Novel Osteoporosis Therapeutic Targets Derived from Medical Biotechnology 2 2 Osteoporosis is known as the most prevalent metabolic bone disease. Population aging and increasing life span are making osteoporosis one of the most challenging age-related diseases 1. Based on reports in 2009, over 500 million people are struggling with this disease. Also, 1 in 5 men and 1 in 3 women over 50 years suffer from osteoporotic fractures during their lifetime. 1.5 million osteoporotic fractures are occurred annually in the United States and it cost the healthcare system in this country about $57 billion in 2018. The disease is caused by an imbalance in bone homeostasis, which is induced by bone formation not compensating for bone resorption. This process lowers the Bone Mineral Density (BMD) and makes bone susceptible to fracture 2,3. The significant burden of osteoporosis can be decreased since the disease is treatable. Based on the mechanisms resulting in osteoporosis, two main conventional drug categories are used for the treatment of the disease including anti-resorptive agents and anabolic agents 2. Conventional treatments consist of Selective Estrogen-Receptor Modulators (SERMs), bisphosphonates, parathyroid hormone analogs, and calcitonin. The need for more advanced therapies with fewer adverse effects arises from complications such as increasing risk of cardiovascular diseases, gastrointestinal problems, altering blood calcium levels, potential risks for women in estrogen-related therapies, etc. 4,5. As mentioned previously, the imbalance of bone metabolism causes osteoporosis. In this dynamic complex process of metabolism which is known as bone remodeling, connection or disruption of each specific intra-cellular or inter-cellular link can cause or treat the disease 6. Hence, this fact represents the remarkable role of medical biotechnology in osteoporosis treatment. Medical biotechnology as an upcoming main pillar of health-related science, has grown so fast in the field of treating disease as well as prevention and diagnosis using a variety of novel approaches 7. In recent years, biotechnology has introduced some novel therapeutic targets to the medical world to provide more efficient and safe therapies followed by diminishing osteoporosis as a burdensome disease. Monoclonal RANK Ligand (RANKL) antibodies and monoclonal sclerostin antibodies including denosumab, romosozumab, and blosozumab as recently rendered treatments to medicine, have achieved better therapeutic outcomes than conventional treatments 2. One of the main novel therapeutic targets is microRNA (miRNA)-based treatment. Modified nucleoside oligomers are the most common miRNA inhibitors used for new approaches. Since the clinical transformation of miRNA inhibitors is less difficult than lentivirus transfection, using them to affect the progress of osteoporosis is more feasible. Cathepsin K inhibitors which are involved in bone resorption in addition to remodeling, have been studied in many trials and are being investigated to find a promising one 8. Another innovation is stem cell therapy which is performed with cell sources from Embryonic Stem Cells (ESCs), induced Pluripotent Stem Cells (iPSCs), and Adult Stem Cells (ASCs). Stem cell therapy provides tissue regeneration which in osteoporosis translates to bone formation. Inducing the growth and differentiation of osteoblasts alongside decreasing activity of osteoclasts via cellular crosstalk are the main roles of stem cell therapy 4. The Wnt signaling pathway components are recently presented as one of the therapeutic targets. So many clinical trials are being conducted for each component to benefit from the bone mass-maintaining ability of these molecules 9. The sphingosine-1-phos-phate (S1P) signaling pathway and leucine zipper motif (APPL1) extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway and the Bone Morphogenetic Protein (BMP) signaling pathway are other targets to point out that play roles in osteogenesis, conducting osteogenic differentiation of stem cells, and promoting angiogenesis 5. A novel RANKL i-body nominated as ADR3 was introduced with features such as a high affinity for binding to human RANKL (hRANKL) and an ability to tolerate many different physical environments 3. Also, integrins as cell-adhesion transmembrane molecules have improved postmenopausal women’s BMD and made bone loss reversed 6. Furthermore, in recent years light has been shed on the bone-related roles of melatonin. Some of its important effects are bone biological rhythm regulatory effects, bone microenvironment modulation, and osteoporosis treatment 10. Population aging is greatly increasing osteoporosis prevalence and burden thus promoting osteoporosis to become an immense concern for the healthcare system. Hence, researching more effective therapies with fewer side effects has become urgent. Although studies introduced innovative targets as effective therapies, more investigations such as high-quality clinical trials are necessary to provide more evidence proving their efficiency. Despite all the mentioned conventional treatments, innovative targets, and upcoming therapeutic strategies, studying and developing molecular targets with more accurate and detailed mechanisms is indispensable. Undoubtedly, cooperation between basic sciences, especially neuroscience and biotechnology, and internal medicine can create a brighter future for the treatment of endocrine diseases 11-14. 1 2 Sepideh Hajivalizadeh Osteoporosis Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical SciencesSchool of Medicine, Tehran University of Medical Sciences Osteoporosis Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical SciencesSchool of Medicine, Tehran University of Medical Sciences IranIran Shahin Akhondzadeh AntibodiesBone densityCathepsin KMicroRNAsMonoclonalOsteogenesisRANK LigandStem cellsWnt signaling pathway 60559.pdf Sözen T, Özışık L, Başaran NÇ. An overview and management of osteoporosis. Eur J Rheumatol 2017 Mar;4(1):46-56.##Jiang Q, Zhang F, Zhu B, Zhang H, Han L, Liu X. Comparative efficacy between monoclonal antibodies and conventional drugs in postmenopausal women with osteoporosis: A network meta-analysis. Ann Palliat Med 2021;10(2):1693-702.##Qiu H, Hosking C, Rothzerg E, Samantha A, Chen K, Kuek V, et al. ADR3, a next generation i-body to human RANKL, inhibits osteoclast formation and bone resorption. J Biol Chem 2023 Feb 1;299(2):102889.##Liang B, Burley G, Lin S, Shi YC. Osteoporosis pathogenesis and treatment: existing and emerging avenues. Cell Mol Biol Lett 2022 Sep 4;27:72.##Zou Z, Liu W, Cao L, Liu Y, He T, Peng S, et al. Advances in the occurrence and biotherapy of osteoporosis. Biochem Soc Trans 2020;48(4):1623-36.##Xu H, Wang W, Liu X, Huang W, Zhu C, Xu Y, et al. Targeting strategies for bone diseases: signaling pathways and clinical studies. Signal Transduct Target Ther 2023 May 17;8(1):202.##Pham PV. Chapter 19 - Medical Biotechnology: Techniques and Applications. In: Barh D, Azevedo V, editors. Omics Technologies and Bio-Engineering [Internet]. Academic Press; 2018 [cited 2023 Nov 12]. p. 449–69.##Pham PV. Chapter 19 - Medical Biotechnology: Techniques and Applications. In: Barh D, Azevedo V, editors. Omics Technologies and Bio-Engineering [Internet]. Academic Press; 2018 [cited 2023 Nov 12]. p. 449–69.##Tonk CH, Shoushrah SH, Babczyk P, El Khaldi-Hansen B, Schulze M, Herten M, et al. Therapeutic treatments for osteoporosis-which combination of pills is the best among the bad? Int J Mol Sci 2022 Jan 26;23(3):1393.##Vlashi R, Zhang X, Wu M, Chen G. Wnt signaling: Essential roles in osteoblast differentiation, bone metabolism and therapeutic implications for bone and skeletal disorders. Genes Dis 2023 Jul 1;10(4):1291-317.##Yang K, Qiu X, Cao L, Qiu S. The role of melatonin in the development of postmenopausal osteoporosis. Front Pharmacol 2022 Oct 7;13:975181. ##Noorbala AA, Akhondzadeh S, Davari-Ashtiani R, Amini-Nooshabadi H. 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One-step and Rapid Identification of SARS-CoV-2 using Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) 2 2 Background: SARS-CoV-2 as the cause of novel coronavirus disease (COVID-19) is a member of the family Coronaviridea that has generated an emerging global health concern. Controlling and preventing the spread of the disease requires a simple, portable, and rapid diagnostic method. Today, a standard method for detecting SARS-CoV-2 is quantitative real-time reverse transcription PCR, which is time-consuming and needs an advanced device. The aim of this study was to evaluate a faster and more cost-effective field-based testing method at the point of risk. We utilized a one-step RT-LAMP assay and developed, for the first time, a simple and rapid screening detection assay targeting the Envelope (E) gene, using specific primers. Methods: For this, the total RNA was extracted from respiratory samples of COVID-19 infected patients and applied to one-step a RT-LAMP reaction. The LAMP products were visualized using green fluorescence (SYBR Green I). Sensitivity testing was conducted using different concentrations of the designed recombinant plasmid (TA-E) as positive control constructs. Additionally, selectivity testing was performed using the influenza H1N1 genome. Finally, the results were compared using with conventional real time RT-PCR. Results: It was shown that the RT-LAMP assay has a sensitivity of approximately 15 ng for the E gene of SARS-CoV-2 when using extracted total RNA. Additionally, a sensitivity of 112 pg was achieved when using an artificially prepared TA-E plasmid. Accordingly, for the detection of SARS-CoV-2 infection, the RT-LAMP had high sensitivity and specificity and also could be an alternative method for real-time RT-PCR. Conclusion: Overall, this method can be used as a portable, rapid, and easy method for detecting SARS-CoV-2 in the field and clinical laboratories. 3 8 Mohammad Shoushtari Department of Virology, Pasteur Institute of Iran Department of Virology, Pasteur Institute of Iran Iran Mehdi Zeinoddini Javad Fathi Hani Keshavarz Alikhani Department of Regenerative Medicine, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR Department of Regenerative Medicine, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR Iran Fatemeh Shiekhi Faculty of Chemistry and Chemical Engineering, Malek Ashtar University of Technology Faculty of Chemistry and Chemical Engineering, Malek Ashtar University of Technology Iran COVID-19DetectionLAMP AssayReal time PCRSARS-CoV-2 60560.pdf Sohrabi C, Alsafi Z, O′ Neill N, Khan M, Kerwan A, Al-Jabir A, et al. World Health Organization declares global emergency: A review of the 2019 novel coronavirus (COVID-19). Int J Surg 2020 Apr;76:71-76.##Chen L, Xiong J, Bao L, Shi Y. Convalescent plasma as a potential therapy for COVID-19. Lancet Infec Dis 2020;20(4):398-400.##Burki T. Outbreak of coronavirus disease 2019. Lancet Infec Dis 2020;20(3):292-3.##Peeri NC, Shrestha N, Rahman MS, Zaki R, Tan Z, Bibi S, et al. The SARS, MERS and novel coronavirus (COVID-19) epidemics, the newest and biggest global health threats: what lessons have we learned? Int J Epidemiol 2020;49(3):717-26.##Sedighimehr N, Fathi J, Hadi N, Rezaeian ZS. Rehabilitation, a necessity in hospitalized and discharged people infected with COVID-19: a narrative review. Physical Therapy Reviews 2021;26(3):202-10.##Branson B, Tavakoli R, Khaledi M, Fathi J, Shafiee SM, Afkhami H, et al. The correlations between epidemiological and clinical characteristics, laboratory tests and CT scan reports in the diagnosis of cases 2019 novel coronavirus pneumonia. A diagnostic accuracy study. Acta Medica Iranica 2021;59(10):578-86.##Liu Z, Xiao X, Wei X, Li J, Yang J, Tan H, et al. Composition and divergence of coronavirus spike proteins and host ACE2 receptors predict potential intermediate hosts of SARS‐CoV‐2. J Med Virol 2020;92(6):595-601.##Cascella M, Rajnik M, Aleem A, Dulebohn SC, Di Napoli R. Features, evaluation, and treatment of coronavirus (COVID-19). In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan.##Kannan S, Shaik Syed Ali P, Sheeza A, Hemalatha K. COVID-19 (Novel Coronavirus 2019) - recent trends. Eur Rev Med Pharmacol Sci 2020 Feb;24(4):2006-2011.##Phan T. Genetic diversity and evolution of SARS-CoV-2. Infect Genet Evol 2020;81:104260. ##Corman V, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu D, et al. Detection of 2019 novel coronavirus 1361 (2019-nCoV) by real-time RT-PCR. Euro Surveill 2020 Jan;25(3):2000045.##Tang A, Tong Z-d, Wang H-l, Dai Y-x, Li K-f, Liu J-n, et al. Detection of novel coronavirus by RT-PCR in stool specimen from asymptomatic child, China. Emerg Infect Dis 2020;26(6):1337-9. ##Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 2000;28(12):E63.##Mori Y, Notomi T. Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. J Infect Chemother 2009;15(2):62-9. ##Monazah A, Zeinoddini M, Saeeidinia A. Evaluation of a rapid detection for Coxsackievirus B3 using one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP). J Virol Methods 2017;246:27-33.##Zeinoddini M, Monazah A, Saeedinia AR. Comparison between RT-PCR, NASBA and RT-LAMP methods for detection of Coxsackievirus B3. Biomacromol J 2017;3(2):100-6.##Monazah A, Zeinoddini M, Saeedinia AR, Xodadadi N. Detection of Coxsackievirus B3 using RT-LAMP assay: Optimization by Taguchi method and comparison of one and two step RT-LAMP. Journal of Bionanoscience 2018;12(4):515-20.##Shoushtari M, Salehi-Vaziri M, Roohvand F, Arashkia A, Jalali T, Azadmanesh K. Taguchi array optimization of the reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for sensitive and rapid detection of dengue virus serotype 2. Biotechnol Lett 2021;43(11):2149-60.##Ganguli A, Mostafa A, Berger J, Aydin MY, Sun F, deRamirez SAS, et al. Rapid isothermal amplification and portable detection system for SARS-CoV-2. Proc Natl Acad Sci USA 2020;117(37):22727-35.##Lau YL, Ismail I, Mustapa NI, Lai MY, Soh TST, Hassan A, et al. Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of SARS-CoV-2. PeerJ. 2020;8:e9278.##Kashir J, Yaqinuddin A. Loop mediated isothermal amplification (LAMP) assays as a rapid diagnostic for COVID-19. Med Hypotheses 2020;141:109786. ##Mousavi SM, Zeinoddini M, Saeeidinia AR, Xodadadi N. Specific and Rapid Detection of Zonula Occludens Toxin-producing Vibrio Cholerae Using LAMP. Biomacromol J 2020;6(2):139-46.##Lamb LE, Bartolone SN, Ward E, Chancellor MB. Rapid detection of novel coronavirus (COVID-19) by reverse transcription-loop-mediated isothermal amplification. PLoS One 2020 Jun 12;15(6):e0234682.##Yu L, Wu S, Hao X, Dong X, Mao L, Pelechano V, et al. Rapid detection of COVID-19 coronavirus using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic platform. Clin Chem 2020;66(7):975-7.##El-Tholoth M, Bau HH, Song J. A single and two-stage, closed-tube, molecular test for the 2019 novel coronavirus (COVID-19) at home, clinic, and points of entry. ChemRxiv 2020.##Zhang Y, Odiwuor N, Xiong J, Sun L, Nyaruaba RO, Wei H, et al. Rapid molecular detection of SARS-CoV-2 (COVID-19) virus RNA using colorimetric LAMP. MedRxiv 2020:2020.02. 26.20028373.##

Recombinant Production of TP4-LYC1, A New Chimeric Peptide with Targeted Cytotoxicity to HeLa Cells 2 2 Background: Tilapia Piscidin 4 (TP4) showed potential anti-tumor effects against various cancer cells. Lycosine-1 (LYC1), is another Antimicrobial Peptides (AMP) from spider venom with targeted penetration to cancer cells without any adverse effects on normal cells. The aim of this study was to produce a soluble recombinant fusion peptide in order to diminish the cytotoxicity of TP4 against normal cells. Methods: In order to express of TP4-LYC-1, TP4, and LYC1 in fusion to the inteins1/2 of pTWIN-1 vector, induction condition was optimized to earn soluble peptides. Auto-cleavage induction of inteins1/2 was performed based on IMPACT® manual and their effect on cell viability of HeLa and HUVEC cells was surveyed by MTT assay. Results: The best condition for accessing the most soluble peptide in fusion to the inteins was approximately similar for all three peptides (0.1 mM of IPTG, at 22°C). After the induction of self-cleavage of inteins, a band in 3, 3, and 6 kDa was observed on tricine-SDS-PAGE. The IC50 values of TP4-LYC1 and TP4 against HeLa cells were calculated as 0.83, and 2.75 µM, respectively. Conclusion: In the present study, a novel chimeric peptide, TP4-LYC1, was successfully produced. This fusion protein can act as a safe bio-molecule with potent cytotoxic effects against cancer cells, but the penetration ability and determination of cell death mechanism must be performed in order to have more precise view on the apoptosis induction of this recombinant peptide.  9 15 Hanieh Mohammad Pour Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences Iran Ali Jahanian-Najafabadi Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences Iran Fatemeh Shafiee Cell survivalHeLa cellsInteinsSpider venomsTilapia 60561.pdf Mcphee JB, Hancock RE. Function and therapeutic potential of host defense peptides. J Pept Sci 2005;11(11):677-87. ##Zanetti M. Cathelicidins, multifunctional peptides of the innate immunity. J Leukoc Biol 2004;75(1):39-48.##Mader JS, Salsman J, Conrad DM, Hoskin DW. Bovine lactoferricin selectively induces apoptosis in human leukemia and carcinoma cell lines. Mol Cancer Ther 2005;4(4):612-24.##Furlong SJ, Mader JS, Hoskin DW. Lactoferricin-induced apoptosis in estrogen-nonresponsive MDA-MB-435 breast cancer cells is enhanced by C6 ceramide or tamoxifen. Oncol Rep 2006;15(5):1385-90. ##Raghuraman H, Chattopadhyay A. Cholesterol inhibits the lytic activity of melittin in erythrocytes. Chem Phys Lipids 2005;134(2):183-9.##Risso A, Zanetti M, Gennaro R. Cytotoxicity and apoptosis mediated by two peptides of innate immunity. Cell Immunol 1998;189(2):107-15.##Chavakis T, Cines DB, Rhee JS, Liang OD, Schubert U, Hammes HP, ET AL. Regulation of neovascularization by human neutrophil peptides (α-defensins): a link between inflammation and angiogenesis. FASEB J 2004;18(11):1306-8. ##Mader JS, Hoskin DW. Cationic antimicrobial peptides as novel cytotoxic agents for cancer treatment. Expert Opin Investig Drugs 2006;15(8):933-46. ##Neshani A, Eidgahi MR, Zare H, Ghazvini K. Extended-Spectrum antimicrobial activity of the Low cost produced Tilapia Piscidin 4 (TP4) marine antimicrobial peptide. J Res Med Dent Sci 2018;6(5):327-4.##Ting CH, Liu YC, Lyu PC, Chen JY. Nile tilapia derived antimicrobial peptide TP4 exerts antineoplastic activity through microtubule disruption. Mar Drugs 2018;16(12):462. ##Ting CH, Chen JY. Nile tilapia derived TP4 shows broad cytotoxicity toward to non-small-cell lung cancer cells. Mar Drugs 2018;16(12):506. ##Ting CH, Chen YC, Wu CJ, Chen JY. Targeting FOSB with a cationic antimicrobial peptide, TP4, for treatment of triple-negative breast cancer. 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Effect of Intra-ovarian Injection of Mesenchymal Stem Cells or its Conditioned Media on Repeated OPU-IVEP Outcomes in Jersey Heifers and Its Relationship with Follicular Fluid Inflammatory Markers 2 2 Background: Repeated Ovum Pick Up (OPU) could have a detrimental effect on ovarian function, reducing In Vitro Embryo Production (IVEP). The present study examined the therapeutic effect of adipose–derived Mesenchymal Stem Cells (MSCs) or its Conditioned Medium (ConM) on ovarian trauma following repeated OPU. Resolvin E1 (RvE1) and Interleukin-12 (IL-12) were investigated as biomarkers. Methods: Jersey heifers (n=8) experienced 11 OPU sessions including 5 pre-treatment and 6 treatment sessions. Heifers received intra-ovarian administration of MSCs or ConM (right ovary) and Dulbecco’s Modified Phosphate Buffer Saline (DMPBS; left ovary) after OPU in sessions 5 and 8 and 2 weeks after session 11. The concentrations of RvE1 and IL-12 in follicular fluid was evaluated on sessions 1, 5, 6, 9, and 4 weeks after session 11. Following each OPU session, the IVEP parameters were recorded. Results: Intra-ovarian administration of MSCs, ConM, and DMPBS did not affect IVEP parameters (p>0.05). The concentration of IL-12 in follicular fluid increased at the last session of pre-treatment (Session 5; p<0.05) and remained elevated throughout the treatment period. There was no correlation between IL-12 and IVEP parameters (p>0.05). However, RvE1 remained relatively high during the pre-treatment and decreased toward the end of treatment period (p<0.05). This in turn was associated with decline in some IVEP parameters (p<0.05). Conclusion: Intra-ovarian administration of MSCs or ConM during repeated OPU did not enhance IVEP outcomes in Bos taurus heifers. The positive association between RvE1 and some of IVEP parameters could nominate RvE1 as a promising biomarker to predict IVEP parameters following repeated OPU. 16 28 Ali Sarvari Department of Theriogenology, Faculty of Veterinary Medicine, University of TehranReproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Department of Theriogenology, Faculty of Veterinary Medicine, University of Tehran IranIran Amir Niasari-Naslaji Department of Theriogenology, Faculty of Veterinary Medicine, University of Tehran Department of Theriogenology, Faculty of Veterinary Medicine, University of Tehran Iran Abolfazl Shirazi Banafsheh Heidari Department of Photo Healing and Regeneration, Medical Laser Research Center, Yara Institute, ACECR Department of Photo Healing and Regeneration, Medical Laser Research Center, Yara Institute, ACECR Iran Sara Borjian Boroujeni Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Mohammad Hossein Moradi Department of Animal Sciences, Faculty of Agriculture and Natural Resources, Arak University Department of Animal Sciences, Faculty of Agriculture and Natural Resources, Arak University Iran Mohammad Mehdi Naderi Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Bahareh Behzadi Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Mohammad-Mahdi Mehrazar Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Mohammad Mehdi Dehghan Department of Surgery and Radiology, Faculty of Veterinary Medicine, University of Tehran Department of Surgery and Radiology, Faculty of Veterinary Medicine, University of Tehran Iran AnimalsBiomarkersCattleConditioned mediumFollicular fluidInflammationInterleukin-12OvaryResolvin E1Stem cells 60562.pdf Nicholas F, Smith C. 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Orexin-1 Receptor Antagonist SB-334867 Enhances Formalin-Induced Nociceptive Behaviors in Adult Male Rats 2 2 Background: Orexin (hypocretin) is one of the hypothalamic neuropeptides that plays a critical role in some behaviors including feeding, sleep, arousal, reward processing, and drug addiction. Neurons that produce orexin are scattered mediolaterally within the Dorsomedial Hypothalamus (DMH) and the lateral hypothalamus. In the current research, we assessed the impact of prolonged application of the antagonist of Orexin Receptor 1 (OXR1) on nociceptive behaviors in adult male rats. Methods: Sixteen Wistar rats received subcutaneous (s.c.) injections of the OXR1 antagonist, SB-334867 (20 mg/kg, i.p.), or its vehicle repetitively from Postnatal Day 1 (PND1)-PND30. On the 30th day following the final application of the OXR1 antagonist formalin-provoked pain was evaluated by injecting formalin. Results: Administration of the OXR1 antagonist in the long-term augmented the formalin-provoked nociceptive behaviors in interphase and phase II of the formalin-induced pain. Conclusion: Current results showed that the continued inhibiting OXR1 might be implicated in formalin-induced nociceptive behaviors. Therefore, the present study highlighted the effect of orexin on analgesia. 29 33 Masoumeh Kourosh-Arami Alireza Komaki Department of Neuroscience, School of Science and Advanced Technologies in Medicine, Hamadan University of Medical Sciences Department of Neuroscience, School of Science and Advanced Technologies in Medicine, Hamadan University of Medical Sciences Iran Masoumeh Gholami Department of Physiology, Medical College, Arak University of Medical Sciences Department of Physiology, Medical College, Arak University of Medical Sciences Iran FormalinNociceptionOrexin receptor 1OrexinSB-334867 60563.pdf De Lecea L, Kilduff T, Peyron C, Gao X-B, Foye P, Danielson P, et al. 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Neurons containing hypocretin (orexin) project to multiple neuronal systems. J Neurosci 1998;18(23):9996-10015. ##Razavi BM, Hosseinzadeh H. A review of the role of orexin system in pain modulation. Biomed Pharmacother 2017;90:187-93. ##Babaie F, Kourosh-Arami M, Farhadi M. Administration of orexin-A into the rat thalamic paraventricular nucleus enhances the naloxone induced morphine withdrawal. Drug Res (Stuttg) 2022;72(04):209-14. ##Erami E, Azhdari-Zarmehri H, Rahmani A, Ghasemi-Dashkhasan E, Semnanian S, Haghparast A. Blockade of orexin receptor 1 attenuates the development of morphine tolerance and physical dependence in rats. Pharmacol Biochem Behav 2012;103(2):212-9.##Kourosh-Arami M, Gholami M, Alavi-Kakhki SS, Komaki A. Neural correlates and potential targets for the contribution of orexin to addiction in cortical and subcortical areas. Neuropeptides 2022;95:102259. ##Majidinezhad M, Amirteymouri H, Karimi-Haghighi S, Kourosh-Arami M, Haghparast A. Orexin system in the ventral tegmental area is implicated in the rewarding properties of methamphetamine. Eur J Pharmacol 2022;930:175170. ##Samani F, Arami MK. Repeated administration of orexin into the thalamic paraventricular nucleus inhibits the development of morphine-induced analgesia. Protein Pept Lett 2022;29(1):57-63. ##Kourosh-Arami M, Javan M, Semnanian S. Inhibition of orexin receptor 1 contributes to the development of morphine dependence via attenuation of cAMP response element-binding protein and phospholipase Cβ3. J Chem Neuroanat 2020;108:101801. ##Kourosh-Arami M, Hajizadeh S. Maturation of NMDA receptor-mediated spontaneous postsynaptic currents in the rat locus coeruleus neurons. Physiol Int 2020 Apr 23;107(1):18-29. ##Babasafari M, Kourosharami M, Behman J, Farhadi M, Komaki A. Alteration of phospholipase C expression in rat visual cortical neurons by chronic blockade of orexin receptor 1. 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Analgesic effect of intrathecally administered orexin‐A in the rat formalin test and in the rat hot plate test. Br J Pharmacol 2002;137(2):170-6.##Rezaei Z, Kourosh-Arami M, Azizi H, Semnanian S. Orexin type-1 receptor inhibition in the rat lateral paragigantocellularis nucleus attenuates development of morphine dependence. Neurosci Lett 2020:134875. ##Mobarakeh JI, Takahashi K, Sakurada S, Nishino S, Watanabe H, Kato M, et al. Enhanced antinociception by intracerebroventricularly administered orexin A in histamine H1 or H2 receptor gene knockout mice. Pain 2005;118(1-2):254-62.##Zarmehri HA, Semnanian S, Fathollahi Y, Erami E, Khakpay R, Azizi H, et al. Intra-periaqueductal gray matter microinjection of orexin-A decreases formalin-induced nociceptive behaviors in adult male rats. J Pain 2011;12(2):280-7. ##Heidary N, Sahraei H, Afarinesh MR, Bahari Z, Meftahi GH. Investigating the inhibition of NMDA glutamate receptors in the basolateral nucleus of the amygdala on the pain and inflammation induced by formalin in male Wistar rats. Frontiers in Biology. 2018;13(2):149-55.##Sofiabad M, Heidari N, Ghasemi E, Esmaeili MH, Haghdoost-Yazdi H, Erami E. Assesment of orexin receptor 1 in stress attenuated nociceptive behaviours in formalin test. Physiol Pharmacol 2011;15(3):395-402.##Kang X, Tang H, Liu Y, Yuan Y, Wang M. Research progress on the mechanism of orexin in pain regulation in different brain regions. Open Life Sci 2021;16(1):46-52. ##Komaki A, Shahidi S, Sarihi A, Hasanein P, Lashgari R, Haghparast A, et al. Effects of neonatal C-fiber depletion on interaction between neocortical short-term and long-term plasticity. Basic Clin Neurosci 2013;4(2):136-45. ##Kourosh-Arami M, Hosseini N, Komaki A. Brain is modulated by neuronal plasticity during postnatal development. J Physiol Sci 2021;71(1):34.##Kourosh-Arami M, Komaki A, Gholami M. 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Annexin-A5 Overexpression Increases Sensitivity of MCF-7 and MCF-7/ADR Cells to Epirubicin 2 2 Background: Multi-drug resistance is an important challenge in the chemotherapy of cancer. The role of annexin A5 (ANXA5) in the biology of cancer has been the focus of many studies. Breast Cancer (BC) is frequent cancer in women with high morbidity and mortality rate. The present study aimed to investigate the effects of ANXA5 overexpression on the anti-tumor activity of Epirubicin (EPI) in MCF-7 and MCF-7/ADR cells. Methods: MCF-7 and MCF-7/ADR cells were transfected with the pAdenoVator-CMV-ANXA5-IRES-GFP plasmid or mock plasmid. The overexpression of ANXA5 was evaluated using qPCR. The effects of ANXA5 overexpression and EPI on the cell viability of MCF-7 and MCF-7/ADR cells were measured using an MTT assay. Cell apoptosis was measured by annexin V/7-AAD flow cytometry assay. Results: Following the overexpression of ANXA5, the viability of MCF-7 and MCF-7/ADR was significantly decreased. Furthermore, the overexpression of ANXA5 in MCF-7 cells increased the cytotoxic effects of EPI in all doses and reduced the IC50 of EPI from 17.69 µM to 4.07 µM. Similarly, the overexpression of ANXA5 in MCF7-ADR cells reduced the IC50 of EPI from 27.3 µM to 6.69 µM. ANXA5 overexpression alone or combined with EPI treatment increased the apoptosis of MCF7 and MCF7-ADR cells.  Conclusion: The results of the present study demonstrate that ANXA5 overexpression increases the sensitivity of MCF-7 and MCF-7/ADR to EPI, suggesting a possible beneficial role of ANXA5 in the therapy of BC. 34 39 Mahshad Ghasemi Division of Medical Biotechnology, Department of Laboratory Sciences, School of Paramedical Sciences, Shiraz University of Medical Sciences Division of Medical Biotechnology, Department of Laboratory Sciences, School of Paramedical Sciences, Shiraz University of Medical Sciences Iran Niloofar Reiazi Division of Medical Biotechnology, Department of Laboratory Sciences, School of Paramedical Sciences, Shiraz University of Medical Sciences Division of Medical Biotechnology, Department of Laboratory Sciences, School of Paramedical Sciences, Shiraz University of Medical Sciences Iran Abbas Behzad-Behbahani Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences Iran Mohammad Ali Takhshid Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences Iran Annexin-A5Antineoplastic agentsBreast neoplasmsDrug resistanceMCF7 cells 60564.pdf Kashyap D, Pal D, Sharma R, Garg VK, Goel N, Koundal D, et al. 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Extracellular L-Asparaginase Synthesis Bacillus niacin Isolation, Optimization, and Characterization from Marine Saltern Sediment Sources 2 2 Background: Asparagine is an amino acid that can be converted into aspartic acid and ammonia by the enzyme L-asparaginase. Some forms of cancer, such Acute Lymphoblastic Leukaemia (ALL) and Non-Hodgkin Lymphoma (NHL), respond well to this enzyme when employed as a chemotherapeutic drug. The purpose of this research was to find bacteria that can manufacture the enzymes L-asparaginasein marine slattern sediment which can be employed in commercial and industrial scale production. Methods: All of the strains were identified as Bacillus niacini spp. by biochemical and molecular testing. The strain belongs to the Bacillus genus, according to nutritional, biochemical, PCR and 16srRNA sequencing data. Results: According to the findings of this research, Bacillus niacin spp. have the potential to create a substance that is helpful in a variety of medical applications. The results of this study hint to the possibility that bacteria have the ability to produce antimicrobial compounds, which have the potential to be successful in a wide variety of environments. Conclusion: Numerous opportunities may arise for researchers interested in utilizing the medical potential of enzyme-producing bacteria if they are successfully isolated and screened from aquatic and terrestrial habitats. 40 48 Mugip Rahaman Abdul Wahab Department of Biotechnology, Dr. M.G.R Educational and Research Institute Department of Biotechnology, Dr. M.G.R Educational and Research Institute Iran Thirunavukkarasu Palaniyandi Department of Anatomy, Biomedical Research Unit and Laboratory Animal Centre, Saveetha Dental College and Hospital, Saveetha Institute of Medical and Technical Science, Saveetha University Department of Anatomy, Biomedical Research Unit and Laboratory Animal Centre, Saveetha Dental College and Hospital, Saveetha Institute of Medical and Technical Science, Saveetha University India John Wyson Department of Food Processing Technology, AMET University, Kanathur Department of Food Processing Technology, AMET University, Kanathur India Asha Sivaji Department of Biochemistry, DKM College for Women, Vellore-632001 Department of Biochemistry, DKM College for Women, Vellore-632001 India Swarnakala Thamada Molecular Systematics Laboratory, Zoological Survey of India, Andaman & Nicobar Regional Centre Molecular Systematics Laboratory, Zoological Survey of India, Andaman & Nicobar Regional Centre Andaman and Nicobar Islands AsparagineAsparaginaseBacillus niaciniPolymerase chain reaction 60565.pdf Castro D, Marques AS, Almeida MR, de Paiva GB, Bento HB, Pedrolli DB, et al. 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Anti-Quorum Sensing and Anti-Biofilm Activity of Ginger (Zingiber officinale) Rhizomes against Multidrug-Resistant Clinical Isolates of Pseudomonas aeruginosa 2 2 Background: The aim of this study was to determination of Anti-Quorum Sensing (AQS) and anti-biofilm potential of the methanol extract of ginger (Zingiber officinale) rhizomes against multidrug-resistant clinical isolates of Pseudomonas aeruginosa (P. aeruginosa). Methods: The AQS activity of ginger was determined against Chromobacterium violaceum (C. violaceum) ATCC 12472 (CV12472), a biosensor strain, in qualitative manner using the agar well diffusion method. The violacein pigment inhibition was assessed to confirm AQS activity of ginger. The AQS potential of sub-minimum Inhibitory Concentrations (sub-MICs) of the ginger extract was determined by targeting different QS regulated virulence factors, including swarming motility (using swarm diameter measurement method), pyocyanin pigment (using chloroform extraction method), Exopolysaccharide (EPS) (using phenol-sulphuric acid method), and biofilm formation (using microtiter plate assay), against clinical isolates (CIs 2, 3, and 4) and standard reference strain of P. aeruginosa (PA01). Results: The AQS activity of methanol extract of ginger was confirmed against C. violaceum (CV12472) as inhibition of violacein pigment formation without effecting the growth of CIs and PA01 of P. aeruginosa. The ginger extract exhibited concentration-dependent inhibition of virulence factors and biofilm formation. The maximum reduction was found in swarming motility, pyocyanin, EPS and biofilm formation against PA01 (51.38%), CI3 (57.91%), PA01 (63.29%) and CI2 (64.37%), respectively at 1/2 MIC of ginger extract. Conclusion: The results of present study revealed the effective AQS and anti-biofilm potential of Zingiber officinale rhizome methanol extract at a reduced dose (sub-MICs). The extract may be explored as an agent of antimicrobial compounds having AQS and anti-biofilm activity for controlling microbial infection and also for reducing the chances of emergence of resistance in P. aeruginosa.   49 56 Pankaj Kumar Sagar Department of Microbiology, Bundelkhand University, Jhansi-284128 Department of Microbiology, Bundelkhand University, Jhansi-284128 India Poonam Sharma Department of Zoology, Indira Gandhi National Tribal University (A Central University), Amarkantak-484886 Department of Zoology, Indira Gandhi National Tribal University (A Central University), Amarkantak-484886 India Rambir Singh Chromobacterium violaceumGingerPseudomonas aeruginosaPyocyaninRhizome 60566.pdf Boyle EM, Ainsworth JR, Levin AV, Campbell AN, Watkinson M. Ophthalmic Pseudomonas infection in infancy. Arch Dis Child Fetal Neonatal Ed 2001;85(2):F139-40. ##Chastre J, Fragon J-Y. Ventilator-associated pneumonia. 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Effective anti-platelet and COX-1 enzyme inhibitors from pungent constituents of ginger. Thromb Res 2003;111(4-5):259-65. ##Chrubasik S, Pittler MH, Roufogalis BD. Zingiberis rhizoma: A comprehensive review on the ginger effect and efficacy profiles. Phytomedicine 2005;12(9):684-701. ##Vattem DA, Mihalik K, Crixell SH, McLean RJ. Dietary phytochemicals as quorum sensing inhibitors. Fitoterapia 2007;78(4):302-10. ##Kim HS, Park HD. Ginger extract inhibits biofilm formation by Pseudomonas aeruginosa PA14. PLoS One 2013;8(9):e76106. ##Asfour HZ. Anti-quorum sensing natural compounds. J Microsc Ultrastruct 2018;6(1):1-10. ##Unuofin JO, Masuku NP, Paimo OK, Lebelo SL. Ginger from Farmyard to Town: Nutritional and Pharmacological Applications. Front Pharmacol 2021;12:779352. ##Ali BH, Blunden G, Tanira MO, Nemmar A. Some phytochemical, pharmacological and toxicological properties of ginger (Zingiber officinale Roscoe): a review of recent research. 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Green Tea Extract Reduced Lipopolysaccharide-Induced Inflammation in L2 Cells as Acute Respiratory Distress Syndrome Model Through Genes and Cytokine Pro-Inflammatory 2 2 Background: Acute Respiratory Distress Syndrome (ARDS) is a severe lung inflammatory condition that has the capacity to impair gas exchange and lead to hypoxemia. This condition is found to have been one of the most prevalent in patients of COVID-19 with a more serious condition. Green tea (Camellia sinensis L.) contains polyphenols that possess many health benefits. The purpose of this study was to assess the anti-inflammatory activities of green tea extract in Lipopolysaccharide (LPS)-induced lung cells as ARDS cells model. Methods: In this study, rat lung cells (L2) were induced by LPS to mimic the inflammation observed in ARDS and later treated with green tea extract. Pro-inflammatory cytokines such as Interleukin (IL)-12, C-Reactive Protein (CRP) as well as Tumor Necrosis Factor-α (TNF-α) were investigated using the ELISA method. Gene expression of NOD-Like Receptor Protein 3 (NLRP-3), Receptor for Advanced Glycation End-product (RAGE), Toll-like Receptor-4 (TLR-4), and Nuclear Factor-kappa B (NF-κB) were evaluated by qRTPCR. Apoptotic cells were measured using flow cytometry. Results: The results showed that green tea extract treatment can reduce inflammation by suppressing gene expressions of NF-κB, NLRP-3, TLR-4, and RAGE, as well as pro-inflammatory cytokines such as IL-12, TNF-α, and CRP, an acute phase protein. Apoptosis levels of inflamed cells also found to be lowered when green tea extract was administered; thus, also increasing live cells compared to non-treated cells. Conclusion: These findings could lead to the future development of supplements from green tea to help alleviate ARDS symptoms, especially during critical moments such as the current pandemic. 57 65 Didik Priyandoko Biology Study Program, Faculty of Mathematics and Natural Sciences, Indonesia University of Education Biology Study Program, Faculty of Mathematics and Natural Sciences, Indonesia University of Education Indonesia Wahyu Widowati Lenny Lenny Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia, BSD Campus Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia, BSD Campus Indonesia Sintya Novianti Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia, BSD Campus Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia, BSD Campus Indonesia Revika Revika Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia, BSD Campus Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia, BSD Campus Indonesia Hanna Sari Widya Kusuma Biomolecular and Biomedical Research Center, Aretha Medika Utama Biomolecular and Biomedical Research Center, Aretha Medika Utama Indonesia Ika Adhani Sholihah Biomolecular and Biomedical Research Center, Aretha Medika UtamaSchool of Life Sciences and Technology, Bandung Institute of Technology Biomolecular and Biomedical Research Center, Aretha Medika UtamaSchool of Life Sciences and Technology, Bandung Institute of Technology IndonesiaIndonesia Acute respiratory distress syndromeCamellia sinensisCytokinesInflammationTea 60567.pdf Han S, Mallampalli RK. 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