Chemotherapy is the most widely recognized technique to regard leukemia and also different sorts of human tumors. In any case, tranquilize protection has stayed as the primary test against the adequacy of medications. Besides, having different unfriendly impacts, chemotherapy drugs are getting to be traded by characteristic modalities for growth treatment. In such manner, natural segments, for example, resveratrol and prednisolone have been recognized to sharpen the leukemic cells to modified cell demise through an arrangement of complex procedures. In this investigation, we have analyzed effect of 15, 50 and 100μM of resveratrol and 700μM of prednisolone on the human multidrug protection quality 1 (MDR1) as a notable marker for cell sedate protection. We assessed the impact of resveratrol and prednisolone on MDR1 protein expression in the CCRF-CEM cell line as an agent for intense lymphoblastic leukemia. The investigation was planned to clear up whether. CCRF-CEM cells linage get under drug treatment with use of resveratrol and prednisolone. Western blot use at 24 and 48 hours with different doses of resveratrol and prednisolone to analysis of MDR1 expression changes. Effect of 15, 50, and 100 micro molar of resveratrol and 700 micro molars of prednisolone on CCRF-CEM cells led to the MDR1 decrease. Western blot use for evaluation of MDR1 protein expression changes. In the present study, we observed that resveratrol and prednisolone, with a dose-dependent effect, can reduce the expression of the MDR1 protein. This reduction of expression demonstrates that resveratrol and prednisolone can overcome to drug resistance created by MDR1. An obtrusive and quickly developing growth of White Blood Cells (WBC) is the Acute Lymphoblastic Leukemia (ALL) which generally starts from bone marrow . ALL is presently considered as the most common threat in youngsters, containing 77% of leukemic cases in this age gathering which is most every now and again happened in offspring of 2-5 years of age . Since the revelation of ALL, few lines of medication specialists have centered to create compelling and safe modalities to keep the development and movement of this threat. An extensive variety of cures were planned with different sorts of systems; however, their danger and antagonistic impacts have been reliably remained the principle hindrance in front of productive treatment. Concerns about the danger and wide range of reactions, these days expanding interests are concentrated to supplant the concoction specialists by more regular or plant-based parts to treat the human diseases . Resveratrol , a constituent of red wine, has been appeared to have against oxidative properties which is found in excess of 70 types of plants, for example, pines, shelled nut, mulberry and grape. Pathogen contamination, condition changes, daylight, overwhelming metals, and ozone presentation are a few sorts of stresses that incite resveratrol creation in plants . Previous examinations have demonstrated that the resveratrol has numerous valuable impacts from chemoprevention of tumors to hostile to oxidative, against aggravation, cardio assurance and immunomodulation, in any case the correct atomic component is yet to be cleared up . Resveratrol has been shown to be prepared to do avoiding tumor angiogenesis and metastasis and stifles three noteworthy phases of tumor extension including tumor start, tumor advancing, and advancement. The utilization of resveratrol to treat the medication protection for chemoprevention has been archived in light of in vitro and in vivo discoveries . a few examinations on human cells have demonstrated that different pathways engaged with cell development, cell passing and irritation are managed by resveratrol. Various examinations from in vitro tries have likewise checked that resveratrol can smother the multiplication of an assorted scope of cells . Broad examinations have upheld additionally the part of glucocorticoids . In this way these specialists are legitimate contender to be used in the treatment of youth intense lymphoblastic leukemia (ALL) and different malignancies influencing lymphoid framework . Among the distinctive instruments by which the malignancy cells may ensure their survival, expansion, and intrusion the MDR-1 has been considered as a key potential giver amid tumor movement. The MDR1 quality encodes the medication transporter P glycoprotein (Pgp) which advances medicate efflux. The most broadly thinks about system by which the human disease what’s more, tumor cell lines oppose to chemotherapy is the over-articulation of Pgp. Different components of medication efflux are intervened by transporter proteins including the multidrug resistance associated protein (MRP)  and over-articulation of the significant vault protein (LRP). Changes in topoisomerase compounds and a modified glutathione/ glutathione -S-transferase (GSH/GST) detoxification framework  are likewise speak to instruments of cell protection from cytotoxic specialists. We have also examined the effect of resveratrol and prednisolone on the gene expression and methylation level of DNA promoters of the MDR1 gene in our previous study and observed that resveratrol and prednisone cause decrease in MDR1 gene expression without altering the methylation level of the MDR1 gene promoter .In another study that was done by Al-Abd et al., (2011)resveratrol inhibited P-gp and reduced the expression of MDR1 in cancer cell lines in vitro. Gautam et al., (2000) illustrated some effects of proliferative inhibition of resveratrol on leukemic cell lines  during a series of laboratory studies. We have also examined the effect of resveratrol and prednisolone on the gene expression and methylation level of DNA promoters of the MDR1 gene in our previous study and observed that resveratrol and prednisone cause decrease in MDR1 gene expression without altering the methylation level of the MDR1 gene promoter .Due to the low toxicity of resveratrol in vivo as well as in vitro and its anti-leukemic effects, being selective of its effects and be useful as an anti-leukemic agent; We decided to assess the effect of resveratrol and prednisolone on overcoming drug resistance caused by increased expression and function of MDR1 by changing its expression and reducing its expression in CCRF-CEM cancer cells.Cell Culture2. Preceding resveratrol treatment the cells were seeded in 60mm culture dishes at a thickness of 4 ×105 cells for each dish. As a harmful cell line speaking to ALL, the CCRF_CEM cell line was bought from Pasteur organization of Tehran, Iran. The cells were refined routinely in RPMI 1640 medium supplemented with 10% warmth inactivated fetal cow-like serum , streptomycin (10mg/ml) and 10,000 U/mL of penicillin. The cells were kept up at 37˚C with 5% COTreatmentResveratrol  was broken up in ethanol what’s more, added to culture dishes at the convergences of 15, 50 and 100μM. Following 24 and 48 hours, the cells were gathered for protein extraction. Prednisolone  was broken up in DMSO and added to CCRF-CEM societies at the centralization of 700 μM  At last, the cells were collected after 24h and 48h for RNA extraction. Assessment of MDR1 protein 6 cells were cultured in 25cm2 flasks in the final volume of 5ml. Subsequently, the cells treated with 15, 50 and 100μM of resveratrol and 700μM of prednisolone for 24 and 48 hours. In order to assess the alteration of protein (MDR1) expression, the Western Blot technique used. Hence, 5×10Extraction and isolation of proteinsoC. Then, the supernatant discarded, and Bradford assay used for evaluation of protein concentration. According to Laemmli assay, the equivalent amounts of protein samples were separated using 10% SDS-PAGE. The cells were harvested and washed by cold phosphate buffer saline. Equivalent amounts of cell lysis buffer, including 1% NP-40, 0.5% SDS, 10mM Tris-HCL (pH=7.4), 5mM EDTA, 150mM NaCl, 100µM PMSF, 0.5% sodium deoxycholate, and protease-phosphatase inhibitor were added to cells. 13000RPM was considered to centrifuge samples for 20 min in -4Western BlotoC for a whole night. Anti-actin antibody was considered as an internal control. Then, the membrane was washed in three steps by TBS-T, exposed with secondary antibody, conjugated with HRP, at room temperature for 1 hour. Eventually, chemiluminescence assay was used to the visualization of protein bands.Transference of bands from gel to nitrocellulose membrane fulfilled Using Mini Trans-Blot Bio-Rad. After that, the membrane was washed and exposed with blocking solution, containing 5% skim milk in TBS-T, for 1 hour. Subsequently, the cells were washed with TBS-T and exposed to the primary monoclonal antibody in the presence of blocking solution at -4The effect of resveratrol and prednisolone on CCRF-CEM cells was significant and reduced MDR1 expression. The Western blot method was applied to the measurement semi quantitatively of MDR1 protein expression .In order to assess changes in MDR1 protein expression, the western blot technique was used. The Actin protein considered as an internal control. Western blot results illustrated decline in MDR1 expression influenced by 15, 50 and 100μM of resveratrol and 700μM of Prednisolone for 24 and 48 hours . As showAdditionally, the western blot technique was used to P-gp (a protein encoded by MDR1) quantitative measurement.Statistically AnalysisIn order to do densitometry and assess the bands, the ImageJ software and data analysis carried out by SPSS 20.0 software were used. The immunoblotting and flow cytometry data were obtained from three distinct experiments and demonstrated as a ± mean standard error. For studying the differences between groups, paired T-test was used and the p values < 0.05 were considered as statistically significant.Resveratrol and prednisolone decrease the expression of MDR1 gene without altering the level of methylation of DNA promotersIn our previous study, to investigate the effect of resveratrol and prednisolone on the level of methylation of MDR1 DNA gene promoters and their effects on MDR1 gene expression in acute lymphoblastic cell CCRF-CEM cells, we measured these cells at doses of 15, 50 and 100 μm of resveratrol and 700 μm of prednisolone and treated them for 24 and 48 hours. Our results in that study showed that resveratrol and prednisone cause decrease in MDR1 gene expression without altering MDR1 gene methylation pattern in a dose and time-dependant manner .Resveratrol and prednisolone cause decrease in MDR1 protein expressionAs indicated by the past looks into, the convergences of resveratrol required for apoptosis enlistment in the CCRF-CEM cells were 15, 50 and 100μM, while 700μM of prednisolone appeared to be ideal. Likewise, Resveratrol and prednisolone cause a diminishing in MDR1 quality articulation in a period and focus subordinate way. Following 24 and 48 hours of treatment, the varieties of expression levels of MDR1 protein were assessed contrasted and untreated control cells. To look at the adjustments in expression levels of MDR1 protein, the CCRF-CEM cells were treated with resveratrol and prednisolone. It was watched that resveratrol and prednisolone have significant effect on expression levels of MDR1 protein CCRF-CEM cells in the measurements and interims utilized as a part of this investigation. Our result demonstrated that resveratrol in the groupings of 15, 50 and 100μM of Resveratrol and prednisolone 700μM can diminish MDR1 quality articulation  followinMoreover, Western blot analysis also showed a significant decrease in MDR1 expression in CCRF-CEM cells treatment with concentration of 15, 50 and 100μM of Resveratrol and 700μM of Prednisolone after 24 and 48 hours . Results are representative of the reduction in MDR1 at doses of 15, 50 and 100μM of Resveratrol and 700μM of Prednisolone after treatment, as compared to the protein level in control group (untreated cells). The band intensity of MDR1 was quantitated and normalized with b-actin band and expressed as relative MDR1 expression. These days, the chemotherapy, surgery and radiotherapy are the most widely recognized methodologies for treatment of tumor. Considering drug protection, one of the normal strategies to battle malignancies is to sharpen the threatening cells to restorative operators . In spite of the fact that a number of novel obtrusive restorative systems are accessible, a difficult issue in hostile to leukemia treatments is protection from apoptosis which is in any event to some degree a reason of treatment protection. Consequently, apoptosis acceptance is a key component to bring wanted medication proficiency.Reducing the expression of MDR1 gene can be effective in decreasing the effects of drug resistance, improving the chemotherapy outcome and rescuing patients who are resistant to treatment from the effects of severe toxic chemotherapy . MDR1 and MRP1 are members of the large ABC family of protein transductors; it is believed that they are an undesirable prognostic factor in the response to and survival of malignancies . In the event of a successful use of natural materials to reduce the effect of drug resistance, we will face some minor side effects. Resveratrol is one of the natural ingredients associated with known anti-cancer effects. These effects associated with the reduction of the expression and function of ABC carriers which reflects a significant reduction in IC50 medications.Resveratrol is a natural part that has an assortment of organic impacts on malignant cells counting against proliferative, calming and chemo preventive impacts . It is cleared up that resveratrol can make the safe harmful cells delicate to treatment through a few systems. It expands the declaration of CD95L and passing receptors interceded apoptosis , changes NF-KB pathway and incites customized cell passing, changes STAT3 articulation by diminishing the capacity of tyrosine kinase Src and directs the survivin acceptance , and fortifies the p53 task and NO generation, Besides, GCs act by means of a ligand initiated translation factor known as GC receptor In a wide range of natural examples, the assessment of changes of protein level is a particular procedure for concentrate of expression levels of qualities. In spite of being non-quantitative, western blot is a valuable procedure especially as a confirmation test for prove changes of expression of a protein by two different drugs or substances. Considering the above-mentioned items, we investigated whether resveratrol and prednisolone can reduce the expression of MDR1 in the level of protein as a strong barrier against anticancer drugs and one of the main causes of drug resistance, as well as overcome the drug resistance, increasing the expression and function of the MDR1 protein by its reducing . In the present study, our results show that resveratrol and prednisolone, with a dose-dependent effect, can reduce the expression of the MDR1 protein. Reducing the expression of the MDR1 illustrates that resveratrol and prednisolone can overcome the drug resistance by MDR1. In our previous research, we demonstrated that resveratrol and prednisolone can reduce the expression of MDR1 gene without changing the methylation pattern of the MDR1 gene . Our observation on these acute lymphoblastic cells CCRF-CEM also showed that resveratrol and prednisolone can increase the apoptosis of these cancer cells by changing the expression of microRNAs miR 15a and miR16-1 and also reducing the expression of BAX gene as well as increasing expression of BCL-2 without changing the pattern of methylation of the promoter of BAX and BCL-2 genes . In the present study, we also found that resveratrol and prednisolone can overcome drug resistance caused by increasing the expression of MDR1 in acute lymphoblastic cell CCRF-CEM via reducing the expression of MDR1 in the protein level. The issue of overcoming drug resistance has always been an important factor in the elimination of cancer resistant cells to cure. In other studies, such as a study carried out by Quan F, resveratrol reduced the expression of MDR1 (P gp gene) and Bcl 2 (anti-apoptotic) in chemotherapy-resistant epidermoid cell line, KB 200 . In another study that was done by Al-Abd et al., (2011) resveratrol inhibited P-gp and reduced the expression of MDR1 in cancer cell lines in vitro. Gautam et al., (2000) illustrated some effects of proliferative inhibition of resveratrol on leukemic cell lines  during a series of laboratory studies.Previous studies have shown that resveratrol causes decrease in MDR1 gene expression by inhibiting NF-kB activation as well as the signaling pathway of p38 MAPK .In a study carried out by Vasil F. Chekhun and Galina I. Kulik on the resistance of the human MCF-7 / R breast cancer cell line to doxorubicin, it was concluded that the development of MCF-7 / R drug resistance to doxorubicin, they concluded that the development of MCF-7 / R drug resistance to doxorubicin is the result of a change in the expression of P gp . Chekhun et al., (2006) reported the relationship between MDR1 promoter methylation and drug resistance in the T-cell line as well as three patients with chronic lymphocytic leukemia. Expression of P-gp is the main barrier in chemotherapy for hematological malignancies, including acute myeloid leukemia .In conclusion, in the present study, ability of resveratrol and prednisolone in reducing the expression of the MDR1, with dose-dependent effect, was observed. Reducing the expression of the MDR1 demonstrates that resveratrol and prednisolone can overcome the drug resistance induced by MDR1.The authors state that there is no conflict of interest.


Purpose: A lot of plants are available which can produce nanoparticles used in medicine, life sciences, and the pharmaceutical industry. The present study aims to introduce safe biological and eco-friendly methods for synthesizing silver nanoparticles (AgNPs) by using Saturaja rechengri Jamzad extract, which can replace traditional chemical methods. In addition, the chemical nature and antimicrobial activities were identified and accordingly the anticancer effects of AgNPs was successfully reported on colon cancer cells (HT-29).Methods: Light and ultrasound, as two green chemistry techniques were first used for AgNPs synthesis. Then, morphological and crystalline structure of AgNPs was evaluated by scanning electron microscopy (SEM) and X-ray diffraction (XRD) analysis, respectively. In addition, functional groups were determined by using the Fourier transform infrared spectroscopy (FTIR) spectrum. Further, a maximum adsorption of AgNPs was observed in UV-visible spectrum. In the next stage, antibacterial activity of green synthesized AgNPs was evaluated against two pathogenic bacteria including Escherichia coli and Staphylococcus aureus. Finally, the cytotoxicity of AgNPs on HT-29 at different concentrations and times of AgNPs was determined by MTT assay.Results: The findings indicated that the synthesis of AgNPs by ultrasonic technique leads to smaller particle size and more distribution. Based on the results of MTT test for calculating the IC50%, the anti-proliferative effects of the light and ultrasound AgNPs were observed on HT-29 cell lines depending on the dose and time. Finally, the AgNPs had the most cytotoxicity HT-29 cell lines at 100 μg/ml concentration although the lowest toxicity effect was reported on HEK-293 cell lines at the same conditions.Conclusion: The change in the concentration, physical and chemical properties of AgNPs including the form and size of particles, and their type of covering and fields can influence the induction of cytotoxicity and morphological change in the treated cells. The present research opens a new horizon on the development of new biological and cytotoxicity agents. In addition, the biomass derived from live or dead plants is used as a renewable energy source to generate electricity, biogas, fuel, and the like. Further, the use of biomass for producing nanoparticles is exciting.3 Generally, this technique helps to protect the environment and reduce the risk of damage for human beings, nature, air and the ecosystem.5 Nanoparticles used in diagnosing and treating diseases are divided into organic and inorganic category. Organic nanoparticles include albumin, lipid nanoparticle and chitosan, and the like while inorganic nanoparticles are often found as metal nanoparticles including magnetic, copper, gold and silver, etc.6The use of nanoparticles is important since they are comparable with cellular components in human cells in term of size. It seems that the nature has used materials at a nano-scale to construct biological systems. If someone is supposed to accompany the nature in treating the diseases, he should use materials at a scale which is similar to the nature in some areas like correcting defective genes,7 preventing the proliferation of viral genomes8 and cancer cell death,9 and restoring to cellular metabolism.10 Among different synthesized nanoparticles, AgNPs are considered as one of the most widely used drug-delivery nanotechnologies for treating diseases. In this regard, a large body of research was conducted on the related anticancer,11 antibacterial,12 antifungal,13 and antiviral effects.14 The results indicated that AgNPs can damage DNA by destructing the mitochondrial respiratory chain in cancer cells, leading to the cell death.16 However, silver, in itself, can play a minor role or lacks the properties. Compared with macro silver particles, this dual effect of nanoparticles is caused by increasing the areas, along with the reactivity of matter and its compliance with quantum physics and chemistry in nano mode.17 Based on green chemical compounds, producing AgNPs by using green chemistry includes choosing the solvent environment, a safe operating environment revivalist, and non-toxic materials for the stability of the nanoparticles . Hera et al. reported the formation of Ag+ from alfalfa biomass in an aqueous solution in a pH-dependent manner.18 Mata et al. examined the cytotoxic effects of AgNPs synthesized by the aqueous extract of Abution indicum leaf in the cancer cell line (COLO 205) (human colon cancer) and MDCK  cell in vitro conditions and observed that concentration-depend AgNPs, inhibit the growth of this cancer cell line. In addition, it was observed that AgNPs cause morphological changes in chromatin densities and the membranes of treated cells, which ultimately lead to the cell death.19 Savory with the scientific name (Satureja hortensis) belongs to the Lamiaceae family. Lamiaceae is one the largest plant families with a global distribution. This genus has 15 species in Iran, among which 9 species such as S. edmondi, S. sahendica, S. kallarica, S. bachtiarica, S. intermedia, S. isophylla, S.atropatana, S. khuzestanica. S. Rechingeri are exclusive to Iran. Some studies reported carvacrol and flavonoids as the main components of Satureja species21 including antioxidant properties.22 During recent years, a number of studies reported anti-viral properties,23 analgesic and anti-inflammatory,24 antibacterial and antifungal,27 antispasmodic and anti-diarrhea,28 vasodilatory properties29 for different species of this plant, among which we can refer to Satureja rechingeri as one of the endemic species of this plant. In this study, the green synthesized AgNPs extracted from S.rechengeri were used to determine the antimicrobial and anti-cancer effects on colon cancer cell lines. However, no study has yet addressed different AgNPs synthesis methods of S.rechingeri by using light and ultrasonic techniques. Based on the review of the literature, this is the first study addressing the quick and simple synthesis of AgNPs by using S.rechingeri extract as a reducing and stable agent to determine its effect on colon cancer cells. It seems that the introduction of drugs used in traditional medicine, especially medicinal herbs, represents a good starting point for the development of research projects to produce new drugs for the treatment of cancer.30Bio-nanotechnology is regarded as one of the most valuable nanotechnology in the modern era. In addition, it is an interdisciplinary area related to physics, electronic materials, biology, chemistry and medicine, which uses these scientific fields to control matter at the molecular scale.S.rechengri were dried in the shade for 10 days and then were powdered. In order to prepare aqueous solution of the plant extract, 20 g of plant powder was weighed and was kept for 24 hours in the laboratory conditions after being ionized into 80 ml water. In the next stage, the solution was placed in water bath with 80oC for 45 min and was filtered by Watman Filter Paper No. 2. Accordingly, the extract was treated by vacuum rotary evaporation  and was kept at 4°C for use and identification by various techniques in order to remove the extra solvent and obtain a good concentration.The aerial parts of the plant were collected at the flowering stage from a farm owned by Khoraman Company in Lorestan Province (Iran) in July 2016 with the Herbarium code MPH-1348. The fresh plants of 3)  0.001 M was prepared in both methods.In order to synthesize AgNPs, the two following methods as green chemistry were used. The solution of silver nitrate salt  and the same was exposed at ultrasound irradiation of 40 Hz in the dark for three times, each time for 30 min, at 40oC and pH= 7 Changes in color from pale yellow to reddish-brown indicated the formation of AgNPs.Ultrasound: One part of AgNOThe color change was observed under light and ultrasound conditions, which was very fast and clear in light conditions. The absorption spectra were measured by using a spectrophotometer (JASCO V-670 Spectrophotometer) in the range of 300-600 as silver absorption occurs in this range.X-ray diffraction analysis XRD was used to identify the phase and determine the crystalline structure of AgNPs in the range of  5> 2Ǿ> 90. Sharp peaks indicated the crystallization properties of the produced sample. The precipitation from the interaction of the aqueous extract of the plant and silver ion was centrifuged three times at 1200 rpm and washed with deionized water. Then, it was dried at 45°C for 24 hours and accordingly used for its identification.-1.In the next stage, the functional groups of AgNPs were determined by ultrasound and light techniques by using Fourier transform spectrometer. This technique involves the identification of functional groups AgNPs in the range of 500-4000 cmScanning electron microscopy SEM is a considered as a technique used for direct observation of AgNPs and estimating their size and surface morphology. This technique is appropriate for identifying the particles with smaller particle size and is regarded as a good standard for determining the validity of other techniques. Further, it provides some information about the structure of particles through electron distribution and dimensions of synthesized nanoparticles. In the next procedure, SEM image of AgNPs was photographed to determine the shape and size of the nanoparticles. To this end, a thin layer of sediment was placed on a gold cloth at a low voltage and under vacuum pressure (5-8 Torr) by using Philips XL30 electron microscope. It was found that the particles are mainly spherical in shape and their diameter is in the range of 44.2 and 65.3 nm, which are stacked together in some areas.Escherichia coli and Staphylococcus aureus was determined by the Mueller-Hinton broth  micro dilution method  on 96-well plates with incubation at 37°C for 24 hours. Further, MIC and higher concentrations were cultured on nutrient agar  plates and incubated at 37°C for 24 hours in order to evaluate the minimum bactericidal concentration (MBC) of AgNPs light and ultrasound. Then, the MIC value was determined for the least dose of synthesized AgNPs, which prevented the growth of tested bacteria. In order to achieve a better result, the test was repeated twice.The Minimum inhibitory concentration (MIC) of synthesized AgNPs light and ultrasound for 4 cell lines were cultured in a 96-well plate and incubated within incubators (Binder) at 37°C and 5% CO2.The HT-29 cells  and Normal HEK-293 (IBRC C10139) were purchased from National Cell Bank in Iranian Biological Resource Center (Cell Bank of Pasteur Institute Tehran-Iran). Then, they were defrozen and cultured in the IMPR environment enriched with 10% SBF and bovine serum and 1% penicillin –streptomycin. After the cell lines were grown sufficiently, they were counted and a total number of 1031 After 24 hours, 100 ml of the cultured cells was covered with 100 ml of AgNPs of salt with a concentration of 0.5 mg ml for 4 hours by using a foil. Then, the cell cytotoxicity was estimated through colorimetric method (MTT) on colon cancer cells HT-29 and healthy cells HEK-293 by measuring the light and ultrasound absorbance based on the concentration of AgNPs. In the next stage, the results were compared with the rate of cell survival. In addition, the cells were cultured in a 96-well plate with a density of 104cells per plant and were incubated in a CO2 incubator (37oC) for 24 h. Further, HT-29 cancer cells were treated with different concentration of AgNPs synthesized from the aqueous extract of the plant for 24 and 48 h, respectively. After treating the samples, 10 μl of MTT solution (5 mg/ml) was added to each plate containing AgNPs-treated cells and incubated at 37°C and 5% CO2 for 5 h. Finally, DMSO (0.5 μl ) was added to each well until dissolving the purple crystals, and the light absorbance was read by a spectrophotometer at 570 nm after 20 min at room temperature.32 In addition, the inhibitory concentration (IC50%) for the samples was determined as follows:MTT assay is a colorimetric method based on the revival and break of yellow crystals with chemical formula 3-  -2, 5-diphenyl-] which are regenerated by tetrazolium bromide enzyme succinate dehydrogenase in the active cellular mitochondria and the formation of insoluble blue crystals. The color is proportional to the cellular activity and the number of living cells.The quantitative data was evaluated using SPSS software (Version 16)  and the results were analyzed by ANOVA at a software using statistical tests at significant level. The data were determined as mean ± SD and p < 0.05 was considered statistically significant.S.rechengri extract based on light and ultrasonic techniques, which are economical and ecofriendly. In addition, the enormous potentials of the nature can be employed for synthesizing nanoparticles in these two techniques, without damaging the environment. Recently, a lot of plant extracts have been studied for AgNPs synthesis. However, in the present study, aqueous extracts were extracted from the aerial parts of dried S.rechengri for AgNPs synthesis and the extracts prepared by using a solution of AgNO3 0.001 M  with a purity of 99/99% were mixed. In order to synthesize the AgNPs from the obtained solution, both light and ultrasound techniques (absence of light) were used. The results showed a change in color of the extract from pale yellow to dark brown at room temperature and the formation of a colloidal mix of AgNPs. The observation of the color change due to the interaction of herbal extracts and silver salt solution was similar to the results of the study by Reddy and Gandhi (2012) and is considered as the first microscopic signs of the production of AgNPs.33 The speed of the reaction reducing Ag+ in the aqueous extract of S.rechengri by using the light technique was found to be higher than that of ultrasound technique. Further, the color change in the light technique was considerably clearer than of the ultrasound technique. In the ultrasound techniques, the silver nitrate solution and the extracts were exposed to ultrasonic irradiation with intensity (40 Hz) at 40oC for 30 minutes while the solution was exposed to direct sunlight (visible waves) for 5 min in the optical techniques. The completion of the synthesis of AgNPs at higher speeds is regarded as one of the benefits of these methods. Furthermore, UV-visible absorption occurred in the 300-600 nm region for both methods. The observation of absorption in the region of x nm confirms the synthesis of AgNPs. In the present study, the presence of AgNPs peak at a wavelength of 430 nm is consistent with the observations made by Song and Kim (2009) and Sathyavathi et al (2010).34 Todays, there are many plants which are used for synthesizing metal nanoparticles such as Ag.36 However, only one of these species, S.rechengri is used for AgNPs synthesis although Satureja has many species. Biological organisms are significant at both capping and oxidant agents in the synthesis of AgNPs. A large body of research indicates that the synthesis of AgNPs by using bioorganic compounds is successful. In addition, the reduction of Ag+ by biomolecules including the cell extract such as enzymes, proteins, vitamins, amino acids, and polysaccharides are compatible with the nature.This study was designed to synthesize AgNPs by using 37 Extensive studies have shown that a combination of phenolic acids and amine acids, flavonoids, enzymes and proteins present in the plant extract may be the main cause for rapid and easy reduction of metallic silver ions,38 which play an important role in the synthesis of AgNPs.40 However, the chemical synthesis of such nanoparticles brings about many environmental disadvantages and problems, which is not environment-friendly.41Dehghanizade et al. (2017) investigated the synthesis of AgNPs using Anthemis atropatana extract and its antimicrobial and anticancer effects, and showed that biosynthesis of AgNPs by green chemistry is easily affordable and there is no require any reducing agents.42 Thus, this spectrum confirms the formation and fineness of AgNPs  spectrometer was used . In geneThe analysis of the structure and morphology of nanoparticles, as well as their size and surface morphology, was performed by SEM microscopy. The synthesized AgNPs in the colloidal solution were exposed to irradiation by using light and ultrasound techniques. The comparison of the produced spherical AgNPs indicated that the synthesized AgNPs produced through ultrasonic irradiation technique are smaller in size and more spherical in shape and have a better distribution. The particle size in the ultrasound technique ranges from 44 to 65.3 nm and is 54.75 nm on average  while thS.rechengri was considered based on two bacterial pathogens including Escherichia coli and Staphylococcus aureus. The results were evaluated for the lowest inhibitory concentration (MIC) of AgNPs in terms of the growth of selected bacteria. The results of some studies indicated that the synthesized AgNPs cause bacterial cell death by destructing the cell membrane of the bacteria such as Staphylococcus and E-coli at a given concentration  by measuring the optical density based on the concentrations (3.125-6.5-12.5-25-50-100 μg/ml) of green synthesized AgNPs through light and ultrasound techniques. Then, the results were compared with cell survival rate. In addition, the effects of synthesized AgNPs on colon cancer cell lines HT-29) and normal cells (HEK-293) were evaluated. In the next procedure, the survival rate of the treated cells with AgNPs was measured, compared to the survival rate of the untreated (control) cells during 24 and 48 hours after the treatment. Based on the results, the reduction of the cell survival rate was observed with increasing concentrations of AgNPs. In addition, at the highest dose (100 µg /ml), the cell viability for the cells treated by synthesized AgNPs by light and ultrasound techniques were found to be at the lowest rate. Tables  and norm50% for the light AgNPs showed that such nanoparticles can inhibit 50% of the cells during 24 and 48 hours at concentrations of 1.3 and 0.71 µg /ml. Furthermore, light AgNPs within 48 hours with lower doses can prevent the growth of 50% of the cancer cells HT-29, indicating a significant difference during 24 hours. Additionally, the results of ultrasound AgNPs in 24 and 48 hours at the concentrations of 4.7 and 2.6 µg /ml indicated that these AgNPs can inhibit 50% of the cells. In addition, the MTT results of synthesized AgNPs through light and ultrasound technique were performed on a healthy HEK-293 cell line. The values were determined for ultrasound AgNPs 3.125-6.5-12.5-25-50-100 μg/ml for 24 hours  and Dr. Amir Mirzaie for useful guidance. This research was sponsored by Islamic Azad University, Science and Research Branch of Tehran, Iran.This project was conducted in accordance with the principles laid down in the Helsinki Protocol and approved by the Ethics Committee of the Council of Science and Research of the Islamic Azad University.The authors report no conflicts of interest in this study.


Hyperspectral sensors, especially the close-range hyperspectral camera, have been widely introduced to detect biological processes of plants in the high-throughput phenotyping platform, to support the identification of biotic and abiotic stress reactions at an early stage. However, the complex geometry of plants and their interaction with the illumination, severely affects the spectral information obtained. Furthermore, plant structure, leaf area, and leaf inclination distribution are critical indexes which have been widely used in multiple plant models. Therefore, the process of combination between hyperspectral images and 3D point clouds is a promising approach to solve these problems and improve the high-throughput phenotyping technique. We proposed a novel approach fusing a low-cost depth sensor and a close-range hyperspectral camera, which extended hyperspectral camera ability with 3D information as a potential tool for high-throughput phenotyping. An exemplary new calibration and analysis method was shown in soybean leaf experiments. The results showed that a 0.99 pixel resolution for the hyperspectral camera and a 3.3 millimeter accuracy for the depth sensor, could be achieved in a controlled environment using the method proposed in this paper. We also discussed the new capabilities gained using this new method, to quantify and model the effects of plant geometry and sensor configuration. The possibility of 3D reflectance models can be used to minimize the geometry-related effects in hyperspectral images, and to significantly improve high-throughput phenotyping. Overall results of this research, indicated that the proposed method provided more accurate spatial and spectral plant information, which helped to enhance the precision of biological processes in high-throughput phenotyping. Accelerating the process of breeding crops and ensuring their production is a critical step to solving the global food security problem by 2050, where a world population of 9.7 billion is projected . For thaCurrently, the research on phenotyping focuses on analysis based on the measurements of hyperspectral information, which can be used to compute various indexes , to indicate plant biochemical contents . However, the complex geometry of plants  and their interaction with the illumination setting, severely affects the spectral information obtained . FurtherHyperspectral sensors are often designed as line scanning cameras that break down the spectral composition of a 1D pixel line onto a 2D CCD (Charge-coupled Device) array, which records one spatial dimension and one spectral dimension. Consequently, a 2D hyperspectral image is composed of consecutively recorded lines . CurrentMost publications based on the line scanning camera model, focus on the scale of observations from air or spacecraft ,18. HoweIn this paper, we added the examination of the reference gauge and proposed a novel and simple geometric calibration method, for a close-range hyperspectral pushbroom camera using the reference objects to combine spectral and spatial information.Capturing the 3D geometry parameters of plants is a common technique in plant science, which can be applied for the measurements of canopy structure, height, leaf angle distributions, root architecture, and shoot structure across various scales, ranging from laboratory to greenhouse and field ,23,24. DLiang et al.  , as shown in Kinect V2 is a second generation range sensor of Microsoft windows, shown in The fusion algorithm was based on the selected reference points, with corresponding projections in the hyperspectral camera and depth sensor. For this purpose, a reference gauge was applied, which consisted of two planes, shown in The reference gauge was assessed using a high precision 3D printer Form 2 , with an accuracy of 0.2 mm. The selected reference points .The system was mainly based on a high-throughput phenotyping platform, consisting of the Middleton Research Via-Spec II Hyperspectral Scanning Station, which can be programmed using Python to perform continuous imaging. The structure of the scanning station could be separated into three parts: A base platform with a designed reference gauge on it, a vertically transformable platform with halogen light source, and an imaging system on the top. As shown in Before imaging, the 3D coordinates of the selected points on the reference gauge were measured using a Vernier caliper. Y-axis was defined as the same direction as the movement of the base platform. The X-axis was defined as the direction vertical to the Y-axis on the horizontal plane. Z-axis was defined as the vertical direction of the X-Y plane. The base platform could move using the motor which was built inside. The motor was controlled by the official Middleton software MSV.UI.Measure, which could move the whole upper platform along the Y-axis with an acceleration-free linear movement, without rotations.After warming up the imaging system, hyperspectral images and the 3D point clouds were captured synchronously. The 3D coordinates of the selected points, the corresponding hyperspectral image coordinates, and the corresponding 3D point clouds were later used as inputs for the fusion model.We used soybean leaf to verify the method proposed in this paper. The soybean variety was Asgrow 30X6, and the growth stage of the plants in the experiment was V3 (V3 is a staging system we used to indicate that the plants had 3 trifoliate leaves open) with 4 nodes. The soybeans were grown in commercial potting media , in 4-inch square pots. Moreover, the soybeans were watered as needed, and fertilized weekly . The greenhouse temperature was maintained between 23 and 29 C. Supplemental light was provided by 600 W high pressure sodium bulbs, set to a 16-h photoperiod.The component of the fusion procedure for the close-range hyperspectral pushbroom camera and the depth sensor, was presented in this section. The reference gauge provided a high number of detectable, homogeneously distributed, and accurate reference points. Based on the selected points, the fusion model of the depth sensor and the close-range hyperspectral camera was applied.The image coordinates of the projection for the selected points of the chess patterns in the hyperspectral image, were the input parameters of the fusion algorithm. The detectCheckerboardPoints function in Matlab 9.2, was used as a corner detector for semi-automatic point detection. Worth noting that the locations of patterns were input manually, and the semi-automated corner selection method was necessary. The points on the upper plane of the reference gauge had to be reliably separated from those on the lower plane, for a correct assignment of the 3D coordinates. Furthermore, the semi-automatic approach was more flexible if only a part of the reference gauge is observed or if the reference gauge is rotated .Since the depth and the hyperspectral image information were captured by different kinds of sensors, the matching between hyperspectral images and 3D point clouds was required. The close-range hyperspectral pushbroom camera could be modelled as a linear camera. Like a perspective camera model, the linear camera model describes the relation between the selected points in 3D coordinates and the pixels in 2D image coordinates, as outlined in References ,21,32,33The resulting hyperspectral images in this paper, were created by the image coordinate u-axis (the movement direction) and the v-axis (the direction of the view plane). Worth noting that the movement of the base platform was an acceleration-free linear movement without rotation. Therefore, the pushbroom camera could be modelled by two parts, one as the view plane section, and the other one, as the movement direction section. The details of the fusion model, are shown in For the view plane section, according to projection role of hyperspectral camera noted by Gupta and Hartly , the equu is the v value of object point in o-uv coordinate, y is the Y axis value of object point in O-XYZ coordinate, and s is the scanning speed of the hyperspectral camera.For the movement direction section, as the movement of the base platform was acceleration-free linear without rotation, the image coordinate u in the moving direction is the result of a parallel projection, which is only determined by scanning speed s and the moving distance y. That can be summed up as:Therefore, to combine the spatial and spectral information between the hyperspectral camera and depth sensor, only the focal length of the hyperspectral camera and the scanning speed in pixel scale are needed.1Y1Z1, which is corresponding to the system 3D coordinate O-XYZ in s, and the movement between two pixel lines. The imaging characteristics of the line scanning camera were parametrized by the principal point (pv) of the sensor line (v-direction) and the focal length A uniform 3D coordinate system was built to implement the fusion model. As shown in Therefore, the fusion equation can be achieved:T was the 3D coordinate of hyperspectral camera optical center in the base platform coordinate O-X1Y1Z1, where the value was T mm in this paper. RH2O is the rotation matrix from hyperspectral camera coordinate H-X2Y2Z2, to the base platform coordinate O-X1Y1Z1. It was easy to know that:According to Savage, Paul G , the 3D 1Y1Z1 coordinate, the corresponding coordinate of reference points in the H-X2Y2Z2 coordinate can be calculated by Equation (5).Depending on the known coordinate in the O-XT was the 3D coordinate of the optical center of the infrared camera in the base platform coordinate O-X1Y1Z1, where the value was T mm in this paper. RK2O is the rotation matrix from the Kinect V2 coordinates K-X3Y3Z3 to the base platform coordinates O-X1Y1Z1, which is equal to RH2O.Same as the 3D transformation relationship between the hyperspectral camera and the base platform, the 3D transformation relationship between the Kinect V2 and the base platform was:The Equations (4) to (6) were used to calculate the 3D transformation relationship, between the hyperspectral camera and the Kinect V2 (Equation (7)):Up to now, the fusion model between the hyperspectral camera and the Kinect V2 can be modified as:Based on at least three non-planar selected points, the components of f and s can be estimated using the pushbroom camera model in The description of pushbroom camera model contained parameters defined in the coordinate system of the selected reference points. The compact representation fusion algorithm, allowed us to apply the camera model to a similar pinhole camera model, and to support the exchange with available frameworks. Distortion, non-linear camera movement, and camera rotations in close-range applications will cause non-linearity. However, in this paper the hyperspectral camera was fixed, and the base platform was moving with an acceleration-free linear movement without rotations. Moreover, 20 images were captured one time for average analysis. Therefore, the non-linearity was ignorable in this paper.The experiment took place in a dark room at Purdue University. After warming up the system, hyperspectral images and 3D point clouds were captured by the computers synchronously. The setting of the Middleton software was shown in The hyperspectral image coordinate and system 3D coordinate of the selected reference points can be achieved, both from the hyperspectral camera and the Kinect V2 depth sensor. Worth noting that the 3D cloud points needed to be captured first because the base platform was moving during the imaging. Since the definition of image coordinate and the system 3D coordinate were both positive, to get the corresponding 3D coordinates from 3D point clouds (providing both positive and negative 3D points), a simple rectification coefficient needed to be calculated before the derivation of the fusion model. These could be abridged using the first quadrant of the Kinect V2 3D coordinate to measure the selected reference points. However, it is a special condition and needs a higher requirement for hardware platform setup. Based on at least six non-planar reference points, the components of rectification coefficients can be estimated using Cartesian coordinates principle. We used the same 8 reference points shown in For example, for X axis corrected coefficients, from reference points 1 and 2 we were able to know:The fusion model between the pushbroom hyperspectral camera and the depth sensor was modified as:The average of the 20 images was used to calculate the fusion model. The processing hyperspectral images and 3D point clouds, were shown in The corresponding image coordinates from the hyperspectral camera and 3D coordinate from Kinect V2 of the selected reference points, were shown in In order to verify the validity and accuracy of the fusion model proposed in this paper, soybean leaf was used to implement the validation experiment. Theoretically, the validation of the fusion model from both depth sensor information to hyperspectral camera information, and hyperspectral camera information to depth sensor information is needed. However, according to the principle of machine vision, image coordinates can be only derivated to 2D coordinate information without depth information. Therefore, we used depth information from Kinect V2 to derivate the pixel information from the close-range hyperspectral camera, to verify the fusion model proposed in this paper.The experiment setup was the same as in After segmenting 3D point clouds of soybean leaf into the fusion model as described in Reference , correspThe imaging system had the ability to reproduce the position of a start point of the system, which was a very important feature as it enabled the direct reuse of the fusion model for multiple images. The proposed method relied on the assumption that all bands of a surface point were projected to the same point in the image . We reviSeveral factors may influence the accuracy of the fusion model, including the sensor resolution, the setup of experiment platform, and the precision of data processing algorithms. Higher resolution hyperspectral cameras can achieve higher quality hyperspectral images, especially in u-axis image coordinate. With a higher resolution depth sensor, for example, 0.1 mm scale accuracy, pixel level hyperspectral image can be matched with corresponding 3D information using the fusion model in this paper. We attached the plants and gauge in the middle of the assigned start point and end point, to guarantee the uniform scanning speed when imaging the objects. Furthermore, we used an average method (captured 20 images and selected 8 reference points evenly) to minimize the error that may be caused by the corner detected algorithm.2 and 63.1° inclination, respectively. With the built of soybean leaf hyperspectral 3D model, more precise (pixel level) hyperspectral analysis can be achieved. From the figure, we could easily find out that leaf angle has a severe influence on the biological processes of soybean leaf. The 3D information of plants is required when using the hyperspectral camera in a high-throughput phenotyping platform.In this section, we discuss approaches that can add the value of the fusion model to be demonstrated in further plant observations, which are based on the linkage between hyperspectral images and 3D point clouds (depth information). This combination can be used to combine the data of more than one hyperspectral camera with depth sensors, which will help to solve the overlapping problems that were not considered in this paper. For example, a normal high-throughput greenhouse conveyor phenotyping platform usually contains two hyperspectral cameras, one is for top view imaging and one is for side view imaging, which helps to overcome overlapping during imaging. Depth sensors can attach to each hyperspectral camera, using the method proposed in this paper and build new high-throughput phenotyping platforms, which can avoid the overlapping impact and jointly analyze plants spatial and spectral features. Leaf structure, leaf area, and leaf inclination distribution can be calculated with a plant’s 3D information, which provides the possibility to build a pixel level hyperspectral 3D plant model ,12,13,14More and more researchers have realized the importance of plants 3D information in hyperspectral imaging. Paulus et al.  pointed The proposed method for combination of a close-range hyperspectral pushbroom camera and a low-cost depth sensor, is based on the pushbroom camera model and the selected reference points. The presented pushbroom camera was modelled using the view plane section and the movement direction section. Eight reference points on both the upper and lower planes were evenly selected, which were used to combine spectral information from the hyperspectral camera and spatial information from depth sensors. The validation soybean leaf indoor experiments, showed that the fusion model between the hyperspectral image system and Kinect V2 was able to fulfill the specified demands for close-range phenotyping application. The results showed that in a controlled environment, 0.99 pixel level accuracy for the hyperspectral camera and 3.3 mm accuracy for the depth sensor could be achieved, using the method proposed in this paper. We also discussed the new capabilities gained using this new method, which can allow the effects of plant geometry and sensor configuration to be quantified and modelled. The possibility of 3D reflectance models can be used to minimize the geometry-related effects in hyperspectral images; and therefore, has the potential to improve high-throughput phenotyping significantly. This method contributes to the potential of combining images from two or more  sensors, and the derivation of hyperspectral 3D models.High throughput phenotyping platforms that integrate depth sensors, hyperspectral cameras, and other cameras (such as thermography images), may fuse the recorded data automatically based on the proposed fusion model in this paper. The 3D plant models may be built by the automated sensing systems, under strictly controlled environmental conditions with a high level of reproducibility. With the 3D plant model library, a 3D white reference system may be built to improve the simulation of illumination significantly. Large amounts of 3D plant model data sets will provide more accurate analysis methods, for a high number of phenotypic traits.


The use of recycled concrete aggregates (RCA) in high performance concrete (HPC) was analyzed. The paper presents the experimental studies of model reinforced concrete beams with a rectangular section using high-performance recycled aggregates. Two variable contents of recycled aggregate concrete were used in this study: 50% and 100%. The experimental analyses conducted as immediate studies concerned the following issues: short time loads-deflection, load-carrying capacity of beams, deformation of concrete, cracks, and long-term loads-deflection. The comparative analysis involves the behavior of beams made of high performance concrete-high strength concrete (HPC-HSC) recycled aggregates with model control elements made of regular concrete based on natural aggregates. The deflection values for the recycled aggregate beams were 20% higher than in the case of the control beams made of HPC-HSC exclusively. Replacement of aggregate with recycled concrete aggregate resulted in a large decrease in the value of these two parameters, i.e., compression strength by about 42% and modulus of elasticity by about 33%. The modern construction industry should meet the requirements of providing durable and ecological structures. There are a number of factors that prevent concrete structures from meeting the basic standards of sustainable development.Special attention should be paid to the problems of climate changes, depletion of natural resources, and non-environmentally-friendly cement production. During the cement production process, a large amount of gases is released into the environment, contributing to the global greenhouse effect and climate changes in many parts of the Earth. Moreover, large quantities of particularly valuable natural resources such as gravel, sand, crushed rock aggregate, and water, are used in the course of concrete production. It is estimated that, in order to produce Portland cement, approximately 1,000,000,000 tons of raw materials are used each year.Over the last 30 years, a rapid development has occurred in terms of designing, optimizing and improving formulas of high-performance concrete. This type of concrete has been increasingly widely used in practice. The future of HPC is high and super-high buildings . NowadayAnother reason for the increased interest in high-performance concrete involves stronger and stronger trends connected to implementation of recycling in construction ,12. In aAs a consequence of ecological practices, a growing tendency for the application of recycled materials can be observed in many areas of industry. Using construction and demolition debris as a substitute for natural aggregate in the production of concrete mixtures is gaining popularity due to both economic and ecological reasons. The use of construction and demolition debris means limiting the use of expensive natural aggregate, and thus minimizing the negative impact on the natural environment. The maximum benefit is achieved when the greatest possible amount of recycled aggregate is used in the production of concrete ,20,21,22It can be assumed that, because of the growing social emphasis on ecological aspects, the recycled aggregate will be increasingly widely applied as a construction concrete component of full value. Even though HPC is used for building structures, they may have to be demolished at some point in the future. The obtained debris may be reused as recycled aggregate made of high-performance concrete of which the structural elements had been made. The issue pertaining to the reuse of building rubble from demolition or reconstruction has been a long-standing concern in industrialized countries ,30,31,32The aspects connected to protecting the natural environment gain importance with time; therefore, the interest in alternative construction solutions has been growing. The idea of reusing concrete debris is increasingly often taken into consideration as people are against establishing another open-pit mine to mine aggregate, since they have a significant negative impact on large areas.Two fundamental reasons leading to the popularization of recycling are the continuous growth of the amount of concrete waste and the constant increase in the use of concrete as a construction material ,33.Górażdże are presented in The concrete was prepared using CEM I 52.5 R Portland cement. The technical parameters of applied Portland cement Natural rinsed quartz sand (0–2 mm), natural basalt aggregate (BA) (2–8 mm), recycled aggregate (2–8 mm), and silica fume (SF) were utilized for concrete preparation. The grain size of BA and RA is shown in Three concrete mixtures were prepared with the composition presented in The chemical composition of the natural quartz is shown in −3. The properties of basalt and recycled aggregate are shown in The density of quartz sand amounted to 2.65g∙cmThe chemical composition of the basalt is shown in The chemical composition of the silica fume is shown in −3. The properties of the superplasticizer are given in The density of silica fume amounted to 2.40 g∙cmIn order to determine the physical properties of each concrete type, 18 cubic samples with an edge length of 150 mm were prepared (6 for each concrete). They were used to determine the apparent density, absorption of water and bulk density. The bulk density was determined according to the PN-EN 12390-7 standard . In ordeConcrete porosity was tested by capillary mercury injection (MICP) using AutoPore IV 9520 t  to test pores between 2 nm and 200 μm in diameter . Cubic sG—deformation at the upper range of the force level, amounting to 250 kN ; the measurement base—100 mm, εD—deformation at the lower range of the force level, amounting to 20 kN ; the measurement base—100 mm, σG—stress at the upper range of the force level, σD—stress at the lower range of the force level.In accordance with the PN-EN 12390-3 standard , the comThe bending tensile strength test was carried out in accordance with the PN-EN 12390-5 standard  on the cCarrying capacity, deformations and deflections were determined on 9 cuboid reinforced concrete beams with the dimensions of 80 mm × 120 mm × 1100 mm after 28 days of samples maturing . The reiThe research elements were placed on the test stand. The tests on the beams were performed using a universal testing machine, with a simple-supported beam as the static diagram. The momentary load consisted of two concentrated forces applied at one-third of the beam spans. After the beams had been placed on the test stand and the appropriate sensors had been set to measure the particular parameters, the measurements began. The force was applied slowly, and it was increased by 5 kN; then, the necessary measurements were conducted. The strength tests of the investigated reinforced concrete beams were performed until their destruction. The deformations of concrete were measured with an extensometer. The measurements were carried out at the two levels: 1 (in the compression zone) and 2 (in the tension zone), each time after the load force value had increased.The measurements of the deflection of the reinforced concrete beams were carried out using dial gauges with 0.001 mm resolution. The gauges were placed at three locations: on the left support, in the beam midspan, and on the right support. The deflection was measured each time after the force level had been increased by 5 kN. The long-term measurements consisted of measuring the deflection of the bent concrete elements. The aim of this research was to analyze the deformability of the beams under study in the function of time, with the constant loading value. The bars with the dimensions of 40 mm × 40 mm × 500 mm were the test samples for the long-term experiments (3 for each concrete). The test elements were placed in the test stand designed specifically for the purpose of this research. A load was applied to the beams in a room maintained under constant temperature conditions. The deflections of the bars under research were read every day over approximately 100 days.The physical properties of the recycled aggregate concrete and the HSC-HPC concrete adopted for the examination are shown in −3 g∙mm−2 after 72 h, while the recycled concrete with 50% and 100% substitutes, showed a value of 1.68–2.83 × 10−3 g∙mm−2 and 1.84–3.43 × 10−3 g∙mm−2 [The use of recycled aggregate resulted in a reduction in the apparent density of REC-1 and REC-2 concrete by 4% and 7%, respectively, compared to the reference concrete. Moreover, a clear influence of the recycled aggregate addition on water absorptivity can be observed; an increase of 18% was recorded for the REC-1 concrete and 49% for REC-2. The use of recycled aggregate has also contributed to an increase in open porosity by 15% for REC-1 and by 38% for REC-2, which affects lower compression strength, as shown in 3 g∙mm−2 , respectIn the water absorption and sorption tests carried out by Zega and Di Maio, these parameters for recycled aggregate concretes (RACs) were 15% higher than those for the reference concrete, while, in the water penetration test, all concrete types show a similar behavior. This is due to the different water transport mechanisms in each of these concretes .According to Zhang et al. , the RACc value of 56 MPa is found in the concrete with the highest recycled aggregate content (REC-2 100%). It is 30% lower than for the concrete with 50% recycled aggregate content and as much as 43% lower than for the basalt concrete. After 14 days and after 28 days of ripening of all three concrete types, the basalt concrete still showed the best compression strength results. The worst compression strength after 7, 14, and 28 days was recorded for the concrete with 100% recycled aggregate content, which was 43%, 35%, and 43% lower than the reference concrete, respectively.Other researchers, e.g., Çakır , also noThe compression strength of RACs was least dependent on the quality and quantity of RAC than in studies carried out by other authors. After 90 days of concrete ripening, RACs showed higher increases in the compression strength than NAC. According to some authors ,19, imprc decreased faster with the increase of the recycling rates of aggregate under autoclaving conditions than under standard conditions of its ripening. In the latest research of Wang et al. [As noted by Zhang et al. , fc decrg et al. , the comg et al. ,56,57,58In the study by Fonseca et al. , it was cm) after 7, 28, and 56 days was 42.8, 49.8, and 51.6 MPa, respectively. In general, after 7 and 28 days of curing, the samples showed about 80% and 95% of their 56-day compression strength [As shown by Fonseca et al. , the com2 = 0.988 and relatively low errors in the intercept. The obtained correlations can be described by the equation: y = 0.325x + 13.90.Flexible modulus of elasticity is closely related to the compressive strength of concrete. The highest parameters were achieved with the concrete comprising the HSC-HPC natural aggregate. Replacement of the aggregate with recycled concrete aggregate resulted in a large decrease in the value of these two parameters—compression strength by about 42% and modulus of elasticity by about 33%.c of concrete barely grows after 28 days. Ec after 50 years is only 6% higher than Ec after 28 days, according to EC2 [c value of 45 MPa with a recycled fine aggregate, the modulus of elasticity E varies between 17 GPa and 35 GPa with a 109% difference, while E varies between 15.6 GPa and 36.2 GPa with a 132% difference for coarse recycled aggregate concrete. In addition, the E value decreases by an average of 23% and 32% when the natural aggregate is replaced by the recycled aggregate in 50 and 100%, respectively [Concrete is generally considered to be a composite material consisting of three phases: aggregate, cement paste and ITZ . Elasticg to EC2 . The litg to EC2  showed tectively ,64,65,66As shown by Fonseca et al. , the 28-Zhang et al. , similarAjmani et al.  tested tThe strength tests of the reinforced concrete beams under investigation were performed until the tested beams were destroyed. At that moment, in each case, a sudden decrease in the rigidity of the beams was observed, as well as a rapid increase in the number of cracks that became much wider, which was indicative of the reinforcement in the tension zone becoming more susceptible to fatigue, and the concrete in the compressed zone was destroyed. The beams were destroyed suddenly, which was accompanied by a characteristic cracking sound. There are many factors that influence the fact that the cracking of reinforced concrete structures is a complex phenomenon. Monitoring of cracking in the reinforced concrete elements is very important because the presence of cracks may indicate the loss of the load-bearing capacity of a structure, which would lead to the concrete being destroyed. Performing the measurements of the width and character of cracks influences the evaluation of the construction safety. The cracking was monitored simultaneously with the deflection and deformation measurements of the tested elements, carried out by the authors of the present paper. Each time, after the loading force had been increased by 5 kN, the appearance and penetration of the formed cracks were marked, with the loading force value inscribed at the end of each crack. The first cracks were observed when the value of the applied loading force was approximately 30% of the critical load, which amounted to 10 kN and 15 kN for the recycled aggregate beams and natural aggregate beams, respectively. The mean experimental values of rupture force and breaking moments of the recycled aggregate concrete and the HSC-HPC concrete adopted for the examination are shown in sd.2 + 0.0143x + 6.53 indicate a good coefficient of determination R2 = 0.954. Bending tensile strength and breaking moment Msd of concrete also depend on the type and quantity of aggregate used. The best strength parameters were achieved by the concrete with basalt aggregate. As the amount of recycled aggregate in the concrete increases, the bending strength and the breaking moment causing the beams to break down decrease. This moment is approximately 10% lower for the REC-2 concrete than for the HSC-HPC concrete.The correlations described by the polynomial formula y = 0.0169xHaving analyzed the above-mentioned results and deformation graphs, it can be concluded that the recycled aggregate beams under study showed greater deformation than the tested natural aggregate beams for the respective values of the loading force. After comparison of the obtained results, it was concluded that the observed mean deformations in the compressed zones of the recycled aggregate beams were, on average, 39% greater than in the case of the natural aggregate beams, while the deformation values in the tension zones were greater by 20% for the recycled aggregate beams in comparison to these for the natural aggregate beams.p takes values < 0.05 . The ANOVA test showed no significant differences in the deflection values between REC-1 and REC-2 (p = 0.765).On the basis of the test results shown in Having analyzed the obtained results of the long-term tests consisting in measuring the deflection of the bent concrete elements, it was concluded that the deflection values of the elements made of the recycled aggregate concrete were, on average, 45% higher than the values of the elements which were made solely of high-performance concrete.Similar observations on deflections and long-term deflections were made by Łapko A. and Grygo . In theiIn contrast to others, Łapko and Grygob  based thIn several studies ,76,77,78Recycled aggregate is viable for use as a substitute of coarse aggregate in concrete structures. Attention has to be paid, however, to the deterioration of some concrete parameters, such as the increased deformability.When designing compositions of concrete mixtures with the use of recycled material, a larger safety margin has to be provided for.The use of recycled aggregate resulted in a reduction in the apparent density of REC-1 and REC-2 concrete by 4% and 7%, respectively, compared to the reference concrete.A clear influence of the recycled aggregate on water absorptivity can be observed; an increase of 18% was recorded for the REC-1 concrete and 49% for REC-2.The use of recycled aggregate contributed to an increase in open porosity by 15% for REC-1 and by 38% for REC-2, which affects the lower compression strength.Replacement of aggregate with the recycled concrete aggregate resulted in a large decrease in the value of these two parameters—in the compression strength by about 42% and modulus of elasticity by about 33%.As the amount of recycled aggregate in the concrete increases, the bending strength decreases.The deflection values for the recycled aggregate beams were 20% higher than in the case of the control beams made solely of HPC-HSC.The long-term tests consisting of measuring the deflection of the bent concrete elements, showed that the deflection values of the elements made of recycled aggregate concrete were on average 45% higher than the values of the elements, which were made of high-performance concrete exclusively.Replacement of basalt with recycled aggregate resulted in the changes in the concrete structure, which affects its physical and strength parameters.It can be assumed that the recycled aggregate will be increasingly commonly used as a construction concrete component of full value because of the increasing social emphasis on the ecological aspects.Hopefully, the continuous development of technologies in the construction sector will lead to a reduction in the amount of waste produced as well as an increase in the number of materials which can be recycled and reused in concrete manufacturing.The following key conclusions can be drawn from this study:


The healing of bone defects in the craniofacial region is an important clinical issue. We aimed to compare the effects of octacalcium phosphate (OCP) and the combination of OCP/gelatin (OCP/Gel) on calvarial bone regeneration in rats.In this study, 72 male Sprague Dawley rats were randomly assigned to the OCP (n=24), OCP/Gel (n=24), and control groups (n=24). Lesions with a diameter of 9 mm were created in the parietal bone and were filled with 9-mg OCP and OCP/Gel disks. In the control group, no substance was implanted in the defect. Sampling was performed on days 10, 14, 21, and 28 after the implantation. After tissue processing, 5-μm sections were prepared and stained by hematoxylin and eosin (H&E) stain. The sections were studied, and the volume fraction of the newly formed bone was assessed by Kruskal-Wallis test at a significance level of 0.05.In the experimental groups, new bone formation was detected at the margins of the defects 10 days after the implantation. With the progression of the healing process, the newly formed bone covered greater areas of the defects and developed a more mature structure. In the control group, the defects were primarily filled with a dense connective tissue with small islands of new bone. The results of histomorphometric assessments showed that the volume of the newly formed bone in the experimental groups had a significant statistical difference with that in the control group (P<0.001).The OCP/Gel composite can be useful in the healing process of calvarial bone defects. Bone defects significantly decrease the quality of life of the affected patients . Differeite (HA) ,12 or amite (HA) . It has ite (HA) ,15 afterite (HA) . It is lite (HA) . Althougite (HA) . A greatite (HA) . Combiniite (HA) . Collageite (HA) ,22.Gelatin, which is a natural material derived from collagen with an almost identical composition, has been widely used as a matrix to obtain biomimetic CaP in bone regeneration and tissue engineering fields ,24. In aIn an experimental study, Saito et al  evaluateIn another study, Saberi et al  reportedSynthetic OCP was prepared by following the methods previously described ,32. The In this study, 72 adult male Sprague Dawley rats aged 6–8 weeks with an average weight of 120–150 g were used. The rats were obtained from the Animal Research Center of Zahedan University of Medical Sciences and were randomly allocated to two experimental groups (OCP and OCP/Gel) and one control group (n=24 in each group) and were kept in standard conditions with light/dark cycles of equal duration. The principles of the laboratory animal care and the national law were followed. All the procedures were approved by the Ethics Committee for Animal Experiments of Zahedan University of Medical Sciences (IR.ZAUMS.REC.1394–351). General anesthesia was induced by intraperitoneal injection of 60 mg/kg of Ketamine hydrochloride  combined with 20 mg/kg of Xylazine  at a ratio of two to one supplemented with ether inhalation. After disinfection of the operation field, skin incisions were made on the parietal bones. The muscle and periosteum of the calvarium were ablated. A full-thickness of standardized critical-sized trephine defect (9 mm in diameter) was made in the calvarium by utilizing a dental handpiece under continuous irrigation with buffered saline. Extreme care was taken to avoid injury to the superior sagittal sinus and dura mater . In our Afterwards, the periosteum, muscle, and skin were repositioned and sutured by using absorbable 04 sutures . To prevent infection, 20 mg/kg of Gentamicin  was injected intramuscularly for 3 days after the surgery. Based on the study design, sampling was performed during days 10, 14, 21, and 28 after the surgery and implanting the materials.2O2), 2.9% citric acid (C6H8O7), and 1.8% trisodium citrate (Na3C6H5O7) [In all the groups, general anesthesia was induced by intraperitoneal injection of 60 mg/kg of Ketamine hydrochloride , and 10% buffered formalin was perfused through the heart for fixation, and in-situ fixation was immediately done. For a better fixation, the respective area along with a margin of the host bone was resected and stored in 10% buffered formalin at room temperature for one week. The obtained samples were decalcified over four weeks at room temperature by using 10% formic acid (CH3C6H5O7) . After p3C6H5O7) , while h3C6H5O7) .For histomorphometric analysis and determining the volume of the newly formed bone in all the groups, the sections obtained on the 28th postoperative day were used; Six sections were randomly selected from each group  and were placed on three slides (three slides and six sections for each sample). Ultimately, 18 slides and 36 sections were selected in each group for histomorphometric evaluation. The sections were stained by H&E stain and were evaluated by using the light microscope equipped with eyepiece graticules at ×40 magnification according to the point counting technique. The mean volume of the newly formed bone was calculated in all the groups and was expressed in percentages .Histomorphometric data were analyzed by using SPSS software program  and were reported based on the mean, standard deviation (SD), mode, and median. Kruskal-Wallis test was used for comparing the mean volume of the newly formed bone among the groups at a significance level of 0.05.In the control group, the defective area was covered with a highly vascular connective tissue 10 days after the implantation .On the 14th day, the connective tissue filling the defect area developed a better organization pattern, and its adhesion to the defect margins was visible in most parts . On the In the OCP group, on the 10th day, a high density of fibrous elements with differentiating osteoclasts were visible along with epithelioid osteoblasts and the eosinophilic bone matrix . On the In the OCP/Gel group, on the 10th day, the newly formed bone could clearly be detected at two areas: at the peripheral area of the defect attached to the host bone, and in the deep and central parts of the defect around the implanted particles . FurtherThe volume of the newly formed bone in the experimental and control groups on the 28th day after the surgery and implanting the materials was calculated by using the point counting technique, and the mean value was reported as a volume fraction up to two decimals, the summary of which is presented in The effect of synthetic materials, used either alone or combined, on the healing of bone defects has been evaluated in many studies ,39. In tThe histomorphometric findings and comparison of the mean volume of the newly formed bone showed that the volume of the new bone in the experimental groups had a significant statistical difference with that in the control group (P<0.001) indicating the positive effects of the implanted materials, which is consistent with the findings of the study by Suzuki and colleagues . On the The present study demonstrated that an OCP/Gel composite containing a high content of OCP granules is able to improve bone formation and defect healing. By adjusting the type of the natural polymer and the OCP form and content, the bone regenerative properties of OCP can be enhanced and biodegradation can be improved, which may be related to the ability of these materials to stimulate osteoclastic activity. Further studies are underway to examine the bone regenerative capacity of OCP/Gel in experimentally created large bone defects in the maxillofacial region.


The prevalence of gastric cancer (GC) with increased expression of the HER2 oncoprotein shows important variations worldwide. Incidence and mortality rates of GC in Costa Rica are among the highest in Latin America and the world; however, the prevalence of HER2-positive cases in this country is unknown. Evaluation of this parameter is important to decide the therapeutic approach for GC patients. The aim of this study was to provide an estimation of the prevalence of GC patients overexpressing the HER2 oncogene in Costa Rica.in situ hybridisation (FISH) analyses were performed in formalin-fixed and paraffin-embedded (FFPE) surgical samples from 50 patients diagnosed with GC between 2012 and 2015.The investigation was carried out in two phases. The first one consisted of a retrospective review of 331 clinical records of patients diagnosed with advanced or metastatic GC from January 2010 to January 2012 in four hospitals in Costa Rica. In the second phase, immunohistochemistry (IHC) and fluorescent Of the 331 clinical files reviewed, the assessment of HER2 status was carried out in 62 patients (18.7%), of which only five (8%) were HER2-positive. In the 50 surgical specimens in which IHC and FISH analyses were performed, two of them (4%) presented overexpression and amplification of the HER2 oncogene.This study suggests that the prevalence of GC cases overexpressing the HER2 oncogene in Costa Rica is less than 8%. This is the first attempt ever undertaken to estimate the prevalence of HER2-positivity in GC in Costa Rica. Gastric cancer (GC) is the fifth most diagnosed neoplasm and the third cause of cancer death worldwide. In 2018 alone, more than 1 million new cases and 782,000 deaths were attributed to this type of malignant tumour . In CostIn 2010, the first prospective, randomised, multicentre phase III study was published (the ToGA study), in which the efficacy and safety of the monoclonal antibody, Trastuzumab was evaluated in advanced gastric adenocarcinoma and cancer of the gastroesophageal junction overexpressing human epidermoid growth factor receptor 2 (HER2) . This stFunctional studies performed in cancer with HER2 overexpression have shown that HER2 binds to other receptors of the epidermal growth factor receptor (EGFR) family, thus resulting in dimerisation and phosphorylation of these, even in the absence of ligands –14. ConsThe prevalence of GC cases with increased expression of the HER2 oncoprotein presents substantial variations worldwide, ranging between 2% and 45% . This wiin situ hybridisation with fluorescence-labelled probes (FISH) [et al [The methods accepted by FDA and EMA for the determination of HER2 status are immunohistochemistry (IHC) analysis and s (FISH) . The sco) [et al . Accordi) [et al .In Costa Rica, no studies have been conducted to determine the prevalence of GC cases with increased expression of HER2. The determination of the prevalence of malignant lesions with overexpression of HER2 in Costa Rican GC patients is important to have a clearer understanding of the relevance of this genetic alteration in the aetiology of the disease, as a potential prognostic biomarker and as a criterion to decide the therapeutic approach of the patients. Since the prevalence of HER-positivity in Costa Rica is not known, the anti-HER2 agents have not been employed so far for the treatment of GC, which means that HER2-positive GC patients are not deriving clinical benefit from these pharmacological agents. In this study, we provide for the first time an estimation of the prevalence of Costa Rican GC patients overexpressing the HER2 oncogene.The investigation was carried out in two phases. The first part consisted of a retrospective review of clinical records of patients diagnosed with advanced or metastatic GC in public Costa Rican hospitals between 2010 and 2012. Through this revision, we identified the patients who underwent evaluation of HER2 status. In the second phase, IHC and FISH tests were performed on formalin-fixed and paraffin-embedded (FFPE) surgical tissue samples from 50 patients diagnosed with GC at the Calderón Guardia Hospital between 2012 and 2015. The study was approved by the Scientific Ethics Committee of the University of Costa Rica (# 817-B2-371), Institutional Scientific Ethics Committee (R013-SABI-00048) of the Caja Costarricense del Seguro Social (CCSS) and Local Bioethics Committee of the Calderón Guardia Hospital (CLOBI-01 January 2015).We retrospectively reviewed the clinical records of 331 patients older than 18 years that were diagnosed with resectable advanced or metastatic GC between January 2010 and January 2012 in the four public hospitals having Clinical Oncology service at the time of the study; namely, Max Peralta Hospital, Mexico Hospital, Calderón Guardia Hospital and San Juan de Dios Hospital. From each file, we retrieved the following information: anatomical location of the tumour, histological subtype and HER2 status . In those cases, in which the evaluation of HER2 had been made, the date of completion and the type of test carried out (IHC and/or FISH) were documented. The information collected was processed, digitised and managed in databases in Excel files (Microsoft Office).The sample required to carry out this study was determined according to the historical data (3 years: 2008–2010) of the number of GC cases diagnosed per year in Costa Rica, and the number of GC cases treated per year. The statistical power for the present study is 80% (α = 0.05).In addition to generating an estimation of the prevalence of positive cases for HER2, we standardised and validated the tests for the determination of the HER2 status in GC . To do tThe 5-μm-thick FFPE tissue sections were deparaffinised with xylene and hydrated in gradual series of ethanol–water dilutions. HER2 immunostaining was carried out with the HercepTestTM kit  following the manufacturer’s instructions, with minor adjustments in the antigen retrieval and incubation time of the anti-HER2 primary antibody. Heat-induced epitope retrieval in a T/T Micromed microwave processor  was performed at 98 °C for 15 min in the antigen retrieval solution provided by the kit. The primary antibody was applied to the tissue sections and incubated overnight at 4 °C. Evaluation of the stainings was made in an Olympus BX43 microscope by two of the researchers participating in the study (WAA and ECG). A third observer (YRM), an experienced pathologist who also collaborated in this study, independently evaluated the sections.FISH was performed by using the HER2 FISH pharmDxTM kit , kindly donated by the company MAKOL (Costa Rica), following the manufacturer’s instructions with some modifications. Specifically, the tissue sections were placed on a heat plate for 30 min at 37 °C to facilitate the deparaffinisation process. Subsequently, the slides were immersed in xylene and hydrated in a gradual series of ethanol–water dilutions. The FISH tests were evaluated on a Nikon Eclipse 80i microscope  between 30 min and 24 h after finalising the experimental procedure. This evaluation was carried out by two of the researchers participating in the study (ABA and ECG). GC cases showing amplification of the gene encoding HER2 (HER2/CEN17 ratio > 2) were considered HER2-positive; those in which the ratio was less than 2 were regarded as negative.As experimental controls, we selected three FFPE tissue blocks from breast cancer in which IHC and FISH for HER2 had been previously performed. Specifically, one of the tissue samples was HER2-positive (score 3+) and two were negative (score 0). Tissue sections of these blocks were included each time we performed an IHC staining for GC samples. Similarly, control slides provided by the manufacturers as part of materials of the HER2 FISH pharmDxTM kit were included each time we performed FISH experimental procedures.For the present investigation, a total of 331 clinical records were reviewed. According to these files, only 62 of the patients were subjected to HER2 IHC/FISH analysis, and five of them (8%) were HER2-positive. Of these five patients, two had a malignant lesion located in the stomach body, one in the antrum and two in the body-antrum. According to histological criteria, three tumours were classified as well-differentiated and two as moderately differentiated; that is, five cases were of the intestinal subtype according to Lauren’s scheme. Regarding the TNM stage at the time of diagnosis, two patients corresponded to stage II-A and three to stage II-B. The average age of the five patients was 73 years (range 66–80 years) .When performing FISH tests in the 50 tissue samples of GC lesions, 100% concordance between them and the IHC was observed. Specifically, amplification of the gene encoding HER2 was observed in the two samples showing overexpression of the HER2 oncoprotein in the IHC analysis . The valFinally, IHC and the FISH were repeated in the two GC samples showing overexpression and amplification for HER2, respectively. This was done independently by a second investigator (ABA) in the facilities of the Molecular Oncology Laboratory at the Calderón Guardia Hospital, obtaining the same result as in the initial determinations, thus confirming the veracity and reproducibility of the IHC and FISH analyses.Overexpression of HER2 in malignant gastric tumours has been described in multiple studies, , 18, 19;According to our review of 331 clinical records, only 62 (18.7%) of the patients diagnosed with advanced GC underwent IHC and FISH analysis, and 8% of those overexpressed HER2. Importantly, according to the accessed clinical records, more than 81% of the 331 patients were not subjected to IHC or FISH analysis, which is due to the fact that these analyses are not routinely carried out for GC in Costa Rica. Although our study provides an estimation that is in agreement with the prevalence reported in other countries , 21, it ERBB2 is a biological indication of a more aggressive cancer phenotype; it has been shown that HER2 overexpression, due to amplification of this gene, in GC is associated with worse prognosis [In an attempt to better define the prevalence of advanced GC cases overexpressing HER2, IHC and FISH analyses were carried out in postoperative tissue samples from 50 patients. The IHC revealed overexpression in 2 of the 50 cases, which corresponds to 4%. These two HER2-positive cases corresponded to the intestinal histological subtype of GC. According to the literature, the HER2 oncoprotein tends to be more frequently overexpressed in intestinal subtype , 12. Alsrognosis –23.Tests for the diagnosis (IHC) and confirmation (FISH) of HER2 overexpression can lead to variability in the obtained results due to factors such as the conditions in which the tests are performed, and interpretation criteria. Therefore, thorough standardisation and validation are of paramount importance when implementing these tests in a laboratory . Thus, oAmong the limitations of this study was the use of breast cancer tissue of known HER2 status as controls, mainly because of the differences in HER2 IHC staining patterns between breast and GC. Notwithstanding this, we utilised breast cancer tissues merely as experimental control; that is, to ensure that the IHC staining procedure was properly done. Also, we did not have GC tissue of known HER2 status at that time to use as the experimental control. The control breast cancer tissue was, in addition, used in the FISH tests, as a confirmatory method of the adequate performance of the test, in addition to the control slides provided by the manufacturers as part of the FISH kit. Another limitation in our study pertains to the data obtained from the clinical records; the low number of patients for whom HER2 analyses were requested and the nonperformance of FISH as a confirmatory test for all cases with IHC 2+. Finally, we did not repeat the IHC and FISH analyses in all 50 GC tissue specimens, but only the two samples in which IHC against HER2 revealed positive staining/amplification; this could also be considered as a limitation of our study.The present study suggests that the prevalence of GC cases with high expression of the HER2 oncogene in Costa Rica could range between 4% and 8%. Although we provide, for the first time, an estimation of the prevalence of HER2-overexpressing cases of advanced GC in Costa Rica, by no means should this be interpreted as the real prevalence. As already alluded to, most of the GC cases were not subjected to evaluation of the HER2 status, as evidenced in the clinical records; also, the clinical records retrieved for this study only represent a specific period of time, and the 50 surgical specimens used to perform IHC and FISH were not randomly chosen. Notwithstanding this, the prevalence here reported is consistent with other reports from various latitudes. Given the very high mortality rate of GC in Costa Rica, we suggest that HER2 testing should be considered at least in those cases that, according to clinical-pathological characteristics, are more likely to be positive for this oncoprotein. We consider the latter as a potential strategy to adopt in countries in which the health budget is limited, as this information is crucial to provide clinical benefit to GC patients with the highest risk of overexpressing HER2.The authors declare that they have no conflicts of interest.Vicerectoria de Investigación, Universidad de Costa Rica, MAKOL.