Journal of Clinical and Diagnostic Research, ISSN - 0973 - 709X

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On Aug 2018




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MBBS, MD (Pathology),
Sanjay Gandhi institute of trauma and orthopedics,
Bengaluru.
On Aug 2018




Dr. Mamta Gupta,
"It gives me great pleasure to be associated with JCDR, since last 2-3 years. Since then I have authored, co-authored and reviewed about 25 articles in JCDR. I thank JCDR for giving me an opportunity to improve my own skills as an author and a reviewer.
It 's a multispecialty journal, publishing high quality articles. It gives a platform to the authors to publish their research work which can be available for everyone across the globe to read. The best thing about JCDR is that the full articles of all medical specialties are available as pdf/html for reading free of cost or without institutional subscription, which is not there for other journals. For those who have problem in writing manuscript or do statistical work, JCDR comes for their rescue.
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Reviewing articles is no less a pain staking process and requires in depth perception, knowledge about the topic for review. It requires time and concentration, yet I enjoy doing it. The JCDR website especially for the reviewers is quite user friendly. My suggestions for improving the journal is, more strict review process, so that only high quality articles are published. I find a a good number of articles in Obst. Gynae, hence, a new journal for this specialty titled JCDR-OG can be started. May be a bimonthly or quarterly publication to begin with. Only selected articles should find a place in it.
An yearly reward for the best article authored can also incentivize the authors. Though the process of finding the best article will be not be very easy. I do not know how reviewing process can be improved. If an article is being reviewed by two reviewers, then opinion of one can be communicated to the other or the final opinion of the editor can be communicated to the reviewer if requested for. This will help one’s reviewing skills.
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Dr. Mamta Gupta
Consultant
(Ex HOD Obs &Gynae, Hindu Rao Hospital and associated NDMC Medical College, Delhi)
Aug 2018




Dr. Rajendra Kumar Ghritlaharey

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Writing is the representation of language in a textual medium i e; into the words and sentences on paper. Quality medical manuscript writing in particular, demands not only a high-quality research, but also requires accurate and concise communication of findings and conclusions, with adherence to particular journal guidelines. In medical field whether working in teaching, private, or in corporate institution, everyone wants to excel in his / her own field and get recognised by making manuscripts publication.


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Salient features of the JCDR: It is a biomedical, multidisciplinary (including all medical and dental specialities), e-journal, with wide scope and extensive author support. At the same time, a free text of manuscript is available in HTML and PDF format. There is fast growing authorship and readership with JCDR as this can be judged by the number of articles published in it i e; in Feb 2007 of its first issue, it contained 5 articles only, and now in its recent volume published in April 2011, it contained 67 manuscripts. This e-journal is fulfilling the commitments and objectives sincerely, (as stated by Editor-in-chief in his preface to first edition) i e; to encourage physicians through the internet, especially from the developing countries who witness a spectrum of disease and acquire a wealth of knowledge to publish their experiences to benefit the medical community in patients care. I also feel that many of us have work of substance, newer ideas, adequate clinical materials but poor in medical writing and hesitation to submit the work and need help. JCDR provides authors help in this regards.
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Every one of us: authors, reviewers, editors, and publisher are responsible for enhancing the stature of the journal. I wish for a great success for JCDR."



Thanking you
With sincere regards
Dr. Rajendra Kumar Ghritlaharey, M.S., M. Ch., FAIS
Associate Professor,
Department of Paediatric Surgery, Gandhi Medical College & Associated
Kamla Nehru & Hamidia Hospitals Bhopal, Madhya Pradesh 462 001 (India)
E-mail: drrajendrak1@rediffmail.com
On May 11,2011




Dr. Shankar P.R.

"On looking back through my Gmail archives after being requested by the journal to write a short editorial about my experiences of publishing with the Journal of Clinical and Diagnostic Research (JCDR), I came across an e-mail from Dr. Hemant Jain, Editor, in March 2007, which introduced the new electronic journal. The main features of the journal which were outlined in the e-mail were extensive author support, cash rewards, the peer review process, and other salient features of the journal.
Over a span of over four years, we (I and my colleagues) have published around 25 articles in the journal. In this editorial, I plan to briefly discuss my experiences of publishing with JCDR and the strengths of the journal and to finally address the areas for improvement.
My experiences of publishing with JCDR: Overall, my experiences of publishing withJCDR have been positive. The best point about the journal is that it responds to queries from the author. This may seem to be simple and not too much to ask for, but unfortunately, many journals in the subcontinent and from many developing countries do not respond or they respond with a long delay to the queries from the authors 1. The reasons could be many, including lack of optimal secretarial and other support. Another problem with many journals is the slowness of the review process. Editorial processing and peer review can take anywhere between a year to two years with some journals. Also, some journals do not keep the contributors informed about the progress of the review process. Due to the long review process, the articles can lose their relevance and topicality. A major benefit with JCDR is the timeliness and promptness of its response. In Dr Jain's e-mail which was sent to me in 2007, before the introduction of the Pre-publishing system, he had stated that he had received my submission and that he would get back to me within seven days and he did!
Most of the manuscripts are published within 3 to 4 months of their submission if they are found to be suitable after the review process. JCDR is published bimonthly and the accepted articles were usually published in the next issue. Recently, due to the increased volume of the submissions, the review process has become slower and it ?? Section can take from 4 to 6 months for the articles to be reviewed. The journal has an extensive author support system and it has recently introduced a paid expedited review process. The journal also mentions the average time for processing the manuscript under different submission systems - regular submission and expedited review.
Strengths of the journal: The journal has an online first facility in which the accepted manuscripts may be published on the website before being included in a regular issue of the journal. This cuts down the time between their acceptance and the publication. The journal is indexed in many databases, though not in PubMed. The editorial board should now take steps to index the journal in PubMed. The journal has a system of notifying readers through e-mail when a new issue is released. Also, the articles are available in both the HTML and the PDF formats. I especially like the new and colorful page format of the journal. Also, the access statistics of the articles are available. The prepublication and the manuscript tracking system are also helpful for the authors.
Areas for improvement: In certain cases, I felt that the peer review process of the manuscripts was not up to international standards and that it should be strengthened. Also, the number of manuscripts in an issue is high and it may be difficult for readers to go through all of them. The journal can consider tightening of the peer review process and increasing the quality standards for the acceptance of the manuscripts. I faced occasional problems with the online manuscript submission (Pre-publishing) system, which have to be addressed.
Overall, the publishing process with JCDR has been smooth, quick and relatively hassle free and I can recommend other authors to consider the journal as an outlet for their work."



Dr. P. Ravi Shankar
KIST Medical College, P.O. Box 14142, Kathmandu, Nepal.
E-mail: ravi.dr.shankar@gmail.com
On April 2011
Anuradha

Dear team JCDR, I would like to thank you for the very professional and polite service provided by everyone at JCDR. While i have been in the field of writing and editing for sometime, this has been my first attempt in publishing a scientific paper.Thank you for hand-holding me through the process.


Dr. Anuradha
E-mail: anuradha2nittur@gmail.com
On Jan 2020

Important Notice

Original article / research
Year : 2022 | Month : May | Volume : 16 | Issue : 5 | Page : AC05 - AC09 Full Version

Histopathological Changes in Kidneys of Developing Chick Embryo on Exposure to Artesunate


Published: May 1, 2022 | DOI: https://doi.org/10.7860/JCDR/2022/53730.16357
Rajesh Kumar, Lavlesh Kumar Mittal, Ghazal Mittal

1. Assistant Professor, Department of Anatomy, Saraswathi Institute of Medical Sciences, Hapur, Uttar Pradesh, India. 2. Reader, Department of Human Anatomy, Institute of Dental Education and Advance Studies, Gwalior, Madhya Pradesh, India. 3. Research Trainee, Department of Cell Biology, Dabur Research Foundation, Ghaziabad, Uttar Pradesh, India.

Correspondence Address :
Dr. Rajesh Kumar,
Saraswathi Institute of Medical Sciences, Pilakhua,
Hapur, Uttar Pradesh-245304 India.
E-mail: rajeshshakarwal99@gmail.com

Abstract

Introduction: Chick embryo is one of the most commonly used animals to study the adverse effects of various drugs for research purpose. In India, Malaria imposes incredible socio-economic burden on humankind. India reports approximately two million cases of malaria yearly, with large number of deaths. Surveys have demonstrated that the rates of treatment failure are higher than 50% due to Chloroquine resistance and poor efficacy of Sulphadoxine Pyrimethamine. Artesunate is a concentrate of Artemisia plant found in China, also called as Qinghaosu. It is a subordinate of a group of drugs artemisinin that have the most rapid action of all current drugs against Chloroquine resistant Plasmodium vivax and Plasmodium falciparum malaria.

Aim: To understand the adverse effects of Artesunate on kidney of developing chick embryo.

Materials and Methods: The present study was an experimental study which comprised of steps like selection and sampling of eggs in groups (control and experimental), selection and preparation of drug (dose titration), drug administration, incubation of eggs, manual hatching to obtain chick embryo, isolation of kidney, sectioning of kidney and staining for slide preparation, microscopical analysis of the slides. In this study, the fertilised eggs used were of White Leghorn chicken and were procured from King and King poultry farm Hapur, Uttar Pradesh, India. Hundred fertilised chicken eggs were divided into five experimental groups denoted by A, B, C, D, and E and five control groups denoted by a, b, c, d and e one for each experimental group respectively. Each experimental and control group had 10 eggs. Experimental groups A, B, C, D and E were exposed to artesunate with dose of 0.0004 mg, 0.0005 mg, 0.0006 mg, 0.0007 mg and 0.0008 mg respectively and control group a, b, c, d and e were treated with same concentration of normal saline as artesunate. The eggs were broken by scalpel on 18th day of incubation and chick embryos were obtained. The kidneys were removed sectioned, stained and studied using light and compound microscope.

Results: Histopathological changes like tubular degeneration, vacuolation in the cytoplasm of epithelium lining of Proximal Convoluted Tubules (PCT) and Distal Convoluted Tubules (DCT), congestion in Glomeruli, haemorrhage in urinary space and mild lymphocytic infiltration were observed.

Conclusion: Exposure to artesunate increases the risk of nephrotoxicity with increase of embryonic age.

Keywords

Congestion, Degeneration, Haemorrhage, Nephrotoxic, Vacuolation

The chick embryo is one of the most commonly used animals to study the adverse effects of various drugs, animal models are commonly considered in research for ethical reasons, as chick has short gestation period (21 days), its eggs are fairly large in size therefore easy to handle, they are available throughout the year, can be incubated artificially and also the chick embryology is much like that of humans in general (1),(2).

In India, Malaria imposes incredible socio-economic burden on humankind along with six other diseases like Diarrhoea, Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS, Tuberculosis, Measles, Hepatitis B and Pneumonia (3). India reports approximately two million cases of malaria yearly, with large number of deaths (4). It has been demonstrated in many surveys that there is more than 50% failure in the treatment rate due to Chloroquine resistance and the poor efficacy of Sulphadoxine and Pyrimethamine combination (5).

Artesunate is derived from Artemisia plant, which is found in China. It is also referred as Qinghaosu. It is one of the subordinate in a group of drugs artemisinin which have the most rapid action against Chloroquine resistant Plasmodium vivax and Plasmodium falciparum Malaria as compared to all other current drugs (6). Toxic effects are less frequent with artesunate than with other antimalarial drugs. The most common side effects are neurotoxicity, embryo toxicity, genotoxicity, haemato and immuno toxicity, cardio toxicity, nephrotoxicity and allergic reactions (7).

Malaria parasite is developing resistance against chloroquine drugs at fast rate (8),(9). Currently, artesunate is the choice of drug for the treatment of chloroquine resistance malaria and malaria falciparum (10). If artesunate is misused, it may prove to be toxic or have side-effects (11). Research on Artesunate is scanty in the current date. Hence, this study was conducted to assess the adverse effects of artesunate on kidney of developing chick embryo.

Material and Methods

This study was an experimental study, conducted in the Department of Anatomy, Saraswathi Institute of Medical Sciences, Hapur road Pilakhua, Uttar Pradesh, India between December 2020-January 2022, after taking ethical clearance from animal ethical committee (SIMS/Ana 3483).

This experimental study was performed with steps like selection and sampling of eggs in groups (control and experimental), selecting the appropriate drug artesunate and preparing it (dose titration) for drug administration, incubation of eggs for embryo development at appropriate conditions, manual hatching of eggs to obtain chick embryo, isolation of kidney organ, sectioning of kidney for slides preparation and staining the slides for microscopical analysis, analysing the findings.

In this study the fertilised eggs of White Leghorn chicken eggs were used which were procured from King and King poultry farm Hapur, Uttar Pradesh, India.

Inclusion criteria: Fertilised eggs of White Leghorn chicken were included.

Exclusion criteria: The unfertilized eggs (detected by candling of the eggs), damaged eggs, eggs not having air cell at broader end and eggs having blood clot in air cell were discarded.

Study procedure: In the main study, the fertilised chicken eggs were divided into five experimental groups denoted by A, B, C, D, and E and five control groups denoted by a, b, c, d and e one for each experimental group, respectively. Each experimental and control group had 10 eggs. The artesunate injection used in the study was Injection Falcigo (Zydus Cadila). Automatic Tissue Processor (Thermo Scientific, Germany) and Rotary Microtome (Thermo Scientific, Germany) were used for tissue processing and the slide preparation. The stains used for staining the tissue were Haematoxylin and Eosin (H&E) (Sigma Aldrich). The slides were studied using Light Microscope and Compound Microscope. The chemicals and instruments used in this study were selected based on availability.

Determination of gross anatomy and histology of normal chicken kidney (Preliminary Study 1): Before starting the main study, a preliminary study was conducted in order to study normal histology of kidneys of chick embryo. Ten fertilised eggs of White Leghorn chicken were developed in incubator without normal saline or distilled water or any drug. On 18th day of incubation eggs were broken and chick embryos were obtained and sacrificed by drowning method. After sacrificing, the chick embryos were dissected and both kidneys were isolated and preserved in 10% formaldehyde solution. The tissues were washed in running water. With aseptic measures, all tissue samples were passed through automatic tissue processor (Thermo Scientific Germany) for 24 hours. Blocks were made and sectioned at 4-6 μm thickness by rotary microtome (Thermo Scientific Germany) and stained with H&E. The sections were studied with light and compound microscope to know the normal histological features of the kidneys.

The kidneys of chicken are located on dorsal wall of trunk on either side of vertebral column immediate behind the lungs and are brownish in colour. The collecting tubules empty into ureters which empty into cloaca. Cloaca is a common vestibule into which the digestive tract and reproductive tract also empties. The kidneys of chicken are multi lobulated and have three types of nephrons, those with loop of Henle (looped) and without loop of Henle (loop less). Loop less (reptilian) nephrons are more in number than looped (mammalian) nephrons (12),(13). Loop less nephrons lie only in cortex while looped nephrons extends up to medulla. Beside reptilian and mammalian type of nephrons another intermediate type of nephrons was also found. These nephrons are intermediate in structure between reptilian and mammalian type nephrons. The chicken kidneys are composed of large and small renal corpuscles which consist of Bowmen’s capsule and glomerulus. The PCT, DCT and collecting ducts are lined by simple cuboidal epithelium. Thick and thin segments of loop of Henle are also lined by simple cuboidal epithelium (Table/Fig 1), (Table/Fig 2).

The drug given was injection Falcigo (Artesunate 120 mg).

Dose titration: The recommended dose of artesunate in human being is 2.4 mg/kg of body weight (14). The weight of chick embryo on the 5th day of incubation was 0.13 gm (15). So, the dose of artesunate for chick embryo at 5th day of incubation will be (2.4 x 0.13)/1000 = 0.000312 mg = 0.0003 mg.

One mL of sodium bicarbonate was injected in 120 mg artesunate vial (provided by the manufacturer), in order to achieve concentrated solution for easy drug administration. The solution was remained to be clear. Solution of 120 mg artesunate and 1 mL of sodium bicarbonate was mixed with 11 mL of normal saline. The solution was mixed until it became clear. This was named as solution A. Final concentration of solution A was 10 mg artesunate present in 1mL of solution A. One mL Solution A was diluted by adding 9 mL normal saline to make 10 mL called Solution B. One mL solution B contain 0.1mg artesunate diluted by adding 9 mL normal saline to make 10 mL called solution C. One mL solution C contains 0 .01 mg artesunate diluted by adding 9 mL normal saline to make 10 mL called solution D. 1 mL solution D contains 0.001 mg artesunate. If, 0.001 mg artesunate was present in one mL of solution D, then 0.0003 mg artesunate was present in (1 x 0.0003) / 0.001= 0.3 mL of solution D.

Estimation of toxic dose of artesunate (Preliminary Study 2): The fertilised eggs were divided into eight groups having 10 eggs each. All the eggs were thoroughly washed with soap water solution and candled. For candling of eggs, a wooden box was made of which the insides were painted with black and given an electric bulb connection. The slots for egg placement were made in the roof of the box. The eggs first were candled to locate the air cell and then put into incubator trays with broad ends up (15). The incubator was maintained at an optimum temperature of 38°C and a relative humidity of 70-80% (16). On 5th day of incubation (17), seven groups were injected by tuberculin syringe with solution D in the dose of 0.0003 mg, 0.0004 mg, 0.0005 mg, 0.0006 mg, 0.0007 mg, 0.0008 mg and 0.0009 mg and no drug was given to the 8th group. The eggs were shaken between two hands for 3 to 4 times. The broad end of eggs was wiped with sterile gauze pad moistened with 70% isopropyl alcohol solution. A hole was drilled in egg shells in the centre of the surface over the air cell with a lancet. It was taken care not to damage the shell membranes with point of drill. The needle of the tuberculin syringe filled with solution D was inserted horizontally into the air cell through point of drill and the solution was injected (18),(19). The needle of the syringe was sterilised after wiping with sterile 70% isopropyl alcohol swab between each injection. The hole of the shell was sealed with melted candle wax immediately after injection. After the injection procedure the eggs were again put into incubator with same due care. The eggs were broken on 18th day of incubation (20). The toxicity of the drug was estimated after manually hatching the eggs and observing the development of chick’s embryos. At the dose of 0.0009 mg chick embryos were dead and the mortality rate was 90%. In contrast with smaller doses 0.0004 mg, 0.0005 mg, 0.0006 mg, 0.0007 mg and 0.0008 the survival rate was 80-90%. However, the embryos obtained from smaller dose of 0.0003 mg and 8th group apparently showed no abnormality. From the above study it was proved that dose of 0.0009 mg of artesunate is lethal dose for chick embryo. Hence five doses 0.0004 mg, 0.0005 mg, 0.0006 mg, 0.0007 mg and 0.0008 mg were selected for assessment.

Drug administration: The candling, drug administration, incubation procedures were performed same as in preliminary study 2. Experimental groups A, B, C, D and E were exposed to artesunate with dose of 0.0004 mg, 0.0005 mg, 0.0006 mg, 0.0007 mg and 0.0008 mg respectively and control group a, b, c, d and were treated with same volume of normal saline with same concentration as artesunate drug. On 18th day of incubation eggs were broken and chick embryos were obtained. The chick embryos were sacrificed through drowning method and dissected through ventral wall of the trunk. The kidneys were removed and preserved in 10% formaldehyde solution.

Sectioning and Staining: The tissues were washed in running water. All aseptic measures were followed to avoid contamination of the tissues. The slides were prepared similarly as mentioned in the preliminary study 1.

Results

The kidneys of chick embryos were found located on dorsal wall of trunk either side of vertebral column just caudal to lungs and brownish in colour.

Histologically three types of nephrons were found in the kidneys, those with loop of Henle (looped) and without loop of Henle (loop less), intermediate type of nephron. Both types of nephrons, loop of Henle (looped) and without loop of Henle (loop less) types of nephrons were more or less similar in number. Loop less nephrons lie in cortex only, while looped nephrons extends up to medulla. Beside Reptilian (without loop of Henle) (12) and Mammalian (with loop of Henle) (13),(21) type of nephron third intermediate type of nephrons was also found. These nephrons were intermediate in structure between reptilian and mammalian type nephron. The chicken kidneys were composed of large and small renal corpuscles consisting Bowman’s capsule and glomerulus. The PCT, DCT and collecting ducts were lined by simple cuboidal epithelium. Thick and thin segments of loop of Henle were also lined by simple by cuboidal epithelium. All the above histological findings observed were more or less same as observed in preliminary study 1.

Mild to moderate tubular fatty degenerative changes were found in all experimental groups except experimental groups A and B. Minute vacuolation with or without fat globules in the cytoplasm of lining epithelium of proximal and distal convoluted tubules were observed in experimental groups C, D and E (Table/Fig 3), (Table/Fig 4). Congestion in glomeruli was found in experimental group D and E (Table/Fig 5), (Table/Fig 6). Some nephrons in experimental group D and E showed haemorrhage in urinary space (Table/Fig 7). In all experimental groups except experimental group A capillaries were found mildly distended. Mild lymphocytic infiltration was observed in renal parenchyma in all experimental groups (Table/Fig 3), (Table/Fig 4). Lymphocytic infiltration is accumulation of lymphocytes (22).

(Table/Fig 8), (Table/Fig 9) shows the summary of chick embryos found with or without histopathological changes per experimental and control groups.

Discussion

Any chemical substance that is administered during time of development and can cause some disturbances during embryonic development is called teratogen (23). A teratogen exerts its influence on the developing tissues of chick embryos resulting in malformations either structural or functional. The initial event in teratogenesis may be brought by a teratogen itself or by its metabolites. A teratogen can influence on an organ primordium which will be malformed later or on the embryonic tissues other than the one going to be malformed and the maternal tissue or placenta (15).

Artesunate can act indirectly through generating high levels of Reactive Oxygen Species (ROS) through its anti-malarial properties or directly as toxin to the nephrons by affecting their cellular integrity and causing defect in membrane permeability and cell volume homeostasis (24).

In the studies conducted by other researchers, the symptoms observed after exposure of chicken embryo to other teratogens were similar as found in present study. Such as lymphoid infiltration in kidney of chick embryo (25), degenerative changes in glomerulus (26), and acute kidney failure and haemoglobinuria (27).

Further immunohistochemistry studies can be conducted in order to have wide knowledge regarding the symptoms observed. Further studies such as effect of artesunate on other organs of chicken embryos can be done for better understanding of the utmost of Artesunate.

Based on present findings, it can be said that the drug should not be misused. It should be used and administered only when genuinely required to the patient. Else it may prove nephrotoxic.

Limitation(s)

The study was performed on chick embryo animal model (aviary group). Although the embryology of chick embryos is somewhat similar to mammal including humans, it only gives the idea regarding the toxicity of the drug. Therefore, the study must be performed in mammal research models for more better understanding of effects of artesunate in humans.

Conclusion

Research on artesunate is scanty and the parasite is developing resistance at a fast rate. Currently, artesunate is the choice of drug for the treatment for chloroquine resistant malaria and life-threatening malaria falciparum.

As it can be seen in the observations (vacuolation in DCT, congestion in glomeruli, lymphocytic infiltration etc.) that the drug has some side effects on the kidney of chick embryo.

Therefore, if we tend to use this life saving drug haphazardly, Malaria parasite may also become resistant to artesunate like chloroquine in near future. Therefore, the drug must be used adequately and only when genuinely required.

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DOI and Others

DOI: 10.7860/JCDR/2022/53730.16357

Date of Submission: Jan 09, 2022
Date of Peer Review: Feb 04, 2022
Date of Acceptance: Mar 21, 2022
Date of Publishing: May 01, 2022

AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? NA
• For any images presented appropriate consent has been obtained from the subjects. NA

PLAGIARISM CHECKING METHODS:
• Plagiarism X-checker: Jan 10, 2022
• Manual Googling: Mar 08, 2022
• iThenticate Software: Mar 29, 202 (22%)

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