”,“Coronavirus+Koronavirüs+Koronavirus ”, “ "Covid 19"+"Covid-19" ”, “Pandemi (pandemic)”, “Salgın (epidemic)”. In this study, plus sign (+) which means “OR” was used to represent the combination of those multiple terms. Then relative search volumes (RSV) were determined for the most searching these keywords in country. Finaly, for the purpose of comparing the difference of public perception and attantion about COVID-19 across Turkey, 1) The similarity between the day when MoH announces the numbers of death and case, and nd the day when RSV reached the apex,2) The similarity between the day when official autority announces the precoution strategy and the day when RSV reached the apex were evaluaded.
RESULTS: Searches related to COVID-19 in Turkey rapidly increased following around January 30, 2020 when the epidemic was announced in China and reached a small peak. Then clear peak was seen around February 26,2020 when the number of infections rapidly increased in Italy. Finally, the apex point was seen around March 11,2020 when the announcements of the first case in the country. The search interest was continued with the massive precaution measures which were taken such as the closure of schools, and mosques, and travel restrictions. The duration of public attention in Turkey was 26 days from the March 11, 2020 to the April 5,2020.

DISCUSSION AND CONCLUSION: It is important that until disease introduction to Turkey, there were lots of COVID-19 related searches. However, after mid-April, queries for all search terms had been dramatically declining. As a conclusion this study shows that the authorities should more strengthen the publicity of COVID-19 nationally and inform public.

5. Comparing the biofilm formation properties of S. maltophilia isolates obtained from the pulmonary and extrapulmonary samples
Kemal BİLGİN, Yeliz TANRIVERDİ ÇAYCI, İlknur BIYIK, Demet GÜR VURAL, Elif GÜLSÜM TORUN, Asuman BİRİNCİ
doi: 10.5505/TurkHijyen.2020.09365  Pages 147 - 152
INTRODUCTION: Stenotrophomonas maltophilia is commonly a nosocomial pathogen with multiple drug resistance, although it has been reported in community-acquired infections. Trimethoprim-sulfamethoxazole is prefered as the first antibiotic in the treatment of S. maltophilia infections. In hospitalized patients, the respiratory tract is the body area that it is most frequently isolated. Although it is known about the bacteria that it causes the formation of biofilms due to its ability to attach to plastic surfaces, relatively little is known in terms of virulence factors. The aim of our study is to compare the property of biofilm formation of pulmonary and extrapulmonary isolates of S. maltophilia.
METHODS: A total of 78 S. maltophilia isolates isolated from 37 pulmonary and 41 extrapulmonary specimens were included in the study. The identfication of the isolates were performed in Vitek MS (bioMérieux, Fransa) automated system. In addition, antibiotic susceptibility was tested using the disc diffusion method. All isolates were investigated for biofilm formation by microtitration plate method. The relation of the pulmonary or extrapulmonary specimens with the biofilm formation was statistically investigated.
RESULTS: All of the isolates were found susceptible to trimetoprim-sulphametaxozole. Biofilm formation was detected in 68 of 78 (87.2%) S. maltophilia isolates. Biofilm formation of S. maltophilia isolates that isolated from pulmonary and extrapulmonary specimens were detected as 35/37 (94.6%), 33/41 (80.5%), respectively. There was no statistical relationship between pulmonary or extrapulmonary samples and biofilm forming capability.
DISCUSSION AND CONCLUSION: It was observed that a significant part (87.2%) of all strains included in the study were formed biofilms. It was seen that the biofilm activity of the pulmonary specimens was proportionally higher than extrapulmonary specimens. However, there was no significant difference in terms of biofilm formation between the strains isolated from pulmonary and extrapulmonary specimens. It was also detected that there was no development of resistance to trimethoprim-sulfamethoxazole. New comprehensive studies on this subject may contribute to a better understanding of the virulence mechanisms of S. maltophilia.

6. Total PSA ve serbest PSA testlerinin analitik performansının 6-sigma yöntemi ile değerlendirilmesi
Çiğdem YÜCEL, Müjgan ERCAN, Murat KIZILGÜN
doi: 10.5505/TurkHijyen.2020.19052  Pages 153 - 158
INTRODUCTION: Sigma Metrics Methodology is a quality measurement method in order to evaluate and compare the analytical performance of laboratories. Six Sigma can be used as a clinical analytical phase assessment methodology to form an internal quality control (IQC) strategy and plan its frequency. In this study, we aimed to evaluate the analytical performance of the tumor markers total and free prostate specific antigen (PSA) by calculating process sigma values.
METHODS: Sigma levels for both tests were analyzed by using IQC values retrieved from laboratory information system for consecutive 3 months. Bias and coefficient of variation (CV) were calculated. Biological variation databases were used for Total Allowable Error (TEa). The calculated sigma values were classified as follows: ">5", "4-5", "3-4" and " <3” as very good, good, minimum and unacceptable respectively.
RESULTS: The sigma values of total PSA and free PSA tests according to the 3-month data of two IQC levels were found to > 5 and 4-5 for total PSA,while those for free PSA were <3 for both levels.
DISCUSSION AND CONCLUSION: Our results showed that performance of total PSA is good while performance of free PSA is unacceptable. It is possible to determine a test with high error probability by evaluating the fine sigma levels and the tests that must be quarded by more stringent quality control applications. Our study has shown that we need to apply a more stringent IQC regime for fPSA test.

7. Bazı hidrazon ve kalkon türevlerinin biyolojik aktivite taraması
Begüm EVRANOS AKSÖZ, Fatma KAYNAK ONURDAĞ, Erkan AKSÖZ, Selda ÖZGEN ÖZGACAR
doi: 10.5505/TurkHijyen.2020.02439  Pages 159 - 166
INTRODUCTION: Today, the development of resistance to antibiotics is increasing rapidly. Loss of antibiotics effectiveness due to the development of resistance causes serious hazards. Especially methicillin-resistant Staphylococcus aureus and Enterococcus faecalis isolate which is resistant to vancomycin create life-threatening conditions in patients with a weak immune system. For this reason, it is important to develop new drug active substances against these microorganisms. In this article, hydrazones and chalcones were synthesized and some biological activities of these compounds such as antimicrobial and antituberculosis were investigated. This study was conducted to investigate new antimicrobial and antituberculous drug-active substances instead of antibiotics that lost their effectiveness due to the rapidly increasing resistance to existing antibiotics.
METHODS: The compounds were obtained by synthesis of chalcones via Claisen Schmidt condensation and then by reacting these chalcones with hydrazides to form hydrazone. Antimicrobial and antituberculosis activities of the compounds were evaluated by microdilution method and microplate alamar blue assays, respectively.
RESULTS: All compounds displayed the same activity against Mycobacterium tuberculosis (MIC: 64 µg/mL). E4, 4’-Bromo-4-methyl chalcone, displayed the best activity against Meticilline resistant-Staphylococcus aureus (MIC: 16 µg/mL). The most effective compounds against Enterococcus faecalis isolate and Enterococcus faecalis were found B26 (N-(1-(4fluorophenyl)-3-p-tolylallylidene)thiophene-2-carbohydrazide) and E8 (4’-fluoro-3-methyl chalcone), respectively (MIC: 32 µg/mL).
DISCUSSION AND CONCLUSION: Hydrazones have been found to be more effective than chalcones against S. aureus ATCC 29213. However, chalcones exhibited a remarkable effect against meticilline resistant-Staphylococcus aureus and Enterococcus faecalis. In terms of tuberculosis bacillus, it did not differ whether the compounds had a chalcone or hydrazone structure. Both chalcones and hydrazones exhibited the same activity against candida species. All compounds were observed to be more effective than gentamicin against methicillin-resistant Staphylococcus aureus.

8. Sıçanlarda epilepsi modelinde vagus sinirinde norepinefrin taşıyıcılarının immünohistokimyası
Kristina Ayşe POLAT, Eslem GÜNEY, Züleyha DOĞANYİĞİT, Enes AKYÜZ
doi: 10.5505/TurkHijyen.2020.74507  Pages 167 - 174
INTRODUCTION: Epileptic seizures occur as a result of the excessive and uncontrolled spread of electrical activity. Studies have reported that epileptic seizures may affect the autonomic function. However, the main molecular mechanism has not been elucidated yet.
Since the vagus nerve (VN) carries 75% of all parasympathetic autonomic system fibers, it is of great importance in the autonomic center associated with epilepsy pathogenesis. Afferent sensory fibers of the VN have connections to associated regions in the brainstem, providing norepinephrine (NE) transmission. Therefore, the suppressive effect of NE on seizures and the role of NE transporters (NET) in neurotransmitter reuptake may occur. Examining the neurotransmitters and related transporters in the autonomic nervous system at the molecular level may help to concede the related mechanism.
In this study, we aimed to determine the NET-associated immunoreactivity in the VN in pentylenetetrazole (PTZ) epilepsy model.

METHODS: Epileptic seizures were induced in Wistar rats (280-380g) by administering PTZ (first 12 injections 35 mg/kg, I. P. and last injection 50 mg/kg I. P.) for 28 days. Subsequently, the VN dissected as two parts, in terms of the thoracic and cervical sections. The NET levels were evaluated by immunohistochemical staining on the tissue samples.
RESULTS: Expression of NETs in the thoracic VN region of the male and female experimental groups significantly increased. In the cervical VN, NET expression was significantly increased in epileptic male rats, while decreased in epileptic female rats compare to control groups.
DISCUSSION AND CONCLUSION: Our study may disclose significant regional enhancement in the NET immunoreactivity. It is assumed that NET, which plays a role in NE regulation, may be characterized by abnormal neural stimulation. The findings provide proof-of-concept that alterations in NET expression may affect the ANS dysfunctions associated with epilepsy.
The data obtained from this study may be crucial to illuminate the pathologies that occur in VS. It is also a preliminary study in the development of new targets for preventing treatment resistance in epilepsy and pathologies associated with autonomic nervous system disorders.


9. Toxoplasma gondii in pregnant women in Kars province: Seroprevalance and possible risk factors
Funda DEMİRCİ, Neriman MOR
doi: 10.5505/TurkHijyen.2021.77528  Pages 175 - 186
INTRODUCTION: In this study, it was aimed to determine the seroprevalence of T.gondii in healthy pregnant women in Kars province, to examine possible risk factors that may be associated with infection and to evaluate the effect of risk factors on seropositivity.
METHODS: The material of the study was composed of 308 healthy pregnant women between the ages of 15-45 and matching 6-39 weeks of gestation between September 2018 and March 2019. After the pregnant women were informed about the study, each pregnant woman filled a questionnaire form consisting of 26 questions related to the infection by adhering to the voluntary principle. In addition, serum samples of blood requested from these individuals during routine examination were examined for anti T.gondii antibodies by commercial micro-ELISA method. Questionnaire and laboratory results were evaluated statistically in SPSS 20 program.
RESULTS: Anti-T gondii IgG seropositivity was detected as 44.8% (138/308) in the pregnant women in the study. While seropositivity of both IgM and IgG of anti-T. gondii was determined as 0.3% (1/308), anti-T.gondii lgM seropositivity alone was not detected. Toxoplasma gondii seropositivity was determined according to the responses of pregnant women to possible risk factors such as socio-demographic characteristics, pregnancy status, social habits, eating habits and hygiene habits. The seropositivity of T.gondii in pregnant women who stated low income, aged between 30 and 34, consumed raw eggs, used tap water as a drinking water source among possible risk factors, was statistically significant compared to other possible risk factors (P˂ 0.05).
DISCUSSION AND CONCLUSION: It was observed that T. gondii seropositivity was significant in Kars province. It has been determined that possible risk factors such as socioeconomic conditions, social, nutritional and hygienic habits are effective on toxoplasmosis in pregnant women and pregnant women do not have information about the infection. For this reason, it has been concluded that it is important and necessary to educate pregnant women about the ways of protection from the infection.

10. Investigation of tick bites and affecting factors in Ezine district of Çanakkale
Buse YÜKSEL, Esen EKER, Taylan ÖNDER, Özgür ÖZERDOĞAN, Alper ŞENER, Sibel OYMAK, Coşkun BAKAR
doi: 10.5505/TurkHijyen.2021.24992  Pages 187 - 196
INTRODUCTION: According to the World Health Organization, approximately 17% of all infectious diseases are vector-borne diseases. Turkey has convinient climate and vegetation conditions for ticks, therefore Turkey is a risky country for diseases transmitted by ticks. The aim of this study is to investigate the prevalence of tick bite and the factors affecting of tick bite in those living in Mahmudiye and Pınarbaşı villages in the rural area of Ezine district of Çanakkale.
METHODS: This cross-sectional study was carried out in 2018 in the villages of Mahmudiye and Pınarbaşı of the Ezine district of Çanakkale. Before the study, permissions were obtained from the Clinical Research Ethics Committee of Çanakkale Onsekiz Mart University. The sample size was calculated as 329 people, by the formula of sample size used to estimate the population rate. A total of 420 people were contacted, including 292 from Mahmudiye and 128 from Pinarbasi. The non-probability sampling method was used to reach the number of samples determined in the villages. The data of the study was obtained by a questionnaire which was applied by face to face interview technique. The questionnaire consisted of 23 questions questioning the demographic characteristics of participants, their knowledge and attitude about tick bite and tick-borne diseases. Logistic regression analysis was performed to analyze the risk factors associated with a tick bite. After the data collection phase of the research, education about prevention from tick bites was given to women and men in both villages to prevent tick bites.
RESULTS: The prevalence of tick bite was 20%. The risk of tick bite was 3.4 times higher for males than females; 3.0 times higher for the people whom level of education below high school than high school and above; 3.4 times higher for occupations of farming, animal husbandry, agricultural technician, butchery than the other occupations.
DISCUSSION AND CONCLUSION: There are protective measures to prevent tick bite and the basis of preventive health services is health education. It is thought that training to be made for raising awareness about tick bites for individuals who live in rural areas, especially those who are engaged in agriculture and animal hunsbandry will be beneficial. In addition, we recommend screening for health problems that may be caused by tick bite in this rural area where one of five people was bitten by a tick.

11. Cinnamomum cassia metanolik ekstraktı ilave edilen diş macunlarının in vitro incelenmesi
Gülşah TOLLU, Elif Ayşe ERDOĞAN ELİUZ
doi: 10.5505/TurkHijyen.2021.25993  Pages 197 - 204
INTRODUCTION: In recent years, many types of bacteria have developed resistance to antibiotics and antibacterial chemicals used in the treatment of diseases caused by microorganisms. Therefore, researchers have sought an alternative product instead of synthetic chemicals. Phytochemicals in traditional medicine and isolated from plants are seen as a good alternative to synthetic chemicals. The aim of this study is to investigate the antibacterial activity of toothpastes of different contents and toothpastes with Cinnamomum cassia methanolic extract added against Staphylococcus aureus bacteria.
METHODS: In the study, the experimental set up of six different toothpastes, a positive control group of a fluoride-free toothpaste and a negative control group of distilled water constituted. Anti-Staphylococcus aureus activities of toothpastes alone and with C. cassia methanol extract were determined according to the disk diffusion method and n=3 repeats. Inhibition zone diameters were determined using caliper and all results were evaluated statistically
RESULTS: According to the GC / MS analysis, Cinnamaldehyde, one of the main ingredients of C. cassia methanolic extract, was found higher than other components. The most effective toothpaste was found different after adding C. cassia methanol extract and the most effective toothpaste after adding C. cassia methanol extract (p <0.05).
DISCUSSION AND CONCLUSION: With this study, it has been shown that toothpaste with low or no anti-Staphylococcus aureus effect can be enriched with herbal extracts to increase their biological activities. Studies that evaluate the effectiveness of toothpaste-oil combinations, which have been proved by us to have antibacterial efficacy, for other components such as remineralization, whitening, desensitization, which form oral and dental health, should also be conducted. In further studies, it has been determined that various studies can be conducted to use the essential oils used in the study in place of various antimicrobials in the content of toothpastes.



REVIEW
12. Cerebral ischemia model created by transient middle cerebral artery occlusion
Güven AKÇAY
doi: 10.5505/TurkHijyen.2021.73693  Pages 205 - 218
Our brain weighs an average of 1500 g and uses approximately 20% of the cardiac output, although it corresponds to approximately 2% of our total body weight. Cerebral blood flow constitutes the most important place in the nutrition of the brain and cerebral ischemia occurs as a result of a decrease in cerebral blood flow in a selected brain region or the whole brain. Cerebral ischemia occurs as a result of blockage or bleeding in the vessels feeding the brain or depending on the characteristics of the blood. There are approximately 17 million stroke cases per year in the world, and approximately 132.000 cases are seen annually in our country. Since stroke cases are increasing every year, it is predicted that it will cause health and economic problems in the future, and the prevention of stroke and the application of effective treatments are of great importance. Experimental animal models are preferred for discovering new agents and developing new treatment protocols in cerebral ischemia treatment research. Transient global cerebral ischemia, transient focal cerebral ischemia and transient forebrain ischemia models are mostly used in rodents such as rats and mice to investigate the pathophysiology of cerebral ischemic diseases in humans.. The most common type of ischemia encountered in the clinic is focal cerebral ischemia and the most important causes are occlusion of the internal carotid artery or its largest branch, the middle cerebral artery. Transient cerebral ischemia animal models (with carotid and/or vertebral artery occlusion) beautifully mimic human ischemia. The ischemia model created by transient middle cerebral artery occlusion is one of the most widely used models in investigating the pathophysiology of stroke. In this review; information on the epidemiology, pathophysiology of cerebral ischemia and middle cerebral artery occlusion method, which is the most used animal models of experimental cerebral ischemia, are presented. In light of this information, cerebral ischemia treatment studies often preferred model of transient middle cerebral ischemia important information about the issues to be considered in the creation will be acquired.

13. Vaccine epidemiology: Observational vaccine safety study designs
Can Hüseyin HEKİMOĞLU
doi: 10.5505/TurkHijyen.2021.29567  Pages 219 - 232
After a vaccine programme introduced into a population, vaccine safety monitoring is an important issue as much as vaccine effectiveness monitoring. Concerns about vaccine safety may continue in a population after licensure, although vaccine safety were proven before licensure. While a vaccination programme implemented, it may need to respond questions about vaccine safety more quickly than questions about vaccine effectiveness. These concerns that will be revealed by pharmacovigilance reports and active surveillance should be eliminated or verified. In addition adverts events that were detected before licensure should be shown more accurately and more precisely. Therefore vaccine safety monitoring with observational studies should be a part of a vaccine programme. In traditional cohort and case-control studies finding unvaccinated groups is difficult due to high vaccine coverage and providing comparability between vaccinated and unvaccinated groups is rarely possible. Therefore alternative study designs have been developed for vaccine safety monitoring at post-licensure stage. However evaluation of long term adverse events associated with vaccines is more difficult than evaluation of short term adverse events. Long term adverse events can only be determined with studying vaccinated and unvaccinated groups over a period of time and comparing them. However alternative designs are used for investigation of short term and rare adverse events. The alternative vaccine safety study designs are ‘risk interval design’, ‘self-controlled risk interval design’, ‘self-controlled case series method’, case-centered approach’, case-crossover design’, ‘case-time-control design’, ‘case-coverage design’ and ‘ecologic studies’. According to the question sought in the vaccine safety study and available data sources, choosing the appropriate one from among these traditional and alternative designs is necessary to make correct conclusions. Therefore limitations, strenghts and assumptions of the vaccine safety study designs should be necessarily known and considered.

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